MYOFIBROBLAST-DERIVED SMALL EXTRACELLULAR VESICLES ARE REGULATORS OF ELECTROPHYSIOLOGY AND MEDIATORS OF HYPERTROPHY IN HUMAN CARDIOMYOCYTES

Abstract

Introduction Hypertrophic cardiomyopathy (HCM) is a common genetic disorder (~1 in 500 individuals) that leads to arrhythmias and heart failure. Despite advances in early diagnosis, treatment remains limited, with surgical myectomy as the primary intervention. Myofibroblasts drive myocardial fibrosis, disrupt cardiomyocyte electrophysiology, and increase arrhythmic risk. Previously, we demonstrated that myofibroblasts influence cardiomyocyte electrophysiology via paracrine signalling. Here, we investigate the role of myofibroblastderived small extracellular vesicles (sEVs) in regulating cardiomyocyte electrophysiology and hypertrophy. Methods Primary cardiac fibroblasts (CFs) from HCM patient biopsy-derived explants were characterized for activation markers (aSMA, COL1A1, COL3A1, IL-11, IL-6, FAP, POSTN) using molecular and imaging techniques (qRT-PCR, flow cytometry, Western blotting, confocal microscopy, and collagen secretion assay). sEVs were isolated and characterized per MISEV 2023 guidelines. Their uptake in CFs and human iPSC-derived cardiomyocytes (hiPSC-CMs) was assessed by confocal microscopy. Functional effects of sEVs on hiPSC-CMs were evaluated via contractility assays, calcium transients (optical mapping), and action potential duration measurements. Molecular changes were analysed by qRT-PCR, RNA sequencing, and label-free quantitative proteomics (LC-MS/MS). Results HCM-derived CFs exhibited significantly increased activation markers compared to controls, with TGF-beta1 receptor inhibition (SD208, 3 muM) reducing this activation. In contrast, TGF-beta1 stimulation (5 ng/mL) significantly enhanced immortalized control CFs (ICFs) activation. sEVs from HCM myofibroblasts, ICFs (+/-TGF-beta1), and HEK293 cells were enriched with EV markers (CD63, CD9, CD81) and lacked calnexin. TEM and NTA confirmed their morphology and size distribution. DiI-labelled sEVs were taken up by recipient CFs and hiPSC-CMs while no uptake of free dye control was observed. Functionally, myofibroblast-derived sEVs activated quiescent CFs, indicating their fibrosis promoting ability, and altered hiPSC-CM electrophysiology. Treated hiPSC-CMs exhibited increased spontaneous beating rates and metabolic activity, altered contractility, and disrupted calcium transients. Hypertrophic changes included increased cell size, nuclear count, and elevated expression of hypertrophy and calcium-handling genes (NPPA, NPPB, MYH6, RYR2, CACNA1C, ITP3R, PLN) suggested by qRT-PCR and RNA-sequencing. Proteomic analysis revealed enrichment of fibrosis-associated proteins, particularly Serpin E1/E2, in HCM myofibroblasts, their sEVs and sEV-treated hiPSC-CMs, indicating their role in driving hypertrophy. Conclusions Myofibroblast-derived sEVs act as key mediators of intercellular signalling in HCM, driving cardiomyocyte hypertrophy and electrophysiological remodelling. These findings highlight their potential as novel therapeutic targets for less invasive interventions in cardiovascular disease.