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J Appl Physiol 87: 1948-1956, 1999;
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Vol. 87, Issue 5, 1948-1956, November 1999

Exercise training improves endothelium-mediated vasorelaxation after chronic coronary occlusion

Kawanza L. Griffin1,2, M. Harold Laughlin1,2,3, and Janet L. Parker1,2,4

1 Dalton Cardiovascular Research Center and 2 Departments of Physiology and 3 Veterinary Biomedical Sciences, University of Missouri, Columbia, Missouri 65211; and 4 Department of Medical Physiology, Texas A & M University, College Station, Texas 77843


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study evaluated combined effects of chronic coronary occlusion and exercise training on endothelial function. Gradual occlusion was produced by placement of an ameroid constrictor around the proximal left circumflex (LCX) coronary artery of female swine. Two months after placement of the ameroid, animals were restricted to their pens or exercise trained for 16 wk. Epicardial arteries (>500 µm ID) were isolated from the collateral-dependent LCX coronary artery distal to the occlusion and the nonoccluded left anterior descending (LAD) coronary artery. Bradykinin- and ADP-mediated relaxation of LCX and LAD coronary arteries was enhanced after exercise training. Inhibition of nitric oxide synthase with NG-nitro-L-arginine methyl ester decreased bradykinin- and ADP-mediated relaxation in LCX and LAD myocardial regions. Importantly, combined inhibition of effects of endothelium-derived hyperpolarizing factor with increased extracellular K+ (20-30 mM) and nitric oxide synthase completely abolished coronary LAD and LCX relaxation to bradykinin. Our data indicate that exercise training improves endothelium-mediated relaxation of arteries isolated after chronic coronary artery occlusion, likely as a result of enhanced production of nitric oxide and endothelium-derived hyperpolarizing factor.

bradykinin; collateral circulation; vasodilation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

OCCLUSION OF A MAJOR coronary artery results in a lack of blood flow and oxygen delivery to the region of the myocardium supplied by the occluded artery. The fate of the myocardium distal to the occlusion will depend, partially, on the restoration of blood flow via the development of the collateral circulation (25, 28, 35). Although the collateral circulation is generally able to maintain sufficient blood flow during resting conditions (12, 25, 26, 35), myocardial ischemia and inotropic dysfunction have been documented in the collateral-dependent myocardium during bouts of exercise (16, 25). Sellke et al. (29, 30) demonstrated that resistance arteries isolated from the collateral-dependent vasculature of untrained animals exhibit an impairment of endothelium-mediated relaxation and enhancement of selected vasoconstrictor responses in the porcine and canine coronary circulation. Similarly, Rapps et al. (23) demonstrated enhanced alpha -adrenergic vasoconstriction and impaired vasorelaxation responses in conduit arteries distal to chronic coronary occlusion in the canine circulation. However, the combined effects of chronic coronary occlusion and exercise training on coronary function remain unclear. In consideration of the finding that exercise training has been shown to enhance endothelium-mediated relaxation (18, 21) while decreasing vasoconstriction (19, 24) in the normal nonoccluded porcine heart, it is essential that the effects of exercise training be determined in the presence of a coronary occlusion.

The purpose of this study was to evaluate exercise-training-induced alterations of coronary endothelial function in porcine hearts exposed to chronic coronary occlusion. Because exercise training has been associated with increases in nitric oxide (NO) (2, 18, 21) and endothelial cell NO synthase (ecNOS) gene expression (31, 36) in arteries isolated from normal nonoccluded hearts, we also sought to evaluate the role of NO in mediating vasorelaxation in our model of chronic coronary occlusion. We hypothesized that exercise training would improve endothelium-mediated relaxation in those arteries isolated after occlusion and, furthermore, that improved relaxation would be due to an increased production of NO. Responses to the endothelium-dependent agonists bradykinin and ADP were evaluated in epicardial arteries isolated from the collateral-dependent left circumflex (LCX) coronary artery and the nonoccluded left anterior descending (LAD) coronary artery of sedentary or exercise-trained animals. All experiments were performed in vitro; therefore, intrinsic coronary vascular relaxation could be studied in the absence of confounding neural, humoral, and myocardial contractile influences on coronary vascular tone. Results obtained from these studies suggest that chronic exercise training enhances endothelium-mediated relaxation to bradykinin and ADP, potentially via an enhanced production of NO and endothelium-derived hyperpolarizing factor (EDHF).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal Instrumentation for Chronic Coronary Occlusion

Adult female miniature swine (~25-45 kg) were sedated with ketamine (20 mg/kg im), midazolam (0.5 mg/kg im), and glycopyrrolate (0.004 mg/kg im). Animals were then intubated and maintained on isoflurane during subsequent surgical procedures. A left thoracotomy was performed through the fifth intercostal space. The heart was exposed, and the pericardium was opened. An ameroid constrictor (2.5-3.5 mm ID; carefully chosen for snug but nonconstrictive fit) was placed around the proximal region of the LCX coronary artery. During surgery and recovery, animals received buprenorphine (0.01 mg/kg iv) as needed for pain. Antibiotics were given immediately before surgery (~30,000 U penicillin im) and for 5 days after surgery (sulfatrimethoprim, ~480 mg/50 pounds). All experimental procedures were in accordance with the "Position of the American Heart Association on Research Animal Use," adopted on 11 November 1984, and were approved by the Animal Care and Use Committee of the University of Missouri.

Exercise Training Program

Animals were allowed to recuperate for 2 mo after surgery. This time period allows adequate formation and development of collaterals and ensures the ability of animals to successfully enter into the training program (25, 27, 34). After this period, animals were randomly divided into equal groups of exercise-trained or sedentary control animals. Exercise-trained animals entered a progressive treadmill training program for 16 wk; sedentary animals remained inactive during this period. This exercise program has been utilized extensively (18, 19) and is similar to that described for dogs (32). Briefly, during the 1st wk of training, the animals ran at 4-5 mph for 15 min (sprint) and 3 mph, 0% grade for 20-30 min (endurance). Speed and duration were progressively increased dependent on the ability of the animal. By week 12 of training, the animals ran 85 min/day, 5 days/wk. This consisted of a 5-min warm-up at 2.5 mph, a 15-min sprint at 6 mph, a 60-min endurance run at 4.5-5 mph, and a 5-min cooldown at 2.5 mph. After each exercise bout, the animals were hosed with water to cool them, and food was provided as positive reinforcement.

Preparation of Coronary Arteries

After completion of the 16-wk exercise training program or period of inactivity, pigs were sedated with ketamine (25 mg/kg im) and xylazine (Rompun, 2.25 mg/kg im) and anesthetized with thiopental sodium (10 mg/kg iv). A left thoracotomy was performed, and the heart was rapidly removed, weighed, and placed into ice-cold Krebs solution (4°C) bubbled with 95% O2-5% CO2. Complete closure of the ameroid around the LCX coronary artery was confirmed in all experiments. We isolated size-matched conduit (~500 µm ID) arteries from the collateral-dependent LCX coronary artery distal to the chronic occlusion and the nonoccluded LAD coronary artery from each heart.

With the aid of a dissection microscope, segments obtained from the LAD and LCX coronary arteries were cleaned of myocardium and connective tissue and cut into rings with axial lengths of 3.5-4.0 mm; outer diameter, inner diameter, wall thickness, and axial length of each vessel ring were measured with a Filar calibrated micrometer eyepiece (Hitschfel Instruments, St. Louis, MO). All arterial rings were prepared in Krebs buffer. Rings were carefully mounted on two stainless steel wires (Rocky Mountain Orthodontics). One wire was attached to a force transducer (model FT.03c, Grass Instrument, Quincy, MA), and the other was attached to a micrometer (Stoelting/Prior Microdrive, Wood Dale, IL). The rings were then lowered into 20-ml tissue baths (Harvard Apparatus, S. Natick, MA) that contained Krebs bicarbonate buffer equilibrated at 37°C with 95% O2-5% CO2. Coronary artery rings were individually stretched to the maximum of the length-developed tension relationship by repeated exposures to 30 mmol/l KCl at increasing vessel stretch. Optimal length was defined as the circumferential length at which the active tension produced was <5% greater than the tension produced at the previous length. All studies were performed with arteries stretched to optimal length, and vessel preparations were allowed to stabilize for 30 min before experimentation.

Experimental Protocols

Concentration-response relationships to bradykinin (10-13-10-6 mol/l), ADP (10-8-10-4 mol/l), and sodium nitroprusside (10-8-10-4 mol/l) were determined by cumulative addition of these agents in half-log increments directly to the tissue bath. All arteries were rinsed and restabilized between agents. Arteries were preconstricted with prostaglandin F2alpha (PGF2alpha ) to approximate half-maximal contraction before assessment of relaxation responses. Drug concentrations were increased when the response to the previous concentration was maximal.

Solutions and Drugs

The Krebs bicarbonate solution for all experiments contained (in mmol/l) 131.5 NaCl, 5.0 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, 25 NaHCO3, and 10.1 glucose (bubbled with 95% O2-5% CO2, pH 7.4). This solution also contained 25 µM EDTA. Solutions with elevated K+ concentrations used for depolarizing smooth muscle were produced by equimolar replacement of NaCl with KCl. Bradykinin, ADP, and sodium nitroprusside were purchased from Sigma Chemical (St. Louis, MO) and PGF2alpha from Upjohn (Kalamazoo, MI). All drugs were prepared fresh daily in Krebs solution.

Oxidative Enzyme Capacity

Immediately after the animals were killed, samples were obtained from the middle of the triceps brachii muscle. The samples were then frozen in liquid nitrogen and stored at -70°C until processed. Citrate synthase activity was measured in the samples by spectrophotometry (18, 32). Increases in skeletal muscle citrate synthase activity are representative of successful exercise training.

Data Analyses

Citrate synthase activities of skeletal muscle and the heart weight-to-body weight ratios were compared using Student's unpaired t-test. Concentration-response curves were compared using two-way ANOVA for repeated measures. Differences between individual points were ascertained using Fisher's test for least significant difference. IC50 values were defined as the concentration of the agonist causing 50% of the maximal response. Differences in IC50 values between groups were compared using nonlinear regression analysis of the concentration-response data. These values were calculated using nonlinear regression analysis of the concentration-response data for each artery. For all statistical analyses, significance was defined as P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of Training

Citrate synthase activity was significantly increased in samples obtained from the medial head of the triceps brachii muscle of exercise-trained animals (18.2 ± 1.2 and 15.1 ± 0.6 in exercise-trained and sedentary animals, respectively; P < 0.05). In samples obtained from other areas of the triceps brachii muscle, citrate synthase activity also tended to be higher after exercise training. Heart weight-to-body weight ratio was also significantly higher in exercise-trained animals than in sedentary animals (6.6 ± 0.4 and 5.0 ± 0.2 g/kg in exercise-trained and sedentary animals, respectively; P < 0.05).

Vessel Characteristics

Conduit artery ring dimensions of LAD and LCX coronary arteries are reported in Table 1. There were no significant differences in vessel dimensions (outer diameter, inner diameter, and axial length) between sedentary and trained groups of LAD or LCX vascular regions; however, wall thickness was slightly higher in LCX than in LAD coronary arteries (P < 0.05) in the sedentary group. Similarly, there were no significant differences in vessel dimensions between LAD and LCX coronary arteries of sedentary and exercise-trained groups; however, resting tension was slightly higher (P < 0.05) in LCX than in LAD coronary arteries from exercise-trained animals. Furthermore, in the presence of PGF2alpha , developed tension was not significantly different between LAD and LCX coronary arteries isolated from sedentary or exercise-trained animals (not shown).

                              
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  		Table 1.   Porcine conduit artery ring dimensions

Effects of Exercise Training on Endothelium- Dependent and -Independent Relaxation Responses

Relaxation responses to bradykinin and ADP. All vessels were preconstricted with PGF2alpha before exposure to increasing concentrations of each vasorelaxation agent. As shown in Fig. 1, bradykinin-induced relaxation was significantly enhanced in arteries isolated from exercise-trained animals in the nonoccluded LAD coronary artery (P < 0.05) and the collateral-dependent LCX coronary artery (P < 0.001). Furthermore, sensitivity to bradykinin was markedly enhanced in the LCX artery after training, as indicated by a shift upward and to the left of the exercise-trained concentration-response curve. The calculated IC50 value of LCX arteries isolated from trained animals was threefold lower than control (3.5 ± 1.8 and 24.9 ± 15.4 nmol/l in exercise-trained and sedentary animals, respectively, P < 0.05). This reduction in IC50 suggests a training-induced increase in sensitivity to bradykinin, with a dominant effect in vessels distal to occlusion (Fig. 1). To further examine our hypothesis, we investigated comparable relaxation responses to the endothelium-dependent vasodilator ADP (10-8-10-4 mol/l), which is believed to act via a mechanism similar to bradykinin but utilizes purinergic, rather than kininergic, receptor activation. Similar to our findings with bradykinin, relaxation responses to ADP were significantly enhanced (P < 0.05) in LCX and LAD arteries after exercise training (Fig. 2). The mean IC50 value after exercise was decreased compared with control in the LCX artery (0.93 ± 0.43 and 9.92 ± 6.90 nmol/l in exercise-trained and sedentary groups, respectively, P < 0.05). These results suggest that exercise training enhances endothelium-mediated relaxation of porcine conduit-sized arteries from the collateral-dependent LCX and the nonoccluded LAD vasculature.


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  		Fig. 1.   Relaxation of conduit arteries from nonoccluded [left anterior descending (LAD), A] and collateral-dependent [left circumflex (LCX), B] regions in response to increasing concentrations of bradykinin. Relaxation responses were significantly enhanced after exercise training in LAD and LCX arteries. Values are means ± SE of number of animals in parentheses. Mean IC50 value for exercise-trained (Ex) animals (4.7 ± 2.0 nM) is significantly less than sedentary (Sed) value (11.2 ± 5.7 nM). PGF2alpha , prostaglandin F2alpha .



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  		Fig. 2.   Relaxation of conduit arteries from nonoccluded (LAD, A) and collateral-dependent (LCX, B) regions in response to increasing concentrations of ADP. Relaxation responses were significantly enhanced after exercise training in LAD and LCX arteries. Values are means ± SE of number of animals in parentheses. Mean IC50 value for exercise-trained animals (0.93 ± 0.43 nM) is significantly less than sedentary value (9.92 ± 6.9 nM).

Effects of sodium nitroprusside. To examine the possibility of effects of chronic occlusion and exercise training on coronary smooth muscle responsiveness to NO, we evaluated LAD and LCX coronary artery relaxation responses to sodium nitroprusside (10-8-10-4 mol/l). Sodium nitroprusside is an NO donor (endothelium-independent vasodilator) that causes direct vasodilation via elevation of smooth muscle cGMP levels. In contrast to our findings with bradykinin and ADP, relaxation responses to sodium nitroprusside were not enhanced by exercise training in LCX or LAD vessels (P > 0.05; Fig. 3).


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  		Fig. 3.   Relaxation of conduit arteries from nonoccluded (LAD, A) and collateral-dependent (LCX, B) regions in response to increasing concentrations of sodium nitroprusside. Relaxation responses were not significantly different after exercise training in LAD or LCX arteries (P > 0.05). Values are means ± SE of number of animals in parentheses.

Role of NO in exercise-induced enhanced relaxation responses. To determine the role of exercise-induced alterations in the synthesis of NO, we evaluated agonist-induced relaxation in the presence of the NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 100 µM), in coronary arteries isolated from occluded hearts. Arteries were continuously incubated in L-NAME 10 min before preconstriction with PGF2alpha and 25-30 min before exposure to bradykinin or ADP. In the presence of L-NAME, there was a decrease in the maximal bradykinin-mediated relaxation response in LAD and LCX arteries (Fig. 4, A and B). Furthermore, bradykinin-mediated relaxation of sedentary and exercise-trained groups was not different between the LAD and LCX arteries in the presence of the inhibitor (Fig. 4, C and D). Furthermore, L-NAME partially reversed training-induced enhanced bradykinin-mediated relaxation in LAD and LCX arteries from exercise-trained animals (Fig. 4, A and B). Training-induced effects on bradykinin-mediated relaxation remained significantly different in LCX (P < 0.05), but not in LAD (P > 0.05), arteries, suggesting an additional role of another mediator (e.g., EDHF) in LCX arteries. We then compared bradykinin-mediated relaxation in the presence of KCl preconstriction. Responses to bradykinin of KCl-preconstricted arteries were not altered by exercise training in LCX or LCX vessels (P > 0.05; Fig. 5). L-NAME also inhibited ADP-mediated relaxation (Fig. 6). L-NAME shifted ADP-induced responses to the right, suggesting a decreased sensitivity to ADP in the presence of NOS inhibition. Relaxation responses were not different between LCX and LAD arteries in the presence of L-NAME; however, ADP relaxation remained enhanced in arteries from exercise-trained animals. Importantly, inhibition of the effects of EDHF with depolarizing levels of increased extracellular K+ preconstriction (25-30 mM) in conjunction with NOS inhibition (L-NAME) completely abolished LAD and LCX artery relaxation to bradykinin (Fig. 7). Thus effects of exercise training on endothelium-mediated relaxation of coronary arteries isolated from occluded hearts appear to be the result of an enhanced production of endothelium-derived NO (EDNO) and EDHF.


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  		Fig. 4.   Effects of NG-nitro-L-arginine methyl ester (L-NAME) on concentration-dependent relaxation responses to bradykinin in nonoccluded (LAD, A) and collateral-dependent (LCX, B) coronary arteries isolated from sedentary and exercise-trained animals. Sedentary and exercise-trained values are also plotted in C and D, respectively, to allow direct comparisons of LAD vs. LCX responses. In presence of L-NAME, there was a decrease in maximal bradykinin-mediated relaxation responses in LCX and LAD arteries. Bradykinin-mediated relaxation was not different in LAD and LCX arteries in presence of L-NAME. Values are means ± SE of number of animals in parentheses.



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  		Fig. 5.   Effects of KCl preconstriction on relaxation of conduit arteries from nonoccluded (LAD, A) and collateral-dependent (LCX, B) regions in response to increasing concentrations of bradykinin. Relaxation responses were not significantly different after exercise training in LAD or LCX arteries (P > 0.05). Values are means ± SE of number of animals in parentheses.



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  		Fig. 6.   Effects of L-NAME on concentration-dependent relaxation responses to ADP in collateral-dependent (LCX) conduit arteries distal to occlusion and nonoccluded (LAD) conduit arteries isolated from sedentary (A) and exercise-trained (B) animals. In presence of L-NAME, there was a decreased sensitivity to ADP. Relaxation was not different in LAD and LCX arteries in presence of L-NAME. Values are means ± SE of number of animals in parentheses.



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  		Fig. 7.   Combined effects of L-NAME and KCl preconstriction on concentration-dependent relaxation responses to bradykinin in collateral-dependent (LCX) conduit arteries distal to occlusion and nonoccluded (LAD) conduit arteries isolated from sedentary (A) and exercise-trained (B) animals. Relaxation responses to bradykinin were completely abolished. Values are means ± SE of number of animals in parentheses.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We believe that this study is the first to examine the effects of exercise training on coronary endothelial function after chronic coronary occlusion. Our findings confirm the results of previous studies indicating that chronic coronary occlusion minimally affects endothelium-mediated relaxation in larger conduit arteries (29, 30); bradykinin and ADP relaxation responses were unaffected by occlusion in porcine coronary conduit arteries isolated 6 mo after ameroid placement. However, our results indicate that chronic exercise training enhances endothelium-mediated responses in conduit arteries isolated from coronary-occluded hearts. Importantly, we determined that exercise training effects were partially due to an enhanced production of NO; however, enhanced production of EDHF and/or increased sensitivity of coronary smooth muscle to EDHF also appears to play a contributory role. In contrast to our findings with bradykinin and ADP, endothelium-independent relaxation responses to sodium nitroprusside were not significantly affected by chronic coronary occlusion or exercise training in this porcine model, indicating that the adaptations are independent of potential training-induced enhanced responsiveness of coronary smooth muscle to NO.

Chronic Coronary Artery Occlusion

Gradual occlusion of a coronary artery has been shown to stimulate development of the collateral circulation in many species (3, 25, 35). Sellke et al. (29, 30) demonstrated that gradual ameroid occlusion of the LCX artery of canine and porcine hearts results in an impairment of endothelium-mediated relaxation in coronary resistance arteries isolated from the collateral-dependent region; however, chronic occlusion did not alter relaxation responses in the larger conduit coronary arteries. Sellke et al. (29) also reported that endothelium-independent relaxation to nitroprusside was enhanced in coronary microvessels distal to chronic occlusion. In our present studies we found no significant difference in responsiveness to bradykinin between arteries isolated from the nonoccluded LAD region and the collateral-dependent LCX arteries, although there was a tendency for impaired bradykinin-mediated relaxation of conduit arteries isolated from the collateral-dependent LCX region. However, in contrast to the findings of Sellke et al., we found no difference in the responses of conduit arteries to the endothelium-independent vasodilator sodium nitroprusside (Fig. 3). These findings are similar to our previous studies in which we used the canine model of chronic coronary occlusion, in which sodium nitroprusside relaxation responses were unaffected by chronic occlusion and no differences were observed in nitroprusside-induced relaxation between LAD and LCX arteries (23). The reasons for the discrepancy in effects of chronic coronary occlusion on nitroprusside responsiveness in our findings and those of Sellke et al. (29) are unclear and may involve differences in vessel sizes studied, time after ameroid placement and levels of collateral development and perfusion, altered guanyl cyclase/cGMP levels, and/or potential differences in basal (rather than agonist-stimulated) release of NO. However, neither study directly measured levels of cGMP or NO. Interestingly, in contrast to lack of effect of chronic occlusion on smooth muscle NO (and associated cGMP) responsiveness, adenosine (cAMP)-mediated smooth muscle relaxation responses appear impaired in canine coronary arteries distal to chronic occlusion (23).

Exercise Training and Chronic Coronary Occlusion

Exercise training has been shown to stimulate increased blood flow through the myocardium via native and developing coronary collaterals (3, 25). Exercise training has also been shown to result in lower myocardial oxygen consumption and blood flow requirements at rest and during submaximal exercise, to slow disease progression, and to improve the cardiovascular risk profile (8, 13). Therefore, exercise training appears to play a major role in the improvement of risk for cardiovascular disease. However, to our knowledge, potential vascular functional effects of exercise training in models of chronic coronary occlusion have not been determined. Thus our findings may provide a new aspect to the beneficial cardiovascular actions of exercise by demonstrating that exercise training improves endothelium-mediated relaxation responses in a porcine model of chronic coronary occlusion.

We found enhanced bradykinin- and ADP-stimulated relaxation of nonoccluded and collateral-dependent coronary arteries from exercise-trained pigs exposed to chronic coronary occlusion. These findings in the nonoccluded LAD artery are very interesting, because in previous studies we found that endothelium-mediated vasodilation was not altered by exercise training in conduit arteries isolated from normal nonoccluded exercise-trained pigs (19), suggesting that changes in vascular responsiveness were dependent on the presence of an occlusion. It is not clear to us why there are differences in effects of exercise training in arteries isolated from coronary-occluded hearts and those from the normal nonoccluded hearts. However, these different effects of exercise training may involve adaptive responses of the nonoccluded artery to chronic occlusion of another major artery, potentially interacting with the effects of exercise. Chronic occlusion of a major coronary artery produces parallel and sustained increases in coronary flow (and, e.g., associated exposure to chronic alterations in vessel wall shear stress) in the nonoccluded "donor" arteries during and after collateral development. Furthermore, chronic occlusion drastically reduces coronary flow and shear forces in the collateral-dependent region. Thus, in this respect, arteries isolated from occluded hearts may not be functionally equivalent to "normal" arteries isolated from nonoccluded hearts. It seems reasonable to propose that these hemodynamic effects of chronic coronary occlusion can interact with the effects of exercise training. Nevertheless, our findings provide the first evidence for beneficial effects of exercise training on endothelial function in arteries isolated from hearts exposed to chronic coronary artery occlusion.

Mediators of Coronary Relaxation in Chronic Coronary-Occluded Hearts: Effects of Training

Vascular endothelial cells play a major role in mediating the transduction of signals from the bloodstream to the underlying vascular smooth muscle cells. Under normal conditions, the major relaxing factors released from the endothelium include prostacyclin (PGI2, an arachidonic acid metabolite), NO (a major regulator of smooth muscle tone and blood flow) (7, 9), and EDHF (5, 17). EDNO is believed to contribute significantly to exercise-induced hyperemia, and inhibition of NOS activity reduces exercise capacity (15). EDHF is a diffusable substance believed to be a cytochrome P-450 metabolite capable of evoking hyperpolarization and, hence, relaxation of the vascular smooth muscle (5, 17). A number of studies have demonstrated preferential enhancement of NO production after exercise training (2, 18, 21). Furthermore, an upregulation of ecNOS mRNA and gene expression after exercise training has been shown in aortic tissue and in large conduit coronary arteries and coronary microvessels (31, 36). Thus it is possible that the improved responses seen after exercise in chronic coronary-occluded hearts may result from an upregulation in the synthesis and/or production of NO (increased NOS activity) and/or increases in levels of the ecNOS protein.

Recently, Traverse et al. (33) reported that inhibition of NOS with N-nitro-L-arginine significantly decreased blood flow to the normal and collateral-dependent myocardium during a single bout of exercise. These results suggest that NO plays an important role in maintaining coronary collateral blood flow during exercise in canine hearts exposed to chronic coronary occlusion. Interestingly, findings in the normal nonoccluded hearts demonstrated no change in blood flow during NOS inhibition in the normal or the collateral-dependent region. These results are similar to our findings in the normal nonoccluded porcine heart of no changes in coronary epicardial endothelium-dependent relaxation and, consequently, a minimal role for NO in adaptations to exercise training in epicardial arteries (19). In combination, these studies suggest that the presence of a chronic coronary occlusion elicits changes in coronary vascular responsiveness, particularly endothelium-dependent relaxation. These changes may result from alterations in the relative contribution of endogenous NO production to vasodilators. In our model the NOS inhibitor L-NAME decreased relaxation responses to bradykinin and ADP (Figs. 4 and 6), shifting the concentration-response relationships to these agonists downward and to the right. These results support the role of NO as a mediator of relaxation in response to bradykinin and ADP as well as the possibility that the training-induced enhanced vasorelaxation of LAD and LCX arteries after chronic coronary occlusion may be partially due to an increased production of NO. However, training-induced enhancement of relaxation to bradykinin and ADP appears to persist in the presence of NOS inhibition with L-NAME (exercise-trained vs. sedentary groups; Figs. 4 and 6), indicating that effects of training on these coronary arteries likely involve another endothelium-derived mediator, potentially EDHF. Inhibition of EDHF with K+ precontractions abolished the training-induced enhancement of relaxation to bradykinin; however, a relaxation response was still present. These results support our belief that more than one mediator was responsible for endothelium-dependent vasorelaxation in our model. Indeed, we observed complete abolishment of LAD and LCX artery relaxation to bradykinin with combined inhibition of NOS and EDHF (K+ depolarization), suggesting a contributory role for EDHF in mediating bradykinin-induced relaxation in our model. However, we recognize that conclusions drawn relative to EDHF with high-K+ (depolarizing) precontractions are subject to complications by known inhibitory activity on NO/cGMP-mediated relaxations (14, 37). The precise role of EDHF in training-induced effects awaits further study with more selective K+ channel interventions and/or measures of membrane potential.

The role of prostaglandin release during periods of increased metabolic demand is controversial. For example, previous findings in our laboratory and others have demonstrated a minimal role for PGI2 during exercise in the normal nonoccluded heart (6, 21). However, in contrast to studies in the normal heart, Altman et al. (1) demonstrated that prostaglandins contribute to increased blood flow through coronary collaterals in dogs exposed to chronic coronary occlusion. In support of this concept, our laboratory has also reported evidence for enhanced release of vasodilator prostanoids distal to chronic coronary occlusion in the canine model (22). However, our present study supports a major role for EDNO and EDHF, since bradykinin-mediated relaxation of porcine coronary arteries was completely abolished in the presence of L-NAME and KCl preconstriction. This result supports the previous findings of Oltman et al. (19) that bradykinin-mediated relaxation is unaffected by indomethacin in porcine epicardial coronary arteries isolated from sedentary or exercise-trained Yucatan miniswine. Thus we believe that a role for prostaglandins in training-induced effects in our porcine model of chronic coronary occlusion, if present, would be minimal compared with that of NO and EDHF.

During bouts of exercise, shear stress likely increases as a result of increased blood flow requirements and results in the production of endothelium-derived vasodilator agents that stimulate relaxation of the underlying vascular smooth muscle cells (7, 20). Shear-induced vasodilation is an endothelium-dependent mechanism that is primarily mediated by endothelial release of NO (11) but may also involve PGI2 (10) and EDHF (5). Although activation of this pathway is proposed for the response to exercise in the normal heart, involvement of these mechanisms in the chronic adaptive response to training in occluded hearts remains speculative. We suggest that these underlying functional responses are present in arteries of chronically occluded hearts and may potentially act in concert with structural adaptations and increased collateral development to optimize myocardial blood flow to the collateral-dependent region during exercise.

In conclusion, our results indicate intrinsic alterations in the vascular responsiveness of conduit arteries isolated from chronic coronary-occluded hearts after exercise training. Exercise training produces an enhanced relaxation to bradykinin and ADP in coronary vasculature distal to chronic coronary occlusion as well as nonoccluded coronary vasculature. Furthermore, this enhancement appears to involve training-induced increased production of NO as well as increased production or responsiveness to EDHF.


    ACKNOWLEDGEMENTS

The authors greatly appreciate the expert technical and surgical contributions of Mildred Mattox and the technical expertise of Qiao Zhong and Arej Sawani.


    FOOTNOTES

These studies were supported by National Heart, Lung, and Blood Institute Program Project PO1 HL-52490. Kawanza Griffin was supported by a predoctoral supplemental fellowship from the National Heart, Lung, and Blood Institute.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. L. Parker, Dept. of Medical Physiology, 336 Reynolds Medical Bldg., Texas A & M University, College Station, TX 77843-1114 (E-mail: parkerj{at}pop.tamu.edu).

Received 17 February 1999; accepted in final form 16 July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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J APPL PHYSIOL 87(5):1948-1956
8570-7587/99 $5.00 Copyright © 1999 the American Physiological Society


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