Small extracellular vesicles released by cardiac fibroblasts from hypertrophic cardiomyopathy patients alter action potential of human cardiomyocytes

Novel Mechanisms of Disease and Arrhythmias (University of Liverpool, UK) (2023) Proc Physiol Soc 53, C03

Oral Communications: Small extracellular vesicles released by cardiac fibroblasts from hypertrophic cardiomyopathy patients alter action potential of human cardiomyocytes

Georgina Thompson1, Elias Suliman1, Konstantinos Savvatis1, Sean Davidson1, Aled Clayton1, John McVey1, Patrizia Camelliti1,

1The Hatter Cardiovascular Institute, University College London United Kingdom, 2School of Biosciences and Medicine, University of Surrey Guildford United Kingdom, 3Inherited Cardiovascular Diseases Unit, Barts Heart Centre London United Kingdom, 4Division of Cancer & Genetics, Cardiff University Cardiff United Kingdom,

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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.



Where applicable, experiments conform with Society ethical requirements.

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