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Angiotensin II and the ERK pathway mediate the induction of myocardin by hypoxia in cultured rat neonatal cardiomyocytes.

ABSTRACT: Hypoxic injury to cardiomyocytes is a stress that causes cardiac pathology through cardiac-restricted gene expression. SRF (serum-response factor) and myocardin are important for cardiomyocyte growth and differentiation in response to myocardial injuries. Previous studies have indicated that AngII (angiotensin II) stimulates both myocardin expression and cardiomyocyte hypertrophy. In the present study, we evaluated the expression of myocardin and AngII after hypoxia in regulating gene transcription in neonatal cardiomyocytes. Cultured rat neonatal cardiomyocytes were subjected to hypoxia, and the expression of myocardin and AngII were evaluated. Different signal transduction pathway inhibitors were used to identify the pathway(s) responsible for myocardin expression. An EMSA (electrophoretic mobility-shift assay) was used to identify myocardin/SRF binding, and a luciferase assay was used to identify transcriptional activity of myocardin/SRF in neonatal cardiomyocytes. Both myocardin and AngII expression increased after hypoxia, with AngII appearing at an earlier time point than myocardin. Myocardin expression was stimulated by AngII and ERK (extracellular-signal-regulated kinase) phosphorylation, but was suppressed by an ARB (AngII type 1 receptor blocker), an ERK pathway inhibitor and myocardin siRNA (small interfering RNA). AngII increased both myocardin expression and transcription in neonatal cardiomyocytes. Binding of myocardin/SRF was identified using an EMSA, and a luciferase assay indicated the transcription of myocardin/SRF in neonatal cardiomyocytes. Increased BNP (B-type natriuretic peptide), MHC (myosin heavy chain) and [(3)H]proline incorporation into cardiomyocytes was identified after hypoxia with the presence of myocardin in hypertrophic cardiomyocytes. In conclusion, hypoxia in cardiomyocytes increased myocardin expression, which is mediated by the induction of AngII and the ERK pathway, to cause cardiomyocyte hypertrophy. Myocardial hypertrophy was identified as an increase in transcriptional activities, elevated hypertrophic and cardiomyocyte phenotype markers, and morphological hypertrophic changes in cardiomyocytes.

SUBMITTER: Chiu CZ

PROVIDER: S-EPMC2890999 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:1. In the present experiments, we investigated the effects of methylecgonidine (MEG) on nitric oxide (NO) production in cultured neonatal rat cardiomyocytes. Incubation of cultured cardiomyocytes with carbachol or MEG for 48 h significantly enhanced NO production. No release was increased from 1.48+/-0.13 microM (mg protein)(-1) for control to 5.73+/-0.19 microM (mg protein)(-1) for 1 microM carbachol treated cells (P<0.001). In addition, incubation with 1 microM MEG enhanced NO production to 5.55+/-0.28 microM (mg protein)(-1). The effects of MEG on NO production were concentration-dependent. The muscarinic antagonist atropine prevented the enhancement of NO production induced by carbachol or MEG. Compared to MEG-induced NO production, cocaine was much less potent. 2. The enhancement of NO production by carbachol or MEG was even greater in cultured cardiomyocytes transfected with the M(2) cDNA. After 48-h incubation with 1 microM carbachol or 1 microM MEG, NO production was increased by 6.5 and 6.7 fold, respectively, in cardiomyocytes overexpressing M(2) receptors. Coincubation with atropine or N(G)-nitro-L-arginine methyl ester abolished the enhancement of NO production. In contrast, NO production enhanced by carbachol or MEG in M(1)- or M(3)-transfected cardiomyocytes was similar to the level in non-transfected cells. 3. Western blot analysis showed that the protein levels of M(1), M(2), and M(3) were significantly increased in cardiomyocytes transfected with the receptor cDNAs, but MEG had no effect on the expressions. It is interesting that both carbachol and MEG caused a significant increase in constitutive endothelial NO synthase (eNOS) only in M(2)-transfected cardiomyocytes, not in non-transfected, M(1)- or M(3)-transfected cells. Again, atropine blocked the MEG-produced induction of eNOS. 4. Our data demonstrate that MEG significantly enhanced NO production in cultured cardiomyocytes and that the enhancement of NO production may result from MEG stimulation of muscarinic M(2) receptors.

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Angiotensin II and the ERK pathway mediate the induction of myocardin by hypoxia in cultured rat neonatal cardiomyocytes.

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