MicroRNA-16 Exerts Anti-fibrotic Effects Via Suppression Of Tgfbr3 In Rats With Heart Failure And Preserved Ejection Fraction

Background: Cardiosphere-derived cells (CDCs) reverse diastolic dysfunction by attenuating ventricular fibrosis in rats with heart failure and preserved ejection fraction (HFpEF). MicroRNAs packaged in exosomes secreted from CDCs (CDCexo), are discovered to mediate the beneficial effects of CDCs. Hypothesis: The anti-fibrotic effects of CDCs are mediated by microRNAs contained in CDCexo. Methods: Dahl salt-sensitive rats were fed either a high-salt diet to induce HFpEF, or a normal-salt diet to serve as controls. Echocardiogram-verified HFpEF rats were randomized to receive human CDCexo (n=37) vs. vehicle (n=47) via tail vein weekly for 4 weeks. Control rats (n=16) served as the normal phenotype. RNA sequencing of CDCexo was performed to search for candidate microRNAs. Single-cell RNA sequencing was performed in isolated ventricular cells to investigate the mechanisms of CDCexo. Five most abundant microRNAs were selected and injected via tail vein to HFpEF rats. MicroRNA-injected HFpEF rat hearts underwent single-cell RNA sequencing to investigate its mechanisms. Results: HFpEF rats injected with CDCexo demonstrated improved diastolic function compared to HFpEF rats injected with vehicle (E/E’ ratio 12.1±2.6 vs. 18.7±4.6 in vehicle, p<0.0001). Masson’s trichrome staining revealed reduced ventricular fibrosis in CDCexo rats (3.5±1.4 vs. 5.4±1.4 % in vehicle rats, p=0.016). Negative regulation of transforming growth factor beta (TGF-β) receptor signaling pathway (p=0.002) was identified in isolated cardiac fibroblasts after CDCexo injection. MicroRNA-16 was revealed as the most abundant microRNA in CDCexo, and intravenous delivery of microRNA-16 recapitulated the beneficial responses (improvement of diastolic function and attenuation of ventricular fibrosis) of CDCexo in rats with HFpEF. Single-cell RNA sequencing of microRNA-16-injected HFpEF hearts confirmed down-regulation of TGF-β receptor activity (p=0.018) in cardiac fibroblasts via suppression of Tgfbr3. Conclusion: CDCexo improved diastolic function by suppressing ventricular fibrosis in rats with HFpEF. MicroRNA-16, the most abundant microRNA in CDCexo, recapitulated the beneficial responses of CDCexo in HFpEF rats, via suppression of Tgfbr3 in cardiac fibroblasts.