Abstract 10183: Augmentation of Cardiac Micrornas Enhanced Cardiac Mesenchymal Cell Differentiation Into Cardiomyocyte Like Cells

Abstract

Introduction: Infusion of cardiac mesenchymal cells (CMCs) showed improved cardiac function in myocardial infarction models. Cell based therapy to treat cardiac injuries have shown modest success due to limitations, including poor retention and differentiation. Current studies are focused to improve engraftment and differentiation potential of the therapeutic cells. Here, we used cardiac miRNAs in CMCs to increase their differentiation potential towards cardiac lineage. Hypothesis: Increased expression of cardiac miRNAs in CMCs could allow them to differentiate into cardiomyocyte like cells. Methods: Mouse CMCs were isolated from the adult mouse heart and analyzed for the expression of cardiac miRNAs. The CMCs were transiently transfected with cardiac miRNAs cocktail (miR-1, miR-133, miR-208 and miR-499) and cultured for 48h. (n=3) The potential miRNAs targets such as HDAC4 (miR-1), Snail-1 (miR-133), myostatin (miR-208) and Sox6 (miR-499) were analyzed using western blot and immunostaining. The miRNAs cocktail transfected CMCs were cultured for 7 days and analyzed for the expression of cardiac specific genes with RT-PCR. (n=3) Results: Quantitative PCR analysis showed that the cardiac miRNAs were either less or not expressed in CMCs, whereas transient transfection significantly elevated them. Western blot analysis showed significant downregulation of cardiac miRNAs targets HDAC4 (0.62±0.06), Snail1 (0.61±0.03), Sox6 (0.75±0.01) and Myostatin (0.3±0.04). Immunostaining confirmed significant reduction of these target proteins. Cardiac miRNAs cocktail transfected CMCs showed significantly increased expression of cardiac specific genes such as mef2c (3.87±1.54), Gata4 (3.73±1.07), Tnni3 (3.31±1.01), Nkx2.5 (3.89±0.37) Actc1 (4.27±1.57), Tnnt2 (2.49±1.12) and Cx40 (2.37±0.61). The expression of fibroblast markers such as Col1a and Thy1 were reduced upon enriching the cardiac miRNAs in the CMCs. Conclusions: Our results demonstrate that enrichment of cardiac miRNAs in CMCs increase their differentiation into cardiomyocyte lineage, which could improve their therapeutic efficacy. Further in vivo experiments would reveal functional benefits from these improved CMCs for clinical use.