Abstract 18870: Direct Reprogramming of Cardiac Fibroblasts to Cardiomyocyte: Insight into Mechanisms and Feasibility in Human

Abstract

Introduction: Direct conversion of injured heart tissue to functional cardiomyocyte in situ represents an exciting approach for cardiac regeneration. We have recently shown that the combination of miRNAs 1, 133, 208 and 499 were able to reprogram mouse cardiac fibroblasts in vitro and in vivo to cardiomyocyte like cells. Here, we investigate the mechanisms involved in these processes as well as explore the feasibility of this approach in reprogramming human fibroblasts towards the cardiomyocyte fate. Methods and Results: Expression analysis miRNA transfected fibroblast demonstrated rapid induction (1-2 days) of primitive cardiac mesodermal marker Mesp2 but no change in the pluripotency markers Oct4 and Nanog suggesting that reprogramming results from a direct switch to cardiomyocyte progenitor state. Further microarray analysis using the Affymetrix GeneChip Mouse Genome 430 2.0 Array revealed that 1200 genes were differentially regulated between miRNA treated and control cardiac fibroblasts (P<0.01). This list was highly enriched for transcription factors and chromatin remodeling modulators (125 genes). HDAC2, a histone deacetylase that was recently associated with reprogramming of fibroblasts to iPS cells, was the only histone deacetylase that showed significant change upon treatment with the miRNA combination (decreased nearly 95%). These results were corroborated by qRT-PCR. Clustering analysis consistently altered expression of a sub-cluster of 5 genes (with functional relevance to HDAC2, further highlighting the potential importance of HDAC in modulating the miRNA effects directly and/or indirectly. Finally, to explore the feasibility of the miRNA approach to reprogram human fibroblasts, we transiently transfected BJ cells with the microRNA combination. Our preliminary results indicate that combination microRNA treatment of human fibroblasts results in the upregulation of early cardiomyocyte markers such as Mef2. Conclusion: Our data provide insights into the mechanisms of microRNA mediated cardiac reprogramming, indicating direct conversion and the potential role of epigenetic modulation.