Abstract 5144: The Obligatory Role of Heat Shock Protein 90 in iNOS Induction in Postischemic Hearts

Inducible nitric oxide synthase (iNOS) plays important roles in the process of myocardial infarction, heart failure, and cardiac preconditioning. The expression of iNOS is mainly induced by inflammatory substances such as endotoxins and cytokines. NF- κ B and STAT-1 are the two transcriptional factors that mediate iNOS expression in stimulated cells. Previous studies showed that heat shock protein 90 (Hsp90) associates with iNOS and this association enhances iNOS activity. Here we show that Hsp90 is also essential for iNOS induction. To determine the role of Hsp90 in iNOS induction, we treated mouse macrophages (RAW 264.7) with lipopolysaccharide (LPS, 1 μ g/ml) and interferon- γ (IFN- γ , 100 units/ml) in the absence and presence of Hsp90 inhibitor geldanamycin (1 μ M). Hsp90 inhibition completely prevented iNOS protein expression. RT-PCR experiments showed that iNOS mRNA transcription was blocked by Hsp90 inhibition. Radicicol (10 μ M), another Hsp90 inhibitor whose structure is different from that of geldanamycin, also blocked iNOS mRNA transcription. Further experiments showed that Hsp90 inhibition prevented NF- κ B activation in LPS-treated cells. In IFN- γ -stimulated cells, STAT-1 activation was also abolished by Hsp90 inhibition. To explore whether these findings from cultured cells occurred in diseases, we performed in vivo myocardium infarction on mice. While no iNOS expression was detected in the heart of mice in sham group, iNOS mRNA and proteins were observed in the infarcted areas of the hearts subjected to 1-hr coronary artery (LAD) occlusion and 24-hr reperfusion. Geldanamycin (4 mg/kg, i.p.) completely prevented iNOS mRNA and protein expression in the infarcted myocardium ( P <0.001, n=5). Taken together, these results demonstrate that besides acting as an allosteric enhancer, Hsp90 is also required for iNOS gene transactivation. Intervening Hsp90 may represent a novel approach to modulate iNOS expression in myocardial infarction and cardiac preconditioning.