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