Chronic Hypoxia Increases Endothelial Nitric Oxide Synthase Generation of Nitric Oxide by Increasing Heat Shock Protein 90 Association and Serine Phosphorylation

Abstract

Abstract-Chronic hypoxia increases endothelial nitric oxide synthase (eNOS) production of nitric oxide (·NO) and cardioprotection in neonatal rabbit hearts. However, the mechanism by which this occurs remains unclear. Recent studies suggest that heat shock protein 90 (hsp90) alters eNOS function. In the present study, we examined the role of hsp90 in eNOS-dependent cardioprotection in neonatal rabbit hearts. Chronic hypoxia increased recovery of postischemic left ventricular developed pressure (LVDP). Geldanamycin (GA), which inhibits hsp90 and increases oxidative stress, decreased functional recovery in normoxic and hypoxic hearts. To determine if a loss in ·NO, afforded by GA, decreased recovery, GA-treated hearts were perfused with S-nitrosoglutathione (GSNO) as a source of ·NO. GSNO increased recovery of postischemic LVDP in GA-treated normoxic and hypoxic hearts to baseline levels. Although chronic hypoxia decreased phosphorylated eNOS (S1177) levels by Ϸ4-to 5-fold and total Akt and phosphorylated Akt by 4-and 5-fold, it also increased hsp90 association with eNOS by more than 3-fold. Using hydroethidine (HEt), a fluorescent probe for superoxide, we found that hypoxic hearts contained less ethidine (Et) staining than normoxic hearts. Normoxic hearts generated 3 times more superoxide by an N -nitro-L-arginine methyl ester (L-NAME)-inhibitable mechanism than hypoxic hearts. Taken together, these data indicate that the association of hsp90 with eNOS is important for increasing ·NO production and limiting eNOS-dependent superoxide anion generation. Such changes in eNOS function appear to play a critical role in protecting the myocardium against ischemic injury. (Circ Res. 2002;91:300-306.) Key Words: chronic hypoxia Ⅲ endothelial NOS Ⅲ heat shock protein 90 Ⅲ superoxide anion Ⅲ nitric oxide N itric oxide plays an important role in protecting the heart against ischemic injury. S-nitrosoglutathione (GSNO), a nitric oxide (·NO) donor, improves functional recovery after ischemia, which is associated with increased cGMP. 1 Chronic hypoxia from birth in a neonatal rabbit model increases recovery of postischemic left ventricular developed pressure (LVDP) compared with recovery in normoxic hearts. 2 It is important to note that nitric oxide synthase (NOS) inhibitors, N -nitro-Larginine methyl ester (L-NAME) and N G -methyl-L-arginine (L-NMA), decrease functional recovery of postischemic LVDP in hypoxic hearts after ischemia but do not decrease recovery in normoxic hearts. 2,3 These findings suggest that chronic hypoxia may alter the function of endothelial nitric oxide synthase (eNOS), the most abundant NOS isozyme in the rabbit heart, to increase cardioprotection. 2 An increase in the association of heat shock protein 90 (hsp90) with eNOS increases production and activity of ·NO in response to growth factor stimulation. 4 Disruption of this protein-protein interaction decreases ·NO and blocks vasodilation in response to agonists. 4 -7 Geldanamycin (GA), which inhibits conformational changes in hsp90 8 and increases ox...

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