title: Aza-induced cardiomyocyte differentiation of P19 EC-cells by epigenetic co-regulation and ERK signaling.
abstract: Stem cells in cell based therapy for cardiac injury is being potentially considered. However, genetic regulatory networks involved in cardiac differentiation are not clearly understood. Among stem cell differentiation models, mouse P19 embryonic carcinoma (EC) cells, are employed for studying (epi)genetic regulation of cardiomyocyte differentiation. Here, we comprehensively assessed cardiogenic differentiation potential of 5-azacytidine (Aza) on P19 EC-cells, associated gene expression profiles and the changes in DNA methylation, histone acetylation and activated-ERK signaling status during differentiation. Initial exposure of Aza to cultured EC-cells leads to an efficient (55%) differentiation to cardiomyocyte-rich embryoid bodies with a threefold (16.8%) increase in the cTnI+ cardiomyocytes. Expression levels of cardiac-specific gene markers i.e., Isl-1, BMP-2, GATA-4, and α-MHC were up-regulated following Aza induction, accompanied by differential changes in their methylation status particularly that of BMP-2 and α-MHC. Additionally, increases in the levels of acetylated-H3 and pERK were observed during Aza-induced cardiac differentiation. These studies demonstrate that Aza is a potent cardiac inducer when treated during the initial phase of differentiation of mouse P19 EC-cells and its effect is brought about epigenetically and co-ordinatedly by hypo-methylation and histone acetylation-mediated hyper-expression of cardiogenesis-associated genes and involving activation of ERK signaling.
authors.affiliation: Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560 012, India.
authors.forename: Deepti
authors.initials: D
authors.lastname: Abbey
authors.forename: Polani B
authors.initials: PB
authors.lastname: Seshagiri
mesh_terms: D000107:Acetylation; D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D018899:CpG Islands; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation; D054278:Embryonal Carcinoma Stem Cells / Q000187:drug effects* / Q000378:metabolism*; D044127:Epigenesis, Genetic; D006321:Heart / Q000196:embryology; D006657:Histones / Q000378:metabolism; D020935:MAP Kinase Signaling System; D008648:Mesoderm / Q000378:metabolism; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects*; D038081:Organogenesis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics
chemical_list: D006657:Histones; D012333:RNA, Messenger; D004248:DNA (Cytosine-5-)-Methyltransferases; D001374:Azacitidine
keywords: 5-Aza cytidine; Aza; Azacytidine; BMP; Bone morphogenetic protein; COBRA; Cardiomyocyte; Combined bisulfite restriction analysis; DMSO; DNA methyl transferase; DNA methylation; DNMT; Differentiation; Dimethyl sulfoxide; EBs; EC-cells; ERK; ES-cells; Embryoid bodies; Embryonal carcinoma cells; Embryonic carcinoma cells; Embryonic stem cells; Extracellular receptor kinase; FGF; Fibroblast growth factor; H; Histone; Human embryonic stem cells; Ig; Immunoglobulin(s); LIF; Leukemia inhibitory factor; MLC 2v; MSCs; MSP; Mesenchymal stem cells; Methylation specific PCR; Myosin light chain 2v; PSCs; Pluripotent stem cells; SDS; Sodium dodecyl sulfate; TRED; Transcriptional regulatory element database; cTnI; cardiac Troponin I; hESC; α-MHC; α-Myosin heavy chain