Abstract 470: Gata4, Mef2c and Tbx5 More Efficiently Transdifferentiate Endothelial Cells into Cardiomyocyte-like Cells Through Endothelial-Mesenchymal Transition Process

Abstract

Background: The cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) have been used to successfully reprogram cardiac fibroblasts into induced cardiomyocyte-like cells (iCMs). However their effects on other cell types remain largely unknown. Endothelial cells are an important population of the heart by Pinto et al. (2016). Given the more profound iCM induction effects observed in vivo, we have speculated that reprogramming of endogenous endothelial cells may also play an important role. Purpose: To investigate the cellular reprograming effects and mechanisms of GMT factors on cardiac endothelial cells and compare the outcomes with that from cardiac fibroblasts. Methods: Adult rat cardiac endothelial cells (REC) and cardiac fibroblasts (RCF) were purchased commercially. The cells were cultured in appropriate media and infected with GMT-expressing lentivirus and maintained with iCM media. Fourteen days later Immunofluorescence staining, FACS, and qRT-PCR were performed. Results: Both GMT treated RCFs and RECs showed positive staining of cardiac marker cTnT and αActinin-positive cells via immunofluorescence staining. FACS analysis showed GMT treated RECs had 16.7% of cTnT positive cells versus 3.3% in GMT treated RCF’s. qRT-PCR results demonstrated significant up-regulation of cTnT in GMT infected RECs (74.4±23.3 times, p<0.05) as compared to the control group. Similar upregulation of cTnT was observed in RCFs but to a lesser degree (16.1±7.2 times, p=NS). Moreover, RECs presented a significant change in cell morphology from rounded cells in a cobblestone arrangement to elongated spindle-shaped cells during 14 days culture. In addition, qRT-PCR analysis demonstrated that endothelial-mesenchymal transition (EndoMT) markers such as Slug, Snail, Oct4, and αSMA were upregulated in the cells with GFP encoded virus infection and those markers were downregulated in the GMT-treated RECs (p<0.01 for Snail, p<0.05 for Slug, and αSMA). Conclusion: This report suggests that RECs are susceptive to GMT induced cardiac reprogramming than in RCFs. Our study also indicate that EMT and MET sequential process are involved in the reprogramming of RECs toward iCM cells. RECs could be targeted towards iCM generation for a higher reprogramming rate.