© 2002 by European Society of Cardiology
Copyright © 2001, European Society of Cardiology
TNF
decreases MHC expression by a NO mediated pathway: role of E-box transcription factors for cardiomyocyte specific gene regulation
aDepartment of Cardiology and Angiology, Medizinische Hochschule Hannover, Carl-Neuberg Strasse 1, 30625 Hannover, Germany
bWinters Center for Heart Failure Research, Baylor College of Medicine, 2002 Holocombe Boulevard, Houston, TX 77030, USA
drexler.helmut{at}mh-hannover.de
* Corresponding author: Tel.: +49-511-532-3840; fax: +49-511-532-5412
Objective: Tumor necrosis factor 
(TNF) is thought to play a key role in the pathogenesis of cardiac failure. In the myocardium, TNF 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 TNF mediated NO release has sustained cardiac effects, by altering expression of cardiomyocyte specific genes such as -myosin heavy chain (MHC). Methods: Neonatal rat ventricular cardiomyocytes (CM) were stimulated with TNF and/or the NOS inhibitor nitro-L-arginine (L-NNA). Protein binding to the E-box enhancer element in the MHC 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 MHC gene were assessed by RT–PCR. In vivo studies were performed in transgenic mice with cardiac specific over-expression of TNF. Results: CM treated with TNF exhibited decreased levels of MHC transcripts (69±8% of control), the effect of TNF was reversed by L-NNA (94±14% of control). As shown by EMSA, TNF reduced protein binding to the MHC 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 MHC E-box. Furthermore, exposure of CM to TNF or SNP decreased transcription from an E-box luciferase-reporter construct (TNF: 74±12%; SNP 250 µM: 72±10%; SNP 500 µM: 66±11% of control). In myocardial tissue of TNF transgenic mice, increased nitrotyrosine staining, decreased protein binding to the MHC E-box motif and reduced expression of MHC (62±26%) were observed. Conclusions: The present study shows that TNF reduces MHC transcript levels in cardiomyocytes. Our data obtained in cultured CM and in TNF transgenic mice support the notion that TNF exerts these effects by NO and E-box dependent mechanisms in vitro and possibly in vivo.
KEYWORDS Cell culture/isolation; Cytokines; Gene expression; Heart failure; Nitric oxide