Effect of NF-kappa B inhibition on TNF-alpha-induced apoptosis and downstream pathways in cardiomyocytes

Heart-specific inhibition of survival pathway gp130 was recently shown to s ensitize transgenic mice towards stress stimuli, resulting in rapid onset o f cardiac dilatation and heart failure. In order to identify further surviv al pathways we evaluated the role of transcription factor nuclear factor-ka ppa beta (NF-kappaB) in tumour necrosis factor-alpha (TNF-alpha)-induced ap optosis of cardiomyocytes. TNF-alpha stimulation (10 ng/ml) of both H9c2 ce lls and primary cardiomyocytes isolated from neonatal Wistar rats resulted in rapid nuclear translocation of NF-kappaB complexes. The NF-kappaB comple xes consisted of rel-proteins p50 and p65, as revealed by supershift analys is. Addition of proteasome inhibitor MG132 or adenoviral expression of a tr uncated I kappaB alpha (I kappaB DeltaN) inhibited TNF-alpha -induced NF-ka ppaB nuclear translocation in a dose-dependent manner. Both neonatal cardio myocytes and H9c2 cells were resistant to TNF-induced apoptosis. However, s pecific inhibition of NF-kappaB activation by Ad5-I kappaB alpha DeltaN (MO I=50) or MG132 (5 muM) increased apoptosis as measured by subG1-assay (H9c2 cells) and annexin V binding/propidium iodide (neonatal cardiomyocytes, FA CS-analysis: 7 +/-2% to 26 +/-5% annexin V positive/PI negative), respectiv ely. TUNEL-assay double-stained with anti-alpha -sarcomeric actin confirmed apoptosis of neonatal cardiomyocytes. Furthermore, caspase-3 activation wa s 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 downregulate d by NF-kappaB inhibition nor upregulated by TNF-alpha stimulation. In summ ary, cardiomyocytes utilize transcription factor NF-kappaB to activate surv ival 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 Pres s.