Bone marrow mesenchymal stem cells transport connexin43 via tunneling nanotubes to alleviate isopreterenol-induced myocardial hypertrophy

Bone marrow mesenchymal stem cells transport connexin43 via tunneling nanotubes to alleviate isopreterenol-induced myocardial hypertrophy | 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 Research Article Bone marrow mesenchymal stem cells transport connexin43 via tunneling nanotubes to alleviate isopreterenol-induced myocardial hypertrophy Jianghui Zhang, Hongfeng Jiang, Sa Liu, Zhong Xian, Limin Zhao, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5069090/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 May, 2025 Read the published version in Stem Cell Research & Therapy → Version 1 posted 5 You are reading this latest preprint version Abstract Background Paracrine signaling plays an important role in stem cell therapy. However, it alonecannot fully explain the therapeuticmechanisms of stem cell therapy in treating heart diseases. Recently, tunneling nanotubes (TNTs)—a novel type of long-distance intercellular connectional structure—have been identified between mesenchymal stem cells (MSCs) and cardiomyocytes (CMs). TNTs mediate the transmission of multiple signaling molecules, enabling cells to exert different biological functions. In the present study, we investigated the role of TNTs in MSC-based therapy for myocardial hypertrophy. Methods MSCs and CMs were co-cultured for 24 h with or without isopreterenol (ISO) to induce myocardial hypertrophy. Confocal microscopy was used to quantify and analyze the number, morphology, composition, and cell source of TNTs between MSCs and CMs. The effects of ISO on CMs were assessed by comparing cell area (measured by confocal microscopy) and expression levels of hypertrophy-related genes (using qRT-PCR) under co-culture and trans-well culture conditions. Flow cytometry was employed to assess the transfer of connexin43 (Cx43) from MSCs to CMs; lentivirus-mediated Cx43 overexpression and Cx43 siRNA were used to investigate the effects of Cx43 on ISO-induced myocardial hypertrophy. Results ISO stimulation significantly increased the number, length, and thickness of TNTs between MSCs and CMs (number: P<0.05; length and thickness: P<0.01). ISO also increased the proportion of TNTs containing microtubules and those derived from MSCs (P<0.05). Co-culture conditions were more effective than trans-well culture in alleviating ISO-induced myocardial hypertrophy (P<0.05). Furthermore, Cx43 was observed in TNTs, and ISO enhanced the transfer of Cx43-mCherry from MSCs to co-cultured CMs (P<0.05). Overexpression of Cx43 in CMs alleviated myocardial hypertrophy, whereas knocking down of Cx43 in MSCs reduced their ability to alleviate myocardial hypertrophy (P<0.05). Conclusions Our results demonstrate that ISO promotes the formation of TNTs, particularly between MSCs and CMs, and induces changes in the morphology of TNTs (thickening and lengthening). Additionally, MSCs transmitted Cx43 to CMs via TNTs, which contributes to the alleviation of ISO-induced myocardial hypertrophy. These results suggest that TNTs represent an important mechanism in MSC-mediated therapy for myocardial hypertrophy. Mesenchymal stem cells Cardiomyocytes tunneling nanotubes Connexin43 myocardial hypertrophy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Pathological myocardial hypertrophy is a major risk factor for heart failure, sudden cardiac death, and various cardiovascular diseases such as arrhythmia and myocardial infarction [ 1 – 3 ]. Both basic and clinical studies show that stem cells have great potential for treating these heart diseases [ 4 – 8 ]. Identifying the mechanism underlying stem cell actions is crucial for promoting their therapeutic applications. Tunneling nanotubes (TNTs) are long-distance cell junctions first reported in 2004 by Rustom et al. between rat pheochromocytoma PC12 cells [ 9 ]. Since then, TNTs have been found in various tissues and cell types [ 10 – 12 ]. They serve not only as connecting structures but also as novel intercellular junctions that allow cells to exchange small molecules and organelles, such as mitochondria, endosomes, and lysosomes, to perform different biological functions [ 13 – 16 ]. In 2014, a study found that TNTs can form between distressed cardiomyocytes and stem cells, demonstrating that damaged cardiomyocytes can promote the paracrine function of stem cells through TNTs [ 17 ]. In agreement, our previous study revealed that stem cells can transport fully functional mitochondria to damaged cardiomyocytes through TNTs, thereby reducing myocardial cell apoptosis [ 18 ]. It is becoming clear that TNTs may represent a new pathway for stem cell therapy in heart disease, alongside paracrine signaling and self-differentiation. Exploring the mechanism underlying TNT action could significantly advance the clinical applications of stem cells. The intercellular hydrophilic channels between adjacent cells, formed by connexin 43 (Cx43), are the main conduits for calcium signal transmission between cardiomyocytes. They play an important role in maintaining normal heart function, including the coordination of heart contractions and relaxation [ 19 , 20 ]. However, increased degradation and/or redistribution of Cx43 has been associated with several cardiac disorders such as myocardial ischemia, hypertrophy, arrhythmia, and heart failure [ 21 – 23 ]. Cx43 is also important in studies of TNTs as it is closely associated with the formation of intercellular TNTs [ 24 , 25 ] and affects their biological functions by promoting mitochondrial transport [ 26 ]. In this study, we observed Cx43 in TNTs between mesenchymal stem cells (MSCs) and cardiomyocytes (CMs). We further demonstrated that MSCs can transmit Cx43 to CMs through TNTs, thereby alleviating isopreterenol (ISO)-induced cardiomyocyte hypertrophy. Methods Isolation and culture of cardiomyocytes (CMs) Neonatal rat CMs were cultured as described previously [ 18 ]. According to the American Veterinary Association, the sensory nerves of newborn rats begin to develop 5–7 days after birth, and the nervous system of newborn rats is still immature at 2–3 days of age, with strong pain tolerance and no need for anesthesia. Therefore, in this study, 2–3-day old newborn rats were soaked in 75% alcohol for 5–10 seconds before conducting experiments. Briefly, the hearts of 2–3-day old Sprague Dawley (SD) Rats (provided by Beijing Huafukang BioScience Company, Beijing, China) were rapidly removed. Ventricles were finely cut and digested in 0.2% collagenase type II (Gibco, Waltham, Massachusetts, USA) at 37°C for eight min, and the isolated cells were neutralized in cell culture medium. Cardiac tissues were digested until they disappeared. All suspensions were pelleted by centrifugation at 1000 rpm for five min and resuspended in Dulbecco’s modified Eagle’s medium nutrient mixture F-12 (DMEM/F-12, Gibco, Waltham, Massachusetts, USA) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin solution (100 U/mL). Cells were plated in 10-cm dishes and transferred to an incubator at 37°C with an atmosphere containing 5% CO 2 . After allowing two hours for fibroblast adherence, the CMs was seeded onto culture plates for subsequent treatments. All animal experimental procedures were performed in compliance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH, revised 1996) and with the approval of Anzhen Hospital (approval no. AZ2023LA014), which is affiliated with the Capital Medical University. This work was reported in accordance with the ARRIVE Guidelines 2.0. Trans-well analysis and co-culturing of MSCs with CMs Bone marrow-derived MSCs, derived from SD rats were purchased from Procell (Wuhan, China). To observe the role of TNTs in alleviating isopreterenol (ISO)-induced myocardial hypertrophy in MSCs, Trans-well culture and co-culture modes of MSCs and CMs were established in vitro, as previously described with some modifications [ 18 ]. Briefly, the Trans-well culture model consisted of indirectly culturing MSCs and CMs in a 2:1 ratio using a semipermeable membrane as a Trans-well insert (pore size, 0.4 µm; Corning, USA), which prevented the formation of TNTs between the two kinds of cells but allowed the diffusion of secreted factors. The co-culture model involved directly mixing and culturing MSCs and CMs in the same dish in a 2:1 ratio. All cells were maintained in DMEM/F-12. Immunofluorescence staining analysis MSCs and CMs were fixed in 4% paraformaldehyde for 20 min. After incubation, cells were penetrated with 0.2% Triton X-100, blocked with 5% BSA in PBS, and then stained with primary antibodies, α-tubulin (Abcam, Cambridge, UK), cardiac troponin T (Abcam) or Cx43 (Cell Signaling Technology, Boston, MA, USA) at 4°C overnight, followed by Alexa Fluor 546/633 secondary antibodies. TRITC Phalloidin (Yeasen, China) and WGA Alexa Fluor® 488/555 conjugates (Invitrogen, Carlsbad, CA, USA) were incubated with the cells for 30 min at room temperature for F-actin or cell membrane staining. For living cell membrane staining, CellTracker Green CMFDA (Invitrogen) was incubated with the cells for 45 min under cell growth condition. Flow cytometry Following labeling with CellTracker Green CMFDA for 45 min at 37℃ in serum-free DMEM/F-12, CMs were co-cultured with MSCs for 24 h. After stimulation with ISO (10 µM for 24 h), CMs were sorted from other co-cultured cells according to CellTracker Green CMFDA immunostaining. Sorted cells were used to detect the expression levels of myocardial hypertrophy markers. Quantitative Real-Time PCR (qRT-PCR) After 24 h of ISO treatment, total RNA was extracted from the sorted CMs using TRIZOL reagent, and reverse transcription reactions were performed using HiScript II Q RT SuperMix for qPCR (Vazyme, NanJing, China). QRT-PCR was performed on a PCR detection system using ChamQ Universal SYBR qPCR Master Mix (Vazyme). PCR conditions were 95°C for 30 s, 40 cycles at 95°C for 10 s, and 60°C for 30 s, 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. The expression levels of the target genes were normalized against GAPDH mRNA levels. Primer sequences are summarized in Table 1. Lentiviral infection To overexpress Cx43, Lentiviral-mCherry-Cx43 (LV-mCherry-Cx43) was constructed using the lentiviral vector pSLenti-CMV-mCherry (LV-mCherry). The Cx43 coding sequence was inserted between the EcoRI and XbaI genes of the vector, and mCherry was fused in frame to the carboxyl terminus of Cx43 with the addition of a ten-amino-acid polylinker (GGGSGGGGGS). After infecting MSCs and CMs with LV-mCherry-Cx43 for eight hours in serum-free medium containing 5 µg/mL polybrene, we replaced the serum-containing medium and continued to culture for 5–7 days. Control cells were infected with an empty vector (LV-mCherry). Transfection of siRNAs We used rat small interfering RNA (siRNA) targeting Cx43 to silence its gene expression. A scramble was used as the non-targeting control. The sequences for the Cx43 siRNA and siRNA control were as follows: Cx43 siRNA#1-F: 5′-GCUUCUGGACAAGGUCCAAGCTT-3,’ Cx43 siRNA#1-R: 5′-GCUUGGACCUUGUCCAGAAGCTT-3′; Cx43 siRNA#2-F: 5′-GGAAGGAAGAGAAGCUAAACATT-3’, Cx43 siRNA#2-R: 5′-UGUUUAGCUUCUCUUCCUUCCTT-3′; siRNA control-F: 5′-UUCUCCGAACGUGUCACGUTT-3’, siRNA control-R: 5′-ACGUGACACGUUCGGAGAATT-3′. Transfection of MSCs with Cx43 siRNA#1 and #2 involved diluting the siRNA with DMEM/F-12 without serum, and incubating the cells with GP-transfect-Mate reagent (Genepharma, Shanghai, China) and the siRNA solution for four hours. The cells were further cultured in complete culture medium for three days before conducting subsequent experiments. Statistical analyses Group values are presented as means ± standard error of the mean (SEM). An unpaired two-tailed t-test or one-way analysis of variance (ANOVA) was applied to determine the significance of differences between two or more groups of parametric data, respectively. Statistical analyses were performed using GraphPad Prism software version 6.01 (GraphPad Software Inc., San Diego, CA, USA), and differences were considered significant at P < 0.05. Results Effect of ISO on the formation and morphology of TNTs between bone marrow (BM)-MSCs and neonatal rat CMs Previously, we discovered that TNTs can form between CMs and MSCs [ 27 ]. In this study, we aimed to determine the biological functions of these TNTs in alleviating MSC-induced CM hypertrophy. First, we used a confocal microscope to observe and quantitatively analyze the effect of ISO, a compound that induces myocardial hypertrophy, on TNT formation between the two cell types. Our results revealed that TNTs were present, and their numbers significantly increased after 24 h of co-culture with ISO stimulation (Fig. 1 A, B), indicating that ISO promoted the formation of intercellular TNTs. Next, we quantified the length and thickness of 92 TNTs in the ISO-treated group and 118 TNTs in the without ISO (control) group. Both measurements significantly increased in the ISO group: length (38.39 ± 1.79 µm vs 26.83 ± 1.52 µm) and thickness (2.19 ± 0.07 µm vs 1.68 ± 0.07 µm) (Fig. 1 C, D, E). ISO stimulation significantly increases TNTs derived from MSCs We further investigated the effect of ISO on the cellular origin of TNTs. Confocal microscopy was employed to observe TNTs derived from CellTracker™ Green/ WGA555-labeled CMs or WGA 555-labeled MSCs (Fig. 2 A). Almost 47% of the 94 TNTs were derived from MSCs, and this proportion significantly increased to about 60% of the 118 TNTs in the with ISO group (Fig. 2 B). However, we did not observe any TNTs that originated from both types of cells simultaneously. These results suggest that ISO promoted the formation of TNTs from MSCs to CMs. Effect of ISO on the cytoskeleton in TNTs Consistent with previous reports, both F-actin and microtubules were observed in the TNTs between MSCs and CMs [ 18 ]. F-actin and microtubules are essential for the formation and function of TNTs [ 9 , 18 ], with microtubules also influencing the morphology of these structures [ 18 ]. To further investigate the effects of ISO on the cytoskeleton in TNTs, we examined the presence of F-actin and microtubules. F-actin was observed in all TNTs regardless of ISO stimulation (Fig. 3 A). However, microtubules were present in only some TNTs, but ISO significantly increased the proportion of TNTs that contained microtubules (Fig. 3 B, C). Potential role of TNTs in MSC-mediated alleviation of ISO-induced myocardial hypertrophy ISO can induce myocardial hypertrophy, and paracrine secretion is an important mechanism by which stem cell therapy addresses this pathological condition [ 4 , 28 ]. As a novel form of intercellular “communication,” TNTs mediate the transfer of multiple signaling molecules and functional proteins between cells [ 29 ], potentially providing a new pathway for MSCs to alleviate ISO-induced myocardial hypertrophy. To verify this hypothesis, we quantified the cellular area of CMs when co-cultured with MSCs under ISO stimulation, using both Trans-well and direct co-culture systems. Our results showed that the area of ISO-treated CMs decreased when cells were trans-well cultured with MSCs, and the anti-hypertrophic effect further enhanced under co-culture conditions (Fig. 4 A, B). We also analyzed the expression level of brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC), both indicators of myocardial hypertrophy, using quantitative RT-PCR (qRT-PCR). We obtained CellTracke Green CMFDA-labeled CMs, which co-cultured with MSCs, through flow cytometry sorting (Fig. 4 C). The results showed that MSCs in trans-well culture inhibited the ISO-induced increase in BNP and β-MHC mRNA expression, with further reduction observed under co-culture conditions (Fig. 4 D, E). These results indicate that, in addition to paracrine secretion, MSCs may alleviate ISO-induced myocardial hypertrophy through TNTs. The list of primers used in qRT-PCR is shown in Table 1. ISO facilitates TNT-mediated Cx43 transfer from MSCs to CMs How do TNTs promote MSC functions in inhibiting myocardial hypertrophy? Previous clinical and scientific studies have shown that Cx43 is closely associated with the occurrence and progression of myocardial hypertrophy [ 22 , 27 , 30 ]. In addition, Cx43 has been detected in various intercellular TNTs [ 25 , 26 , 31 ]. Based on these findings, we hypothesized that MSCs alleviate myocardial hypertrophy by transferring Cx43 via TNTs. To test this hypothesis, we investigated whether TNTs mediated the transfer of Cx43 from MSCs to CMs. Using immunofluorescence, we labeled Cx43 with red fluorescence and observed it under a confocal microscope. We observed that Cx43 was present in some intercellular TNTs, and the proportion of TNTs containing Cx43 significantly increased following ISO stimulation (Fig. 5 A, B). Additionally, we constructed a lentivirus (LV)-mCherry-Cx43 and expressed it in MSCs. After co-culturing LV-mCherry-Cx43-expressing MSCs with CellTracke Green CMFDA-labeled CMs for 24 h, we detected mCherry-Cx43 in some CMs via flow cytometry (Fig. 6 A). This proportion increased significantly with ISO stimulation (Fig. 6 B). However, no mCherry-Cx43-positive CMs were detected in the Trans-well culture system. These results suggest that Cx43 is transferred from MSCs to CMs via TNTs, and ISO promoted this process. Similar to the characteristics of endogenous Cox43, we also observed aggregation of mCherry-Cx43 at cell-cell junctions. Additionally, the presence of Cx43-mCherry in TNTs was verified (Supplementary Fig. 1). TNTs inhibit ISO-induced myocardial hypertrophy by transmitting Cx43 To define the role of Cx43 in ISO-induced myocardial hypertrophy, we overexpressed LV-mCherry-Cx43 in CMs and quantitatively analyzed the area of Cx43-positive CMs under ISO stimulation. The results showed that upregulating Cx43 expression significantly alleviated ISO-induced myocardial hypertrophy (Fig. 7 A, B). Building on previous results that TNTs may provide a novel pathway for MSCs to alleviate ISO-induced myocardial hypertrophy (Fig. 4 ), we next examined the effect of Cx43 depletion on TNT-mediated MSC activation. We transfected MSCs with a specific siRNA targeting Cx43 and co-cultured them with CMs for 24 h. Cx43 silencing strongly impaired the ability of MSCs to alleviate myocardial hypertrophy under co-culture conditions (Fig. 8 B, C). The efficiency of siRNA-induced Cx43 silencing was detected by western blot analysis (Fig. 8 A). Additionally, depletion of Cx43 did not affect the ability of ISO to promote TNT formation (Supplementary Figs. 2A, B). These data suggest that MSCs may alleviate ISO-induced myocardial hypertrophy by transmitting Cx43 to CMs via TNTs. Discussion Five decades after the discovery of MSCs, these cells have become leading candidates for cell-based therapies [ 32 ]. Currently, MSCs are widely used to treat various heart diseases, including myocardial infarction, myocardial hypertrophy, and dilated cardiomyopathy [ 32 – 34 ]. Significant progress has been made in understanding the mechanisms through which MSCs exert their therapeutic effects on cardiovascular diseases, but it mainly focuses on self-differentiation and paracrine signaling pathways [ 35 ]. Despite increasing interest and extensive research, many questions remain regarding the underlying biological mechanisms of MSCs. TNTs are long-distance filamentous junctional structures that connect various types of cells, such as T cells, B cells, nerve cells (neurons), and cancer cells. These structures can mediate the transport of signaling molecules and organelles, thereby exerting certain biological effects [ 36 – 39 ]. Both our research group and Rodriguez et al. have reported that TNTs form between MSCs and damaged CMs[ 18 , 31 ]. Furthermore, our research group revealed that MSCs can transport fully functional mitochondria along microtubules to damaged CMs through TNTs, thereby alleviating myocardial cell apoptosis [ 17 , 18 ]. This observation indicates that TNTs may serve as a novel pathway by which MSCs contribute to the treatment of heart diseases. Pathological myocardial hypertrophy, induced by stimuli such as hypertension and valvular disease, is a leading risk factor for heart failure, sudden cardiac death, and triggers various cardiovascular conditions such as arrhythmia and myocardial infarction [ 1 , 2 , 40 ]. The role of stem cells in treating myocardial hypertrophy has received extensive attention and considerable in-depth research has been devoted to understanding their mechanisms of action in this context [ 41 , 42 ]. In this study, we investigated whether TNTs play an important role in alleviating myocardial hypertrophy in MSCs. To clarify this, we investigated the effects of ISO, a β-receptor agonist that is widely used to induce myocardial hypertrophy [ 43 , 44 ], on the quantity and morphology of intercellular TNTs. Our quantitative analysis showed that ISO stimulation significantly increased the number of TNTs formed between the cells (MSCs or CMs). Furthermore, a significant elongation and thickening of TNTs was also noticed. We also found that ISO significantly upregulated the ratio of TNTs originating from MSCs, suggesting that ISO enhances the ability of MSCs to influence CM function via TNTs. While the exact mechanism by which ISO regulates the formation and extension of TNTs remains unclear, recent studies have suggested that the F-actin cytoskeleton is essential for TNT formation in virtually all cell types [ 39 , 45 ]. Several well-characterized proteins that modulate cellular actin dynamics play key roles in TNT formation, such as the Arp2/3 complex, small GTPases, Cdc42, and M-sec [ 46 – 50 ]. Moreover, ISO-induced activation of β-2 adrenergic receptors affects actin cytoskeleton organization [ 51 ], which may in turn affect TNT formation and its extension. We propose that future studies should explore the influence of ISO on actin dynamics and, subsequently, TNT formation. Considering that ISO induces myocardial hypertrophy, whereas MSCs can treat it, we further explored whether TNTs are involved in MSC-mediated protection against myocardial hypertrophy. By analyzing the effects of ISO on CM area under different cultivation conditions, we found that direct co-culture with MSCs, as compared to transwell cultures, more effectively alleviated ISO-induced myocardial hypertrophy. This observation suggests that in addition to paracrine secretion, TNTs play an important role in the ability of MSCs to treat myocardial hypertrophy. This conclusion was further confirmed by qRT PCR analysis, which showed that MSC co-culture reduced the expression of BNP and β-MHC, two well-established markers of myocardial hypertrophy in CMs. Cx43 has been implicated in the pathogenesis of myocardial hypertrophy. Studies have shown that the Cx43 expression is significantly reduced in ISO-induced hypertrophic CMs, and that maintaining or upregulating Cx43 levels can alleviate myocardial hypertrophy and improve cardiac function [ 22 , 52 ]. Additionally, in patients with myocardial hypertrophy, Cx43 expression in the myocardium is significantly decreased [ 30 ]. These findings highlight the close association between Cx43 and the occurrence and development of myocardial hypertrophy. Cx43 is also present in TNTs, including those formed by pluripotent stem cell (iPSC)-derived MSCs, epithelial cells, and even cancer cells [ 25 , 26 , 53 ]. We observed the presence of Cx43 in some TNTs between MSCs and CMs and found that ISO significantly increased the proportion of TNTs containing Cx43. Using flow cytometry, we further demonstrated that MSCs could transmit Cx43 to CMs via TNTs, and this process was facilitated by ISO. These results suggest that ISO promotes Cx43 transmission from MSCs to CMs via TNTs. The cytoskeleton, motor proteins, and autophagy are known to be involved in cargo transport in TNTs [ 54 , 55 ]. However, the precise mechanism by which ISO promotes Cx43 transmission remains unclear and warrants further investigation. To further demonstrate the role of TNT-mediated Cx43 in MSC-mediated alleviation of myocardial hypertrophy, we depleted Cx43 in MSCs using siRNA. The results showed that depletion of Cx43 significantly impaired the ability of MSCs to reduce myocardial hypertrophy under co-culture conditions. Furthermore, we confirmed that overexpression of Cx43 in CMs inhibited ISO-induced myocardial hypertrophy. While these findings support the hypothesis that Cx43 plays a critical role in MSC-mediated protection against hypertrophy, several questions remain unanswered: how does TNT-mediated Cx43 transfer occur? How is Cx43 internalized by CMs? What specific mechanisms through which Cx43 alleviates myocardial hypertrophy remain to be clarified? Cx43 is an important protein that forms gap junctions between CMs, facilitating intracellular communication and calcium signaling. The localization and expression of Cx43 are critical for proper calcium signal transmission between CMs. Therefore, it is important to explore whether TNT-mediated Cx43 transfer affects calcium signaling in the heart, as this could provide important insights into how Cx43 contributes to the alleviation of myocardial hypertrophy. Conclusion ISO stimulation caused significant changes in the quantity, morphology, composition, and origin of TNTs formed between MSCs and CMs. MSCs use TNTs to transport Cx43 to CMs, which plays a key role in alleviating ISO-induced myocardial hypertrophy. Our study suggests that, in addition to self-differentiation and paracrine secretion, TNTs provide a novel pathway for MSCs to treat pathological myocardial hypertrophy. Future investigations should focus on uncovering the molecular mechanisms underlying TNT formation, cargo transport, and the role of Cx43 in calcium signaling and myocardial remodeling. These lines of investigations will be essential for optimizing the therapeutic potential of TNTs in regenerative cardiovascular medicine and enhancing the effectiveness of MSC-based therapies for myocardial hypertrophy and other cardiac disorders. Abbreviations TNTs Tunneling nanotubes MSCs Mesenchymal stem cells CMs Cardiomyocytes ISO Isopreterenol Cx43 Connexin43 LV-Cx43- mCherry Lentiviral-Connexin 43-mCherry LV- mCherry pSLenti-CMV-mCherry BNP Brain natriuretic peptide β-MHC β-myosin heavy chain TnT Troponin T Declarations Acknowledgements We would like to acknowledge Peking University International Hospital Center for their financial support and guidance. The authors declare that artificial intelligence is not used in this study. Author contributions JZ, SL, LZ performed all experiments. ZX, YL, WL and CS participated in the data collection, data analysis. JZ and SC supervised the study and edited the manuscript. HJ and SC acquired the funding. All authors have read and approved the current version of the manuscript. Funding This work was supported by a grant from the National Natural Science Foundation of China (No: 82370440) and the Natural Science Foundation of Beijing Municipality (7232226). Availability of data and materials The authors confirm that all data generated or analyzed during this study are included in this published article and its supplementary file. Ethics approval and consent to participate All animal experimental procedures were performed in adherence with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH, revised 1996) and with the approval of the Capital medical university affiliated Anzhen hospital (Approval No. AZ2023LA014, Data of Approval: February 11, 2023). And the title of the approved project is “The role of Tunneling Nanotubes between mesenchymal stem cells and cardiomyocytes in alleviating isoproterenol induced cardiac hypertrophy”. This study did not involve human subjects. Consent for publication All authors and institutions have confirmed this manuscript for publication Competing interests The authors declare that they have no competing interests. References Kehat I, Molkentin JD. Molecular pathways underlying cardiac remodeling during pathophysiological stimulation. Circulation. 2010;122(25):2727–35. Kang YJ. Cardiac hypertrophy: a risk factor for QT-prolongation and cardiac sudden death. Toxicol Pathol. 2006;34(1):58–66. Kavey RE. Left ventricular hypertrophy in hypertensive children and adolescents: predictors and prevalence. Curr Hypertens Rep. 2013;15(5):453–7. Klinger JR, Pereira M, Del Tatto M, Brodsky AS, Wu KQ, Dooner MS, Borgovan T, Wen S, Goldberg LR, Aliotta JM, et al. 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Cell bioscience. 2021;11(1):132. Drab M, Stopar D, Kralj-Iglič V, Iglič A. Inception Mechanisms of Tunneling Nanotubes. Cells 2019, 8(6). Sartori-Rupp A, Cordero Cervantes D, Pepe A, Gousset K, Delage E, Corroyer-Dulmont S, Schmitt C, Krijnse-Locker J, Zurzolo C. Correlative cryo-electron microscopy reveals the structure of TNTs in neuronal cells. Nat Commun. 2019;10(1):342. Pollard TD, Beltzner CC. Structure and function of the Arp2/3 complex. Curr Opin Struct Biol. 2002;12(6):768–74. Ljubojevic N, Henderson JM, Zurzolo C. The Ways of Actin: Why Tunneling Nanotubes Are Unique Cell Protrusions. Trends Cell Biol. 2021;31(2):130–42. Hase K, Kimura S, Takatsu H, Ohmae M, Kawano S, Kitamura H, Ito M, Watarai H, Hazelett CC, Yeaman C, et al. M-Sec promotes membrane nanotube formation by interacting with Ral and the exocyst complex. Nat Cell Biol. 2009;11(12):1427–32. Pergu R, Dagar S, Kumar H, Kumar R, Bhattacharya J, Mylavarapu SVS. The chaperone ERp29 is required for tunneling nanotube formation by stabilizing MSec. J Biol Chem. 2019;294(18):7177–93. Porter AC, Svensson SP, Stamer WD, Bahl JJ, Richman JG, Regan JW. Alpha-2 adrenergic receptors stimulate actin organization in developing fetal rat cardiac myocytes. Life Sci. 2003;72(13):1455–66. Tang Y, Wang M, Le X, Meng J, Huang L, Yu P, Chen J, Wu P. Antioxidant and cardioprotective effects of Danshensu (3-(3, 4-dihydroxyphenyl)-2-hydroxy-propanoic acid from Salvia miltiorrhiza) on isoproterenol-induced myocardial hypertrophy in rats. Phytomedicine: Int J phytotherapy phytopharmacology. 2011;18(12):1024–30. Antanavičiūtė I, Rysevaitė K, Liutkevičius V, Marandykina A, Rimkutė L, Sveikatienė R, Uloza V, Skeberdis VA. Long-distance communication between laryngeal carcinoma cells. PLoS ONE. 2014;9(6):e99196. Sadeghsoltani F, Avci ÇB, Hassanpour P, Haiaty S, Rahmati M, Mota A, Rahbarghazi R, Nemati M, Mahdipour M, Talebi M, et al. Autophagy modulation effect on homotypic transfer of intracellular components via tunneling nanotubes in mesenchymal stem cells. Stem Cell Res Ther. 2024;15(1):189. Medina LY, Serda RE. Intercellular Communication Through Microtubular Highways. Result Probl Cell Differ. 2024;73:155–71. Tables Table 1 is available in the Supplementary Files section. Supplementary Files table1.pdf Movie1.wmv S1.pdf S2.pdf SupplementaryInformation.docx additionalfile1.pdf supplementaryfile.docx Cite Share Download PDF Status: Published Journal Publication published 06 May, 2025 Read the published version in Stem Cell Research & Therapy → Version 1 posted Reviewers agreed at journal 08 Nov, 2024 Reviewers invited by journal 08 Nov, 2024 Editor assigned by journal 08 Nov, 2024 First submitted to journal 07 Nov, 2024 Editorial decision: Minor Revision 19 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-5069090","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":375736467,"identity":"62f73a33-f6e6-457d-8e82-967974bac510","order_by":0,"name":"Jianghui Zhang","email":"data:image/png;base64,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","orcid":"https://orcid.org/0009-0000-1368-9036","institution":"Beijing Institute of Heart Lung and Blood Vessel Diseases: Capital Medical University Affiliated Anzhen Hospital","correspondingAuthor":true,"prefix":"","firstName":"Jianghui","middleName":"","lastName":"Zhang","suffix":""},{"id":375736468,"identity":"e5d2e8dd-3440-4aeb-aecb-ec51ce363db2","order_by":1,"name":"Hongfeng Jiang","email":"","orcid":"","institution":"Beijing Institute of Heart Lung and Blood Vessel Diseases: Capital Medical University Affiliated Anzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hongfeng","middleName":"","lastName":"Jiang","suffix":""},{"id":375736469,"identity":"aeb0d387-bbd8-4c76-8c20-6667fc968fe2","order_by":2,"name":"Sa Liu","email":"","orcid":"","institution":"Beijing Institute of Heart Lung and Blood Vessel Diseases: Capital Medical University Affiliated Anzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Sa","middleName":"","lastName":"Liu","suffix":""},{"id":375736470,"identity":"0dbf211c-173f-4771-8239-599623163efe","order_by":3,"name":"Zhong Xian","email":"","orcid":"","institution":"Beijing Institute of Heart Lung and Blood Vessel Diseases: Capital Medical University Affiliated Anzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhong","middleName":"","lastName":"Xian","suffix":""},{"id":375736471,"identity":"d112bcc4-236e-4096-ae0f-6f534b9c5240","order_by":4,"name":"Limin Zhao","email":"","orcid":"","institution":"Beijing Institute of Heart Lung and Blood Vessel Diseases: Capital Medical University Affiliated Anzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Limin","middleName":"","lastName":"Zhao","suffix":""},{"id":375736472,"identity":"6a87d92e-66fa-4466-b2de-b32516e5a331","order_by":5,"name":"Yue Li","email":"","orcid":"","institution":"Beijing Institute of Heart Lung and Blood Vessel Diseases: Capital Medical University Affiliated Anzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Li","suffix":""},{"id":375736473,"identity":"5b7ac2ad-3426-4c1f-af6b-2ac7d6e5b939","order_by":6,"name":"Wenxiu Lu","email":"","orcid":"","institution":"Beijing Institute of Heart Lung and Blood Vessel Diseases: Capital Medical University Affiliated Anzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wenxiu","middleName":"","lastName":"Lu","suffix":""},{"id":375736474,"identity":"8bab2ff2-9ad1-4f5a-bd50-47a4bf182e2a","order_by":7,"name":"Changrong Shao","email":"","orcid":"","institution":"Beijing Institute of Heart Lung and Blood Vessel Diseases: Capital Medical University Affiliated Anzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Changrong","middleName":"","lastName":"Shao","suffix":""},{"id":375736475,"identity":"7a8453be-2fad-44d7-a942-78485ab0762f","order_by":8,"name":"Sanbao Chai","email":"","orcid":"","institution":"Department of Endocrinology and Metabolism, Beiking University International Hospital","correspondingAuthor":false,"prefix":"","firstName":"Sanbao","middleName":"","lastName":"Chai","suffix":""}],"badges":[],"createdAt":"2024-09-11 07:35:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5069090/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5069090/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13287-025-04339-w","type":"published","date":"2025-05-06T15:57:39+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68747211,"identity":"79f9a5f0-44ff-41db-882d-5bc2e49512ea","added_by":"auto","created_at":"2024-11-11 15:37:27","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":743997,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of the effect of ISO on the formation and morphology of TNTs. Co-cultured MSCs and CMs were treated with ISO for 24 h, Cell membranes were labeled with WGA488 (green), and CMs were labeled by CM-specific marker protein Troponin T (TnT) (red). (A, B) TNTs (indicated by arrow heads) were observed under a 63 lens of a microscope, and TNT numbers per 100 CMs were evaluated by quantifying 10 randomly selected fields. The number of replicates: N=4. (C, D, E) Quantifying the length and thickness of 95 and 112 TNTs (indicated by arrow tips) between 24 h-co-cultured cells with or without ISO treatment, respectively. N=4, scale bar: 100 μm. Data are shown as the mean ± S.E.M. *P \u0026lt; 0.05 and ***P \u0026lt; 0.001 vs Ctrl.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5069090/v1/66d6278e3ce6df42b0416bc7.jpg"},{"id":68747217,"identity":"a925ca97-d8f9-40c7-a381-f189d2bc1f19","added_by":"auto","created_at":"2024-11-11 15:37:27","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":447753,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative evaluation of the effect of ISO on the cellular source of TNTs. (A) CellTracker Green CMFDA-labeled CMs (green) and MSCs were co-cultured for 24h, followed by WGA555 (red). TNTs derived from CMs or MSCs, which were indicated by arrow heads and arrow tips, respectively, were checked under a 40× lens. (B) The ratio of different cellular originations of TNTs was quantified by randomly observing 94 and 118 TNTs with or without ISO. N=4, scale bar: 50 μm. Data are shown as the mean ± S.E.M. *P \u0026lt; 0.05 vs Ctrl.\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5069090/v1/779ffe91e1f1ab36fcf0a48f.jpg"},{"id":68748871,"identity":"6afd116f-c3c5-4cbc-bb3c-1e5169235823","added_by":"auto","created_at":"2024-11-11 15:53:27","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":663842,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of ISO on the cytoskeleton in TNTs. 24h-co-cultured cells were stimulated by ISO, followed labelled by WGA 488 (green) and TnTs (blue). (A) F-actin were labeled with TRITC Phalloidin (red), 175 TNTs were randomly observed under a 40× lens. Arrows heads indicate TNTs containing F-actin, and yellow triangles indicate F-actin in TNTs. N=4, Scale bar: 50 μm. (B) Microtubules were stained by α-tubulin antibodies (red). TNTs with (+) or without (-) microtubules were indicated by arrows heads and arrow tips, respectively. White triangles indicate microtubules in TNTs. (C) Randomly observe 132 TNTs, and quantitative percentage of TNTs containing microtubules. N=5, Scale bar: 75 μm. Data are shown as the mean ± S.E.M.** 0.001\u0026lt;P \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5069090/v1/c41679cf2f8c57e88583cbdf.jpg"},{"id":68748269,"identity":"7386c523-0011-4f22-b6c6-eb95c1195177","added_by":"auto","created_at":"2024-11-11 15:45:27","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":528449,"visible":true,"origin":"","legend":"\u003cp\u003eCompare the suppressing effects of MSCs on ISO induced myocardial hypertrophy under different culture conditions. (A, B) The images represent immunostaining with TnT (Green), and the average cell surface area were quantified by detecting CMs in 10 randomly selected fields in four separate experiments. Scale bar: 100 μm. (C, D) Sorting CellTracker Green CMFDA-labeled CMs (Green) in co-culture system by flow cytometry, and the mRNA level of hypertrophic genes BNP, and β-MHC in CMs were analyzed by qPCR. N=4, Data are shown as the mean ± S.E.M. * P \u0026lt; 0.05, ** 0.001\u0026lt;P \u0026lt; 0.05 and ***P \u0026lt; 0.001 vs Ctrl.\u003c/p\u003e","description":"","filename":"14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5069090/v1/a205111f4ff19dabdacb6b27.jpg"},{"id":68748274,"identity":"37554cff-1aa7-4964-b9c7-3237cb7d8e9f","added_by":"auto","created_at":"2024-11-11 15:45:27","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":479667,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ISO on the localization of Cx43 in TNTs. (A) Co-cultured MSCs and CMs were triple labeled with WGA, Cx43, and TnT. Regardless of whether there was ISO, some TNTs contained Cx43 (indicated by arrow heads), and yellow triangles indicate Cx43 in TNTs. TNTs without Cx43 were indicated by arrow tips. N=4, cale bar: 75 μm. (B) Randomly observe 125 TNTs and quantify the percentage of TNTs containing Cx43 with and without ISO stimulation. N=4, Data are shown as the mean ± S.E.M. * P \u0026lt; 0.05 vs Ctrl.\u003c/p\u003e","description":"","filename":"15.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5069090/v1/cd17c02581d2fc5118c8c9c1.jpg"},{"id":68748272,"identity":"8e22c794-9f1f-4ea6-9b02-8c7c577e49c3","added_by":"auto","created_at":"2024-11-11 15:45:27","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":430998,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ISO on the transfer of Cx43 from MSCs to CMs. (A) Transfecting Lentivirus (LV)-mCherry-Cx43 into MSCs (mCherry-Cx43 MSCs, red) for 7 days, and then co-culturing with CellTracker Green CMFDA-labeled CMs (CMFDA-labeled CMs, green) for 24 h. CMs (green) containing mCherry-Cx43 (red) were detected by flow cytometry regardless ISO treatment. N=3. (B) Comparison of the percentage of CMFDA-labeled CMs (green) containing mCherry-Cx43 (red) in all CMFDA-labeledCMs (green) under co-culture conditions with and without ISO stimulation. N=3, Data are shown as the mean ± S.E.M. * P \u0026lt; 0.05 vs Ctrl.\u003c/p\u003e","description":"","filename":"16.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5069090/v1/1ae4221a3b40d9137de9ddaa.jpg"},{"id":68747222,"identity":"ad6b90ba-6df8-4709-b3dd-ac779b88dcf3","added_by":"auto","created_at":"2024-11-11 15:37:28","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":266317,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of overexpression of Cx43 on ISO induced myocardial hypertrophy. (A) Transfecting LV-mCherry or LV-mCherry-Cx43 (Red) into CMs for 5 days, the image shows transfected CMs stained with TnT (Green). N=6 Scale bar: 75 μm. (B) Quantitative comparison of the area of transfected CMs. N=6, Data are shown as the mean ± S.E.M. ***P \u0026lt; 0.001 vs Ctrl.\u003c/p\u003e","description":"","filename":"17.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5069090/v1/5c51f80e6845a9ef40a3ee9c.jpg"},{"id":68748875,"identity":"e9b44f18-39c2-4987-a814-3993ef1f5c66","added_by":"auto","created_at":"2024-11-11 15:53:27","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":313179,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of knocking down Cx43 on MSCs to alleviate myocardial hypertrophy function. (A) Detecting the knock down efficiency of Cx43 SiRNA#1 and #2 （Si#1 and Si#2）in MSCs by western blot (WB). N=3. (B) MSCs, pretreated with SiRNA control or Cx43 SiRNA, co-cultured with CMs for 24 h. CMs were labeled by TnT (Green). N=4, Scale bar: 100 μm. (C) Quantifying the average surface area of CMs in 10 randomly selected fields. N=4, Data are shown as the mean ± S.E.M. * P \u0026lt; 0.05 vs Ctrl, ** 0.001\u0026lt;P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"18.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5069090/v1/1b5ab0e86ad3e25ae1735453.jpg"},{"id":82537631,"identity":"830832f9-d4c1-4aca-a383-a286ab056736","added_by":"auto","created_at":"2025-05-12 16:09:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4766838,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5069090/v1/37368f36-93a2-4fc1-a5bb-8df66b249572.pdf"},{"id":68749795,"identity":"f309c600-5ea4-42fa-a9cf-1f1c66ebbc96","added_by":"auto","created_at":"2024-11-11 16:05:03","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1024052,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"table1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5069090/v1/27f385b1119d3687d9bf5465.pdf"},{"id":68748275,"identity":"3302fd45-5171-4360-b1a0-32a61e436f12","added_by":"auto","created_at":"2024-11-11 15:45:28","extension":"wmv","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":617937,"visible":true,"origin":"","legend":"","description":"","filename":"Movie1.wmv","url":"https://assets-eu.researchsquare.com/files/rs-5069090/v1/2363834e5be73b7cbab47a6f.wmv"},{"id":68748882,"identity":"c3334501-8583-4e33-8444-4249342e0ff3","added_by":"auto","created_at":"2024-11-11 15:53:28","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":6374308,"visible":true,"origin":"","legend":"","description":"","filename":"S1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5069090/v1/a93da34bff18616b1cdc7cd2.pdf"},{"id":68747224,"identity":"269b2b78-5a7d-44f7-a8cc-406df846cfcd","added_by":"auto","created_at":"2024-11-11 15:37:28","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":7449295,"visible":true,"origin":"","legend":"","description":"","filename":"S2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5069090/v1/d8c7492859c5fec57db8ffa9.pdf"},{"id":68747221,"identity":"b0cc8838-f223-47f5-98e0-42be9b5f0f83","added_by":"auto","created_at":"2024-11-11 15:37:27","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":23797,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5069090/v1/d43dc44e08a1aeb0775744f6.docx"},{"id":68748276,"identity":"250fdd9e-9679-4332-9d17-761c5f8b6475","added_by":"auto","created_at":"2024-11-11 15:45:28","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":7349326,"visible":true,"origin":"","legend":"","description":"","filename":"additionalfile1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5069090/v1/6a4fc575b56fa54bb9ceab96.pdf"},{"id":68747220,"identity":"14e126c6-e139-4bb2-947a-188260b6d3b1","added_by":"auto","created_at":"2024-11-11 15:37:27","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":21047,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-5069090/v1/d38263a3a8a95f10577892fb.docx"}],"financialInterests":"","formattedTitle":"Bone marrow mesenchymal stem cells transport connexin43 via tunneling nanotubes to alleviate isopreterenol-induced myocardial hypertrophy","fulltext":[{"header":"Background","content":"\u003cp\u003ePathological myocardial hypertrophy is a major risk factor for heart failure, sudden cardiac death, and various cardiovascular diseases such as arrhythmia and myocardial infarction [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Both basic and clinical studies show that stem cells have great potential for treating these heart diseases [\u003cspan additionalcitationids=\"CR5 CR6 CR7\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Identifying the mechanism underlying stem cell actions is crucial for promoting their therapeutic applications.\u003c/p\u003e \u003cp\u003eTunneling nanotubes (TNTs) are long-distance cell junctions first reported in 2004 by Rustom et al. between rat pheochromocytoma PC12 cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Since then, TNTs have been found in various tissues and cell types [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. They serve not only as connecting structures but also as novel intercellular junctions that allow cells to exchange small molecules and organelles, such as mitochondria, endosomes, and lysosomes, to perform different biological functions [\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In 2014, a study found that TNTs can form between distressed cardiomyocytes and stem cells, demonstrating that damaged cardiomyocytes can promote the paracrine function of stem cells through TNTs [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In agreement, our previous study revealed that stem cells can transport fully functional mitochondria to damaged cardiomyocytes through TNTs, thereby reducing myocardial cell apoptosis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. It is becoming clear that TNTs may represent a new pathway for stem cell therapy in heart disease, alongside paracrine signaling and self-differentiation. Exploring the mechanism underlying TNT action could significantly advance the clinical applications of stem cells.\u003c/p\u003e \u003cp\u003eThe intercellular hydrophilic channels between adjacent cells, formed by connexin 43 (Cx43), are the main conduits for calcium signal transmission between cardiomyocytes. They play an important role in maintaining normal heart function, including the coordination of heart contractions and relaxation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, increased degradation and/or redistribution of Cx43 has been associated with several cardiac disorders such as myocardial ischemia, hypertrophy, arrhythmia, and heart failure [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Cx43 is also important in studies of TNTs as it is closely associated with the formation of intercellular TNTs [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and affects their biological functions by promoting mitochondrial transport [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we observed Cx43 in TNTs between mesenchymal stem cells (MSCs) and cardiomyocytes (CMs). We further demonstrated that MSCs can transmit Cx43 to CMs through TNTs, thereby alleviating isopreterenol (ISO)-induced cardiomyocyte hypertrophy.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and culture of cardiomyocytes (CMs)\u003c/h2\u003e \u003cp\u003eNeonatal rat CMs were cultured as described previously [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. According to the American Veterinary Association, the sensory nerves of newborn rats begin to develop 5\u0026ndash;7 days after birth, and the nervous system of newborn rats is still immature at 2\u0026ndash;3 days of age, with strong pain tolerance and no need for anesthesia. Therefore, in this study, 2\u0026ndash;3-day old newborn rats were soaked in 75% alcohol for 5\u0026ndash;10 seconds before conducting experiments. Briefly, the hearts of 2\u0026ndash;3-day old Sprague Dawley (SD) Rats (provided by Beijing Huafukang BioScience Company, Beijing, China) were rapidly removed. Ventricles were finely cut and digested in 0.2% collagenase type II (Gibco, Waltham, Massachusetts, USA) at 37\u0026deg;C for eight min, and the isolated cells were neutralized in cell culture medium. Cardiac tissues were digested until they disappeared. All suspensions were pelleted by centrifugation at 1000 rpm for five min and resuspended in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium nutrient mixture F-12 (DMEM/F-12, Gibco, Waltham, Massachusetts, USA) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin solution (100 U/mL). Cells were plated in 10-cm dishes and transferred to an incubator at 37\u0026deg;C with an atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e. After allowing two hours for fibroblast adherence, the CMs was seeded onto culture plates for subsequent treatments. All animal experimental procedures were performed in compliance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH, revised 1996) and with the approval of Anzhen Hospital (approval no. AZ2023LA014), which is affiliated with the Capital Medical University. This work was reported in accordance with the ARRIVE Guidelines 2.0.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTrans-well analysis and co-culturing of MSCs with CMs\u003c/h3\u003e\n\u003cp\u003eBone marrow-derived MSCs, derived from SD rats were purchased from Procell (Wuhan, China). To observe the role of TNTs in alleviating isopreterenol (ISO)-induced myocardial hypertrophy in MSCs, Trans-well culture and co-culture modes of MSCs and CMs were established in vitro, as previously described with some modifications [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Briefly, the Trans-well culture model consisted of indirectly culturing MSCs and CMs in a 2:1 ratio using a semipermeable membrane as a Trans-well insert (pore size, 0.4 \u0026micro;m; Corning, USA), which prevented the formation of TNTs between the two kinds of cells but allowed the diffusion of secreted factors. The co-culture model involved directly mixing and culturing MSCs and CMs in the same dish in a 2:1 ratio. All cells were maintained in DMEM/F-12.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence staining analysis\u003c/h3\u003e\n\u003cp\u003eMSCs and CMs were fixed in 4% paraformaldehyde for 20 min. After incubation, cells were penetrated with 0.2% Triton X-100, blocked with 5% BSA in PBS, and then stained with primary antibodies, α-tubulin (Abcam, Cambridge, UK), cardiac troponin T (Abcam) or Cx43 (Cell Signaling Technology, Boston, MA, USA) at 4\u0026deg;C overnight, followed by Alexa Fluor 546/633 secondary antibodies. TRITC Phalloidin (Yeasen, China) and WGA Alexa Fluor\u0026reg; 488/555 conjugates (Invitrogen, Carlsbad, CA, USA) were incubated with the cells for 30 min at room temperature for F-actin or cell membrane staining. For living cell membrane staining, CellTracker Green CMFDA (Invitrogen) was incubated with the cells for 45 min under cell growth condition.\u003c/p\u003e\n\u003ch3\u003eFlow cytometry\u003c/h3\u003e\n\u003cp\u003eFollowing labeling with CellTracker Green CMFDA for 45 min at 37℃ in serum-free DMEM/F-12, CMs were co-cultured with MSCs for 24 h. After stimulation with ISO (10 \u0026micro;M for 24 h), CMs were sorted from other co-cultured cells according to CellTracker Green CMFDA immunostaining. Sorted cells were used to detect the expression levels of myocardial hypertrophy markers.\u003c/p\u003e\n\u003ch3\u003eQuantitative Real-Time PCR (qRT-PCR)\u003c/h3\u003e\n\u003cp\u003eAfter 24 h of ISO treatment, total RNA was extracted from the sorted CMs using TRIZOL reagent, and reverse transcription reactions were performed using HiScript II Q RT SuperMix for qPCR (Vazyme, NanJing, China). QRT-PCR was performed on a PCR detection system using ChamQ Universal SYBR qPCR Master Mix (Vazyme). PCR conditions were 95\u0026deg;C for 30 s, 40 cycles at 95\u0026deg;C for 10 s, and 60\u0026deg;C for 30 s, 95\u0026deg;C for 15 s, 60\u0026deg;C for 60 s, and 95\u0026deg;C for 15 s. The expression levels of the target genes were normalized against GAPDH mRNA levels. Primer sequences are summarized in Table\u0026nbsp;1.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLentiviral infection\u003c/h2\u003e \u003cp\u003eTo overexpress Cx43, Lentiviral-mCherry-Cx43 (LV-mCherry-Cx43) was constructed using the lentiviral vector pSLenti-CMV-mCherry (LV-mCherry). The Cx43 coding sequence was inserted between the EcoRI and XbaI genes of the vector, and mCherry was fused in frame to the carboxyl terminus of Cx43 with the addition of a ten-amino-acid polylinker (GGGSGGGGGS).\u003c/p\u003e \u003cp\u003eAfter infecting MSCs and CMs with LV-mCherry-Cx43 for eight hours in serum-free medium containing 5 \u0026micro;g/mL polybrene, we replaced the serum-containing medium and continued to culture for 5\u0026ndash;7 days. Control cells were infected with an empty vector (LV-mCherry).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTransfection of siRNAs\u003c/h3\u003e\n\u003cp\u003eWe used rat small interfering RNA (siRNA) targeting Cx43 to silence its gene expression. A scramble was used as the non-targeting control. The sequences for the Cx43 siRNA and siRNA control were as follows: Cx43 siRNA#1-F: 5\u0026prime;-GCUUCUGGACAAGGUCCAAGCTT-3,\u0026rsquo; Cx43 siRNA#1-R: 5\u0026prime;-GCUUGGACCUUGUCCAGAAGCTT-3\u0026prime;; Cx43 siRNA#2-F: 5\u0026prime;-GGAAGGAAGAGAAGCUAAACATT-3\u0026rsquo;, Cx43 siRNA#2-R: 5\u0026prime;-UGUUUAGCUUCUCUUCCUUCCTT-3\u0026prime;; siRNA control-F: 5\u0026prime;-UUCUCCGAACGUGUCACGUTT-3\u0026rsquo;, siRNA control-R: 5\u0026prime;-ACGUGACACGUUCGGAGAATT-3\u0026prime;.\u003c/p\u003e \u003cp\u003eTransfection of MSCs with Cx43 siRNA#1 and #2 involved diluting the siRNA with DMEM/F-12 without serum, and incubating the cells with GP-transfect-Mate reagent (Genepharma, Shanghai, China) and the siRNA solution for four hours. The cells were further cultured in complete culture medium for three days before conducting subsequent experiments.\u003c/p\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eGroup values are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). An unpaired two-tailed t-test or one-way analysis of variance (ANOVA) was applied to determine the significance of differences between two or more groups of parametric data, respectively. Statistical analyses were performed using GraphPad Prism software version 6.01 (GraphPad Software Inc., San Diego, CA, USA), and differences were considered significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEffect of ISO on the formation and morphology of TNTs between bone marrow (BM)-MSCs and neonatal rat CMs\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePreviously, we discovered that TNTs can form between CMs and MSCs [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In this study, we aimed to determine the biological functions of these TNTs in alleviating MSC-induced CM hypertrophy. First, we used a confocal microscope to observe and quantitatively analyze the effect of ISO, a compound that induces myocardial hypertrophy, on TNT formation between the two cell types. Our results revealed that TNTs were present, and their numbers significantly increased after 24 h of co-culture with ISO stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B), indicating that ISO promoted the formation of intercellular TNTs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we quantified the length and thickness of 92 TNTs in the ISO-treated group and 118 TNTs in the without ISO (control) group. Both measurements significantly increased in the ISO group: length (38.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79 \u0026micro;m vs 26.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52 \u0026micro;m) and thickness (2.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 \u0026micro;m vs 1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 \u0026micro;m) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D, E).\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eISO stimulation significantly increases TNTs derived from MSCs\u003c/h2\u003e \u003cp\u003eWe further investigated the effect of ISO on the cellular origin of TNTs. Confocal microscopy was employed to observe TNTs derived from CellTracker\u0026trade; Green/ WGA555-labeled CMs or WGA 555-labeled MSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Almost 47% of the 94 TNTs were derived from MSCs, and this proportion significantly increased to about 60% of the 118 TNTs in the with ISO group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). However, we did not observe any TNTs that originated from both types of cells simultaneously. These results suggest that ISO promoted the formation of TNTs from MSCs to CMs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffect of ISO on the cytoskeleton in TNTs\u003c/h2\u003e \u003cp\u003eConsistent with previous reports, both F-actin and microtubules were observed in the TNTs between MSCs and CMs [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. F-actin and microtubules are essential for the formation and function of TNTs [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], with microtubules also influencing the morphology of these structures [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. To further investigate the effects of ISO on the cytoskeleton in TNTs, we examined the presence of F-actin and microtubules. F-actin was observed in all TNTs regardless of ISO stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, microtubules were present in only some TNTs, but ISO significantly increased the proportion of TNTs that contained microtubules (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePotential role of TNTs in MSC-mediated alleviation of ISO-induced myocardial hypertrophy\u003c/h2\u003e \u003cp\u003eISO can induce myocardial hypertrophy, and paracrine secretion is an important mechanism by which stem cell therapy addresses this pathological condition [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. As a novel form of intercellular \u0026ldquo;communication,\u0026rdquo; TNTs mediate the transfer of multiple signaling molecules and functional proteins between cells [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], potentially providing a new pathway for MSCs to alleviate ISO-induced myocardial hypertrophy. To verify this hypothesis, we quantified the cellular area of CMs when co-cultured with MSCs under ISO stimulation, using both Trans-well and direct co-culture systems. Our results showed that the area of ISO-treated CMs decreased when cells were trans-well cultured with MSCs, and the anti-hypertrophic effect further enhanced under co-culture conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). We also analyzed the expression level of brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC), both indicators of myocardial hypertrophy, using quantitative RT-PCR (qRT-PCR). We obtained CellTracke Green CMFDA-labeled CMs, which co-cultured with MSCs, through flow cytometry sorting (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The results showed that MSCs in trans-well culture inhibited the ISO-induced increase in BNP and β-MHC mRNA expression, with further reduction observed under co-culture conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E). These results indicate that, in addition to paracrine secretion, MSCs may alleviate ISO-induced myocardial hypertrophy through TNTs. The list of primers used in qRT-PCR is shown in Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eISO facilitates TNT-mediated Cx43 transfer from MSCs to CMs\u003c/h2\u003e \u003cp\u003eHow do TNTs promote MSC functions in inhibiting myocardial hypertrophy? Previous clinical and scientific studies have shown that Cx43 is closely associated with the occurrence and progression of myocardial hypertrophy [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In addition, Cx43 has been detected in various intercellular TNTs [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Based on these findings, we hypothesized that MSCs alleviate myocardial hypertrophy by transferring Cx43 via TNTs. To test this hypothesis, we investigated whether TNTs mediated the transfer of Cx43 from MSCs to CMs. Using immunofluorescence, we labeled Cx43 with red fluorescence and observed it under a confocal microscope. We observed that Cx43 was present in some intercellular TNTs, and the proportion of TNTs containing Cx43 significantly increased following ISO stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Additionally, we constructed a lentivirus (LV)-mCherry-Cx43 and expressed it in MSCs. After co-culturing LV-mCherry-Cx43-expressing MSCs with CellTracke Green CMFDA-labeled CMs for 24 h, we detected mCherry-Cx43 in some CMs via flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). This proportion increased significantly with ISO stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). However, no mCherry-Cx43-positive CMs were detected in the Trans-well culture system. These results suggest that Cx43 is transferred from MSCs to CMs via TNTs, and ISO promoted this process. Similar to the characteristics of endogenous Cox43, we also observed aggregation of mCherry-Cx43 at cell-cell junctions. Additionally, the presence of Cx43-mCherry in TNTs was verified (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eTNTs inhibit ISO-induced myocardial hypertrophy by transmitting Cx43\u003c/h2\u003e \u003cp\u003eTo define the role of Cx43 in ISO-induced myocardial hypertrophy, we overexpressed LV-mCherry-Cx43 in CMs and quantitatively analyzed the area of Cx43-positive CMs under ISO stimulation. The results showed that upregulating Cx43 expression significantly alleviated ISO-induced myocardial hypertrophy (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBuilding on previous results that TNTs may provide a novel pathway for MSCs to alleviate ISO-induced myocardial hypertrophy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), we next examined the effect of Cx43 depletion on TNT-mediated MSC activation. We transfected MSCs with a specific siRNA targeting Cx43 and co-cultured them with CMs for 24 h. Cx43 silencing strongly impaired the ability of MSCs to alleviate myocardial hypertrophy under co-culture conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, C). The efficiency of siRNA-induced Cx43 silencing was detected by western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Additionally, depletion of Cx43 did not affect the ability of ISO to promote TNT formation (Supplementary Figs.\u0026nbsp;2A, B). These data suggest that MSCs may alleviate ISO-induced myocardial hypertrophy by transmitting Cx43 to CMs via TNTs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eFive decades after the discovery of MSCs, these cells have become leading candidates for cell-based therapies [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Currently, MSCs are widely used to treat various heart diseases, including myocardial infarction, myocardial hypertrophy, and dilated cardiomyopathy [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Significant progress has been made in understanding the mechanisms through which MSCs exert their therapeutic effects on cardiovascular diseases, but it mainly focuses on self-differentiation and paracrine signaling pathways [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Despite increasing interest and extensive research, many questions remain regarding the underlying biological mechanisms of MSCs.\u003c/p\u003e \u003cp\u003eTNTs are long-distance filamentous junctional structures that connect various types of cells, such as T cells, B cells, nerve cells (neurons), and cancer cells. These structures can mediate the transport of signaling molecules and organelles, thereby exerting certain biological effects [\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Both our research group and Rodriguez et al. have reported that TNTs form between MSCs and damaged CMs[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Furthermore, our research group revealed that MSCs can transport fully functional mitochondria along microtubules to damaged CMs through TNTs, thereby alleviating myocardial cell apoptosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This observation indicates that TNTs may serve as a novel pathway by which MSCs contribute to the treatment of heart diseases.\u003c/p\u003e \u003cp\u003ePathological myocardial hypertrophy, induced by stimuli such as hypertension and valvular disease, is a leading risk factor for heart failure, sudden cardiac death, and triggers various cardiovascular conditions such as arrhythmia and myocardial infarction [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The role of stem cells in treating myocardial hypertrophy has received extensive attention and considerable in-depth research has been devoted to understanding their mechanisms of action in this context [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In this study, we investigated whether TNTs play an important role in alleviating myocardial hypertrophy in MSCs.\u003c/p\u003e \u003cp\u003eTo clarify this, we investigated the effects of ISO, a β-receptor agonist that is widely used to induce myocardial hypertrophy [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], on the quantity and morphology of intercellular TNTs. Our quantitative analysis showed that ISO stimulation significantly increased the number of TNTs formed between the cells (MSCs or CMs). Furthermore, a significant elongation and thickening of TNTs was also noticed. We also found that ISO significantly upregulated the ratio of TNTs originating from MSCs, suggesting that ISO enhances the ability of MSCs to influence CM function via TNTs. While the exact mechanism by which ISO regulates the formation and extension of TNTs remains unclear, recent studies have suggested that the F-actin cytoskeleton is essential for TNT formation in virtually all cell types [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Several well-characterized proteins that modulate cellular actin dynamics play key roles in TNT formation, such as the Arp2/3 complex, small GTPases, Cdc42, and M-sec [\u003cspan additionalcitationids=\"CR47 CR48 CR49\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Moreover, ISO-induced activation of β-2 adrenergic receptors affects actin cytoskeleton organization [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], which may in turn affect TNT formation and its extension. We propose that future studies should explore the influence of ISO on actin dynamics and, subsequently, TNT formation.\u003c/p\u003e \u003cp\u003eConsidering that ISO induces myocardial hypertrophy, whereas MSCs can treat it, we further explored whether TNTs are involved in MSC-mediated protection against myocardial hypertrophy. By analyzing the effects of ISO on CM area under different cultivation conditions, we found that direct co-culture with MSCs, as compared to transwell cultures, more effectively alleviated ISO-induced myocardial hypertrophy. This observation suggests that in addition to paracrine secretion, TNTs play an important role in the ability of MSCs to treat myocardial hypertrophy. This conclusion was further confirmed by qRT PCR analysis, which showed that MSC co-culture reduced the expression of BNP and β-MHC, two well-established markers of myocardial hypertrophy in CMs.\u003c/p\u003e \u003cp\u003eCx43 has been implicated in the pathogenesis of myocardial hypertrophy. Studies have shown that the Cx43 expression is significantly reduced in ISO-induced hypertrophic CMs, and that maintaining or upregulating Cx43 levels can alleviate myocardial hypertrophy and improve cardiac function [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Additionally, in patients with myocardial hypertrophy, Cx43 expression in the myocardium is significantly decreased [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These findings highlight the close association between Cx43 and the occurrence and development of myocardial hypertrophy. Cx43 is also present in TNTs, including those formed by pluripotent stem cell (iPSC)-derived MSCs, epithelial cells, and even cancer cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. We observed the presence of Cx43 in some TNTs between MSCs and CMs and found that ISO significantly increased the proportion of TNTs containing Cx43. Using flow cytometry, we further demonstrated that MSCs could transmit Cx43 to CMs via TNTs, and this process was facilitated by ISO. These results suggest that ISO promotes Cx43 transmission from MSCs to CMs via TNTs. The cytoskeleton, motor proteins, and autophagy are known to be involved in cargo transport in TNTs [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. However, the precise mechanism by which ISO promotes Cx43 transmission remains unclear and warrants further investigation.\u003c/p\u003e \u003cp\u003eTo further demonstrate the role of TNT-mediated Cx43 in MSC-mediated alleviation of myocardial hypertrophy, we depleted Cx43 in MSCs using siRNA. The results showed that depletion of Cx43 significantly impaired the ability of MSCs to reduce myocardial hypertrophy under co-culture conditions. Furthermore, we confirmed that overexpression of Cx43 in CMs inhibited ISO-induced myocardial hypertrophy. While these findings support the hypothesis that Cx43 plays a critical role in MSC-mediated protection against hypertrophy, several questions remain unanswered: how does TNT-mediated Cx43 transfer occur? How is Cx43 internalized by CMs? What specific mechanisms through which Cx43 alleviates myocardial hypertrophy remain to be clarified? Cx43 is an important protein that forms gap junctions between CMs, facilitating intracellular communication and calcium signaling. The localization and expression of Cx43 are critical for proper calcium signal transmission between CMs. Therefore, it is important to explore whether TNT-mediated Cx43 transfer affects calcium signaling in the heart, as this could provide important insights into how Cx43 contributes to the alleviation of myocardial hypertrophy.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eISO stimulation caused significant changes in the quantity, morphology, composition, and origin of TNTs formed between MSCs and CMs. MSCs use TNTs to transport Cx43 to CMs, which plays a key role in alleviating ISO-induced myocardial hypertrophy. Our study suggests that, in addition to self-differentiation and paracrine secretion, TNTs provide a novel pathway for MSCs to treat pathological myocardial hypertrophy. Future investigations should focus on uncovering the molecular mechanisms underlying TNT formation, cargo transport, and the role of Cx43 in calcium signaling and myocardial remodeling. These lines of investigations will be essential for optimizing the therapeutic potential of TNTs in regenerative cardiovascular medicine and enhancing the effectiveness of MSC-based therapies for myocardial hypertrophy and other cardiac disorders.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"573\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eTNTs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eTunneling nanotubes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eMSCs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eMesenchymal stem cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCMs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCardiomyocytes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eISO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eIsopreterenol\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCx43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eConnexin43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eLV-Cx43- mCherry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eLentiviral-Connexin 43-mCherry\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eLV- mCherry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003epSLenti-CMV-mCherry\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBNP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBrain natriuretic peptide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026beta;-MHC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026beta;-myosin heavy chain\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eTnT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eTroponin T\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge Peking University International Hospital Center for their financial support and guidance.\u0026nbsp;The authors declare that artificial intelligence is not used in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJZ, SL,\u0026nbsp;LZ\u0026nbsp;performed all\u0026nbsp;experiments.\u0026nbsp;ZX, YL, WL\u0026nbsp;and CS\u0026nbsp;participated in the data collection, data analysis. JZ and SC supervised the study and edited the manuscript. HJ and SC acquired the funding. All\u0026nbsp;authors have read and approved the\u0026nbsp;current version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a grant from the National Natural Science Foundation of China (No: 82370440) and the Natural Science Foundation of Beijing Municipality (7232226).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that all data generated or analyzed during this study are included in this published article and its supplementary file.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experimental procedures were performed in adherence with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH, revised 1996) and with the approval of the\u0026nbsp;Capital medical university affiliated Anzhen hospital (Approval No.\u0026nbsp;AZ2023LA014, Data of Approval:\u0026nbsp;February 11, 2023). And the title of the approved project is \u0026ldquo;The role of Tunneling Nanotubes between mesenchymal stem cells and cardiomyocytes in alleviating isoproterenol induced cardiac hypertrophy\u0026rdquo;.\u0026nbsp;This study did not involve human\u0026nbsp;subjects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors and institutions have confirmed this manuscript for publication\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKehat I, Molkentin JD. Molecular pathways underlying cardiac remodeling during pathophysiological stimulation. 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Result Probl Cell Differ. 2024;73:155\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mesenchymal stem cells, Cardiomyocytes, tunneling nanotubes, Connexin43, myocardial hypertrophy","lastPublishedDoi":"10.21203/rs.3.rs-5069090/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5069090/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParacrine signaling plays an important role in stem cell therapy. However, it alonecannot fully explain the therapeuticmechanisms of stem cell therapy in treating heart diseases. Recently, tunneling nanotubes (TNTs)—a novel type of long-distance intercellular connectional structure—have been identified between mesenchymal stem cells (MSCs) and cardiomyocytes (CMs). TNTs mediate the transmission of multiple signaling molecules, enabling cells to exert different biological functions. In the present study, we investigated the role of TNTs in MSC-based therapy for myocardial hypertrophy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMSCs and CMs were co-cultured for 24 h with or without isopreterenol (ISO) to induce myocardial hypertrophy. Confocal microscopy was used to quantify and analyze the number, morphology, composition, and cell source of TNTs between MSCs and CMs. The effects of ISO on CMs were assessed by comparing cell area (measured by confocal microscopy) and expression levels of hypertrophy-related genes (using qRT-PCR) under co-culture and trans-well culture conditions. Flow cytometry was employed to assess the transfer of connexin43 (Cx43) from MSCs to CMs; lentivirus-mediated Cx43 overexpression and Cx43 siRNA were used to investigate the effects of Cx43 on ISO-induced myocardial hypertrophy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eISO stimulation significantly increased the number, length, and thickness of TNTs between MSCs and CMs (number: P\u0026lt;0.05; length and thickness: P\u0026lt;0.01). ISO also increased the proportion of TNTs containing microtubules and those derived from MSCs (P\u0026lt;0.05). Co-culture conditions were more effective than trans-well culture in alleviating ISO-induced myocardial hypertrophy (P\u0026lt;0.05). Furthermore, Cx43 was observed in TNTs, and ISO enhanced the transfer of Cx43-mCherry from MSCs to co-cultured CMs (P\u0026lt;0.05). Overexpression of Cx43 in CMs alleviated myocardial hypertrophy, whereas knocking down of Cx43 in MSCs reduced their ability to alleviate myocardial hypertrophy (P\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur results demonstrate that ISO promotes the formation of TNTs, particularly between MSCs and CMs, and induces changes in the morphology of TNTs (thickening and lengthening). Additionally, MSCs transmitted Cx43 to CMs via TNTs, which contributes to the alleviation of ISO-induced myocardial hypertrophy. These results suggest that TNTs represent an important mechanism in MSC-mediated therapy for myocardial hypertrophy.\u003c/p\u003e","manuscriptTitle":"Bone marrow mesenchymal stem cells transport connexin43 via tunneling nanotubes to alleviate isopreterenol-induced myocardial hypertrophy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-11 15:37:23","doi":"10.21203/rs.3.rs-5069090/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-11-08T12:16:45+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-08T11:49:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-08T07:49:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Stem Cell Research \u0026 Therapy","date":"2024-11-08T02:37:12+00:00","index":"","fulltext":""},{"type":"decision","content":"Minor Revision","date":"2024-09-19T11:06:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2ce62280-262f-41be-818f-d60b735d8bee","owner":[],"postedDate":"November 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-12T16:06:00+00:00","versionOfRecord":{"articleIdentity":"rs-5069090","link":"https://doi.org/10.1186/s13287-025-04339-w","journal":{"identity":"stem-cell-research-and-therapy","isVorOnly":false,"title":"Stem Cell Research \u0026 Therapy"},"publishedOn":"2025-05-06 15:57:39","publishedOnDateReadable":"May 6th, 2025"},"versionCreatedAt":"2024-11-11 15:37:23","video":"","vorDoi":"10.1186/s13287-025-04339-w","vorDoiUrl":"https://doi.org/10.1186/s13287-025-04339-w","workflowStages":[]},"version":"v1","identity":"rs-5069090","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5069090","identity":"rs-5069090","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}