Stoichiometry of Gata4, Mef2c, and Tbx5 Influences the Efficiency and Quality of Induced Cardiac Myocyte Reprogramming

Rationale:Generation of induced cardiac myocytes (iCMs) directly from fibroblasts offers great opportunities for cardiac disease modeling and cardiac regeneration. A major challenge of iCM generation is the low conversion rate of fibroblasts to fully reprogrammed iCMs, which could in part be attributed to unbalanced expression of reprogramming factors Gata4 (G), Mef2c (M), and Tbx5 (T) using the current gene delivery approach. Objective:We aimed to establish a system to express distinct ratios of G, M, T proteins in fibroblasts and determine the effect of G, M, T stoichiometry on iCM reprogramming. Methods and Results:We took advantage of the inherent feature of the polycistronic system and generated all possible combinations of G, M, T with identical 2A sequences in a single transgene. We demonstrated that each splicing order of G, M, T gave rise to distinct G, M, T protein expression levels. Combinations that resulted in higher protein level of Mef2c with lower levels of Gata4 and Tbx5 significantly enh...