Transcription Factors MYOCD, SRF, Mesp1 and SMARCD3 Enhance the Cardio-Inducing Effect of GATA4, TBX5, and MEF2C during Direct Cellular Reprogramming

Abstract

Transient overexpression of defined combinations of master regulator genes can effectively induce cellular reprogramming: the acquisition of an alternative predicted phenotype from a differentiated cell lineage. This can be of particular importance in cardiac regenerative medicine wherein the heart lacks the capacity to heal itself, but simultaneously contains a large pool of fibroblasts. In this study we determined the cardio-inducing capacity of ten transcription factors to actuate cellular reprogramming of mouse embryonic fibroblasts into cardiomyocyte-like cells. Overexpression of transcription factors MYOCD and SRF alone or in conjunction with Mesp1 and SMARCD3 enhanced the basal but necessary cardio-inducing effect of the previously reported GATA4, TBX5, and MEF2C. In particular, combinations of five or seven transcription factors enhanced the activation of cardiac reporter vectors, and induced an upregulation of cardiac-specific genes. Global gene expression analysis also demonstrated a significantly greater cardio-inducing effect when the transcription factors MYOCD and SRF were used. Detection of cross-striated cells was highly dependent on the cell culture conditions and was enhanced by the addition of valproic acid and JAK inhibitor. Although we detected Ca2+ transient oscillations in the reprogrammed cells, we did not detect significant changes in resting membrane potential or spontaneously contracting cells. This study further elucidates the cardio-inducing effect of the transcriptional networks involved in cardiac cellular reprogramming, contributing to the ongoing rational design of a robust protocol required for cardiac regenerative therapies.

Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors

“…Various combinations of transcription factors have been shown to activate aspects of the cardiac phenotype when expressed in fibroblasts Christoforou et al, 2013;Fu et al, 2013;Ieda, 2013;Nam et al, 2013;Song et al, 2012;Wada et al, 2013), but the potential diversity of cardiac cell types generated by this reprogramming approach has not been rigorously explored. Here, we established a multiplex immunostaining assay in combination with a PM-specific reporter mouse to distinguish individual endogenous cell types -atrial, ventricular and PM.…”

“…These cells display many of the key features of bona fide cardiomyocytes (CMs), including expression of sarcomeric genes, spontaneous beating activity, calcium oscillations and characteristic action potentials. Interestingly, follow-up studies have revealed that many different combinations of transcription factors, including GHMT (H; Hand2), can function to reprogram fibroblasts into CM-like cells in vitro Christoforou et al, 2013;Fu et al, 2013;Nam et al, 2013;Protze et al, 2012;Song et al, 2012;Wada et al, 2013). Based on these results, several groups subsequently demonstrated direct reprogramming of activated fibroblasts toward a cardiac phenotype in vivo (Inagawa et al, 2012;Jayawardena et al, 2012;Mathison et al, 2012;Qian et al, 2012;Song et al, 2012).…”

Strategies for Heart Regeneration

“…Through global gene expression analysis, they demonstrated the significantly greater cardiac-inducing effects of Myocd and Srf. 17) Very recently, we demonstrated that miR-133 overexpression paired with GMT generated seven-fold more beating iCMs from mouse embryonic fibroblasts, compared to GMT treatment alone; this treatment also shortened the duration required to induce beating iCMs (from 30 days to 10 days). Furthermore, we found that miR-133-mediated Snai1 repression was critical for cardiac reprogramming in adult mouse (and human cardiac) fibroblasts, and that silencing fibroblast signatures via miR-133/Snai1 was a key molecular roadblock during cardiac reprogramming.…”

Reprogramming Approaches to Cardiovascular Disease: From Developmental Biology to Regenerative Medicine

“…Since then, other labs around the world have reported success in reprogramming mouse fibroblasts into iCMs with similar cocktails of reprogramming factors [6][7][8][9][10]. We and others have also directly converted human cardiac and dermal fibroblasts into cardiac cells [11][12][13].…”