Exercise Training Shifts Dependency from NADPH Oxidase 2 to NADPH Oxidase 4 in Endothelium-Dependent Dilation in Coronary Arterioles Distal to Chronic Occlusion.

Previously, we have reported that exercise training enhances endothelium-dependent dilation in coronary arterioles distal to chronic occlusion, through increased contribution of the redox signaling molecule, hydrogen peroxide (H O ). In the present study, we tested the hypothesis that enhanced endothelium-dependent dilation in collateral-dependent arterioles after exercise training would be attributable to an increased contribution of NADPH oxidase (NOX) 4-derived reactive oxygen species. Yucatan miniature swine were surgically instrumented with an ameroid constrictor around the proximal left circumflex artery, which gradually induced occlusion of the artery and created a collateral-dependent vascular bed. Eight weeks postoperatively, swine were assigned to either a sedentary protocol consisting of normal pen activity or a progressive exercise-training regimen comprised of treadmill running 5 days a week, for 14 weeks. Coronary arterioles were isolated from the nonoccluded and collateral-dependent myocardial regions and bradykinin-mediated dilation via pressure myography was performed in the presence and absence of ML-171, gp91ds-tat and GKT136901, inhibiting NOX1, NOX2, and NOX1/4 isoforms, respectively. Additionally, using the superoxide probe, dihydroethidium, HPLC analysis was performed on isolated microvascular endothelial cells, pretreated with NOX inhibitors, to determine candidate sources of superoxide. Inhibition of NOX2 produced a significant rightward shift in EC50 values of arterioles from sedentary animals, independent of occlusion, as well as the nonoccluded arterioles of exercise-trained pigs. EC50 values of collateral-dependent arterioles from exercise-trained swine were not significantly altered by NOX2 inhibition. Contrastingly, collateral-dependent arterioles from exercise-trained swine exhibited a significant shift in EC50 values in the presence NOX1/4 inhibition, which was absent in other arteriole treatment groups. Importantly, NOX1 inhibition did not significantly shift EC50 values in any treatment group, suggesting responses to the NOX1/4 inhibitor, GKT136901, reflect the contribution of NOX4. Interestingly, microvascular endothelial cell superoxide levels were not different across arteriole treatment groups. Our present findings suggest that exercise training produces adaptations in collateral-dependent arterioles that appear to shift dependence from NOX2- to NOX4-derived reactive oxygen species for endothelium-dependent dilation. Taken together with our previous findings, our data support the assertion that enhanced dilation of collateral-dependent arterioles after exercise training is driven by enhanced NOX4-derived H O levels.