Nitrite confers protection against myocardial infarction: role of xanthine oxidoreductase, NADPH oxidase and K(ATP) channels

Abstract

Reduction of nitrite to nitric oxide during ischemia protects the heart against injury from ischemia/reperfusion. However the optimal dose of nitrite and the mechanisms underlying nitrite-induced cardioprotection are not known. We determined the ability of nitrite and nitrate to confer protection against myocardial infarction in two rat models of ischemia/reperfusion injury and the role of xanthine oxidoreductase, NADPH oxidase, nitric oxide synthase and K(ATP) channels in mediating nitrite-induced cardioprotection. In vivo and in vitro rat models of myocardial ischemia/reperfusion injury were used to cause infarction. Hearts (n=6/group) were treated with nitrite or nitrate for 15 min prior to 30 min regional ischemia and 180 min reperfusion. Xanthine oxidoreductase activity was measured after 15 min aerobic perfusion and 30 min ischemia. Nitrite reduced myocardial necrosis and decline in ventricular function following ischemia/reperfusion in the intact and isolated rat heart in a dose- or concentration-dependent manner with an optimal dose of 4 mg/kg in vivo and concentration of 10 microM in vitro. Nitrate had no effect on protection. Reduction in infarction by nitrite was abolished by the inhibition of flavoprotein reductases and the molybdenum site of xanthine oxidoreductase and was associated with an increase in activity of xanthine dehydrogenase and xanthine oxidase during ischemia. Inhibition of nitric oxide synthase had no effect on nitrite-induced cardioprotection. Inhibition of NADPH oxidase and K(ATP) channels abolished nitrite-induced cardioprotection. Nitrite but not nitrate protects against infarction by a mechanism involving xanthine oxidoreductase, NADPH oxidase and K(ATP) channels.

Figure 1

Nitrite-induced cardioprotection in vivo. (A.) Dose-response study. Rats were treated intravenously with either saline or increasing doses of nitrite (■) or nitrate (□) administered as an IV bolus at 15 minutes prior to ischemia, followed by 30 minutes regional ischemia and 120 minutes reperfusion. Infarct size was determined at the end of 120 minutes of reperfusion. (B.) Phase of action of nitrite. Infarct size was determined after 30 minutes of regional ischemia and 120 minutes of reperfusion. Data are mean ± SD, n=6/group. IS, infarct size * = p<0.05, nitrite vs. drug-free control.

Figure 2

Nitrite and cardioprotection in vitro. Rats were perfused for 15 minutes with nitrite (■) or nitrate (□) prior to 30 minutes regional ischemia and 180 minutes reperfusion. Infarct size was determined at the end of 180 minutes of reperfusion (A). Percent infarction after treatment with nitrite or nitrate (B). Recovery of left ventricular developed pressure (LVDP). Data are mean ± SD, n=6/group. * = p<0.05, nitrite vs. drug-free control.

Figure 3

Inhibition of molybdenum and FAD site in xanthine oxidoreductase prevents nitrite-induced cardioprotection. Rat hearts were perfused with and without nitrite (10 μM) for 15 minutes prior to 30 minutes regional ischemia and 180 minutes of reperfusion. Oxypurinol (OXY) (10 μM) or DPI (2 μM) were present in the coronary perfusate for 30 minutes prior to ischemia (A). Percent infarction (B). Recovery of left ventricular developed pressure (LVDP). Data are mean ± SD, n=6/group. * = p<0.05, vs. control. (C) Ischemia is required for nitrite to increase xanthine oxidase and xanthine dehydrogenase activity. Rat hearts were perfused with nitrite (10 μM) for 15 minutes and subjected to 30 minutes regional ischemia with and without DPI (2μM). Xanthine oxidase (□) and xanthine dehydrogenase (■) activity was then determined. Data are mean ± SD, n=6/group. # = p<0.05, vs. perfusion. + = p<0.05 ischemia plus nitrite, vs. ischemia.

Figure 4

Mechanisms underlying nitrite-induced cardioprotection. Rat hearts were perfused with and without nitrite (10 μM) and either L-NMA (100 μM) or CPTIO (100 μM) for 15 minutes prior to 30 minutes regional ischemia and 180 minutes of reperfusion. (A) Percent infarction (B) Recovery of left ventricular developed pressure (LVDP). Data are mean ± SD, n=6/group. * = p<0.05, vs. control.

Figure 5

Nitrite-induced cardioprotection is mediated by NADPH oxidase. Rat hearts were perfused with and without nitrite (10 μM) for 15 minutes prior to 30 minutes regional ischemia and 180 minutes of reperfusion. Apocynin (30 μM) was present in the coronary perfusate for 30 minutes prior to ischemia. (A) Percent infarction (B) Recovery of left ventricular developed pressure (LVDP). Data are mean ± SD, n=6/group. * = p<0.05, vs. control.

Figure 6

Nitrite-induced cardioprotection is mediated by K_ATP channels. Rats were treated with and without nitrite (4 mg/kg) 15 minutes prior to 30 minutes regional ischemia and 180 minutes of reperfusion. HMR 1098 (3 mg/kg) or 5-HD (10 mg/kg) were administered either 20 minutes prior to ischemia or 5 minutes prior to reperfusion. Data are mean ± SD, n=6/group. * = p<0.05, vs. control.