Abstract 475: Human Cardiac Progenitor Cells Derived Exosomes Stimulate Cardiomyocytes Proliferation by MicroRNA-Hippo Pathway

Abstract

Background: We identified that human neonatal cardiac progenitor cells (CPCs) improve cardiac function and attenuate adverse left ventricular remodeling after myocardial infarction through their exosomes (nEXOs). RNAseq analysis of nEXOs identified miRs which may be responsible for modulation of Hippo signaling, thereby promoting cardiomyocytes proliferation. Hypothesis: nEXOs effectively stimulate endogenous cardiomyocyte (CM) proliferation by targeting the Hippo pathway to restore cardiac function in an injured heart. Methods and results: nCPCs were conditioned for 48 hours in serum free nutrition mix (Ham’sF12) and nEXOs were purified from supernatant using size exclusion chromatography (SEC; CL2B) coupled with ultracentrifugation. nEXOs were quantified by Nanosight (NS300) and characterized by transmission electron microscopy and flow cytometry for the presence of CD63 and CD9. Our results show recovery of cardiac function (ejection fraction = 63.4% vs 40.5%, n=10, p<0.001)) and generation of myocardial mass in a rodent MI model following nEXOs’ intra-myocardial injection. We further show that nEXOs are preferentially acquired by CMs in the border zone of the infarction (m-cherry-Alix labeled EXOs). Using deep RNAseq (sequencing depth: 25 million reads) coupled with literature based in silico analysis, we identified a candidate panel of miRNAs, predicted to promote CM proliferation by targeting the Hippo Pathway. Our In vitro experiments show that miR-528-3p and miR-7641 (25nM) are the most effective miRs to induce proliferation of quiescent cardiomyocytes (40.1% and 48.8%, respectively, n=3). Increased miR-7641 expression led to a profound increase in quiescent CMs proliferation, in part through repression of the Hippo signal transduction pathway (increased YAP/pYAP ratio). By immunoblotting we show that LATs1/2 (a protein in the Hippo pathway) is directly targeted by miR-7641. nEXOs enriched with miR 7641 promote CM proliferation by 3 folds as compared to non-enriched nEXOs. Conclusion: Our data for the first time demonstrates the ability of EXOs derived micro-RNA based therapeutic approaches to improve cardiac function through the activation of cardiomyocyte proliferation.