Exercise training-enhanced, endothelium-dependent dilation mediated by altered regulation of BK(Ca) channels in collateral-dependent porcine coronary arterioles

Abstract

Objective: Test the hypothesis that exercise training increases the contribution of BK(Ca) channels to endothelium-mediated dilation in coronary arterioles from collateral-dependent myocardial regions of chronically occluded pig hearts and may function downstream of H2O2.

Methods: An ameroid constrictor was placed around the proximal left circumflex coronary artery to induce gradual occlusion in Yucatan miniature swine. Eight weeks postoperatively, pigs were randomly assigned to sedentary or exercise training (treadmill; 14 week) regimens.

Results: Exercise training significantly enhanced bradykinin-mediated dilation in collateral-dependent arterioles (~125 μm diameter) compared with sedentary pigs. The BK(Ca) -channel blocker, iberiotoxin alone or in combination with the H2O2 scavenger, polyethylene glycol catalase, reversed exercise training-enhanced dilation in collateral-dependent arterioles. Iberiotoxin-sensitive whole-cell K+ currents (i.e., BK(Ca)-channel currents) were not different between smooth muscle cells of nonoccluded and collateral-dependent arterioles of sedentary and exercise trained groups.

Conclusions: These data provide evidence that BK(Ca)-channel activity contributes to exercise training-enhanced endothelium-dependent dilation in collateral-dependent coronary arterioles despite no change in smooth muscle BK(Ca)-channel current. Taken together, our findings suggest that a component of the bradykinin signaling pathway, which stimulates BK(Ca) channels, is enhanced by exercise training in collateral-dependent arterioles and suggest a potential role for H2O2 as the mediator.

Figure 1. Effect of chronic occlusion and exercise training on bradykinin-mediated vasodilation in porcine coronary arterioles

Bradykinin-mediated dilation was not significantly altered by exercise training in arterioles of the nonoccluded region (A). In contrast, exercise training significantly enhanced bradykinin-mediated dilation in the collateral-dependent region (B). IC₅₀ values (insets) revealed that sensitivity to bradykinin was not altered by exercise training in the nonoccluded region, but was significantly increased in collateral-dependent arterioles of exercise trained (EX) compared with sedentary (SED) pigs. Bradykinin-mediated dilation and IC₅₀ values (insets) were significantly attenuated in collateral-dependent compared with nonoccluded coronary arterioles of sedentary, but not exercise-trained pigs. Values are means ± S.E.M. of the number of animals in parentheses. A subset of these data has been published previously (53). * P ≤ 0.05.

Figure 2. Effect of BK_Ca channel blockade on bradykinin-mediated dilation of nonoccluded and collateral-dependent arterioles

The BK_Ca channel blocker, iberiotoxin (IbTx; 100 nM), significantly attenuated dilation in nonoccluded (A) and collateral-dependent (B) arterioles of sedentary (SED) and exercise trained (EX) pigs. In the presence of iberiotoxin, dilation in nonoccluded arterioles was similar between sedentary and exercise-trained pigs. In contrast, exercise training-enhanced dilation in collateral-dependent arterioles was reverse in the presence of iberiotoxin. In the presence of iberiotoxin, IC₅₀ values were similar in arterioles from the nonoccluded region of sedentary and exercise-trained pigs (2A, inset). Treatment with iberiotoxin reversed the enhanced sensitivity to bradykinin that was observed under control conditions in arterioles from the collateral-dependent region of exercise-trained pigs (2B, inset). Values are means ± S.E.M. of the number of animals in parentheses. * P ≤ 0.05.

Figure 3. Effect of combined BK_Ca channel blockade and scavenging of H₂O₂ on bradykinin-mediated dilation in nonoccluded and collateral-dependent arterioles

Combined pretreatment with iberiotoxin and the H₂O₂ scavenger, PEG-catalase (PEG-CAT; 1000 U/ml), significantly attenuated dilation in nonoccluded (A) and collateral-dependent (B) arterioles of sedentary (SED) and exercise trained (EX) pigs. In the presence of iberiotoxin plus PEG-CAT, dilation in nonoccluded arterioles was similar between sedentary and exercise-trained pigs. In contrast, exercise training-enhanced dilation in collateral-dependent arterioles was reverse in the presence of the combined inhibitors. In the presence of iberiotoxin plus PEG-CAT, IC₅₀ values were similar in arterioles from the nonoccluded region of sedentary and exercise-trained pigs (3A, inset). Treatment with iberiotoxin plus PEG-CAT reversed the enhanced sensitivity to bradykinin that was observed under control conditions in arterioles from the collateral-dependent region of exercise-trained pigs (3B, inset). Values are means ± S.E.M. of the number of animals in parentheses. * P ≤ 0.05.

Figure 4. Effect of chronic occlusion and exercise training on whole-cell K⁺ current in coronary arteriolar smooth muscle cells

A. As illustrated by the voltage template, currents were elicited by 500-ms step depolarizations (tp) to potentials ranging from –70 to +100 (in 10 mV increments) from a holding potential (hp) of –80 mV. Representative current traces for whole-cell K⁺ current of cells from nonoccluded and collateral-dependent arterioles of sedentary and exercise-trained pigs. B and C. Comparison of I-V relationships obtained by plotting mean current at the end of the steps as a function of the indicated test potential. Whole-cell K⁺ current was significantly diminished in cells from exercise-trained pigs from both nonoccluded (B) and collateral-dependent (C) arterioles. K⁺ current was also significantly reduced in cells from collateral-dependent compared with nonoccluded arterioles of both sedentary and exercise trained animals. Numbers in parentheses indicate number of pigs, cells. Values are means ± S.E.M. of the number of cells in parentheses; *P≤0.05.

Figure 5. Effect of chronic occlusion and exercise training on iberiotoxin-sensitive K⁺ channel currents in coronary arteriolar smooth muscle cells

A. Iberiotoxin-sensitive K⁺ channel currents were obtained by subtraction of currents in the presence of iberiotoxin (100 nM) from control currents from cells of both nonoccluded and collateral-dependent arterioles of sedentary and exercise trained pigs. B. Comparison of iberiotoxin-sensitive I-V relationships obtained by plotting mean iberiotoxin (100 nM)-sensitive current at the end of the steps as a function of the indicated step potential. Values are means ± S.E.M. of the number of cells in parentheses; No significant differences existed.

Figure 6. Effect of chronic occlusion and exercise training on superoxide dismutase protein and activity levels in coronary arterioles

Immunoblot analyses of the superoxide dismutase (SOD) family of isoforms revealed that neither ecSOD (A), MnSOD (B), nor Cu/ZnSOD (C) protein levels were significantly altered by occlusion or exercise training. Protein was quantified by densitometry analysis, normalized to β-actin, and expressed relative to the density of nonoccluded arterioles of sedentary (SED) pigs. Evaluation of SOD activity also demonstrated no significant different between arteriolar treatment groups. Furthermore, bradykinin (BK; 10 nM) treatment had no effect on SOD activity compared with control (PSS; conditions). EX, exercise-trained. Values are means ± S.E.M. of the number of animals indicated.

Figure 7. Effect of chronic occlusion and exercise training on catalase protein levels in coronary arterioles

Immunoblot analyses demonstrated that catalase protein levels were not significantly altered by occlusion or exercise training. Protein was quantified as in Figure 6 legend. SED, sedentary; EX, exercise-trained. Values are means ± S.E.M. of the number of animals in parentheses. No significant differences existed.