Gene Transfer of Inducible Nitric Oxide Synthase Affords Cardioprotection by Upregulating Heme Oxygenase-1 Via a Nuclear Factor-κB-Dependent Pathway

Background-Although inducible nitric oxide synthase (iNOS) is known to impart powerful protection against myocardial infarction, the mechanism for this salubrious action remains unclear. Methods and Results-Adenovirus-mediated iNOS gene transfer in mice resulted 48 to 72 hours later in increased expression not only of iNOS protein but also of heme oxygenase (HO)-1 mRNA and protein; HO-2 protein expression did not change. iNOS gene transfer markedly reduced infarct size in wild-type mice, but this effect was completely abrogated in HO-1(-/-) mice. At 48 hours after iNOS gene transfer, nuclear factor-kappa B was markedly activated. In transgenic mice with cardiomyocyte-restricted expression of a dominant negative mutant of I kappa B alpha (I kappa B alpha(S32A,S36A)), both basal HO-1 levels and upregulation of HO-1 by iNOS gene transfer were suppressed. Chromatin immunoprecipitation analysis of mouse hearts provided direct evidence that nuclear factor-kappa B subunits p50 and p65 were recruited to the HO-1 gene promoter (-468 to -459 bp) 48 hours after iNOS gene transfer. Conclusions-This study demonstrates for the first time the existence of a close functional coupling between cardiac iNOS and cardiac HO-1: iNOS upregulates HO-1 by augmenting nuclear factor-kappa B binding to the region of the HO-1 gene promoter from -468 to -459 bp, and HO-1 then mediates the cardioprotective effects of iNOS. These results also reveal an important role of nuclear factor-kappa B in both basal and iNOS-induced expression of cardiac HO-1. Collectively, the present findings significantly expand our understanding of the regulation of cardiac HO-1 and of the mechanism whereby iNOS exerts its cardioprotective actions. (Circulation. 2009; 120: 1222-1230.)