Effect of NF-κB inhibition on TNF-α-induced apoptosis and downstream pathways in cardiomyocytes

Heart-specific inhibition of survival pathway gp130 was recently shown to sensitize transgenic mice towards stress stimuli, resulting in rapid onset of cardiac dilatation and heart failure. In order to identify further survival pathways we evaluated the role of transcription factor nuclear factor-kappa beta (NF-kappaB) in tumour necrosis factor-alpha (TNF-alpha)-induced apoptosis of cardiomyocytes. TNF-alpha stimulation (10 ng/ml) of both H9c2 cells and primary cardiomyocytes isolated from neonatal Wistar rats resulted in rapid nuclear translocation of NF-kappaB complexes. The NF-kappaB complexes consisted of rel-proteins p50 and p65, as revealed by supershift analysis. Addition of proteasome inhibitor MG132 or adenoviral expression of a truncated I kappaB alpha (I kappaB DeltaN) inhibited TNF-alpha -induced NF-kappaB nuclear translocation in a dose-dependent manner. Both neonatal cardiomyocytes and H9c2 cells were resistant to TNF-induced apoptosis. However, specific inhibition of NF-kappaB activation by Ad5-I kappaB alpha DeltaN (MOI=50) or MG132 (5 muM) increased apoptosis as measured by subG1-assay (H9c2 cells) and annexin V binding/propidium iodide (neonatal cardiomyocytes, FACS-analysis: 7 +/-2% to 26 +/-5% annexin V positive/PI negative), respectively. TUNEL-assay double-stained with anti-alpha -sarcomeric actin confirmed apoptosis of neonatal cardiomyocytes. Furthermore, caspase-3 activation was increased by 52 +/-7% in neonatal cardiomyocytes after TNF alpha + Ad5-I kappaB alpha DeltaN compared to TNF alpha + Ad5-control treatment, Protein levels of hiAP1, hiAP2, x-iAP. bcl-2 and bcl-x(L) were neither downregulated by NF-kappaB inhibition nor upregulated by TNF-alpha stimulation. In summary, cardiomyocytes utilize transcription factor NF-kappaB to activate survival factors in the context of TNF-alpha stimulation. As locally increased levels of TNF-alpha have been detected in heart failure, NF-kappaB activity is essential for cellular homeostasis in the heart. (C) 2001 Academic Press.