The late phase of ischemic preconditioning is abrogated by targeted disruption of the inducible NO synthase gene

The goal of this study was to interrogate the role of inducible NO synthase (iNOS) in the late phase of ischemic preconditioning (PC) irt vivo. A tota l of 321 mice were used. Wild-type mice preconditioned 24 h earlier with si x cycles of 4-min coronary occlusion/4-min reperfusion exhibited a signific ant (P < 0.05) increase in myocardial iNOS protein content, iNOS activity ( assessed as calcium-independent L-citrulline formation), and nitrite + nitr ate tissue levels. In contrast, endothelial NOS protein content and calcium -dependent NOS activity remained unchanged. No immunoreactive neuronal NOS was detected. When wild-type mice were preconditioned 24 h earlier with six 4-min occlusion/4-min reperfusion cycles, the size of the infarcts produce d by a 30-min coronary occlusion followed by 24 h of reperfusion was reduce d markedly (by 67%; P < 0.05) compared with sham-preconditioned controls, i ndicating a late PC effect. In contrast, when mice homozygous for a null iN OS allele were preconditioned 24 h earlier with the same protocol, infarct size was not reduced. Disruption of the iNOS gene had no effect on early PC or on infarct size in the absence of PC. These results demonstrate that (i ) the late phase of ischemic PC is associated with selective up-regulation of iNOS, and (ii) targeted disruption of the iNOS gene completely abrogates the infarct-sparing effect of late PC (but not of early PC), providing une quivocal molecular genetic evidence for an obligatory role of iNOS in the c ardioprotection afforded by the late phase of ischemic PC. Thus, this study identifies a specific protein that mediates late PC in vivo.