TNFα decreases αMHC expression by a NO mediated pathway: role of E-box transcription factors for cardiomyocyte specific gene regulation

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Abstract
Objective: Tumor necrosis factor α (TNFα) is thought to play a key role in the pathogenesis of cardiac failure. In the myocardium, TNFα enhances the expression of inducible nitric oxide synthase (iNOS). Nitric oxide (NO) has been shown to affect β-agonist-dependent cardiac contractility and relaxation. It is not clear, however, whether TNFα mediated NO release has sustained cardiac effects, by altering expression of cardiomyocyte specific genes such as α-myosin heavy chain (αMHC). Methods: Neonatal rat ventricular cardiomyocytes (CM) were stimulated with TNFα and/or the NOS inhibitor nitro-l-arginine (L-NNA). Protein binding to the E-box enhancer element in the αMHC promoter was evaluated by electrophoretic mobility shift assay (EMSA) and transcriptional activity of the E-box consensus motif was determined by luciferase assay. mRNA levels of the endogenous αMHC gene were assessed by RT–PCR. In vivo studies were performed in transgenic mice with cardiac specific over-expression of TNFα. Results: CM treated with TNFα exhibited decreased levels of αMHC transcripts (69±8% of control), the effect of TNFα was reversed by L-NNA (94±14% of control). As shown by EMSA, TNFα reduced protein binding to the αMHC E-box enhancer motif via NO dependent pathways. Addition of the NO-donor sodium nitroprusside (SNP) to CM nuclear extracts dose dependently disrupted protein binding to the αMHC E-box. Furthermore, exposure of CM to TNFα or SNP decreased transcription from an E-box luciferase-reporter construct (TNFα: 74±12%; SNP 250 μM: 72±10%; SNP 500 μM: 66±11% of control). In myocardial tissue of TNFα transgenic mice, increased nitrotyrosine staining, decreased protein binding to the αMHC E-box motif and reduced expression of αMHC (62±26%) were observed. Conclusions: The present study shows that TNFα reduces αMHC transcript levels in cardiomyocytes. Our data obtained in cultured CM and in TNFα transgenic mice support the notion that TNFα exerts these effects by NO and E-box dependent mechanisms in vitro and possibly in vivo.

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