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Stoichiometry of Gata4, Mef2c, and Tbx5 influences the efficiency and quality of induced cardiac myocyte reprogramming. Circ Res 2015 Jan 16;116(2):237-44

Date

11/25/2014

Pubmed ID

25416133

Pubmed Central ID

PMC4299697

DOI

10.1161/CIRCRESAHA.116.305547

Scopus ID

2-s2.0-84922105989   150 Citations

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 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.

CONCLUSIONS: 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.

Author List

Wang L, Liu Z, Yin C, Asfour H, Chen O, Li Y, Bursac N, Liu J, Qian L

Author

Ziqing Liu PhD Assistant Professor in the Physiology department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Animals
Cells, Cultured
Cellular Reprogramming
GATA4 Transcription Factor
MEF2 Transcription Factors
Mice
Mice, Transgenic
Myocytes, Cardiac
T-Box Domain Proteins