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Inhibition of DNA Methyltransferase 1 Promotes Proliferation and Alters Gene Expression in Mouse Embryonic Cardiomyocytes
Author(s) -
Fang Xiefan,
Rivkees Scott,
Wendler Christopher
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.557.1
Subject(s) - dnmt1 , myh6 , embryonic stem cell , gata4 , biology , gene expression , microbiology and biotechnology , dna methylation , dna methyltransferase , homeobox protein nanog , gene , myh7 , myosin , myosin light chain kinase , genetics , induced pluripotent stem cell
Our previous research demonstrated that in utero caffeine exposure from embryonic day (E) 6.5‐10.5 down‐regulates the expression of DNA methyltransferases ( Dnmts ) and enhances cardiac gene expression in embryonic ventricles. Hypothesis We hypothesize that inhibition or down‐regulation of Dnmts promotes proliferation and differentiation of embryonic cardiomyocytes. Method Ventricles were dissociated from mouse E13.5 embryos and treated with 0.5‐1 mM 5‐azacytidine, a non‐specific DNMT inhibitor, or Dnmt1 siRNA. Immunofluorescence staining was performed to identify cardiomyocytes and illustrate morphological changes. Total RNA was isolated and used for quantitative real‐time PCR (qPCR). Results Knock down of Dnmt1 significantly decreased global DNA methylation in cultured cells. Immunofluorescence staining showed that the percentage of α‐actinin and connexin 43 positive cells was increased in cultures treated with Dnmt1 siRNA compared to control. Dnmt1 knocked down cardiomyocytes formed larger beating units compared with control cells. 5‐azacytidine treatment increased the expression of cardiac structural genes ( Myh7 and Myh7b ), hormonal genes ( Bnp ), and transcription factors ( Gata4 and Nfatc1 ) in HL‐1 cardiomyocytes. Dnmt1 knock down by siRNA increased the expression of Myh6 , Tnni3 , Tnnt2 , Tnnc1 , Bnp , Gata4 , Mef2c , Nfatc1 , Camta1 , and Camta2 in primary embryonic cardiomyocytes. Ongoing studies will evaluate cardiomyocyte function after Dnmt1 knock down and identify CpG sites that regulate cardiac gene expression. Conclusion Reduced Dnmt1 expression increases cell proliferation and cardiac gene expression of embryonic cardiomyocytes in culture.