cIAP-2 protects cardiac fibroblasts from oxidative damage: an obligate regulatory role for ERK1/2 MAPK and NF-κB.

Cardiac fibroblasts are resistant to several pro-apoptotic factors that prevail in the diseased myocardium. Resistance to death signals may, in the short-term, enable these cells to play a central role in tissue repair following myocyte loss but, in the long-term, facilitate their persistence in the infarct scar, resulting in disproportionate stromal growth and pump dysfunction. Surprisingly, the molecular basis of apoptosis resistance in cardiac fibroblasts remains unclear. We explored the recruitment of anti-apoptotic mechanisms in cardiac fibroblasts subjected to oxidative stress, a major component of ischemia-reperfusion injury and heart failure. Cardiac fibroblasts exposed to H2O2 expressed enhanced levels of anti-apoptotic cIAP-2 mRNA and protein, revealed by real time PCR and western blot analysis, respectively. Pulmonary fibroblasts did not express cIAP-2 and were more susceptible than cardiac fibroblasts to H2O2. cIAP-2 knockdown by RNA interference promoted apoptosis in H2O2-treated cardiac fibroblasts. Electrophoretic mobility shift assay showed NF-κB activation in cells under oxidative stress. NF-κB inhibition in H2O2-treated cells resulted in significant attenuation of cIAP-2 mRNA and protein expression and apoptosis, indicating involvement of NF-κB in cell survival via regulation of cIAP-2. Further, pCMV promoter-driven constitutive expression of cIAP-2 reduced viability loss in NF-κB-inhibited cardiac fibroblasts exposed to oxidative stress. H2O2 also caused ERK1/2 activation, which, upon inhibition, prevented IκBα degradation and nuclear translocation of NF-κB. Moreover, ERK1/2 inhibition attenuated H2O2-induced cIAP-2 expression and compromised viability in H2O2-treated cardiac fibroblasts. We propose for the first time that ERK1/2-dependent activation of NF-κB and consequent induction of cIAP-2 protects cardiac fibroblasts from oxidative damage.

NF-κB inhibition compromises cardiac fibroblast viability under hypoxia.

Cardiac fibroblasts are reported to be relatively resistant to stress stimuli compared to cardiac myocytes and fibroblasts of non-cardiac origin. However, the mechanisms that facilitate their survival under conditions of stress remain unclear. We explored the possibility that NF-κB protects cardiac fibroblasts from hypoxia-induced cell death. Further, we examined the expression of the antiapoptotic cIAP-2 and Bcl-2 in hypoxic cardiac fibroblasts, and their possible regulation by NF-κB. Phase contrast microscopy and propidium iodide staining revealed that cardiac fibroblasts are more resistant than pulmonary fibroblasts to hypoxia. Electrophoretic Mobility Shift Assay showed that hypoxia activates NF-κB in cardiac fibroblasts. Supershift assay indicated that the active NF-κB complex is a p65/p50 heterodimer. An I-κB-super-repressor was constructed that prevented NF-κB activation and compromised cell viability under hypoxic but not normoxic conditions. Similar results were obtained with Bay 11-7085, an inhibitor of NF-κB. Western blot analysis showed constitutive levels of Bcl-2 and hypoxic induction of cIAP-2 in these cells. NF-κB inhibition reduced cIAP-2 but not Bcl-2 levels in hypoxic cardiac fibroblasts. The results show for the first time that NF-κB is an important effector of survival in cardiac fibroblasts under hypoxic stress and that regulation of cIAP-2 expression may contribute to its pro-survival role.