Exercise induces superoxide and NOX4 contribution in endothelium-dependent dilation in coronary arterioles from a swine model of chronic myocardial ischemia.

Exercise training is an effective, nonpharmacologic therapy and preventative measure for ischemic heart disease. While recent studies have examined reactive oxygen species (ROS) as mediators of exercise training-enhanced coronary blood flow, specific oxidants and their sources have yet to be fully elucidated. We investigated the hypothesis that NADPH oxidase (NOX)-derived superoxide anion would contribute to vasodilation effects in the coronary microcirculation of swine and that these effects would be impaired by chronic ischemia and rescued with exercise training. Adult Yucatan miniature swine were instrumented with an ameroid occluder around the proximal left circumflex coronary artery, resulting in a collateral-dependent myocardial region. Eight weeks post-operatively, swine were randomly assigned to either a sedentary or exercise training (treadmill run; 5 days/week for 14 weeks) protocol. Coronary arterioles were isolated from nonoccluded and collateral-dependent myocardial regions and pressure myography was performed. Exercise training resulted in enhanced endothelium-dependent dilation after occlusion. Scavenging of superoxide via the superoxide dismutase (SOD)-mimetic, tempol, attenuated dilation in both nonoccluded and collateral-dependent arterioles of exercise-trained, but not sedentary swine. NOX1/4 inhibition with GKT136901 attenuated dilation after exercise training but only in collateral-dependent arterioles. High performance liquid chromatography revealed that neither ischemia nor exercise training significantly altered basal or bradykinin-stimulated superoxide levels. Furthermore, superoxide production was not attributable to NOX isoforms nor mitochondria. Immunoblot analyses revealed significantly decreased NOX2 protein after exercise with no differences in NOX1, NOX4, p22phox, SOD proteins. Taken together, these data provide evidence that superoxide and NOX4 independently contribute to enhanced endothelium-dependent dilation following exercise training.

Exercise training rescues impaired H2O2-mediated vasodilation in porcine collateral-dependent coronary arterioles through enhanced K+ channel activation.

We previously reported that exercise training drives enhanced agonist-stimulated hydrogen peroxide (H2O2) levels and restores endothelium-dependent dilation via an increased reliance on H2O2 in arterioles isolated from ischemic porcine hearts. In this study, we tested the hypothesis that exercise training would correct impaired H2O2-mediated dilation in coronary arterioles isolated from ischemic myocardium through increases in protein kinase G (PKG) and protein kinase A (PKA) activation and subsequent colocalization with sarcolemmal K+ channels. Female adult Yucatan miniature swine were surgically instrumented with an ameroid constrictor around the proximal left circumflex coronary artery, gradually inducing a collateral-dependent vascular bed. Arterioles (∼125 µm) supplied by the left anterior descending artery served as nonoccluded control vessels. Pigs were separated into exercise (treadmill; 5 days/wk for 14 wk) and sedentary groups. Collateral-dependent arterioles isolated from sedentary pigs were significantly less sensitive to H2O2-induced dilation compared with nonoccluded arterioles, whereas exercise training reversed the impaired sensitivity. Large conductance calcium-activated potassium (BKCa) channels and 4AP-sensitive voltage-gated (Kv) channels contributed significantly to dilation in nonoccluded and collateral-dependent arterioles of exercise-trained but not sedentary pigs. Exercise training significantly increased H2O2-stimulated colocalization of BKCa channels and PKA, but not PKG, in smooth muscle cells of collateral-dependent arterioles compared with other treatment groups. Taken together, our studies suggest that with exercise training, nonoccluded and collateral-dependent coronary arterioles better use H2O2 as a vasodilator through increased coupling with BKCa and 4AP-sensitive Kv channels; changes that are mediated in part by enhanced colocalization of PKA with BKCa channels.NEW & NOTEWORTHY The current study reveals that coronary arterioles distal to stenosis display attenuated dilation responses to H2O2 that are restored with endurance exercise training. Enhanced H2O2 dilation after exercise is dependent on Kv and BKCa channels and at least in part on in colocalization of BKCa channel and PKA and independent of PKA dimerization. These findings expand our earlier studies which demonstrated that exercise training drives beneficial adaptive responses of reactive oxygen species in the microvasculature of the ischemic heart.

Enhanced KCl-mediated contractility and Ca2+ sensitization in porcine collateral-dependent coronary arteries persist after exercise training.

We have previously reported enhanced Ca2+ sensitivity of coronary arteries that is dependent upon collateral circulation for their blood supply. For the current study, we hypothesized that small collateral-dependent arteries would exhibit an enhanced KCl-mediated contractile response attributable to Ca2+ sensitization and increased Ca2+ channel current. Ameroid constrictors were surgically placed around the left circumflex (LCX) artery of female Yucatan miniature swine. Eight weeks postoperatively, pigs were randomized into sedentary or exercise-trained (treadmill run; 5 days/wk; 14 wk) groups. Small coronary arteries (150-300 µm luminal diameter) were isolated from myocardial regions distal to the collateral-dependent LCX and the nonoccluded left anterior descending arteries. Contractile tension and simultaneous measures of both tension and intracellular free Ca2+ levels (fura-2) were measured in response to increasing concentrations of KCl. In addition, whole cell Ca2+ currents were also obtained. Chronic occlusion enhanced contractile responses to KCl and increased Ca2+ sensitization in collateral-dependent compared with nonoccluded arteries of both sedentary and exercise-trained pigs. In contrast, smooth muscle cell Ca2+ channel current was not altered by occlusion or exercise training. Ca2+/calmodulin-dependent protein kinase II (CaMKII; inhibited by KN-93, 0.3-1 µM) contributed to the enhanced contractile response in collateral-dependent arteries of sedentary pigs, whereas both CaMKII and Rho-kinase (inhibited by hydroxyfasudil, 30 µM or Y27632, 10 µM) contributed to increased contraction in exercise-trained animals. Taken together, these data suggest that chronic occlusion leads to enhanced contractile responses to KCl in collateral-dependent coronary arteries via increased Ca2+ sensitization, a response that is further augmented with exercise training.NEW & NOTEWORTHY Small coronary arteries distal to chronic occlusion displayed enhanced contractile responses, which were further augmented after exercise training and attributable to enhanced calcium sensitization without alterations in calcium channel current. The calcium sensitization mediators Rho-kinase and CaMKII significantly contributed to enhanced contraction in collateral-dependent arteries of exercise-trained, but not sedentary, pigs. Exercise-enhanced contractile responses may increase resting arterial tone, creating an enhanced coronary flow reserve that is accessible during periods of increased metabolic demand.