title: Stoichiometry of Gata4, Mef2c, and Tbx5 influences the efficiency and quality of induced cardiac myocyte reprogramming.
abstract: BACKGROUND
  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.
  
  
  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.
authors.affiliation: 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.).
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.affiliation: 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.).
authors.forename: Ziqing
authors.initials: Z
authors.lastname: Liu
authors.affiliation: 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.).
authors.forename: Chaoying
authors.initials: C
authors.lastname: Yin
authors.affiliation: 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.).
authors.forename: Huda
authors.initials: H
authors.lastname: Asfour
authors.affiliation: 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.).
authors.forename: Olivia
authors.initials: O
authors.lastname: Chen
authors.affiliation: 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.).
authors.forename: Yanzhen
authors.initials: Y
authors.lastname: Li
authors.affiliation: 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.).
authors.forename: Nenad
authors.initials: N
authors.lastname: Bursac
authors.affiliation: 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.).
authors.forename: Jiandong
authors.initials: J
authors.lastname: Liu
authors.affiliation: 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.). li_qian@med.unc.edu.
authors.forename: Li
authors.initials: L
authors.lastname: Qian
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000502:physiology*; D050991:GATA4 Transcription Factor / Q000096:biosynthesis* / Q000235:genetics; D064326:MEF2 Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D032383:Myocytes, Cardiac / Q000502:physiology*; D020825:T-Box Domain Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D050991:GATA4 Transcription Factor; C484171:Gata4 protein, mouse; D064326:MEF2 Transcription Factors; C578041:Mef2c protein, mouse; D020825:T-Box Domain Proteins; C103172:T-box transcription factor 5
keywords: Gata4 protein; Mef2c protein; Tbx5 protein; fibroblasts; gene expression/regulation; myocytes, cardiac; regeneration; transcription factors