BS50 ALK5 inhibitor SD-208 exhibits antifibrotic activity by reverting myofibroblasts to fibroblasts in hypertrophic cardiomyopathy and inhibits small extracellular vesicle secretion

Hypertrophic cardiomyopathy (HCM) is the most prevalent genetic cardiomyopathy that results due to mutations of sarcomeric proteins. It manifests as left ventricular hypertrophy and fibrosis as the heart wall thickens and become stiff, leading to heart failure and stroke. While myocardial fibrosis is observed as an early manifestation of HCM, the role of cardiac fibroblasts (CFs) in HCM prognosis and effects on cardiomyocyte function remain poorly understood. We have earlier reported a paracrine signalling mediated regulation of human induced pluripotent stem cell derived cardiomyocyte electrophysiology by myofibroblasts (Johnson et al. 2023). All eukaryotic cells are known to secrete nanosized membrane bound small extracellular vesicles (sEVs) which can regulate recipient cell's phenotype. To further understand the role of CFs and their EVs in regulating cardiomyocyte function, in the current study, we used ALK5 inhibitor (ALK5i) SD-208 to characterise its effects on HCM CFs. HCM patient tissue biopsies were processed to culture the explants ex vivo for CF isolation. CFs were cultured with or without SD-208 (3 μM) followed by molecular characterisation with RT-qPCR, flow cytometry, Western blotting, immunostaining, and other in vitro assays. sEVs were collected from CF conditioned media ultrafiltration and size exclusion chromatography (SEC) and characterised with nanoparticle tracking analysis (NTA) and EV marker analysis. SD-208 treatment did not significantly alter the viability and metabolic potential of HCM CFs. However, molecular characterisation suggested significant reduction in mRNA levels of activation markers ACTA2, POSTN, FAP, COL1A1, IL-6 and IL-11 (p<0.0001) followed by SD-208 treatment. Flow cytometry analysis suggested a significant decrease in α-SMA positive cells (p=0.0043). These observations were accompanied by significant reduction in total collagen secreted by CFs following SD-208 treatment (p=0.00062). CF EVs collected using SEC were observed to be in the small EV range as suggested by NTA and were enriched with EV marker proteins CD63, CD81 and CD9. Interestingly, SD-208 treatment led to a 6.5-fold reduction in EV particles secreted by HCM CFs (p<0.0001). Similarly, SD-208 treatment had no significant impact on viability and metabolic potential of HEK 293 cells but led to a dose dependent reduction in EV yield (<0.01). Overall, these findings demonstrate the antifibrotic activity of ALK5i drug SD-208, thus establishing a model to understand the role of CFs in HCM. As SD-208 significantly reduced expression of myofibroblast activation markers in HCM CFs, reverting myofibroblasts to fibroblasts, this would allow for further studies to understand the role of CF sEVs in regulating cardiomyocyte function through paracrine signalling. Lastly, these findings have also highlighted the potential role of SD-208 as a regulator of EV biogenesis and secretion. Reference Johnson RD, Lei M, McVey JH, Camelliti P. Human myofibroblasts increase the arrhythmogenic potential of human induced pluripotent stem cell-derived cardiomyocytes. Cell Mol Life Sci 2023;80(9):276. Conflict of Interest None