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

Abstract

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 enhanced reprogramming efficiency compared to 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 iCMs loci. Molecular characterization revealed that more optimal G, M, T stoichiometry correlated with higher expression of mature cardiac myocyte markers. Conclusion Our 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.

Stoichiometry of Transcription Factors Is Critical for Cardiac Reprogramming

“…13 In this study, Wang et al generated 6 polycistronic constructs with identical self-cleaving 2A peptides (P2A and T2A) to include all possible splicing orders of G, M, and T in a single transgene (ie, MGT, MTG, GMT, GTM, TMG, and TGM; Figure). 15 The total and endogenous transcript levels of G, M, and T were not significantly different in the CFs transduced with the 6 polycistronic vectors, whereas the G, M, and T protein expression was high when the reprogramming factor was placed at the 5′ end of a polycistronic construct, upstream of the P2A peptide. This is consistent with the results of a previous study demonstrating that the P2A peptide is more efficiently cleaved than other 2A peptides for efficient protein translation from polycistronic vectors.…”

“…In this issue, Wang et al 15 generated a complete set of polycistronic constructs encoding G, M, and T with all possible splicing orders and analyzed the cardiac reprogramming efficiency using these vectors. They found that each polycistronic vector gave rise to distinct G, M, and T protein expression levels, and that an optimal balance of G, M, and T protein expression greatly improved both the efficiency and the quality of cardiac reprogramming.…”

Direct Cardiac Reprogramming

“…29 The low and variable reprogramming efficiency by GMT may be because of the stoichiometry of reprogramming factors in the cells transduced with separate vectors. 80,82 To address this, Wang et al 83 recently generated all possible polycistronic vectors of GMT and demonstrated that the stoichiometry of reprogramming factors is indeed critical for cardiac reprogramming. They found that the 2 polycistronic vectors, MGT and MTG, which expressed a high level of Mef2c and low levels of Gata4 and Tbx5, enhanced cardiac reprogramming, whereas the other vectors reduced reprogramming efficiency (Figure 2).…”

The Proangiogenic Potential of Mesenchymal Stem Cells and Their Therapeutic Applications

“…The reprogramming efficiency of the retrovirus-mediated gene delivery approach is partially dependent on the stoichiometry of the delivered transcription factors [102]. It was reported that a higher reprogramming efficiency than that achieved in the original GMT experiment when the stoichiometry of the transcription factors is altered [102]. In this investigation, six different polycistronic lentiviral vectors were constructed to cover all possible combinations of G, M, T with identical internal 2A sequences.…”

“…This method has been used by several groups and the efficiency of reprogramming has been enhanced by using alternative transcription factors or small molecules (summarized in Tables 2 and 3). The reprogramming efficiency of the retrovirus-mediated gene delivery approach is partially dependent on the stoichiometry of the delivered transcription factors [102]. It was reported that a higher reprogramming efficiency than that achieved in the original GMT experiment when the stoichiometry of the transcription factors is altered [102].…”