Cardiac fibroblasts (CFs) are abundant in the heart, where they play a major role in cardiac homeostasis and disease development (1). Recently, small extracellular vesicles (sEV) released by mouse CFs have been implicated in cardiac hypertrophy (2). However, if human CF sEV have a role in cardiac disease remains unclear. Here we isolate CFs from hypertrophic cardiomyopathy (HCM) patients, purify and characterise their sEV and study the effect of these sEV on Ca²⁺ cycling and action potentials of human cardiomyocytes (CMs).

CFs isolated from HCM patients’ biopsies were characterised by RT-qPCR, flow cytometry and immunohistochemistry. Media conditioned by CFs over 72 hours was concentrated by ultrafiltration. sEV were purified by size exclusion chromatography and characterised by nanoparticle tracking analysis and tetraspanin staining, purity was determined using microBCA. CMs were differentiated from human induced pluripotent stem cells and cultured for 28 days before purification using MACS bead separation. Purified CMs were cultured with sEV from HCM CFs for 48 hours. Action potentials and calcium (Ca²⁺) transients were studied using optical mapping, with calcium dye (Fluo-4) or voltage dye (FluoVolt), during field stimulation at 0.7Hz, 1Hz and 1.5Hz. Signals were analysed using OPTIQ software. Unpaired T-tests were used for statistical analysis of 2 groups.

CFs from HCM patient biopsies had a myofibroblast pheno/genotype determined by the presence of myofibroblast markers: α-SMA, COL1A1, FAP. Pure sEV were found in fractions 7-9 (>2×10¹⁰ particles/µg), had a modal size of 109±3.5nm and were positive for the tetraspanins CD63, CD81 and CD9. On average HCM CFs secreted 104,676 sEV per cell (n=3). CM Ca²⁺ transient parameters, time to peak and time to 50/80% Ca²⁺ decay, were unaffected by HCM CF sEV at a concentration of 5×10¹⁰ particles/ml (p>0.05 vs untreated CM, n=4). However, CM action potentials were prolonged after culture with HCM CF sEV at a concentration of 5×10¹⁰ particles/ml with a significant increase in action potential duration (APD)50 at 1Hz (p<0.01 vs untreated CM), 0.7Hz and 1.5Hz (p<0.05); and significant increase in APD80 at 1Hz and 0.7Hz (p<0.01) and 1.5Hz (p<0.05, n=4).

Our results indicate that CFs isolated from HCM patients have a myofibroblast phenotype and secrete a large number sEV. Treating human CMs with highly pure sEV secreted by HCM CFs prolongs CM action potential duration, highlighting a potential role of CF sEV in heart disease.