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

Abstract

The goal of this study was to interrogate the role of inducible NO synthase (iNOS) in the late phase of ischemic preconditioning (PC) in vivo. A total of 321 mice were used. Wild-type mice preconditioned 24 h earlier with six cycles of 4-min coronary occlusion͞4-min reperfusion exhibited a significant (P < 0.05) increase in myocardial iNOS protein content, iNOS activity (assessed as calciumindependent L-citrulline formation), and nitrite ؉ nitrate 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 occlusion͞4-min reperfusion cycles, the size of the infarcts produced by a 30-min coronary occlusion followed by 24 h of reperfusion was reduced markedly (by 67%; P < 0.05) compared with sham-preconditioned controls, indicating a late PC effect. In contrast, when mice homozygous for a null iNOS 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 unequivocal molecular genetic evidence for an obligatory role of iNOS in the cardioprotection afforded by the late phase of ischemic PC. Thus, this study identifies a specific protein that mediates late PC in vivo.

Role of nitric oxide synthases in the infarct size‐reducing effect conferred by heat stress in isolated rat hearts

“…Moreover, iNOS seems to be the principal NOS isoform involved in this cardioprotective phenomenon, since a selective iNOS inhibitor completely abolished the delayed protection induced by the ischaemic preconditioning in vivo in the rabbit (Imagawa et al, 1999). This is in accordance with a study from Guo et al (1999) which demonstrates in vivo in the mouse that the late phase of ischaemic preconditioning is associated with a selective up-regulation of myocardial iNOS. NO seems to also trigger the delayed protective e ect of monophosphoryl lipid A (MLA) in the isolated rat heart, since co-administration of NOS inhibitors and MLA abolished the preservation of ventricular function induced by MLA alone (Tosaki et al, 1998;GyoÈ rgy et al, 1999).…”

The late phase of preconditioning and its natural clinical application—gene therapy

“…As mentioned above, iNOS was the first protein to be identified as a mediator of late PC [8,9]. Approximately 10 years ago, we postulated that iNOS could participate in late PC, because this enzyme is known to be induced by stress and since NO possesses a host of cardioprotective actions that would be expected to be beneficial during myocardial ischemia/reperfusion [11].…”

“…The first gene to be identified as a mediator of late PC was the inducible isoform of NOS (iNOS) [8,9]; subsequently, other genes have been discovered, including cyclooxy-genase-2 (COX-2), heme oxygenase-1 (HO-1), and antioxidant enzymes such as extracellular SOD (ecSOD), aldose reductase, manganese SOD, etc. [3,[10][11][12].…”

“…To determine, whether iNOS mediates the late phase of ischemic PC, we undertook a study in mice [9]. First, we asked whether iNOS is upregulated by ischemic PC.…”

“…Myriad proteins are up-regulated following ischemic PC, and most of them have nothing to do with the protection (i.e., they represent epi-phenomena). To determine whether the upregulation of iNOS was causally related to protection, we performed the second phase of the study in which either wild-type (WT) or iNOS −/− mice that were subjected to myocardial infarction (30-min coronary occlusion followed by 24 h of rep-erfusion) [9]. We found that under control (unstressed) conditions, infarct size was similar in WT and iNOS −/− mice, implying that iNOS does not modulate myocardial ischemia/reperfusion injury in the absence of stress.…”