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Stoichiometry of Gata4, Mef2c, and Tbx5 influences the efficiency and quality of induced cardiac myocyte reprogramming.

ABSTRACT: 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.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.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 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.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.

SUBMITTER: Wang L

PROVIDER: S-EPMC4299697 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Stoichiometry of Gata4, Mef2c, and Tbx5 influences the efficiency and quality of induced cardiac myocyte reprogramming.

Wang Li L Liu Ziqing Z Yin Chaoying C Asfour Huda H Chen Olivia O Li Yanzhen Y Bursac Nenad N Liu Jiandong J Qian Li L

Circulation research 20141121 2

<h4>Rationale</h4>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.<h4>Objective</h4>We aimed to establish a system to express di ...[more]

PMID: 25416133

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Project description:Cardiac transcription factors are master regulators during heart development. Some were shown to transdifferentiate tail tip and cardiac fibroblasts into cardiomyocytes. However, recent studies have showed that controversies exist. Potential difference in tail tip and cardiac fibroblast isolation may possibly confound the observations. Moreover, due to the use of a cardiac reporter (Myh6) selection strategy for induced cardiomyocyte enrichment, and the lack of tracking signals for each transcription factors, individual roles of each transcription factors in activating cardiac gene expression in mammalian non-myoblastic cells have never been elucidated. Answers to these questions are an important step toward cardiomyocyte regeneration. Because mouse 10T1/2 fibroblasts are non-myoblastic in nature and can be induced to express genes of all three types of muscle cells, they are an ideal model for the analysis of cardiac and non-cardiac gene activation after induction. We constructed bi-cistronic lentiviral vectors, capable of expressing cardiac transcription factors along with different fluorescent tracking signals. By infecting 10T1/2 fibroblasts with Nkx2-5, Tbx5, Gata4 or Myocd cardiac transcription factor lentivirus alone or different combinations, we found that only Tbx5+Myocd and Tbx5+Gata4+Myocd combinations induced Myh6 and Tnnt2 cardiac marker protein expression. Microarray-based gene ontology analysis revealed that Tbx5 alone activated genes involved in the Wnt receptor signaling pathway and inhibited genes involved in a number of cardiac-related processes. Myocd alone activated genes involved in a number of cardiac-related processes and inhibited genes involved in the Wnt receptor signaling pathway and non-cardiac processes. Gata4 alone inhibited genes involved in non-cardiac processes. Tbx5+Gata4+Myocd was the most effective activator of genes associated with cardiac-related processes. Unlike Tbx5, Gata4, Myocd alone or Tbx5+Myocd, Tbx5+Gata4+Myocd activated the fewest genes associated with non-cardiac processes. Conclusively, Tbx5, Gata4 and Myocd play different roles in cardiac gene activation in mammalian non-myoblastic cells. Tbx5+Gata4+Myocd activates the most cardiac and the least non-cardiac gene expression.

Dataset Information

Stoichiometry of Gata4, Mef2c, and Tbx5 influences the efficiency and quality of induced cardiac myocyte reprogramming.

Publications

Stoichiometry of Gata4, Mef2c, and Tbx5 influences the efficiency and quality of induced cardiac myocyte reprogramming.

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