Abstract 13646: Electrical Stimulation Upregulates the miR-99 Family to Promote Cardiac Differentiation and Maturation of Human iPS Cells

Abstract

Background: Electrical stimulation (ES) can efficiently promote cardiomyocyte differentiation and maturation of several cardiac progenitors, but the underlying mechanism has not yet been identified. This study aims to determine whether microRNAs (miRs) contribute to ES-induced cardiomyogenesis of human induced pluripotent stem cells (hiPSCs). Methods and Results: HiPSCs were induced into cardiomyocytes by our defined ES approaches (a biphasic square pulse (5ms) at 5 Hz frequency and with voltage of 0.5-1.0V/cm), and non-ES as treatment control. Cardiac markers (cTnT, Nkx2-5 and α-MHC) were significantly upregulated in hiPSCs with ES treatment as determined by qPCR (Fig.A), Western Blot, and immunostaining (Fig.B). The profile of miRs responsible for ES-induced cardiomyogenesis was analyzed by microarray and qPCR. Importantly, miR-99 family (Fig.C) (i.e., miR-99a and miR-100) were significantly upregulated in hiPSC-derived cardiac cells with ES treatment. Expression of cardiac genes in differentiated hiPSCs with ES treatment was decreased by miR-99a inhibition. Conversely, miR-99a overexpression enhanced sarcomeric maturation of hiPSC-derived cardiac cells under ES, as observed with immunostaining (Fig.E) and transmission electron microscopy. MiR-99a also enhanced the cardiac electrophysiological maturation as determined by patch clamp and calcium influx measurements. MiR-99a putatively targeted the 3'-UTR of BMPR2 mRNA (Fig.D) to facilitate cardiac maturation in hiPSCs, as identified by qPCR and the luciferase reporter assay. Conclusion: The miR-99 family appears to act as a crucial mediator for cardiogenic maturation of hiPSCs treated with ES. ES can enhance cardiac gene expression and structural maturation of hiPSCs for in vitro heart regeneration.