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Hypertrophic cardiomyopathy (HCM) is a common genetic disorder, often leading to arrhythmias and heart failure. Despite advances in diagnostics, current treatments remain limited, primarily involving invasive procedures. Myofibroblasts, a key cell-type in HCM progression, contribute to myocardial fibrosis and disrupt cardiomyocyte electrophysiology. In this study, we explored the role of myofibroblast-derived small extracellular vesicles (sEVs) in regulating cardiomyocyte electrophysiology and promoting hypertrophic phenotypes. Primary cardiac fibroblasts from HCM patient biopsies were characterised for fibroblast activation markers and their sEVs were isolated. These sEVs were characterised according to MISEV 2023 guidelines, confirming their size range, morphology, and presence of typical sEV markers. Myofibroblast sEV uptake was observed in recipient fibroblasts and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Furthermore, they were shown to activate control fibroblasts and influence the electrophysiology of hiPSC-CMs, enhancing spontaneous beating activity, metabolic potential and altering contractility and calcium transients. Additionally, sEV treatment resulted in significant hypertrophic changes in hiPSC-CMs, including increased cell size, nuclear count, and expression of hypertrophic markers. Molecular analysis with qRT-PCR and RNA sequencing revealed that sEVs from myofibroblasts modulated the expression of genes involved in hypertrophy and calcium handling, including NPPA, NPPB, MYH6, RYR2, CACNA1C, ITP3R and PLN. Proteomics further identified enrichment of protease inhibitor proteins Serpin E1 and E2, associated with fibrosis and hypertrophy in HCM myofibroblasts and their sEVs. Significantly higher abundance of proteins including Serpin E1 and E2 among others was observed upon treatment of hiPSC CMs with myofibroblasts sEVs. Our findings highlight the pivotal role of myofibroblast sEVs as mediators of intercellular signalling, promoting fibrosis and hypertrophy in cardiomyocytes, thus providing potential targets for novel, less invasive therapeutic interventions in cardiovascular diseases.Introduction Hypertrophic cardiomyopathy (HCM) is the most prevalent genetic cardiomyopathy, affecting approximately one in 500 individuals. Despite advancements in early diagnosis, treatment options remain limited, with surgical myectomy being the primary intervention for symptom relief. 1 2 Myofibroblasts play a critical role in HCM progression and poor prognosis, contributing to myocardial fibrosis, disrupting cardiomyocyte electrophysiology, impairing cardiac function, and increasing the risk of arrhythmias.3–9 Previously, we demonstrated that myofibroblasts influence human cardiomyocyte electrophysiology through paracrine signalling, increasing arrhythmogenic potential.10 However, the precise mechanisms underlying these effects remain unclear, necessitating further investigation to identify novel, less invasive therapeutic strategies.Extracellular vesicles (EVs) have emerged as key mediators of intercellular communication in cardiovascular pathophysiology.11–13 In this study, we explore the role of myofibroblast-derived small EVs (sEVs) in modulating cardiomyocyte electrophysiology and promoting a hypertrophic phenotype, providing new insights into their potential contribution to cardiomyocyte phenotype regulation and HCM progression.Methods Primary cardiac fibroblasts (CFs) were isolated from HCM patient biopsy-derived explants and characterised for fibroblast activation marker expression using qRT-PCR, flow cytometry, Western blotting, confocal microscopy, and collagen secretion assay. sEVs were isolated via differential centrifugation and size exclusion chromatography and characterised according to MISEV 2023 guidelines with sticky plate assay for EV markers, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). 14 EV uptake was assessed in recipient cells using confocal microscopy.To evaluate functional impact of myofibroblast-derived EVs, human iPSC-derived cardiomyocytes (hiPSC-CMs) were treated with sEVs, and their effects on contractility, calcium transients (via optical mapping), and action potential durations were assessed. Additionally, qRT-PCR and OMICS analyses (RNA sequencing and label-free quantitative proteomics (LC/MS-MS)) was performed to examine changes in ion channel and hypertrophy marker expression and elucidate the molecular mechanisms underlying myofibroblast EV-mediated effects in hiPSC-CMs.Results CFs from HCM patient biopsies exhibited high expression of activation markers (αSMA, COL1A1, COL3A1, IL-11, IL-6, FAP, and POSTN), confirmed by qRT-PCR and immunofluorescence. TGF-β1 receptor inhibitor SD208 (3 μM) treatment exerted antifibrotic effects, reducing the expression of activation markers, validated by qRT-PCR, Western blotting, flow cytometry, immunostaining, and collagen secretion assays. Contrarily, immortalised control CFs (ICFs) exhibited significantly increased expression of activation markers upon TGF-β1 stimulation (5 ng/mL) ( figure 1).sEVs isolated from HCM myofibroblasts, ICFs (with and without TGF-β1 treatment), and HEK293 cells were enriched with sEV markers (CD63, CD9, and CD81) and depleted with cell-abundant protein calnexin, demonstrated by sticky plate analysis. TEM analysis confirmed presence of EV particles, while NTA analysis identified particles within sEV size-range. Time-course immunofluorescence studies confirmed the uptake of DiI-labelled myofibroblast sEVs in recipient CFs and hiPSC-CMs as early as 2 hours post-administration, whereas free-dye (DiI in PBS) was not detected in cells even after 24 hours (figure 2).HCM myofibroblast-derived sEVs exhibited autocrine effects in healthy donor-derived CFs, significantly increasing the expression of activation markers, suggesting their role in myocardial fibrosis. Additionally, myofibroblast sEVs influenced hiPSC-CM phenotype in a dose-dependent manner, significantly increasing spontaneous beating activity and metabolic potential. Contractility analysis revealed a reduction in time-to-peak, relaxation, and contraction duration. Optical mapping further demonstrated alterations in calcium handling, with shorter calcium time-to-peak and reduced time to 50% and 80% calcium decay. Notably, hiPSC-CMs treated with myofibroblast sEVs exhibited a hypertrophic phenotype, characterised by increased cell area, perimeter, and nuclear count (figure 3). Contrastingly, HEK293 and ICF sEVs did not significantly affect spontaneous beating or contractility parameters. However, ICF sEVs (with and without TGF-β1 treatment) induced shorter calcium time-to-peak and decay, similar to myofibroblast sEVs.To elucidate the molecular mechanisms dictating these effects, qRT-PCR and RNA sequencing were performed. Myofibroblast-derived sEVs induced upregulation of hypertrophy markers (NPPA, NPPB, and MYH6) and key ion channel genes (RYR2, CACNA1C, ITP3R, and PLN) in hiPSC-CMs (figure 4). Proteomics analysis revealed a significantly higher abundance of fibrosis- and hypertrophy-associated proteins in HCM myofibroblasts compared to SD208-treated cells. Interestingly, compared to SD208 treated controls, myofibroblast and their sEVs were enriched in protease inhibitor SERPIN E1 and E2, proteins known to contribute to fibrosis and hypertrophy. Upon treatment of hiPSC-CMs with sEVs, increased abundance of pro-hypertrophic (SERPIN E1, SERPIN E2, COL1A1) and reduction in cardioprotective proteins were observed (figure 5).Discussion and Conclusions Our study identifies myofibroblast-derived sEVs as key regulators of hiPSC-CM electrophysiology and inducers of cardiomyocyte hypertrophy. By promoting cardiac fibrosis and hypertrophy through autocrine and paracrine signalling, myofibroblast sEVs contribute to disease progression. Further investigations are needed to characterise the effects of their molecular cargo and elucidate the specific cardiomyocyte signalling pathways they influence. Although preliminary, these insights enhance our understanding of HCM pathophysiology and pave the way for novel, targeted therapeutic strategies to mitigate myocardial fibrosis and hypertrophy.Abstract D Figure 1Characterisation of CFs to assess expression of activation markers in HCM myofibroblasts with and without SD208 treatment using (A) qRT-pcr, (B) Flow cytometry, (C) Collagen secretion assay, (D) Western blotting and (E) Immunofluorescence and (F) ICFs with and without TGF-β1 treatment with qRT-PCR. Scale bars represent 50 μm, error bars respresent S.E.M, *<0.05, **<0.01, ***<0.001Abstract D Figure 2Characterisation and uptake of myofibroblast sEVs. (A) Sticky plate analysis for EV markers, (B) Nanoparticle tracking analysis, (C) Transmission electron microscopy. Uptake of DiI labelled sEVs was studied in (D) CFs, (E) hiPSC-CMs using confocal microcopy and free dye control. Scale bars represent 50 μm, error bars respresent S.E.MAbstract D Figure 3Characterisation of myofibroblast sEV effects on hiPSC-CM. (A) Spontaneous beating, (B) Metabolic activity, (C-F) Contractiliy, (G) Cell area, (H) Perimeter, (I, J) Nucleation, (K-M) Calcium transients assessed with optical mapping. Scale bars represent 50 μm, error bars respresent S.E.M, *<0.05, **<0.01, ***<0.001Abstract D Figure 4Molecular effects of myofibroblast sEV treatement in hiPSC-CMs were characterised using qRT-PCR and RNA sequencing to measure changes in expression of (A-D) hypertrophic genes and (E-K) genes which regulate Ca2+ handling. Error bars respresent S.E.M, *<0.05, **<0.01, ***<0.001Abstract D Figure 5Volcano plots generated using proteomic analysis to compare abundance of genes 2-fold upregulated and downregulated in (A) HCM myofibroblasts with and without SD208 treatment. (B) hiPSC-CM with and without sEV treatmentReferences Maron BJ, et al. Management of hypertrophic cardiomyopathy. JACC. 2022;79(4):390–414.Maron BJ, Maron MS. Hypertrophic cardiomyopathy. The Lancet 2013;381(9862):242–255.Bageghni SA, et al. Cardiac fibroblast-specific p38α MAP kinase promotes cardiac hypertrophy via a putative paracrine interleukin-6 signaling mechanism. Faseb j. 2018;32(9):4941–4954.Fujiu K, Nagai R. Fibroblast-mediated pathways in cardiac hypertrophy. J Mol Cell Cardiol. 2014;70:64–73.Fredj S, et al. Interactions between cardiac cells enhance cardiomyocyte hypertrophy and increase fibroblast proliferation. J Cell Physiol. 2005;202(3):891–9.Xie Y, et al. 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J Vet Med Sci. 2023;85(11):1157–1164.Wang BX, et al. Extracellular vesicles from human cardiac fibroblasts modulate calcium cycling in human stem cell-derived cardiomyocytes. Cells 2022;11(7):1171.Welsh JA, et al. Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. J Extracell Vesicles. 2024;13(2):e12404.Author and Collaborator Contributions Applicant- Rahul Sanwlani (R.S) R.S and P.C isolated primary HCM myofibroblasts from patient biopsies. R.S and G.H.T cultured and characterised primary HCM myofibroblasts and ICFs. R.S and G.H.T isolated and characterised sEVs from HCM myofibroblasts, ICFs and HEK 293 cells. R.S performed EV uptake studies using confocal microscopy. R.S and G.H.T isolated and purified hiPSC-CMs and performed studies to assess functional effects of sEVs; contractility, spontaneous beating, metabolic potential, cell size, perimeter, nucleation and calcium handling. RNA sequencing was performed by Novogene, and results were analysed by G.H.T. R.S performed proteomics sample preparations and analyses for fibroblasts, sEVs and hiPSC-CM samples. Figures were prepared by R.S. and G.H.T. Abstract and communication drafted by R.S. Collaborators- E.S and S.D provided access to equipment at UCL to perform NTA analyses for sEVs by R.S and G.H.T.K.S provided HCM patient biopsy samples At Barts Heart Centre, London.A.C assisted in sticky plate analyses at Cardiff University to assess enrichment of EV markers.K.B, C.S.A and S.M assisted in running LC-MS/MS for samples prepared by R.S at La Trobe University and University of Melbourne.