Peripheral Blood Mesenchymal Stem Cell–Derived Exosomes Improve Renal Sympathetic Denervation Efficacy Through β-Catenin-Mediated Cardiac Reprogramming

Peripheral Blood Mesenchymal Stem Cell–Derived Exosomes Improve Renal Sympathetic Denervation Efficacy Through β-Catenin-Mediated Cardiac Reprogramming | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Peripheral Blood Mesenchymal Stem Cell–Derived Exosomes Improve Renal Sympathetic Denervation Efficacy Through β-Catenin-Mediated Cardiac Reprogramming Shaoheng zhang, Lan Zhao, Chen Li, Zhichuan Huang, Jianshuo Wang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6409278/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objectives To explore the role of self-peripheral blood mesenchymal stem cell (PBMSC)-derived exosomes (Exos) in enhancing renal sympathetic denervation (RD)-mediated cardiac repair following myocardial infarction (MI) in a porcine model. Methods Pigs (EF < 40% post-MI) were randomized to early sham RD or RD. At 2 weeks post-MI, autologous PBMSC-Exos were collected. At 30 days post-MI, pigs received either PBMSC-Exos (2 × 10¹³ particles) or PBS and were followed until 90 days. Another cohort underwent myocardial biopsy at 14 days post-MI to assess PBMSC-Exos effects on ischemic cardiomyocyte (CM) reprogramming, followed by AAV therapy with miR-141-200-429 sponges or negative control (NC) sponges to explore the role of miR-141-200-429 clusters in reprogramming. Results Two weeks post-MI, RD hearts showed increased Exos uptake and inhibited the sympathetic nervous system. By 90 days, the RD + Exos group had 11–26% higher EF than single-treatment groups (all P < 0.001), with improved survival and reduced fibrosis. Exos therapy enhanced RD effects by inhibiting the renin-angiotensin-aldosterone system and transferring the miR-141-200-429 cluster into ischemic CMs. CMs from RD-treated hearts co-cultured with PBMSC-Exos RD exhibited a more immature state, promoting reprogramming. β-catenin overexpression further enhanced PBMSC-Exos RD effects, while miR-141-200-429 inhibition blocked RD-induced CM reprogramming and survival. Ultimately, PBMSC-Exos RD reduced Dkk1 expression and activated GSK3β phosphorylation, thereby stimulating the Wnt/β-catenin pathway. Conclusions PBMSC-Exos RD enhances RD-mediated cardiac repair by activating the Wnt/β-catenin pathway via miR-141-200-429 cluster, offering a novel therapeutic strategy for MI-induced heart failure. Our findings unveil a novel therapeutic strategy, highlighting that RD maintains its efficacy and safety when integrated with complementary approaches over extended periods. Health sciences/Cardiology/Interventional cardiology Biological sciences/Stem cells/Mesenchymal stem cells Health sciences/Diseases/Cardiovascular diseases/Heart failure acute myocardial infarction exosome microRNA heart failure sympathetic nervous system Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 BACKGROUND Percutaneous coronary intervention has the potential to enhance treatment outcomes for myocardial infarction (MI) by enabling timely revascularization and myocardial salvage; however, it may also lead to cardiomyocyte (CM) loss and activation of sympathetic nervous system (SNS), 1 which often contributes to the development of heart failure (HF), with a 5-year mortality rate of approximately 11%. 2 Therefore, there is a critical need to develop therapeutic strategies that can suppress SNS activation and promote endogenous CM regeneration following MI. 3 , 4 Both cell therapy and renal sympathetic denervation (RD) hold promise for promoting myocardial repair by counteracting functional CM loss and mitigating SNS activation. 5 RD, a catheter-based procedure first introduced in 2009 for the treatment of resistant hypertension, involves the ablation of renal sympathetic nerve activity in the kidneys. 6 Recent studies have further demonstrated that RD can enhance myocardial salvage and improve cardiac function by suppressing SNS activation following MI. 7 The potential mechanisms underlying these benefits include the attenuation of inflammation, inhibition of the renin–angiotensin–aldosterone system (RAAS), increased levels of protective circulating natriuretic peptide levels, and reduction in cardiac fibrosis, all of which contribute to its cardioprotective effects. 8 However, to date, there is no evidence supporting RD's ability to modulate cardiac regeneration after MI. Sustained ischemia leads to myocardial cell apoptosis and necrosis. In the past 20 years, the field of heart regeneration has entered a renaissance period with remarkable progress in the understanding of the putative capacity of stem/progenitor cells to generate clinically significant quantities of functional CMs- whether through exogenous cell administration or endogenous regeneration activation. 9 Mesenchymal stem cells (MSCs) can differentiate into cardiac, endothelial, and smooth muscle cells. MSCs have strong paracrine effects, making them promising candidates for endogenous regeneration and repair pathways. Relatively, peripheral blood mesenchymal stem cells (PBMSCs), demonstrate superior advantages over conventional adipose-derived MSCs and bone marrow-derived MSCs, including easier accessibility, reduced invasiveness, and enhanced clinical feasibility. Recent trends demonstrate that PBMSCs are a promising source for regenerative medicine favoring ambulatory cell sourcing, as endorsed in the ISCT 2023 Position Statement on translational MSC protocols. We recently revealed that PBMSCs are efficacious regenerative materials, exerting their benefits on ischemic myocardium via their paracrine effects. 10 However, transplanted stem cells exhibit limited survival in damaged myocardium, suggesting that mechanisms other than trans-differentiation, such as paracrine factors, may play a critical role in heart regeneration. Apart from cytokines and growth factors, MSCs secrete small, single-membrane organelles called exosomes (Exos). MSC-derived Exos are key players in communication with local and distant tissues. 11 Accumulating evidence confirms that MSC-derived Exos and their active molecules, such as microRNAs (miRNAs), play a pivotal role in regulating signaling pathways associated with heart repair and regeneration. Furthermore, studies have shown that very few new myocytes are generated in the hearts of adult rodents following ischemic injury, 12 and the proliferation of preexisting CMs primarily drives endogenous cardiac regeneration. 13 However, little is known about the regulation of endogenous CMs regeneration mechanism by PBMSC-Exos. Both Exos therapy and RD have exhibited encouraging results in laboratory studies, though intrinsic challenges such as short half-life and lack of clear targets hinder the clinical feasibility. 14 , 15 Exos treatments aim to restore compromised CMs integrity, whereas RD targets aberrant neural signaling mechanisms that drive heart failure progression after MI. This research examines innovative strategies to optimize regenerative interventions for post-infarction cardiac repair. Here, we explored preclinical therapeutic strategies combining RD with exosome-based therapy to enhance endogenous cardiac regeneration following MI. First, we investigated the efficacy of RD in a pig model of MI. RD primarily modulates early sympathetic overactivation (days 0–14 post-MI), leading short-term improvement of cardiac performance. However, this improvement did not remain significant afterward. Second, because exosomal therapy reduces fibrotic tissue formation and enhances cardiomyocyte proliferation, 14 which may ameliorate cardiac remodeling during the late-stage phase of MI, we hypothesized that RD administered in the early phase of MI in combination with delayed autologous PBMSC-derived Exos (PBMSC-Exos) transplantation affords sustained cardiac regeneration and repair compared with either therapy alone. Third, through in vitro molecular mechanistic and in vivo animal experiments, we provided the first evidence that after RD, PBMSC-Exos carry miR-141-200-429 clusters from renal artery (RA) endothelial cells (RAECs) to ischemic CMs, resulting in CM reprogramming and improving heart function. Targeting beneficial communication mediated by PBMSC-Exos or miR-141-200-429 clusters between RAs and damaged hearts may be a novel strategy for improving RD-initiated cardiac repair. RESULTS RD Inhibits SNS/RAAS and Boosts PBMSC Exosomal Cardioprotective microRNAs in Post-MI HFrEF Pigs To investigate RD's cardioprotective role post-MI, we assessed temporal changes in catecholamine and RAAS levels in HFrEF pigs after RD. RA nerve tyrosine hydroxylase (TH) staining revealed that gradual renal nerve viability increased after RD-Sham but significantly decreased after RD (Fig. 1 A). In particular, TH staining intensity was the lowest in RD pigs after 2 weeks of MI (Fig. 1 B); this observation was confirmed by the occurrence of the largest reduction in kidney dopamine and norepinephrine (NE) levels at this time point—a quantitative index for the sympathetic nerve function (Fig. 1 C and 1 D). The most significant reduction in circulating NE levels was also noted 2 weeks after RD (Fig. 1 E). Next, we assessed RAAS activity based on plasma angiotensin I and angiotensin II levels and noted a significant reduction in their levels in RD pigs, with the lowest levels noted 2 weeks after MI (Fig. 1 F and 1 G). After 2 weeks of RD, no significant changes were observed in the SNS and RAAS of any HFrEF pig. At later time points (e.g., 4 weeks post-RD), we still detected no significant improvements in SNS activity or RAAS markers, suggesting that the treatment's regulatory effects were both early and transient. Despite the absence of sustained SNS/RAAS modulation, previous studies indicate that MSC-based therapies may exert cardioprotection through indirect mechanisms. Given that MSC-mediated cardioprotection is independent of direct cell–cell contact, 16 we hypothesized that Exos transfer biological molecules, thereby contributing to the remote cardioprotective effects of RD. To investigate the impact of RD on PBMSC-Exos, we isolated PBMSC-Exos from RD or RD-Sham pigs via ultracentrifugation and analyzed them using transmission electron microscopy (TEM) at 1, 2, and 4 weeks post-MI (Fig. 1 H). Then, we performed western blotting to confirm the presence of the Exos-associated proteins CD9, CD63, TSG101and HSP70, and the absence of negative marker Calnexinin in PBMSC-Exos preparations from RD and RD-Sham pigs at 1, 2, and 4 weeks post-MI (Fig. 1 I). CD9 and CD63 were selected as canonical exosomal markers based on MISEV2018 guidelines, 16 with TSG101 immunoblotting confirming endosomal origin. 17 These tetraspanins were prioritized given their established role in cardiomyocyte-exosome interactions. These results were consistent with our nanoparticle tracking analysis (NTA) results: RD increased the expression of these proteins, peaking 2 weeks after MI; in contrast, RD-Sham gradually reduced their expression, with the greatest decrease occurring 2 weeks after MI (Fig. 1 J and 2 K). Next, PBMSC-Exos size distribution, measured using NanoSight, demonstrated no significant differences between these two groups at any timepoint. In both groups, we observed a unimodal distribution of isolated particles with an average diameter of 100 nm (Fig. 1 J and 1 L)—consistent with the definition of Exos. 19 To confirm that Exos were derived from PBMSCs, we first validate the identity of PBMSCs using cell surface marker profiling by FACS. FACS showed that PBMSCs harvested from the animals at 2 weeks post-MI expressed ≥ 95% of MSC-associated cell surface markers CD44, CD71, CD90, CD105 (mesenchymal markers), while expressing ≤ 2% of both hematopoietic markers HSC markers CD 34 and CD45, and endothelial progenitor cell (EPC) molecular markers CD31 andCD133 (Fig. 1 M). Next, we performed co-immunostaining of the MSC marker protein CD105 and the Exos marker protein TSG101 to confirm that the Exos were derived from PBMSCs. Overexpression of MSC-specific molecule CD105 in Exos (Fig. 1 N) confirmed their derivation from PBMSCs. Taken together, these results indicated that RD increases Exos release originated from PBMSCs in a time-dependent manner, with the greatest increase occurring 2 weeks after MI. As such, we selected autologous PBMSC-Exos from the RD and RD-Sham group (denoted as PBMSC- Exos RD−Sham or PBMSC-Exos RD , respectively) at 2 weeks after MI as the Exos source to investigate the additive effects of delayed exosomal therapy (i.e., intravenous injection of PBMSC-Exos 30 days after MI) with RD-induced myocardial repair in our HFrEF pigs. Given that numerous miRNAs are known to stimulate cardiac repair by promoting CM dedifferentiation and proliferation after MI, 20 we conducted small RNA sequencing (RNA-seq) to characterize miRNAs present in PBMSC-Exos RD . First, to identify miRNAs within PBMSC-Exos RD that potentially enhance cardiac proliferation and regeneration, we compared 66 miRNAs enriched in PBMSCs with datasets of miRNAs implicated in cardiac proliferation 4 , 20 and regeneration. 21 As illustrated in the Venn diagram (Fig. 2 A), we identified 15 common miRNAs. Next, we analyzed the expression of these 15 miRNAs in PBMSC-Exos RD and PBMSC-Exos RD−Sham and noted that PBMSC-Exos RD significantly upregulated the expression of miR199 (miR-199a-3p; 7.73-fold increase), miR-200a (miR-200a-3p; 8.03-fold increase), and miR-200b (miR-200b-3p; 9.01-fold increase; Fig. 2 B). Third, to determine whether the observed increase in miRNA expression was due to a higher number of PBMSC-Exos RD or an increase in their miRNA content, we quantified miRNA expression per equal number of PBMSC-Exos RD using RT-qPCR. The results revealed that miR-200a-3p and miR-200b-3p expression was significantly higher in PBMSC-Exos RD compared to an equivalent amount amount of PBMSC-Exos RD−Sham (Fig. 2 C). Fourth, miR-200a and miR-200b belong to the kidney-enriched miRNA family, 22 which also includes miR141, miR200, and miR429 (miR-141-200-429). 23 To investigate whether RD-treated RAs release PBMSC-Exos RD with elevated miR-200 content, we measured the expression of the miR-141-200-429 expression in Exos derived from RA (RA-Exos). Compared to RA-Exos RD−Sham , RA-Exos RD exhibited significantly higher levels of all miR-141-200-429 cluster members (Fig. 2 D). Finally, we compared the expression of the miR-141-200-429 cluster in RAs and hearts from RD-treated pigs. The expression of miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-141, and miR-429 expression was considerably higher in RAECs than in CMs (Fig. 2 E). Notably, the expression of these five miRNAs was significantly increased in RACEs following RD, with miR-200a and miR-200b showing the most pronounced upregulation (6.52- and 4.23-fold increase, respectively; Fig. 2 F). However, in RD-treated pigs, the expression of miR-200c-3p and miR-429 was significantly upregulated in RAECs (Fig. 2 F) but not in CMs (Fig. 2 G). miR-200b-3p-encapsulated MSCs-derived exosomes (MSCs-Exos) have been shown to protect against MI-induced cardiomyocyte apoptosis and inflammation. 24 These findings collectively suggest that RD stimulates the release of PBMSC-Exos from RAECs, delivering cardioprotective microRNA cargo to infarcted hearts. RD Improves Short-term Cardiac Performance and Delayed Exosomal Therapy Affords Long-term Benefit Confirming our earlier report, PBMSCs (5×10 6 cells) improved cardiac function. 10 By applying this cell count, we had obtained 2 ×10 13 PBMSCs-Exos from MI pigs. Next, we further tested this dosage in MI rats. PBMSC-Exos were injected into caudal vein of MI rats at dosage of 5×10 12 , 2×10 13 , or 5×10 13 in 100µL PBS). PBMSCs-Exos improved cardiac function (LVEF) and expression levels of β-catenin and Oct4, and reduced CMs injury (plasmahs-cTnT) to a similar level at doses of 2×10 13 and 5×10 13 , but not at 5×10 12 ( Figure S1 ). Based on these data we proceeded to test the effects of PBMSC-Exos injection on cardiac repair at the dose of 2×10 13 in the following pig experiments. Figure 3 A presents a schematic timeline integrating RD/Exos intervention windows with pathophysiological milestones. Based the above results, we harvested PBMSC-Exos at day 14 post-MI, we aimed to capture Exos with maximal reparative cargo before potential functional exhaustion. We performed RD immediately after MI for modulating early sympathetic overactivation (days 0–14 post-MI), which exacerbates inflammation. Then, Exos therapy focuses on the transition period between proliferation and scar formation (day 30 post-MI). 25 In this study, MI was induced in 82 out of 95 experimental pigs by ligation of the left anterior descending branch of a coronary artery. From these, 40 pigs with LVEF < 40% were selected and randomly assigned to receive early (within 2 h after MI) RD-Sham or RD followed by delayed (30 days after MI) PBS or Exos injection, with 10 pigs per group. These groups were labeled as RD-Sham + PBS, RD-Sham + PBMSC-Exos RD−Sham (denoted as RD-Sham + Exos), RD + PBS, and RD + PBMSC-Exos RD (denoted as RD + Exos). No deaths occurred in this cohort of pigs during any treatment. However, 10 pigs died during follow-up. Finally, 30 pigs survived to undergo serial functional studies 90 days after MI ( Figure S2 ). Functional data, measured through echocardiography, demonstrated that early RD administration after MI significantly improved LVEF 30 days after MI compared with early RD-Sham; however, this improvement did not remain significant afterward. Although delayed Exos injection significantly improved LVEF in the RD-Sham + Exos group by 60 days post-MI, the therapeutic effect failed to persist until 90 days post-MI. However, sequential exosome therapy maintained these therapeutic benefits throughout the study period (Figs. 3 B and 3 C). At 90 days post-MI, the RD + Exos group exhibited 11–26% greater preservation of LVEF compared to other three groups, with all intergroup differences achieving statistical significance (P < 0.001; Fig. 3 C). Similarly, at 90 days after MI, left ventricular stroke volume was larger in the RD + Exos group than in the other three groups (Fig. 3 D). This was mainly due to significant reductions in left ventricular end-diastolic (Fig. 3 E) and end-systolic (Fig. 3 F) volumes and the recovery of left ventricle (LV) anterior wall thickness (Fig. 3 G) in the combination groups, but no similar significant differences were observed between the RD + PBS and RD-Sham + PBS groups (Fig. 3 E– 3 G). Moreover, all pigs receiving early RD plus delayed Exos injection were alive 90 days after MI; however, 5 of 10 pigs in the RD-Sham + PBS group died within 90 days after MI (Fig. 3 H). Moreover, mirroring functional recovery patterns, monotherapy with either RD or Exos significantly upregulated myocardial expression of miR-200a-3p, miR-220b-3p, and miR-141, with the most pronounced upregulation observed in RD + Exos pigs receiving combination therapy at 90 days post-MI (Fig. 3 K). Importantly, no significant differential expression was detected for miR-200c-3p and miR-429 across experimental groups. These findings aligned with our previous observations in CM-Exos derived from MI models, strongly suggesting that the myocardial exosomal miR-141-200 cluster mediates the therapeutic effects of both RD and Exos interventions on cardiac functional restoration. We assessed renal function by measuring plasma levels of blood urea nitrogen (BUN, Fig. 3 I) and creatinine (Fig. 3 J) at 0, 30, 60, and 90 days after MI. The results demonstrated that as MI-induced HFrEF progressed, plasma BUN and creatinine levels became increasingly elevated in the RD-Sham + PBS group; nevertheless, RD did not impair renal function. Moreover, plasma BUN and creatinine levels were significantly lower in the RD + Exos group than in the RD-Sham + PBS group 30, 60, and 90 days after MI, indicating that RD plus Exos may further reduce plasma BUN and creatinine levels. Furthermore, the dynamic hemodynamics were monitored in all animals (Table 1 ). Compared with the baseline level, MI-induced HF caused a significant increase in heart rate, which persisted in the RD-Sham + PBS group, but were restored to the baseline levels in the RD-Sham + Exos group and all RD groups at 90 d post-MI. MABP gradually decreased in the RD-Sham + PBS group, but there was no significant change in the other three groups. We also observed that RD did not significantly affect cardiac function, blood pressure and heart rate in pigs received sham surgery ( Table S3 ). Following RD, no significant alterations were detected in plasma norepinephrine (NE) or RAAS components (angiotensin I and II), with no biochemical evidence of heart failure (BNP < 100 pg/mL) or myocardial necrosis (cTnI < 0.01 ng/mL). All these data suggested that RD-mediated cardiac repair specifically targets MI-induced sympathetic hyperactivation rather than non-ischemic surgical effects. Table 1 Heart Rate, MABP, BNP, and hs-cTnT (means ± SD) Time n Heart rate (beats/min) MABP ( mmHg) Plasma BNP (pg/ml) hs-cTnT (ng/l) Baseline RD-Sham + PBS 10 85 ± 8 87 ± 4 94.6 ± 19.7 6.4 ± 1.9 RD-Sham + Exos 10 80 ± 6 85 ± 6 88.6 ± 17.0 7.4 ± 1.3 RD + PBS 10 81 ± 6 85 ± 6 87.0 ± 15.9 7.3 ± 1.4 RD + Exos 10 83 ± 7 84 ± 4 83.1 ± 15.1 7.2 ± 1.3 On the day of myocardial infarction RD-Sham + PBS 10 105 ± 10 83 ± 5 432.5 ± 84.0 § 1191.6 ± 209.7 § RD-Sham + Exos 10 108 ± 10 86 ± 6 425.7 ± 83.4 § 1154.5 ± 248.7 § RD + PBS 10 100 ± 9 83 ± 5 436.7 ± 78.2 § 1150.7 ± 165.6 § RD + Exos 10 99 ± 12 81 ± 5 433.4 ± 83.2 § 1119.6 ± 159.8 § 30 d post-MI RD-Sham + PBS 7 115 ± 11 §║ 83 ± 6 528.9 ± 83.0 § 42.1 ± 12.3 §║ RD-Sham + Exos 8 102 ± 13 *§ 85 ± 6 779.4 ± 94.4 *§║ 14.7 ± 4.4 *║ RD + PBS 9 93 ± 9 *#§ 85 ± 6 687.6 ± 118.5 §║ 17.0 ± 5.9 *║ RD + Exos 10 95 ± 9 *§ 80 ± 7 829.1 ± 128.8 *§║ 11.3 ± 4.9 *║ 60 d post-MI RD-Sham + PBS 5 116 ± 8 §║ 81 ± 6 § 577.8 ± 79.0 § 35.0 ± 7.1 §║ RD-Sham + Exos 8 100 ± 11 *§ 83 ± 5 800.0 ± 83.3 *§║ 10.7 ± 3.4 *║ RD + PBS 9 87 ± 10 *#║ 81 ± 5 730.6 ± 71.9 §║ 13.8 ± 5.2 *║ RD + Exos 10 89 ± 10 *# 81 ± 7 1142.3 ± 181.6 *#†§║¶ 9.7 ± 2.1 *║ 90 d post-MI RD-Sham + PBS 5 116 ± 9 §║ 76 ± 6 §║¶ 598.4 ± 89.2 § 26.8 ± 6.4 §║ RD-Sham + Exos 7 98 ± 10 *§║ 82 ± 5 754.3 ± 117.8 §║ 13.5 ± 5.6 ║ RD + PBS 8 88 ± 7 *#║ 81 ± 5 803.4 ± 95.4 §║ 13.3 ± 3.2 §║ RD + Exos 10 84 ± 10 *#║¶ 79 ± 5 1409.0 ± 122.3 *#†§║¶ 8.4 ± 1.7 ║ Delayed Exosomal Therapy Attenuates Cardiac Fibrosis via RD-Mediated RAAS Inhibition and Enhanced Cardiomyocyte Proliferation and Regeneration We used tetrazolium chloride (TTC), Masson’s trichrome, and hematoxylin–eosin (H&E) staining for histological assessment 90 days after MI. The scar size (Fig. 4 A and 4 E) and fibrosis (Fig. 4 B and 4 F) demonstrated a considerable reduction in the RD + Exos group compared with the other groups. Moreover, the RD + Exos group showed the greatest increase in the number of viable CMs (Fig. 4 C and 4 G). Wheat germ agglutinin (WGA) staining was carried out to measure the cross-sectional area of CMs. Both macroscopic postmortem analysis (Fig. 4 D) and CM cross-sectional area quantification (Fig. 4 H) demonstrated that either RD or Exos monotherapy increased CM size, while combination therapy (RD + Exos) resulted in a further significant increase in cross-sectional area at 90 days post-MI. All these data suggest that both RD and Exos monotherapy inhibited cardiac pathological remodeling, and their combination therapy resulted in an enhanced therapeutic effect. To elucidate the underlying mechanisms by which the combined RD + delayed Exos regimen prevents LV remodeling, we performed a series of four experiments. First, we explored some molecular correlates of cardiac repair and cardiac function improvement. Peripheral blood samples were obtained at baseline and 0, 30, 60, and 90 days after MI to measure plasma B-type natriuretic peptide (BNP) and high-sensitivity cardiac troponin T (hs-cTnT) levels (Table 1 ). The results consistently demonstrated that RD significantly increases plasma BNP levels while reducing plasma hs-cTnT levels. Delayed exosomal therapy further amplified these effects, suggesting that it enhances RD-mediated elevation in BNP bioavailability while concurrently attenuating CM injury. Second, we assessed the effects of early RD plus delayed exosomal therapy on the SNS of our pigs, specifically efferent renal nerve activity markers, including renal TH and NE. At 90 days after MI, we observed equally reduced TH staining intensity in the RD + PBS and RD + Exos groups than in the RD-Sham + PBS and RD-Sham + Exos groups (Fig. 4 I). In particular, both RD + PBS and RD + Exos groups demonstrated an approximately 65% decrease in TH stain intensity compared with the RD-Sham + PBS and RD-Sham + Exos groups (all P < 0.001; Fig. 4 J). Moreover, we noted an approximately 80% reduction in the renal levels of the kidney SNS activity markers dopamine ( P < 0.001) and NE ( P < 0.001) 90 days after MI in the RD + Exos group (Fig. 4 K and 4 L), indicating equivalent reductions in kidney SNS activity. RD-Sham + Exos could not reduce kidney dopamine and NE levels below those in the RD-Sham + PBS group, indicating that Exos did not significantly alter kidney SNS activity. Similarly, a decrease in plasma NE levels was achieved in both RD + PBS and RD + Exos groups, indicating that RD reduced renal afferent nerve stimulation (Fig. 4 M). Moreover, the RD + PBS and RD + Exos groups demonstrated significantly lower kidney angiotensin I and angiotensin II levels at 90 days after MI than the RD-Sham + PBS and RD-Sham + Exos group ( Figure S3A and S3B ). Moreover, alterations in angiotensin levels resulting from RD extended to changes in plasma angiotensin: plasma angiotensin I and angiotensin II levels were significantly lower in the RD + PBS and RD + Exos groups than in the RD-Sham + PBS and RD-Sham + Exos groups (all P < 0.001, Figure S3D and S3E ). Similarly, plasma renin activity was significantly lower in both RD groups than in both RD-Sham groups (both P < 0.001, Figure S3C ). Kidney or plasma angiotensin I and angiotensin II levels showed no significant difference between the RD-Sham + PBS and RD-Sham + Exos groups or between the RD + PBS and RD + Exos groups. In contrast, plasma aldosterone levels were significantly lower in the RD + PBS and RD + Exos groups than in the RD-Sham + PBS and RD-Sham + Exos groups, respectively (both P < 0.01); moreover, they were significantly lower in the RD-Sham + Exos and RD + Exos groups than in the RD-Sham + PBS and RD + PBS groups, respectively ( P < 0.05 and P < 0.001, respectively; Figure S3F ). However, plasma NE levels did not differ significantly between the RD + PBS and RD + Exos groups ( Fig. 4 M), suggesting that the key protective effects of delayed Exos therapy are primarily mediated through RD-induced RAAS inhibition rather than modulation of SNS activation. Third, we observed cardiomyocyte proliferation following RD and Exos therapy. Ki67 is a well-established marker of cellular proliferation, as it is expressed during all active phases of the cell cycle (G1, S, G2, and M) but is absent in quiescent cells (G0). 26 In the context of cardiac repair, Ki67 immunolabeling is widely used to assess CMs proliferation, which is a critical process for myocardial regeneration and functional recovery after injury. 27 In this study, we used Ki67 immunolabeling to evaluate whether early RD (remote conditioning) and delayed Exos therapy could promote cardiomyocyte proliferation. Dual immunofluorescence co-staining of Ki67 (proliferation marker) and MHC (myosin heavy chain, cardiomyocyte marker) demonstrated that early RD treatment significantly increased Ki67 + cardiomyocyte populations in both peri-infarct and remote myocardial regions. Moreover, delayed Exos administration further enhanced this proliferative response, confirming the therapeutic potential of sequential interventions for cardiomyocyte cycle re-activation. (Fig. 5 A and 5 E). Similarly, RD led to an increase in the number of Aurora B-positive CMs within the myocardium, and delayed exosomal therapy increased this number further (Fig. 5 B and 5 F). Next, we assessed cardiomyocyte (CM) dedifferentiation via α-actin staining to confirm cell-cycle reentry in mature CMs. RD treatment induced CM dedifferentiation, as evidenced by α-smooth muscle actin (α-SMA) expression in a subset of CMs, with this trend being more pronounced in the RD + Exos group than in the RD-Sham group (Fig. 5 C and 5 G). Western blot analysis further demonstrated that RD upregulated Aurora B, α-SMA, and α-actin expression, and delayed Exos administration further enhanced their levels (Fig. 5 I). Collectively, these findings indicate that delayed Exos injection amplifies RD-induced CM proliferation and dedifferentiation. Fourth, we assessed the uptake of PBMSC-Exos by CMs in the RD-Sham-treated and RD-treated hearts. To examine whether PBMSC-Exos were differentially taken up by HFrEF hearts receiving RD-Sham or RD, PKH26 was used to label the exosomes before injection. PKH26 detection in the MHC through immunofluorescence staining (Fig. 5 D). PKH26 signals were absent in the CMs of the RD-Sham + PBS and RD + PBS groups. However, strong PKH26 signals were observed in the CMs of RD-Sham + Exos animals, and these signals were further enhanced by delayed Exos injection (Fig. 5 H). In addition, related analysis demonstrated that CM Exos uptake was positively correlated with the expression of Aurora B (r = 0.817, P = 0.004; Fig. 5 J) and α-SMA (r = 0.905, P < 0.001; Fig. 5 K) in the RD + Exos group. Thus, RD-induced proliferation and dedifferentiation of CMs may be related to the content and delivery of Exos, conferring regenerative capacity to an ischemic heart. PBMSC-Exos RD Reprogram Ischemic CMs The induction of adult CM proliferation is typically associated with three hallmark cellular transitions: (1) modulation of differentiation markers, (2) metabolic remodeling, and (3) re-activation of embryonic gene programs. While this process has been historically termed "dedifferentiation", emerging evidence supports its characterization as "partial reprogramming" - a controlled transition to a fetal-like proliferative state where adult CMs regain mitotic capacity while maintaining core functional characteristics. 28 Notably, MSC-Exos have been shown to orchestrate this regenerative process through their miRNA cargo, which simultaneously modulates tissue repair pathways and immune responses, thereby creating a permissive microenvironment for ischemic myocardial regeneration. 29 To comprehensively evaluate the effects of PBMSC-Exos RD enriched with the miR-141-200-429 cluster on ischemic cardiomyocyte reprogramming, we conducted myocardial biopsies on animals treated with either RD-Sham or RD at 14 days post-MI (Fig. 3 A). The biopsied myocardial tissues were then used to implement an integrated experimental strategy, combining complementary in vitro and in vivo approaches, to systematically assess the therapeutic outcomes. For in vitro functional assessment, CMs isolated from biopsied tissues of post-MI hearts at 2 weeks were exposed to hypoxia-mimicking conditions and co-cultured with either PBMSC-Exos RD−Sham or PBMSC-Exos RD (1×10 8 particles/mL) for 48 hours to systematically evaluate the composition-dependent effects on CM functional recovery. The results demonstrated that 39.1% of CMs cultured with PBMSC-Exos RD−Sham lacked cardiac troponin (cTnI), whereas this proportion significantly decreased in CMs cultured with PBMSC-Exos RD , suggesting that PBMSC-Exos RD increased CM dedifferentiation ( Figure S4A ). Moreover, compared with PBMSC-Exos RD−Sham , PBMSC-Exos RD increased the levels of the dedifferentiation markers Runx1 and Dab2 ( Figure S4B and S4C ). Cell-cycle reentry of CMs from PBMSC-Exos RD treated-CM cultures was confirmed by analysis of purified MHC-expressing CMs positive for Ki67 ( Figure S4Da ) and PH3 ( Figure S4Ea ). Compared with CMs treated with PBMSC-Exos RD−Sham , all cases of CMs receiving PBMSC-Exos RD showed a much higher Ki67 proliferation index ( Figure S4Db ) along with a higher mitotic index, calculated as the fraction of PH3-positive cells ( Figure S4Eb ). The redifferentiation of these dedifferentiated CMs evaluated by the levels of cTnT led to a similar result ( Figure S4F ): cTnT expression was 16.2-fold higher in PBMSC-Exo RD -treated CMs than in those treated with PBMSC-Exos RD−Sham . These data strongly suggested that PBMSC-Exos RD favor cardiac dedifferentiation, proliferation, and redifferentiation under hypoxia. To evaluate in vivo therapeutic effects, we performed comprehensive transcriptional profiling of CMs isolated via cardiac biopsy from RD-treated and RD-Sham control groups at 14 days post-MI, revealing significant PBMSC-Exo RD -mediated molecular reprogramming. Consistent with the functional and morphological assessments, these changes were related to cell proliferation, division, and differentiation. Of the 10 genes significantly altered by PBMSC-Exos RD ( Figure S5A ), the expression of genes encoding six reprogramming factors involved with cardiovascular system development, cell specification, cycle, division, and proliferation was upregulated, whereas four genes associated with inflammation and apoptosis were downregulated ( Figure S5B ). These findings collectively suggest that PBMSC-Exos RD treatment potentially induces molecular reprogramming in ischemic CMs, facilitating their transition towards a regenerative state through activation of endogenous repair mechanisms. β-Catenin Promotes Exos Uptake, Proliferation, and Redifferentiation by CMs PBMSC-Exos RD were noted to reprogram CMs—as evidenced by overexpression of the pluripotent transcript factors Oct4 , Sox2 , and Klf4 ( Figure S5A and S5B ). Moreover, among all genes investigated, the β-catenin was the most significantly upregulated molecule by PBMSC-Exos RD ( Figure S5B ), suggesting that β-catenin underlies CMs potency under hypoxic conditions. 30 Because the β-catenin pathway is a key regulator of CMs differentiation, 31 , 32 we next investigated the effects of β-catenin on PBMSC-Exo RD -mediated CMs proliferation and redifferentiation. First, we assessed the β-catenin-promoted direct CMs uptake of MSC-Exos. CMs were randomly transfected with an empty vector, oe β-catenin, si β-catenin, or WAY-262611 (1 µmol/L; a β-catenin agonist; ab145229, Abcam, USA) and then cocultured with PBMSC-Exos RD under hypoxic conditions (Fig. 6 A). CMs uptake of PBMSC-Exos RD was evaluated as the percent proportion of cells double-positive for PKH26 and MHC to those completely positive for MHC (Fig. 6 Ba ). As shown in Fig. 6Bb , quantitative analysis indicated that the myocardial uptake of PBMSC-Exos RD was the greatest in the oe β-catenin-transfected CMs, followed by WAY-262611-treated CMs. The least uptake levels were noted in si β-catenin-transfected CMs. Thus, β-catenin may improve the Exos uptake of CMs. Since transfection with oe β-catenin resulted in higher CM uptake compared to the β-catenin agonist, we exclusively employed oe β-catenin in the subsequent experiments. Second, we determined whether β-catenin induces a proliferative dedifferentiated state in PBMSC-Exo RD -treated CMs. CMs were pretreated with transfection of empty vector, oe β-catenin, or si β-catenin and then cultured for 7 days with PBMSC-Exos RD under hypoxic conditions. We observed a considerable increase in CMs double-positive for EdU and cTnT after oe β-catenin transfection. After 7 days of culture, oe β-catenin-transfected CMs were rod-shaped CMs, undergoing mitosis and cytokinesis (Fig. 6 Ca ). Compared with empty vector transfection, oe β-catenin transfection led to a continual increase in CM proliferation rates (Fig. 6 D): 7.1% of CMs proliferated (mononucleated: 0.7%; binucleated or multinucleated: 6.4%) after 7 days of culture. However, si β-catenin transfection abrogated this benefit (Fig. 6 C, and 6 D-F). Multiple proliferation patterns were observed in the oe β-catenin-transfected cells. Binucleated CMs could divide into two or three mononucleated cells or into one mononucleated cell and one binucleated or multinucleated cell (Fig. 6 E and 6 F). Thus, in the presence of PBMSC-Exos RD , β-catenin may stimulate terminally differentiated CMs to reenter the cell cycle and proliferate in mononucleated and binucleated cells. Third, considering the aforementioned findings, the apoptosis rate of CMs after 7 days of culture under hypoxic conditions was evaluated using annexin V–APC and propidium iodide (PI) staining, followed by flow cytometry. The cell death rate was significantly lower in oe β-catenin-transfected CMs than in control CMs; nevertheless, the greatest cell death rate was noted in the si β-catenin group (Fig. 6 G). Thus, β-catenin may reduce CM apoptosis, promoting CM proliferation. Fourth, after 7 days of culture, CMs were immunostained for cTnI to determine whether dedifferentiated CMs had redifferentiated (Fig. 6 Ha ). Of the dedifferentiated CMs (i.e., α-SMA-positive CMs), the cTnI-positive rate was 53.5%, 23.9%, and 5.8% in oe β-catenin-transfected, empty vector-transfected, and si β-catenin-transfected CMs, respectively (Fig. 6 Hb ). These results indicated that dedifferentiated CMs form new functional CMs. Finally, our quantitative reverse transcription polymerase chain reaction (RT-qPCR) results demonstrated considerably higher expression of the Yamanaka factors Oct4 , Sox2 , Klf4 , and c- Myc in PBMSC-Exo RD -treated CMs in empty vector-transfected CMs than in si β-catenin-transfected CMs (Fig. 6 I)—consistent with their expression changes mentioned above ( Figure S5A ). Overexpression of β-catenin ( oe β-Catenin) further increased the mRNA expression of Oct4, Sox2, Klf4, and Myc (Fig. 6 I), indicating that β-catenin enhances PBMSC-Exos RD -induced CM reprogramming and exerts anti-apoptotic effects. PBMSC-Exos Transfer miR-141-200-429 Clusters to Induce Heart Regeneration via β-Catenin Signaling As established in our prior research, RD achieves cardioprotection by facilitating the release of Exos miR-141-200-429 clusters from RAECs, which are transported to cardiomyocytes through PBMSCs. Next, we investigated the mechanism through which miR-141-200-429 clusters in PBMSC-Exos RD initiate myocardial reprogramming via the β-catenin signaling pathway. To further confirm that CMs miR-141-200-429 clusters originate from RD-induced RAECs, we used the endoglin promoter to generate RAECs adeno-associated virus (AAV)-expressing miR-141-200-429 cluster sponges (miR-141-200-429 sponges) or NC sponges (Fig. 7 A). Following the biopsy conducted at 14 days post-MI, AAV therapy was immediately administered to the pigs treated with either RD-Sham or RD. Four weeks later, PBMSC-Exos were isolated, and CMs were collected. BODIPY TR ceramide–labeled Exos uptake in hearts was detected through fluorescence microscopy. miR-141-200-429 sponges significantly reduced CM uptake of these Exos in RD-treated hearts (Fig. 7 B). These sponges significantly reduced miR-141-200-429 levels in PBMSC-Exos derived from the same amounts of cells (Fig. 7 C). Notably, miR-141-200-429 sponges significantly reduced miR-200a-3p, miR-200b-3p, and miR-141 levels after RD but did not alter miR-200c-3p and miR-429 levels in CM-Exos from the RD pigs receiving miR-141-200-429 sponges (Fig. 7 D). Taken together, these results indicated that CM miR-141-200-429 cluster is produced by RD-treated RAECs and transferred to CMs by Exos. To confirm that miR-141-200-429 cluster induces heart regeneration, we next conducted RNA sequencing (RNA-seq) on CMs isolated from the aforementioned pigs, which were divided into the following groups: RD-Sham, RD-Sham + miR-141-200-429 sponge, RD + NC sponge, and RD + miR-141-200-429 sponge. A total of 1,634 and 1,745 differentially expressed genes (DEGs; all P < 0.05) were identified in hearts treated with RD-Sham + miR-141-200-429 sponges and RD + miR-141-200-429 sponges, respectively. MSC-derived Exos and their active molecule miRNAs have been recently reported to regulate signaling pathways involved in heart repair and regeneration. 23 , 33 , 34 Gene set expression analysis revealed that miR-141-200-429 sponges caused a significant reduction in the expression of genes and pathways related to cell proliferation and differentiation but an increase in the expression of genes and pathways involved in apoptosis and contraction, concomitant with dedifferentiation marker downregulation (Fig. 7 E-G). Downregulated genes were noted to be mainly involved in CM proliferation and differentiation and heart development, suggesting a less immature state of CM miR-141-200-429 sponges. This observation was further confirmed through WGA staining (Fig. 7 H) and CM cross-sectional area quantification (Fig. 7 I), which confirmed a reduction in sarcomere density. Moreover, RT-qPCR revealed increased expression of the proapoptotic genes Bax , Casp3 , Casp9 , and p53 but reduced that of immature CM-specific genes involved in proliferation (i.e., c-Myc , Cdk1 , Yap1 , and Mcl-1 ) and dedifferentiation markers ( Nppa and Acta1 ; Fig. 7 J) after miR-141-200-429 sponge treatment. Integrative analysis of RNA-seq data sets from RD-Sham and RD miR-141-200-429 sponge CMs revealed 226 and 305 genes jointly upregulated or downregulated in both HFrEF pig groups, respectively (Fig. 7 K). Most of the downregulated genes were those involved in heart development and CM proliferation and differentiation (Fig. 7 L). Taken together, these results indicated that miR-141-200-429 cluster abrogation initiates a cascade of events inhibiting RDCMs into a more immature state, inhibiting CM proliferation and dedifferentiation. Notably, we observed a significant inhibition of antiapoptosis and reprogramming, as indicated by the decreased expression of β-catenin and its target genes Bcl2 , survivin , and Oct4 in CMs from the RD + miR-141-200-429 sponge group compared to those from the RD + NC sponge group CMs (Fig. 7 G and 7 J). These findings suggest that miR-141-200-429 sponges inactivates β-catenin signaling, thereby suppressing a cascade of RD-induced events that convert mature CMs into a more immature state, ultimately inhibiting CMs survival and reprogramming. However, we did not detect differences in the levels of the inflammation factor Tgf-β1 between RD + NC and RD + miR-141-200-429 sponge group hearts, indicating that inhibition of miR-141-200-429 cluster does not alter inflammation (Fig. 7 G). Dickkopf-related protein 1 (Dkk1), a suppressor of β-catenin, and bFGF, a neurotrophic factor, 19 were upregulated in miR-141-200-429 sponge group CMs. However, the expression of Dkk1 and bFgf was significantly reduced in CMs from the RD + NC sponge group (Fig. 7 G and 7 J), indicating RAAS inhibition in RD-treated hearts ( Figure S3A-F ). The abrogation of Dkk1 caused by miR-141-200-429 sponges led to the activation of β-catenin (Fig. 7 G and 7 J), which in turn upregulated the expression of bFGF, a factor required for prorenin synthesis. 35 This mechanism ultimately negated the beneficial effects of PBMSC-Exos RD related to RAAS inhibition. We next performed loss- and gain-of-function experiments to determine the potential target of the miR-141-200-429 cluster derived from PBMSC-Exos RD. RDCMs were transfected with miR-141-200-429 mimics (to overexpress the miRNA), sponges (to inhibit endogenous miRNA), or scrambled miRNA/NC sponges (as negative controls). After 48 hours of hypoxic culture, qRT-PCR analysis demonstrated that Dkk1 mRNA levels were significantly decreased or increased with miRNA overexpression or inhibition, respectively ( Figure S6A ). Western blot analysis further revealed that Dkk1 protein expression followed the same trend, decreasing with miRNA overexpression and increasing with miRNA suppression ( Figure S6B ). These findings provide evidence that the miR-141-200-429 cluster directly targets Dkk1. PBMSC-Exos RD Activate the βCatenin Pathway and Improve the Biological Behavior of CMs by Negatively Targeting Dkk1 Since miR-141-200-429 sponges were found to inhibit the β-catenin pathway and impede the RD-induced reprogramming of mature CMs into a more immature state, we next analyzed whether these processes are regulated by Dkk1. To explore this, RDCMs were treated with PBMSCs-Exos RD either alone or in combination with recombinant Dkk1. The RDCMs were then cultured for 7 days under hypoxic conditions to assess the effects. Although PBMSCs-Exos RD ameliorated the proliferation of RDCMs, this benefit was abrogated when RDCMs cotreated with recombinant Dkk1, as demonstrated by Ki67 staining (Fig. 8 A) and CCK-8 assay results (Fig. 8 B). Immunoblot analysis revealed that PBMSC-Exos RD enhanced Oct4 and the cyclin-dependent kinase 1 gene ( Cdk1 ) expression, while notably, c-Myc expression remained largely unaltered (Fig. 8 C and 8 D). However, overexpression of Dkk1 attenuated the PBMSC-Exo RD -induced enhancement of Aurora B incorporation (Fig. 8 E-H), which had previously been observed in hearts subjected to early RD followed by delayed Exos therapy (Fig. 5 B and 5 F). Additionally, Dkk1 overexpression diminished the marked re-expression of Nppa and Acta1 (Fig. 8 G and 8 H)—both markers highly expressed during embryonic and fetal developmental stages. 36 These findings suggest that Dkk1 disrupts the PBMSC-ExosRD-mediated activation of β-catenin signaling, thereby negating its cardioprotective effects linked to CM reprogramming. In contrast to these findings, TUNEL assay revealed that the cell death rate was significantly lower in PBMSC-Exo RD -treated CMs compared to control CMs; however, this protective effect was reversed by Dkk1 overexpression (Fig. 8 I and 8 K). Furthermore, we found that Dkk1 abolished the prosurvival effects of PBMSC-Exos RD on CMs, as evidenced by the upregulation of the proapoptotic factors caspase-3, cleaved caspase 3 and p53, and downregulation of the antiapoptotic factors survivin and Bcl2 (Fig. 8 J and 8 L). These results suggest that Dkk1 may either suppress RD-induced proliferation or induce apoptosis in RD-treated CMs. Consequently, targeted inhibition of Dkk1 in CMs likely represents a key mechanism through which PBMSC-Exos RD exerts its therapeutic benefits. To confirm that RD-induced β-catenin activation is mediated by Dkk1 inhibition, we analyzed β-catenin-associated protein expression in RDCMs receiving PBMSC-Exos RD , recombinant Dkk1, or both, using Western blotting. The results demonstrated that PBMSC-Exos RD significantly suppressed Dkk1 expression while upregulating phosphorylated GSK-3β at serine 9 (p-GSK-3β S9), cyclin D1, Tcf4 (Fig. 8 M and 8 P), and Oct4 (Fig. 8 C and 8 D) compared to the control group. RDCMs cotreated with Dkk1 produced results contrasting those observed in the PBMSC-Exos RD group, suggesting that PBMSC-Exos RD activated the Wnt/β-catenin pathway via repression of Dkk1, thereby enhancing proliferation in RDCMs. This aligns with our prior findings demonstrating that nuclear β-catenin accumulation serves as a hallmark of canonical Wnt pathway activation. 10 Hypoxia injury during lung development leads to excessive Wnt/β-catenin activation through β-catenin accumulation. GSK3 phosphorylation can promote β-catenin transfer from the cytoplasm to the nucleus. 37 Consistent with the changing trend of GSK3 phosphorylation, PBMSC-Exos RD significantly increased and Dkk1 significantly reduced the expression of β-catenin, especially nuclear β-catenin, compared with those in the CTRL group (Fig. 8 N and 8 Q). Immunofluorescence staining demonstrated a stronger β-catenin fluorescence intensity in the PBMSC-Exos RD group compared to the CTRL group, which was significantly attenuated upon Dkk1 overexpression (Fig. 8 O and 8 R). Collectively, these findings suggest that miR-141-200-429-enriched PBMSC-Exos RD likely suppresses Dkk1—a negative regulatory factor of Wnt signaling—thereby activating GSK3β phosphorylation. This mechanism promotes β-catenin accumulation, nuclear translocation, and transcriptional activity, ultimately driving activation of the Wnt/β-catenin pathway. To investigate the potential role of the Wnt/β-catenin pathway in promoting cardiac reprogramming following RD and Exos administration, we evaluated the expression of β-catenin-relative signaling molecules in cardiac tissue. As shown in Figure S7A and S7B , hearts from pigs treated with RD or Exos (PBMSC-Exo RD−Sham or PBMSC-Exos RD ) ) exhibited a significant upregulation of β-catenin and phosphorylated Gsk-3β S9 at serine 9 (p-GSK-3β S9) compared to the RD-Sham + PBS group. The combination treatments further amplified these expression levels relative to either treatment alone. Notably, both RD and Exos monotherapy significantly suppressed Dkk1 expression compared to PBS treatment in the RD-Sham group, with the RD + Exos combination demonstrating an additive inhibitory effect on Dkk1 ( Figure S7C ). These findings are corroborated by immunohistochemical evidence, demonstrating that RD and Exos both individually and combinatorially upregulate β-catenin signaling through suppression of the pathway's negative regulator Dkk1. Finally, to confirm that PBMSC-Exo RD -induced β-catenin activation and subsequent CMs reprogramming contribute to heart regeneration, we employed a human pluripotent stem cell antibody array to assess the relative expression of β-catenin transcriptional activation downstream targets 38 in the hearts of pigs treated with RD-Sham + PBS, RD + PBS, or RD + Exos. The expression of three crucial pluripotency factors (i.e., Oct4, Klf4, and Nanog), two β-catenin nuclear retention factors (i.e., YAP1 and TCF4), 39 and a cardiac lineage marker (i.e., Gata4) was elevated in the RD and RD-Sham + Exos group, with the highest expression observed in the RD + Exos group ( Figure S7D ). In contrast, hearts of pigs receiving the combination treatment exhibited significantly reduced levels of β-catenin destruction regulators LEF1 and Axin2 ( Figure S7E ). 40 No significant differences were observed in the expression of other Wnt/β-catenin-related transcript factors SMAD2, TCF7, Sox2, and c-Myc across these groups ( Figure S7E ). These findings were corroborated by immunoblot assays, which revealed that hearts from pigs co-treated with RD and Exos displayed the highest expression of Oct4, Klf4, Nanog, and Gata4 but the lowest expression of LEF1 and Axin2 ( Figure S7F) , suggesting a reprogramming state in HF hearts following RD + Exos treatment. Immunohistochemical staining further validated these results, demonstrating increased density of the cardiac β-catenin-relative reprogramming transcription factors Oct4, Klf4, Nanog,YAP1, TCF4, and Gata4 ( Figure S7G ). DISCUSSION Our study demonstrates that RD combined with PBMSC-derived Exos promotes cardiac repair in a porcine model of MI by enhancing cardiomyocyte reprogramming. This process is mediated via miR-141-200-429 cluster-dependent suppression of Dkk1, leading to activation of the Wnt/β-catenin pathway. Key findings include: (1) PBMSC-derived Exos significantly improved cardiac functional recovery and reduced infarct size; (2) miR-141-200-429 clusters within Exos drove the reprogramming of mature cardiomyocytes into a more immature, regenerative state; and (3) this cellular reprogramming process was mechanistically linked to Dkk1 inhibition and subsequent β-catenin activation, as evidenced by genetic suppression experiments utilizing AAV-mediated delivery of miR-141-200-429 sponge constructs. These results highlight a novel therapeutic strategy targeting Wnt/β-catenin signaling to mitigate post-MI remodeling. RD was initially developed as a treatment for resistant hypertension. Given the sympathoinhibitory effects of RD, Polhemus et al. demonstrated that RD represents a novel therapeutic strategy to reduce SNS activation and enhance left ventricular performance, with benefits extending beyond blood pressure lowering. 41 Building on this, we explored the remote, direct cardioprotective effects of RD on CMs. Given the clinical importance of CM proliferation in MI treatment, we employed a pig MI model to evaluate the impact of RD on cardiac injury under HFrEF conditions. Our findings revealed that elevated SNS activity under MI conditions exacerbates myocardial ischemic stress, suppresses proliferation signaling, and activates apoptosis pathway. In contrast, RD-treated pigs exhibited a significant reduction in kidney SNS activity. Furthermore, RD administered after MI onset improved LVEF, facilitated recovery of left ventricular end-systolic and diastolic volumes, and inhibited adverse LV remodeling. However, this local attenuation of SNS activity was transient, lasting only up to 2 weeks post-MI (Fig. 1 ), and did not sustain long-term, sustained improvements in cardiac function, remodeling, or survival rates (Fig. 3 ). These results underscore the need to enhance the remote cardioprotective effects of RD to achieve sustained cardiac repair and improved outcomes following MI-induced HF. 41 In the current study, we identified five novel findings. First, we provided direct evidence that RD-derived PBMSC-Exos therapy promotes the beneficial effects of RD on LV performance, resulting in long-term, sustained improvement in the cardiac outcomes of our HFrEF pig model (Fig. 3 ). The clinical application of RD for hypertension treatment remains controversial. Furthermore, it is still unclear whether RD exerts lasting effects or can be effectively applied to other SNS-related conditions, such as HF, atrial fibrillation, and sleep apnea syndrome. 42 In our HFrEF pig model, the hearts demonstrated distinct therapeutic effects at various timepoints and stages of the process. Specifically, we observed a significant reduction in SNS and RAAS activities in our RD pigs, with peak effects occurring 2 weeks post- MI (Fig. 1 ). In addition to disrupting renal afferent signaling pathways, RD demonstrated significant attenuation of ventricular fibrosis (Fig. 4 B) and downregulation of myocardial pro-inflammatory mediators (IL-6 and NF-κB; Figures S5A-B ), which effectively mitigate macrophage-driven fibrotic cascades. 43 These findings suggest RD exerts regulatory effects on ventricular fibrosis progression in post-MI remodeling. In contrast, the release of PBMSC-Exos increased significantly in a time-dependent manner, peaking 2 weeks after MI in the RD group. However, the magnitude of these changes diminished during subsequent follow-up time. This observation may explain why the Symplectity HTN-3 trial results failed to demonstrate further improvement in clinical outcomes 6 months after RD. 44 Delayed MSC-Exos administration resulted in sustained HF-preventive effects in RD pigs, persisting up to 30 days post-MI. Specifically, LV function and dimensions were preserved, infarct expansion was suppressed, and post-MI survival rates were significantly improved. These findings provide critical insights into potential therapeutic approaches for HFrEF (Fig. 3 ). Second, we obtained the first evidence that PBMSC-Exos RD induce reprogramming of ischemic CMs. Several recent studies have indicated that RD may have beneficial effects on ventricular remodeling in post-MI HF. 8 , 41 These results have confirmed robust attenuation of kidney SNS and RAAS activation, coupled with oxidative stress inhibition, G-protein-coupled receptor kinase 2 (GRK2) inhibition, and increased nitric oxide signaling, as a key beneficial effect of RD in HF. Nevertheless, therapeutic mechanisms through which RD improves outcomes have yet to be fully elucidated in preclinical models of HFrEF and remain the subject of ongoing research efforts. 45 In the current study, we observed transient SNS and RAAS activity–lowering effects in HFrEF pigs; however, this attenuation could not be sustained with no significant changes in plasma and kidney SNS and RAAS activity 15 days after MI (Figs. 1 A-E and Fig. 4 I-M). As such, these short-term effects cannot sufficiently explain long-term improvements in cardiac function after RD + Exos. We then focused on the other mechanisms through which Exos may promote cardiac repair by preserving LV function independent of the attenuation of kidney SNS and RAA activities. Thus far, Exos have been applied to enhance human induced pluripotent stem cell viability and differentiation into mature CMs. 46 Exos-mediated intercellular communication plays a considerable role in myocardial repair and signaling transduction after MI. 47 Here, we, for the first time, investigated the effects of PBMSC-Exos RD in a large animal model of post-MI HFrEF. The increased proliferation of CMs in RD-treated hearts, Exos-treated hearts, and PBMSC-Exos RD treated-CM cultures was demonstrated through Ki67 (Fig. 5 A, Fig. 8 A, and Figure S4D ) and pH3 ( Figure S4E ) staining-established biomarkers for cell cycle activity. This observed proliferation indicates enhanced CMs division capacity, a crucial mechanism for myocardial regeneration. 48 As shown in Fig. 5 A and 5 E, early RD alone significantly increased the number of Ki67-positive CMs in both peri-infarct and remote zones compared to RD-Sham. This suggests that RD stimulates endogenous regenerative mechanisms, including CMs proliferation. Delayed Exos injection further enhanced this effect, leading to a greater number of Ki67-positive CMs in the RD + Exos group. This additive interaction suggests that delayed Exos therapy amplifies the regenerative benefits of early RD. Terminally differentiated CMs cultured with PBMSC-Exos RD reentered the cell cycle and proliferated under hypoxic conditions, with CM proliferation occurring through redifferentiation under hypoxia ( Figure S4F ). Consistent with our immunofluorescence staining results, gene detection analysis revealed that PBMSC-Exos RD upregulated the expression of factors related to proliferation ( Cdk1 and Yap1 ) and reprogramming ( Oct4 , Sox2 , and Klf4 ; Figure S5 ). Similar findings were noted in our in vivo animal study. In HFrEF pigs, RD enhanced mature CM proliferation and dedifferentiation, and subsequent delayed exosomal therapy further amplified these effects (Fig. 5 A-C). These results aligned with our histological findings, which demonstrated a significant reduction in scar size and fibrosis, correlating with the greatest increase in the number of viable CMs (Fig. 4 C and 4 D). This is consistent with Gallet et al.’s observation that cardiosphere-derived cell Exos therapy reduce scarring, mitigate adverse remodeling, and improve LVEF in HFrEF pigs. 49 Furthermore, we observed a significant increase in the Exos uptake rate of hearts of HFrEF pigs treated with RD compared to those receiving RD-Sham, and delayed exosomal therapy further increased this uptake in HFrEF pigs (Fig. 5 D and 5 H). These findings were corroborated by recent studies showing a robust increase in the Exos level in the blood of RD-treated mice. 30 Additionally, Exos uptake was positively correlative with cardiac dedifferentiation in the HFrEF pigs receiving the combination of RD and Exos therapy (Fig. 5 J and 5 K). Collectively, these results advance our understanding of the mechanisms underlying the inhibition of LV remodeling in HFrEF pigs following effective RD. They also facilitate the identification of novel therapeutic targets aimed at reducing MI mortality by maintaining circulating Exos levels and their cardioprotective effect. Third, we identified β-catenin as the most important molecule in RD-mediated myocardial reprogramming. Exos play a role in the cardiorenal syndrome. Cardiorenal Exos regulate proangiogenic paracrine signaling in adipose MSCs after MI. 50 Endothelial cells can transfer caveolin-1-containing Exos to adipocytes in newly generated mouse models. 51 Here, we demonstrated, for the first time, that RAECs released functional circulating PBMSC-Exos and became transferred to injury CMs after RD (Fig. 2 ). Moreover, RD significantly upregulates the myocardial expression of β-catenin ( Figure S5 )—a major signal transduction molecule in MSC-Exos widely involved in regeneration or repair-related biological processes. 52 This finding aligns with our observation that RD-treated cardiomyocytes exhibited a marked upregulation of reprogramming factors Oct4, Sox2, and Klf4 ( Figure S5 ), a phenomenon associated with β-catenin activation. 53 Notably, β-catenin overexpression led to a continuous increase in CMs uptake of PBMSC-Exos RD and proliferation, redifferentiation, and apoptosis prevention in hypoxia-cultured CMs (Fig. 6 ). In cancers, Exos are exchanged between cancer cells and the tumor stroma, promoting the transfer of various oncogenes (e.g., β-catenin , Ceacam1 , Her2 , Melan-A / Mart-1 , and Lmp1 ) from one cell to another, leading to recipient cell reprogramming. 54 Taken together, these findings further highlighted the importance of β-catenin upregulation by circulating PBMSC-Exos for RD-mediated myocardial reprogramming. Fourth, we identified miR-141-200-429 cluster as the most essential molecule in myocardial reprogramming induced by PBMSC-Exos RD . Emerging evidence indicates that Exos contain specific miRNAs contributing to tissue repair and immunomodulation. 55 Rodent studies have demonstrated that the potential therapeutic effects of RD on LV fibrosis were partly mediated by miRNA regulation. 56 As such, we performed high-throughput screening and found that PBMSC-Exos RD contained higher levels of miR-141-200-429 clusters than PBMSC-Exos RD-Sham did (Fig. 2 D). miR-141-200-429 clusters are generated by RD-treated RAECs and carried by PBMSCs from RAECs to CMs (Fig. 2 E). In particular, although all 5 genes in miR-141-200-429 clusters were significantly upregulated in RAECs (Fig. 2 F), only miR-200a-3p, miR-200b-3p, and miR-141 were significantly upregulated in CMs (Fig. 2 G). Furthermore, RAEC-specific miR-141-200-429 cluster sponge administration to RD-Sham and RD pigs significantly reduced CMs Exos uptake in MI hearts (Fig. 7 B) and prevented miR-200a-3p, miR-200b-3p, and miR-141 elevation in RD-treated CMs (Fig. 7 D). Notably, miR-141-200-429 sponges significantly reduced expression of the β-catenin-related genes and pathways related to cell proliferation, apoptosis, and dedifferentiation (Fig. 7 G and 7 J). Exos-encapsulated miRNAs can increase CMs proliferation. 57 In particular, a few human miRNAs can stimulate the entry of rodent CMs into the cell cycle and cardiac regeneration after MI in mice. 58 Taken together, these findings provide novel insights into the positive feedback mechanisms of RD: RD promotes the release and uptake of Exos rich in miRNAs. miRNAs then enhance the remote cardioprotective effects of RD, improving CM proliferation, antiapoptosis, and regeneration, as well as systolic LV function through activation of β-catenin signaling under HF conditions ( the Graphical Abstract Image ). However, further validation through additional studies, including clinical studies, is essential to confirm these findings and explore the potential therapeutic applications. Finally, by utilizing bioinformatic analysis followed by multiple experimental validation, we identified Dkk1 as a target gene of the novel miR-141-200-429 cluster. Dkk1 stands for Dickkopf-related protein 1, which is a secreted protein that acts as an inhibitor of the Wnt/β-catenin signaling pathway. 59 The Wnt/β-catenin pathway is crucial for various cellular processes, including cell proliferation, differentiation, and survival. Various miRNAs target Dkk1 , which is silenced by miRNAs in the early stages of osteogenic differentiation. In contrast, miRNA levels decrease and Dkk1 expression is upregulated in the later stages of differentiation. 60 We demonstrated, for the first time, that Dkk1 is a direct target of miR-141-200-429 clusters, and its inhibition after RD is pivotal for CMs proliferation and re-differentiation. Since miR-141-200-429 sponges inhibit the β-catenin pathway and hinder the RD-induced reprogramming pathways ( 7E and 7F ), it is essential to investigate whether Dkk1, as an inhibitor of this pathway, plays a regulatory role in these processes. By analyzing Dkk1, we have gained insights into multiple mechanisms through which it modulates the effects of miR-141-200-429 sponges on the β-catenin pathway and the subsequent reprogramming of CMs (Fig. 7 G and 7 J), including (1) preventing CM proliferation and expression of its markers Cdk1 and Mcl-1; (2) preventing CM dedifferentiation and expression of its markers Acta1 and Nppa; (3) increasing CM apoptosis and expressions of the apoptotic proteins caspase3 and p53 along with decreased expression of antiapoptotic proteins survivin and Bcl2; (4) reducing pluripotency transcription factors Oct4 and c-Myc functionally associated with impaired β-catenin signaling activity. 61 Moreover, Dkk1 overexpression was noted to significantly attenuate PBMSC-Exo RD -induced increase in phosphorylated GSK-3β, nuclear translocation of β-catenin, and expression of cyclin D1 and Tcf4 (Fig. 8 M-R), which are miR-200a targets. 62 Pretreatment with Dkk1 inhibits Wnt1-stimulated differentiation of human periodontal ligament fibroblasts by suppressing GSK-3β phosphorylation and nuclear translocation of β-catenin, 63 leading to upregulation of the Wnt/β-catenin target genes cyclin D1 , Tcf4 , and Lef1 . 64 This could help elucidate the molecular mechanisms underlying the observed phenomena and potentially identify Dkk1 as a key player in the regulation of cardiomyocyte reprogramming. In the present study, the expression of RAEC miR-141-200-429 clusters was significantly increased in PBMSC-Exos. miR-141-200-429 clusters are packaged in Exos and transferred from PBMSCs to CMs, where they suppress the expression of Dkk1, a critical β-catenin suppressor, causing β-catenin activation (Fig. 7 G and 7 J). Increasing PBMSC-Exos production, enhancing CMs biogenesis, or activating β-catenin target genes (particularly Tcf4, Fig. 8 M and 8 P) may be novel, effective therapeutic avenues to improve PBMSC-Exos-mediated beneficient communication between RAs and MI hearts after RD, enhancing cardiac regeneration in acute MI ( Figure S8 ). Taken together, these results suggested that PBMSC-Exos participate in the protective effects of RD against ischemic injury. Existing studies demonstrate that the post-MI inflammatory response progressively transitions into the reparative phase, followed by the remodeling phase. 65 Therefore, the treatment for MI must address both acute-phase anti-inflammatory interventions and chronic-phase management of myocardial fibrosis. This study demonstrates that the sequential therapeutic approach integrating acute-phase RD intervention with later-phase Exos administration achieves comprehensive functional recovery in infarcted hearts throughout the post-MI repair process. This translation aligns with current guidelines emphasizing stage-specific treatment strategies in MI. We also found that delayed Exos injection significantly reduced plasma aldosterone levels (Figure S3) . It also increased circulating BNP levels sustainably with RD (Table 1 ). RD-induced increases in circulating BNP lead to improvements in cardiac function and protection against ischemia-reperfusion injury, 8 suggesting that long-term cardioprotective effects may arise from the additive actions of RAAS inhibition and enhanced cardiac function. Study Limitations While our findings demonstrate the potential of PBMSC-Exos miRNAs to enhance cardiac repair of RD after MI, this study has several limitations. First, the porcine model of MI-induced HFrEF was established solely through the ligation of the left anterior descending coronary artery. However, clinical MI patients with HFrEF frequently present with multivessel disease and undergo revascularization therapies, whereas our model does not account for these complexities. Consequently, this experimental system fails to fully replicate the multifactorial pathophysiology of human HFrEF. Second, early cardiac biopsy specimens collected at 14 days post-MI from RD-treated and RD-Sham hearts demonstrated that PBMSC-Exos RD significantly upregulated pluripotency transcription factors (Oct4, Klf4, Sox2) in RDCMs ( Figure S5 ). However, longitudinal analysis revealed no detectable increase in Sox2 or c-Myc expression in ischemic cardiac tissues from either group, even at the 90-day post-MI endpoint ( Figure S7 ). This temporal discrepancy—where transient transcriptional activation occurs acutely but dissipates chronically—may reflect dynamic shifts in epigenetic regulation, microenvironmental crosstalk (e.g., inflammatory or fibrotic signaling), or unresolved compensatory feedback loops. While these findings underscore the context-dependent nature of cellular reprogramming, their clinical extrapolation necessitates rigorous consideration of spatiotemporal biological constraints and interspecies translational gaps. Third, while in vitro analyses identified β-catenin as a pivotal regulator of hypoxia-induced endogenous reprogramming in mature mammalian CMs, we were unable to corroborate this mechanism through functional gain- or loss-of-function experiments (e.g., β-catenin overexpression or knockdown) in vivo . The absence of animal-level validation substantially undermines the translational validity of our proposed molecular pathway. Fourth, while in vitro sponge-mediated miR-141-200-429 knockdown blocked RA-Exos' cardioprotection under hypoxia, validation in an MI model via RA-Exos-specific miR-141-200-429 overexpression is lacking. Future studies employing targeted activation of miR-141-200-429 in RA-Exos, followed by functional assessments in MI models, would clarify its specific role and enhance the translational relevance of our findings. Conclusions This study provides the first evidence that RD confers protection against myocardial ischemia-reperfusion injury in a porcine model of HFrEF post-MI, mediated through increased delivery of PBMSC-Exos enriched with miR-141-200-429 clusters. These miRNAs orchestrate multiphasic cardioprotection by attenuating neurohormonal activation (reduced circulating catecholamines and suppressed renin-angiotensin-aldosterone system [RAAS] activity), inhibiting apoptosis pathways (downregulation of Bax/Caspase-3), enhancing myocardial stress adaptation (elevated BNP expression), and activating β-catenin-mediated proliferative signaling. Notably, the RD + Exos combinatorial strategy enabled sustained miRNA persistence (Up to 90 days) in ischemic myocardium, overcoming the transient bioavailability limitations of conventional miRNA therapies. While RD's therapeutic potential was initially attributed to blood pressure modulation, our findings reveal broader mechanistic implications, including epigenetic reprogramming and paracrine crosstalk. This positions RD not merely as a hemodynamic intervention but as a platform for targeted organ protection, warranting exploration in other chronic conditions characterized by oxidative stress and maladaptive remodeling (e.g., diabetic cardiomyopathy, chronic kidney disease). Furthermore, we propose a translational framework integrating RD with precision medicine approaches: (1) Temporal synergy: Co-administration with guideline-directed antihypertensive therapies to optimize hemodynamic and cellular repair phases; (2) Spatial control: Nanoparticle engineering of Exos for tissue-specific miRNA delivery; (3) Safety escalation: Biomarker-guided titration to minimize off-target epigenetic effects. These preclinical insights bridge the gap between observational cardioprotection and mechanism-driven therapeutic innovation, offering a roadmap for clinical translation in HFrEF management. MATERIALS AND METHODS Antibodies and other reagents The sequences of the primers used in this study are provided in Table S1 , and the antibody details are listed in Table S2 . 4′,6-Diamidino-2- phenylindole (DAPI; catalog 28718-90-3) was purchased from Sigma-Aldrich. Animals and Study Design Rat-To investigate the appropriate dose of PBMSC-Exos used for injection, a preliminary dose ranging experiment was performed on adult male Sprague-Dawley rats (200–250). Ligation of the left anterior descending coronary artery was ligated as our previously described, 10 which was subsequently removed after 45 minutes to allow for reperfusion. Twenty minutes later, the rats were injected with 100µl PBS or PBMSC-Exos from MI pigs (5×10 12 , 2×10 13 , or 5×10 13 in 100µL PBS) via caudal vein, over a period of 20 seconds. Thirty days later, all the rats underwent echocardiography, and then were euthanized for serological and molecular biology testing. Pig-Domestic pigs were housed indoors and provided with a commercial diet and fresh water ad libitum to maintain body weight and support growth. 66 All domestic pigs (weight, 40–50 kg) were sedated with ketamine (15–20 mg/kg) and diazepam (1.5–2 mg/kg), followed by anesthesia maintenance with intravenous thiopental (1–2 mg/kg/min). Preanesthetic atropine (30–50 µg/kg, intramuscular) was also administered. MI was induced by ligating the left anterior descending coronary artery midway between its origin and the apex, as previously described. 67 After 45 minutes of ischemia, reperfusion was initiated and maintained until the endpoint. Pigs with LVEF < 40% within 2 hours post-MI were selected for a 90-day follow-up (Fig. 3 A). A total of 95 pigs underwent surgery, with 82 surviving the procedure. Among the survivors, 22 MI pigs were excluded due to ventricular fibrillation or LVEF ≥ 40%. The remaining 60 pigs with LVEF < 40% within 2 hours post-MI were selected as our HFrEF model and randomly assigned to either RD-Sham or radiofrequency RD treatment. Twenty pigs from each group were further divided into four subgroups (n = 10 per group) based on treatment: RD-Sham + PBS (phosphate buffered saline), RD-Sham + Exos, RD + PBS, and RD + Exos (n = 10 per group). At 30 days post-MI, surviving animals received either 2 × 10¹³ PBMSC-derived exosomes (Exos) or an equivalent volume of PBS and were monitored for 90 days ( Figure S2 ). No deaths occurred during RD or Exos administration. For endpoint analysis, pigs were euthanized with phenobarbital sodium at 90 days post-MI. An additional 10 pigs from the RD/RD-Sham groups underwent myocardial biopsy and Exos collection 14 days post-MI, followed by injection of BODIPY TR ceramide-prelabeled RAEC-derived Exos. These pigs were euthanized with intravenous pentobarbital sodium at 44 days post-MI (observation endpoint; Figure S2 ). As the negative control of MI surgery, sham-operated controls (n = 5) underwent identical surgical procedures (thoracotomy, pericardial exposure) without coronary artery ligation. These pigs received RD treatment and were followed for 30 days. Monitoring In all experiments, heart rate was monitored through electrocardiography (ECG), and mean aortic blood pressure was measured at baseline and 0, 30, 60, and 90 days after MI. Echocardiography Two-dimensional echocardiography was performed at midpapillary and apical levels. Left ventricular end-diastolic volume (LVEDV) and end-systolic volume (LVESV) were measured using the biplane area-length method, with ejection fraction (LVEF) calculated by the modified Simpson's method. Radiofrequency RD Pigs were randomly grouped into an RD or RD-Sham group within 2 h after MI. Radiofrequency RD was performed, as described previously. 41 For RD, pigs received bilateral denervation of the aorta and RA with a Celsius electrophysiology catheter (Stockert 70; Biosense Webster, Diamond Bar, CA, USA). Ground pads were placed to protect the underlying tissue between the shoulder blades. The tip of the radiofrequency probe was placed in four quadrants (circles) of RAs, with 10 W for each quadrant in the RD group and 0 W for the RD-Sham group, lasting 20 s. Cardiac Biopsy Transmural biopsy specimens (10 mm wide) were collected from RD-treated pigs at 14 days post-MI. From each heart, five samples were obtained from distinct peri-infarct regions and analyzed for ischemic CM proliferation. These myocardial specimens were processed through an integrated experimental platform employing both in vitro and in vivo approaches to systematically evaluate the therapeutic effects and mechanistic basis of PBMSC-Exo RD -induced cardiac reprogramming. Plasma Biochemistry Assessment Peripheral blood was collected at baseline and at 0, 30, 60, and 90 days post-MI. Circulating levels of BNP, hs-cTnT, BUN, creatinine, norepinephrine, and epinephrine were quantified using commercial ELISA kits (Abnova) per manufacturer's protocol. Plasma renin and aldosterone levels, along with aldosterone-renin ratios, were assessed at 90 days post-MI using established methods. 68 Kidney Catecholamine and Angiotensin Measurements To evaluate RD efficacy, kidney catecholamine and angiotensin levels were quantified at 90 days post-MI. Renal cortex tissues were rapidly harvested, flash-frozen in liquid nitrogen, and homogenized. Catecholamine concentrations were determined by high-performance liquid chromatography (HPLC) and expressed as nanograms per gram of wet tissue weight. Angiotensin levels were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) at Attoquant Diagnostics (Vienna, Austria), as previously described. 8 PBMSCs and RAEC Isolation and Collection Peripheral blood was collected from RD- and RD-Sham-treated pigs at 14 days post-MI. PBMSCs were isolated using established methods, 10 with second-passage cells utilized for Exos collection and subsequent experiments. RAs and myocardial tissues were collected from pigs at 14 days post-MI. RAECs were isolated from RAs and cultured in endothelial cell growth medium (ScienCell) containing 5% heat-inactivated FBS, 1% penicillin-streptomycin, and 1% endothelial cell growth supplement (all from ScienCell), maintained at 37°C with 5% CO₂in a humidified incubator. 69 Only RAECs with > 96% purity (confirmed by flow cytometry/immunostaining) were used for Exos production. Exos Isolation, Characterization, Transfection, Labeling, and Tracing Cells were expanded in serum-free conditions (MesenCult-ACF-XFAttachment Substrate; STEMCELL Technologies) following the manufacturer’s instructions. To ensure Exos derived exclusively from PBMSCs and exclude contamination by other extracellular vesicles or protein aggregates, we performed sequential ultracentrifugation and density gradient purification. After 96 h of culture, the conditioned medium was harvested and subjected to: (1) Low-speed centrifugation (800×g, 5 min) to remove cells and debris; (2) Intermediate centrifugation (2,000×g, 10 min) to eliminate apoptotic bodies and large microvesicles; (3) Filtration through a 0.22-µm pore-size membrane to exclude particles > 200 nm; (4) Ultracentrifugation (100,000×g, 2 h, 4°C) to pellet Exos. The final Exos pellet was resuspended in sterile PBS and stored at − 80°C for downstream applications. 70 Next, Exos morphology was verified by TEM. Briefly, purified Exos were diluted in PBS, negatively stained with 2% uranyl acetate, and adsorbed onto carbon-coated copper grids. Vesicles exhibiting characteristic cup-shaped morphology were visualized at varying magnifications using a Hitachi H-7500 TEM. To further validate Exos quality, we performed: (1) NTA to determine particle size distribution and concentration; (2) Western blotting to confirm the presence of exosomal markers (CD9, CD63, TSG101, and HSP70). 71 For Exos labeling, PBMSC-derived Exos from RD-treated pigs were incubated with 4 µM PKH26 fluorescent dye (Sigma-Aldrich) in Diluent C, following the manufacturer's protocol. 72 The labeled Exos were subsequently administered via peripheral intravenous injection to MI pigs at 30 days post-MI. To assess Exos uptake, Exos-treated CMs were fixed and immunostained with primary antibodies against myosin heavy chain (MHC), followed by Alexa Fluor 488-conjugated secondary antibodies (1:200 dilution; A32723, Thermo Fisher Scientific). Nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole). Fluorescent images were acquired using an FV1000 confocal microscope (Olympus) and analyzed with FV10-ASW software (Olympus). CMs exhibiting colocalization of MHC and the Exos tracker (PKH26) were identified as Exos-positive cells. To assess the role of miR-141-200-429 clusters in RD outcomes, RAECs isolated from RD-treated pigs were expanded as previously described. We constructed miR-141-200-429 sponge vectors (and corresponding negative control [NC] sponges; Syngentech, Shanghai) under the control of the endoglin promoter. At 60–80% confluence, RAECs were transfected with 50 nM sponge constructs using Lipofectamine 3000 (Invitrogen) per manufacturer's protocol. After 48 h, conditioned medium was collected, and Exos were isolated by ultracentrifugation for downstream analyses. Exos miRNA Analysis Total RNA was isolated from PBMSC-derived Exos using TRIzol reagent (Invitrogen). Small RNAs (18–50 nt) were size-selected for miRNA sequencing. miRNA libraries were prepared with the QIAseq miRNA Library Kit (Qiagen) and sequenced on an Illumina NovaSeq 6000 platform. Bioinformatic analysis included: (1) Raw read processing: Demultiplexing, adapter trimming, and quality filtering; (2) Alignment to miRBase v21 using bowtie2; (3) Quantification with HTSeq (v0.6.1); (4) Differential expression analysis and hierarchical clustering (pheatmap R package); 61 (5) Functional annotation including GO and KEGG pathway analysis (clusterProfiler), Visualization of KEGG pathways (Bioinformatics Network platform), and Venn diagram analysis ( http://bioinformatics.psb.ugent.be/webtools/ Venn). 73 Core miRNAs regulating cardiac proliferation/regeneration were identified through integrative analysis of these datasets. Primary CMs Isolation and Culture To assess cardiomyocyte CM proliferation and differentiation potential, we isolated primary CMs from endomyocardial biopsies. Tissue samples were enzymatically digested in Tyrode buffer containing 1.5 mg/mL collagenase type II and 0.1 mg/mL hyaluronidase for 50 min. 74 Following digestion and filtration, cells were sequentially equilibrated inCalcium-free Tyrode solution (5 min) and Gradual calcium reintroduction (30 min). Isolated CMs were plated at 1.5×10⁵cells/cm² in complete culture medium (DMEM supplemented with 10% FBS (Biosera), 4,500 mg/L glucose (Sigma-Aldrich), 4 mM L-glutamine (Biosera), 100 IU/mL penicillin (Biosera), and 100 µg/mL streptomycin (Biosera)) Loss-and Gain-of-Function Experiments For genetic manipulation of β-catenin expression, we used pMXs retroviral vectors carrying β-catenin cDNA in pReceiver-LV233 lentiviral backbone (GeneCopoeia) and pSiLVRU6GP vectors expressing Ctnnb1-targeting shRNAs with puromycin resistance (GeneCopoeia) for overexpression and knockdown, respectively. Transfection was performed in PBMSCs using FuGENE HD transfection reagent according to manufacturer's protocol. This generated β-catenin-overexpressing cells ( oe β-catenin), β-catenin-deficient cells ( si β-catenin) and Vector-transfected controls To identify the potential target of PBMSC-Exo RD -derived miR-141-200-429 cluster, RDCMs were transfected with miR-141-200-429 mimics (to overexpress the miRNA, synthesized by Syngentech, Shanghai, China) or sponges (to suppress endogenous miRNA, synthesized by Syngentech, Shanghai, China). Scrambled miRNA or NC sponges were used as negative controls. Hypoxic Experiments We next investigated the effects of PBMSC-Exos, β-catenin modulation, and Dkk1 on CM behavior under hypoxia. RDCMs were subjected to the following interventions: transfection with oe β-catenin, sh β-catenin, or empty vectors; treatment with the β-catenin agonist WAY-262611 (1 µmol/L; ab145229; Abcam, USA); administration of PBMSCs-Exos (2 × 10 13 ) isolated from RD or RD-Sham pigs; or supplementation with recombinant Dkk1 (100 ng/mL; R&D Systems #5497-A6-050). 75 CMs were cultured at 1.5×10⁵cells/cm²in complete medium under hypoxic conditions (1% O₂) for 7 days. CM proliferation and cardiomyogenesis were assessed on days 3 and 7 of culture. Immunofluorescence Staining Cell samples and myocardial tissues were fixed with 4% paraformaldehyde for 10 minutes. Myocardial tissues were embedded in optimum cutting temperature (OCT) compound and sectioned into 6-µm-thick slices using a cryostat (Leica CM1520). For immunofluorescence staining, sections were sequentially processed as follows: overnight incubation with primary antibodies at 4°C, 1-hour incubation with fluorescent secondary antibodies at room temperature, and nuclear counterstaining with DAPI (2 minutes). Finally, sections were mounted with antifade medium and imaged. Five independent fields per section were analyzed across representative tissue regions. Cell Proliferation Assay Following 24-hour transfection, CMs were co-cultured with Exos under hypoxic conditions for 48 hours. Cell metabolic activity was quantified using a Cell Counting Kit-8 (CCK-8; Sigma) according to the manufacturer’s protocol. Proliferation was assessed through two parallel methods: (1) Immunofluorescence analysis of cell cycle markers (Ki67, BrdU, and phospho-histone H3 [pH3]), with proliferation rates calculated as the percentage of Ki67-positive nuclei relative to total DAPI-stained nuclei in matched fields; (2) EdU incorporation assay performed via Click-iT™ EdU Alexa Fluor® 488 Imaging Kit (Thermo Fisher, #C10337), utilizing the standardized Click-iT reaction protocol. 76 Flow Cytometry Detection of Cell Apoptosis To assess CM apoptosis, we conducted Annexin V-APC/PI dual staining using a commercial apoptosis detection kit (FACS Annexin V Assay Kit; Trevigen, USA) according to the manufacturer's protocol. Briefly, CMs were harvested after 7-day hypoxic co-culture with either 2×10¹³ empty AAV6 particles or PBMSC-Exos. Cells were resuspended in 400 µL 1× binding buffer (adjusted to 1 × 10⁵ cells/mL), then incubated with 5 µL Annexin V-FITC and 10 µL PI in 100 µL binding buffer for 30 minutes at room temperature (protected from light). Apoptotic rates were quantified using a FACScan flow cytometer (Becton Dickinson, USA), with Annexin V⁺/PI⁻ (early apoptosis) and Annexin V⁺/PI⁺ (late apoptosis) populations combined for total apoptosis analysis. Pluripotent Stem Cell Antibody Array Next, we defined pluripotent stem cell marker levels from RDCMs in the absence or presence of RD or Exos. In brief, we used a human pluripotent stem cell antibody array (ARY010; MN 55413; USA) to measure the relative levels of pluripotent stem cell markers in hearts from HF pigs receiving early RD-sham or RD plus delayed injection of PBS or Exos. Fluorescence signals were detected using Axon GenePix, and the relative levels of the factors were calculated and analyzed. qRT-PCR Total RNA was isolated from cultured cells and myocardial tissue samples using RNA-Stat reagent (Iso-Tex Diagnostics, USA), followed by cDNA synthesis with 500 ng RNA template using the TaqMan Reverse Transcription Kit (Applied Biosystems, USA). Target-specific primers and probes ( listed in Table S1 ) were designed with Primer Express® software (v3.0, Applied Biosystems). Quantitative RT-PCR was performed on an ABI Prism 7500 system (Applied Biosystems) under standardized cycling conditions. Fluorescence signals from 6-carboxyfluorescein (6-FAM)-labeled probes were monitored in real time, and cycle threshold (Ct) values were determined using ABI Prism SDS software (v2.0, Applied Biosystems). Relative mRNA expression levels were normalized to GAPDH and calculated via the 2 −ΔΔ CT method. Western Blotting Western blot analysis was performed to evaluate protein expression across experimental groups. Tissue/cell lysates were prepared using Pierce™ RIPA buffer (Thermo Scientific™, #78510) supplemented with protease inhibitors. Protein extracts were denatured in Laemmli buffer (Beyotime, #P0015) by boiling at 95°C for 5 min, followed by ice-cooling and centrifugation (12,000×g, 10 min). Proteins (30 µg/lane) were resolved on 10% SDS-PAGE gels and transferred to PVDF membranes (Millipore) using semi-dry electrophoretic transfer. Membranes were blocked with 5% non-fat milk in TBST for 1 hr at RT, then incubated overnight at 4°C with primary antibodies (see Table S2 ) and anti-GAPDH (1:1000; Abcam #ab9485). After three 10-min TBST washes, membranes were probed with HRP-conjugated goat anti-mouse IgG secondary antibody (1:2000; ComWin Biotech #CW0102) for 1 hr at RT. Signal detection was achieved using Clarity™ Western ECL Substrate (Bio-Rad #170–5060) and visualized on a ChemiDoc™ MP Imaging System (Bio-Rad) miRNA Inhibition In Vivo by AAV-Carrying miRNA Sponges miR-141-200-429 sponge inhibitors and NC sponges were purchased from Genechem Co., Ltd. (Shanghai, China). Adeno-associated virus (AAV) vectors were constructed using blood vascular cell–specific promoter elements from the chicken endoglin promoter. The miR-141-200-429 sponges were designed with bulged or imperfect complementarity to target miRNA sequences, including conserved seed regions, as previously described. 77 Each sponge sequence was inserted triplicate. BODIPY TR ceramide (Cat# B34400, Thermo Fisher Scientific, Carlsbad, CA, USA) was utilized to label sponge sequences and validate promoter activity. Recombinant AAV particles (serotype GV618) were generated using the edited AAV vector. Two weeks post-retinal detachment (RD) or RD-Sham surgery, pigs were anesthetized with 2% isoflurane in oxygen, underwent myocardial biopsy and Exos collection, and followed by the administration of 10 µL of viral solution containing approximately 3 × 10 11 vector genomes (vg) per animal via the jugular vein. 78 At 30 days post-AAV administration, frozen cardiac tissues were cryosectioned into 7-µm-thick slices. Sponge expression efficacy was assessed by detecting BODIPY TR ceramide-labeled Exos in cardiac tissues. Fluorescent images were captured using an Olympus BX51 microscope equipped with a DP74 camera (Olympus Corporation, Tokyo, Japan). Wheat Germ Agglutinin (WGA) Staining To measure CM cross sectional area, WGA (TheromFisher) staining was performed as previously described. 33 Cross sectional area was calculated by summing the areas of the high-resolution images at 20X magnification, which were quantified by blinded researchers. Histology and Immunohistochemistry Immunohistochemical staining for tyrosine hydroxylase (TH; HSRL, MA, USA) was performed on RAs from pigs at 1 week, 2 weeks, 4 weeks, and 90 days post-MI, following previously described methods. 73 Briefly, RA segments were harvested from the mid-main and distal branch regions of the RA, fixed in formalin for at least 7 days, and subsequently washed. The tissues were then dehydrated in 70% ethanol, embedded in paraffin, and sectioned into 5-µm-thick slices. Finally, the sections were processed for TH immunohistochemical staining. Hearts from pigs were excised, weighed, and cross-sectioned at the maximal LV area. The sections were immersed in 0.09 M phosphate-buffered saline (PBS, pH 7.4) containing 1.0% triphenyl tetrazolium chloride (TTC; Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 20 minutes to delineate the infarcted area. Additionally, Masson’s trichrome staining was performed on these sections according to the manufacturer’s protocol. Infarct size was quantified by calculating the sum of the infarcted and collagen areas as a percentage of the total LV area using ImageJ software (v1.53, National Institutes of Health, USA). Peri-infarct regions dissected from necropsy specimens were either paraffin-embedded or snap-frozen for cryosectioning. Sections were stained with hematoxylin and eosin (H&E) or processed for immunofluorescence and immunohistochemical analyses. Statistical Analysis The surgical team and investigators were blinded to treatment assignments and outcome data until completion of the study analysis. All experimental results were systematically tabulated, and final reports were generated following unblinding. Quantitative data are presented as mean ± standard deviation (SD), while categorical variables are expressed as frequencies and proportions. Normality was assessed using the Shapiro-Wilk test, and homogeneity of variance was verified via Levene's test. Continuous variables meeting both normality and homoscedasticity assumptions were analyzed using one-way ANOVA. For normally distributed data with heteroscedasticity, Welch’s ANOVA was applied. Categorical variables were compared using the chi-square test or Fisher’s exact test, as appropriate. A threshold of P < 0.05 was defined for statistical significance. Abbreviations α-SMA=alpha-smooth muscle actin BNP = B-type natriuretic peptide CMs=cardiomyocytes Dkk1=dickkopf-1 Exos=exosomes HF = heart failure HFrEF=heart failure with reduced ejection fraction LV = left ventricle/ventricular LVEF=left ventricularejection fraction MHC =myosin heavy chain MI = myocardial infarction MSCs=mesenchymal stem cells; NE=norepinephrine PBMSCs=peripheral blood MSCs mesenchymal stem cells PBS=phosphate buffered saline RA=renal artery; RAECs=renal artery endothelial cells RAAS=renin aniotension aldosterone system RD=renal sympathetic denervation RDCMs=renal sympathetic denervation-treated cardiomyocytes SNS = sympathetic nervous system TH=tyrosine hydroxylase Declarations ACKNOWLEDGEMENTS We thank Renmin Yao (Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai, China) and Baihe Zhang (GuangZhou Red Cross Hospital Medical College of Ji-Nan University) for technical assistance. FUNDING This work was supported by the National Natural Sciences Foundation of China (grant number 81770291, to Zhang S), the Guangzhou Science and Technology Planning Project (grant number 202002030081 to SZ), Guangzhou Science and Technology Program Project funded by the Guangzhou Science and Technology Bureau (Grant No.2023A03J0982). CONFLICT OF INTEREST There is no conflict of interest between the authors. Availability of Data and Material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Code Availability Not applicable Authors' Contributions Z. : Conceptualization, Methodology, Writing-Original draft preparation, preparing figures 1-5. L. C. : Supplement data for later revisions. Z.H. : Data curation, preparing figures 6-10. J . W . : Visualization, investigation, preparing table1. Z . D . :Supervision, preparing figures S1-6; S.Z. : Software, Validation, preparing figures S7 and table S1-2. Y.D. : preparing figures S8 and Graphical Abstract Image. S.Z. :Writing-Reviewing and Editing. All authors reviewed the manuscript. Ethics Approval The Institute for Animal Care and Use Committee at Dahua Hospital approved all the animal experiments, which were carried out in compliance with the Guide for the Care and Use of Laboratory Animals published by The National Academies Press (http://www.nap.edu/). Consent to Participate Not applicable Consent for Publication All authors read and approved the final manuscript. References Azizi, M., Sanghvi, K., Saxena, M. et al. Ultrasound renal denervation for hypertension resistant to a triple medication pill (RADIANCE-HTN TRIO): a randomised, multicentre, single-blind, sham-controlled trial. Lancet 397 , 2476-2486 (2021). Zhou, Z., Liu, C., Xu, S. et al. Metabolism regulator adiponectin prevents cardiac remodeling and ventricular arrhythmias via sympathetic modulation in a myocardial infarction model. Basic. Res. Cardiol. 117 , 34 (2022). Kwon, J.S., Schumacher, S.M., Gao, E. et al. Characterization of βARKct engineered cellular extracellular vesicles and model specific cardioprotection. Am. J. Physiol. Heart. Circ. Physiol. 320 , H1276-1289 (2021). Kimura, K., Ieda, M. & Fukuda, K. Development, maturation, and transdifferentiation of cardiac sympathetic nerves. Circ. Res. 110 , 325-336 (2012). Polhemus, D.J., Trivedi, R.K., Sharp, T.E. et al. Repeated cell transplantation and adjunct renal denervation in ischemic heart failure: exploring modalities for improving cell therapy efficacy. Basic. Res. Cardiol. 114 , 9 (2019). Böhm, M., Ewen, S & Wolf, M. Renal Denervation Halts Left Ventricular Remodeling and Dysfunction in Heart Failure: New Shores Ahead. J. Am. Coll. Cardiol. 72 , 2622-2624 (2018). Sepe, J.J., Gardner, R.T., Blake, M.R. et al. Therapeutics That Promote Sympathetic Reinnervation Modulate the Inflammatory Response After Myocardial Infarction. JACC. Basic. Transl. Sci. 7 , 915-930 (2022). Sharp, T.E. 3rd., Polhemus, D.J., Li, Z. et al. Renal Denervation Prevents Heart Failure Progression Via Inhibition of the Renin-Angiotensin System. J. Am. Coll. Cardiol. 72 , 2609-26021 (2018). Garbern, J.C. & Lee, R.T. Heart regeneration: 20 years of progress and renewed optimism. Dev. Cell. 57 , 424-439 (2022). Wang, P., Deng, Z., Li, A. et al. β-Catenin promotes long-term survival and angiogenesis of peripheral blood mesenchymal stem cells via the Oct4 signaling pathway. Exp. Mol. Med. 54 , 1434-1449 (2022). Sahoo, S. & Losordo, D.W. Exosomes and cardiac repair after myocardial infarction. Circ. Res. 114 , 333-344 (2014). Sharp, T.E. III, Polhemus, D.J., Li, Z. et al. Hypoxia induces heart regeneration in adult mice. Nature 541 , 222-227 (2017). Wang, W.E., Li, L., Xia, X. et al. Dedifferentiation, proliferation, and redifferentiation of adult mammalian cardiomyocytes after ischemic injury. Circulatio n 136 , 834-848 (2017). Li, J., Sun, S., Zhu, D. et al. Inhalable Stem Cell Exosomes Promote Heart Repair After Myocardial Infarction. Circulation 150 , 710-723 (2024). Messerli, F.H., Bavishi, C., Brguljan, J. et al. Renal denervation in the antihypertensive arsenal - knowns and known unknowns. J. Hypertens. 40 , 1859-1875 (2022). Peng, Y., Zhao, J.L., Peng, Z.Y. et al. Exosomal miR-25-3p from mesenchymal stem cells alleviates myocardial infarction by targeting pro-apoptotic proteins and EZH2. Cell Death Dis. 11 , 317 (2020). Théry, C., Witwer, K.W., Aikawa, E. et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 7 , 1535750 (2018). Hessvik, N.P., Sagini, K., Romero, S. et al. siRNA screening reveals that SNAP29 contributes to exosome release. Cell. Mol. Life Sci. 80 , 177 (2023). Kumar, R., Tang, Q., Müller, S.A. et al. Fibroblast Growth Factor 2-Mediated Regulation of Neuronal Exosome Release Depends on VAMP3/Cellubrevin in Hippocampal Neurons. Adv. Sci. (Weinh.) 7 , 1902372 (2020). Tian, Y., Liu, Y., Wang, T. et al. A microRNA-Hippo pathway that promotes cardiomyocyte proliferation and cardiac regeneration in mice. Sci. Transl. Med. 7 , 279ra38 (2015). Patel, V., Hajarnis, S., Williams, D. et al. MicroRNAs regulate renal tubule maturation through modulation of Pkd1. J. Am. Soc. Nephrol. 23 , 1941-1948 (2012). Pecot, C.V., Rupaimoole, R., Yang, D. et al. Tumour angiogenesis regulation by the miR-200 family. Nat. Commun. 4 , 2427 (2013). Akbar, N., Razzaq, S.S., Salim, A. & Haneef, K. Mesenchymal Stem Cell-Derived Exosomes and Their MicroRNAs in Heart Repair and Regeneration. J. Cardiovasc. Transl. Res. 17 , 505-522 (2024). Wan, J., Lin, S., Yu, Z. et al. Protective Effects of MicroRNA-200b-3p Encapsulated by Mesenchymal Stem Cells-Secreted Extracellular Vesicles in Myocardial Infarction Via Regulating BCL2L11. J. Am. Heart Assoc. 11 , e024330 (2022). Barrère-Lemaire, S., Vincent, A., Jorgensen, C. et al. Mesenchymal stromal cells for improvement of cardiac function following acute myocardial infarction: a matter of timing. Physiol. Rev. 104 , 659-725 (2024). Miller, I., Min, M., Yang, C. et al. Ki67 is a Graded Rather than a Binary Marker of Proliferation versus Quiescence. Cell Rep. 24 , 1105-1112.e5 (2018). Cho, K.W., Andrade, M., Bae, S. et al. Polycomb Group Protein CBX7 Represses Cardiomyocyte Proliferation Through Modulation of the TARDBP/RBM38 Axis. Circulation 147 , 1823-1842 (2023). Chen, Y., Lüttmann, F.F., Schoger, E. et al. Reversible reprogramming of cardiomyocytes to a fetal state drives heart regeneration in mice. Science 373 , 1537-1540 (2021). Wang, J., Pei, B., Yan, J. et al. hucMSC-Derived Exosomes Alleviate the Deterioration of Colitis via the miR-146a/SUMO1 Axis. Mol. Pharm. 19 , 484–493 (2022). Ibrahim, A.G.E., Li, C., Rogers, R. et al. Augmenting canonical Wnt signalling in therapeutically inert cells converts them into therapeutically potent exosome factories. Nat. Biomed. Eng. 3 , 695-705 (2019). Lian, X., Zhang, J., Azarin, S.M. et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions. Nat. Protoc. 8 , 162–175 (2013). Zhu, C., Yuan, T. & Krishnan, J. Targeting cardiomyocyte cell cycle regulation in heart failure. Basic Res. Cardiol. 119 , 349-369 (2024). Gabisonia, K., Prosdocimo, G., Aquaro, G.D. et al. MicroRNA therapy stimulates uncontrolled cardiac repair after myocardial infarction in pigs. Nature 569 , 418-422 (2019). Dzau, V.J. & Hodgkinson, C.P. RNA Therapeutics for the Cardiovascular System. Circulation 149 , 707-716 (2024). Fiebeler, A., Schmidt, F., Müller, D.N. et al. Mineralocorticoid receptor affects AP-1 and nuclear factor-kappab activation in angiotensin II-induced cardiac injury. Hypertension 37 , 787–793 (2001). Li, X., Wu, F., Günther, S. et al. Inhibition of fatty acid oxidation enables heart regeneration in adult mice. Nature 622 , 619–626 (2023). Liu, J., Xiao, Q., Xiao, J. et al. Wnt/β-catenin signalling: function, biological mechanisms, and therapeutic opportunities. Signal Transduct. Target. Ther. 7 , 3 (2022). Huang, M., Wang, X., Banerjee, M. et al. Regulation of myogenesis by a Na/K-ATPase α1 caveolin-binding motif. Stem Cells 40 , 133-148 (2022). Deng, F., Peng, L., Li, Z. et al. YAP triggers the Wnt/β-catenin signalling pathway and promotes enterocyte self-renewal, regeneration and tumorigenesis after DSS-induced injury. Cell Death Dis . 9 , 153 (2018). Kim, S., Song, G., Lee, T. et al. PARsylated transcription factor EB (TFEB) regulates the expression of a subset of Wnt target genes by forming a complex with β-catenin-TCF/LEF1. Cell Death Differ . 28 , 2555–2570 (2021). Polhemus, D.J., Gao, J., Scarborough, A.L. et al. Radiofrequency Renal Denervation Protects the Ischemic Heart via Inhibition of GRK2 and Increased Nitric Oxide Signaling. Circ. Res. 119 , 470-480 (2016). Okamura, K., Shimada, H., Imazato, K. et al. Impact of renal denervation on quality of life (How does renal denervation contribute to improving hypertension treatment affected by poor medication adherence?). Hypertens. Res . 47 , 2652-2658 (2024). Taru, V., Szabo, G., Mehal, W. & Reiberger, T. Inflammasomes in chronic liver disease: Hepatic injury, fibrosis progression and systemic inflammation. J. Hepatol . 81 , 895-910 (2024). Bhatt, D.L., Kandzari, D.E., O'Neill, W.W. et al. A controlled trial of renal denervation for resistant hypertension. N. Engl. J. Med . 370 , 1393-1401 (2014). Sharp, T.E. III & Lefer, D.J. Renal Denervation to Treat Heart Failure. Annu. Rev. Physiol . 83 , 39-58 (2021). Davidson, S.M., Padró, T., Bollini, S. et al. Progress in cardiac research: from rebooting cardiac regeneration to a complete cell atlas of the heart. Cardiovasc. Res. 117 , 2161-2174 (2021). Feng, Y., Wang, Y., Li, L. et al. Exosomes Induce Crosstalk Between Multiple Types of Cells and Cardiac Fibroblasts: Therapeutic Potential for Remodeling After Myocardial Infarction. Int. J. Nanomedicine 19 , 10605-10621 (2024). Mohamed, T.M.A., Ang, Y.S., Radzinsky, E. et al. Regulation of Cell Cycle to Stimulate Adult Cardiomyocyte Proliferation and Cardiac Regeneration. Cell 173 , 104-116.e12 (2018). Gallet, R., Dawkins, J., Valle, J. et al. Exosomes secreted by cardiosphere-derived cells reduce scarring, attenuate adverse remodelling, and improve function in acute and chronic porcine myocardial infarction. Eur. Heart J. 38 , 201-211 (2017). Gao, L., Mei, S., Zhang, S. et al. Cardio-renal Exosomes in Myocardial Infarction Serum Regulate Proangiogenic Paracrine Signaling in Adipose Mesenchymal Stem Cells. Theranostics 10 , 1060-1073 (2020). Crewe, C., Joffin, N., Rutkowski, J.M. et al. An Endothelial-to-Adipocyte Extracellular Vesicle Axis Governed by Metabolic State. Cell 175 , 695-708.e13 (2018). Liang, X., Li, C., Song, J. et al. HucMSC-Exo Promote Mucosal Healing in Experimental Colitis by Accelerating Intestinal Stem Cells and Epithelium Regeneration via Wnt Signaling Pathway. Int. J. Nanomedicine 18 , 2799–2818 (2023). Feng, D., Yan, K., Liang, H. et al. CBP-mediated Wnt3a/β-catenin signaling promotes cervical oncogenesis initiated by Piwil2. Neoplasia 23 , 1-11 (2021). Kharaziha, P., Ceder, S., Li, Q. & Panaretakis, T. Tumor cell-derived exosomes: a message in a bottle. Biochim. Biophys. Acta 1826 , 103–111 (2012). Wang, J., Pei, B., Yan, J. et al. hucMSC-Derived Exosomes Alleviate the Deterioration of Colitis via the miR-146a/SUMO1 Axis. Mol. Pharm . 19 , 484-493 (2022). Zheng, X., Li, X., Lyu, Y. et al. Renal Sympathetic Denervation in Rats Ameliorates Cardiac Dysfunction and Fibrosis Post-Myocardial Infarction Involving MicroRNAs. Med. Sci. Monit . 22 , 2751-2760 (2016). Gao, L., Qiu, F., Cao, H. et al. Therapeutic delivery of microRNA-125a-5p oligonucleotides improves recovery from myocardial ischemia/reperfusion injury in mice and swine. Theranostics 13 , 685-703 (2023). Braga, L., Ali, H., Secco, I. & Giacca, M. Non-coding RNA therapeutics for cardiac regeneration. Cardiovasc. Res. 117 , 674-693 (2021). Papukashvili, D., Rcheulishvili, N., Liu, C. et al. Perspectives on miRNAs Targeting DKK1 for Developing Hair Regeneration Therapy. Cells 10 , 2957 (2021). Li, S., Yin, Y., Yao, L. et al. TNF-α treatment increases DKK1 protein levels in primary osteoblasts via upregulation of DKK1 mRNA levels and downregulation of miR-335-5p. Mol. Med. Rep . 22 , 1017-1025 (2020). Zhao, L., Wang, J., Wang, P. et al. Oct4 cooperates with c-Myc to improve mesenchymal-to-endothelial transition and myocardial repair of cardiac-resident mesenchymal stem cells. Stem Cell Res. Ther . 13 , 445 (2022). Liang, X.L., Wang, Y.L. & Wang, P.R. MiR-200a with CDC7 as a direct target declines cell viability and promotes cell apoptosis in Wilm's tumor via Wnt/β-catenin signaling pathway. Mol. Cell. Biochem . 476 , 2409-2420 (2021). Kook, S.H., Heo, J.S. & Lee, J.C. Crucial roles of canonical Runx2-dependent pathway on Wnt1-induced osteoblastic differentiation of human periodontal ligament fibroblasts. Mol. Cell. Biochem . 402 , 213-223 (2015). Dharavath, B., Butle, A., Chaudhary, A. et al. Recurrent UBE3C-LRP5 translocations in head and neck cancer with therapeutic implications. NPJ Precis. Oncol . 8 , 63 (2024). Frangogiannis, N.G. The inflammatory response in myocardial injury, repair, and remodelling. Nat. Rev. Cardiol . 11 , 255-265 (2014). Liu, S., Li, K., Wagner Florencio, L. et al. Gene therapy knockdown of Hippo signaling induces cardiomyocyte renewal in pigs after myocardial infarction. Sci. Transl. Med . 13 , eabd6892 (2021). Zhang, S., Zhao, L., Shen, L. et al. Comparison of various niches for endothelial progenitor cell therapy on ischemic myocardial repair: coexistence of host collateralization and Akt-mediated angiogenesis produces a superior microenvironment. Arterioscler. Thromb. Vasc. Biol . 3 2, 910-923 (2012). Mahfoud, F., Townsend, R.R., Kandzari, D.E. et al. Changes in Plasma Renin Activity After Renal Artery Sympathetic Denervation. J. Am. Coll. Cardiol . 77 , 2909-2919 (2021). Lu, H., Jiang, X., He, L. et al. Endothelial Sp1/Sp3 are essential to the effect of captopril on blood pressure in male mice. Nat. Commun . 14 , 5891 (2023). Villamizar, O., Waters, S.A., Scott, T. et al. Mesenchymal Stem Cell exosome delivered Zinc Finger Protein activation of cystic fibrosis transmembrane conductance regulator. J. Extracell. Vesicles 10 , e12053 (2021). Chen, K., Wang, Q., Liu, X. et al. Hypoxic pancreatic cancer derived exosomal miR-30b-5p promotes tumor angiogenesis by inhibiting GJA1 expression. Int. J. Biol. Sci . 18 , 1220-1237 (2022). Wang, N., Li, X., Zhong, Z. et al. 3D hESC exosomes enriched with miR-6766-3p ameliorates liver fibrosis by attenuating activated stellate cells through targeting the TGFβRII-SMADS pathway. J. Nanobiotechnology . 19 , 437 (2021). Bervoets, S., Wei, N., Erfurth, M.L. et al. Transcriptional dysregulation by a nucleus-localized aminoacyl-tRNA synthetase associated with Charcot-Marie-Tooth neuropathy. Nat. Commun . 10 , 5045 (2019). Zhao, M., Nakada, Y., Wei, Y. et al. Cyclin D2 Overexpression Enhances the Efficacy of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Myocardial Repair in a Swine Model of Myocardial Infarction. Circulation 144 , 210-228 (2021). Suda, T., Yamashita, T., Sunagozaka, H. et al. Dickkopf-1 Promotes Angiogenesis and is a Biomarker for Hepatic Stem Cell-like Hepatocellular Carcinoma. Int. J. Mol. Sci . 23 , 2801 (2022). Shi, X.B., Xue, L., Ma, A.H. et al. Tumor suppressive miR-124 targets androgen receptor and inhibits proliferation of prostate cancer cells. Oncogene 32 , 4130-4138 (2013). Gan, L., Liu, D., Xie, D. et al. Ischemic Heart-Derived Small Extracellular Vesicles Impair Adipocyte Function. Circ. Res. 130 , 48-66 (2022). Andres-Mateos, E., Landegger, L.D., Unzu, C. et al. Choice of vector and surgical approach enables efficient cochlear gene transfer in nonhuman primate. Nat. Commun . 13 , 1359 (2022). Additional Declarations There is NO Competing Interest. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6409278","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":440514511,"identity":"41491aa0-6a10-4048-8682-7a1047630e60","order_by":0,"name":"Shaoheng zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYDACCQYGZgiD+fiPDwY2dqRoYUuQnFGQlkyKFh4FaZ4PhxgbCOmQn9388HFBxR27+bN7GIxtDA4wM7AfProBnxaDO8eMjWeceZbcOOfsgeQcgzt8DDxpaTfwapFIMJPmbTuczCyRl3A4x+AZM9CFZni1yM9I/ybN++9wMptEjmGzhcFhxgZCWhhu5ABtaThsxyORY8zMQIwWgxs5xcY8xw4nSEikpTH2GKQlsxHyC9BhGx/z1By2l5+RfIzhxx8bO372w8fwOwwKEhtgLDZilIOAPbEKR8EoGAWjYAQCAJs3SOxkqffqAAAAAElFTkSuQmCC","orcid":"","institution":"Guangzhou Red Cross Hospital Medical College of Ji-Nan University","correspondingAuthor":true,"prefix":"","firstName":"Shaoheng","middleName":"","lastName":"zhang","suffix":""},{"id":440514512,"identity":"10fba141-ffe4-4827-bd18-264d07a00c94","order_by":1,"name":"Lan Zhao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Lan","middleName":"","lastName":"Zhao","suffix":""},{"id":440514513,"identity":"0ed4e9cc-4778-4526-8387-98e63a1b3027","order_by":2,"name":"Chen Li","email":"","orcid":"","institution":"Guangzhou Red Cross Hospital Medical College of Ji-Nan University","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Li","suffix":""},{"id":440514514,"identity":"1cd6e0bf-828b-432d-a389-9ae3edc0a948","order_by":3,"name":"Zhichuan Huang","email":"","orcid":"","institution":"Guangzhou Red Cross Hospital Medical College of Ji-Nan University","correspondingAuthor":false,"prefix":"","firstName":"Zhichuan","middleName":"","lastName":"Huang","suffix":""},{"id":440514515,"identity":"c4521b6e-8988-48bb-9633-914e71ca04d4","order_by":4,"name":"Jianshuo Wang","email":"","orcid":"","institution":"Guangzhou Red Cross Hospital Medical College of Ji-Nan University","correspondingAuthor":false,"prefix":"","firstName":"Jianshuo","middleName":"","lastName":"Wang","suffix":""},{"id":440514516,"identity":"92be3823-ac3c-4938-944c-1a5c93de8f83","order_by":5,"name":"Zhanyu Deng","email":"","orcid":"","institution":"Guangzhou Red Cross Hospital Medical College of Ji-Nan University","correspondingAuthor":false,"prefix":"","firstName":"Zhanyu","middleName":"","lastName":"Deng","suffix":""},{"id":440514517,"identity":"dec02250-d0f3-4a49-ad33-7188bf3eacf4","order_by":6,"name":"Yanwen Deng","email":"","orcid":"","institution":"Guangzhou Red Cross Hospital Medical College of Ji-Nan University","correspondingAuthor":false,"prefix":"","firstName":"Yanwen","middleName":"","lastName":"Deng","suffix":""},{"id":440514518,"identity":"318decca-5a79-4fdd-a332-dc3bd03a7edd","order_by":7,"name":"Pengzhen Wang","email":"","orcid":"","institution":"Guangzhou Red Cross Hospital Medical College of Ji-Nan University","correspondingAuthor":false,"prefix":"","firstName":"Pengzhen","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-04-09 07:45:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6409278/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6409278/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80288036,"identity":"2055c439-8fd6-44fe-a2c4-9ca0a6c821ac","added_by":"auto","created_at":"2025-04-10 07:15:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6772204,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDynamic Changes in SNS/RAAS Activity and PB-MSC-Exose Viability Following RD in HFrEF Porcine Models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRD induced early-phase transient suppression of SNS and RAAS activity in MI swine models. (\u003cstrong\u003eA\u003c/strong\u003e) Representative images of TH staining from serial RA samples from RD-Sham or RD pigs obtained 1, 2, and 4 weeks after MI. Scale bar: 50 μm. (\u003cstrong\u003eB\u003c/strong\u003e) TH staining intensity. (\u003cstrong\u003eC\u003c/strong\u003e) Dopamine and (\u003cstrong\u003eD\u003c/strong\u003e) NE concentrations per gram of kidney cortex tissue. (\u003cstrong\u003eE\u003c/strong\u003e-\u003cstrong\u003eG\u003c/strong\u003e) Quantitative analysis of plasma NE (\u003cstrong\u003eE\u003c/strong\u003e), angiotensin I (\u003cstrong\u003eF\u003c/strong\u003e), and angiotensin II (\u003cstrong\u003eG\u003c/strong\u003e) 1, 2, and 4 weeks after MI in RD-Sham or RD pigs. \u0026nbsp;All data, presented as means ± standard deviations, were analyzed using one-way analysis of variance. \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05: \u003csup\u003e*\u003c/sup\u003evs. RD-Sham at each timepoint, \u003csup\u003e#\u003c/sup\u003evs. 1 week after MI, \u003csup\u003e†\u003c/sup\u003evs. 2 weeks after MI (n = 3 per group). RD augments the secretion of PBMSC-originated Exos in porcine models of HFrEF. (\u003cstrong\u003eH\u003c/strong\u003e) Serial representative TEM images of PBMSC-Exos from RD or RD-Sham pigs 1, 2, and 4 weeks after MI. Scale bar: 100 nm. (\u003cstrong\u003eI\u003c/strong\u003e), Western blotting for Exos markers CD9, CD63,TSG101and HSP70, and negative marker Calnexin in PBMSC-Exos from RD or RD-Sham pigs 1, 2, 4 weeks after MI. (\u003cstrong\u003eJ\u003c/strong\u003e) Representative size distribution of PBMSC-Exos by Nanosight 2 weeks after MI. (\u003cstrong\u003eK\u003c/strong\u003e) Quantitative analysis of the particle number of PBMSC-Exos in culture medium of PBMSCs from RD or RD-Sham pigs through NTA at 1, 2, and 4 weeks post-MI. (\u003cstrong\u003eL\u003c/strong\u003e) RD or RD-Sham treatment did not significantly affect the particle size of PBMSC-Exos from RD or RD-Sham pigs 1, 2, and 4 weeks after MI. (\u003cstrong\u003eM\u003c/strong\u003e) Flow cytometry analysis of PBMSC-positive surface markers for the MSC markers CD44, CD71, CD90, and CD105 and negative markers for the HSC markers CD34 and CD45 and EPC markers CD31 and CD133. (\u003cstrong\u003eN\u003c/strong\u003e) PBMSC-Exos were examined via immunofluorescence for the expression of the MSC marker CD105 (green) and Exos markers TSG101 (red).Scale bar: 10 μm. All data, presented as means ± standard deviations, were analyzed using one-way analysis of variance. \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05: \u003csup\u003e*\u003c/sup\u003evs RD-Sham at each timepoint, \u003csup\u003e#\u003c/sup\u003evs. 1 week after MI, \u003csup\u003e†\u003c/sup\u003evs. 2 weeks after MI (n = 3 per group). Exos=exosoms; HFrEF=heart failure with reduced ejection fraction; HSC=hematopoietic stem cells; MI=myocardialinfarction; NE=norepinephrine; NTA=nanoparticle tracking analysis; PBMSCs= peripheral blood mesenchymal stem cells; RAAS=renin aniotension aldosterone system; RD=renal denervation; SNS = sympathetic nervous system; TEM= transmission electron microscope; TH=tyrosine hydroxylase.\u003c/p\u003e","description":"","filename":"Paper10Figure120254.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/d2f6ad6f17a6cf8522169fec.jpg"},{"id":80287329,"identity":"404deb14-f5ba-47e6-b459-7a9e27cbde08","added_by":"auto","created_at":"2025-04-10 07:07:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2125920,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-141-200-429 Clusters Are Upregulated in RD RAECs and Transferred to CMs by PBMSC-Exos\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRD enhances the release of PBMSC-Exos from RAECs, delivering cardioprotective microRNA cargo to infarcted hearts. (\u003cstrong\u003eA\u003c/strong\u003e) Venn diagram identifying 15 miRNAs associated with cardiac proliferation and regeneration enriched in the heart. (\u003cstrong\u003eB\u003c/strong\u003e) Heatmap generated using correlation distance based on average miRNA expression detected by RT-qPCR. RT-qPCR revealed significant miR-200a-3p and miR-200b-3p elevation in PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e compared with PBMSC-Exos\u003csup\u003eRD-Sham\u003c/sup\u003e from equal amounts of PBMSCs. Relative abundance of each miRNA is indicated by a gradient of color from red (highest abundance) to white (lowest abundance). \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, multiple \u003cem\u003et\u003c/em\u003e tests; n = 10. (\u003cstrong\u003eC\u003c/strong\u003e) RT-qPCR revealing significant elevation of miR-200a-3p and miR-200b-3p expression in PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e compared with equal number of PBMSC-Exos\u003csup\u003eRD-Sham\u003c/sup\u003e. \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, unpaired \u003cem\u003et\u003c/em\u003e tests; n = 10. (\u003cstrong\u003eD\u003c/strong\u003e) Significant elevation of 141-200-429 cluster expression in RA-Exos\u003csup\u003eRD\u003c/sup\u003e compared with an equal number of RA-Exos\u003csup\u003eRD-Sham\u003c/sup\u003e. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, unpaired \u003cem\u003et\u003c/em\u003e tests; n = 10. (\u003cstrong\u003eE\u003c/strong\u003e) Increased miR-141-200-429 cluster expression in RAECs than CMs after RD treatment. \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, unpaired \u003cem\u003et\u003c/em\u003e tests; n = 10. (\u003cstrong\u003eF\u003c/strong\u003e and \u003cstrong\u003eG\u003c/strong\u003e) Relative expression of miR-141-200-429 cluster in RAs (\u003cstrong\u003eF\u003c/strong\u003e) and hearts (\u003cstrong\u003eG\u003c/strong\u003e) from RD and RD-Sham pigs. \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, unpaired \u003cem\u003et\u003c/em\u003e tests, compared with RD-Sham; n = 10. CMs=cardiomyocytes; Exos=exosoms; RA=renal artery; RAECs=renal artery endothelial cells; RD=renal denervation; RT-qPCR=reverse transcription quantitative real-time polymerase chain reaction.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Paper10Figure220254.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/57ba5e3b19c8b966fe7cb63f.jpg"},{"id":80287335,"identity":"f26709b8-6636-4a49-996a-b9568213795b","added_by":"auto","created_at":"2025-04-10 07:07:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2610841,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDelayed Exosomal Therapy Promotes Improved Outcome in the RD-Treated Pigs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMyocardial exosomal miR-141-200 cluster mediates the therapeutic effects of both RD and Exos interventions on cardiac functional restoration. (\u003cstrong\u003eA\u003c/strong\u003e) Ischemic HFrEF protocol. (\u003cstrong\u003eB\u003c/strong\u003e) Representative echocardiography images of hearts from pigs receiving RD-Sham or RD with or without delayed MSC-derived Exos transplantation 30, 60, and 90 days after MI. (\u003cstrong\u003eC\u003c/strong\u003e-\u003cstrong\u003eG\u003c/strong\u003e) Echocardiography for LVEF (\u003cstrong\u003eC\u003c/strong\u003e), stroke volume (\u003cstrong\u003eD\u003c/strong\u003e), LVESV (\u003cstrong\u003eE\u003c/strong\u003e), LVEDV (\u003cstrong\u003eF\u003c/strong\u003e), and LVAW (\u003cstrong\u003eG\u003c/strong\u003e) 0 days, 30 days, 60 days, and 90 days after MI. (\u003cstrong\u003eH\u003c/strong\u003e) Kaplan–Meier survival curves to compare mortality between the four pig groups. Significance was determined by log-rank (Mantel–Cox) test. (\u003cstrong\u003eI\u003c/strong\u003e and \u003cstrong\u003eJ\u003c/strong\u003e) Circulating BUN (\u003cstrong\u003eI\u003c/strong\u003e) and creatinine (\u003cstrong\u003eJ\u003c/strong\u003e) levels on 0 days, 30 days after MI, 60 days after MI, and 90 days after MI. All data, presented as means ± standard deviations, were analyzed using one-way analysis of variance. \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05: \u003csup\u003e*\u003c/sup\u003evs. RD-Sham+PBS\u003cem\u003e, \u003c/em\u003e\u003csup\u003e#\u003c/sup\u003evs. RD-Sham+Exos, \u003csup\u003e†\u003c/sup\u003evs. RD+PBS, \u003csup\u003e§ \u003c/sup\u003evs. 0 days after MI, \u003csup\u003e║\u003c/sup\u003evs. 30 days after MI, \u003csup\u003e¶\u003c/sup\u003e\u003cem\u003evs\u003c/em\u003e.60 d post-MI, ns, Non-Significant (0 days after MI, n=10 pigs in each group; 30 days after MI, n = 7 for RD-Sham+PBS, 8 for RD-Sham+Exos, 9 for RD-Sham+PBS, and 10 for RD+Exos; 60 days after MI, n = 5 for RD-Sham+PBS, 8 for RD-Sham+Exos, 9 for RD-Sham+PBS, and 10 for RD+Exos; 90 days after MI, n = 5 for RD-Sham+PBS, 7 for RD-Sham+Exos, 8 for RD-Sham+PBS, and 10 for RD+Exos). (\u003cstrong\u003eK\u003c/strong\u003e) Relative expression of miR-141-200-429 cluster in hearts from pigs receiving RD-Sham or RD with or without delayed MSC-derived Exos transplantation 90 days after MI. All data, shown as means ± standard deviations, were analyzed using one-way ANOVA. \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001; ns, Non-Significant, n=5 for RD-Sham+PBS, 7 for RD-Sham+Exos, 8 for RD-Sham+PBS, and 10 for RD+Exos). BUN=blood urea nitrogen; Exos=exosoms; HFrEF=heart failure with reduced ejection fraction; LVAW=left ventricular anterior wall; LVEDV=left ventricular end-systolic volume; LVEF=left ventricular ejection fraction; LVESV=left ventricular end-systolic volume; MI=myocardial infarction; PBS=phosphate buffered saline; PBMSCs= peripheral blood mesenchymal stem cells; RD=renal denervation.\u003c/p\u003e","description":"","filename":"Paper10Figure320254.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/49970a75291435115ea6481b.jpg"},{"id":80288035,"identity":"40e6435b-a5b8-469d-aadd-fbde564eac8b","added_by":"auto","created_at":"2025-04-10 07:15:15","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5920289,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRD Combined with Delayed Exos Therapy Attenuates Pathological Cardiac Remodeling and Modulates SNS Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e–\u003cstrong\u003eC\u003c/strong\u003e) Heart sections were stained subjected to TTC (\u003cstrong\u003eA\u003c/strong\u003e), Masson’s trichrome (\u003cstrong\u003eB\u003c/strong\u003e), or H\u0026amp;E (\u003cstrong\u003eC\u003c/strong\u003e) staining. Representative images (\u003cstrong\u003eA\u003c/strong\u003e) and quantification (\u003cstrong\u003eE\u003c/strong\u003e) of infarct size appearing pale white. Representative images (\u003cstrong\u003eB\u003c/strong\u003e) and quantification (\u003cstrong\u003eF\u003c/strong\u003e) of fibrosis (blue). Scale bar: 20 μm. Representative images (\u003cstrong\u003eC\u003c/strong\u003e) and quantification (\u003cstrong\u003eG\u003c/strong\u003e) of viable CMs. Scale bar: 20 μm. (\u003cstrong\u003eD\u003c/strong\u003e) WGA staining. (\u003cstrong\u003eD\u003c/strong\u003e) Representative images of immunofluoroscence staining of WGA-stained heart cross-section of HFrEF pigs receiving RD-Sham or RD in combination with or without delayed transplantation of PBMSC-Exos 90 days after MI; nuclei (DAPI) appear blue, and WGA appears green. Scale bar: 50 μm. (\u003cstrong\u003eH\u003c/strong\u003e) Graph showing cross-sectional area measurements of cells showing WGA-positive cytoplasmic localization. (\u003cstrong\u003eI\u003c/strong\u003e) Representative RA TH-stained photomicrographs from RD-Sham pigs receiving PBS or Exos and RD pigs receiving PBS or Exos. Scale bar: 50 µm. (\u003cstrong\u003eK\u003c/strong\u003e) TH staining intensity. (\u003cstrong\u003eL\u003c/strong\u003e and \u003cstrong\u003eM\u003c/strong\u003e) Kidney dopamine (\u003cstrong\u003eL\u003c/strong\u003e) and NE (\u003cstrong\u003eM\u003c/strong\u003e) concentration per gram of kidney cortex tissue. (\u003cstrong\u003eM\u003c/strong\u003e) Plasma NE levels 90 days after MI. All data, presented as means ± standard deviations, were analyzed using one-way analysis of variance.\u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001; ns, Non-Significant. n = 5 for RD-Sham+PBS, 7 for RD-Sham+Exos, 8 for RD-Sham+PBS, and 10 for RD+Exos. DAPI=4′,6-Diamidino-2-phenylindole; Exos=exosoms; HFrEF=heart failure with reduced ejection fraction; MHC=myosin heavy chain; MI=myocardial infarction; PBS=phosphate buffered saline; PBMSCs= peripheral blood mesenchymal stem cells; RD=renal denervation; TH=tyrosine hydroxylase; TTC=triphenyl tetrazolium chloride; WGA= wheat\u0026nbsp;germ\u0026nbsp;agglutinin. All data, presented as means ± standard deviations, were analyzed using one-way analysis of variance. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.005, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001; n = 5 for RD-Sham+PBS, 7 for RD-Sham+Exos, 8 for RD-Sham+PBS, and 10 for RD+Exos.\u003c/p\u003e","description":"","filename":"Paper10Figure420254.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/3467056bc748234cf55137b0.jpg"},{"id":80287327,"identity":"aeba8781-d729-4a06-8c09-786afb5d59ed","added_by":"auto","created_at":"2025-04-10 07:07:15","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4653575,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExosomal Therapy Promotes RD-Induced CM Proliferation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Representative images of immunofluorescencetri-staining for Ki67 (red), MHC (green), and DAPI (nucleri, blue) in the peri-infarct (upper) and remote (lower) zone 90 days after MI, and its relative quantification (\u003cstrong\u003eE\u003c/strong\u003e). Arrows point to proliferating CMs. Scale bar:25 µm. (\u003cstrong\u003eB\u003c/strong\u003e) Representative images of immunofluorescencetri-staining for Aurora B (red), MHC (green), and DAPI (nucleri, blue) in the infarct borderzone 90 days after MI, and its relative quantification (\u003cstrong\u003eF\u003c/strong\u003e). White arrows indicate related CMs co-expressing Aurora. Scale bar: 25 µm. (\u003cstrong\u003eC\u003c/strong\u003e) Immunofluorescence staining for α-SMA (red, marked by arrows) and α-actin (green)in CMs from hearts cross-sections. Scale bar: 20 µm. (\u003cstrong\u003eG\u003c/strong\u003e) Quantification of CMs dedifferentiation for immunofluorescence staining results in (\u003cstrong\u003eC\u003c/strong\u003e). (\u003cstrong\u003eD\u003c/strong\u003e and \u003cstrong\u003eH\u003c/strong\u003e) CMs uptake analysis of Exos. At 90 days after MI, Exos were detected in myocardial tissues (MHC, red) of pigs with or without RD or exosomal therapy through immunofluorescence staining (\u003cstrong\u003eD\u003c/strong\u003e). Exos were pre-labeled with PKH26 (red). Nuclei (DAPI) and MHC appear blue and green, respectively. White arrows indicate related CMs co-stained with PKH26. Scale bar: 25 μm. (\u003cstrong\u003eH\u003c/strong\u003e) Quantification of CM uptake for Exos from immunofluorescence staining results in (\u003cstrong\u003eD\u003c/strong\u003e). All data, presented as means ± standard deviations, were analyzed using one-way analysis of variance.\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.005, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001; n = 5 for RD-Sham+PBS, 7 for RD-Sham+Exos, 8 for RD-Sham+PBS, and 10 for RD+Exos. (\u003cstrong\u003eI\u003c/strong\u003e) Western blotting for Aurora B, α-SMA, and α-actin expression were confirmed. (\u003cstrong\u003eJ\u003c/strong\u003e and \u003cstrong\u003eK\u003c/strong\u003e) Relative correlation CM uptake for Exos with Aurora B (\u003cstrong\u003eJ\u003c/strong\u003e) or α-SMA (\u003cstrong\u003eK\u003c/strong\u003e) expression evaluated by Western blotting in the RD+Exos group (n =10). α-SMA=α-smooth muscle actin; CMs=cardiomyocytes; Exos=exosoms; DAPI=4′,6-Diamidino-2-phenylindole; MHC=myosin heavy chain; MI=myocardial infarction; PBS=phosphate buffered saline; RD=renal denervation.\u003c/p\u003e","description":"","filename":"Paper10Figure520253.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/ccc4144c1c9d42ce1d580165.jpg"},{"id":80288041,"identity":"ca0a1081-ff6a-4518-a7e5-55592775f99a","added_by":"auto","created_at":"2025-04-10 07:15:15","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1202753,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eβ-Catenin Promotes CMs Exos-Induced Reprogramming\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Experimental design for investigating CM reprogramming \u003cem\u003ein vitro\u003c/em\u003e. (\u003cstrong\u003eB\u003c/strong\u003e) CMs uptake rate of PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e evaluated by calculating proportion of cells expressing both PKH26 (red) and MHC (green) relative to all MHC-positive cells. Representative images (\u003cstrong\u003eBa\u003c/strong\u003e) and quantification (\u003cstrong\u003eBb\u003c/strong\u003e) of CMs positively stained for PKH26 and MHC 7 days after coculture. Nuclei (DAPI) appear blue. Scale bar: 50 µm. (\u003cstrong\u003eC\u003c/strong\u003e) Identification of CM proliferation. (\u003cstrong\u003eCa\u003c/strong\u003e) Morphological remodeling of CMs in the coculture system observed in a time-relative manner. Binucleated cells among CMs treated with both PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e and \u003cem\u003eoe\u003c/em\u003eβ-catenin (white arrows) became spherical during the first 3 days of coculture and then proliferated into several daughter cells over the next few days. Explosive symbols represent apoptotic cells. Nuclei (DAPI) appear blue. Scale bar: 50 µm. (\u003cstrong\u003eCb\u003c/strong\u003e) Schematic of CM division patterns. (\u003cstrong\u003eCc\u003c/strong\u003e) Mononucleated and binucleated or multinucleated CM proliferation rates. (\u003cstrong\u003eD\u003c/strong\u003e) Quantification curves of CMs expressing EdU at 1d, 3d, and 7d after hypoxic coculture. \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05: \u003csup\u003e*\u003c/sup\u003evs. CON\u003cem\u003e, \u003c/em\u003e\u003csup\u003e#\u003c/sup\u003evs. \u003cem\u003eoe\u003c/em\u003eβ-Catenin, \u003csup\u003e†\u003c/sup\u003e1d after hypoxic coculture, \u003csup\u003e§ \u003c/sup\u003evs. 3d after hypoxic coculture (n=10 each group). (\u003cstrong\u003eE\u003c/strong\u003e and \u003cstrong\u003eF\u003c/strong\u003e) Proliferation rates of mononucleated (\u003cstrong\u003eE\u003c/strong\u003e) and multinucleated (\u003cstrong\u003eF\u003c/strong\u003e) CMs. (\u003cstrong\u003eG\u003c/strong\u003e) Representative scatter plot (\u003cstrong\u003eGa\u003c/strong\u003e) and apoptosis levels (\u003cstrong\u003eGb\u003c/strong\u003e) of CMs assessed through flow cytometry after annexin V–PI staining after 7 days of hypoxic culture. (\u003cstrong\u003eG\u003c/strong\u003e) Characteristics of dedifferentiated cells after CM cytokinesis immunostained for cTnI (red).(\u003cstrong\u003eHa\u003c/strong\u003e) Representative images of CM-derived dedifferentiated cells (α-SMA, green) that regained sarcomeric structure (cTnI, red). Nuclei (DAPI) appeared blue. Scale bar: 50 µm. (\u003cstrong\u003eHb\u003c/strong\u003e) Percentage of dedifferentiated cells with different properties. (\u003cstrong\u003eI\u003c/strong\u003e) Reprogramming factors’ mRNA expression levels in the CMs pretreated with empty vector, \u003cem\u003eoe\u003c/em\u003eβ-catenin, or \u003cem\u003esi\u003c/em\u003eβ-catenin after induction with PBMSC-Exos\u003csup\u003eRD \u003c/sup\u003efor 7 days under hypoxic conditions. All data, presented as means ± standard deviations, were analyzed using one-way analysis of variance. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.005, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001; ns, Non-Significant. (n=10 each group) in (\u003cstrong\u003eBb\u003c/strong\u003e), (\u003cstrong\u003eD\u003c/strong\u003e), (\u003cstrong\u003eE\u003c/strong\u003e), (\u003cstrong\u003eF\u003c/strong\u003e), (\u003cstrong\u003eGb\u003c/strong\u003e), (\u003cstrong\u003eHb\u003c/strong\u003e), and (\u003cstrong\u003eI\u003c/strong\u003e). α-SMA=α-smooth muscle actin; CMs=cardiomyocytes; CON stands for transfection of empty vector; cTnI=cardiac troponin I; Exos=exosoms; DAPI=4′,6-Diamidino-2-phenylindole; MHC=myosin heavy chain.\u003c/p\u003e","description":"","filename":"Paper10Figure620253.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/4f9954060c6813a6bb9e0aa2.jpg"},{"id":80287359,"identity":"2d8ca303-1e48-4d7e-b446-0e4ecd241ba4","added_by":"auto","created_at":"2025-04-10 07:07:16","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3603037,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-141-200-429 Cluster Abrogation Inhibits RD-Induced Cardiac Regeneration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic of protocol for miR-141-200-429 or NC sponge administration. Two weeks after RD or RD-Sham, AAV9 injection was performed. (\u003cstrong\u003eB\u003c/strong\u003e) miR-141-200-429 and NC sponges with endoglin-AAV were injected via the jugular vein. Four weeks later, BODIPY TR ceramide–labeled Exos uptake in hearts was detected through fluorescence microscopy. Scale bar: 50 μm. (\u003cstrong\u003eC\u003c/strong\u003e and \u003cstrong\u003eD\u003c/strong\u003e) Downregulation of miR-141-200-429 in PBMSC-Exos (\u003cstrong\u003eC\u003c/strong\u003e) and CMs-Exos (\u003cstrong\u003eD\u003c/strong\u003e) by RAEC-specific miRNAs sponges. miR-141-200-429 or NC sponges were carried by AAV9 with the endoglin promoter. \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001; ns, Non-Significant. One-way analysis of variance and Tukey’s multiple comparisons test to compare the means in each group; n = 5. (\u003cstrong\u003eE\u003c/strong\u003e and \u003cstrong\u003eF\u003c/strong\u003e) GO enrichment analysis of DEGs in CMs with RD-Sham+miR-141-200-429 sponges and RD+miR-141-200-429 sponges. Enriched biological processes for upregulated and downregulated DEGs are shown in red and blue, respectively. (\u003cstrong\u003eG\u003c/strong\u003e) Heatmap of selected DEGs involved in apoptosis (deep red), contraction (red), nervous growth (light red), dedifferentiation (deep blue), proliferation (blue), and β-catenin signaling (light blue). (\u003cstrong\u003eH\u003c/strong\u003e) WGA immunofluoroscence staining of hearts receiving RD-Sham, RD, NC sponges, or miR-141-200-429 sponges 44 days after MI; nuclei (DAPI) appear blue, and WGA appears green. Scale bar: 50 μm. (\u003cstrong\u003eI\u003c/strong\u003e) Graph showing cross-sectional area measurements of cells showing WGA-positive cytoplasmic localization. Data are shown as means ± standard deviations. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, multiple \u003cem\u003et\u003c/em\u003e tests; n = 5. (\u003cstrong\u003eJ\u003c/strong\u003e) qRT-qPCR on selected genes in hearts of pigs receiving RD-Sham, RD, NC sponges, or miR-141-200-429 sponges. Data, shown as mean ± SD, were analyzed using one-way analysis of variance; n = 5 per group. \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05: \u003csup\u003e*\u003c/sup\u003evs. RD-Sham+NC sponges, \u003csup\u003e#\u003c/sup\u003evs. RD-Sham+miR-141-200-429 sponges, \u003csup\u003e†\u003c/sup\u003evs. RD+NC sponges. (\u003cstrong\u003eK\u003c/strong\u003e) Overlap of upregulated (left panel) and downregulated (right panel) DEGs in CMs with RD-Sham and RD miR-141-200-429 sponges compared with CMs with NC sponges. (\u003cstrong\u003eL\u003c/strong\u003e) GO enrichment analysis of DEGs from the overlap in (\u003cstrong\u003eK\u003c/strong\u003e). CMs=cardiomyocytes; DAPI=4′,6-Diamidino-2-phenylindole; MHC= myosin heavy chain; MI=myocardial infarction; NC= negative control; RD=renal denervation; RT-qPCR=real- time quantitative polymerase chain reaction; WGA=wheat\u0026nbsp;germ\u0026nbsp;agglutinin.\u003c/p\u003e","description":"","filename":"Paper10Figure720253.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/7138e561438125535a66684d.jpg"},{"id":80288042,"identity":"cb9fc69a-fc10-4807-b5ba-6b9ec1d90080","added_by":"auto","created_at":"2025-04-10 07:15:15","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2756469,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePBMSC-Exos\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eRD\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e Improve RDCM Proliferation and Dedifferentiation by Targeting Dkk1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Cell proliferation measured through Ki67 staining. Scale bar: 200 µm. \u003cstrong\u003eB\u003c/strong\u003e, Cell growth evaluated using CCK-8 assay as optical density values. (\u003cstrong\u003eC\u003c/strong\u003e) Western blotting for Cdk1, Oct4, and c-Myc in RDCMs treated with or without PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e or cotreated with PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e and Dkk1. (\u003cstrong\u003eD\u003c/strong\u003e) Quantitative analysis of results in (\u003cstrong\u003eC\u003c/strong\u003e). (\u003cstrong\u003eE\u003c/strong\u003e) Immunostaining for the cell proliferation marker Aurora B. Scale bar: 50 µm. (\u003cstrong\u003eF\u003c/strong\u003e) Quantification of results in (\u003cstrong\u003eE\u003c/strong\u003e) showing reduced proliferation in RDCMs cotreated with PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e and Dkk1, as indicated by the percentage of weak, diffuse, or no Aurora B expression. (\u003cstrong\u003eG\u003c/strong\u003e) Western blotting for Acta1, Nppa, and Aurora B in RDCMs. (\u003cstrong\u003eH\u003c/strong\u003e) Quantitative analysis of results in (\u003cstrong\u003eG\u003c/strong\u003e). (\u003cstrong\u003eI\u003c/strong\u003e) Representative images of TUNEL assay for apoptosis in all RDCM groups. (\u003cstrong\u003eJ\u003c/strong\u003e) Western blotting for apoptosis-related proteins caspase-3, cleaved caspase 3, p53, survivin, and Bcl2 in RDCMs. (\u003cstrong\u003eK\u003c/strong\u003e) Quantitative analysis of results in (\u003cstrong\u003eI\u003c/strong\u003e). Scale bar: 50 µm. (\u003cstrong\u003eL\u003c/strong\u003e) Quantitative analysis of results in (\u003cstrong\u003eJ\u003c/strong\u003e). (\u003cstrong\u003eM\u003c/strong\u003e) Western blotting for Dkk1, Gsk-3β, p-Gsk-3β S9, cyclin D1, and Tcf4 expression in RDCMs. (\u003cstrong\u003eN\u003c/strong\u003e) Western blotting fornuclear or cytoplasmic β-catenin. (\u003cstrong\u003eO\u003c/strong\u003e) Representative fluorescence images of β-catenin. Scale bar: 50 µm. (\u003cstrong\u003eP\u003c/strong\u003e) Quantitative analysis of results in (\u003cstrong\u003eM\u003c/strong\u003e). (\u003cstrong\u003eQ\u003c/strong\u003e) Quantitative analysis of results in (\u003cstrong\u003eN\u003c/strong\u003e). (\u003cstrong\u003eR\u003c/strong\u003e) Relative luciferase activity of β-catenin. All data, shown as means ± standard deviations, were analyzed using one-way ANOVA. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001; ns, Non-Significant, n = 5 per group. CTRL=control; Exos=exosoms; DAPI=4′,6-Diamidino-2-phenylindole; MHC= myosin heavy chain; NC= negative control; PBMSCs= peripheral blood mesenchymal stem cells; RD=renal denervation; RDCMs=renal sympathetic denervation-treated cardiomyocytes.\u003c/p\u003e","description":"","filename":"Paper10Figure820253.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/a0825d519c903751faa99e69.jpg"},{"id":80816246,"identity":"f0b954ae-f3d2-4041-a14d-776e3a360e2a","added_by":"auto","created_at":"2025-04-17 11:08:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":32160448,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/962218cc-821c-4da3-85e0-bdfaf0b36431.pdf"},{"id":80287326,"identity":"9a36e423-2d57-4234-ba94-e760d81426ed","added_by":"auto","created_at":"2025-04-10 07:07:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":372633,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstractText.docx","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/9d8486e616d93f92f8e9fb6b.docx"},{"id":80288034,"identity":"59804fc5-41c1-4975-b6c8-0475674b0038","added_by":"auto","created_at":"2025-04-10 07:15:15","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":931260,"visible":true,"origin":"","legend":"","description":"","filename":"CentralIllustration.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/2e0b9dbf5164ccdfae0c9b1f.jpg"},{"id":80288033,"identity":"f06d7eef-ca53-4b29-817c-23da5f694142","added_by":"auto","created_at":"2025-04-10 07:15:15","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":48963,"visible":true,"origin":"","legend":"Research design, methods, and supplemental data","description":"","filename":"SupplementalDataResearchdesignandmethods202542.docx","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/741c1f70f4519ac2e6fbf063.docx"},{"id":80288037,"identity":"b324a7c1-b431-4780-8252-7137e8628d5f","added_by":"auto","created_at":"2025-04-10 07:15:15","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1409796,"visible":true,"origin":"","legend":"Figure S1","description":"","filename":"Paper10FigureS120253.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/b8368d891642e889ed449a7e.jpg"},{"id":80287338,"identity":"91fda5e5-3bf2-4be9-8c00-d608548b7d3b","added_by":"auto","created_at":"2025-04-10 07:07:15","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":588484,"visible":true,"origin":"","legend":"Figure S2","description":"","filename":"Paper10FigureS220253.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/2b5884c68af6ef190472f8c5.jpg"},{"id":80288049,"identity":"64ccb2bd-01c2-4e92-bb53-5203febb9ece","added_by":"auto","created_at":"2025-04-10 07:15:16","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1051866,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S3\u003c/p\u003e","description":"","filename":"Paper10FigureS320252.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/c04bac62d634ce9e21c2c05b.jpg"},{"id":80288795,"identity":"811d7aef-a957-4521-8fde-9e1301f07014","added_by":"auto","created_at":"2025-04-10 07:23:15","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":2331566,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S4\u003c/p\u003e","description":"","filename":"Paper10FigureS420253.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/53ea36f0ebc60a164c14c39d.jpg"},{"id":80287354,"identity":"9c37de3e-d1e0-4df1-a19e-4d10d8d1fa59","added_by":"auto","created_at":"2025-04-10 07:07:15","extension":"jpg","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":992726,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S5\u003c/p\u003e","description":"","filename":"Paper10FigureS520253.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/e4a7d01181f5ad94b12e9c9e.jpg"},{"id":80287346,"identity":"8d24e2c6-e542-4352-97b8-8315f4b66794","added_by":"auto","created_at":"2025-04-10 07:07:15","extension":"jpg","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":768327,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S6\u003c/p\u003e","description":"","filename":"Paper10FigureS620253.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/997f4bd04714b71eb2c2f8a8.jpg"},{"id":80287373,"identity":"f6479a89-a3ad-41f8-8a16-9b6066c66565","added_by":"auto","created_at":"2025-04-10 07:07:16","extension":"jpg","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":6135269,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S7\u003c/p\u003e","description":"","filename":"Paper10FigureS720253.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/f6171ceeaebb7a3ca1931448.jpg"},{"id":80287387,"identity":"851760bf-59f7-48ad-b321-80064ec6d5ab","added_by":"auto","created_at":"2025-04-10 07:07:16","extension":"jpg","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":1052463,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S8\u003c/p\u003e","description":"","filename":"Paper10FigureS820253.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/3a836aea7fb3823322ad293d.jpg"},{"id":80288045,"identity":"aa563916-0465-4ad3-849f-819067125f7b","added_by":"auto","created_at":"2025-04-10 07:15:15","extension":"doc","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":33792,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1\u003c/p\u003e","description":"","filename":"Paper10TableS1202411.doc","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/4343456e25339fffed15c9fe.doc"},{"id":80288059,"identity":"3613d806-6e70-4cb0-bc1a-6616073d03df","added_by":"auto","created_at":"2025-04-10 07:15:16","extension":"doc","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":118784,"visible":true,"origin":"","legend":"\u003cp\u003eTable S2\u003c/p\u003e","description":"","filename":"Paper10TableS220252.doc","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/d0111b0facd220bbd8798908.doc"},{"id":80287343,"identity":"86302707-6727-46e3-ad36-19e45a0c9438","added_by":"auto","created_at":"2025-04-10 07:07:15","extension":"doc","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":38400,"visible":true,"origin":"","legend":"Table S3","description":"","filename":"Paper10TableS320252.doc","url":"https://assets-eu.researchsquare.com/files/rs-6409278/v1/076646ff0618f8e72b002fab.doc"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Peripheral Blood Mesenchymal Stem Cell–Derived Exosomes Improve Renal Sympathetic Denervation Efficacy Through β-Catenin-Mediated Cardiac Reprogramming","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003ePercutaneous coronary intervention has the potential to enhance treatment outcomes for myocardial infarction (MI) by enabling timely revascularization and myocardial salvage; however, it may also lead to cardiomyocyte (CM) loss and activation of sympathetic nervous system (SNS),\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e which often contributes to the development of heart failure (HF), with a 5-year mortality rate of approximately 11%.\u003csup\u003e2\u003c/sup\u003e Therefore, there is a critical need to develop therapeutic strategies that can suppress SNS activation and promote endogenous CM regeneration following MI.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eBoth cell therapy and renal sympathetic denervation (RD) hold promise for promoting myocardial repair by counteracting functional CM loss and mitigating SNS activation.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e RD, a catheter-based procedure first introduced in 2009 for the treatment of resistant hypertension, involves the ablation of renal sympathetic nerve activity in the kidneys.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Recent studies have further demonstrated that RD can enhance myocardial salvage and improve cardiac function by suppressing SNS activation following MI.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e The potential mechanisms underlying these benefits include the attenuation of inflammation, inhibition of the renin\u0026ndash;angiotensin\u0026ndash;aldosterone system (RAAS), increased levels of protective circulating natriuretic peptide levels, and reduction in cardiac fibrosis, all of which contribute to its cardioprotective effects.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e However, to date, there is no evidence supporting RD's ability to modulate cardiac regeneration after MI.\u003c/p\u003e \u003cp\u003eSustained ischemia leads to myocardial cell apoptosis and necrosis. In the past 20 years, the field of heart regeneration has entered a renaissance period with remarkable progress in the understanding of the putative capacity of stem/progenitor cells to generate clinically significant quantities of functional CMs- whether through exogenous cell administration or endogenous regeneration activation.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Mesenchymal stem cells (MSCs) can differentiate into cardiac, endothelial, and smooth muscle cells. MSCs have strong paracrine effects, making them promising candidates for endogenous regeneration and repair pathways. Relatively, peripheral blood mesenchymal stem cells (PBMSCs), demonstrate superior advantages over conventional adipose-derived MSCs and bone marrow-derived MSCs, including easier accessibility, reduced invasiveness, and enhanced clinical feasibility. Recent trends demonstrate that PBMSCs are a promising source for regenerative medicine favoring ambulatory cell sourcing, as endorsed in the ISCT 2023 Position Statement on translational MSC protocols. We recently revealed that PBMSCs are efficacious regenerative materials, exerting their benefits on ischemic myocardium via their paracrine effects.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e However, transplanted stem cells exhibit limited survival in damaged myocardium, suggesting that mechanisms other than trans-differentiation, such as paracrine factors, may play a critical role in heart regeneration.\u003c/p\u003e \u003cp\u003eApart from cytokines and growth factors, MSCs secrete small, single-membrane organelles called exosomes (Exos). MSC-derived Exos are key players in communication with local and distant tissues.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Accumulating evidence confirms that MSC-derived Exos and their active molecules, such as microRNAs (miRNAs), play a pivotal role in regulating signaling pathways associated with heart repair and regeneration. Furthermore, studies have shown that very few new myocytes are generated in the hearts of adult rodents following ischemic injury,\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and the proliferation of preexisting CMs primarily drives endogenous cardiac regeneration.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e However, little is known about the regulation of endogenous CMs regeneration mechanism by PBMSC-Exos.\u003c/p\u003e \u003cp\u003eBoth Exos therapy and RD have exhibited encouraging results in laboratory studies, though intrinsic challenges such as short half-life and lack of clear targets hinder the clinical feasibility.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Exos treatments aim to restore compromised CMs integrity, whereas RD targets aberrant neural signaling mechanisms that drive heart failure progression after MI. This research examines innovative strategies to optimize regenerative interventions for post-infarction cardiac repair. Here, we explored preclinical therapeutic strategies combining RD with exosome-based therapy to enhance endogenous cardiac regeneration following MI. First, we investigated the efficacy of RD in a pig model of MI. RD primarily modulates early sympathetic overactivation (days 0\u0026ndash;14 post-MI), leading short-term improvement of cardiac performance. However, this improvement did not remain significant afterward. Second, because exosomal therapy reduces fibrotic tissue formation and enhances cardiomyocyte proliferation,\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e which may ameliorate cardiac remodeling during the late-stage phase of MI, we hypothesized that RD administered in the early phase of MI in combination with delayed autologous PBMSC-derived Exos (PBMSC-Exos) transplantation affords sustained cardiac regeneration and repair compared with either therapy alone. Third, through \u003cem\u003ein vitro\u003c/em\u003e molecular mechanistic and \u003cem\u003ein vivo\u003c/em\u003e animal experiments, we provided the first evidence that after RD, PBMSC-Exos carry miR-141-200-429 clusters from renal artery (RA) endothelial cells (RAECs) to ischemic CMs, resulting in CM reprogramming and improving heart function. Targeting beneficial communication mediated by PBMSC-Exos or miR-141-200-429 clusters between RAs and damaged hearts may be a novel strategy for improving RD-initiated cardiac repair.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eRD Inhibits SNS/RAAS and Boosts PBMSC Exosomal Cardioprotective microRNAs in Post-MI HFrEF Pigs\u003c/h2\u003e \u003cp\u003eTo investigate RD's cardioprotective role post-MI, we assessed temporal changes in catecholamine and RAAS levels in HFrEF pigs after RD. RA nerve tyrosine hydroxylase (TH) staining revealed that gradual renal nerve viability increased after RD-Sham but significantly decreased after RD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In particular, TH staining intensity was the lowest in RD pigs after 2 weeks of MI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB); this observation was confirmed by the occurrence of the largest reduction in kidney dopamine and norepinephrine (NE) levels at this time point\u0026mdash;a quantitative index for the sympathetic nerve function (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The most significant reduction in circulating NE levels was also noted 2 weeks after RD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Next, we assessed RAAS activity based on plasma angiotensin I and angiotensin II levels and noted a significant reduction in their levels in RD pigs, with the lowest levels noted 2 weeks after MI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). After 2 weeks of RD, no significant changes were observed in the SNS and RAAS of any HFrEF pig. At later time points (e.g., 4 weeks post-RD), we still detected no significant improvements in SNS activity or RAAS markers, suggesting that the treatment's regulatory effects were both early and transient.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDespite the absence of sustained SNS/RAAS modulation, previous studies indicate that MSC-based therapies may exert cardioprotection through indirect mechanisms. Given that MSC-mediated cardioprotection is independent of direct cell\u0026ndash;cell contact,\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e we hypothesized that Exos transfer biological molecules, thereby contributing to the remote cardioprotective effects of RD. To investigate the impact of RD on PBMSC-Exos, we isolated PBMSC-Exos from RD or RD-Sham pigs via ultracentrifugation and analyzed them using transmission electron microscopy (TEM) at 1, 2, and 4 weeks post-MI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Then, we performed western blotting to confirm the presence of the Exos-associated proteins CD9, CD63, TSG101and HSP70, and the absence of negative marker Calnexinin in PBMSC-Exos preparations from RD and RD-Sham pigs at 1, 2, and 4 weeks post-MI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). CD9 and CD63 were selected as canonical exosomal markers based on MISEV2018 guidelines,\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e with TSG101 immunoblotting confirming endosomal origin.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e These tetraspanins were prioritized given their established role in cardiomyocyte-exosome interactions. These results were consistent with our nanoparticle tracking analysis (NTA) results: RD increased the expression of these proteins, peaking 2 weeks after MI; in contrast, RD-Sham gradually reduced their expression, with the greatest decrease occurring 2 weeks after MI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). Next, PBMSC-Exos size distribution, measured using NanoSight, demonstrated no significant differences between these two groups at any timepoint. In both groups, we observed a unimodal distribution of isolated particles with an average diameter of 100 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL)\u0026mdash;consistent with the definition of Exos.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e To confirm that Exos were derived from PBMSCs, we first validate the identity of PBMSCs using cell surface marker profiling by FACS. FACS showed that PBMSCs harvested from the animals at 2 weeks post-MI expressed\u0026thinsp;\u0026ge;\u0026thinsp;95% of MSC-associated cell surface markers CD44, CD71, CD90, CD105 (mesenchymal markers), while expressing\u0026thinsp;\u0026le;\u0026thinsp;2% of both hematopoietic markers HSC markers CD 34 and CD45, and endothelial progenitor cell (EPC) molecular markers CD31 andCD133 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM). Next, we performed co-immunostaining of the MSC marker protein CD105 and the Exos marker protein TSG101 to confirm that the Exos were derived from PBMSCs. Overexpression of MSC-specific molecule CD105 in Exos (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN) confirmed their derivation from PBMSCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken together, these results indicated that RD increases Exos release originated from PBMSCs in a time-dependent manner, with the greatest increase occurring 2 weeks after MI. As such, we selected autologous PBMSC-Exos from the RD and RD-Sham group (denoted as PBMSC- Exos\u003csup\u003eRD\u0026minus;Sham\u003c/sup\u003e or PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e, respectively) at 2 weeks after MI as the Exos source to investigate the additive effects of delayed exosomal therapy (i.e., intravenous injection of PBMSC-Exos 30 days after MI) with RD-induced myocardial repair in our HFrEF pigs.\u003c/p\u003e \u003cp\u003eGiven that numerous miRNAs are known to stimulate cardiac repair by promoting CM dedifferentiation and proliferation after MI,\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e we conducted small RNA sequencing (RNA-seq) to characterize miRNAs present in PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e. First, to identify miRNAs within PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e that potentially enhance cardiac proliferation and regeneration, we compared 66 miRNAs enriched in PBMSCs with datasets of miRNAs implicated in cardiac proliferation\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e and regeneration.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e As illustrated in the Venn diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), we identified 15 common miRNAs. Next, we analyzed the expression of these 15 miRNAs in PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e and PBMSC-Exos\u003csup\u003eRD\u0026minus;Sham\u003c/sup\u003e and noted that PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e significantly upregulated the expression of miR199 (miR-199a-3p; 7.73-fold increase), miR-200a (miR-200a-3p; 8.03-fold increase), and miR-200b (miR-200b-3p; 9.01-fold increase; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Third, to determine whether the observed increase in miRNA expression was due to a higher number of PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e or an increase in their miRNA content, we quantified miRNA expression per equal number of PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e using RT-qPCR. The results revealed that miR-200a-3p and miR-200b-3p expression was significantly higher in PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e compared to an equivalent amount amount of PBMSC-Exos\u003csup\u003eRD\u0026minus;Sham\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Fourth, miR-200a and miR-200b belong to the kidney-enriched miRNA family,\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e which also includes miR141, miR200, and miR429 (miR-141-200-429).\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e To investigate whether RD-treated RAs release PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e with elevated miR-200 content, we measured the expression of the miR-141-200-429 expression in Exos derived from RA (RA-Exos). Compared to RA-Exos\u003csup\u003eRD\u0026minus;Sham\u003c/sup\u003e, RA-Exos\u003csup\u003eRD\u003c/sup\u003e exhibited significantly higher levels of all miR-141-200-429 cluster members (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Finally, we compared the expression of the miR-141-200-429 cluster in RAs and hearts from RD-treated pigs. The expression of miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-141, and miR-429 expression was considerably higher in RAECs than in CMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Notably, the expression of these five miRNAs was significantly increased in RACEs following RD, with miR-200a and miR-200b showing the most pronounced upregulation (6.52- and 4.23-fold increase, respectively; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). However, in RD-treated pigs, the expression of miR-200c-3p and miR-429 was significantly upregulated in RAECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF) but not in CMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). miR-200b-3p-encapsulated MSCs-derived exosomes (MSCs-Exos) have been shown to protect against MI-induced cardiomyocyte apoptosis and inflammation.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e These findings collectively suggest that RD stimulates the release of PBMSC-Exos from RAECs, delivering cardioprotective microRNA cargo to infarcted hearts.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRD Improves Short-term Cardiac Performance and Delayed Exosomal Therapy Affords Long-term Benefit\u003c/h3\u003e\n\u003cp\u003eConfirming our earlier report, PBMSCs (5\u0026times;10\u003csup\u003e6\u003c/sup\u003ecells) improved cardiac function.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e By applying this cell count, we had obtained 2 \u0026times;10\u003csup\u003e13\u003c/sup\u003e PBMSCs-Exos from MI pigs. Next, we further tested this dosage in MI rats. PBMSC-Exos were injected into caudal vein of MI rats at dosage of 5\u0026times;10\u003csup\u003e12\u003c/sup\u003e, 2\u0026times;10\u003csup\u003e13\u003c/sup\u003e, or 5\u0026times;10\u003csup\u003e13\u003c/sup\u003e in 100\u0026micro;L PBS). PBMSCs-Exos improved cardiac function (LVEF) and expression levels of β-catenin and Oct4, and reduced CMs injury (plasmahs-cTnT) to a similar level at doses of 2\u0026times;10\u003csup\u003e13\u003c/sup\u003e and 5\u0026times;10\u003csup\u003e13\u003c/sup\u003e, but not at 5\u0026times;10\u003csup\u003e12\u003c/sup\u003e (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Based on these data we proceeded to test the effects of PBMSC-Exos injection on cardiac repair at the dose of 2\u0026times;10\u003csup\u003e13\u003c/sup\u003e in the following pig experiments. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA presents a schematic timeline integrating RD/Exos intervention windows with pathophysiological milestones. Based the above results, we harvested PBMSC-Exos at day 14 post-MI, we aimed to capture Exos with maximal reparative cargo before potential functional exhaustion. We performed RD immediately after MI for modulating early sympathetic overactivation (days 0\u0026ndash;14 post-MI), which exacerbates inflammation. Then, Exos therapy focuses on the transition period between proliferation and scar formation (day 30 post-MI).\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this study, MI was induced in 82 out of 95 experimental pigs by ligation of the left anterior descending branch of a coronary artery. From these, 40 pigs with LVEF\u0026thinsp;\u0026lt;\u0026thinsp;40% were selected and randomly assigned to receive early (within 2 h after MI) RD-Sham or RD followed by delayed (30 days after MI) PBS or Exos injection, with 10 pigs per group. These groups were labeled as RD-Sham\u0026thinsp;+\u0026thinsp;PBS, RD-Sham\u0026thinsp;+\u0026thinsp;PBMSC-Exos\u003csup\u003eRD\u0026minus;Sham\u003c/sup\u003e (denoted as RD-Sham\u0026thinsp;+\u0026thinsp;Exos), RD\u0026thinsp;+\u0026thinsp;PBS, and RD\u0026thinsp;+\u0026thinsp;PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e (denoted as RD\u0026thinsp;+\u0026thinsp;Exos). No deaths occurred in this cohort of pigs during any treatment. However, 10 pigs died during follow-up. Finally, 30 pigs survived to undergo serial functional studies 90 days after MI (\u003cb\u003eFigure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eFunctional data, measured through echocardiography, demonstrated that early RD administration after MI significantly improved LVEF 30 days after MI compared with early RD-Sham; however, this improvement did not remain significant afterward. Although delayed Exos injection significantly improved LVEF in the RD-Sham\u0026thinsp;+\u0026thinsp;Exos group by 60 days post-MI, the therapeutic effect failed to persist until 90 days post-MI. However, sequential exosome therapy maintained these therapeutic benefits throughout the study period (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). At 90 days post-MI, the RD\u0026thinsp;+\u0026thinsp;Exos group exhibited 11\u0026ndash;26% greater preservation of LVEF compared to other three groups, with all intergroup differences achieving statistical significance (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Similarly, at 90 days after MI, left ventricular stroke volume was larger in the RD\u0026thinsp;+\u0026thinsp;Exos group than in the other three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). This was mainly due to significant reductions in left ventricular end-diastolic (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) and end-systolic (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) volumes and the recovery of left ventricle (LV) anterior wall thickness (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG) in the combination groups, but no similar significant differences were observed between the RD\u0026thinsp;+\u0026thinsp;PBS and RD-Sham\u0026thinsp;+\u0026thinsp;PBS groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Moreover, all pigs receiving early RD plus delayed Exos injection were alive 90 days after MI; however, 5 of 10 pigs in the RD-Sham\u0026thinsp;+\u0026thinsp;PBS group died within 90 days after MI (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Moreover, mirroring functional recovery patterns, monotherapy with either RD or Exos significantly upregulated myocardial expression of miR-200a-3p, miR-220b-3p, and miR-141, with the most pronounced upregulation observed in RD\u0026thinsp;+\u0026thinsp;Exos pigs receiving combination therapy at 90 days post-MI (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK). Importantly, no significant differential expression was detected for miR-200c-3p and miR-429 across experimental groups. These findings aligned with our previous observations in CM-Exos derived from MI models, strongly suggesting that the myocardial exosomal miR-141-200 cluster mediates the therapeutic effects of both RD and Exos interventions on cardiac functional restoration.\u003c/p\u003e \u003cp\u003eWe assessed renal function by measuring plasma levels of blood urea nitrogen (BUN, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI) and creatinine (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ) at 0, 30, 60, and 90 days after MI. The results demonstrated that as MI-induced HFrEF progressed, plasma BUN and creatinine levels became increasingly elevated in the RD-Sham\u0026thinsp;+\u0026thinsp;PBS group; nevertheless, RD did not impair renal function. Moreover, plasma BUN and creatinine levels were significantly lower in the RD\u0026thinsp;+\u0026thinsp;Exos group than in the RD-Sham\u0026thinsp;+\u0026thinsp;PBS group 30, 60, and 90 days after MI, indicating that RD plus Exos may further reduce plasma BUN and creatinine levels.\u003c/p\u003e \u003cp\u003eFurthermore, the dynamic hemodynamics were monitored in all animals (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Compared with the baseline level, MI-induced HF caused a significant increase in heart rate, which persisted in the RD-Sham\u0026thinsp;+\u0026thinsp;PBS group, but were restored to the baseline levels in the RD-Sham\u0026thinsp;+\u0026thinsp;Exos group and all RD groups at 90 d post-MI. MABP gradually decreased in the RD-Sham\u0026thinsp;+\u0026thinsp;PBS group, but there was no significant change in the other three groups. We also observed that RD did not significantly affect cardiac function, blood pressure and heart rate in pigs received sham surgery (\u003cb\u003eTable S3\u003c/b\u003e). Following RD, no significant alterations were detected in plasma norepinephrine (NE) or RAAS components (angiotensin I and II), with no biochemical evidence of heart failure (BNP\u0026thinsp;\u0026lt;\u0026thinsp;100 pg/mL) or myocardial necrosis (cTnI\u0026thinsp;\u0026lt;\u0026thinsp;0.01 ng/mL). All these data suggested that RD-mediated cardiac repair specifically targets MI-induced sympathetic hyperactivation rather than non-ischemic surgical effects.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHeart Rate, MABP, BNP, and hs-cTnT (means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHeart rate\u003c/p\u003e \u003cp\u003e(beats/min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMABP\u003c/p\u003e \u003cp\u003e( mmHg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePlasma BNP\u003c/p\u003e \u003cp\u003e(pg/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ehs-cTnT\u003c/p\u003e \u003cp\u003e(ng/l)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eBaseline\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD-Sham\u0026thinsp;+\u0026thinsp;PBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e94.6\u0026thinsp;\u0026plusmn;\u0026thinsp;19.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD-Sham\u0026thinsp;+\u0026thinsp;Exos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e80\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e88.6\u0026thinsp;\u0026plusmn;\u0026thinsp;17.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD\u0026thinsp;+\u0026thinsp;PBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e81\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e87.0\u0026thinsp;\u0026plusmn;\u0026thinsp;15.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD\u0026thinsp;+\u0026thinsp;Exos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e83\u0026thinsp;\u0026plusmn;\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e84\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e83.1\u0026thinsp;\u0026plusmn;\u0026thinsp;15.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eOn the day of myocardial infarction\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD-Sham\u0026thinsp;+\u0026thinsp;PBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e105\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e432.5\u0026thinsp;\u0026plusmn;\u0026thinsp;84.0\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1191.6\u0026thinsp;\u0026plusmn;\u0026thinsp;209.7\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD-Sham\u0026thinsp;+\u0026thinsp;Exos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e108\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e86\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e425.7\u0026thinsp;\u0026plusmn;\u0026thinsp;83.4\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1154.5\u0026thinsp;\u0026plusmn;\u0026thinsp;248.7\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD\u0026thinsp;+\u0026thinsp;PBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e436.7\u0026thinsp;\u0026plusmn;\u0026thinsp;78.2\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1150.7\u0026thinsp;\u0026plusmn;\u0026thinsp;165.6\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD\u0026thinsp;+\u0026thinsp;Exos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99\u0026thinsp;\u0026plusmn;\u0026thinsp;12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e81\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e433.4\u0026thinsp;\u0026plusmn;\u0026thinsp;83.2\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1119.6\u0026thinsp;\u0026plusmn;\u0026thinsp;159.8\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e30 d post-MI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD-Sham\u0026thinsp;+\u0026thinsp;PBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e115\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003csup\u003e\u0026sect;║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e528.9\u0026thinsp;\u0026plusmn;\u0026thinsp;83.0\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e42.1\u0026thinsp;\u0026plusmn;\u0026thinsp;12.3\u003csup\u003e\u0026sect;║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD-Sham\u0026thinsp;+\u0026thinsp;Exos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e102\u0026thinsp;\u0026plusmn;\u0026thinsp;13\u003csup\u003e*\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e779.4\u0026thinsp;\u0026plusmn;\u0026thinsp;94.4\u003csup\u003e*\u0026sect;║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4\u003csup\u003e*║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD\u0026thinsp;+\u0026thinsp;PBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e93\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003csup\u003e*#\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e687.6\u0026thinsp;\u0026plusmn;\u0026thinsp;118.5\u003csup\u003e\u0026sect;║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17.0\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9\u003csup\u003e*║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD\u0026thinsp;+\u0026thinsp;Exos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003csup\u003e*\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80\u0026thinsp;\u0026plusmn;\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e829.1\u0026thinsp;\u0026plusmn;\u0026thinsp;128.8\u003csup\u003e*\u0026sect;║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003csup\u003e*║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e60 d post-MI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD-Sham\u0026thinsp;+\u0026thinsp;PBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e116\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003csup\u003e\u0026sect;║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e81\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e577.8\u0026thinsp;\u0026plusmn;\u0026thinsp;79.0\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e35.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1\u003csup\u003e\u0026sect;║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD-Sham\u0026thinsp;+\u0026thinsp;Exos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003csup\u003e*\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e800.0\u0026thinsp;\u0026plusmn;\u0026thinsp;83.3\u003csup\u003e*\u0026sect;║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003csup\u003e*║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD\u0026thinsp;+\u0026thinsp;PBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e87\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003csup\u003e*#║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e81\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e730.6\u0026thinsp;\u0026plusmn;\u0026thinsp;71.9\u003csup\u003e\u0026sect;║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2\u003csup\u003e*║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD\u0026thinsp;+\u0026thinsp;Exos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003csup\u003e*#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e81\u0026thinsp;\u0026plusmn;\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1142.3\u0026thinsp;\u0026plusmn;\u0026thinsp;181.6\u003csup\u003e*#\u0026dagger;\u0026sect;║\u0026para;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003csup\u003e*║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e90 d post-MI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD-Sham\u0026thinsp;+\u0026thinsp;PBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e116\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003csup\u003e\u0026sect;║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003csup\u003e\u0026sect;║\u0026para;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e598.4\u0026thinsp;\u0026plusmn;\u0026thinsp;89.2\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4\u003csup\u003e\u0026sect;║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD-Sham\u0026thinsp;+\u0026thinsp;Exos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003csup\u003e*\u0026sect;║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e754.3\u0026thinsp;\u0026plusmn;\u0026thinsp;117.8\u003csup\u003e\u0026sect;║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6\u003csup\u003e║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD\u0026thinsp;+\u0026thinsp;PBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e88\u0026thinsp;\u0026plusmn;\u0026thinsp;7\u003csup\u003e*#║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e81\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e803.4\u0026thinsp;\u0026plusmn;\u0026thinsp;95.4\u003csup\u003e\u0026sect;║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003csup\u003e\u0026sect;║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRD\u0026thinsp;+\u0026thinsp;Exos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e84\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003csup\u003e*#║\u0026para;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e79\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1409.0\u0026thinsp;\u0026plusmn;\u0026thinsp;122.3\u003csup\u003e*#\u0026dagger;\u0026sect;║\u0026para;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003csup\u003e║\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDelayed Exosomal Therapy Attenuates Cardiac Fibrosis via RD-Mediated RAAS Inhibition and Enhanced Cardiomyocyte Proliferation and Regeneration\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe used tetrazolium chloride (TTC), Masson\u0026rsquo;s trichrome, and hematoxylin\u0026ndash;eosin (H\u0026amp;E) staining for histological assessment 90 days after MI. The scar size (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) and fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) demonstrated a considerable reduction in the RD\u0026thinsp;+\u0026thinsp;Exos group compared with the other groups. Moreover, the RD\u0026thinsp;+\u0026thinsp;Exos group showed the greatest increase in the number of viable CMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Wheat germ agglutinin (WGA) staining was carried out to measure the cross-sectional area of CMs. Both macroscopic postmortem analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) and CM cross-sectional area quantification (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH) demonstrated that either RD or Exos monotherapy increased CM size, while combination therapy (RD\u0026thinsp;+\u0026thinsp;Exos) resulted in a further significant increase in cross-sectional area at 90 days post-MI. All these data suggest that both RD and Exos monotherapy inhibited cardiac pathological remodeling, and their combination therapy resulted in an enhanced therapeutic effect.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo elucidate the underlying mechanisms by which the combined RD\u0026thinsp;+\u0026thinsp;delayed Exos regimen prevents LV remodeling, we performed a series of four experiments. First, we explored some molecular correlates of cardiac repair and cardiac function improvement. Peripheral blood samples were obtained at baseline and 0, 30, 60, and 90 days after MI to measure plasma B-type natriuretic peptide (BNP) and high-sensitivity cardiac troponin T (hs-cTnT) levels (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The results consistently demonstrated that RD significantly increases plasma BNP levels while reducing plasma hs-cTnT levels. Delayed exosomal therapy further amplified these effects, suggesting that it enhances RD-mediated elevation in BNP bioavailability while concurrently attenuating CM injury.\u003c/p\u003e \u003cp\u003eSecond, we assessed the effects of early RD plus delayed exosomal therapy on the SNS of our pigs, specifically efferent renal nerve activity markers, including renal TH and NE. At 90 days after MI, we observed equally reduced TH staining intensity in the RD\u0026thinsp;+\u0026thinsp;PBS and RD\u0026thinsp;+\u0026thinsp;Exos groups than in the RD-Sham\u0026thinsp;+\u0026thinsp;PBS and RD-Sham\u0026thinsp;+\u0026thinsp;Exos groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). In particular, both RD\u0026thinsp;+\u0026thinsp;PBS and RD\u0026thinsp;+\u0026thinsp;Exos groups demonstrated an approximately 65% decrease in TH stain intensity compared with the RD-Sham\u0026thinsp;+\u0026thinsp;PBS and RD-Sham\u0026thinsp;+\u0026thinsp;Exos groups (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). Moreover, we noted an approximately 80% reduction in the renal levels of the kidney SNS activity markers dopamine (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and NE (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) 90 days after MI in the RD\u0026thinsp;+\u0026thinsp;Exos group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL), indicating equivalent reductions in kidney SNS activity. RD-Sham\u0026thinsp;+\u0026thinsp;Exos could not reduce kidney dopamine and NE levels below those in the RD-Sham\u0026thinsp;+\u0026thinsp;PBS group, indicating that Exos did not significantly alter kidney SNS activity. Similarly, a decrease in plasma NE levels was achieved in both RD\u0026thinsp;+\u0026thinsp;PBS and RD\u0026thinsp;+\u0026thinsp;Exos groups, indicating that RD reduced renal afferent nerve stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM). Moreover, the RD\u0026thinsp;+\u0026thinsp;PBS and RD\u0026thinsp;+\u0026thinsp;Exos groups demonstrated significantly lower kidney angiotensin I and angiotensin II levels at 90 days after MI than the RD-Sham\u0026thinsp;+\u0026thinsp;PBS and RD-Sham\u0026thinsp;+\u0026thinsp;Exos group (\u003cb\u003eFigure S3A\u003c/b\u003e and \u003cb\u003eS3B\u003c/b\u003e). Moreover, alterations in angiotensin levels resulting from RD extended to changes in plasma angiotensin: plasma angiotensin I and angiotensin II levels were significantly lower in the RD\u0026thinsp;+\u0026thinsp;PBS and RD\u0026thinsp;+\u0026thinsp;Exos groups than in the RD-Sham\u0026thinsp;+\u0026thinsp;PBS and RD-Sham\u0026thinsp;+\u0026thinsp;Exos groups (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cb\u003eFigure S3D\u003c/b\u003e and \u003cb\u003eS3E\u003c/b\u003e). Similarly, plasma renin activity was significantly lower in both RD groups than in both RD-Sham groups (both \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cb\u003eFigure S3C\u003c/b\u003e). Kidney or plasma angiotensin I and angiotensin II levels showed no significant difference between the RD-Sham\u0026thinsp;+\u0026thinsp;PBS and RD-Sham\u0026thinsp;+\u0026thinsp;Exos groups or between the RD\u0026thinsp;+\u0026thinsp;PBS and RD\u0026thinsp;+\u0026thinsp;Exos groups. In contrast, plasma aldosterone levels were significantly lower in the RD\u0026thinsp;+\u0026thinsp;PBS and RD\u0026thinsp;+\u0026thinsp;Exos groups than in the RD-Sham\u0026thinsp;+\u0026thinsp;PBS and RD-Sham\u0026thinsp;+\u0026thinsp;Exos groups, respectively (both \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01); moreover, they were significantly lower in the RD-Sham\u0026thinsp;+\u0026thinsp;Exos and RD\u0026thinsp;+\u0026thinsp;Exos groups than in the RD-Sham\u0026thinsp;+\u0026thinsp;PBS and RD\u0026thinsp;+\u0026thinsp;PBS groups, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively; \u003cb\u003eFigure S3F\u003c/b\u003e). However, plasma NE levels did not differ significantly between the RD\u0026thinsp;+\u0026thinsp;PBS and RD\u0026thinsp;+\u0026thinsp;Exos groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM), suggesting that the key protective effects of delayed Exos therapy are primarily mediated through RD-induced RAAS inhibition rather than modulation of SNS activation.\u003c/p\u003e \u003cp\u003eThird, we observed cardiomyocyte proliferation following RD and Exos therapy. Ki67 is a well-established marker of cellular proliferation, as it is expressed during all active phases of the cell cycle (G1, S, G2, and M) but is absent in quiescent cells (G0).\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e In the context of cardiac repair, Ki67 immunolabeling is widely used to assess CMs proliferation, which is a critical process for myocardial regeneration and functional recovery after injury.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e In this study, we used Ki67 immunolabeling to evaluate whether early RD (remote conditioning) and delayed Exos therapy could promote cardiomyocyte proliferation. Dual immunofluorescence co-staining of Ki67 (proliferation marker) and MHC (myosin heavy chain, cardiomyocyte marker) demonstrated that early RD treatment significantly increased Ki67\u003csup\u003e+\u003c/sup\u003e cardiomyocyte populations in both peri-infarct and remote myocardial regions. Moreover, delayed Exos administration further enhanced this proliferative response, confirming the therapeutic potential of sequential interventions for cardiomyocyte cycle re-activation. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Similarly, RD led to an increase in the number of Aurora B-positive CMs within the myocardium, and delayed exosomal therapy increased this number further (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Next, we assessed cardiomyocyte (CM) dedifferentiation via α-actin staining to confirm cell-cycle reentry in mature CMs. RD treatment induced CM dedifferentiation, as evidenced by α-smooth muscle actin (α-SMA) expression in a subset of CMs, with this trend being more pronounced in the RD\u0026thinsp;+\u0026thinsp;Exos group than in the RD-Sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Western blot analysis further demonstrated that RD upregulated Aurora B, α-SMA, and α-actin expression, and delayed Exos administration further enhanced their levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). Collectively, these findings indicate that delayed Exos injection amplifies RD-induced CM proliferation and dedifferentiation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFourth, we assessed the uptake of PBMSC-Exos by CMs in the RD-Sham-treated and RD-treated hearts. To examine whether PBMSC-Exos were differentially taken up by HFrEF hearts receiving RD-Sham or RD, PKH26 was used to label the exosomes before injection. PKH26 detection in the MHC through immunofluorescence staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). PKH26 signals were absent in the CMs of the RD-Sham\u0026thinsp;+\u0026thinsp;PBS and RD\u0026thinsp;+\u0026thinsp;PBS groups. However, strong PKH26 signals were observed in the CMs of RD-Sham\u0026thinsp;+\u0026thinsp;Exos animals, and these signals were further enhanced by delayed Exos injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). In addition, related analysis demonstrated that CM Exos uptake was positively correlated with the expression of Aurora B (r\u0026thinsp;=\u0026thinsp;0.817, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ) and α-SMA (r\u0026thinsp;=\u0026thinsp;0.905, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK) in the RD\u0026thinsp;+\u0026thinsp;Exos group. Thus, RD-induced proliferation and dedifferentiation of CMs may be related to the content and delivery of Exos, conferring regenerative capacity to an ischemic heart.\u003c/p\u003e\n\u003ch3\u003e\u003cp\u003e\u003cstrong\u003ePBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e Reprogram Ischemic CMs\u003c/strong\u003e\u003c/p\u003e\u003c/h3\u003e\n\u003cp\u003eThe induction of adult CM proliferation is typically associated with three hallmark cellular transitions: (1) modulation of differentiation markers, (2) metabolic remodeling, and (3) re-activation of embryonic gene programs. While this process has been historically termed \"dedifferentiation\", emerging evidence supports its characterization as \"partial reprogramming\" - a controlled transition to a fetal-like proliferative state where adult CMs regain mitotic capacity while maintaining core functional characteristics.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Notably, MSC-Exos have been shown to orchestrate this regenerative process through their miRNA cargo, which simultaneously modulates tissue repair pathways and immune responses, thereby creating a permissive microenvironment for ischemic myocardial regeneration.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e To comprehensively evaluate the effects of PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e enriched with the miR-141-200-429 cluster on ischemic cardiomyocyte reprogramming, we conducted myocardial biopsies on animals treated with either RD-Sham or RD at 14 days post-MI (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The biopsied myocardial tissues were then used to implement an integrated experimental strategy, combining complementary \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e approaches, to systematically assess the therapeutic outcomes.\u003c/p\u003e \u003cp\u003eFor \u003cem\u003ein vitro\u003c/em\u003e functional assessment, CMs isolated from biopsied tissues of post-MI hearts at 2 weeks were exposed to hypoxia-mimicking conditions and co-cultured with either PBMSC-Exos\u003csup\u003eRD\u0026minus;Sham\u003c/sup\u003e or PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e (1\u0026times;10\u003csup\u003e8\u003c/sup\u003e particles/mL) for 48 hours to systematically evaluate the composition-dependent effects on CM functional recovery. The results demonstrated that 39.1% of CMs cultured with PBMSC-Exos\u003csup\u003eRD\u0026minus;Sham\u003c/sup\u003e lacked cardiac troponin (cTnI), whereas this proportion significantly decreased in CMs cultured with PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e, suggesting that PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e increased CM dedifferentiation (\u003cb\u003eFigure S4A\u003c/b\u003e). Moreover, compared with PBMSC-Exos\u003csup\u003eRD\u0026minus;Sham\u003c/sup\u003e, PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e increased the levels of the dedifferentiation markers Runx1 and Dab2 (\u003cb\u003eFigure S4B\u003c/b\u003e and \u003cb\u003eS4C\u003c/b\u003e). Cell-cycle reentry of CMs from PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e treated-CM cultures was confirmed by analysis of purified MHC-expressing CMs positive for Ki67 (\u003cb\u003eFigure S4Da\u003c/b\u003e) and PH3 (\u003cb\u003eFigure S4Ea\u003c/b\u003e). Compared with CMs treated with PBMSC-Exos\u003csup\u003eRD\u0026minus;Sham\u003c/sup\u003e, all cases of CMs receiving PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e showed a much higher Ki67 proliferation index (\u003cb\u003eFigure S4Db\u003c/b\u003e) along with a higher mitotic index, calculated as the fraction of PH3-positive cells (\u003cb\u003eFigure S4Eb\u003c/b\u003e). The redifferentiation of these dedifferentiated CMs evaluated by the levels of cTnT led to a similar result (\u003cb\u003eFigure S4F\u003c/b\u003e): cTnT expression was 16.2-fold higher in PBMSC-Exo\u003csup\u003eRD\u003c/sup\u003e-treated CMs than in those treated with PBMSC-Exos\u003csup\u003eRD\u0026minus;Sham\u003c/sup\u003e. These data strongly suggested that PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e favor cardiac dedifferentiation, proliferation, and redifferentiation under hypoxia.\u003c/p\u003e \u003cp\u003eTo evaluate \u003cem\u003ein vivo\u003c/em\u003e therapeutic effects, we performed comprehensive transcriptional profiling of CMs isolated via cardiac biopsy from RD-treated and RD-Sham control groups at 14 days post-MI, revealing significant PBMSC-Exo\u003csup\u003eRD\u003c/sup\u003e-mediated molecular reprogramming. Consistent with the functional and morphological assessments, these changes were related to cell proliferation, division, and differentiation. Of the 10 genes significantly altered by PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e (\u003cb\u003eFigure S5A\u003c/b\u003e), the expression of genes encoding six reprogramming factors involved with cardiovascular system development, cell specification, cycle, division, and proliferation was upregulated, whereas four genes associated with inflammation and apoptosis were downregulated (\u003cb\u003eFigure S5B\u003c/b\u003e). These findings collectively suggest that PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e treatment potentially induces molecular reprogramming in ischemic CMs, facilitating their transition towards a regenerative state through activation of endogenous repair mechanisms.\u003c/p\u003e\n\u003ch3\u003eβ-Catenin Promotes Exos Uptake, Proliferation, and Redifferentiation by CMs\u003c/h3\u003e\n\u003cp\u003ePBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e were noted to reprogram CMs\u0026mdash;as evidenced by overexpression of the pluripotent transcript factors \u003cem\u003eOct4\u003c/em\u003e, \u003cem\u003eSox2\u003c/em\u003e, and \u003cem\u003eKlf4\u003c/em\u003e (\u003cb\u003eFigure S5A\u003c/b\u003e and \u003cb\u003eS5B\u003c/b\u003e). Moreover, among all genes investigated, the β-catenin was the most significantly upregulated molecule by PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e (\u003cb\u003eFigure S5B\u003c/b\u003e), suggesting that β-catenin underlies CMs potency under hypoxic conditions.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eBecause the β-catenin pathway is a key regulator of CMs differentiation,\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e we next investigated the effects of β-catenin on PBMSC-Exo\u003csup\u003eRD\u003c/sup\u003e-mediated CMs proliferation and redifferentiation. First, we assessed the β-catenin-promoted direct CMs uptake of MSC-Exos. CMs were randomly transfected with an empty vector, \u003cem\u003eoe\u003c/em\u003eβ-catenin, \u003cem\u003esi\u003c/em\u003eβ-catenin, or WAY-262611 (1 \u0026micro;mol/L; a β-catenin agonist; ab145229, Abcam, USA) and then cocultured with PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e under hypoxic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). CMs uptake of PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e was evaluated as the percent proportion of cells double-positive for PKH26 and MHC to those completely positive for MHC (Fig.\u0026nbsp;6\u003cb\u003eBa\u003c/b\u003e). As shown in \u003cb\u003eFig.\u0026nbsp;6Bb\u003c/b\u003e, quantitative analysis indicated that the myocardial uptake of PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e was the greatest in the \u003cem\u003eoe\u003c/em\u003eβ-catenin-transfected CMs, followed by WAY-262611-treated CMs. The least uptake levels were noted in \u003cem\u003esi\u003c/em\u003eβ-catenin-transfected CMs. Thus, β-catenin may improve the Exos uptake of CMs. Since transfection with \u003cem\u003eoe\u003c/em\u003eβ-catenin resulted in higher CM uptake compared to the β-catenin agonist, we exclusively employed \u003cem\u003eoe\u003c/em\u003eβ-catenin in the subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSecond, we determined whether β-catenin induces a proliferative dedifferentiated state in PBMSC-Exo\u003csup\u003eRD\u003c/sup\u003e-treated CMs. CMs were pretreated with transfection of empty vector, \u003cem\u003eoe\u003c/em\u003eβ-catenin, or \u003cem\u003esi\u003c/em\u003eβ-catenin and then cultured for 7 days with PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e under hypoxic conditions. We observed a considerable increase in CMs double-positive for EdU and cTnT after \u003cem\u003eoe\u003c/em\u003eβ-catenin transfection. After 7 days of culture, \u003cem\u003eoe\u003c/em\u003eβ-catenin-transfected CMs were rod-shaped CMs, undergoing mitosis and cytokinesis (Fig.\u0026nbsp;6\u003cb\u003eCa\u003c/b\u003e). Compared with empty vector transfection, \u003cem\u003eoe\u003c/em\u003eβ-catenin transfection led to a continual increase in CM proliferation rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD): 7.1% of CMs proliferated (mononucleated: 0.7%; binucleated or multinucleated: 6.4%) after 7 days of culture. However, \u003cem\u003esi\u003c/em\u003eβ-catenin transfection abrogated this benefit (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-F). Multiple proliferation patterns were observed in the \u003cem\u003eoe\u003c/em\u003eβ-catenin-transfected cells. Binucleated CMs could divide into two or three mononucleated cells or into one mononucleated cell and one binucleated or multinucleated cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Thus, in the presence of PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e, β-catenin may stimulate terminally differentiated CMs to reenter the cell cycle and proliferate in mononucleated and binucleated cells.\u003c/p\u003e \u003cp\u003eThird, considering the aforementioned findings, the apoptosis rate of CMs after 7 days of culture under hypoxic conditions was evaluated using annexin V\u0026ndash;APC and propidium iodide (PI) staining, followed by flow cytometry. The cell death rate was significantly lower in \u003cem\u003eoe\u003c/em\u003eβ-catenin-transfected CMs than in control CMs; nevertheless, the greatest cell death rate was noted in the \u003cem\u003esi\u003c/em\u003eβ-catenin group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Thus, β-catenin may reduce CM apoptosis, promoting CM proliferation.\u003c/p\u003e \u003cp\u003eFourth, after 7 days of culture, CMs were immunostained for cTnI to determine whether dedifferentiated CMs had redifferentiated (Fig.\u0026nbsp;6\u003cb\u003eHa\u003c/b\u003e). Of the dedifferentiated CMs (i.e., α-SMA-positive CMs), the cTnI-positive rate was 53.5%, 23.9%, and 5.8% in \u003cem\u003eoe\u003c/em\u003eβ-catenin-transfected, empty vector-transfected, and \u003cem\u003esi\u003c/em\u003eβ-catenin-transfected CMs, respectively (Fig.\u0026nbsp;6\u003cb\u003eHb\u003c/b\u003e). These results indicated that dedifferentiated CMs form new functional CMs.\u003c/p\u003e \u003cp\u003eFinally, our quantitative reverse transcription polymerase chain reaction (RT-qPCR) results demonstrated considerably higher expression of the Yamanaka factors \u003cem\u003eOct4\u003c/em\u003e, \u003cem\u003eSox2\u003c/em\u003e, \u003cem\u003eKlf4\u003c/em\u003e, and c-\u003cem\u003eMyc\u003c/em\u003e in PBMSC-Exo\u003csup\u003eRD\u003c/sup\u003e-treated CMs in empty vector-transfected CMs than in \u003cem\u003esi\u003c/em\u003eβ-catenin-transfected CMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI)\u0026mdash;consistent with their expression changes mentioned above (\u003cb\u003eFigure S5A\u003c/b\u003e). Overexpression of β-catenin (\u003cem\u003eoe\u003c/em\u003eβ-Catenin) further increased the mRNA expression of Oct4, Sox2, Klf4, and Myc (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI), indicating that β-catenin enhances PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e-induced CM reprogramming and exerts anti-apoptotic effects.\u003c/p\u003e\n\u003ch3\u003ePBMSC-Exos Transfer miR-141-200-429 Clusters to Induce Heart Regeneration via β-Catenin Signaling\u003c/h3\u003e\n\u003cp\u003eAs established in our prior research, RD achieves cardioprotection by facilitating the release of Exos miR-141-200-429 clusters from RAECs, which are transported to cardiomyocytes through PBMSCs. Next, we investigated the mechanism through which miR-141-200-429 clusters in PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e initiate myocardial reprogramming via the β-catenin signaling pathway.\u003c/p\u003e \u003cp\u003eTo further confirm that CMs miR-141-200-429 clusters originate from RD-induced RAECs, we used the endoglin promoter to generate RAECs adeno-associated virus (AAV)-expressing miR-141-200-429 cluster sponges (miR-141-200-429 sponges) or NC sponges (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Following the biopsy conducted at 14 days post-MI, AAV therapy was immediately administered to the pigs treated with either RD-Sham or RD. Four weeks later, PBMSC-Exos were isolated, and CMs were collected. BODIPY TR ceramide\u0026ndash;labeled Exos uptake in hearts was detected through fluorescence microscopy. miR-141-200-429 sponges significantly reduced CM uptake of these Exos in RD-treated hearts (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). These sponges significantly reduced miR-141-200-429 levels in PBMSC-Exos derived from the same amounts of cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Notably, miR-141-200-429 sponges significantly reduced miR-200a-3p, miR-200b-3p, and miR-141 levels after RD but did not alter miR-200c-3p and miR-429 levels in CM-Exos from the RD pigs receiving miR-141-200-429 sponges (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Taken together, these results indicated that CM miR-141-200-429 cluster is produced by RD-treated RAECs and transferred to CMs by Exos.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm that miR-141-200-429 cluster induces heart regeneration, we next conducted RNA sequencing (RNA-seq) on CMs isolated from the aforementioned pigs, which were divided into the following groups: RD-Sham, RD-Sham\u0026thinsp;+\u0026thinsp;miR-141-200-429 sponge, RD\u0026thinsp;+\u0026thinsp;NC sponge, and RD\u0026thinsp;+\u0026thinsp;miR-141-200-429 sponge. A total of 1,634 and 1,745 differentially expressed genes (DEGs; all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were identified in hearts treated with RD-Sham\u0026thinsp;+\u0026thinsp;miR-141-200-429 sponges and RD\u0026thinsp;+\u0026thinsp;miR-141-200-429 sponges, respectively. MSC-derived Exos and their active molecule miRNAs have been recently reported to regulate signaling pathways involved in heart repair and regeneration.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Gene set expression analysis revealed that miR-141-200-429 sponges caused a significant reduction in the expression of genes and pathways related to cell proliferation and differentiation but an increase in the expression of genes and pathways involved in apoptosis and contraction, concomitant with dedifferentiation marker downregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-G). Downregulated genes were noted to be mainly involved in CM proliferation and differentiation and heart development, suggesting a less immature state of CM miR-141-200-429 sponges. This observation was further confirmed through WGA staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH) and CM cross-sectional area quantification (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI), which confirmed a reduction in sarcomere density. Moreover, RT-qPCR revealed increased expression of the proapoptotic genes \u003cem\u003eBax\u003c/em\u003e, \u003cem\u003eCasp3\u003c/em\u003e, \u003cem\u003eCasp9\u003c/em\u003e, and \u003cem\u003ep53\u003c/em\u003e but reduced that of immature CM-specific genes involved in proliferation (i.e., \u003cem\u003ec-Myc\u003c/em\u003e, \u003cem\u003eCdk1\u003c/em\u003e, \u003cem\u003eYap1\u003c/em\u003e, and \u003cem\u003eMcl-1\u003c/em\u003e) and dedifferentiation markers (\u003cem\u003eNppa\u003c/em\u003e and \u003cem\u003eActa1\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ) after miR-141-200-429 sponge treatment. Integrative analysis of RNA-seq data sets from RD-Sham and RD miR-141-200-429 sponge CMs revealed 226 and 305 genes jointly upregulated or downregulated in both HFrEF pig groups, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK). Most of the downregulated genes were those involved in heart development and CM proliferation and differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eL). Taken together, these results indicated that miR-141-200-429 cluster abrogation initiates a cascade of events inhibiting RDCMs into a more immature state, inhibiting CM proliferation and dedifferentiation.\u003c/p\u003e \u003cp\u003eNotably, we observed a significant inhibition of antiapoptosis and reprogramming, as indicated by the decreased expression of β-catenin and its target genes \u003cem\u003eBcl2\u003c/em\u003e, \u003cem\u003esurvivin\u003c/em\u003e, and \u003cem\u003eOct4\u003c/em\u003e in CMs from the RD\u0026thinsp;+\u0026thinsp;miR-141-200-429 sponge group compared to those from the RD\u0026thinsp;+\u0026thinsp;NC sponge group CMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ). These findings suggest that miR-141-200-429 sponges inactivates β-catenin signaling, thereby suppressing a cascade of RD-induced events that convert mature CMs into a more immature state, ultimately inhibiting CMs survival and reprogramming. However, we did not detect differences in the levels of the inflammation factor \u003cem\u003eTgf-β1\u003c/em\u003e between RD\u0026thinsp;+\u0026thinsp;NC and RD\u0026thinsp;+\u0026thinsp;miR-141-200-429 sponge group hearts, indicating that inhibition of miR-141-200-429 cluster does not alter inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eDickkopf-related protein 1 (Dkk1), a suppressor of β-catenin, and bFGF, a neurotrophic factor,\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e were upregulated in miR-141-200-429 sponge group CMs. However, the expression of \u003cem\u003eDkk1\u003c/em\u003e and \u003cem\u003ebFgf\u003c/em\u003e was significantly reduced in CMs from the RD\u0026thinsp;+\u0026thinsp;NC sponge group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ), indicating RAAS inhibition in RD-treated hearts (\u003cb\u003eFigure S3A-F\u003c/b\u003e). The abrogation of Dkk1 caused by miR-141-200-429 sponges led to the activation of β-catenin (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ), which in turn upregulated the expression of bFGF, a factor required for prorenin synthesis.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e This mechanism ultimately negated the beneficial effects of PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e related to RAAS inhibition.\u003c/p\u003e \u003cp\u003eWe next performed loss- and gain-of-function experiments to determine the potential target of the miR-141-200-429 cluster derived from PBMSC-Exos RD. RDCMs were transfected with miR-141-200-429 mimics (to overexpress the miRNA), sponges (to inhibit endogenous miRNA), or scrambled miRNA/NC sponges (as negative controls). After 48 hours of hypoxic culture, qRT-PCR analysis demonstrated that Dkk1 mRNA levels were significantly decreased or increased with miRNA overexpression or inhibition, respectively (\u003cb\u003eFigure S6A\u003c/b\u003e). Western blot analysis further revealed that Dkk1 protein expression followed the same trend, decreasing with miRNA overexpression and increasing with miRNA suppression (\u003cb\u003eFigure S6B\u003c/b\u003e). These findings provide evidence that the miR-141-200-429 cluster directly targets Dkk1.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePBMSC-Exos\u003c/b\u003e \u003csup\u003e \u003cb\u003eRD\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eActivate the βCatenin Pathway and Improve the Biological Behavior of CMs by Negatively Targeting Dkk1\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSince miR-141-200-429 sponges were found to inhibit the β-catenin pathway and impede the RD-induced reprogramming of mature CMs into a more immature state, we next analyzed whether these processes are regulated by Dkk1. To explore this, RDCMs were treated with PBMSCs-Exos\u003csup\u003eRD\u003c/sup\u003e either alone or in combination with recombinant Dkk1. The RDCMs were then cultured for 7 days under hypoxic conditions to assess the effects. Although PBMSCs-Exos\u003csup\u003eRD\u003c/sup\u003e ameliorated the proliferation of RDCMs, this benefit was abrogated when RDCMs cotreated with recombinant Dkk1, as demonstrated by Ki67 staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA) and CCK-8 assay results (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Immunoblot analysis revealed that PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e enhanced \u003cem\u003eOct4\u003c/em\u003e and the cyclin-dependent kinase 1 gene (\u003cem\u003eCdk1\u003c/em\u003e) expression, while notably, \u003cem\u003ec-Myc\u003c/em\u003e expression remained largely unaltered (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). However, overexpression of Dkk1 attenuated the PBMSC-Exo\u003csup\u003eRD\u003c/sup\u003e-induced enhancement of Aurora B incorporation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE-H), which had previously been observed in hearts subjected to early RD followed by delayed Exos therapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Additionally, Dkk1 overexpression diminished the marked re-expression of \u003cem\u003eNppa\u003c/em\u003e and \u003cem\u003eActa1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH)\u0026mdash;both markers highly expressed during embryonic and fetal developmental stages.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e These findings suggest that Dkk1 disrupts the PBMSC-ExosRD-mediated activation of β-catenin signaling, thereby negating its cardioprotective effects linked to CM reprogramming. In contrast to these findings, TUNEL assay revealed that the cell death rate was significantly lower in PBMSC-Exo\u003csup\u003eRD\u003c/sup\u003e-treated CMs compared to control CMs; however, this protective effect was reversed by Dkk1 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eK). Furthermore, we found that Dkk1 abolished the prosurvival effects of PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e on CMs, as evidenced by the upregulation of the proapoptotic factors caspase-3, cleaved caspase 3 and p53, and downregulation of the antiapoptotic factors survivin and Bcl2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eJ and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eL). These results suggest that \u003cem\u003eDkk1\u003c/em\u003e may either suppress RD-induced proliferation or induce apoptosis in RD-treated CMs. Consequently, targeted inhibition of \u003cem\u003eDkk1\u003c/em\u003e in CMs likely represents a key mechanism through which PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e exerts its therapeutic benefits.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm that RD-induced β-catenin activation is mediated by Dkk1 inhibition, we analyzed β-catenin-associated protein expression in RDCMs receiving PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e, recombinant Dkk1, or both, using Western blotting. The results demonstrated that PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e significantly suppressed Dkk1 expression while upregulating phosphorylated GSK-3β at serine 9 (p-GSK-3β S9), cyclin D1, Tcf4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eM and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eP), and Oct4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD) compared to the control group. RDCMs cotreated with Dkk1 produced results contrasting those observed in the PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e group, suggesting that PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e activated the Wnt/β-catenin pathway via repression of Dkk1, thereby enhancing proliferation in RDCMs. This aligns with our prior findings demonstrating that nuclear β-catenin accumulation serves as a hallmark of canonical Wnt pathway activation.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Hypoxia injury during lung development leads to excessive Wnt/β-catenin activation through β-catenin accumulation. GSK3 phosphorylation can promote β-catenin transfer from the cytoplasm to the nucleus.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e Consistent with the changing trend of GSK3 phosphorylation, PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e significantly increased and Dkk1 significantly reduced the expression of β-catenin, especially nuclear β-catenin, compared with those in the CTRL group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eN and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eQ). Immunofluorescence staining demonstrated a stronger β-catenin fluorescence intensity in the PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e group compared to the CTRL group, which was significantly attenuated upon Dkk1 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eO and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eR). Collectively, these findings suggest that miR-141-200-429-enriched PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e likely suppresses Dkk1\u0026mdash;a negative regulatory factor of Wnt signaling\u0026mdash;thereby activating GSK3β phosphorylation. This mechanism promotes β-catenin accumulation, nuclear translocation, and transcriptional activity, ultimately driving activation of the Wnt/β-catenin pathway.\u003c/p\u003e \u003cp\u003eTo investigate the potential role of the Wnt/β-catenin pathway in promoting cardiac reprogramming following RD and Exos administration, we evaluated the expression of β-catenin-relative signaling molecules in cardiac tissue. As shown in \u003cb\u003eFigure S7A\u003c/b\u003e and \u003cb\u003eS7B\u003c/b\u003e, hearts from pigs treated with RD or Exos (PBMSC-Exo\u003csup\u003eRD\u0026minus;Sham\u003c/sup\u003e or PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e) ) exhibited a significant upregulation of β-catenin and phosphorylated Gsk-3β S9 at serine 9 (p-GSK-3β S9) compared to the RD-Sham\u0026thinsp;+\u0026thinsp;PBS group. The combination treatments further amplified these expression levels relative to either treatment alone. Notably, both RD and Exos monotherapy significantly suppressed Dkk1 expression compared to PBS treatment in the RD-Sham group, with the RD\u0026thinsp;+\u0026thinsp;Exos combination demonstrating an additive inhibitory effect on Dkk1 (\u003cb\u003eFigure S7C\u003c/b\u003e). These findings are corroborated by immunohistochemical evidence, demonstrating that RD and Exos both individually and combinatorially upregulate β-catenin signaling through suppression of the pathway's negative regulator Dkk1.\u003c/p\u003e \u003cp\u003eFinally, to confirm that PBMSC-Exo\u003csup\u003eRD\u003c/sup\u003e-induced β-catenin activation and subsequent CMs reprogramming contribute to heart regeneration, we employed a human pluripotent stem cell antibody array to assess the relative expression of β-catenin transcriptional activation downstream targets\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e in the hearts of pigs treated with RD-Sham\u0026thinsp;+\u0026thinsp;PBS, RD\u0026thinsp;+\u0026thinsp;PBS, or RD\u0026thinsp;+\u0026thinsp;Exos. The expression of three crucial pluripotency factors (i.e., Oct4, Klf4, and Nanog), two β-catenin nuclear retention factors (i.e., YAP1 and TCF4),\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e and a cardiac lineage marker (i.e., Gata4) was elevated in the RD and RD-Sham\u0026thinsp;+\u0026thinsp;Exos group, with the highest expression observed in the RD\u0026thinsp;+\u0026thinsp;Exos group (\u003cb\u003eFigure S7D\u003c/b\u003e). In contrast, hearts of pigs receiving the combination treatment exhibited significantly reduced levels of β-catenin destruction regulators LEF1 and Axin2 (\u003cb\u003eFigure S7E\u003c/b\u003e).\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e No significant differences were observed in the expression of other Wnt/β-catenin-related transcript factors SMAD2, TCF7, Sox2, and c-Myc across these groups (\u003cb\u003eFigure S7E\u003c/b\u003e). These findings were corroborated by immunoblot assays, which revealed that hearts from pigs co-treated with RD and Exos displayed the highest expression of Oct4, Klf4, Nanog, and Gata4 but the lowest expression of LEF1 and Axin2 (\u003cb\u003eFigure S7F)\u003c/b\u003e, suggesting a reprogramming state in HF hearts following RD\u0026thinsp;+\u0026thinsp;Exos treatment. Immunohistochemical staining further validated these results, demonstrating increased density of the cardiac β-catenin-relative reprogramming transcription factors Oct4, Klf4, Nanog,YAP1, TCF4, and Gata4 (\u003cb\u003eFigure S7G\u003c/b\u003e).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur study demonstrates that RD combined with PBMSC-derived Exos promotes cardiac repair in a porcine model of MI by enhancing cardiomyocyte reprogramming. This process is mediated via miR-141-200-429 cluster-dependent suppression of Dkk1, leading to activation of the Wnt/β-catenin pathway. Key findings include: (1) PBMSC-derived Exos significantly improved cardiac functional recovery and reduced infarct size; (2) miR-141-200-429 clusters within Exos drove the reprogramming of mature cardiomyocytes into a more immature, regenerative state; and (3) this cellular reprogramming process was mechanistically linked to Dkk1 inhibition and subsequent β-catenin activation, as evidenced by genetic suppression experiments utilizing AAV-mediated delivery of miR-141-200-429 sponge constructs. These results highlight a novel therapeutic strategy targeting Wnt/β-catenin signaling to mitigate post-MI remodeling.\u003c/p\u003e \u003cp\u003eRD was initially developed as a treatment for resistant hypertension. Given the sympathoinhibitory effects of RD, Polhemus et al. demonstrated that RD represents a novel therapeutic strategy to reduce SNS activation and enhance left ventricular performance, with benefits extending beyond blood pressure lowering.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e Building on this, we explored the remote, direct cardioprotective effects of RD on CMs. Given the clinical importance of CM proliferation in MI treatment, we employed a pig MI model to evaluate the impact of RD on cardiac injury under HFrEF conditions. Our findings revealed that elevated SNS activity under MI conditions exacerbates myocardial ischemic stress, suppresses proliferation signaling, and activates apoptosis pathway. In contrast, RD-treated pigs exhibited a significant reduction in kidney SNS activity. Furthermore, RD administered after MI onset improved LVEF, facilitated recovery of left ventricular end-systolic and diastolic volumes, and inhibited adverse LV remodeling. However, this local attenuation of SNS activity was transient, lasting only up to 2 weeks post-MI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and did not sustain long-term, sustained improvements in cardiac function, remodeling, or survival rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results underscore the need to enhance the remote cardioprotective effects of RD to achieve sustained cardiac repair and improved outcomes following MI-induced HF.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn the current study, we identified five novel findings. First, we provided direct evidence that RD-derived PBMSC-Exos therapy promotes the beneficial effects of RD on LV performance, resulting in long-term, sustained improvement in the cardiac outcomes of our HFrEF pig model (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The clinical application of RD for hypertension treatment remains controversial. Furthermore, it is still unclear whether RD exerts lasting effects or can be effectively applied to other SNS-related conditions, such as HF, atrial fibrillation, and sleep apnea syndrome.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e In our HFrEF pig model, the hearts demonstrated distinct therapeutic effects at various timepoints and stages of the process. Specifically, we observed a significant reduction in SNS and RAAS activities in our RD pigs, with peak effects occurring 2 weeks post- MI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In addition to disrupting renal afferent signaling pathways, RD demonstrated significant attenuation of ventricular fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) and downregulation of myocardial pro-inflammatory mediators (IL-6 and NF-κB; \u003cb\u003eFigures S5A-B\u003c/b\u003e), which effectively mitigate macrophage-driven fibrotic cascades.\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e These findings suggest RD exerts regulatory effects on ventricular fibrosis progression in post-MI remodeling. In contrast, the release of PBMSC-Exos increased significantly in a time-dependent manner, peaking 2 weeks after MI in the RD group. However, the magnitude of these changes diminished during subsequent follow-up time. This observation may explain why the Symplectity HTN-3 trial results failed to demonstrate further improvement in clinical outcomes 6 months after RD.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e Delayed MSC-Exos administration resulted in sustained HF-preventive effects in RD pigs, persisting up to 30 days post-MI. Specifically, LV function and dimensions were preserved, infarct expansion was suppressed, and post-MI survival rates were significantly improved. These findings provide critical insights into potential therapeutic approaches for HFrEF (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSecond, we obtained the first evidence that PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e induce reprogramming of ischemic CMs. Several recent studies have indicated that RD may have beneficial effects on ventricular remodeling in post-MI HF.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e These results have confirmed robust attenuation of kidney SNS and RAAS activation, coupled with oxidative stress inhibition, G-protein-coupled receptor kinase 2 (GRK2) inhibition, and increased nitric oxide signaling, as a key beneficial effect of RD in HF. Nevertheless, therapeutic mechanisms through which RD improves outcomes have yet to be fully elucidated in preclinical models of HFrEF and remain the subject of ongoing research efforts.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e In the current study, we observed transient SNS and RAAS activity\u0026ndash;lowering effects in HFrEF pigs; however, this attenuation could not be sustained with no significant changes in plasma and kidney SNS and RAAS activity 15 days after MI (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-E and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-M). As such, these short-term effects cannot sufficiently explain long-term improvements in cardiac function after RD\u0026thinsp;+\u0026thinsp;Exos. We then focused on the other mechanisms through which Exos may promote cardiac repair by preserving LV function independent of the attenuation of kidney SNS and RAA activities. Thus far, Exos have been applied to enhance human induced pluripotent stem cell viability and differentiation into mature CMs.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e Exos-mediated intercellular communication plays a considerable role in myocardial repair and signaling transduction after MI.\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e Here, we, for the first time, investigated the effects of PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e in a large animal model of post-MI HFrEF. The increased proliferation of CMs in RD-treated hearts, Exos-treated hearts, and PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e treated-CM cultures was demonstrated through Ki67 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, and \u003cb\u003eFigure S4D\u003c/b\u003e) and pH3 (\u003cb\u003eFigure S4E\u003c/b\u003e) staining-established biomarkers for cell cycle activity. This observed proliferation indicates enhanced CMs division capacity, a crucial mechanism for myocardial regeneration.\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, early RD alone significantly increased the number of Ki67-positive CMs in both peri-infarct and remote zones compared to RD-Sham. This suggests that RD stimulates endogenous regenerative mechanisms, including CMs proliferation. Delayed Exos injection further enhanced this effect, leading to a greater number of Ki67-positive CMs in the RD\u0026thinsp;+\u0026thinsp;Exos group. This additive interaction suggests that delayed Exos therapy amplifies the regenerative benefits of early RD.\u003c/p\u003e \u003cp\u003eTerminally differentiated CMs cultured with PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e reentered the cell cycle and proliferated under hypoxic conditions, with CM proliferation occurring through redifferentiation under hypoxia (\u003cb\u003eFigure S4F\u003c/b\u003e). Consistent with our immunofluorescence staining results, gene detection analysis revealed that PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e upregulated the expression of factors related to proliferation (\u003cem\u003eCdk1\u003c/em\u003e and \u003cem\u003eYap1\u003c/em\u003e) and reprogramming (\u003cem\u003eOct4\u003c/em\u003e, \u003cem\u003eSox2\u003c/em\u003e, and \u003cem\u003eKlf4\u003c/em\u003e; \u003cb\u003eFigure S5\u003c/b\u003e). Similar findings were noted in our \u003cem\u003ein vivo\u003c/em\u003e animal study. In HFrEF pigs, RD enhanced mature CM proliferation and dedifferentiation, and subsequent delayed exosomal therapy further amplified these effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). These results aligned with our histological findings, which demonstrated a significant reduction in scar size and fibrosis, correlating with the greatest increase in the number of viable CMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). This is consistent with Gallet et al.\u0026rsquo;s observation that cardiosphere-derived cell Exos therapy reduce scarring, mitigate adverse remodeling, and improve LVEF in HFrEF pigs.\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e Furthermore, we observed a significant increase in the Exos uptake rate of hearts of HFrEF pigs treated with RD compared to those receiving RD-Sham, and delayed exosomal therapy further increased this uptake in HFrEF pigs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). These findings were corroborated by recent studies showing a robust increase in the Exos level in the blood of RD-treated mice.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Additionally, Exos uptake was positively correlative with cardiac dedifferentiation in the HFrEF pigs receiving the combination of RD and Exos therapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). Collectively, these results advance our understanding of the mechanisms underlying the inhibition of LV remodeling in HFrEF pigs following effective RD. They also facilitate the identification of novel therapeutic targets aimed at reducing MI mortality by maintaining circulating Exos levels and their cardioprotective effect.\u003c/p\u003e \u003cp\u003eThird, we identified β-catenin as the most important molecule in RD-mediated myocardial reprogramming. Exos play a role in the cardiorenal syndrome. Cardiorenal Exos regulate proangiogenic paracrine signaling in adipose MSCs after MI.\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e Endothelial cells can transfer caveolin-1-containing Exos to adipocytes in newly generated mouse models.\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e Here, we demonstrated, for the first time, that RAECs released functional circulating PBMSC-Exos and became transferred to injury CMs after RD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Moreover, RD significantly upregulates the myocardial expression of β-catenin (\u003cb\u003eFigure S5\u003c/b\u003e)\u0026mdash;a major signal transduction molecule in MSC-Exos widely involved in regeneration or repair-related biological processes.\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e This finding aligns with our observation that RD-treated cardiomyocytes exhibited a marked upregulation of reprogramming factors Oct4, Sox2, and Klf4 (\u003cb\u003eFigure S5\u003c/b\u003e), a phenomenon associated with β-catenin activation.\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e Notably, β-catenin overexpression led to a continuous increase in CMs uptake of PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e and proliferation, redifferentiation, and apoptosis prevention in hypoxia-cultured CMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In cancers, Exos are exchanged between cancer cells and the tumor stroma, promoting the transfer of various oncogenes (e.g., \u003cem\u003eβ-catenin\u003c/em\u003e, \u003cem\u003eCeacam1\u003c/em\u003e, \u003cem\u003eHer2\u003c/em\u003e, \u003cem\u003eMelan-A\u003c/em\u003e/\u003cem\u003eMart-1\u003c/em\u003e, and \u003cem\u003eLmp1\u003c/em\u003e) from one cell to another, leading to recipient cell reprogramming.\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e Taken together, these findings further highlighted the importance of β-catenin upregulation by circulating PBMSC-Exos for RD-mediated myocardial reprogramming.\u003c/p\u003e \u003cp\u003eFourth, we identified miR-141-200-429 cluster as the most essential molecule in myocardial reprogramming induced by PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e. Emerging evidence indicates that Exos contain specific miRNAs contributing to tissue repair and immunomodulation.\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e Rodent studies have demonstrated that the potential therapeutic effects of RD on LV fibrosis were partly mediated by miRNA regulation.\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e As such, we performed high-throughput screening and found that PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e contained higher levels of miR-141-200-429 clusters than PBMSC-Exos\u003csup\u003eRD-Sham\u003c/sup\u003e did (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). miR-141-200-429 clusters are generated by RD-treated RAECs and carried by PBMSCs from RAECs to CMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In particular, although all 5 genes in miR-141-200-429 clusters were significantly upregulated in RAECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), only miR-200a-3p, miR-200b-3p, and miR-141 were significantly upregulated in CMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Furthermore, RAEC-specific miR-141-200-429 cluster sponge administration to RD-Sham and RD pigs significantly reduced CMs Exos uptake in MI hearts (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) and prevented miR-200a-3p, miR-200b-3p, and miR-141 elevation in RD-treated CMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Notably, miR-141-200-429 sponges significantly reduced expression of the β-catenin-related genes and pathways related to cell proliferation, apoptosis, and dedifferentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ). Exos-encapsulated miRNAs can increase CMs proliferation.\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e In particular, a few human miRNAs can stimulate the entry of rodent CMs into the cell cycle and cardiac regeneration after MI in mice.\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e Taken together, these findings provide novel insights into the positive feedback mechanisms of RD: RD promotes the release and uptake of Exos rich in miRNAs. miRNAs then enhance the remote cardioprotective effects of RD, improving CM proliferation, antiapoptosis, and regeneration, as well as systolic LV function through activation of β-catenin signaling under HF conditions (\u003cb\u003ethe Graphical Abstract Image\u003c/b\u003e). However, further validation through additional studies, including clinical studies, is essential to confirm these findings and explore the potential therapeutic applications.\u003c/p\u003e \u003cp\u003eFinally, by utilizing bioinformatic analysis followed by multiple experimental validation, we identified \u003cem\u003eDkk1\u003c/em\u003e as a target gene of the novel miR-141-200-429 cluster. Dkk1 stands for Dickkopf-related protein 1, which is a secreted protein that acts as an inhibitor of the Wnt/β-catenin signaling pathway.\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e The Wnt/β-catenin pathway is crucial for various cellular processes, including cell proliferation, differentiation, and survival. Various miRNAs target \u003cem\u003eDkk1\u003c/em\u003e, which is silenced by miRNAs in the early stages of osteogenic differentiation. In contrast, miRNA levels decrease and Dkk1 expression is upregulated in the later stages of differentiation.\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e We demonstrated, for the first time, that Dkk1 is a direct target of miR-141-200-429 clusters, and its inhibition after RD is pivotal for CMs proliferation and re-differentiation. Since miR-141-200-429 sponges inhibit the β-catenin pathway and hinder the RD-induced reprogramming pathways (\u003cb\u003e7E\u003c/b\u003e and \u003cb\u003e7F\u003c/b\u003e), it is essential to investigate whether Dkk1, as an inhibitor of this pathway, plays a regulatory role in these processes. By analyzing Dkk1, we have gained insights into multiple mechanisms through which it modulates the effects of miR-141-200-429 sponges on the β-catenin pathway and the subsequent reprogramming of CMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ), including (1) preventing CM proliferation and expression of its markers Cdk1 and Mcl-1; (2) preventing CM dedifferentiation and expression of its markers Acta1 and Nppa; (3) increasing CM apoptosis and expressions of the apoptotic proteins caspase3 and p53 along with decreased expression of antiapoptotic proteins survivin and Bcl2; (4) reducing pluripotency transcription factors Oct4 and c-Myc functionally associated with impaired β-catenin signaling activity.\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e Moreover, Dkk1 overexpression was noted to significantly attenuate PBMSC-Exo\u003csup\u003eRD\u003c/sup\u003e-induced increase in phosphorylated GSK-3β, nuclear translocation of β-catenin, and expression of cyclin D1 and Tcf4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eM-R), which are miR-200a targets.\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e Pretreatment with Dkk1 inhibits Wnt1-stimulated differentiation of human periodontal ligament fibroblasts by suppressing GSK-3β phosphorylation and nuclear translocation of β-catenin,\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e leading to upregulation of the Wnt/β-catenin target genes \u003cem\u003ecyclin D1\u003c/em\u003e, \u003cem\u003eTcf4\u003c/em\u003e, and \u003cem\u003eLef1\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e This could help elucidate the molecular mechanisms underlying the observed phenomena and potentially identify Dkk1 as a key player in the regulation of cardiomyocyte reprogramming. In the present study, the expression of RAEC miR-141-200-429 clusters was significantly increased in PBMSC-Exos. miR-141-200-429 clusters are packaged in Exos and transferred from PBMSCs to CMs, where they suppress the expression of Dkk1, a critical β-catenin suppressor, causing β-catenin activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ). Increasing PBMSC-Exos production, enhancing CMs biogenesis, or activating β-catenin target genes (particularly Tcf4, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eM and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eP) may be novel, effective therapeutic avenues to improve PBMSC-Exos-mediated beneficient communication between RAs and MI hearts after RD, enhancing cardiac regeneration in acute MI (\u003cb\u003eFigure S8\u003c/b\u003e). Taken together, these results suggested that PBMSC-Exos participate in the protective effects of RD against ischemic injury.\u003c/p\u003e \u003cp\u003eExisting studies demonstrate that the post-MI inflammatory response progressively transitions into the reparative phase, followed by the remodeling phase.\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e Therefore, the treatment for MI must address both acute-phase anti-inflammatory interventions and chronic-phase management of myocardial fibrosis. This study demonstrates that the sequential therapeutic approach integrating acute-phase RD intervention with later-phase Exos administration achieves comprehensive functional recovery in infarcted hearts throughout the post-MI repair process. This translation aligns with current guidelines emphasizing stage-specific treatment strategies in MI.\u003c/p\u003e \u003cp\u003eWe also found that delayed Exos injection significantly reduced plasma aldosterone levels \u003cb\u003e(Figure S3)\u003c/b\u003e. It also increased circulating BNP levels sustainably with RD (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). RD-induced increases in circulating BNP lead to improvements in cardiac function and protection against ischemia-reperfusion injury,\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e suggesting that long-term cardioprotective effects may arise from the additive actions of RAAS inhibition and enhanced cardiac function.\u003c/p\u003e\n\u003ch3\u003eStudy Limitations\u003c/h3\u003e\n\u003cp\u003eWhile our findings demonstrate the potential of PBMSC-Exos miRNAs to enhance cardiac repair of RD after MI, this study has several limitations. First, the porcine model of MI-induced HFrEF was established solely through the ligation of the left anterior descending coronary artery. However, clinical MI patients with HFrEF frequently present with multivessel disease and undergo revascularization therapies, whereas our model does not account for these complexities. Consequently, this experimental system fails to fully replicate the multifactorial pathophysiology of human HFrEF. Second, early cardiac biopsy specimens collected at 14 days post-MI from RD-treated and RD-Sham hearts demonstrated that PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e significantly upregulated pluripotency transcription factors (Oct4, Klf4, Sox2) in RDCMs (\u003cb\u003eFigure S5\u003c/b\u003e). However, longitudinal analysis revealed no detectable increase in Sox2 or c-Myc expression in ischemic cardiac tissues from either group, even at the 90-day post-MI endpoint (\u003cb\u003eFigure S7\u003c/b\u003e). This temporal discrepancy\u0026mdash;where transient transcriptional activation occurs acutely but dissipates chronically\u0026mdash;may reflect dynamic shifts in epigenetic regulation, microenvironmental crosstalk (e.g., inflammatory or fibrotic signaling), or unresolved compensatory feedback loops. While these findings underscore the context-dependent nature of cellular reprogramming, their clinical extrapolation necessitates rigorous consideration of spatiotemporal biological constraints and interspecies translational gaps. Third, while \u003cem\u003ein vitro\u003c/em\u003e analyses identified β-catenin as a pivotal regulator of hypoxia-induced endogenous reprogramming in mature mammalian CMs, we were unable to corroborate this mechanism through functional gain- or loss-of-function experiments (e.g., β-catenin overexpression or knockdown) \u003cem\u003ein vivo\u003c/em\u003e. The absence of animal-level validation substantially undermines the translational validity of our proposed molecular pathway. Fourth, while \u003cem\u003ein vitro\u003c/em\u003e sponge-mediated miR-141-200-429 knockdown blocked RA-Exos' cardioprotection under hypoxia, validation in an MI model via RA-Exos-specific miR-141-200-429 overexpression is lacking. Future studies employing targeted activation of miR-141-200-429 in RA-Exos, followed by functional assessments in MI models, would clarify its specific role and enhance the translational relevance of our findings.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study provides the first evidence that RD confers protection against myocardial ischemia-reperfusion injury in a porcine model of HFrEF post-MI, mediated through increased delivery of PBMSC-Exos enriched with miR-141-200-429 clusters. These miRNAs orchestrate multiphasic cardioprotection by attenuating neurohormonal activation (reduced circulating catecholamines and suppressed renin-angiotensin-aldosterone system [RAAS] activity), inhibiting apoptosis pathways (downregulation of Bax/Caspase-3), enhancing myocardial stress adaptation (elevated BNP expression), and activating β-catenin-mediated proliferative signaling.\u003c/p\u003e \u003cp\u003eNotably, the RD\u0026thinsp;+\u0026thinsp;Exos combinatorial strategy enabled sustained miRNA persistence (Up to 90 days) in ischemic myocardium, overcoming the transient bioavailability limitations of conventional miRNA therapies. While RD's therapeutic potential was initially attributed to blood pressure modulation, our findings reveal broader mechanistic implications, including epigenetic reprogramming and paracrine crosstalk. This positions RD not merely as a hemodynamic intervention but as a platform for targeted organ protection, warranting exploration in other chronic conditions characterized by oxidative stress and maladaptive remodeling (e.g., diabetic cardiomyopathy, chronic kidney disease). Furthermore, we propose a translational framework integrating RD with precision medicine approaches: (1) Temporal synergy: Co-administration with guideline-directed antihypertensive therapies to optimize hemodynamic and cellular repair phases; (2) Spatial control: Nanoparticle engineering of Exos for tissue-specific miRNA delivery; (3) Safety escalation: Biomarker-guided titration to minimize off-target epigenetic effects. These preclinical insights bridge the gap between observational cardioprotection and mechanism-driven therapeutic innovation, offering a roadmap for clinical translation in HFrEF management.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies and other reagents\u003c/h2\u003e \u003cp\u003eThe sequences of the primers used in this study are provided in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e, and the antibody details are listed in \u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e. 4\u0026prime;,6-Diamidino-2- phenylindole (DAPI; catalog 28718-90-3) was purchased from Sigma-Aldrich.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and Study Design\u003c/h2\u003e \u003cp\u003eRat-To investigate the appropriate dose of PBMSC-Exos used for injection, a preliminary dose ranging experiment was performed on adult male Sprague-Dawley rats (200\u0026ndash;250). Ligation of the left anterior descending coronary artery was ligated as our previously described,\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e which was subsequently removed after 45 minutes to allow for reperfusion. Twenty minutes later, the rats were injected with 100\u0026micro;l PBS or PBMSC-Exos from MI pigs (5\u0026times;10\u003csup\u003e12\u003c/sup\u003e, 2\u0026times;10\u003csup\u003e13\u003c/sup\u003e, or 5\u0026times;10\u003csup\u003e13\u003c/sup\u003e in 100\u0026micro;L PBS) via caudal vein, over a period of 20 seconds. Thirty days later, all the rats underwent echocardiography, and then were euthanized for serological and molecular biology testing.\u003c/p\u003e \u003cp\u003ePig-Domestic pigs were housed indoors and provided with a commercial diet and fresh water ad libitum to maintain body weight and support growth.\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e All domestic pigs (weight, 40\u0026ndash;50 kg) were sedated with ketamine (15\u0026ndash;20 mg/kg) and diazepam (1.5\u0026ndash;2 mg/kg), followed by anesthesia maintenance with intravenous thiopental (1\u0026ndash;2 mg/kg/min). Preanesthetic atropine (30\u0026ndash;50 \u0026micro;g/kg, intramuscular) was also administered. MI was induced by ligating the left anterior descending coronary artery midway between its origin and the apex, as previously described.\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e After 45 minutes of ischemia, reperfusion was initiated and maintained until the endpoint. Pigs with LVEF\u0026thinsp;\u0026lt;\u0026thinsp;40% within 2 hours post-MI were selected for a 90-day follow-up (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eA total of 95 pigs underwent surgery, with 82 surviving the procedure. Among the survivors, 22 MI pigs were excluded due to ventricular fibrillation or LVEF\u0026thinsp;\u0026ge;\u0026thinsp;40%. The remaining 60 pigs with LVEF\u0026thinsp;\u0026lt;\u0026thinsp;40% within 2 hours post-MI were selected as our HFrEF model and randomly assigned to either RD-Sham or radiofrequency RD treatment. Twenty pigs from each group were further divided into four subgroups (n\u0026thinsp;=\u0026thinsp;10 per group) based on treatment: RD-Sham\u0026thinsp;+\u0026thinsp;PBS (phosphate buffered saline), RD-Sham\u0026thinsp;+\u0026thinsp;Exos, RD\u0026thinsp;+\u0026thinsp;PBS, and RD\u0026thinsp;+\u0026thinsp;Exos (n\u0026thinsp;=\u0026thinsp;10 per group). At 30 days post-MI, surviving animals received either 2 \u0026times; 10\u0026sup1;\u0026sup3; PBMSC-derived exosomes (Exos) or an equivalent volume of PBS and were monitored for 90 days (\u003cb\u003eFigure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). No deaths occurred during RD or Exos administration. For endpoint analysis, pigs were euthanized with phenobarbital sodium at 90 days post-MI. An additional 10 pigs from the RD/RD-Sham groups underwent myocardial biopsy and Exos collection 14 days post-MI, followed by injection of BODIPY TR ceramide-prelabeled RAEC-derived Exos. These pigs were euthanized with intravenous pentobarbital sodium at 44 days post-MI (observation endpoint; \u003cb\u003eFigure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eAs the negative control of MI surgery, sham-operated controls (n\u0026thinsp;=\u0026thinsp;5) underwent identical surgical procedures (thoracotomy, pericardial exposure) without coronary artery ligation. These pigs received RD treatment and were followed for 30 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMonitoring\u003c/h2\u003e \u003cp\u003eIn all experiments, heart rate was monitored through electrocardiography (ECG), and mean aortic blood pressure was measured at baseline and 0, 30, 60, and 90 days after MI.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEchocardiography\u003c/h2\u003e \u003cp\u003eTwo-dimensional echocardiography was performed at midpapillary and apical levels. Left ventricular end-diastolic volume (LVEDV) and end-systolic volume (LVESV) were measured using the biplane area-length method, with ejection fraction (LVEF) calculated by the modified Simpson's method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRadiofrequency RD\u003c/h2\u003e \u003cp\u003ePigs were randomly grouped into an RD or RD-Sham group within 2 h after MI. Radiofrequency RD was performed, as described previously.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e For RD, pigs received bilateral denervation of the aorta and RA with a Celsius electrophysiology catheter (Stockert 70; Biosense Webster, Diamond Bar, CA, USA). Ground pads were placed to protect the underlying tissue between the shoulder blades. The tip of the radiofrequency probe was placed in four quadrants (circles) of RAs, with 10 W for each quadrant in the RD group and 0 W for the RD-Sham group, lasting 20 s.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCardiac Biopsy\u003c/h2\u003e \u003cp\u003eTransmural biopsy specimens (10 mm wide) were collected from RD-treated pigs at 14 days post-MI. From each heart, five samples were obtained from distinct peri-infarct regions and analyzed for ischemic CM proliferation. These myocardial specimens were processed through an integrated experimental platform employing both in vitro and in vivo approaches to systematically evaluate the therapeutic effects and mechanistic basis of PBMSC-Exo\u003csup\u003eRD\u003c/sup\u003e-induced cardiac reprogramming.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePlasma Biochemistry Assessment\u003c/h2\u003e \u003cp\u003ePeripheral blood was collected at baseline and at 0, 30, 60, and 90 days post-MI. Circulating levels of BNP, hs-cTnT, BUN, creatinine, norepinephrine, and epinephrine were quantified using commercial ELISA kits (Abnova) per manufacturer's protocol. Plasma renin and aldosterone levels, along with aldosterone-renin ratios, were assessed at 90 days post-MI using established methods.\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eKidney Catecholamine and Angiotensin Measurements\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTo evaluate RD efficacy, kidney catecholamine and angiotensin levels were quantified at 90 days post-MI. Renal cortex tissues were rapidly harvested, flash-frozen in liquid nitrogen, and homogenized. Catecholamine concentrations were determined by high-performance liquid chromatography (HPLC) and expressed as nanograms per gram of wet tissue weight. Angiotensin levels were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) at Attoquant Diagnostics (Vienna, Austria), as previously described.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003ePBMSCs and RAEC Isolation and Collection\u003c/h2\u003e \u003cp\u003ePeripheral blood was collected from RD- and RD-Sham-treated pigs at 14 days post-MI. PBMSCs were isolated using established methods,\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e with second-passage cells utilized for Exos collection and subsequent experiments.\u003c/p\u003e \u003cp\u003eRAs and myocardial tissues were collected from pigs at 14 days post-MI. RAECs were isolated from RAs and cultured in endothelial cell growth medium (ScienCell) containing 5% heat-inactivated FBS, 1% penicillin-streptomycin, and 1% endothelial cell growth supplement (all from ScienCell), maintained at 37\u0026deg;C with 5% CO₂in a humidified incubator.\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e Only RAECs with \u0026gt;\u0026thinsp;96% purity (confirmed by flow cytometry/immunostaining) were used for Exos production.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eExos Isolation, Characterization, Transfection, Labeling, and Tracing\u003c/h2\u003e \u003cp\u003eCells were expanded in serum-free conditions (MesenCult-ACF-XFAttachment Substrate; STEMCELL Technologies) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eTo ensure Exos derived exclusively from PBMSCs and exclude contamination by other extracellular vesicles or protein aggregates, we performed sequential ultracentrifugation and density gradient purification. After 96 h of culture, the conditioned medium was harvested and subjected to: (1) Low-speed centrifugation (800\u0026times;g, 5 min) to remove cells and debris; (2) Intermediate centrifugation (2,000\u0026times;g, 10 min) to eliminate apoptotic bodies and large microvesicles; (3) Filtration through a 0.22-\u0026micro;m pore-size membrane to exclude particles\u0026thinsp;\u0026gt;\u0026thinsp;200 nm; (4) Ultracentrifugation (100,000\u0026times;g, 2 h, 4\u0026deg;C) to pellet Exos. The final Exos pellet was resuspended in sterile PBS and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for downstream applications.\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e Next, Exos morphology was verified by TEM. Briefly, purified Exos were diluted in PBS, negatively stained with 2% uranyl acetate, and adsorbed onto carbon-coated copper grids. Vesicles exhibiting characteristic cup-shaped morphology were visualized at varying magnifications using a Hitachi H-7500 TEM. To further validate Exos quality, we performed: (1) NTA to determine particle size distribution and concentration; (2) Western blotting to confirm the presence of exosomal markers (CD9, CD63, TSG101, and HSP70).\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eFor Exos labeling, PBMSC-derived Exos from RD-treated pigs were incubated with 4 \u0026micro;M PKH26 fluorescent dye (Sigma-Aldrich) in Diluent C, following the manufacturer's protocol.\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e The labeled Exos were subsequently administered via peripheral intravenous injection to MI pigs at 30 days post-MI.\u003c/p\u003e \u003cp\u003eTo assess Exos uptake, Exos-treated CMs were fixed and immunostained with primary antibodies against myosin heavy chain (MHC), followed by Alexa Fluor 488-conjugated secondary antibodies (1:200 dilution; A32723, Thermo Fisher Scientific). Nuclei were counterstained with DAPI (4\u0026prime;,6-diamidino-2-phenylindole). Fluorescent images were acquired using an FV1000 confocal microscope (Olympus) and analyzed with FV10-ASW software (Olympus). CMs exhibiting colocalization of MHC and the Exos tracker (PKH26) were identified as Exos-positive cells.\u003c/p\u003e \u003cp\u003eTo assess the role of miR-141-200-429 clusters in RD outcomes, RAECs isolated from RD-treated pigs were expanded as previously described. We constructed miR-141-200-429 sponge vectors (and corresponding negative control [NC] sponges; Syngentech, Shanghai) under the control of the endoglin promoter. At 60\u0026ndash;80% confluence, RAECs were transfected with 50 nM sponge constructs using Lipofectamine 3000 (Invitrogen) per manufacturer's protocol. After 48 h, conditioned medium was collected, and Exos were isolated by ultracentrifugation for downstream analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eExos miRNA Analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from PBMSC-derived Exos using TRIzol reagent (Invitrogen). Small RNAs (18\u0026ndash;50 nt) were size-selected for miRNA sequencing. miRNA libraries were prepared with the QIAseq miRNA Library Kit (Qiagen) and sequenced on an Illumina NovaSeq 6000 platform. Bioinformatic analysis included: (1) Raw read processing: Demultiplexing, adapter trimming, and quality filtering; (2) Alignment to miRBase v21 using bowtie2; (3) Quantification with HTSeq (v0.6.1); (4) Differential expression analysis and hierarchical clustering (pheatmap R package);\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e (5) Functional annotation including GO and KEGG pathway analysis (clusterProfiler), Visualization of KEGG pathways (Bioinformatics Network platform), and Venn diagram analysis (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e Venn).\u003csup\u003e73\u003c/sup\u003e Core miRNAs regulating cardiac proliferation/regeneration were identified through integrative analysis of these datasets.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003ePrimary CMs Isolation and Culture\u003c/h2\u003e \u003cp\u003eTo assess cardiomyocyte CM proliferation and differentiation potential, we isolated primary CMs from endomyocardial biopsies. Tissue samples were enzymatically digested in Tyrode buffer containing 1.5 mg/mL collagenase type II and 0.1 mg/mL hyaluronidase for 50 min.\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eFollowing digestion and filtration, cells were sequentially equilibrated inCalcium-free Tyrode solution (5 min) and Gradual calcium reintroduction (30 min). Isolated CMs were plated at 1.5\u0026times;10⁵cells/cm\u0026sup2; in complete culture medium (DMEM supplemented with 10% FBS (Biosera), 4,500 mg/L glucose (Sigma-Aldrich), 4 mM L-glutamine (Biosera), 100 IU/mL penicillin (Biosera), and 100 \u0026micro;g/mL streptomycin (Biosera))\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eLoss-and Gain-of-Function Experiments\u003c/h2\u003e \u003cp\u003eFor genetic manipulation of β-catenin expression, we used pMXs retroviral vectors carrying β-catenin cDNA in pReceiver-LV233 lentiviral backbone (GeneCopoeia) and pSiLVRU6GP vectors expressing Ctnnb1-targeting shRNAs with puromycin resistance (GeneCopoeia) for overexpression and knockdown, respectively. Transfection was performed in PBMSCs using FuGENE HD transfection reagent according to manufacturer's protocol. This generated β-catenin-overexpressing cells (\u003cem\u003eoe\u003c/em\u003eβ-catenin), β-catenin-deficient cells (\u003cem\u003esi\u003c/em\u003eβ-catenin) and Vector-transfected controls\u003c/p\u003e \u003cp\u003eTo identify the potential target of PBMSC-Exo\u003csup\u003eRD\u003c/sup\u003e -derived miR-141-200-429 cluster, RDCMs were transfected with miR-141-200-429 mimics (to overexpress the miRNA, synthesized by Syngentech, Shanghai, China) or sponges (to suppress endogenous miRNA, synthesized by Syngentech, Shanghai, China). Scrambled miRNA or NC sponges were used as negative controls.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eHypoxic Experiments\u003c/h2\u003e \u003cp\u003eWe next investigated the effects of PBMSC-Exos, β-catenin modulation, and Dkk1 on CM behavior under hypoxia. RDCMs were subjected to the following interventions: transfection with \u003cem\u003eoe\u003c/em\u003eβ-catenin, \u003cem\u003esh\u003c/em\u003eβ-catenin, or empty vectors; treatment with the β-catenin agonist WAY-262611 (1 \u0026micro;mol/L; ab145229; Abcam, USA); administration of PBMSCs-Exos (2 \u0026times; 10\u003csup\u003e13\u003c/sup\u003e) isolated from RD or RD-Sham pigs; or supplementation with recombinant Dkk1 (100 ng/mL; R\u0026amp;D Systems #5497-A6-050).\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e CMs were cultured at 1.5\u0026times;10⁵cells/cm\u0026sup2;in complete medium under hypoxic conditions (1% O₂) for 7 days. CM proliferation and cardiomyogenesis were assessed on days 3 and 7 of culture.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eImmunofluorescence Staining\u003c/h2\u003e \u003cp\u003eCell samples and myocardial tissues were fixed with 4% paraformaldehyde for 10 minutes. Myocardial tissues were embedded in optimum cutting temperature (OCT) compound and sectioned into 6-\u0026micro;m-thick slices using a cryostat (Leica CM1520). For immunofluorescence staining, sections were sequentially processed as follows: overnight incubation with primary antibodies at 4\u0026deg;C, 1-hour incubation with fluorescent secondary antibodies at room temperature, and nuclear counterstaining with DAPI (2 minutes). Finally, sections were mounted with antifade medium and imaged. Five independent fields per section were analyzed across representative tissue regions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eCell Proliferation Assay\u003c/h2\u003e \u003cp\u003eFollowing 24-hour transfection, CMs were co-cultured with Exos under hypoxic conditions for 48 hours. Cell metabolic activity was quantified using a Cell Counting Kit-8 (CCK-8; Sigma) according to the manufacturer\u0026rsquo;s protocol. Proliferation was assessed through two parallel methods: (1) Immunofluorescence analysis of cell cycle markers (Ki67, BrdU, and phospho-histone H3 [pH3]), with proliferation rates calculated as the percentage of Ki67-positive nuclei relative to total DAPI-stained nuclei in matched fields; (2) EdU incorporation assay performed via Click-iT\u0026trade; EdU Alexa Fluor\u0026reg; 488 Imaging Kit (Thermo Fisher, #C10337), utilizing the standardized Click-iT reaction protocol. \u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eFlow Cytometry Detection of Cell Apoptosis\u003c/h2\u003e \u003cp\u003eTo assess CM apoptosis, we conducted Annexin V-APC/PI dual staining using a commercial apoptosis detection kit (FACS Annexin V Assay Kit; Trevigen, USA) according to the manufacturer's protocol. Briefly, CMs were harvested after 7-day hypoxic co-culture with either 2\u0026times;10\u0026sup1;\u0026sup3; empty AAV6 particles or PBMSC-Exos. Cells were resuspended in 400 \u0026micro;L 1\u0026times; binding buffer (adjusted to 1 \u0026times; 10⁵ cells/mL), then incubated with 5 \u0026micro;L Annexin V-FITC and 10 \u0026micro;L PI in 100 \u0026micro;L binding buffer for 30 minutes at room temperature (protected from light). Apoptotic rates were quantified using a FACScan flow cytometer (Becton Dickinson, USA), with Annexin V⁺/PI⁻ (early apoptosis) and Annexin V⁺/PI⁺ (late apoptosis) populations combined for total apoptosis analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003ePluripotent Stem Cell Antibody Array\u003c/h2\u003e \u003cp\u003eNext, we defined pluripotent stem cell marker levels from RDCMs in the absence or presence of RD or Exos. In brief, we used a human pluripotent stem cell antibody array (ARY010; MN 55413; USA) to measure the relative levels of pluripotent stem cell markers in hearts from HF pigs receiving early RD-sham or RD plus delayed injection of PBS or Exos. Fluorescence signals were detected using Axon GenePix, and the relative levels of the factors were calculated and analyzed.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eqRT-PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated from cultured cells and myocardial tissue samples using RNA-Stat reagent (Iso-Tex Diagnostics, USA), followed by cDNA synthesis with 500 ng RNA template using the TaqMan Reverse Transcription Kit (Applied Biosystems, USA). Target-specific primers and probes (\u003cb\u003elisted in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) were designed with Primer Express\u0026reg; software (v3.0, Applied Biosystems). Quantitative RT-PCR was performed on an ABI Prism 7500 system (Applied Biosystems) under standardized cycling conditions. Fluorescence signals from 6-carboxyfluorescein (6-FAM)-labeled probes were monitored in real time, and cycle threshold (Ct) values were determined using ABI Prism SDS software (v2.0, Applied Biosystems). Relative mRNA expression levels were normalized to \u003cem\u003eGAPDH\u003c/em\u003e and calculated \u003cem\u003evia\u003c/em\u003e the 2\u003csup\u003e\u0026minus;ΔΔ\u003c/sup\u003e CT method.\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blotting\u003c/h2\u003e \u003cp\u003eWestern blot analysis was performed to evaluate protein expression across experimental groups. Tissue/cell lysates were prepared using Pierce\u0026trade; RIPA buffer (Thermo Scientific\u0026trade;, #78510) supplemented with protease inhibitors. Protein extracts were denatured in Laemmli buffer (Beyotime, #P0015) by boiling at 95\u0026deg;C for 5 min, followed by ice-cooling and centrifugation (12,000\u0026times;g, 10 min). Proteins (30 \u0026micro;g/lane) were resolved on 10% SDS-PAGE gels and transferred to PVDF membranes (Millipore) using semi-dry electrophoretic transfer.\u003c/p\u003e \u003cp\u003eMembranes were blocked with 5% non-fat milk in TBST for 1 hr at RT, then incubated overnight at 4\u0026deg;C with primary antibodies (see \u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e) and anti-GAPDH (1:1000; Abcam #ab9485). After three 10-min TBST washes, membranes were probed with HRP-conjugated goat anti-mouse IgG secondary antibody (1:2000; ComWin Biotech #CW0102) for 1 hr at RT. Signal detection was achieved using Clarity\u0026trade; Western ECL Substrate (Bio-Rad #170\u0026ndash;5060) and visualized on a ChemiDoc\u0026trade; MP Imaging System (Bio-Rad)\u003c/p\u003e \u003cp\u003e \u003cb\u003emiRNA Inhibition\u003c/b\u003e \u003cb\u003eIn Vivo\u003c/b\u003e \u003cb\u003eby AAV-Carrying miRNA Sponges\u003c/b\u003e\u003c/p\u003e \u003cp\u003emiR-141-200-429 sponge inhibitors and NC sponges were purchased from Genechem Co., Ltd. (Shanghai, China). Adeno-associated virus (AAV) vectors were constructed using blood vascular cell\u0026ndash;specific promoter elements from the chicken endoglin promoter. The miR-141-200-429 sponges were designed with bulged or imperfect complementarity to target miRNA sequences, including conserved seed regions, as previously described.\u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e Each sponge sequence was inserted triplicate. BODIPY TR ceramide (Cat# B34400, Thermo Fisher Scientific, Carlsbad, CA, USA) was utilized to label sponge sequences and validate promoter activity. Recombinant AAV particles (serotype GV618) were generated using the edited AAV vector. Two weeks post-retinal detachment (RD) or RD-Sham surgery, pigs were anesthetized with 2% isoflurane in oxygen, underwent myocardial biopsy and Exos collection, and followed by the administration of 10 \u0026micro;L of viral solution containing approximately 3 \u0026times; 10\u003csup\u003e11\u003c/sup\u003e vector genomes (vg) per animal via the jugular vein.\u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e At 30 days post-AAV administration, frozen cardiac tissues were cryosectioned into 7-\u0026micro;m-thick slices. Sponge expression efficacy was assessed by detecting BODIPY TR ceramide-labeled Exos in cardiac tissues. Fluorescent images were captured using an Olympus BX51 microscope equipped with a DP74 camera (Olympus Corporation, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eWheat Germ Agglutinin (WGA) Staining\u003c/h2\u003e \u003cp\u003eTo measure CM cross sectional area, WGA (TheromFisher) staining was performed as previously described.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Cross sectional area was calculated by summing the areas of the high-resolution images at 20X magnification, which were quantified by blinded researchers.\u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eHistology and Immunohistochemistry\u003c/h2\u003e \u003cp\u003eImmunohistochemical staining for tyrosine hydroxylase (TH; HSRL, MA, USA) was performed on RAs from pigs at 1 week, 2 weeks, 4 weeks, and 90 days post-MI, following previously described methods.\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e Briefly, RA segments were harvested from the mid-main and distal branch regions of the RA, fixed in formalin for at least 7 days, and subsequently washed. The tissues were then dehydrated in 70% ethanol, embedded in paraffin, and sectioned into 5-\u0026micro;m-thick slices. Finally, the sections were processed for TH immunohistochemical staining.\u003c/p\u003e \u003cp\u003eHearts from pigs were excised, weighed, and cross-sectioned at the maximal LV area. The sections were immersed in 0.09 M phosphate-buffered saline (PBS, pH 7.4) containing 1.0% triphenyl tetrazolium chloride (TTC; Sigma-Aldrich, St. Louis, MO, USA) at 37\u0026deg;C for 20 minutes to delineate the infarcted area. Additionally, Masson\u0026rsquo;s trichrome staining was performed on these sections according to the manufacturer\u0026rsquo;s protocol. Infarct size was quantified by calculating the sum of the infarcted and collagen areas as a percentage of the total LV area using ImageJ software (v1.53, National Institutes of Health, USA).\u003c/p\u003e \u003cp\u003ePeri-infarct regions dissected from necropsy specimens were either paraffin-embedded or snap-frozen for cryosectioning. Sections were stained with hematoxylin and eosin (H\u0026amp;E) or processed for immunofluorescence and immunohistochemical analyses.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe surgical team and investigators were blinded to treatment assignments and outcome data until completion of the study analysis. All experimental results were systematically tabulated, and final reports were generated following unblinding.\u003c/p\u003e \u003cp\u003eQuantitative data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD), while categorical variables are expressed as frequencies and proportions. Normality was assessed using the Shapiro-Wilk test, and homogeneity of variance was verified via Levene's test. Continuous variables meeting both normality and homoscedasticity assumptions were analyzed using one-way ANOVA. For normally distributed data with heteroscedasticity, Welch\u0026rsquo;s ANOVA was applied. Categorical variables were compared using the chi-square test or Fisher\u0026rsquo;s exact test, as appropriate. A threshold of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was defined for statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u0026alpha;-SMA=alpha-smooth muscle actin\u003c/p\u003e\n\u003cp\u003eBNP = B-type natriuretic peptide\u003c/p\u003e\n\u003cp\u003eCMs=cardiomyocytes\u003c/p\u003e\n\u003cp\u003eDkk1=dickkopf-1\u003c/p\u003e\n\u003cp\u003eExos=exosomes\u003c/p\u003e\n\u003cp\u003eHF = heart failure\u003c/p\u003e\n\u003cp\u003eHFrEF=heart failure with reduced ejection fraction\u003c/p\u003e\n\u003cp\u003eLV = left ventricle/ventricular\u003c/p\u003e\n\u003cp\u003eLVEF=left ventricularejection fraction\u003c/p\u003e\n\u003cp\u003eMHC =myosin heavy chain\u003c/p\u003e\n\u003cp\u003eMI = myocardial infarction\u003c/p\u003e\n\u003cp\u003eMSCs=mesenchymal stem cells;\u003c/p\u003e\n\u003cp\u003eNE=norepinephrine\u003c/p\u003e\n\u003cp\u003ePBMSCs=peripheral blood MSCs mesenchymal stem cells\u003c/p\u003e\n\u003cp\u003ePBS=phosphate buffered saline\u003c/p\u003e\n\u003cp\u003eRA=renal artery; RAECs=renal artery endothelial cells\u003c/p\u003e\n\u003cp\u003eRAAS=renin aniotension aldosterone system\u003c/p\u003e\n\u003cp\u003eRD=renal sympathetic denervation\u003c/p\u003e\n\u003cp\u003eRDCMs=renal sympathetic denervation-treated cardiomyocytes\u003c/p\u003e\n\u003cp\u003eSNS = sympathetic nervous system\u003c/p\u003e\n\u003cp\u003eTH=tyrosine hydroxylase\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Renmin Yao (Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai, China) and Baihe Zhang (GuangZhou Red Cross Hospital Medical College of Ji-Nan University) for technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Sciences Foundation of China (grant number 81770291, to Zhang S), the Guangzhou Science and Technology Planning Project (grant number 202002030081 to SZ), Guangzhou Science and Technology Program Project funded by the Guangzhou Science and Technology Bureau (Grant No.2023A03J0982).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no conflict of interest between the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Material\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZ.\u003c/strong\u003e: Conceptualization, Methodology, Writing-Original draft preparation, preparing figures 1-5. \u003cstrong\u003eL. C.\u003c/strong\u003e: Supplement data for later revisions. \u003cstrong\u003eZ.H.\u003c/strong\u003e: Data curation, preparing figures 6-10. \u003cstrong\u003eJ\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cstrong\u003eW\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e: Visualization, investigation, preparing table1. \u003cstrong\u003eZ\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e:Supervision, preparing figures S1-6; \u003cstrong\u003eS.Z.\u003c/strong\u003e: Software, Validation, preparing figures S7 and table S1-2. \u003cstrong\u003eY.D.\u003c/strong\u003e: preparing figures S8 and Graphical Abstract Image. \u003cstrong\u003eS.Z.\u003c/strong\u003e:Writing-Reviewing and Editing. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Institute for Animal Care and Use Committee at Dahua Hospital approved all the animal experiments, which were carried out in compliance with the Guide for the Care and Use of Laboratory Animals published by The National Academies Press (http://www.nap.edu/).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAzizi, M., Sanghvi, K., Saxena, M. et al. Ultrasound renal denervation for hypertension resistant to a triple medication pill (RADIANCE-HTN TRIO): a randomised, multicentre, single-blind, sham-controlled trial. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e397\u003c/strong\u003e, 2476-2486 (2021).\u003c/li\u003e\n\u003cli\u003eZhou, Z., Liu, C., Xu, S. et al. Metabolism regulator adiponectin prevents cardiac remodeling and ventricular arrhythmias via sympathetic modulation in a myocardial infarction model.\u003cem\u003e Basic. Res. Cardiol.\u003c/em\u003e \u003cstrong\u003e117\u003c/strong\u003e, 34 (2022).\u003c/li\u003e\n\u003cli\u003eKwon, J.S., Schumacher, S.M., Gao, E. et al. Characterization of \u0026beta;ARKct engineered cellular extracellular vesicles and model specific cardioprotection. \u003cem\u003eAm. J. Physiol. Heart. Circ. Physiol.\u003c/em\u003e \u003cstrong\u003e320\u003c/strong\u003e, H1276-1289 (2021).\u003c/li\u003e\n\u003cli\u003eKimura, K., Ieda, M. \u0026amp; Fukuda, K. Development, maturation, and transdifferentiation of cardiac sympathetic nerves. \u003cem\u003eCirc. Res.\u003c/em\u003e \u003cstrong\u003e110\u003c/strong\u003e, 325-336 (2012).\u003c/li\u003e\n\u003cli\u003ePolhemus, D.J., Trivedi, R.K., Sharp, T.E. et al. Repeated cell transplantation and adjunct renal denervation in ischemic heart failure: exploring modalities for improving cell therapy efficacy. \u003cem\u003eBasic. Res. Cardiol. \u003c/em\u003e\u003cstrong\u003e114\u003c/strong\u003e, 9 (2019).\u003c/li\u003e\n\u003cli\u003eB\u0026ouml;hm, M., Ewen, S \u0026amp; Wolf, M. Renal Denervation Halts Left Ventricular Remodeling and Dysfunction in Heart Failure: New Shores Ahead. \u003cem\u003eJ. Am. Coll. Cardiol.\u003c/em\u003e \u003cstrong\u003e72\u003c/strong\u003e, 2622-2624 (2018).\u003c/li\u003e\n\u003cli\u003eSepe, J.J., Gardner, R.T., Blake, M.R. et al. Therapeutics That Promote Sympathetic Reinnervation Modulate the Inflammatory Response After Myocardial Infarction. \u003cem\u003eJACC. Basic. Transl. Sci.\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 915-930 (2022).\u003c/li\u003e\n\u003cli\u003eSharp, T.E. 3rd., Polhemus, D.J., Li, Z. et al. Renal Denervation Prevents Heart Failure Progression Via Inhibition of the Renin-Angiotensin System. \u003cem\u003eJ. Am. Coll. Cardiol. \u003c/em\u003e\u003cstrong\u003e72\u003c/strong\u003e, 2609-26021 (2018).\u003c/li\u003e\n\u003cli\u003eGarbern, J.C. \u0026amp; Lee, R.T. Heart regeneration: 20 years of progress and renewed optimism. \u003cem\u003eDev. Cell.\u003c/em\u003e \u003cstrong\u003e57\u003c/strong\u003e, 424-439 (2022).\u003c/li\u003e\n\u003cli\u003eWang, P., Deng, Z., Li, A. et al. \u0026beta;-Catenin promotes long-term survival and angiogenesis of peripheral blood mesenchymal stem cells via the Oct4 signaling pathway. \u003cem\u003eExp. Mol. Med. \u003c/em\u003e\u003cstrong\u003e54\u003c/strong\u003e, 1434-1449 (2022).\u003c/li\u003e\n\u003cli\u003eSahoo, S. \u0026amp; Losordo, D.W. Exosomes and cardiac repair after myocardial infarction. \u003cem\u003eCirc. Res.\u003c/em\u003e \u003cstrong\u003e114\u003c/strong\u003e, 333-344 (2014).\u003c/li\u003e\n\u003cli\u003eSharp, T.E. III, Polhemus, D.J., Li, Z. et al. Hypoxia induces heart regeneration in adult mice. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e541\u003c/strong\u003e, 222-227 (2017).\u003c/li\u003e\n\u003cli\u003eWang, W.E., Li, L., Xia, X. et al. Dedifferentiation, proliferation, and redifferentiation of adult mammalian cardiomyocytes after ischemic injury. \u003cem\u003eCirculatio\u003c/em\u003en \u003cstrong\u003e136\u003c/strong\u003e, 834-848 (2017).\u003c/li\u003e\n\u003cli\u003eLi, J., Sun, S., Zhu, D. et al. Inhalable Stem Cell Exosomes Promote Heart Repair After Myocardial Infarction. \u003cem\u003eCirculation\u003c/em\u003e \u003cstrong\u003e150\u003c/strong\u003e, 710-723 (2024).\u003c/li\u003e\n\u003cli\u003eMesserli, F.H., Bavishi, C., Brguljan, J. et al. Renal denervation in the antihypertensive arsenal - knowns and known unknowns. \u003cem\u003eJ. Hypertens.\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 1859-1875 (2022).\u003c/li\u003e\n\u003cli\u003ePeng, Y., Zhao, J.L., Peng, Z.Y. et al. Exosomal miR-25-3p from mesenchymal stem cells alleviates myocardial infarction by targeting pro-apoptotic proteins and EZH2. \u003cem\u003eCell Death Dis.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 317 (2020).\u003c/li\u003e\n\u003cli\u003eTh\u0026eacute;ry, C., Witwer, K.W., Aikawa, E. et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. \u003cem\u003eJ. Extracell. Vesicles\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 1535750 (2018).\u003c/li\u003e\n\u003cli\u003eHessvik, N.P., Sagini, K., Romero, S. et al. siRNA screening reveals that SNAP29 contributes to exosome release. \u003cem\u003eCell. Mol. Life Sci.\u003c/em\u003e \u003cstrong\u003e80\u003c/strong\u003e, 177 (2023).\u003c/li\u003e\n\u003cli\u003eKumar, R., Tang, Q., M\u0026uuml;ller, S.A. et al. Fibroblast Growth Factor 2-Mediated Regulation of Neuronal Exosome Release Depends on VAMP3/Cellubrevin in Hippocampal Neurons. \u003cem\u003eAdv. Sci. (Weinh.)\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 1902372 (2020).\u003c/li\u003e\n\u003cli\u003eTian, Y., Liu, Y., Wang, T. et al. A microRNA-Hippo pathway that promotes cardiomyocyte proliferation and cardiac regeneration in mice.\u003cem\u003e Sci. Transl. Med.\u003c/em\u003e\u003cstrong\u003e 7\u003c/strong\u003e, 279ra38 (2015).\u003c/li\u003e\n\u003cli\u003ePatel, V., Hajarnis, S., Williams, D. et al. MicroRNAs regulate renal tubule maturation through modulation of Pkd1. \u003cem\u003eJ. Am. Soc. Nephrol.\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 1941-1948 (2012).\u003c/li\u003e\n\u003cli\u003ePecot, C.V., Rupaimoole, R., Yang, D. et al. Tumour angiogenesis regulation by the miR-200 family. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 2427 (2013).\u003c/li\u003e\n\u003cli\u003eAkbar, N., Razzaq, S.S., Salim, A. \u0026amp; Haneef, K. Mesenchymal Stem Cell-Derived Exosomes and Their MicroRNAs in Heart Repair and Regeneration. \u003cem\u003eJ. Cardiovasc. Transl. Res.\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 505-522 (2024).\u003c/li\u003e\n\u003cli\u003eWan, J., Lin, S., Yu, Z. et al. Protective Effects of MicroRNA-200b-3p Encapsulated by Mesenchymal Stem Cells-Secreted Extracellular Vesicles in Myocardial Infarction Via Regulating BCL2L11. \u003cem\u003eJ. Am. Heart Assoc.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e024330 (2022).\u003c/li\u003e\n\u003cli\u003eBarr\u0026egrave;re-Lemaire, S., Vincent, A., Jorgensen, C. et al. Mesenchymal stromal cells for improvement of cardiac function following acute myocardial infarction: a matter of timing. \u003cem\u003ePhysiol. Rev.\u003c/em\u003e \u003cstrong\u003e104\u003c/strong\u003e, 659-725 (2024).\u003c/li\u003e\n\u003cli\u003eMiller, I., Min, M., Yang, C. et al. Ki67 is a Graded Rather than a Binary Marker of Proliferation versus Quiescence. \u003cem\u003eCell Rep.\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 1105-1112.e5 (2018).\u003c/li\u003e\n\u003cli\u003eCho, K.W., Andrade, M., Bae, S. et al. Polycomb Group Protein CBX7 Represses Cardiomyocyte Proliferation Through Modulation of the TARDBP/RBM38 Axis. \u003cem\u003eCirculation\u003c/em\u003e \u003cstrong\u003e147\u003c/strong\u003e, 1823-1842 (2023).\u003c/li\u003e\n\u003cli\u003eChen, Y., L\u0026uuml;ttmann, F.F., Schoger, E. et al. Reversible reprogramming of cardiomyocytes to a fetal state drives heart regeneration in mice. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e373\u003c/strong\u003e, 1537-1540 (2021).\u003c/li\u003e\n\u003cli\u003eWang, J., Pei, B., Yan, J. et al. hucMSC-Derived Exosomes Alleviate the Deterioration of Colitis via the miR-146a/SUMO1 Axis. \u003cem\u003eMol. Pharm.\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 484\u0026ndash;493 (2022).\u003c/li\u003e\n\u003cli\u003eIbrahim, A.G.E., Li, C., Rogers, R. et al. Augmenting canonical Wnt signalling in therapeutically inert cells converts them into therapeutically potent exosome factories. \u003cem\u003eNat. Biomed. Eng.\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 695-705 (2019).\u003c/li\u003e\n\u003cli\u003eLian, X., Zhang, J., Azarin, S.M. et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/\u0026beta;-catenin signaling under fully defined conditions. \u003cem\u003eNat. Protoc.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 162\u0026ndash;175 (2013).\u003c/li\u003e\n\u003cli\u003eZhu, C., Yuan, T. \u0026amp; Krishnan, J. Targeting cardiomyocyte cell cycle regulation in heart failure. \u003cem\u003eBasic Res. Cardiol.\u003c/em\u003e \u003cstrong\u003e119\u003c/strong\u003e, 349-369 (2024).\u003c/li\u003e\n\u003cli\u003eGabisonia, K., Prosdocimo, G., Aquaro, G.D. et al. MicroRNA therapy stimulates uncontrolled cardiac repair after myocardial infarction in pigs. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e569\u003c/strong\u003e, 418-422 (2019).\u003c/li\u003e\n\u003cli\u003eDzau, V.J. \u0026amp; Hodgkinson, C.P. RNA Therapeutics for the Cardiovascular System. \u003cem\u003eCirculation \u003c/em\u003e\u003cstrong\u003e149\u003c/strong\u003e, 707-716 (2024).\u003c/li\u003e\n\u003cli\u003eFiebeler, A., Schmidt, F., M\u0026uuml;ller, D.N. et al. Mineralocorticoid receptor affects AP-1 and nuclear factor-kappab activation in angiotensin II-induced cardiac injury. \u003cem\u003eHypertension\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 787\u0026ndash;793 (2001).\u003c/li\u003e\n\u003cli\u003eLi, X., Wu, F., G\u0026uuml;nther, S. et al. Inhibition of fatty acid oxidation enables heart regeneration in adult mice. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e622\u003c/strong\u003e, 619\u0026ndash;626 (2023).\u003c/li\u003e\n\u003cli\u003eLiu, J., Xiao, Q., Xiao, J. et al. Wnt/\u0026beta;-catenin signalling: function, biological mechanisms, and therapeutic opportunities. \u003cem\u003eSignal Transduct. Target. Ther.\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 3 (2022).\u003c/li\u003e\n\u003cli\u003eHuang, M., Wang, X., Banerjee, M. et al. Regulation of myogenesis by a Na/K-ATPase \u0026alpha;1 caveolin-binding motif. \u003cem\u003eStem Cells\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 133-148 (2022).\u003c/li\u003e\n\u003cli\u003eDeng, F., Peng, L., Li, Z. et al. YAP triggers the Wnt/\u0026beta;-catenin signalling pathway and promotes enterocyte self-renewal, regeneration and tumorigenesis after DSS-induced injury. \u003cem\u003eCell Death Dis\u003c/em\u003e. \u003cstrong\u003e9\u003c/strong\u003e, 153 (2018).\u003c/li\u003e\n\u003cli\u003eKim, S., Song, G., Lee, T. et al. PARsylated transcription factor EB (TFEB) regulates the expression of a subset of Wnt target genes by forming a complex with \u0026beta;-catenin-TCF/LEF1. \u003cem\u003eCell Death Differ\u003c/em\u003e. \u003cstrong\u003e28\u003c/strong\u003e, 2555\u0026ndash;2570 (2021).\u003c/li\u003e\n\u003cli\u003ePolhemus, D.J., Gao, J., Scarborough, A.L. et al. Radiofrequency Renal Denervation Protects the Ischemic Heart via Inhibition of GRK2 and Increased Nitric Oxide Signaling. \u003cem\u003eCirc. Res.\u003c/em\u003e \u003cstrong\u003e119\u003c/strong\u003e, 470-480 (2016).\u003c/li\u003e\n\u003cli\u003eOkamura, K., Shimada, H., Imazato, K. et al. Impact of renal denervation on quality of life (How does renal denervation contribute to improving hypertension treatment affected by poor medication adherence?).\u003cem\u003e Hypertens. Res\u003c/em\u003e. \u003cstrong\u003e47\u003c/strong\u003e, 2652-2658 (2024).\u003c/li\u003e\n\u003cli\u003eTaru, V., Szabo, G., Mehal, W. \u0026amp; Reiberger, T. Inflammasomes in chronic liver disease: Hepatic injury, fibrosis progression and systemic inflammation. \u003cem\u003eJ. Hepatol\u003c/em\u003e. \u003cstrong\u003e81\u003c/strong\u003e, 895-910 (2024).\u003c/li\u003e\n\u003cli\u003eBhatt, D.L., Kandzari, D.E., O\u0026apos;Neill, W.W. et al. A controlled trial of renal denervation for resistant hypertension. \u003cem\u003eN. Engl. J. Med\u003c/em\u003e. \u003cstrong\u003e370\u003c/strong\u003e, 1393-1401 (2014).\u003c/li\u003e\n\u003cli\u003eSharp, T.E. III \u0026amp; Lefer, D.J. Renal Denervation to Treat Heart Failure. \u003cem\u003eAnnu. Rev. Physiol\u003c/em\u003e. \u003cstrong\u003e83\u003c/strong\u003e, 39-58 (2021).\u003c/li\u003e\n\u003cli\u003eDavidson, S.M., Padr\u0026oacute;, T., Bollini, S. et al. Progress in cardiac research: from rebooting cardiac regeneration to a complete cell atlas of the heart. \u003cem\u003eCardiovasc. Res.\u003c/em\u003e \u003cstrong\u003e117\u003c/strong\u003e, 2161-2174 (2021).\u003c/li\u003e\n\u003cli\u003eFeng, Y., Wang, Y., Li, L. et al. Exosomes Induce Crosstalk Between Multiple Types of Cells and Cardiac Fibroblasts: Therapeutic Potential for Remodeling After Myocardial Infarction.\u003cem\u003e Int. J. Nanomedicine\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 10605-10621 (2024).\u003c/li\u003e\n\u003cli\u003eMohamed, T.M.A., Ang, Y.S., Radzinsky, E. et al. Regulation of Cell Cycle to Stimulate Adult Cardiomyocyte Proliferation and Cardiac Regeneration. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e173\u003c/strong\u003e, 104-116.e12 (2018).\u003c/li\u003e\n\u003cli\u003eGallet, R., Dawkins, J., Valle, J. et al. Exosomes secreted by cardiosphere-derived cells reduce scarring, attenuate adverse remodelling, and improve function in acute and chronic porcine myocardial infarction. \u003cem\u003eEur. Heart J.\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 201-211 (2017).\u003c/li\u003e\n\u003cli\u003eGao, L., Mei, S., Zhang, S. et al. Cardio-renal Exosomes in Myocardial Infarction Serum Regulate Proangiogenic Paracrine Signaling in Adipose Mesenchymal Stem Cells. \u003cem\u003eTheranostics\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 1060-1073 (2020).\u003c/li\u003e\n\u003cli\u003eCrewe, C., Joffin, N., Rutkowski, J.M. et al. An Endothelial-to-Adipocyte Extracellular Vesicle Axis Governed by Metabolic State. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e175\u003c/strong\u003e, 695-708.e13 (2018).\u003c/li\u003e\n\u003cli\u003eLiang, X., Li, C., Song, J. et al. HucMSC-Exo Promote Mucosal Healing in Experimental Colitis by Accelerating Intestinal Stem Cells and Epithelium Regeneration via Wnt Signaling Pathway. \u003cem\u003eInt. J. Nanomedicine\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 2799\u0026ndash;2818 (2023).\u003c/li\u003e\n\u003cli\u003eFeng, D., Yan, K., Liang, H. et al. CBP-mediated Wnt3a/\u0026beta;-catenin signaling promotes cervical oncogenesis initiated by Piwil2. \u003cem\u003eNeoplasia\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 1-11 (2021).\u003c/li\u003e\n\u003cli\u003eKharaziha, P., Ceder, S., Li, Q. \u0026amp; Panaretakis, T. Tumor cell-derived exosomes: a message in a bottle. \u003cem\u003eBiochim. Biophys. Acta\u003c/em\u003e \u003cstrong\u003e1826\u003c/strong\u003e, 103\u0026ndash;111 (2012).\u003c/li\u003e\n\u003cli\u003eWang, J., Pei, B., Yan, J. et al. hucMSC-Derived Exosomes Alleviate the Deterioration of Colitis via the miR-146a/SUMO1 Axis. \u003cem\u003eMol. Pharm\u003c/em\u003e. \u003cstrong\u003e19\u003c/strong\u003e, 484-493 (2022).\u003c/li\u003e\n\u003cli\u003eZheng, X., Li, X., Lyu, Y. et al. Renal Sympathetic Denervation in Rats Ameliorates Cardiac Dysfunction and Fibrosis Post-Myocardial Infarction Involving MicroRNAs. \u003cem\u003eMed. Sci. Monit\u003c/em\u003e. \u003cstrong\u003e22\u003c/strong\u003e, 2751-2760 (2016).\u003c/li\u003e\n\u003cli\u003eGao, L., Qiu, F., Cao, H. et al. Therapeutic delivery of microRNA-125a-5p oligonucleotides improves recovery from myocardial ischemia/reperfusion injury in mice and swine. \u003cem\u003eTheranostics\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 685-703 (2023).\u003c/li\u003e\n\u003cli\u003eBraga, L., Ali, H., Secco, I. \u0026amp; Giacca, M. Non-coding RNA therapeutics for cardiac regeneration. \u003cem\u003eCardiovasc. Res.\u003c/em\u003e \u003cstrong\u003e117\u003c/strong\u003e, 674-693 (2021).\u003c/li\u003e\n\u003cli\u003ePapukashvili, D., Rcheulishvili, N., Liu, C. et al. Perspectives on miRNAs Targeting DKK1 for Developing Hair Regeneration Therapy. \u003cem\u003eCells\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 2957 (2021).\u003c/li\u003e\n\u003cli\u003eLi, S., Yin, Y., Yao, L. et al. TNF-\u0026alpha; treatment increases DKK1 protein levels in primary osteoblasts via upregulation of DKK1 mRNA levels and downregulation of miR-335-5p. \u003cem\u003eMol. Med. Rep\u003c/em\u003e. \u003cstrong\u003e22\u003c/strong\u003e, 1017-1025 (2020).\u003c/li\u003e\n\u003cli\u003eZhao, L., Wang, J., Wang, P. et al. Oct4 cooperates with c-Myc to improve mesenchymal-to-endothelial transition and myocardial repair of cardiac-resident mesenchymal stem cells. \u003cem\u003eStem Cell Res. Ther\u003c/em\u003e. \u003cstrong\u003e13\u003c/strong\u003e, 445 (2022).\u003c/li\u003e\n\u003cli\u003eLiang, X.L., Wang, Y.L. \u0026amp; Wang, P.R. MiR-200a with CDC7 as a direct target declines cell viability and promotes cell apoptosis in Wilm\u0026apos;s tumor via Wnt/\u0026beta;-catenin signaling pathway. \u003cem\u003eMol. Cell. Biochem\u003c/em\u003e. \u003cstrong\u003e476\u003c/strong\u003e, 2409-2420 (2021).\u003c/li\u003e\n\u003cli\u003eKook, S.H., Heo, J.S. \u0026amp; Lee, J.C. Crucial roles of canonical Runx2-dependent pathway on Wnt1-induced osteoblastic differentiation of human periodontal ligament fibroblasts. \u003cem\u003eMol. Cell. Biochem\u003c/em\u003e. \u003cstrong\u003e402\u003c/strong\u003e, 213-223 (2015).\u003c/li\u003e\n\u003cli\u003eDharavath, B., Butle, A., Chaudhary, A. et al. Recurrent UBE3C-LRP5 translocations in head and neck cancer with therapeutic implications. \u003cem\u003eNPJ Precis. Oncol\u003c/em\u003e. \u003cstrong\u003e8\u003c/strong\u003e, 63 (2024).\u003c/li\u003e\n\u003cli\u003eFrangogiannis, N.G. The inflammatory response in myocardial injury, repair, and remodelling. \u003cem\u003eNat. Rev. Cardiol\u003c/em\u003e. \u003cstrong\u003e11\u003c/strong\u003e, 255-265 (2014).\u003c/li\u003e\n\u003cli\u003eLiu, S., Li, K., Wagner Florencio, L. et al. Gene therapy knockdown of Hippo signaling induces cardiomyocyte renewal in pigs after myocardial infarction. \u003cem\u003eSci. Transl. Med\u003c/em\u003e. \u003cstrong\u003e13\u003c/strong\u003e, eabd6892 (2021).\u003c/li\u003e\n\u003cli\u003eZhang, S., Zhao, L., Shen, L. et al. Comparison of various niches for endothelial progenitor cell therapy on ischemic myocardial repair: coexistence of host collateralization and Akt-mediated angiogenesis produces a superior microenvironment. \u003cem\u003eArterioscler. Thromb. Vasc. Biol\u003c/em\u003e. \u003cstrong\u003e3\u003c/strong\u003e2, 910-923 (2012).\u003c/li\u003e\n\u003cli\u003eMahfoud, F., Townsend, R.R., Kandzari, D.E. et al. Changes in Plasma Renin Activity After Renal Artery Sympathetic Denervation. \u003cem\u003eJ. Am. Coll. Cardiol\u003c/em\u003e. \u003cstrong\u003e77\u003c/strong\u003e, 2909-2919 (2021).\u003c/li\u003e\n\u003cli\u003eLu, H., Jiang, X., He, L. et al. Endothelial Sp1/Sp3 are essential to the effect of captopril on blood pressure in male mice. \u003cem\u003eNat. Commun\u003c/em\u003e. \u003cstrong\u003e14\u003c/strong\u003e, 5891 (2023).\u003c/li\u003e\n\u003cli\u003eVillamizar, O., Waters, S.A., Scott, T. et al. Mesenchymal Stem Cell exosome delivered Zinc Finger Protein activation of cystic fibrosis transmembrane conductance regulator. \u003cem\u003eJ. Extracell. Vesicles\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, e12053 (2021).\u003c/li\u003e\n\u003cli\u003eChen, K., Wang, Q., Liu, X. et al. Hypoxic pancreatic cancer derived exosomal miR-30b-5p promotes tumor angiogenesis by inhibiting GJA1 expression. \u003cem\u003eInt. J. Biol. Sci\u003c/em\u003e. \u003cstrong\u003e18\u003c/strong\u003e, 1220-1237 (2022).\u003c/li\u003e\n\u003cli\u003eWang, N., Li, X., Zhong, Z. et al. 3D hESC exosomes enriched with miR-6766-3p ameliorates liver fibrosis by attenuating activated stellate cells through targeting the TGF\u0026beta;RII-SMADS pathway. \u003cem\u003eJ. Nanobiotechnology\u003c/em\u003e. \u003cstrong\u003e19\u003c/strong\u003e, 437 (2021).\u003c/li\u003e\n\u003cli\u003eBervoets, S., Wei, N., Erfurth, M.L. et al. Transcriptional dysregulation by a nucleus-localized aminoacyl-tRNA synthetase associated with Charcot-Marie-Tooth neuropathy. \u003cem\u003eNat. Commun\u003c/em\u003e. \u003cstrong\u003e10\u003c/strong\u003e, 5045 (2019).\u003c/li\u003e\n\u003cli\u003eZhao, M., Nakada, Y., Wei, Y. et al. Cyclin D2 Overexpression Enhances the Efficacy of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Myocardial Repair in a Swine Model of Myocardial Infarction. \u003cem\u003eCirculation\u003c/em\u003e \u003cstrong\u003e144\u003c/strong\u003e, 210-228 (2021).\u003c/li\u003e\n\u003cli\u003eSuda, T., Yamashita, T., Sunagozaka, H. et al. Dickkopf-1 Promotes Angiogenesis and is a Biomarker for Hepatic Stem Cell-like Hepatocellular Carcinoma. \u003cem\u003eInt. J. Mol. Sci\u003c/em\u003e. \u003cstrong\u003e23\u003c/strong\u003e, 2801 (2022).\u003c/li\u003e\n\u003cli\u003eShi, X.B., Xue, L., Ma, A.H. et al. Tumor suppressive miR-124 targets androgen receptor and inhibits proliferation of prostate cancer cells. \u003cem\u003eOncogene\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 4130-4138 (2013).\u003c/li\u003e\n\u003cli\u003eGan, L., Liu, D., Xie, D. et al. Ischemic Heart-Derived Small Extracellular Vesicles Impair Adipocyte Function. \u003cem\u003eCirc. Res.\u003c/em\u003e \u003cstrong\u003e130\u003c/strong\u003e, 48-66 (2022).\u003c/li\u003e\n\u003cli\u003eAndres-Mateos, E., Landegger, L.D., Unzu, C. et al. Choice of vector and surgical approach enables efficient cochlear gene transfer in nonhuman primate. \u003cem\u003eNat. Commun\u003c/em\u003e. \u003cstrong\u003e13\u003c/strong\u003e, 1359 (2022).\u003c/li\u003e\n\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"acute myocardial infarction, exosome, microRNA, heart failure, sympathetic nervous system","lastPublishedDoi":"10.21203/rs.3.rs-6409278/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6409278/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eObjectives\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo explore the role of self-peripheral blood mesenchymal stem cell (PBMSC)-derived exosomes (Exos) in enhancing renal sympathetic denervation (RD)-mediated cardiac repair following myocardial infarction (MI) in a porcine model.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePigs (EF\u0026thinsp;\u0026lt;\u0026thinsp;40% post-MI) were randomized to early sham RD or RD. At 2 weeks post-MI, autologous PBMSC-Exos were collected. At 30 days post-MI, pigs received either PBMSC-Exos (2 \u0026times; 10\u0026sup1;\u0026sup3; particles) or PBS and were followed until 90 days. Another cohort underwent myocardial biopsy at 14 days post-MI to assess PBMSC-Exos effects on ischemic cardiomyocyte (CM) reprogramming, followed by AAV therapy with miR-141-200-429 sponges or negative control (NC) sponges to explore the role of miR-141-200-429 clusters in reprogramming.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTwo weeks post-MI, RD hearts showed increased Exos uptake and inhibited the sympathetic nervous system. By 90 days, the RD\u0026thinsp;+\u0026thinsp;Exos group had 11\u0026ndash;26% higher EF than single-treatment groups (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with improved survival and reduced fibrosis. Exos therapy enhanced RD effects by inhibiting the renin-angiotensin-aldosterone system and transferring the miR-141-200-429 cluster into ischemic CMs. CMs from RD-treated hearts co-cultured with PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e exhibited a more immature state, promoting reprogramming. β-catenin overexpression further enhanced PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e effects, while miR-141-200-429 inhibition blocked RD-induced CM reprogramming and survival. Ultimately, PBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e reduced Dkk1 expression and activated GSK3β phosphorylation, thereby stimulating the Wnt/β-catenin pathway.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePBMSC-Exos\u003csup\u003eRD\u003c/sup\u003e enhances RD-mediated cardiac repair by activating the Wnt/β-catenin pathway via miR-141-200-429 cluster, offering a novel therapeutic strategy for MI-induced heart failure. Our findings unveil a novel therapeutic strategy, highlighting that RD maintains its efficacy and safety when integrated with complementary approaches over extended periods.\u003c/p\u003e","manuscriptTitle":"Peripheral Blood Mesenchymal Stem Cell–Derived Exosomes Improve Renal Sympathetic Denervation Efficacy Through β-Catenin-Mediated Cardiac Reprogramming","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-10 07:07:10","doi":"10.21203/rs.3.rs-6409278/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"be1fb5ce-824a-4096-b738-9b0732eebb3e","owner":[],"postedDate":"April 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46898328,"name":"Health sciences/Cardiology/Interventional cardiology"},{"id":46898329,"name":"Biological sciences/Stem cells/Mesenchymal stem cells"},{"id":46898330,"name":"Health sciences/Diseases/Cardiovascular diseases/Heart failure"}],"tags":[],"updatedAt":"2025-04-17T11:00:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-10 07:07:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6409278","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6409278","identity":"rs-6409278","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}