TBX20 Improves Contractility and Mitochondrial Function During Direct Human Cardiac Reprogramming

Abstract

Background: Direct cardiac reprogramming of fibroblasts into cardiomyocytes has emerged as a promising strategy to remuscularize injured myocardium. However, it is insufficient to generate functional induced cardiomyocytes from human fibroblasts using conventional reprogramming cocktails, and the underlying molecular mechanisms are not well studied. Methods: To discover potential missing factors for human direct reprogramming, we performed transcriptomic comparison between human induced cardiomyocytes and functional cardiomyocytes. Results: We identified TBX20 (T-box transcription factor 20) as the top cardiac gene that is unable to be activated by the MGT133 reprogramming cocktail ( MEF2C , GATA4 , TBX5 , and miR-133 ). TBX20 is required for normal heart development and cardiac function in adult cardiomyocytes, yet its role in cardiac reprogramming remains undefined. We show that the addition of TBX20 to the MGT133 cocktail (MGT+TBX20) promotes cardiac reprogramming and activates genes associated with cardiac contractility, maturation, and ventricular heart. Human induced cardiomyocytes produced with MGT+TBX20 demonstrated more frequent beating, calcium oscillation, and higher energy metabolism as evidenced by increased mitochondria numbers and mitochondrial respiration. Mechanistically, comprehensive transcriptomic, chromatin occupancy, and epigenomic studies revealed that TBX20 colocalizes with MGT reprogramming factors at cardiac gene enhancers associated with heart contraction, promotes chromatin binding and co-occupancy of MGT factors at these loci, and synergizes with MGT for more robust activation of target gene transcription. Conclusions: TBX20 consolidates MGT cardiac reprogramming factors to activate cardiac enhancers to promote cardiac cell fate conversion. Human induced cardiomyocytes generated with TBX20 showed enhanced cardiac function in contractility and mitochondrial respiration.

A Novel Transcription Factor Combination for Direct Reprogramming to a Spontaneously Contracting Human Cardiomyocyte-like State

“…While this manuscript was under review, Tang et. al independently identified TBX20 as a cardiac reprogramming factor 45 . In their study, addition of TBX20 to GMT + miR133 resulted in cells with more fully activated cardiac conduction systems.…”

Regeneration of the heart: from molecular mechanisms to clinical therapeutics

“…Alternatively, Lalit et al [ 161 ] showed that mesoderm posterior bHLH transcription factor 1 (MESP1), GATA4, TBX5, NK2 homeobox 5 (NKX2-5), and BAF60C (SMARCD3, SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily D, member 3) expressed in fibroblasts produce a progenitor population that gives rise to cardiomyocytes, endothelial cells, and mural cells in myocardial infarction mice models. Recent study also suggests that the cardiac gene TBX20 (T-box transcription factor 20) enhances myocardial reprogramming induced by the MGT133 reprogramming cocktail (MEF2C, GATA4, TBX5, and miR-133) [ 162 ]. In summary, transcription factor combinations play an important role in transforming fibroblasts into cardiomyocytes in mice.…”

Two decades of heart regeneration research: Cardiomyocyte proliferation and beyond

“…Since the development of GMT-derived iCMs, many have tried to further optimize the transdifferentiation protocol and conditions. The addition of other transcription factors associated with cardiomyocyte function and maturation has been a common approach to further optimize the transdifferentiation process (Addis et al, 2013;Singh et al, 2021;Song et al, 2012;Y. Tang et al, 2022).…”