• Li Wang, Ziqing Liu, Chaoying Yin, Huda Asfour, Olivia Chen, Yanzhen Li, Nenad Bursac, Jiandong Liu, and Li Qian.
• From the Department of Pathology and Laboratory Medicine (L.W., Z.L., C.Y., O.C., J.L., L.Q.), McAllister Heart Institute (L.W., Z.L., C.Y., O.C., J.L., L.Q,), and Lineberger Comprehensive Cancer Center (L.W., Z.L., C.Y., O.C., J.L., L.Q.), University of North Carolina, Chapel Hill; and Department of Biomedical Engineering, Duke University, Durham, NC (H.A., Y.L., N.B.).
• Circ. Res. 2015 Jan 16; 116 (2): 237-44.

RationaleGeneration 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.ObjectiveWe 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 ResultsWe 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 enhanced reprogramming efficiency compared with separate G, M, T transduction. Importantly, after further optimization, the MGT vector resulted in more than 10-fold increase in the number of mature beating iCM loci. Molecular characterization revealed that more optimal G, M, T stoichiometry correlated with higher expression of mature cardiac myocyte markers.ConclusionsOur results demonstrate that stoichiometry of G, M, T protein expression influences the efficiency and quality of iCM reprogramming. The established optimal G, M, T expression condition will provide a valuable platform for future iCM studies.© 2014 American Heart Association, Inc.

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