Absence of inducible nitric oxide synthase modulates early reperfusion‐induced NF‐κB and AP‐1 activation and enhances myocardial damage

Abstract

The role of nitric oxide (NO) generated by the inducible NO synthase (iNOS) during myocardial ischemia and reperfusion is not understood. We investigated the role of iNOS during early reperfusion damage induced in genetically deficient iNOS (iNOS-/-) mice and wild-type littermates. In wild-type mice, ischemia (60 min) and reperfusion (60 min) induced an elevation in serum levels of creatine phosphokinase and myocardial injury characterized by the presence of scattered apoptotic myocytes and mild neutrophil infiltration. Northern blot analysis showed increased expression of iNOS, whose activity was markedly elevated after reperfusion. Immunohistochemistry showed staining for nitrotyrosine; Western blot analysis showed elevated expression of heat shock protein 70 (HSP70), a putative cardioprotective mediator. Plasma levels of nitrite and nitrate, tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and IL-10 were also increased. These events were preceded by degradation of inhibitor kappaBalpha (IkappaBalpha), activation of IkappaB kinase complex (IKK) and c-Jun-NH2-terminal kinase (JNK), and subsequently activation of nuclear factor-kappaB (NF-kappaB) and activator protein 1 (AP-1) as early as 15 min after reperfusion. In contrast, iNOS-/- mice experienced 35% mortality after reperfusion. The extensive myocardial injury was associated with marked apoptosis and infiltration of neutrophils whereas expression of HSP70 was less pronounced. Nitrotyrosine formation and plasma levels of nitrite and nitrate were undetectable. TNF-alpha and IL-6 were increased and IL-10 was reduced in earlier stages of reperfusion. Activation of IKK and JNK and binding activity of NF-kappaB and AP-1 were significantly reduced. Thus, we conclude that iNOS plays a beneficial role in modulating the early defensive inflammatory response against reperfusion injury through regulation of signal transduction.

Inducible Nitric-oxide Synthase Is an Important Contributor to Prolonged Protective Effects of Ischemic Preconditioning in the Mouse Kidney

“…We also reported that the overexpression of HSP-25 protein in renal epithelial LLC-PK1 cells using adenoviral vectors protects cells from injury due to oxidants and chemical anoxia (6). HSPs suppress cytokine-induced IL-8 and TNF-␣ expression and the translocation of the p65 subunit of NF-B, which regulates iNOS expression (13,55). In iNOS Ϫ/Ϫ mice, however, where the preconditioning effect is mitigated, the expression of HSP-25 is elevated in both iNOS Ϫ/Ϫ and iNOS ϩ/ϩ animals.…”

Endothelial Progenitor Cells Are Rapidly Recruited to Myocardium and Mediate Protective Effect of Ischemic Preconditioning via “Imported” Nitric Oxide Synthase Activity

“…Recently, many investigators have documented the role of eNOS 10,37 or iNOS 18,38,39 in the cardioprotective effect of IP. Our data suggest that in the case of I-R injury after IP, activated eNOS produced from recruited EPCs plays an essential role in cardioprotection.…”

“…Previous studies have documented a favorable role of iNOS in IP. 18,38,39 In particular, Wang et al 40 reported that IP upregulated iNOS in cardiomyocytes and played a protective role against myocardial damage. These authors showed iNOS mRNA expression in ischemic myocardium followed IP, and diffuse iNOS signals were detected with in situ hybridization and immunohistochemistry in the cytoplasmic space of cardiomyocytes.…”

Deficiency of iNOS Does Not Prevent Isoproterenol-induced Cardiac Hypertrophy in Mice

“…Moreover, the mRNA expression of TGF-β in isoproterenolinfused iNOS knockout mice was increased more than 10 times suggesting that a deficiency of iNOS may make subjects more vulnerable to isoproterenol-induced heart failure. Sustained increases of IL-6 and TGF-β in iNOS knockout mice may be caused by modulation of signal transduction mediated by nuclear factor-κB or/and activator protein 1 in iNOS knockout mice (Zingarelli et al, 2002). Cytokines expression such as TNF-α and IL-6 in the heart following ischemia and reperfusion damage was more augmented in iNOS knockout mice than wild-type mice (Zingarelli et al, 2002).…”