Oxidative stress is a major cause of I/R injury. NADPH oxidase is an important source of oxidative stress during I/R, and it consists of NOX1‐5 and DOUX1‐2 isoforms. The role of NOX1 in myocardial I/R injury is unclear. In this study, a specific NOX1 inhibitor, ML171 (MW=241.31 g/mol, IC50 for NOX1=0.25 μM), was tested in isolated perfused rat hearts following I (30 min)/R (45 min). We found that left ventricular developed pressure (LVDP) and the peak of the first derivative of left ventricular pressure (dP/dtmax) only recovered to 42 ± 4% and 30 ± 3% of baseline values, respectively, at 45 min post‐reperfusion in control I/R hearts (n=8). By contrast, ML171 (1 μM, n=5) given at reperfusion for 5 min significantly restored LVDP and dP/dtmax to 90 ± 6% and 67 ± 7% of baseline values, respectively (both p<0.01). ML171 also significantly reduced infarct size to 18 ± 3% compared to 41 ± 2% in control I/R hearts (p<0.01). Furthermore, a selective mitochondrial ATP‐dependent K+ channel (mitoKatp) inhibitor, 5‐ hydroxydecanoate (100 μM, n=4); and a heme oxygenase‐1 inhibitor, SnPP (20 μM, n=3), significantly abolished the cardioprotective effects of ML171 (all p<0.05). We also confirmed that ML171 (0.1‐1 μM, n=4) did not inhibit phorbol 12‐myristate 13‐acetate (30 nM) induced superoxide release from isolated rat neutrophils. These results suggest that NOX1 inhibition improves post‐reperfused contractile function and reduces infarct size, possibly by opening mitoKatp channels and activating heme oxygenase‐1.

Acute hyperglycemia impairs vascular dilatory function in normal subjects. Normally, vascular endothelial function depends on nitric oxide (NO) production from coupled eNOS in the presence of cofactor 5,6,7,8‐tetrahydrobiopterin (BH4). By contrast, 7,8‐dihydrobiopterin (BH2, oxidized form of BH4) and/or lack of L‐arginine (eNOS substrate) causes eNOS uncoupling to produce superoxide (SO), which can be quickly converted to hydrogen peroxide (H2O2). The role of eNOS coupling status in mediating acute hyperglycemia‐induced vascular dysfunction is not known. We simultaneously measured blood NO and H2O2 during the infusion of saline (control), or 20% glucose with or without BH4 (MW=314 g/mol, 6.5 mg/kg), or L‐arginine (MW=211 g/mol, 600 mg/kg), or BH2 (MW=239 g/mol, 4 mg/kg) by NO or H2O2 microsensors (100 µm, WPI Inc.) from the femoral veins of male Sprague‐Dawley rats. We found that acute hyperglycemia (200 mg/dL, n=6) significantly increased blood H2O2 and reduced blood NO levels compared to the saline group over a 3 hr. period (n=7, p<0.05). By contrast, BH4 (n=6), or L‐arginine (n=5‐6), not BH2 (n=6‐7), significantly reduced blood H2O2 and improved vascular NO levels (p<0.05). This study provides novel evidence indicating that eNOS uncoupling during acute hyperglycemia causes endothelial dysfunction and oxidative stress. Promotion of eNOS coupling may be beneficial to maintain normal vascular function. Grant Funding Source: Supported by Center for Chronic Disorders of Aging and Department of Bio‐Medical Sciences at PCOM