Myocardial ischemia and reperfusion (MI/R) initiates a cascade of polymorphonuclear neutrophil (PMN)-mediated injury, the magnitude of which may be influenced by the bioavailability of nitric oxide (NO). We investigated the role of endothelial cell nitric oxide synthase (ecNOS) in MI/R injury by subjecting wild-type and ecNOS-deficient (−/−) mice to 20 min of coronary artery occlusion and 120 min of reperfusion. Myocardial infarct size represented 20.9 ± 2.9% of the ischemic zone in wild-type mice, whereas the ecNOS −/− mice had significantly ( P < 0.01) larger infarcts measuring 46.0 ± 3.8% of the ischemic zone. Because P-selectin is thought to be involved with the pathogenesis of neutrophil-mediated I/R injury, we assessed the effects of MI/R on P-selectin expression in the myocardium of wild-type and ecNOS −/− mice. P-selectin expression measured with a radiolabeled monoclonal antibody (MAb) technique after MI/R in wild-type mice was 0.037 ± 0.009 μg MAb/g tissue, whereas ecNOS −/− coronary vasculature was characterized by significantly ( P < 0.05) higher P-selectin expression (0.080 ± 0.013 μg MAb/g tissue). Histological examination of the postischemic myocardium revealed significantly ( P < 0.01) more neutrophils in the ecNOS −/− (29.5 ± 2.5 PMN/field) compared with wild-type (5.0 ± 0.9 PMN/field) mice. A similar trend in infarct size and neutrophil accumulation was observed when wild-type and ecNOS −/− mice were subjected to 30 min of ischemia and 120 min of reperfusion. These novel in vivo findings demonstrate a cardioprotective role for ecNOS-derived NO in the ischemic-reperfused mouse heart.

Previous studies have demonstrated that circulating neutrophils (PMNs) contribute to the pathophysiology of myocardial ischemia-reperfusion (MI/R) injury. PMN-endothelial cell interactions are highly regulated by adhesive interactions between PMN CD11/CD18 and coronary endothelial cell intercellular adhesion molecule-1 (ICAM-1). We investigated the effects of MI/R in wild-type, CD18-, and ICAM-1-deficient (−/−) mice. Wild-type ( n = 6), CD18 −/− ( n = 6), and ICAM-1 −/− ( n = 6) mice were subjected to 30 min of myocardial ischemia and 120 min of reperfusion to determine the extent of PMN infiltration and myocardial cell necrosis. Myocardial infarction (% of the area at risk) was 45.1 ± 5.9 in wild-type mouse hearts. In contrast, the extent of myocardial infarction was significantly ( P < 0.05) reduced in the CD18 (19.3 ± 5.1%)- and ICAM-1 (17.9 ± 3.2%)-deficient mice. Similarly, PMN infiltration into the ischemic-reperfused myocardium was attenuated by 54% in the CD18 −/− mice and by 32% in ICAM-1 −/− mice compared with wild-type hearts. Deficiency in either CD18 or ICAM-1 expression results in a marked reduction in PMN accumulation and myocardial necrosis after acute MI/R.

We investigated in vivo coronary P-selectin expression and its pathophysiological consequences in a murine model of myocardial ischemia-reperfusion (MI/R) using wild-type and P-selectin deficient (−/−) mice. Coronary P-selectin expression [μg monoclonal antibody (MAb)/g tissue] was measured using a radiolabeled MAb method after 30 min of myocardial ischemia and 20 min of reperfusion. P-selectin expression in wild-type mice was significantly ( P< 0.01) elevated in the ischemic zone (0.070 ± 0.010) compared with the nonischemic zone (0.037 ± 0.008). Myocardial P-selectin expression was nearly undetectable in P-selectin −/− mice after MI/R. Furthermore, myocardial infarct size (% of area at risk) after 30 min of myocardial ischemia and 120 min of reperfusion was 42.5 ± 4.4 in wild-type mice and 24.4 ± 4.0 in P-selectin −/− mice ( P < 0.05). In additional experiments of prolonged myocardial ischemia (60 min) and reperfusion (120 min), myocardial infarct size was similar in P-selectin −/− mice and wild-type mice. Our results clearly demonstrate the involvement of coronary P-selectin in the development of myocardial infarction after MI/R.

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