Fang Xiefan, Robinson Jourdon, Wang-Hu John, Jiang Lingli, Freeman Daniel A, Rivkees Scott A, Wendler Christopher C
Department of Pediatrics, Child Health Research Institute, College of Medicine, University of Florida, Gainesville, Florida
Department of Pediatrics, Child Health Research Institute, College of Medicine, University of Florida, Gainesville, Florida.
Am J Physiol Cell Physiol. 2015 Sep 15;309(6):C425-36. doi: 10.1152/ajpcell.00058.2015. Epub 2015 Jul 29.
cAMP is a highly regulated secondary messenger involved in many biological processes. Chronic activation of the cAMP pathway by catecholamines results in cardiac hypertrophy and fibrosis; however, the mechanism by which elevated cAMP leads to cardiomyopathy is not fully understood. To address this issue, we increased intracellular cAMP levels in HL-1 cardiomyocytes, a cell line derived from adult mouse atrium, using either the stable cAMP analog N(6),2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP) or phosphodiesterase (PDE) inhibitors caffeine and theophylline. Elevated cAMP levels increased cell size and altered expression levels of cardiac genes and micro-RNAs associated with hypertrophic cardiomyopathy (HCM), including Myh6, Myh7, Myh7b, Tnni3, Anp, Bnp, Gata4, Mef2c, Mef2d, Nfatc1, miR208a, and miR208b. In addition, DBcAMP altered the expression of DNA methyltransferases (Dnmts) and Tet methylcytosine dioxygenases (Tets), enzymes that regulate genomic DNA methylation levels. Changes in expression of DNA methylation genes induced by elevated cAMP led to increased global DNA methylation in HL-1 cells. In contrast, inhibition of DNMT activity with 5-azacytidine treatment decreased global DNA methylation levels and blocked the increased expression of several HCM genes (Myh7, Gata4, Mef2c, Nfatc1, Myh7b, Tnni3, and Bnp) observed with DBcAMP treatment. These results demonstrate that cAMP induces cardiomyocyte hypertrophy and altered HCM gene expression in vitro and that DNA methylation patterns mediate the upregulation of HCM genes induced by cAMP. These data identify a previously unknown mechanism by which elevated levels of cAMP lead to increased expression of genes associated with cardiomyocyte hypertrophy.
环磷酸腺苷（cAMP）是一种参与多种生物过程的受到高度调控的第二信使。儿茶酚胺对cAMP信号通路的慢性激活会导致心脏肥大和纤维化；然而，cAMP升高导致心肌病的机制尚未完全阐明。为了解决这个问题，我们使用稳定的cAMP类似物N(6),2'-O-二丁酰腺苷3',5'-环磷酸（DBcAMP）或磷酸二酯酶（PDE）抑制剂咖啡因和茶碱，提高了HL-1心肌细胞（一种源自成年小鼠心房的细胞系）内的cAMP水平。升高的cAMP水平增加了细胞大小，并改变了与肥厚型心肌病（HCM）相关的心脏基因和微小RNA的表达水平，这些基因包括Myh6、Myh7、Myh7b、Tnni3、Anp、Bnp、Gata4、Mef2c、Mef2d、Nfatc1、miR208a和miR208b。此外，DBcAMP改变了DNA甲基转移酶（Dnmts）和Tet甲基胞嘧啶双加氧酶（Tets）的表达，这两种酶可调节基因组DNA甲基化水平。cAMP升高诱导的DNA甲基化基因表达变化导致HL-1细胞中整体DNA甲基化增加。相反，用5-氮杂胞苷处理抑制DNMT活性可降低整体DNA甲基化水平，并阻断DBcAMP处理后观察到的几种HCM基因（Myh7、Gata4、Mef2c、Nfatc1、Myh7b、Tnni3和Bnp）的表达增加。这些结果表明，cAMP在体外诱导心肌细胞肥大并改变HCM基因表达，且DNA甲基化模式介导了cAMP诱导的HCM基因上调。这些数据确定了一种此前未知的机制，即cAMP水平升高导致与心肌细胞肥大相关的基因表达增加。
