Exercise Training Increases the Contribution of NADPH Oxidase 4 to Endothelium‐Dependent Dilation in Collateral‐Dependent Coronary Arterioles

Abstract

We have previously reported that exercise training enhances endothelium-dependent dilation in arterioles distal to chronic coronary artery occlusion through an increased contribution of the reactive oxygen species, hydrogen peroxide (H2O2). In the current study, we began to test the hypothesis that exercise training increases the role of NADPH oxidase (NOX) 4 in bradykinin-mediated, endothelium-dependent dilation, which mediates the increase in H2O2, independent of changes in superoxide. Yucatan miniature swine were instrumented with an ameroid constrictor around the proximal left circumflex coronary artery to induce gradual occlusion and create a collateral-dependent vascular bed. Eight weeks postoperatively, swine were assigned to a progressive exercise-training regimen (treadmill run; 5 days/week for 14 weeks) or remained sedentary and limited to normal pen activity. Coronary arterioles were isolated from both nonoccluded and collateral-dependent myocardial regions and cannulated and pressurized to measure vascular reactivity. Bradykinin-mediated dilation was significantly attenuated in the presence of the NOX 1/4 inhibitor, GKT136901 (1 μM), in collateral-dependent arterioles from exercise-trained pigs, which had no significant influence in other arteriole treatment groups. However, immunoblot analysis revealed no effects of occlusion or exercise training on NOX4 protein levels. In addition, superoxide levels in isolated microvascular endothelial cells, measured by HPLC and dihydroethidium (DHE; 50 μM), revealed that bradykinin-stimulated superoxide levels were not altered by occlusion or exercise training. Taken together, our data suggest that exercise training increases the contribution of H2O2 to endothelium-dependent dilation in collateral-dependent arterioles independent of changes in superoxide levels. Additional evidence suggests that increased H2O2 levels may be produced by NOX4, despite no change in NOX4 protein levels.