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 occlusion/4-min reperfusion exhibited a significant (P < 0.05) increase in myocardial iNOS protein content, iNOS activity (assessed as calcium-independent 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 occlusion/4-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.

Figure 1

Western immunoblots showing the effects of ischemic PC on iNOS (A), eNOS (B), and nNOS (C) expression in WT and iNOS^−/− mice. Myocardial samples were obtained from the anterior LV wall of WT control mice and iNOS^−/− control mice that underwent a sham operation (1 h of open-chest state without coronary occlusion/reperfusion; groups I and II, respectively) and from the ischemic-reperfused region of WT and iNOS^−/− mice that underwent an ischemic PC protocol consisting of six 4-min occlusion/4-min reperfusion cycles (groups III and IV, respectively). Tissue samples were taken 24 h after sham surgery or ischemic PC. (A) iNOS immunoreactivity (cytosolic fraction) increased after ischemic PC in WT mice but was undetectable in iNOS^−/− mice, either after sham operation or after ischemic PC. (B) eNOS immunoreactivity in total homogenates (cytosolic + membranous fractions) remained unchanged after ischemic PC both in WT and iNOS^−/− mice. (C) No nNOS immunoreactivity was detected in total homogenates (cytosolic + membranous fractions) of myocardial samples, either after sham operation or after ischemic PC. In contrast, a robust expression of nNOS was detected in mouse brain and rat pituitary tissue (used as positive controls). This blot was performed by using mAbs (Transduction Laboratories). Similar results were obtained by using polyclonal anti-nNOS antibodies (Transduction Laboratories) (data not shown). Rat pituitary extracts were obtained from Transduction Laboratories.

Figure 2

Effect of ischemic PC on iNOS expression and activity in WT and iNOS^−/− mice. Tissue samples were obtained as described in the legend to Fig. 1. (A) Myocardial content of iNOS (cytosolic fraction). In WT mice, the iNOS protein content in the ischemic-reperfused region was increased by 47 ± 12% 24 h after ischemic PC. In iNOS^−/− mice, immunoreactive iNOS protein was undetectable, either after sham surgery or after ischemic PC. (B) Calcium-independent NOS (iNOS) activity in the cytosolic fraction. In WT mice, iNOS activity in the ischemic-reperfused region increased by 227% 24 h after ischemic PC. In iNOS^−/− mice, low levels of calcium-independent NOS activity were noted in iNOS^−/− mice, either after sham operation or after ischemic PC. These levels probably represent cNOS activity (20, 21). (C) Total myocardial content of nitrite and nitrate (NO_x). In WT mice, NO_x levels in the ischemic-reperfused region increased significantly 24 h after ischemic PC. In iNOS^−/− mice, ischemic PC did not bring about any increase in NO_x levels; furthermore, NO_x levels were significantly lower in iNOS^−/− mice compared with WT mice. Data are means ± SEM.

Figure 3

Effect of ischemic PC on eNOS expression and cNOS (eNOS and/or nNOS) activity in WT and iNOS^−/− mice. Tissue samples were obtained as described in the legend to Fig. 1. (A) Total (cytosolic + membranous fractions) myocardial content of eNOS. (B) cNOS (eNOS and/or nNOS) activity in the cytosolic fraction. (C) cNOS activity in the membranous fraction. eNOS protein and cNOS activity did not increase after ischemic PC, either in WT or iNOS^−/− mice. Data are means ± SEM.

Figure 4

Myocardial infarct size in groups V (WT, control), VI (iNOS^−/−, control), VII (WT, late PC sham), VIII (iNOS^−/−, late PC sham), IX (WT, late PC), X (iNOS^−/−, late PC), XI (WT, early PC), and XII (iNOS^−/−, early PC). ○, individual mice; ●, means ± SEM.