Abstract
Hypertrophic cardiomyopathy (HCM) is a heart disease in which a genetic mutation in the cardiac sarcomere leads to a thickened left ventricular septum and fibrosis. However, the link between cardiac fibroblasts (CFs) and HCM has not been extensively explored. Small extracellular vesicles (sEV) are small membrane bound vesicles secreted by virtually all cell types that have been shown to cause functional changes on recipient cells inducing pathological responses such as hypertrophy of cardiomyocytes. CFs are known to be sensitive to culture and to isolate sEV serum must be removed from culture media which can lead to premature activation of CFs. SD-208, a TGF-β receptor inhibitor, has been proposed to inhibit and reverse CF differentiation into myofibroblasts. In this study we cultured CFs from HCM patient biopsies and CFs from healthy human donors with 3µM SD-208 to determine if this activation can be blocked/reversed and studied how this affects their sEV secretion.CFs isolated from HCM patients’ biopsies and healthy donors CFs were cultured in the absence or presence of 3µM SD-208. CFs were then 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.SD-208 treatment prevented activation of healthy donor CFs in culture as demonstrated by lower levels of myofibroblast markers e.g., ACTA2 (p=0.0013; + SD-208 vs ‘ SD-208). Furthermore, HCM CFs were reverted to a less activated phenotype after treatment with SD-208, with a significant reduction in myofibroblast markers ACTA2, POSTN, IL-6, IL-11 (p<0.0001) and reduction in the number of α-SMA positive cells (p=0.043; figure 1) compared to serum starved HCM CFs. Differences were seen in the concentration of sEV secreted by CFs either cultured with or without SD-208 in both healthy donor and HCM CFs. There was a significant decrease in sEV secretion when HCM CFs were cultured with SD-208 compared to HCM CFs (6.75-fold change, p<0.0001) whereas for healthy donor CFs there was a 1.37-fold decrease after SD-208 compared CFs alone. The size of sEV remained consistent between the two groups (mode: 91.5 nm vs 93.7 nm; p>0.05). Differences in sEV secretion was also observed between HCM CFs and healthy donor CFs with HCM CFs secreting on average 2.5x more sEV per cell than healthy donor CFs.Our results indicate that SD-208 is very effective at reverting HCM CFs to a less activated state as well as inhibiting the activation seen when healthy donor CFs are cultured without serum. Additionally, SD-208 significantly reduces the concentration of sEV secreted without affecting the size or purity of the populations, indicating that sEV secretion is dependent on the activation status of the cell.Abstract BS34 Figure 1SD-208 reduces percent of α-SMA positive HCM CFs. A)CFs from 6 HCM patients were cultured with no serum in the presence or absence of SD-208. All HCM patient CFs had reduced percentage of α-SMA expressing cells, n=1 per patient, B) Average of α-SMA percentages from all HHCM patient CFs showed a significant decrease in percentage of -SMA expressing cells when HCM CFs were cultured with SD-208 compared to HCM CFs with no serum(p-0.0043). Bars represent mean ± SEM, n-=6, unpaired t-test.Conflict of InterestN/A