Abstract 34: Acetylation of GATA 4 Enhanced Direct Cardiac Reprogramming of Induced Cardiomyocytes

Abstract

Objective: Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) by forced expression of cardiomyogenic factors, GMT (GATA4, Mef2c and Tbx5), has recently been demonstrated, suggesting a promising statregy for cardiac regeneration. However, the efficiency of direct reprogramming is usually relatively low and requires extensive epigenetic redesigning, although the underlying mechanism are largely unknown. Methods: In a recent study, we created a novel mutation in rat GATA 4 by replacing lysine residue with glutamine at position 299 i.e. (K299Q), to mimic constitutive acetylation and examined whether constitutive acetylation of GATA4, when compared with wild type GATA4, further enhance GMT-mediated direct reprogramming efficiency of induced cardiomyocytes in vitro and accordingly ventricular function after myocardial infarction in rat, in vivo . Results: We found that acetylated GATA 4 (K299Q), in the presence of Mef2c and Tbx5 upregulated cardiac-specific markers, suppressed fibroblast genes, in rat cardiac fibroblasts (RCFs) more efficiently when compared with Mef2c, Tbx5 plus wild type GATA4. FACS analyses revealed that G(K299Q) MT induced significantly more cardiomyocyte marker cardiac troponin T (cTnT) expression compared with GMT alone. Mechanistic studies demonstrated that the K299Q substitution, resulting in enriched p300 occupancy at the GATA 4 promoter, induced acetylation of Histine 3, decreased HDAC expression. In addition, substitution augmented the increase in an acetylated form of GATA-4 and its DNA binding and transcriptional activity, compared with wildtype GATA 4. In agreement with upregulated cTNT gene expression in vitro , echocardiographic analysis demonstrate that the acetylated G(K299Q) MT vectors have improved effect in enhancing ventricular function than GMT vectors from postinfarct baselines as compared to negative control [G(K299Q) MT, 15.6% ± 2.7%; G(WT)MT, 12.8% ± 1.7%; GFP, -2.3% ± 1.1%]. Conclusions: Collectivily, these data indicate that acetylated GATA4 (K299Q) significantly increases reprogramming efficiency of induced cardiomyocytes (iCMs), in vitro and in vivo, and provide new insight into the molecular mechanism underlying cardiac regeneration.