Exosomal miR-20a-5p Promotes Cardiac Remodeling After Myocardial Infarction by Regulating mTORC1/S6 Pathway

Exosomal miR-20a-5p Promotes Cardiac Remodeling After Myocardial Infarction by Regulating mTORC1/S6 Pathway | 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 Exosomal miR-20a-5p Promotes Cardiac Remodeling After Myocardial Infarction by Regulating mTORC1/S6 Pathway Jingjing Wang, Meihua Jin, Ying Guo, Lin He, Lili Zhang, Guangyuan Gao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9333795/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 18 You are reading this latest preprint version Abstract Background Maladaptive ventricular remodeling is the major pathological process linking myocardial infarction (MI) to heart failure, yet its upstream molecular drivers remain incompletely defined. Exosomal microRNAs (miRNAs) are emerging regulators of intercellular communication and cardiac pathology. This study sought to investigate the contribution of exosome-enriched miR-20a-5p in post-MI remodeling. Methods An acute myocardial infarction (MI) model was induced in SD rats through coronary ligation. Exosomes were isolated from infarcted and sham hearts, and expression of miR-20a-5p was quantified. Neonatal cardiomyocytes and fibroblasts were used for exosome uptake and functional assays. Functional enhancement and impairment assays with miR-20a-5p mimic/inhibitor, combined with rapamycin, were performed. Molecular mechanisms were assessed by RT-qPCR, Western blot, flow cytometry, and dual-luciferase reporter assays, while cardiac function and remodeling were evaluated by echocardiography and histology in vivo. Results Exosomal miR-20a-5p was significantly enriched after MI and preferentially internalized by cardiomyocytes and fibroblasts. Its upregulation promoted cardiomyocyte apoptosis, impaired autophagy, and induced fibroblast-to-myofibroblast transition via direct suppression of Ddit4 and activation of the mTORC1/S6 pathway. Inhibition of miR-20a-5p or mTORC1 partially reversed these effects, while combined therapy produced synergistic benefits, including reduced fibrosis, improved cardiomyocyte survival, and restored ventricular function in MI rats. Conclusions Exosomal miR-20a-5p drives adverse ventricular remodeling after MI through Ddit4-mediated mTORC1/S6 activation. Dual inhibition of miR-20a-5p and mTORC1 exerts synergistic cardioprotection, highlighting this axis as a promising target for preventing post-MI heart failure. Health sciences/Cardiology Biological sciences/Cell biology Health sciences/Diseases myocardial infarction ventricular remodeling miR-20a-5p mTORC1/S6 exosome myocardial fibrosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Heart failure (HF), a common convergent point for multiple cardiovascular conditions, is most frequently triggered by acute myocardial infarction (MI) 1 , 2 . Despite advances in early diagnosis and reperfusion therapy, the incidence of post-MI HF continues to rise. Maladaptive ventricular remodeling—the pathological link between MI and HF 3 —remains only partially addressed by current neuroendocrine therapies, while imaging detects remodeling only after irreversible injury 4 . The lack of reliable molecular markers and incomplete mechanistic understanding thus remain major barriers to early intervention. As a class of small non-coding RNAs, microRNAs (miRNAs) post-transcriptionally silence gene expression by inhibiting the translation of target mRNAs and promoting their degradation 5 , and circulate in stable forms bound to proteins or encapsulated in exosomes 6 . Their dynamic expression in MI, HF, and other cardiovascular diseases highlights their promise as minimally invasive biomarkers 7 – 9 . Exosomal miRNAs, in particular, mediate intercellular communication by transferring regulatory signals between cells 10 , 11 , and are increasingly recognized as modulators of cardiac pathology. The mTORC1 pathway, a master regulator of growth, apoptosis, and autophagy, is central to ventricular remodeling 12 . However, systemic mTOR inhibition disrupts essential cardiac functions 13 , Exosomal miRNAs may provide a more precise and reversible mode of regulation, with growing evidence supporting their therapeutic potential 14 , 15 . As evidenced in our prior publication, a rat model of post-myocardial infarction HF was constructed via permanent ligation of the left anterior descending coronary artery 16 . We used high-throughput sequencing technology to screen out the differential expression profiles of miRNAs in the myocardium and plasma of rats with post-myocardial infarction heart failure. Through qRT-PCR technology, we conducted batch verification of the expression levels of differentially expressed miRNAs in rat myocardium, plasma, the rat myocardial cell apoptosis model induced by angiotensin II, and the plasma of patients with post-myocardial infarction heart failure. Finally, through specificity and sensitivity analysis, we screened out miRNAs with myocardial expression specificity, which were dynamically related to the pathological process of ventricular remodeling and existed in plasma exosomes and supernatants of cultured myocardial cells: miR-20a-5p 16 . Our previous studies using rapamycin demonstrated that mTORC1 inhibition in cardiomyocytes enhances protective autophagy, reduces apoptosis, attenuates ventricular remodeling, and enhances cardiac function in rats following myocardial infarction. Bioinformatic analyses identified miR-20a-5p targets, including Ddit4, Sesn1, and Nfkbia, as key regulators of the mTORC1 pathway, influencing cell growth, apoptosis, autophagy, inflammation, and fibrosis. Notably, DDIT4 suppresses mTORC1 activity via the TSC1/TSC2 complex 17 – 20 . Mechanistically, these findings implicate miR-20a-5p-mediated mTORC1 activation as a pathway that exacerbates remodeling following myocardial infarction.. Here, we explore the involvement of the miR-20a-5p/mTORC1 axis in cardiomyocyte and fibroblast remodeling after MI, and evaluate the therapeutic potential of targeting this pathway. 2. Materials and methods Animal models and groups All experimental procedures were conducted in accordance with institutional and national guidelines for the care and use of laboratory animals and were approved by the Ethical Review Board of General Hospital of Ningxia Medical University (Approval No. KYLL-2022-0877). Male Sprague–Dawley rats (8 weeks old, weighing 230–250 g) were supplied by the Center for Laboratory Animals, Ningxia Medical University, Yinchuan, China. The animals were housed in a specific pathogen-free facility where the environment was kept at 22 ± 2°C with 55 ± 5% relative humidity and a 12 h light/dark rhythm. Standard rodent diet and water were provided ad libitum. Myocardial infarction was triggered through the permanent occlusion of the left anterior descending coronary artery. Briefly, the rats were anesthetized with 100% oxygen containing 3% isoflurane supplied by a rodent respirator. Following anesthetization, the thorax was opened in the left parasternal area, and myocardial infarction was induced by ligating the left anterior descending coronary artery using 3 − 0 suture between the pulmonary cone and the left atrium. Control animals in the sham-operated group experienced the same surgical protocol, with the exception of the coronary artery ligation. The animals' conditions were observed within 24 hours after the operation. The animals were given medication immediately after the operation and were injected with miR-20a-5p inhibitor (20 nmol per rat, tail vein) or Rapamycin (1 mg/kg, intraperitoneal injection, HY-10219R, MCE) once a week for 4 weeks. Animals were then anesthetized using 100% oxygen containing 3% isoflurane and euthanized via a rapid exsanguination from the abdominal aorta and the removal of the hearts. Exsanguination was performed via an abdominal aortic catheter, which permitted the free flow of blood, ,and blood with a total volume of 7–9 ml per rat was rapidly removed until no longer bleeding. The hearts were then quickly harvested and washed with ice-cold PBS. TTC + Evans Blue staining This is used to evaluate the construction of the myocardial infarction model. After blocking the left coronary artery bifurcation for 24 hours, animals were then euthanized, 2% Evans Blue dye (E2129, Sigma-Aldrich, USA) was injected into the ascending aorta. Immediately after that, the heart was collected and frozen at -80°C for 10 minutes. Then, the heart was transverse into 1–2 mm thin slices, and stained by incubation under light-protected conditions at 37°C with a 1% TTC (17779, Sigma-Aldrich, USA) solution for 20 minutes. After 24 hours of fixation with 4% paraformaldehyde, the heart slices were photographed. The images were collected by a binocular microscope (Nikon, Japan), and the infarct area was measured using ImageJ software (NIH, USA), and the percentages of viable myocardium in the left ventricle (deep blue), AAR (red and white), and IA (white) were calculated. Isolation and Cultivation of Cardiac Cells Four weeks post-surgery, rats that experienced abdominal aortic bleeding were euthanized, and their hearts were immediately removed and washed in ice-cold PBS. The hearts were minced to approximately 2 mm³, and then completely digested for several minutes at 37°C using 0.1% type IV collagenase (17104019, Gibco, USA). After sedimentation, they were adhered to the precoated fibronectin-coated dishes, and the 2-4th generation cells were collected. 1% penicillin-streptomycin Dulbecco modified Eagle's medium/F-12 (12634010, Gibco, USA) containing 10% fetal bovine serum (A5670701, FBS, Gibco, USA) was added, and the cardiac cells were placed in a 37°C, 5% CO₂ constant temperature incubator. After 3–5 days, the cardiac cells began to adhere to the walls. Extraction and labeling of extracellular vesicles from cardiac cells Cardiac cells were then maintained in IMDM medium lacking serum, and the supernatants were continuously collected for exosome isolation. Extracellular vesicles originating from pericardial adipose tissue were purified using a commercial isolation kit (UR52161, Umibio, China). In brief, the tissue was chopped into small pieces, washed with PBS, and centrifuged at 300 × g for 10 minutes to remove residual debris. The resulting pellet was subsequently treated with solution A2 for 20 minutes and then centrifuged at 8,000 × g for 10 minutes. The collected supernatant was mixed with solution B2, incubated for 20 min, and subjected to another centrifugation at 8000 × g for 20 min at 4°C. The pellet was resuspended in PBS, centrifuged again at 11,000 × g for 2 minutes, and the resulting supernatant was passed through an exosome purification filter before undergoing a final centrifugation at 3,000 × g for 10 minutes at 4°C. Purified exosomes were divided into aliquots and cryopreserved at − 80°C for subsequent analysis. Exosomes derived from sham and MI cardiac cells (Sham-EXO and MI-EXO) were employed for uptake assays. For tracking, vesicles were labeled with DiI (Beyotime, China) and monitored in real time under a confocal microscope (Zeiss LSM880), with images captured every 5 min for 6 h. Transmission Electron Microscope (TEM) The extracted Sham-EXO and MI-EXO were placed on a copper mesh, exposed to light discharge in air for 1 minute, then 2% phosphotungstic acid was applied to achieve negative staining, and the sample was air-dried under ambient conditions. The morphology was imaged by a Hitachi H-7650 microscope (Hitachi H-7650, Japan) at 80 kV. Cell culture and grouping Neonatal cardiomyocytes and cardiac fibroblasts were harvested from 1 to 2-day-old SD rats. Pups underwent pre-anesthesia via hypothermia (achieved by indirect contact with ice while wrapped in gauze) to minimize distress. Following abdominal sterilization with 70% ethanol for local anesthesia, euthanasia was performed by decapitation using sterile sharp surgical scissors by a trained researcher. The interval between pre-anesthesia induction and euthanasia was controlled within 10 minutes. Thoracotomy was performed using sharp scissors, and the beating heart was rapidly excised and transferred into calcium- and magnesium-free Hank's Balanced Salt Solution (HBSS), then the ventricles were dissected, mechanically dissociated, and digested enzymatically using 0.25% trypsin at 37°C for 15 minutes. The digested suspension was neutralized with fetal bovine serum (FBS), centrifuged, and the isolated cells were resuspended in complete DMEM/F12 medium supplemented with 90% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. Subsequent to filtration using a 40 µm mesh, the suspension was plated on uncoated 100 mm culture dishes and cultured under standard conditions for 90 min to facilitate fibroblast adherence. The cardiomyocyte-enriched non-adherent fraction was harvested and pelleted. Both cardiomyocytes and fibroblasts were subsequently cultured in DMEM/F12 supplemented 2% FBS and 1% penicillin–streptomycin under standard culture conditions. To induce hypertrophy, cardiomyocytes were serum-starved for 24 h before exposure to 150 nM angiotensin II (Ang II, HY-13948, MCE). For functional assays, cells were treated as follows: miR-20a-5p mimic (GenePharma), miR-20a-5p inhibitor (GenePharma), or rapamycin (HY-10219R, MCE). Cell transfection The Lipofectamine 3000 transfection reagent (Invitrogen, L3000015, Carlsbad, CA) was used to transfect miR-20a-5p mimic, inhibitor and their negative controls into neonatal rat cardiomyocytes and cardiac fibroblasts. After 48 hours of culture, the cells were harvested for downstream applications. Cell Counting Kit-8 assay A total of 5 × 10⁴ cells per well were seeded in 96-well plates and cultured for 48 h. Ten microliters of CCK-8 reagent (Beyotime, C0038) was then introduced into each well, and plates were incubated for 2 hours. Cell viability was determined by measuring the absorbance at 450 nm on a microplate reader (Molecular Devices, USA). Flow cytometry The apoptosis of cells was detected by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit (C1062S, Beyotime, China). The cells were harvested with EDTA-free trypsin, washed, and resuspended in binding buffer. Following a 15-minute incubation in the dark with Annexin V-FITC and propidium iodide, apoptotic cells were subsequently quantified using a flow cytometer (Beckman Coulter, USA). Western blot Proteins were extracted from the samples using RIPA lysis buffer (P0013B, Beyotime, China) and protease inhibitors (C0001, TargetMol, USA), and its content was assessed using the bicinchoninic acid (BCA) method. (P0010, Beyotime, China). Protein samples (20–40 µg) was electrophoresed on an SDS-polyacrylamide gel, electroblotted onto a PVDF membrane (IPVH00010, Millipore, USA), followed by incubation with the specified primary antibodies: p-S6 (1:2000, 80130-2-RR, Proteintech), LC3B (1:6000, 14600-1-AP, Proteintech), p62 (1:10000, 84826-1-RR, Proteintech), Cleaved Caspase-3 (1:1000, #9661, CST), Bcl-2 (1: 500, 26593-1-AP, Proteintech), α-SMA (1:1000, 19245S, CST), Gal-3 (1:1000, #89572, CST), and reference GAPDH (1:5000, 10494-1-AP, Proteintech). Then, the reaction was incubated with HRP-labeled secondary antibody (1:3000, RGAR001, Proteintech), and developed with ECL reagent (34577, Thermo Fisher Scientific, USA). Quantitative analysis was performed using Image Lab software. Quantitative RealTime-PCR Following extraction with the RNA extraction kit (AM1912, Invitrogen, USA), total RNA was reverse-transcribed using the PrimeScript RT Reagent Kit (RR047A, Takara, Japan). Quantitative PCR was carried out on the QuantStudio 5 system (Applied Biosystems, USA) using SYBR Green Master Mix (A6001, Promega). The thermal cycling program consisted of an initial denaturation at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds, 57°C for 30 seconds, and 72°C for 30 seconds. For normalization, endogenous controls included U6 for miRNA and GAPDH for mRNA. Relative quantification was performed using the 2^–ΔΔCT method. All oligonucleotide primers were custom-synthesized by Sangon Biotech: miR-20a-5p forward 5’-AAAGTGCTTATAGTGCAGG-3’, reverse 5’-GAACATGTCTGCGTATCTC-3’; U6 forward 5’-CTCGCTTCGGCAGCACATATACT-3’, reverse 5’-CGCTTCACGAATTTGCGTGT-3’. Dual luciferase reporter gene assay Luciferase reporter plasmids containing either the wild-type (Ddit4-wt) or mutant (Ddit4-mut) 3′UTR of Ddit4 were co-transfected into HEK-293T cells with miR-20a-5p mimic or NC mimic (2 µg). The cell density was 7.5×104 per well (ATCC, USA). Transfection was carried out with Lipofectamine 3000 (Invitrogen, L3000015, Carlsbad, CA). Following a 48-h incubation, the fluorescence values were measured using the Dual-Luciferase Assay System (E1910, Promega, USA), with the fluorescence ratio being Renilla luciferase/Firefly luciferase. Echocardiography examination At week 4, cardiac function was assessed by transthoracic echocardiography using a high-frequency imaging system (Vevo 2100, VisualSonics, Canada). Following anesthesia, rats were positioned for M-mode imaging, and parameters including left ventricular ejection fraction (EF), fractional shortening (FS), end-diastolic diameter (LVEDD), and end-systolic diameter (LVIDs) were recorded. Measurements were performed independently by two blinded operators, and mean values were used for analysis. HE staining After echocardiography, rats were euthanized and myocardial tissues from the infarct border zone were fixed by immersion in 4% PFA (pH 7.4) overnight at 4°C. Following dehydration through graded ethanol, tissues were paraffin-embedded and sectioned. Sections were subjected to H&E staining (Beyotime, C0105S) for the assessment of tissue morphology. Collagen deposition was analyzed through Masson’s trichrome staining (Beyotime, C0189S). I Whole-slide images were acquired using a panoramic slide scanner, and fibrotic areas were quantified with ImageJ software. Statistical analyses GraphPad Prism 9.5.0 was used for all statistical analyses. Continuous data are expressed as the mean ± standard deviation. Comparisons between two groups utilized unpaired Student's t-tests, while comparisons across more than two groups were performed using one-way variance (ANOVA) with Tukey’s HSD post hoc analysis. Statistical significance was defined as P < 0.05. 3. Results 3.1 Cardiac exosomes exhibit a significant enrichment of miR-20a-5p after myocardial infarction. An acute myocardial infarction model was established in 8-week-old male SD rats via ligation of the left anterior descending coronary artery. Rats were assigned to sham (Sham, n = 4) or MI (MI, n = 4) groups. In the Sham group, the artery was sutured without ligation, whereas complete ligation was performed in the MI group. Survival was monitored for 24 hours, and hearts were collected on day 3 for histological analysis. TTC and Evans Blue staining showed uniform red-blue coloration in Sham hearts, indicating no infarction. In MI hearts, distinct pale regions unstained by TTC marked deactivated myocardial cells with clear boundaries, sharply contrasting with surrounding tissue (Fig. 1 A). Quantification confirmed a significant increase in necrotic area in MI hearts versus Sham (Fig. 1 B), validating successful model induction. At four weeks post-myocardial infarction, rats were sacrificed, and cardiac tissues were harvested. Cardiomyocytes from Sham and MI groups were isolated using enzymatic digestion combined with adherent culture. Cells were maintained in serum-free IMDM, and culture supernatants were collected for extracellular vesicle (EV) isolation. EVs (Sham-EXO and MI-EXO) were purified via 0.22 µm filtration and ultrafiltration. Transmission electron microscopy confirmed characteristic double-membrane, spherical vesicles of uniform size in both groups, confirming successful EV isolation (Fig. 2 A). Exosomal miR-20a-5p levels following myocardial infarction were assessed using RT-qPCR. miR-20a-5p expression was significantly elevated in MI-EXO relative to Sham-EXO (Fig. 2 B), indicating that myocardial infarction alters exosomal miRNA profiles. 3.2 MI-EXO exhibits enhanced cellular uptake and exerts detrimental effects on cardiomyocytes and fibroblasts DiI-labeled Sham-EXO and MI-EXO were added to neonatal rat cardiomyocytes and cardiac fibroblasts to assess exosome uptake. Fluorescence microscopy showed that both exosome types were internalized and distributed in the cytoplasm. Image analysis revealed significantly higher intracellular red fluorescence in cells treated with MI-EXO compared with Sham-EXO, indicating enhanced uptake of MI-EXO (Fig. 3 A). Functional assays demonstrated that MI-EXO altered cell status. CCK-8 analysis showed reduced cardiomyocyte viability but increased fibroblast viability compared with Sham-EXO (Fig. 3 B). Annexin V-FITC flow cytometry revealed that MI-EXO significantly increased cardiomyocyte apoptosis, whereas fibroblast apoptosis remained unchanged (Fig. 3 C). Western blot analysis indicated that MI-EXO upregulated α-SMA and Gal-3 expression in fibroblasts, suggesting fibroblast-to-myofibroblast transformation (Fig. 3 D). Collectively, these results indicate that MI-EXO enhances intracellular delivery, promotes cardiomyocyte injury, and accelerates cardiac fibrosis. miR-20a-5p acts as a key regulator to aggravate Ang II-induced apoptosis and fibrogenesis via mTORC1/S6 activation An Ang II-induced apoptosis model in mouse cardiomyocytes was established to mimic the stress environment of myocardial infarction, and miR-20a-5p expression was modulated to assess its effects on the mTORC1/S6 pathway and cell fate. RT-qPCR confirmed effective overexpression and inhibition of miR-20a-5p (Fig. 4 A). Western blot analysis demonstrated that Ang II treatment promoted the expression of p-S6, caspase-3, and p62, and decreased Bcl-2 and the LC3B-II/I ratio, indicating mTORC1 activation, enhanced apoptosis, and suppressed autophagy. Compared with Ang II alone, miR-20a-5p overexpression further elevated p-S6, caspase-3, and p62, while reducing Bcl-2 and LC3B-II/I, whereas miR-20a-5p inhibition produced the opposite effect (Fig. 4 B). Flow cytometry confirmed that miR-20a-5p overexpression increased cardiomyocyte apoptosis, while inhibition reduced it (Fig. 4 C). The same approach was applied to cardiac fibroblasts. RT-qPCR verified effective modulation of miR-20a-5p (Fig. 4 D). Western blot showed that Ang II upregulated p-S6, α-SMA, Gal-3, caspase-3, and p62, while downregulating Bcl-2 and LC3B-II/I. miR-20a-5p overexpression further enhanced pro-fibrotic and pro-apoptotic protein expression, whereas inhibition reversed these effects (Fig. 4 E). Flow cytometry confirmed corresponding changes in fibroblast apoptosis (Fig. 4 F). Together, these findings indicate that miR-20a-5p activates the mTORC1/S6 pathway in cardiomyocytes and fibroblasts, promoting apoptosis, inhibiting autophagy, and exacerbating cardiac fibrosis. 3.4 The miR-20a-5p/Ddit4/mTORC1-S6 axis is elucidated as a key mechanistic driver of angiotensin II-induced cardiac cell dysfunction To determine whether miR-20a-5p acts through the mTORC1/S6 pathway, Ang II-induced injury models were established in neonatal rat cardiomyocytes, with interventions using miR-20a-5p inhibitor and the mTORC1 inhibitor rapamycin. Cells were assigned to Ang II + miR-20a-5p inhibitor, Ang II + rapamycin, and Ang II + miR-20a-5p inhibitor + rapamycin groups. RT-qPCR confirmed effective miRNA modulation: miR-20a-5p was upregulated in the Ang II + rapamycin group but unchanged in the combined treatment group (Fig. 5 A). Western blotting showed that both rapamycin-treated groups reduced p-S6, caspase-3, and p62, while increasing Bcl-2 and LC3B-II/I ratios, with the combined treatment producing the strongest effect (Fig. 5 B). Flow cytometry confirmed that apoptosis was lowest in the Ang II + miR-20a-5p inhibitor + rapamycin group (Fig. 5 C), indicating that miR-20a-5p promotes cardiomyocyte apoptosis via mTORC1/S6 activation and that rapamycin exerts a synergistic protective effect. The same interventions in cardiac fibroblasts showed similar trends. RT-qPCR confirmed miR-20a-5p inhibition in relevant groups (Fig. 5 D). Western blotting revealed that rapamycin and combined treatment reduced p-S6, α-SMA, Gal-3, caspase-3, and p62, while increasing Bcl-2 and LC3B-II/I ratios, with the combination producing the most pronounced effects (Fig. 5 E). Flow cytometry corroborated these findings, showing maximal reduction in fibroblast apoptosis in the combined treatment group (Fig. 5 F). To investigate whether miR-20a-5p directly regulates Ddit4, a dual-luciferase reporter assay was conducted in HEK-293T cells. Plasmids containing either the native 3’ untranslated region (Ddit4-wt) or a mutated version (Ddit4-mut) of Ddit4 were co-introduced with a miR-20a-5p mimic or a negative control. In cells transfected with Ddit4-wt, luciferase activity was markedly reduced by miR-20a-5p, whereas no appreciable effect was detected in cells carrying the Ddit4-mut construct. These results demonstrate that miR-20a-5p directly interacts with the 3’ UTR of Ddit4, thereby downregulating its expression (Fig. 5 G, H). Collectively, these results indicate that miR-20a-5p activates the mTORC1/S6 pathway, promotes fibroblast fibrotic transformation and apoptosis, and that pathway inhibition—particularly combined with miR-20a-5p suppression—effectively reverses these pathological processes. 3.5 Synergistic therapeutic effect of miR-20a-5p inhibition and rapamycin on cardiac remodeling via mTORC1/S6 suppression in a rat MI model In the rat MI model, the involvement of the miR-20a-5p/mTORC1/S6 pathway in ventricular remodeling was further evaluated. Male SD rats (8 weeks old) were randomly assigned to five groups: Sham, MI, MI + miR-20a-5p inhibitor, MI + rapamycin, and MI + miR-20a-5p inhibitor + rapamycin. Interventions were administered weekly for four weeks starting immediately post-surgery. Echocardiography revealed that MI reduced left ventricular EF and FS while LVEDD and LVIDs diameters, indicating impaired cardiac function and remodeling. miR-20a-5p inhibition or rapamycin partially restored EF and FS and reduced LVEDD and LVEDV, with the combined treatment showing the most pronounced improvement (Fig. 6 A). HE and Masson staining confirmed disordered myocardial architecture and extensive fibrosis in MI hearts, which were alleviated by miR-20a-5p inhibition and rapamycin, particularly in the combination group (Fig. 6 B, C). Molecular analyses of the infarct border zone showed that MI upregulated miR-20a-5p, while inhibitor interventions effectively reduced its expression (Fig. 6 D). Western blotting demonstrated that MI increased p-S6, α-SMA, Gal-3, caspase-3, and p62, and decreased Bcl-2 and LC3B-II/I ratios, indicating mTORC1/S6 activation, enhanced apoptosis, increased fibrosis, and suppressed autophagy. miR-20a-5p inhibition and rapamycin reversed these changes, with the combined intervention exerting the strongest effect (Fig. 6 E). Collectively, the data indicate that MI-driven upregulation of miR-20a-5p activates the mTORC1/S6 signaling cascade, promoting apoptosis and ventricular remodeling, while concurrent miR-20a-5p inhibition and rapamycin treatment attenuate these adverse effects. 4. Discussion In cardiovascular research, microRNAs (miRNAs) are recognized as key regulators of cardiac development, homeostasis, and disease 21 . By fine-tuning the expression of multiple target genes, miRNAs contribute to the pathophysiology of various cardiovascular disorders—including myocardial hypertrophy, heart failure, atherosclerosis, and myocardial infarction 22 . Notably, miRNAs are not confined to the intracellular space; they can also be secreted and transported via vesicles such as exosomes, facilitating intercellular communication and functional modulation of recipient cells 23 . This intercellular shuttle mechanism underscores the potential of exosomal miRNAs as both minimally invasive biomarkers and novel therapeutic vehicles. Following MI, miRNAs help regulate critical processes such as inflammation, fibrosis, angiogenesis, and apoptosis 24 . Among these, miR-20a-5p, a member of the miR-17-92 cluster, has been implicated in diverse physiological and pathological pathways 25 . The mammalian target of rapamycin complex 1 (mTORC1) serves as a central regulator of cell metabolism, autophagy, and protein synthesis, and plays a vital role in cardiac stress response and adaptation 26 . Its activity is modulated by numerous extracellular and intracellular signals, such as growth factors, nutrient availability, oxygen levels, and amino acid concentrations. Dysregulated mTORC1 signaling has been strongly linked to pathological cardiac remodeling; however, the upstream triggers, especially those mediated by exosomal miRNAs in the ischemic heart, are not well characterized. In the present study, we demonstrated that exosome-derived miR-20a-5p is markedly upregulated following myocardial infarction and exerts detrimental effects on both cardiomyocytes and cardiac fibroblasts. Our data showed that MI-derived exosomes are more efficiently internalized by target cells, thereby amplifying the pathological contribution of miR-20a-5p. This enhanced uptake may be attributed to alterations in exosomal surface composition under ischemic conditions, a phenomenon warranting further investigation. Mechanistically, miR-20a-5p directly targets Ddit4, resulting in sustained activation of the mTORC1/S6 pathway, which promotes cardiomyocyte apoptosis, impairs autophagy, and drives fibroblast-to-myofibroblast transition. These findings establish miR-20a-5p/mTORC1/S6 as a critical regulatory axis in post-MI ventricular remodeling(Figure 7 ). Our study bridges an important knowledge gap by elucidating how an exosomal miRNA can serve as an upstream orchestrator of mTORC1-driven pathology in the heart. Previous research has underscored the role of mTORC1 signaling in cardiomyocyte survival and fibrosis; however, its upstream regulatory mechanisms have remained insufficiently defined. Our results extend this knowledge by identifying exosomal miR-20a-5p as a novel upstream modulator of mTORC1/S6 signaling, bridging extracellular communication and intracellular remodeling responses. Importantly, pharmacological inhibition of mTORC1 using Rapamycin, in combination with miR-20a-5p suppression, produced synergistic cardioprotective effects, supporting the translational relevance of this pathway. This combination strategy potentially overcomes the limitations of systemic mTOR inhibition by providing a more targeted and multi-level therapeutic approach. Although significant progress has been made in the research of miRNAs as therapeutic targets for myocardial infarction, several important challenges and limitations still need to be addressed. Firstly, the environmental dependence of miRNA functions makes their regulatory networks extremely complex. Take miR-20a-5p as an example, it plays a protective role in diabetic cardiomyopathy, but shows a damaging effect in this study and some other models. This context-dependent duality highlights the importance of disease-specific miRNA profiling and cautions against broad extrapolation of miRNA functions across different pathologies. This difference may be related to the differences in the expression profiles of target genes under different disease conditions. Secondly, the specificity and efficiency of drug delivery systems still need to be improved. An ideal delivery system should be able to specifically deliver miRNA inhibitors or mimics to target cells without affecting other tissues and organs. The emerging exosomes as natural delivery carriers may provide new ideas for solving this problem. Future efforts should focus on engineering exosomes or other nanovehicles for cardiac-specific delivery of miR-20a-5p antagonists. Thirdly, temporal dynamic changes and dose effects are also key considerations. Ventricular remodeling after myocardial infarction is a dynamic process, and different stages may involve different dominant mechanisms. Therefore, the choice of intervention timing may be crucial. Our research team has found that inhibiting miR-20a-5p in the early stage of myocardial infarction can significantly improve long-term prognosis, but its function in late remodeling still needs further study. This suggests the existence of a therapeutic window for miRNA-based interventions, which must be precisely defined in future clinical translation. In conclusion, this study not only reveals a new mechanism by which exosomal miR-20a-5p activates the mTORC1/S6 signaling pathway by targeting Ddit4, thereby promoting ventricular remodeling, but also provides potential targets for the precise treatment of myocardial infarction. By delineating this novel exosome-miRNA-mTORC1 axis, we offer a comprehensive mechanistic framework that integrates intercellular communication with intracellular signaling in post-infarction remodeling. Future research should focus on addressing these challenges, promoting the clinical translation of this discovery, and ultimately advancing patient prognosis and overall quality of life with myocardial infarction. 5. Conclusion In summary, our study demonstrates that exosome-derived miR-20a-5p is significantly upregulated following myocardial infarction and serves as a critical mediator of maladaptive ventricular remodeling. We provide compelling evidence that miR-20a-5p directly targets the 3' UTR of Ddit4, a known suppressor of the mTORC1 pathway, thereby relieving its inhibitory effect and leading to sustained activation of mTORC1/S6 signaling. This activation promotes cardiomyocyte apoptosis, impairs autophagic flux, and drives fibroblast-to-myofibroblast transition, collectively contributing to fibrosis and functional deterioration of the heart. Furthermore, our findings highlight the therapeutic potential of targeting this pathway. Both genetic inhibition of miR-20a-5p and pharmacological blockade of mTORC1 with rapamycin attenuated these adverse effects, improving cell survival, reducing fibrosis, and restoring cardiac function in a rat model of MI. Notably, the combination of miR-20a-5p inhibition and rapamycin treatment yielded synergistic benefits, underscoring the clinical relevance of dual-pathway intervention. These results not only elucidate a novel mechanism by which exosomal miRNAs regulate post-infarction remodeling but also position miR-20a-5p as a promising circulating biomarker for early detection of remodeling risk and a potential therapeutic target for preventing the progression to heart failure. Future studies should focus on validating these findings in human samples, optimizing delivery strategies for miRNA-based therapeutics, and exploring the temporal dynamics of miR-20a-5p expression throughout different stages of post-MI remodeling to guide timed interventions. Declarations Acknowledgments Not applicable. Authors' contributions GG and JW conceived and designed the study. MJ and YG performed the experiments. JW, LH and LZ collected the data and prepared the Figures. JW and GG analyzed the data and drafted the manuscript. All authors have read and approved the final manuscript. Human subjects/informed consent statement No human studies were carried out by the authors for this article. Ethics approval and consent to participate All experimental procedures were conducted in accordance with institutional and national guidelines for the care and use of laboratory animals and were approved by the Ethical Review Board of General Hospital of Ningxia Medical University (Approval No. KYLL-2022-0877). Sources of Funding This work was supported by the Key Research and Development Program of Ningxia (grant number: 2022BSB03091); and the Ningxia Natural Science Foundation Project (grant number: 2023AAC03623). Conflict of interest The authors declare that they have no conflict of interest. Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. References Guo, Z. et al. NEU1 Regulates Mitochondrial Energy Metabolism and Oxidative Stress Post-myocardial Infarction in Mice via the SIRT1/PGC-1 Alpha Axis. Front. Cardiovasc. Med. 9 , 821317. 10.3389/fcvm.2022.821317 (2022). Noorabadi, P., Shahabi Rabori, V., Jamali, S., Jafari, N. & Saberiyan, M. An overview on cardiac regeneration revolution: exploring the promise of stem cell therapies. Mol Biol. Rep May . 28 (1), 511. 10.1007/s11033-025-10580-6 (2025). Yin, W. et al. Macrophage-mediated heart repair and remodeling: A promising therapeutic target for post-myocardial infarction heart failure. J Cell. Physiol Nov . 239 (11), e31372. 10.1002/jcp.31372 (2024). Vinten-Johansen, J. & Shi, W. Perconditioning and postconditioning: current knowledge, knowledge gaps, barriers to adoption, and future directions. J Cardiovasc. Pharmacol. Ther Sep-Dec . 16 (3–4), 260–266. 10.1177/1074248411415270 (2011). Kok, V. C. & Yu, C. C. Cancer-Derived Exosomes: Their Role in Cancer Biology and Biomarker Development. Int. J. Nanomed. 15 , 8019–8036. 10.2147/ijn.S272378 (2020). Solé, C. & Lawrie, C. H. MicroRNAs and Metastasis. Cancers (Basel) . Dec 30 (1). 10.3390/cancers12010096 (2019). Liu, N. & Olson, E. N. MicroRNA regulatory networks in cardiovascular development. Dev Cell Apr . 20 (4), 510–525. 10.1016/j.devcel.2010.03.010 (2010). Small, E. M. & Olson, E. N. Pervasive roles of microRNAs in cardiovascular biology. Nature Jan . 20 (7330), 336–342. 10.1038/nature09783 (2011). Abdellatif, M. Differential expression of microRNAs in different disease states. Circ Res Feb . 17 (4), 638–650. 10.1161/circresaha.111.247437 (2012). Raposo, G. & Stoorvogel, W. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell. Biol Feb . 18 (4), 373–383. 10.1083/jcb.201211138 (2013). Wang, L. et al. Exosomal miRNAs in pancreatitis: Mechanisms and potential applications (Review). Mol Med. Rep Aug . 32 (2). 10.3892/mmr.2025.13575 (2025). Bayly-Jones, C. et al. Structure of the human TSC:WIPI3 lysosomal recruitment complex. Sci Adv Nov . 22 (47), eadr5807. 10.1126/sciadv.adr5807 (2024). Zhang, P., Shan, T., Liang, X., Deng, C. & Kuang, S. Mammalian target of rapamycin is essential for cardiomyocyte survival and heart development in mice. Biochem Biophys. Res. Commun Sep. 12 (1), 53–59. 10.1016/j.bbrc.2014.08.046 (2014). Chopp, M. & Zhang, Z. G. Emerging potential of exosomes and noncoding microRNAs for the treatment of neurological injury/diseases. Expert Opin. Emerg. Drugs . 20 (4), 523–526. 10.1517/14728214.2015.1061993 (2015). Wang, X. et al. The Role of Exosomal microRNAs and Oxidative Stress in Neurodegenerative Diseases. Oxid. Med. Cell. Longev. 2020 , 3232869. 10.1155/2020/3232869 (2020). Gao, G., Chen, W., Liu, M., Yan, X. & Yang, P. Circulating MicroRNAs as Novel Potential Biomarkers for Left Ventricular Remodeling in Postinfarction Heart Failure. Dis. Markers . 2019 , 5093803. 10.1155/2019/5093803 (2019). Dehghan Manshadi, M. et al. Lower cytoplasmic expression of DDIT4 is associated with poor prognosis in gastric cancer patients. Discov Oncol Mar. 22 (1), 374. 10.1007/s12672-025-02065-6 (2025). Todosenko, N. et al. Causal Links between Hypovitaminosis D and Dysregulation of the T Cell Connection of Immunity Associated with Obesity and Concomitant Pathologies. Biomedicines Nov . 23 (12). 10.3390/biomedicines9121750 (2021). Panuzzo, C. et al. mTORC2 Is Activated under Hypoxia and Could Support Chronic Myeloid Leukemia Stem Cells. Int J. Mol. Sci Jan . 8 (2). 10.3390/ijms24021234 (2023). Hwang, D. et al. YAP promotes global mRNA translation to fuel oncogenic growth despite starvation. Exp Mol. Med Oct. 56 (10), 2202–2215. 10.1038/s12276-024-01316-w (2024). Ro, W. B. et al. Identification and Characterization of Circulating MicroRNAs as Novel Biomarkers in Dogs With Heart Diseases. Front. Vet. Sci. 8 , 729929. 10.3389/fvets.2021.729929 (2021). Fic, P., Kowalczuk, K., Grabarska, A. & Stepulak, A. [MicroRNA–a new diagnostic tool in coronary artery disease and myocardial infarction]. Postepy Hig Med. Dosw (Online) Apr . 28 , 68:410–418. 10.5604/17322693.1100348 (2014). Mikro-RNA–nowe szanse diagnostyczne w chorobie niedokrwiennej i zawale serca. Zheng, P. et al. Exosomal transfer of tumor-associated macrophage-derived miR-21 confers cisplatin resistance in gastric cancer cells. J Exp. Clin. Cancer Res Apr . 13 (1), 53. 10.1186/s13046-017-0528-y (2017). Hao, Y. et al. Identification and validation of mitophagy-related genes in acute myocardial infarction and ischemic cardiomyopathy and study of immune mechanisms across different risk groups. Front. Immunol. 16 , 1486961. 10.3389/fimmu.2025.1486961 (2025). Zhou, J. et al. A Novel Regulatory Circuit C/EBPα/miR-20a-5p/TOB2 Regulates Adipogenesis and Lipogenesis. Front. Endocrinol. (Lausanne) . 10 , 894. 10.3389/fendo.2019.00894 (2019). Leone, R. D. & Amaravadi, R. K. Autophagy: a targetable linchpin of cancer cell metabolism. Trends Endocrinol. Metab Apr . 24 (4), 209–217. 10.1016/j.tem.2013.01.008 (2013). Additional Declarations No competing interests reported. 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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-9333795","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":626827738,"identity":"d04499b9-cf1e-40a5-815a-da8d8ef914d0","order_by":0,"name":"Jingjing Wang","email":"","orcid":"","institution":"General Hospital of Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jingjing","middleName":"","lastName":"Wang","suffix":""},{"id":626827740,"identity":"44cae66a-1544-4f52-b725-ba74ac8fcd6c","order_by":1,"name":"Meihua Jin","email":"","orcid":"","institution":"General Hospital of Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Meihua","middleName":"","lastName":"Jin","suffix":""},{"id":626827741,"identity":"c97f13a2-215f-4d47-b421-ee0331193258","order_by":2,"name":"Ying Guo","email":"","orcid":"","institution":"General Hospital of Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Guo","suffix":""},{"id":626827742,"identity":"ccf46c67-7bc7-4c37-9264-f2b8cba5b4d9","order_by":3,"name":"Lin He","email":"","orcid":"","institution":"General Hospital of Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"He","suffix":""},{"id":626827744,"identity":"7554fb73-873e-417e-9dd5-eac8c958e4f1","order_by":4,"name":"Lili Zhang","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lili","middleName":"","lastName":"Zhang","suffix":""},{"id":626827747,"identity":"e8747eac-fa57-4093-9df6-840b56e3947d","order_by":5,"name":"Guangyuan Gao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYBACAwbmxgNAmoefvbHx4QfitDA2gLTISfYcbjaWIEWLscGN9DYBHmK0mLM3Nhz48Ks2ccPNh20MEgx2croNBLRY9hxsODiz73jizNuJbQ8KGJKNzQ4QctiNxIbDvD3HEvtuJ7YbSDAcSNxGUMv9hxAtDTcPtknwEKXlBmPDYZ4fNcYCNxiJ1XImEeiXhgPAQE4EBrIBMX45fvjggw9/6oBRefzhww8VdnIEtYABY9thmAnEKAeDP3VEKx0Fo2AUjIIRCABR51FftW+cRQAAAABJRU5ErkJggg==","orcid":"","institution":"General Hospital of Ningxia Medical University","correspondingAuthor":true,"prefix":"","firstName":"Guangyuan","middleName":"","lastName":"Gao","suffix":""}],"badges":[],"createdAt":"2026-04-06 12:11:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9333795/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9333795/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107635330,"identity":"918b6fd3-e50c-4b14-91b4-02870b0d27e4","added_by":"auto","created_at":"2026-04-23 12:25:52","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":474748,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction and validation of an acute myocardial infarction model in rats based on ligation of the left anterior descending coronary artery\u003c/strong\u003e. A: Morphological assessment of rat heart sections was performed using TTC and Evans Blue staining to verify the effectiveness of myocardial infarction model induction The Sham group showed uniform blue-red color, while the MI group presented obvious infarction areas. B: Rat heart tissue was stained with TTC and Evans Blue to evaluate necrotic regions and measure the infarcted area quantitatively. There were 5 animals in each group. *** indicates comparison between the two groups, and P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9333795/v1/b81cf5265565b2a36efbdef1.jpg"},{"id":107634775,"identity":"11383da1-42c0-438d-b92b-9b0a550d0209","added_by":"auto","created_at":"2026-04-23 12:23:25","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":428967,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCardiac Cell-Derived Exosome Extraction and miR-20a-5p Expression Assessment. \u003c/strong\u003eA: Transmission electron microscopy (TEM) was used to observe the structure and morphology of exosomes derived from cardiac cells in the Sham and MI groups, with a 100 nm scale bar. B: Exosomal miR-20a-5p expression in the Sham and MI groups was assessed via RT-qPCR. Data are from three independent experiments, *** indicates a comparison between the two groups, and P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9333795/v1/219c2fd8162de82ebffdf85c.jpg"},{"id":107634772,"identity":"68de6f50-081d-449c-87de-2113200a8480","added_by":"auto","created_at":"2026-04-23 12:23:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2245410,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe uptake and functional impact of myocardial infarction-derived exosomes on neonatal rat cardiomyocytes and cardiac fibroblasts\u003c/strong\u003e. A: Fluorescence microscopy was used to examine the uptake of DiI-labeled Sham-EXO and MI-EXO in neonatal rat cardiomyocytes and cardiac fibroblasts. The red fluorescence indicated the distribution of exosomes. Bar: 50 μm; B: CCK-8 assay to assess cellular metabolic activity of neonatal rat cardiomyocytes and fibroblasts after exosome treatment; C: Evaluation of Cardiomyocyte and Fibroblast Apoptosis Post-Exosome Treatment via Annexin V-FITC and PI Flow Cytometry; D: Western blot analysis was used to measure α-SMA and Gal-3 expression. Data are from three independent experiments, ** indicates comparison between the two groups, P \u0026lt; 0.01, *** P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9333795/v1/c244dcfb2aa8df0b09427b91.jpg"},{"id":107634786,"identity":"56909963-f3b4-46e1-adc9-c6b02150037a","added_by":"auto","created_at":"2026-04-23 12:23:27","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3395960,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModulation of the mTORC1/S6 pathway by miR-20a-5p alters the functional properties of both cardiomyocytes and cardiac fibroblasts following Ang II stimulation.\u003c/strong\u003e A: The expression of miR-20a-5p in neonatal rat cardiomyocytes across groups was quantified using RT-qPCR; B: Protein abundance of p-S6, LC3B-I/II, p62, caspase-3, and Bcl-2 was assessed via Western blot; C: Cardiomyocyte apoptosis was evaluated by Annexin V-FITC flow cytometry; D: miR-20a-5p expression in neonatal rat cardiac fibroblasts from each group was determined through RT-qPCR; E: Western blot analysis was performed to examine the levels of p-S6, LC3B-I/II, p62, caspase-3, Bcl-2, α-SMA, and Gal-3 proteins; F: Apoptosis rates in cardiac fibroblasts were measured using Annexin V-FITC flow cytometry. All cellular experiments were conducted in triplicate. * indicates comparison between two groups, P \u0026lt; 0.05, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9333795/v1/a29ed5c47cf69adf23bf64b6.jpg"},{"id":107635093,"identity":"878f365c-bc54-46b5-a606-141ae503c5f5","added_by":"auto","created_at":"2026-04-23 12:25:05","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6005892,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVerify that miR-20a-5p modulates myocardial cell and myocardial fibroblast damage through the mTORC1/S6 pathway. \u003c/strong\u003eA: The relative expression of miR-20a-5p in neonatal rat cardiomyocytes across different treatment groups were assessed using RT-qPCR; B: Protein abundance of p-S6, LC3B-I/II, p62, caspase-3, and Bcl-2 was assessed via Western blot; C: Flow cytometry employing Annexin V-FITC was utilized to determine myocardial cell apoptosis; D: miR-20a-5p expression in neonatal rat cardiac fibroblasts under various interventions was measured by qPCR; E: Western blot was performed to examine the expressions of p-S6, LC3B-I/II, p62, caspase-3, Bcl-2, α-SMA, and Gal-3; F: Apoptosis of cardiac fibroblasts was detected using Annexin V-FITC flow cytometry; G: Bioinformatic analysis revealed a complementary binding site for miR-20a-5p within the Ddit4 sequence; H: A dual-luciferase reporter assay was performed in HEK293T cells co-transfected with pmirGLO-Ddit4-Wt or pmirGLO-Ddit4-Mut plasmids together with miR-20a-5p mimic. All cellular experiments were conducted in triplicate, ** indicates comparison between the two groups, P \u0026lt; 0.01, *** P \u0026lt; 0.001, **** P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9333795/v1/1b5b9e5fccd7f9f8f4c69828.jpg"},{"id":107634782,"identity":"8a02c316-d8f1-4e04-a041-cab47e69d04d","added_by":"auto","created_at":"2026-04-23 12:23:26","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":12192270,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTo elucidate the crucial role of the miR-20a-5p/mTORC1/S6 pathway in ventricular remodeling within the body. \u003c/strong\u003eA: The echocardiography was used to detect the changes in left ventricular function in different intervention groups of SD rats, including EF (ejection fraction), FS (short-axis shortening rate), LVEDD (diastolic end diameter), and LVIDs (systolic end diameter). B-C: Hematoxylin and eosin (HE) staining, along with Masson’s trichrome staining, was employed to examine tissue architecture and assess the extent of fibrosis at the infarct border zone, bar: 2mm/50 μm; D: RT-qPCR was performed to determine the mRNA \u0026nbsp;expression level of miR-20a-5p in myocardial tissue; E: Western blot was performed to examine the expressions of p-S6, LC3B-I/II, p62, caspase-3, Bcl-2, α-SMA, and Gal-3. Each group had 5 animals, * indicates comparison between two groups, P \u0026lt; 0.05, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9333795/v1/9b577b6362037988d35de12b.jpg"},{"id":107634778,"identity":"28108a46-dc96-49d7-9282-2c9611af2e48","added_by":"auto","created_at":"2026-04-23 12:23:25","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2661904,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-20a-5p exacerbates cardiomyocyte apoptosis and myocardial fibroblast fibrosis through activating the mTORC1/S6 pathway after myocardial infarction, thereby promoting ventricular remodeling.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9333795/v1/05fef8887a162067b966bcff.jpg"},{"id":108180821,"identity":"4b1bc94e-e692-4f0b-b6dd-304f5a33e13f","added_by":"auto","created_at":"2026-04-30 08:54:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":27689383,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9333795/v1/54e0147f-0ba6-4538-b6a0-7cc98e50df4e.pdf"},{"id":107634780,"identity":"152dc1bc-43bb-4c5f-9900-41138a8b18c1","added_by":"auto","created_at":"2026-04-23 12:23:26","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":9513351,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalImagesforBlots.zip","url":"https://assets-eu.researchsquare.com/files/rs-9333795/v1/f0d7cd2ae7575dcde9cb3a46.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exosomal miR-20a-5p Promotes Cardiac Remodeling After Myocardial Infarction by Regulating mTORC1/S6 Pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHeart failure (HF), a common convergent point for multiple cardiovascular conditions, is most frequently triggered by acute myocardial infarction (MI)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Despite advances in early diagnosis and reperfusion therapy, the incidence of post-MI HF continues to rise. Maladaptive ventricular remodeling\u0026mdash;the pathological link between MI and HF\u003csup\u003e3\u003c/sup\u003e\u0026mdash;remains only partially addressed by current neuroendocrine therapies, while imaging detects remodeling only after irreversible injury\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The lack of reliable molecular markers and incomplete mechanistic understanding thus remain major barriers to early intervention.\u003c/p\u003e \u003cp\u003eAs a class of small non-coding RNAs, microRNAs (miRNAs) post-transcriptionally silence gene expression by inhibiting the translation of target mRNAs and promoting their degradation\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, and circulate in stable forms bound to proteins or encapsulated in exosomes\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Their dynamic expression in MI, HF, and other cardiovascular diseases highlights their promise as minimally invasive biomarkers\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Exosomal miRNAs, in particular, mediate intercellular communication by transferring regulatory signals between cells\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, and are increasingly recognized as modulators of cardiac pathology.\u003c/p\u003e \u003cp\u003eThe mTORC1 pathway, a master regulator of growth, apoptosis, and autophagy, is central to ventricular remodeling\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, systemic mTOR inhibition disrupts essential cardiac functions\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, Exosomal miRNAs may provide a more precise and reversible mode of regulation, with growing evidence supporting their therapeutic potential\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. As evidenced in our prior publication, a rat model of post-myocardial infarction HF was constructed via permanent ligation of the left anterior descending coronary artery\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. We used high-throughput sequencing technology to screen out the differential expression profiles of miRNAs in the myocardium and plasma of rats with post-myocardial infarction heart failure. Through qRT-PCR technology, we conducted batch verification of the expression levels of differentially expressed miRNAs in rat myocardium, plasma, the rat myocardial cell apoptosis model induced by angiotensin II, and the plasma of patients with post-myocardial infarction heart failure. Finally, through specificity and sensitivity analysis, we screened out miRNAs with myocardial expression specificity, which were dynamically related to the pathological process of ventricular remodeling and existed in plasma exosomes and supernatants of cultured myocardial cells: miR-20a-5p\u003csup\u003e16\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur previous studies using rapamycin demonstrated that mTORC1 inhibition in cardiomyocytes enhances protective autophagy, reduces apoptosis, attenuates ventricular remodeling, and enhances cardiac function in rats following myocardial infarction. Bioinformatic analyses identified miR-20a-5p targets, including Ddit4, Sesn1, and Nfkbia, as key regulators of the mTORC1 pathway, influencing cell growth, apoptosis, autophagy, inflammation, and fibrosis. Notably, DDIT4 suppresses mTORC1 activity via the TSC1/TSC2 complex\u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Mechanistically, these findings implicate miR-20a-5p-mediated mTORC1 activation as a pathway that exacerbates remodeling following myocardial infarction..\u003c/p\u003e \u003cp\u003eHere, we explore the involvement of the miR-20a-5p/mTORC1 axis in cardiomyocyte and fibroblast remodeling after MI, and evaluate the therapeutic potential of targeting this pathway.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e \u003cb\u003eAnimal models and groups\u003c/b\u003e \u003c/p\u003e \u003cp\u003e All experimental procedures were conducted in accordance with institutional and national guidelines for the care and use of laboratory animals and were approved by the Ethical Review Board of General Hospital of Ningxia Medical University (Approval No. KYLL-2022-0877). Male Sprague\u0026ndash;Dawley rats (8 weeks old, weighing 230\u0026ndash;250 g) were supplied by the Center for Laboratory Animals, Ningxia Medical University, Yinchuan, China. The animals were housed in a specific pathogen-free facility where the environment was kept at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C with 55\u0026thinsp;\u0026plusmn;\u0026thinsp;5% relative humidity and a 12 h light/dark rhythm. Standard rodent diet and water were provided ad libitum. Myocardial infarction was triggered through the permanent occlusion of the left anterior descending coronary artery. Briefly, the rats were anesthetized with 100% oxygen containing 3% isoflurane supplied by a rodent respirator. Following anesthetization, the thorax was opened in the left parasternal area, and myocardial infarction was induced by ligating the left anterior descending coronary artery using 3\u0026thinsp;\u0026minus;\u0026thinsp;0 suture between the pulmonary cone and the left atrium. Control animals in the sham-operated group experienced the same surgical protocol, with the exception of the coronary artery ligation. The animals' conditions were observed within 24 hours after the operation. The animals were given medication immediately after the operation and were injected with miR-20a-5p inhibitor (20 nmol per rat, tail vein) or Rapamycin (1 mg/kg, intraperitoneal injection, HY-10219R, MCE) once a week for 4 weeks. Animals were then anesthetized using 100% oxygen containing 3% isoflurane and euthanized via a rapid exsanguination from the abdominal aorta and the removal of the hearts. Exsanguination was performed via an abdominal aortic catheter, which permitted the free flow of blood, ,and blood with a total volume of 7\u0026ndash;9 ml per rat was rapidly removed until no longer bleeding. The hearts were then quickly harvested and washed with ice-cold PBS.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTTC\u0026thinsp;+\u0026thinsp;Evans Blue staining\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis is used to evaluate the construction of the myocardial infarction model. After blocking the left coronary artery bifurcation for 24 hours, animals were then euthanized, 2% Evans Blue dye (E2129, Sigma-Aldrich, USA) was injected into the ascending aorta. Immediately after that, the heart was collected and frozen at -80\u0026deg;C for 10 minutes. Then, the heart was transverse into 1\u0026ndash;2 mm thin slices, and stained by incubation under light-protected conditions at 37\u0026deg;C with a 1% TTC (17779, Sigma-Aldrich, USA) solution for 20 minutes. After 24 hours of fixation with 4% paraformaldehyde, the heart slices were photographed. The images were collected by a binocular microscope (Nikon, Japan), and the infarct area was measured using ImageJ software (NIH, USA), and the percentages of viable myocardium in the left ventricle (deep blue), AAR (red and white), and IA (white) were calculated.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIsolation and Cultivation of Cardiac Cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFour weeks post-surgery, rats that experienced abdominal aortic bleeding were euthanized, and their hearts were immediately removed and washed in ice-cold PBS. The hearts were minced to approximately 2 mm\u0026sup3;, and then completely digested for several minutes at 37\u0026deg;C using 0.1% type IV collagenase (17104019, Gibco, USA). After sedimentation, they were adhered to the precoated fibronectin-coated dishes, and the 2-4th generation cells were collected. 1% penicillin-streptomycin Dulbecco modified Eagle's medium/F-12 (12634010, Gibco, USA) containing 10% fetal bovine serum (A5670701, FBS, Gibco, USA) was added, and the cardiac cells were placed in a 37\u0026deg;C, 5% CO₂ constant temperature incubator. After 3\u0026ndash;5 days, the cardiac cells began to adhere to the walls.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExtraction and labeling of extracellular vesicles from cardiac cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCardiac cells were then maintained in IMDM medium lacking serum, and the supernatants were continuously collected for exosome isolation. Extracellular vesicles originating from pericardial adipose tissue were purified using a commercial isolation kit (UR52161, Umibio, China). In brief, the tissue was chopped into small pieces, washed with PBS, and centrifuged at 300 \u0026times; g for 10 minutes to remove residual debris. The resulting pellet was subsequently treated with solution A2 for 20 minutes and then centrifuged at 8,000 \u0026times; g for 10 minutes. The collected supernatant was mixed with solution B2, incubated for 20 min, and subjected to another centrifugation at 8000 \u0026times; g for 20 min at 4\u0026deg;C. The pellet was resuspended in PBS, centrifuged again at 11,000 \u0026times; g for 2 minutes, and the resulting supernatant was passed through an exosome purification filter before undergoing a final centrifugation at 3,000 \u0026times; g for 10 minutes at 4\u0026deg;C. Purified exosomes were divided into aliquots and cryopreserved at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent analysis. Exosomes derived from sham and MI cardiac cells (Sham-EXO and MI-EXO) were employed for uptake assays. For tracking, vesicles were labeled with DiI (Beyotime, China) and monitored in real time under a confocal microscope (Zeiss LSM880), with images captured every 5 min for 6 h.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTransmission Electron Microscope (TEM)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe extracted Sham-EXO and MI-EXO were placed on a copper mesh, exposed to light discharge in air for 1 minute, then 2% phosphotungstic acid was applied to achieve negative staining, and the sample was air-dried under ambient conditions. The morphology was imaged by a Hitachi H-7650 microscope (Hitachi H-7650, Japan) at 80 kV.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell culture and grouping\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNeonatal cardiomyocytes and cardiac fibroblasts were harvested from 1 to 2-day-old SD rats. Pups underwent pre-anesthesia via hypothermia (achieved by indirect contact with ice while wrapped in gauze) to minimize distress. Following abdominal sterilization with 70% ethanol for local anesthesia, euthanasia was performed by decapitation using sterile sharp surgical scissors by a trained researcher. The interval between pre-anesthesia induction and euthanasia was controlled within 10 minutes. Thoracotomy was performed using sharp scissors, and the beating heart was rapidly excised and transferred into calcium- and magnesium-free Hank's Balanced Salt Solution (HBSS), then the ventricles were dissected, mechanically dissociated, and digested enzymatically using 0.25% trypsin at 37\u0026deg;C for 15 minutes. The digested suspension was neutralized with fetal bovine serum (FBS), centrifuged, and the isolated cells were resuspended in complete DMEM/F12 medium supplemented with 90% FBS, 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin. Subsequent to filtration using a 40 \u0026micro;m mesh, the suspension was plated on uncoated 100 mm culture dishes and cultured under standard conditions for 90 min to facilitate fibroblast adherence. The cardiomyocyte-enriched non-adherent fraction was harvested and pelleted. Both cardiomyocytes and fibroblasts were subsequently cultured in DMEM/F12 supplemented 2% FBS and 1% penicillin\u0026ndash;streptomycin under standard culture conditions. To induce hypertrophy, cardiomyocytes were serum-starved for 24 h before exposure to 150 nM angiotensin II (Ang II, HY-13948, MCE). For functional assays, cells were treated as follows: miR-20a-5p mimic (GenePharma), miR-20a-5p inhibitor (GenePharma), or rapamycin (HY-10219R, MCE).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell transfection\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe Lipofectamine 3000 transfection reagent (Invitrogen, L3000015, Carlsbad, CA) was used to transfect miR-20a-5p mimic, inhibitor and their negative controls into neonatal rat cardiomyocytes and cardiac fibroblasts. After 48 hours of culture, the cells were harvested for downstream applications.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell Counting Kit-8 assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA total of 5 \u0026times; 10⁴ cells per well were seeded in 96-well plates and cultured for 48 h. Ten microliters of CCK-8 reagent (Beyotime, C0038) was then introduced into each well, and plates were incubated for 2 hours. Cell viability was determined by measuring the absorbance at 450 nm on a microplate reader (Molecular Devices, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFlow cytometry\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe apoptosis of cells was detected by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit (C1062S, Beyotime, China). The cells were harvested with EDTA-free trypsin, washed, and resuspended in binding buffer. Following a 15-minute incubation in the dark with Annexin V-FITC and propidium iodide, apoptotic cells were subsequently quantified using a flow cytometer (Beckman Coulter, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern blot\u003c/b\u003e \u003c/p\u003e \u003cp\u003eProteins were extracted from the samples using RIPA lysis buffer (P0013B, Beyotime, China) and protease inhibitors (C0001, TargetMol, USA), and its content was assessed using the bicinchoninic acid (BCA) method. (P0010, Beyotime, China). Protein samples (20\u0026ndash;40 \u0026micro;g) was electrophoresed on an SDS-polyacrylamide gel, electroblotted onto a PVDF membrane (IPVH00010, Millipore, USA), followed by incubation with the specified primary antibodies: p-S6 (1:2000, 80130-2-RR, Proteintech), LC3B (1:6000, 14600-1-AP, Proteintech), p62 (1:10000, 84826-1-RR, Proteintech), Cleaved Caspase-3 (1:1000, #9661, CST), Bcl-2 (1: 500, 26593-1-AP, Proteintech), α-SMA (1:1000, 19245S, CST), Gal-3 (1:1000, #89572, CST), and reference GAPDH (1:5000, 10494-1-AP, Proteintech). Then, the reaction was incubated with HRP-labeled secondary antibody (1:3000, RGAR001, Proteintech), and developed with ECL reagent (34577, Thermo Fisher Scientific, USA). Quantitative analysis was performed using Image Lab software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantitative RealTime-PCR\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFollowing extraction with the RNA extraction kit (AM1912, Invitrogen, USA), total RNA was reverse-transcribed using the PrimeScript RT Reagent Kit (RR047A, Takara, Japan). Quantitative PCR was carried out on the QuantStudio 5 system (Applied Biosystems, USA) using SYBR Green Master Mix (A6001, Promega). The thermal cycling program consisted of an initial denaturation at 95\u0026deg;C for 30 seconds, followed by 40 cycles of 95\u0026deg;C for 5 seconds, 57\u0026deg;C for 30 seconds, and 72\u0026deg;C for 30 seconds. For normalization, endogenous controls included U6 for miRNA and GAPDH for mRNA. Relative quantification was performed using the 2^\u0026ndash;ΔΔCT method. All oligonucleotide primers were custom-synthesized by Sangon Biotech: miR-20a-5p forward 5\u0026rsquo;-AAAGTGCTTATAGTGCAGG-3\u0026rsquo;, reverse 5\u0026rsquo;-GAACATGTCTGCGTATCTC-3\u0026rsquo;; U6 forward 5\u0026rsquo;-CTCGCTTCGGCAGCACATATACT-3\u0026rsquo;, reverse 5\u0026rsquo;-CGCTTCACGAATTTGCGTGT-3\u0026rsquo;.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDual luciferase reporter gene assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eLuciferase reporter plasmids containing either the wild-type (Ddit4-wt) or mutant (Ddit4-mut) 3\u0026prime;UTR of Ddit4 were co-transfected into HEK-293T cells with miR-20a-5p mimic or NC mimic (2 \u0026micro;g). The cell density was 7.5\u0026times;104 per well (ATCC, USA). Transfection was carried out with Lipofectamine 3000 (Invitrogen, L3000015, Carlsbad, CA). Following a 48-h incubation, the fluorescence values were measured using the Dual-Luciferase Assay System (E1910, Promega, USA), with the fluorescence ratio being Renilla luciferase/Firefly luciferase.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEchocardiography examination\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAt week 4, cardiac function was assessed by transthoracic echocardiography using a high-frequency imaging system (Vevo 2100, VisualSonics, Canada). Following anesthesia, rats were positioned for M-mode imaging, and parameters including left ventricular ejection fraction (EF), fractional shortening (FS), end-diastolic diameter (LVEDD), and end-systolic diameter (LVIDs) were recorded. Measurements were performed independently by two blinded operators, and mean values were used for analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHE staining\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter echocardiography, rats were euthanized and myocardial tissues from the infarct border zone were fixed by immersion in 4% PFA (pH 7.4) overnight at 4\u0026deg;C. Following dehydration through graded ethanol, tissues were paraffin-embedded and sectioned. Sections were subjected to H\u0026amp;E staining (Beyotime, C0105S) for the assessment of tissue morphology. Collagen deposition was analyzed through Masson\u0026rsquo;s trichrome staining (Beyotime, C0189S). I Whole-slide images were acquired using a panoramic slide scanner, and fibrotic areas were quantified with ImageJ software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analyses\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGraphPad Prism 9.5.0 was used for all statistical analyses. Continuous data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Comparisons between two groups utilized unpaired Student's t-tests, while comparisons across more than two groups were performed using one-way variance (ANOVA) with Tukey\u0026rsquo;s HSD post hoc analysis. Statistical significance was defined as P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Cardiac exosomes exhibit a significant enrichment of miR-20a-5p after myocardial infarction.\u003c/h2\u003e \u003cp\u003eAn acute myocardial infarction model was established in 8-week-old male SD rats via ligation of the left anterior descending coronary artery. Rats were assigned to sham (Sham, n\u0026thinsp;=\u0026thinsp;4) or MI (MI, n\u0026thinsp;=\u0026thinsp;4) groups. In the Sham group, the artery was sutured without ligation, whereas complete ligation was performed in the MI group. Survival was monitored for 24 hours, and hearts were collected on day 3 for histological analysis.\u003c/p\u003e \u003cp\u003eTTC and Evans Blue staining showed uniform red-blue coloration in Sham hearts, indicating no infarction. In MI hearts, distinct pale regions unstained by TTC marked deactivated myocardial cells with clear boundaries, sharply contrasting with surrounding tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Quantification confirmed a significant increase in necrotic area in MI hearts versus Sham (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), validating successful model induction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt four weeks post-myocardial infarction, rats were sacrificed, and cardiac tissues were harvested. Cardiomyocytes from Sham and MI groups were isolated using enzymatic digestion combined with adherent culture. Cells were maintained in serum-free IMDM, and culture supernatants were collected for extracellular vesicle (EV) isolation. EVs (Sham-EXO and MI-EXO) were purified via 0.22 \u0026micro;m filtration and ultrafiltration. Transmission electron microscopy confirmed characteristic double-membrane, spherical vesicles of uniform size in both groups, confirming successful EV isolation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExosomal miR-20a-5p levels following myocardial infarction were assessed using RT-qPCR. miR-20a-5p expression was significantly elevated in MI-EXO relative to Sham-EXO (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), indicating that myocardial infarction alters exosomal miRNA profiles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3.2 MI-EXO exhibits enhanced cellular uptake and exerts detrimental effects on cardiomyocytes and fibroblasts\u003c/h2\u003e \u003cp\u003eDiI-labeled Sham-EXO and MI-EXO were added to neonatal rat cardiomyocytes and cardiac fibroblasts to assess exosome uptake. Fluorescence microscopy showed that both exosome types were internalized and distributed in the cytoplasm. Image analysis revealed significantly higher intracellular red fluorescence in cells treated with MI-EXO compared with Sham-EXO, indicating enhanced uptake of MI-EXO (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFunctional assays demonstrated that MI-EXO altered cell status. CCK-8 analysis showed reduced cardiomyocyte viability but increased fibroblast viability compared with Sham-EXO (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Annexin V-FITC flow cytometry revealed that MI-EXO significantly increased cardiomyocyte apoptosis, whereas fibroblast apoptosis remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Western blot analysis indicated that MI-EXO upregulated α-SMA and Gal-3 expression in fibroblasts, suggesting fibroblast-to-myofibroblast transformation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Collectively, these results indicate that MI-EXO enhances intracellular delivery, promotes cardiomyocyte injury, and accelerates cardiac fibrosis.\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003emiR-20a-5p acts as a key regulator to aggravate Ang II-induced apoptosis and fibrogenesis via mTORC1/S6 activation\u003c/b\u003e \u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eAn Ang II-induced apoptosis model in mouse cardiomyocytes was established to mimic the stress environment of myocardial infarction, and miR-20a-5p expression was modulated to assess its effects on the mTORC1/S6 pathway and cell fate. RT-qPCR confirmed effective overexpression and inhibition of miR-20a-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Western blot analysis demonstrated that Ang II treatment promoted the expression of p-S6, caspase-3, and p62, and decreased Bcl-2 and the LC3B-II/I ratio, indicating mTORC1 activation, enhanced apoptosis, and suppressed autophagy. Compared with Ang II alone, miR-20a-5p overexpression further elevated p-S6, caspase-3, and p62, while reducing Bcl-2 and LC3B-II/I, whereas miR-20a-5p inhibition produced the opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Flow cytometry confirmed that miR-20a-5p overexpression increased cardiomyocyte apoptosis, while inhibition reduced it (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe same approach was applied to cardiac fibroblasts. RT-qPCR verified effective modulation of miR-20a-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Western blot showed that Ang II upregulated p-S6, α-SMA, Gal-3, caspase-3, and p62, while downregulating Bcl-2 and LC3B-II/I. miR-20a-5p overexpression further enhanced pro-fibrotic and pro-apoptotic protein expression, whereas inhibition reversed these effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Flow cytometry confirmed corresponding changes in fibroblast apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eTogether, these findings indicate that miR-20a-5p activates the mTORC1/S6 pathway in cardiomyocytes and fibroblasts, promoting apoptosis, inhibiting autophagy, and exacerbating cardiac fibrosis.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4 The miR-20a-5p/Ddit4/mTORC1-S6 axis is elucidated as a key mechanistic driver of angiotensin II-induced cardiac cell dysfunction\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine whether miR-20a-5p acts through the mTORC1/S6 pathway, Ang II-induced injury models were established in neonatal rat cardiomyocytes, with interventions using miR-20a-5p inhibitor and the mTORC1 inhibitor rapamycin. Cells were assigned to Ang II\u0026thinsp;+\u0026thinsp;miR-20a-5p inhibitor, Ang II\u0026thinsp;+\u0026thinsp;rapamycin, and Ang II\u0026thinsp;+\u0026thinsp;miR-20a-5p inhibitor\u0026thinsp;+\u0026thinsp;rapamycin groups. RT-qPCR confirmed effective miRNA modulation: miR-20a-5p was upregulated in the Ang II\u0026thinsp;+\u0026thinsp;rapamycin group but unchanged in the combined treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Western blotting showed that both rapamycin-treated groups reduced p-S6, caspase-3, and p62, while increasing Bcl-2 and LC3B-II/I ratios, with the combined treatment producing the strongest effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Flow cytometry confirmed that apoptosis was lowest in the Ang II\u0026thinsp;+\u0026thinsp;miR-20a-5p inhibitor\u0026thinsp;+\u0026thinsp;rapamycin group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), indicating that miR-20a-5p promotes cardiomyocyte apoptosis via mTORC1/S6 activation and that rapamycin exerts a synergistic protective effect.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe same interventions in cardiac fibroblasts showed similar trends. RT-qPCR confirmed miR-20a-5p inhibition in relevant groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Western blotting revealed that rapamycin and combined treatment reduced p-S6, α-SMA, Gal-3, caspase-3, and p62, while increasing Bcl-2 and LC3B-II/I ratios, with the combination producing the most pronounced effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Flow cytometry corroborated these findings, showing maximal reduction in fibroblast apoptosis in the combined treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). To investigate whether miR-20a-5p directly regulates Ddit4, a dual-luciferase reporter assay was conducted in HEK-293T cells. Plasmids containing either the native 3\u0026rsquo; untranslated region (Ddit4-wt) or a mutated version (Ddit4-mut) of Ddit4 were co-introduced with a miR-20a-5p mimic or a negative control. In cells transfected with Ddit4-wt, luciferase activity was markedly reduced by miR-20a-5p, whereas no appreciable effect was detected in cells carrying the Ddit4-mut construct. These results demonstrate that miR-20a-5p directly interacts with the 3\u0026rsquo; UTR of Ddit4, thereby downregulating its expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, H).\u003c/p\u003e \u003cp\u003eCollectively, these results indicate that miR-20a-5p activates the mTORC1/S6 pathway, promotes fibroblast fibrotic transformation and apoptosis, and that pathway inhibition\u0026mdash;particularly combined with miR-20a-5p suppression\u0026mdash;effectively reverses these pathological processes.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.5 Synergistic therapeutic effect of miR-20a-5p inhibition and rapamycin on cardiac remodeling via mTORC1/S6 suppression in a rat MI model\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn the rat MI model, the involvement of the miR-20a-5p/mTORC1/S6 pathway in ventricular remodeling was further evaluated. Male SD rats (8 weeks old) were randomly assigned to five groups: Sham, MI, MI\u0026thinsp;+\u0026thinsp;miR-20a-5p inhibitor, MI\u0026thinsp;+\u0026thinsp;rapamycin, and MI\u0026thinsp;+\u0026thinsp;miR-20a-5p inhibitor\u0026thinsp;+\u0026thinsp;rapamycin. Interventions were administered weekly for four weeks starting immediately post-surgery.\u003c/p\u003e \u003cp\u003eEchocardiography revealed that MI reduced left ventricular EF and FS while LVEDD and LVIDs diameters, indicating impaired cardiac function and remodeling. miR-20a-5p inhibition or rapamycin partially restored EF and FS and reduced LVEDD and LVEDV, with the combined treatment showing the most pronounced improvement (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). HE and Masson staining confirmed disordered myocardial architecture and extensive fibrosis in MI hearts, which were alleviated by miR-20a-5p inhibition and rapamycin, particularly in the combination group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMolecular analyses of the infarct border zone showed that MI upregulated miR-20a-5p, while inhibitor interventions effectively reduced its expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Western blotting demonstrated that MI increased p-S6, α-SMA, Gal-3, caspase-3, and p62, and decreased Bcl-2 and LC3B-II/I ratios, indicating mTORC1/S6 activation, enhanced apoptosis, increased fibrosis, and suppressed autophagy. miR-20a-5p inhibition and rapamycin reversed these changes, with the combined intervention exerting the strongest effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eCollectively, the data indicate that MI-driven upregulation of miR-20a-5p activates the mTORC1/S6 signaling cascade, promoting apoptosis and ventricular remodeling, while concurrent miR-20a-5p inhibition and rapamycin treatment attenuate these adverse effects.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn cardiovascular research, microRNAs (miRNAs) are recognized as key regulators of cardiac development, homeostasis, and disease\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. By fine-tuning the expression of multiple target genes, miRNAs contribute to the pathophysiology of various cardiovascular disorders\u0026mdash;including myocardial hypertrophy, heart failure, atherosclerosis, and myocardial infarction\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Notably, miRNAs are not confined to the intracellular space; they can also be secreted and transported via vesicles such as exosomes, facilitating intercellular communication and functional modulation of recipient cells\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. This intercellular shuttle mechanism underscores the potential of exosomal miRNAs as both minimally invasive biomarkers and novel therapeutic vehicles. Following MI, miRNAs help regulate critical processes such as inflammation, fibrosis, angiogenesis, and apoptosis \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Among these, miR-20a-5p, a member of the miR-17-92 cluster, has been implicated in diverse physiological and pathological pathways\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe mammalian target of rapamycin complex 1 (mTORC1) serves as a central regulator of cell metabolism, autophagy, and protein synthesis, and plays a vital role in cardiac stress response and adaptation\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Its activity is modulated by numerous extracellular and intracellular signals, such as growth factors, nutrient availability, oxygen levels, and amino acid concentrations. Dysregulated mTORC1 signaling has been strongly linked to pathological cardiac remodeling; however, the upstream triggers, especially those mediated by exosomal miRNAs in the ischemic heart, are not well characterized.\u003c/p\u003e \u003cp\u003eIn the present study, we demonstrated that exosome-derived miR-20a-5p is markedly upregulated following myocardial infarction and exerts detrimental effects on both cardiomyocytes and cardiac fibroblasts. Our data showed that MI-derived exosomes are more efficiently internalized by target cells, thereby amplifying the pathological contribution of miR-20a-5p. This enhanced uptake may be attributed to alterations in exosomal surface composition under ischemic conditions, a phenomenon warranting further investigation. Mechanistically, miR-20a-5p directly targets Ddit4, resulting in sustained activation of the mTORC1/S6 pathway, which promotes cardiomyocyte apoptosis, impairs autophagy, and drives fibroblast-to-myofibroblast transition. These findings establish miR-20a-5p/mTORC1/S6 as a critical regulatory axis in post-MI ventricular remodeling(Figure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur study bridges an important knowledge gap by elucidating how an exosomal miRNA can serve as an upstream orchestrator of mTORC1-driven pathology in the heart. Previous research has underscored the role of mTORC1 signaling in cardiomyocyte survival and fibrosis; however, its upstream regulatory mechanisms have remained insufficiently defined. Our results extend this knowledge by identifying exosomal miR-20a-5p as a novel upstream modulator of mTORC1/S6 signaling, bridging extracellular communication and intracellular remodeling responses. Importantly, pharmacological inhibition of mTORC1 using Rapamycin, in combination with miR-20a-5p suppression, produced synergistic cardioprotective effects, supporting the translational relevance of this pathway. This combination strategy potentially overcomes the limitations of systemic mTOR inhibition by providing a more targeted and multi-level therapeutic approach.\u003c/p\u003e \u003cp\u003eAlthough significant progress has been made in the research of miRNAs as therapeutic targets for myocardial infarction, several important challenges and limitations still need to be addressed. Firstly, the environmental dependence of miRNA functions makes their regulatory networks extremely complex. Take miR-20a-5p as an example, it plays a protective role in diabetic cardiomyopathy, but shows a damaging effect in this study and some other models. This context-dependent duality highlights the importance of disease-specific miRNA profiling and cautions against broad extrapolation of miRNA functions across different pathologies. This difference may be related to the differences in the expression profiles of target genes under different disease conditions. Secondly, the specificity and efficiency of drug delivery systems still need to be improved. An ideal delivery system should be able to specifically deliver miRNA inhibitors or mimics to target cells without affecting other tissues and organs. The emerging exosomes as natural delivery carriers may provide new ideas for solving this problem. Future efforts should focus on engineering exosomes or other nanovehicles for cardiac-specific delivery of miR-20a-5p antagonists. Thirdly, temporal dynamic changes and dose effects are also key considerations. Ventricular remodeling after myocardial infarction is a dynamic process, and different stages may involve different dominant mechanisms. Therefore, the choice of intervention timing may be crucial. Our research team has found that inhibiting miR-20a-5p in the early stage of myocardial infarction can significantly improve long-term prognosis, but its function in late remodeling still needs further study. This suggests the existence of a therapeutic window for miRNA-based interventions, which must be precisely defined in future clinical translation.\u003c/p\u003e \u003cp\u003eIn conclusion, this study not only reveals a new mechanism by which exosomal miR-20a-5p activates the mTORC1/S6 signaling pathway by targeting Ddit4, thereby promoting ventricular remodeling, but also provides potential targets for the precise treatment of myocardial infarction. By delineating this novel exosome-miRNA-mTORC1 axis, we offer a comprehensive mechanistic framework that integrates intercellular communication with intracellular signaling in post-infarction remodeling. Future research should focus on addressing these challenges, promoting the clinical translation of this discovery, and ultimately advancing patient prognosis and overall quality of life with myocardial infarction.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, our study demonstrates that exosome-derived miR-20a-5p is significantly upregulated following myocardial infarction and serves as a critical mediator of maladaptive ventricular remodeling. We provide compelling evidence that miR-20a-5p directly targets the 3' UTR of Ddit4, a known suppressor of the mTORC1 pathway, thereby relieving its inhibitory effect and leading to sustained activation of mTORC1/S6 signaling. This activation promotes cardiomyocyte apoptosis, impairs autophagic flux, and drives fibroblast-to-myofibroblast transition, collectively contributing to fibrosis and functional deterioration of the heart.\u003c/p\u003e \u003cp\u003eFurthermore, our findings highlight the therapeutic potential of targeting this pathway. Both genetic inhibition of miR-20a-5p and pharmacological blockade of mTORC1 with rapamycin attenuated these adverse effects, improving cell survival, reducing fibrosis, and restoring cardiac function in a rat model of MI. Notably, the combination of miR-20a-5p inhibition and rapamycin treatment yielded synergistic benefits, underscoring the clinical relevance of dual-pathway intervention.\u003c/p\u003e \u003cp\u003eThese results not only elucidate a novel mechanism by which exosomal miRNAs regulate post-infarction remodeling but also position miR-20a-5p as a promising circulating biomarker for early detection of remodeling risk and a potential therapeutic target for preventing the progression to heart failure. Future studies should focus on validating these findings in human samples, optimizing delivery strategies for miRNA-based therapeutics, and exploring the temporal dynamics of miR-20a-5p expression throughout different stages of post-MI remodeling to guide timed interventions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\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\u003eGG and JW conceived and designed the study. MJ and YG performed the experiments. JW, LH and LZ collected the data and prepared the Figures. JW and GG analyzed the data and drafted the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman subjects/informed consent statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo human studies were carried out by the authors for this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were conducted in accordance with institutional and national guidelines for the care and use of laboratory animals and were approved by the Ethical Review Board of General Hospital of Ningxia Medical University (Approval No. KYLL-2022-0877).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSources of Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Key Research and Development Program of Ningxia (grant number: 2022BSB03091); and the Ningxia Natural Science Foundation Project (grant number: 2023AAC03623).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGuo, Z. et al. 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Metab Apr\u003c/em\u003e. \u003cb\u003e24\u003c/b\u003e (4), 209\u0026ndash;217. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tem.2013.01.008\u003c/span\u003e\u003cspan address=\"10.1016/j.tem.2013.01.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"myocardial infarction, ventricular remodeling, miR-20a-5p, mTORC1/S6, exosome, myocardial fibrosis","lastPublishedDoi":"10.21203/rs.3.rs-9333795/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9333795/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMaladaptive ventricular remodeling is the major pathological process linking myocardial infarction (MI) to heart failure, yet its upstream molecular drivers remain incompletely defined. Exosomal microRNAs (miRNAs) are emerging regulators of intercellular communication and cardiac pathology. This study sought to investigate the contribution of exosome-enriched miR-20a-5p in post-MI remodeling.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eAn acute myocardial infarction (MI) model was induced in SD rats through coronary ligation. Exosomes were isolated from infarcted and sham hearts, and expression of miR-20a-5p was quantified. Neonatal cardiomyocytes and fibroblasts were used for exosome uptake and functional assays. Functional enhancement and impairment assays with miR-20a-5p mimic/inhibitor, combined with rapamycin, were performed. Molecular mechanisms were assessed by RT-qPCR, Western blot, flow cytometry, and dual-luciferase reporter assays, while cardiac function and remodeling were evaluated by echocardiography and histology in vivo.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eExosomal miR-20a-5p was significantly enriched after MI and preferentially internalized by cardiomyocytes and fibroblasts. Its upregulation promoted cardiomyocyte apoptosis, impaired autophagy, and induced fibroblast-to-myofibroblast transition via direct suppression of Ddit4 and activation of the mTORC1/S6 pathway. Inhibition of miR-20a-5p or mTORC1 partially reversed these effects, while combined therapy produced synergistic benefits, including reduced fibrosis, improved cardiomyocyte survival, and restored ventricular function in MI rats.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eExosomal miR-20a-5p drives adverse ventricular remodeling after MI through Ddit4-mediated mTORC1/S6 activation. Dual inhibition of miR-20a-5p and mTORC1 exerts synergistic cardioprotection, highlighting this axis as a promising target for preventing post-MI heart failure.\u003c/p\u003e","manuscriptTitle":"Exosomal miR-20a-5p Promotes Cardiac Remodeling After Myocardial Infarction by Regulating mTORC1/S6 Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-23 12:23:00","doi":"10.21203/rs.3.rs-9333795/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-15T05:01:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-08T05:47:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T02:35:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-05T10:58:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"255729174818693655485321676748479386342","date":"2026-04-20T17:02:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"147738789720065468285638199306310714526","date":"2026-04-20T12:49:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-19T13:28:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"271037693021187543202187794047589279081","date":"2026-04-17T21:11:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"280262839397326898856822990504179088573","date":"2026-04-17T17:02:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"181916726887785955897963359142260230823","date":"2026-04-17T13:19:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"10315016547582642132635957765731491030","date":"2026-04-15T16:18:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"67527745557410610134673729121374465001","date":"2026-04-15T15:44:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"53668957599344040702757155086769948845","date":"2026-04-15T12:28:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-15T12:10:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-13T10:29:11+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-13T09:49:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-10T17:04:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-10T15:17:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"55c11ab7-19c2-4bf8-beaf-269ced288acc","owner":[],"postedDate":"April 23rd, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-15T05:01:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-08T05:47:48+00:00","index":128,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T02:35:36+00:00","index":126,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-05T10:58:03+00:00","index":125,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":66708796,"name":"Health sciences/Cardiology"},{"id":66708797,"name":"Biological sciences/Cell biology"},{"id":66708798,"name":"Health sciences/Diseases"}],"tags":[],"updatedAt":"2026-05-15T05:09:13+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-23 12:23:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9333795","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9333795","identity":"rs-9333795","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}