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1College of Health, University of Utah, Salt Lake City, Utah 84112; 2School of Human Sciences, Waseda University, Tokyo, Japan 169-8050; and3Department of Nutrition, University of California, Davis, California 95616
Submitted 18 December 2002 ; accepted in final form 6 May 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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105 µm ID) function was evaluated by using isometric techniques. LV developed pressure, the first derivative of LV pressure at a developed pressure of 40 mmHg, and systolic blood pressure were not different between Etr (n = 14) and Sed (n = 7) rats before or after the Sham protocol. Furthermore, hemodynamic variables were similar in Etr (n = 14) and Sed (n = 13) animals before the Isc protocol and were depressed to the same degree by the three O-R cycles. Therefore, Etr did not alter myocardial contractile function in rats that were (i.e., Isc) or were not (i.e., Sham) exposed to ischemia and reperfusion. Acetylcholine-evoked relaxation (10-8 to 3 x 10-5 M) was greater (P < 0.05) in coronary arteries from Sham-Etr vs. Sham-Sed animals (5 of 8 doses tested) and Isc-Etr vs. Isc-Sed rats (3 of 8 doses tested). Maximal relaxation produced by sodium nitroprusside (10-4 M) was similar among groups. Vasocontractile responses produced by KCl (10-100 mM) and endothelin-1 (10-11-10-4 M) were greater (P < 0.05) in the presence vs. the absence of nitric oxide synthase inhibition (10-6 M NG-monomethyl-L-arginine) in vessels from Sham-Etr but not Sham-Sed rats and from Isc-Etr but not Isc-Sed rats. These findings suggest that Etr-evoked improvements in coronary function are maintained in small arteries even when exposed to ischemia and reperfusion. endothelium; vascular smooth muscle; exercise; myocardial function; endothelin-1; sodium nitroprusside; acetylcholine; NG-monomethyl-L-arginine; rats
In a previous study, our laboratory assessed whether treadmill running preserved myocardial contractility and the function of small arteries in response to ischemia and reperfusion in rats (32). Our findings indicated that ischemia-induced reductions in LV contractile performance and coronary endothelial function were similar in animals regardless of their training status. The lack of a beneficial effect of exercise in that study may be related to the intensity of the training stimulus that was employed. In this regard, Bowles et al. (1) treadmill trained rats by using three protocols of differing intensity. At the conclusion of training, coronary flow responses during reperfusion after global ischemia were evaluated in these animals by using the isolated working heart preparation. It was reported that coronary flow during early reperfusion was greatest in animals that completed high-intensity treadmill running compared with a lower intensity regimen similar to the one implemented in our laboratory's previous study (32). It is possible that greater flow after ischemia and reperfusion in animals trained by high-intensity vs. relatively lower intensity treadmill running could have resulted from a beneficial effect on the coronary vasculature.
The primary purpose of the present study was to test the hypothesis that coronary arterial function and myocardial contractile function are greater after ischemia and reperfusion in high-intensity treadmill-trained vs. sedentary rats. Our findings indicate that high-intensity treadmill training improves coronary endothelial function in sham-operated animals and that this exercise-induced benefit is maintained even when arteries are exposed to ischemia and reperfusion. High-intensity treadmill running did not alter myocardial contractile function in either sham-operated animals or those subjected to ischemia and reperfusion.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Myocardial blood flow during exercise. To verify that myocardial blood flow increases when rats progress from running at relatively low to relatively high treadmill speeds, animals (n = 7, 297 ± 12 g) were anesthetized (2-5% isoflurane) and instrumented surgically to measure regional blood flow as previously described (31, 34). After recovery from surgery, rats ran on a motorized treadmill (5% grade) at 15 and 53 m/min. Intensities were performed in random order and separated by 60 min. Approximately 500,000 microspheres (Sn, Sr) were injected during the last minute of each run. Animals then were euthanized (pentobarbital sodium, >50 mg/kg ia), the thorax was opened, the heart was excised, and the right ventricles and LVs were trimmed and placed in counting vials. Finally, several bilateral tissues (e.g., mixed gastrocnemius muscle, kidney) were removed, weighed, and placed in counting vials. These tissues were used to verify adequate microsphere mixing during each blood flow determination.
Experimental animals and exercise training regimen. Rats (n = 48) were familiarized for 1-2 wk with human handling and running on the treadmill (model 42-15, Quinton) for 5-10 min at 13-16 m/min and 5% grade. Rats then were separated into a sedentary (Sed; n = 20) and high-intensity interval trained group (Etr; n = 28). Training sessions consisted of 10 x 4-min bouts of treadmill running at 5% grade, 5 days/wk, for 12 wk (1). The 10 x 4-min bouts were performed consecutively with no rest periods. Each 4-min bout consisted of 2 min at 16 m/min + 2 min at 20-45 m/min. The high-intensity segment of each bout (i.e., 20-45 m/min) was increased from week 0 to week 5. From week 5 to week 12, the high-intensity segment was increased from 45 to 60 m/min. Sed animals were handled daily and ran on the treadmill 1 day/wk for 5-15 min. At the end of week 12, all rats completed a graded intensity treadmill test (5, 17). The performance test was terminated when the animal was unable to maintain the treadmill pace and was unable to right itself after being placed in a dorsal recumbant position when removed from the treadmill. Performance was quantified as total work expressed as kilogram times meters (kg · m), where kg · m = body weight (kg) · treadmill speed (m/min) · exercise duration (min) · treadmill elevation (%) (10).
Surgical procedures. At least 24 h after the performance test, rats were anesthetized with ketamine (30-50 mg/kg im) and xylazine (3-5 mg/kg im). Supplemental doses of this mixture were given as required. A small incision was made in the neck, a tracheotomy was performed, and respiration was maintained artificially (model 661, Harvard Apparatus) by using room air supplemented with 100% oxygen. Next, catheters were inserted into the carotid artery for infusing drugs and fluids and into the caudal artery to measure arterial pressure and obtain blood samples for gas analyses (model ABL-3, Radiometer, Westlake, OH). After the heart was exposed through a lateral thoracotomy and the pericardium was opened, a pressure transducer-tipped catheter (model 2F, Millar Instruments, Houston, TX) was inserted through the apex into the LV to measure LV pressures and the first derivative of LV pressure (LV dP/dt). A 6-0 suture was then passed loosely under the proximal portion of the left coronary artery, and both ends were threaded through a vinyl tube. This tube served as a snare to occlude the coronary artery and evoke reversible myocardial ischemia. Throughout the surgical procedures and experimental protocols, rectal temperature was maintained at 37°C by using a heating pad and lamp, and blood pH was maintained between 7.35 and 7.45 by adjusting the respiration rate, tidal volume, and inspired PO2 concentration and/or by administering sodium bicarbonate.
In situ assessment of myocardial contractile function. When surgical instrumentation was complete and arterial blood gases, pH, and hemodynamic variables were stable, Sed and Etr animals completed a 65-min protocol that did (Isc) or did not (Sham) include ischemia and reperfusion. In 14 trained (Isc-Etr) and 13 sedentary (Isc-Sed) rats, the snare occluder was tightened to evoke three 15-min periods of ischemia. Five minutes of reperfusion followed the first two bouts of ischemia, whereas 10 min of reperfusion followed the third. A previous study verified that this protocol significantly depresses both myocardial and microvascular function compared with sham-operated animals (32). Immediately after coronary artery occlusion, distortion of the electrocardiogram (e.g., T-wave and/or ST-segment alterations) and a cyanotic appearance of the region distal to the occlusion was observed in every animal. In 14 trained (Sham-Etr) and 7 sedentary (Sham-Sed) animals, the snare occluder was placed around the left coronary artery but never tightened. Hemodynamic variables were measured in all rats at 5-min intervals and were processed by a computer through an analog-to-digital interface card (Biopac Systems, Santa Barbara, CA) that allowed for subsequent off-line quantitative analyses (32, 33). Continuously measured variables included systolic and diastolic arterial pressure, LV systolic and enddiastolic pressure, positive and negative LV dP/dt, and the electrocardiogram. Calculated variables included mean arterial pressure, LV developed pressure (LVDP; LV systolic minus LV end-diastolic pressure), and LV dP/dt at a developed pressure of 40 mmHg (LV dP/dt@DP40). LV dP/dt@DP40 was used to estimate global myocardial function because it is more independent of changes in preload and afterload than maximal dP/dt (19). Arterial blood gases (PO2 and PCO2) and pH were measured at baseline, 30 min, and 65 min. The volume required for each analysis (0.25 ml) was replaced by using 0.6% dextran.
In vitro assessment of coronary vascular function. After the 65-min in situ protocol, blood samples were obtained, and the heart, both adrenal glands, and two sections of liver were excised (see Plasma and liver analyses below). Hearts were placed in oxygenated, ice-cold normal physiological saline solution (NPSS; pH 7.40). The adrenal glands were dissected free of adherent tissue, blotted, weighed, and used to estimate training and/or handling-induced stress. Sections of liver were placed in liquid nitrogen and stored for later quantification of indicators of oxidant stress (see Plasma and liver analyses). Regarding the heart, a dissecting microscope (Leica Stereo Zoom 5) was used to confirm placement of the snare occluder around the left coronary artery. The vessel was traced toward the apex of the heart, and second- and third-order branches of this artery were isolated, removed, and prepared for mounting on a myograph (Jules Osher, Pomona, CA) (20, 28, 30, 32, 33). This apparatus allows direct determination of vessel wall force in the absence of influences from neural, humoral, metabolic, and mechanical sources. Two tungsten wires (OD = 20 µm) were inserted in a parallel manner through the lumen of the vessel. One wire was attached to a force transducer (Fort10 Transducer, World Precision Instruments, Sarasota, FL) to measure tension development, and the other was fixed to a micrometer that was used to stretch the vessel in small increments. Tension data were recorded continuously (Biopac Systems, Santa Barbara, CA). Vessels were immersed in a temperature-controlled, 8.5-ml reservoir (i.e., a tissue "bath") containing oxygenated (95% O2-5% CO2) NPSS (pH 7.40). Samples from all buffers and each tissue bath were analyzed frequently for PO2, PCO2, and pH. After the small coronary arteries were mounted, the tissue bath was warmed gradually to 37°C and the vessels equilibrated at zero tension for 30 min. Ten milligrams of tension then were applied to the artery, and the distance between the wires was measured to calculate its internal diameter using the following formula: Lc = (2 + 
) · Wt + 2G, where Lc is internal circumference, Wt is wire thickness, G is the distance between wires, and vessel internal diameter is Lc/ (20). This formula assumes that the walls of the vessel are flat between the wires after application of a slight stretch. Next, a series of internal circumference-active tension curves were constructed to determine the vessel diameter that evoked the greatest tension development (Lmax) to 100 mM KCl as our laboratory has described (30, 32, 33). Lmax was determined for every vessel, and this optimal resting tension was maintained throughout the study. An equilibration period of 30 min preceded assessment of vascular reactivity.
ACh-evoked vasorelaxation. When tension development to precontraction using 45 mM KCl was stable, a concentration-relaxation curve to cumulative additions of the muscarinic-receptor agonist ACh (10-8 to 3 x 10-5 M) was performed. ACh stimulates nitric oxide (NO) production and provides an estimate of endothelium-dependent relaxation. Relaxation responses are presented as a percentage of KCl-induced precontraction. After the response to the final dose of ACh was recorded, NPSS was reintroduced into the tissue bath, and a 30-min equilibration period was initiated during which the bathing medium was rinsed with NPSS at 10- to 15-min intervals.
KCl-evoked vasocontraction in the absence and presence of NO synthase inhibition. Non-receptor-mediated vasocontraction in response to KCl (15-100 mM) is presented as milligrams of developed tension. When responses to the final dose were recorded, the vessel bathing medium was rinsed twice with NPSS and again at 10- to 15-min intervals during a 30-min washout period. Next, identical doses of KCl were administered 10 min after addition of either the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 10-6 M) or its inactive enantiomer NG-monomethyl-D-arginine (D-NMMA) to the vessel bathing medium. Responses in the presence of L-NMMA were used to determine the extent to which basal NO synthase opposed KCl-induced vasocontraction. Responses in the presence of D-NMMA were used to assess repeatability of KCl-induced vasocontraction.
Endothelin-1-evoked vasocontraction in the absence and presence of NO synthase inhibition. Receptor-mediated vasocontraction in response to endothelin-1 (ET-1; 10-11-10-4 M) is presented as milligrams of developed tension. Previous findings from our laboratory showed that ET-1 has a high binding affinity and cannot be washed from its receptors even with repeated NPSS rinses (32, 33) Therefore, one dose-response curve was performed in the absence (D-NMMA) or presence (L-NMMA) of NO synthase inhibition in different vessels. Although this procedure precluded the assessment of repeatability in response to ET-1, the objective was to determine among groups the extent to which basal NO synthase opposed ET-1-evoked vasocontraction.
Sodium nitroprusside-evoked vasorelaxation. In vessels that were not exposed to ET-1 or L-NMMA, concentration-relaxation curves to sodium nitroprusside (SNP; 10-9-10-4 M) were performed when precontraction to 45 mM KCl was stable. This procedure evokes smooth muscle relaxation via mechanisms that are independent of a functional endothelium. Relaxation to the maximal dose of SNP is presented as a percentage of KCl-induced precontraction.
Drugs and solutions. NPSS contained (in mM) 125 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2.5 CaCl2, 18 NaHCO3, 0.026 Na2EDTA, and 11.2 glucose. All solutions were maintained at 37°C and aerated with 95% O2-5% CO2 at a rate sufficient to maintain pH at 7.40. NPSS and KCl solutions were prepared daily from concentrated stock. ACh, SNP, L-NMMA, D-NMMA (Sigma Chemical, St. Louis, MO), and ET-1 (Peninsula Laboratories, San Carlos, CA) were purchased commercially and prepared daily from stock solutions using distilled deionized water. All doses are expressed as the final concentration of each drug in the vessel bath.
Plasma and liver analyses. Blood collected into a prechilled tube containing EDTA was centrifuged at 2,500 g for 10 min at 4°C. One aliquot of plasma was placed in a tube containing 4% butylated hydroxytoluene (BHT) and used to estimate lipid oxidation by determining thiobarbituric acid-reactive substances (fluorescence detection of malondialdehyde equivalents) (8, 22, 30, 36), and additional plasma was used to estimate protein oxidation by quantifying protein carbonyls (spectrophotometric quantification of the dinitrophenylhydrazine adduct) (16).
To estimate lipid oxidation in liver, samples were homogenized, supernatants were combined with BHT and measured as described above (8, 22, 30, 36). For blood and tissue, lipid oxidation is expressed as micromoles per liter of malondialdehyde and as nanomoles of malondialdehyde per milligram of protein, respectively. Protein oxidation in liver was estimated by using methods described above for plasma (16). For blood and plasma, results are expressed as nanomoles of protein carbonyls per milligram of protein.
Liver tissue used to quantify total (Cu/Zn + Mn) superoxide dismutase (SOD) and Mn SOD activities was homogenized, sonicated 3 x 5 s on ice and centrifuged at 10,000 g for 30 min at 4°C, and a kinetic spectrophotometric enzyme assay was performed. Results are expressed as units of SOD per milligram of protein, where one unit of SOD activity is defined as the amount of enzyme required to inhibit the autooxidation of pyrogallol by 50% (18, 29). For all assays, protein concentrations were determined using bovine serum albumin as the standard (2).
Statistical analyses. Animal and vessel characteristics, results from the exercise performance test, and plasma and liver indexes of oxidant or antioxidant status were compared between Isc-Etr and Isc-Sed animals, and between Sham-Etr and Sham-Sed rats by using an unpaired t-test.
Directly measured and/or calculated hemodynamic variables and vasorelaxation and vasocontraction responses were analyzed by using a two-way (time or drug dose vs. experimental group) repeated-measures ANOVA. If significance was observed, planned comparisons were made at each time point or drug dose to determine the location of differences between the Isc-Etr and Isc-Sed animals, and Sham-Etr and Sham-Sed rats. When the same protocol was performed on more than one vessel from a single animal, responses were averaged and counted as one observation.
In rats used to determine regional blood flow, results from the respective circulations were compared between the two treadmill speeds with a paired t-test. All values are presented as means ± SE. Statistical significance was accepted when P < 0.05 (9).
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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46% greater in rats running at 53 m/min (1,863 ± 271 ml · 100 g-1 · min-1) vs. 15 m/min (1,278 ± 218 ml · 100 g-1 · min-1). Blood flow was similar between the right and left kidneys at 53 m/min (449 ± 60 and 449 ± 54 ml · 100 g-1 · min-1) and 15 m/min (586 ± 64 and 586 ± 72 ml · 100 g-1 · min-1), respectively. Furthermore, blood flow was not different between the right and left mixed gastrocnemius muscles at 53 m/min (159 ± 21 and 155 ± 20 ml · 100 g-1 · min-1) and 15 m/min (95 ± 12 and 93 ± 13 ml · 100 g-1 · min-1), respectively. Data from these two bilateral tissues confirmed adequate mixing of the radioactive microspheres. Animal and vessel characteristics. All animal and vessel characteristics were similar between the Sham-Sed and Sham-Etr groups (Table 1) and between the Isc-Sed and Isc-Etr groups (Table 2). An anticipated exception was that Isc-Etr and Sham-Etr rats completed more total work on the exercise performance test compared with Isc-Sed and Sham-Sed animals (Tables 1 and 2).
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Arterial blood gases during in situ protocols. No differences existed between the Sham-Sed and Sham-Etr groups at baseline for arterial pH (7.45 ± 0.02 and 7.43 ± 0.01), PO2 (93 ± 9 and 102 ± 9 Torr), and PCO2 (39 ± 2 and 41 ± 2 Torr). Results were similar at baseline for Isc-Sed and Isc-Etr animals concerning arterial pH (7.41 ± 0.02 and 7.42 ± 0.01), PO2 (123 ± 17 and 127 ± 11 Torr), and PCO2 (38 ± 2 and 37 ± 2 Torr). For all four groups of animals, data did not differ from baseline when measured at 30 and 65 min (data not shown).
Hemodynamic responses during in situ protocols. No differences existed between Sham-Sed and Sham-Etr rats, respectively, at baseline regarding systolic blood pressure (79 ± 2 and 85 ± 3 mmHg), LVDP (78 ± 3 and 84 ± 4 mmHg), LV dP/dt@DP40 (4,207 ± 164 and 4,218 ± 141 mmHg/s) or heart rate (280 ± 16 and 271 ± 14 beats/min). Furthermore, these variables were not different within groups throughout the protocol or between groups at any time point.
All measured and/or calculated indexes of myocardial function were similar between Isc-Sed and Isc-Etr animals at baseline (Fig. 1). Ischemia-induced reductions (all P < 0.05 except heart rate) during the first occlusion in systolic blood pressure (62 ± 15 and 66 ± 8 mmHg), LVDP (51 ± 18 and 49 ± 11 mmHg), LV dP/dt@DP40 (2,680 ± 1,092 and 2,603 ± 991 mmHg/s), and heart rate (251 ± 20 and 230 ± 18 beats/min) were not different between Isc-Sed and Isc-Etr animals, respectively. Values at 15 min of occlusion 1 were similar both within and between groups at 15 min of occlusion 2 and occlusion 3 (data not shown). When measured during reperfusion, LVDP and LV dP/dt@DP40 were lower relative to baseline values at 40 min (reperfusion 2) and 65 min (reperfusion 3), whereas SBP was attenuated at 65 min (reperfusion 3; Fig. 1).
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Coronary vascular reactivity. ACh-evoked relaxation was greater in arteries from Sham-Etr vs. Sham-Sed rats (Fig. 2A), whereas no differences existed between groups concerning vasorelaxation produced by 10-4 M SNP (81 ± 8 vs. 86 ± 9%, respectively). Additionally, ACh-evoked relaxation was greater in arteries from Isc-Etr vs. Isc-Sed animals (Fig. 2B), whereas vasorelaxation caused by 10-4 M SNP was similar between groups (103 ± 7 vs. 91 ± 6%, respectively). Therefore, Etr-evoked increases in endothelial function were maintained in vessels exposed to ischemia and reper-fusion. An anticipated finding was that maximal ACh-evoked relaxation was blunted (P < 0.05) by ischemia and reperfusion in the Isc-Etr and Isc-Sed groups (Fig. 2B) compared with the Sham-Etr and Sham-Sed animals (Fig. 2A).
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Figure 3 indicates that KCl-induced vasocontractile responses were greater in the presence vs. the absence of NO synthase inhibition in arteries from Sham-Etr (Fig. 3B) but not Sham-Sed (Fig. 3A) rats. For both groups, dose-response curves in the absence of L-NMMA were repeatable when separated by 30 min (data not shown). Figure 4 shows greater KCl-induced vasocontractile responses in the presence vs the absence of L-NMMA in arteries from Isc-Etr (Fig. 4B) but not Isc-Sed (Fig. 4A) animals. For both groups, dose-response curves in the absence of L-NMMA were repeatable when separated by 30 min (data not shown).
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Figure 5 illustrates that ET-1-induced vasocontraction was potentiated by L-NMMA in arteries from Sham-Etr (Fig. 5B) but not Sham-Sed (Fig. 5A) rats. Similarly, Figure 6 indicates that ET-1-induced vasocontraction was potentiated by NO synthase inhibition in arteries from Isc-Etr (B) but not Isc-Sed (A) rats.
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KCl-evoked vasocontraction also was greater (P < 0.05 at 40, 60, 80, and 100 mM) in the absence of L-NMMA in the Isc-Sed and Isc-Etr groups (Fig. 4, A and B) compared with the respective Sham animals (Fig. 3, A and B). Similar findings were not observed for ET-1-evoked vasocontractile responses when the two Isc groups (Fig. 6) were compared with their respective Sham groups (Fig. 5).
Plasma and liver analyses. Plasma and liver samples from a subset of animals (n = 5-6 per group) were used to quantify indexes of systemic oxidant stress and antioxidant status. Plasma and liver protein carbonyls and thiobarbituric acid-reactive substances, and three isoforms of liver SOD were similar between the Sham-Etr and Sham-Sed rats and between Isc-Etr and Isc-Sed animals (Table 3).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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105 µm ID) from rats, but they are not consistent with our laboratory's earlier findings (32) or those reported by Parker et al. (23). Specifically, even though standardized treadmill-running programs produced both central and peripheral training adaptations in both of those studies (23, 32), coronary vascular function was not enhanced relative to Sed animals. Therefore, it appears that a training regimen consisting of repeated bouts of high-intensity treadmill running (e.g., present study) is more beneficial than steady-state, moderate-intensity treadmill-running (e.g., 28-32 m/min for 60-90 min) (23, 32) with regard to improving the function of small coronary arteries in rats. Concerning indexes of LV contractility, we observed no differences in Sham animals regardless of their training status. These findings are consistent with our laboratory's previous study (32) and two other investigations that examined the influence of Etr on myocardial function (1, 17). Effects of Etr on coronary vascular and myocardial contractile function in animals exposed to ischemia and reperfusion. Our main finding is that Etr-evoked improvements in coronary vascular function from sham-operated animals are maintained even in arteries from rats exposed to ischemia and reperfusion. Data supporting this statement are that ACh-evoked vasorelaxation was greater, whereas SNP-induced vasorelaxation was unchanged, in small coronary arteries from Isc-Etr vs Isc-Sed rats. These findings suggest that endothelium-dependent vasorelaxation is impaired less, whereas NO- and cGMP-dependent intracellular signaling pathways within vascular smooth muscle are unchanged after ischemia and reperfusion in Isc-Etr vs Isc-Sed animals. Our results may have been different had another endothelium-dependent agent and/or precontractor substance been used. For example, the potential contribution to vasorelaxation from endothelium-derived hyperpolarizing factor (EDHF) cannot be evaluated in vessels that have been precontracted using KCl. In this regard, EDHF has been shown to contribute to ACh-evoked relaxation in canine coronary microvessels (21) and rat mesenteric arteries (35). Because of this potential limitation, we refer to our assessment of endothelial function as "ACh-evoked vasorelaxation" and have shown that NO release evoked by muscarinic-receptor stimulation is largely responsible for the observed vasorelaxation of rat coronary arteries (32, 33).
Results regarding ET-1 and KCl-evoked tension development further support our statement that Etr improves coronary vascular function and that this benefit is sustained in vessels exposed to ischemia and reper-fusion. We hypothesized originally that vasocontraction in response to these agents would be exaggerated in the presence vs the absence of NO synthase inhibition to a greater extent in Etr compared with Sed animals. The data indicate that, although KCl evoked dose-dependent increases in tension development, responses were more pronounced in the presence of L-NMMA in vessels from Etr (both Sham and Isc) than Sed (both Sham and Isc) rats. A similar pattern of results was obtained by using ET-1. These findings suggest strongly that enhanced opposition from endothelium-derived factors (e.g., NO) in response to a receptor-mediated (e.g., ET-1) and non-receptor-mediated agent (e.g., KCl) existed in Sham-Etr but not Sham-Sed rats. Furthermore, this training-induced adaptation is maintained in the presence of an ischemic challenge i.e., in Isc-Etr but not Isc-Sed animals. Therefore, both stimulated (i.e., ACh evoked) and basal (i.e., L-NMMA inhibited) NO production is improved by Etr, and this beneficial alteration persists even in vessels exposed to ischemia and reperfusion.
"Control" vasocontractile responses (i.e., in the absence of L-NMMA) also were compared among groups. In this regard, KCl-evoked tension development was greater in vessels exposed to ischemia and reperfusion (i.e., both Isc-Sed and Isc-Etr; Fig. 4) compared with the respective Sham animals (i.e., Sham-Sed and Sham-Etr; Fig. 3). Whereas NO synthase inhibition had no effect on KCl-induced vasocontraction in Sham-Sed rats (Fig. 3A), L-NMMA was capable of unmasking the contribution from NO in Sham-Etr animals (Fig. 3B). These findings confirm and extend earlier studies that Etr increases NO-related opposition to KCl-evoked contraction. Most important, however, is our new finding that Etr-related improvements are maintained in vessels exposed to ischemia and reperfusion (Fig. 4B). In contrast to our findings with KCl, ET-1-evoked vasocontractile responses in the absence of L-NMMA were similar in vessels that were (Fig. 6) or were not (Fig. 5) exposed to ischemia and reperfusion. Similar to KCl, however, NO synthase inhibition did unmask an Etr-induced increase in the contribution from NO in Sham animals (Fig. 5B) that was maintained in vessels exposed to ischemia and reperfusion (Fig. 6B). Therefore, our data demonstrate the ability of Etr to reduce the contractile potential of receptor (e.g., ET-1) and non-receptor-mediated (KCl) agents in sham-operated animals and, most importantly, extends these findings to include vessels exposed to ischemia and reperfusion.
It appears that training intensity may contribute importantly to the potential for vascular improvements to extend into the pathophysiological setting of myocardial ischemia. For example, our laboratory (32) and others (1) treadmill trained rats at a moderate intensity and observed that myocardial contractile function (1, 32), endothelial function of small coronary arteries (32), and coronary flow (1) were similar after ischemia and reperfusion compared with sedentary animals. In contrast, when rats were trained by using a high-intensity interval-type regimen, coronary flow during reperfusion after global ischemia was greater compared with animals that completed a lower intensity protocol (1). Because resistance to myocardial blood flow is regulated primarily by small coronary arteries (4), greater flow after ischemia and reperfusion in animals trained by high-intensity treadmill running may have resulted from a beneficial effect on the coronary vasculature. In the present study, coronary endothelial function was greater in vessels from Isc-Etr vs. Isc-Sed rats. These results, together with our laboratory's previous findings (32), suggest that stimulus intensity is important when considering whether training-induced vascular improvements are realized after exposure to ischemia and reperfusion.
One explanation for our results that Etr improves coronary vascular function involves the relationship among elevated blood flow, shear stress, and endothelial cell NO synthase (ecNOS). For example, isolated porcine coronary arterioles subjected to high flow and/or shear stress show increased ecNOS mRNA expression compared with those exposed to no flow and/or shear stress and low flow/shear stress (38, 39). Furthermore, exercise training elevates ecNOS mRNA in porcine coronary resistance vessels (37). We determined the extent that LV blood flow increases when rats progress from running at relatively low (i.e., 15 m/min) to relatively high (i.e., 53 m/min) treadmill speeds. Although it is intuitive that myocardial blood flow increases to meet exercise-induced elevations in myocardial oxygen demand, existing data suggest that such elevations are much less in rodents (7) compared with pigs (29). We observed 46% increase in LV blood flow in rats running at 53 vs. 15 m/min. These data suggest that Etr rats were exposed to repeated increases in LV blood flow, and presumably coronary vascular shear stress, during the 12-wk experimental protocol. With the use of past studies as rationale (37-39), repeated exposure to elevated blood flow and/or shear stress may have upregulated ecNOS and, at least in part, could explain the attenuation of ischemia-induced coronary endothelial dysfunction that we observed. Although this possibility is not unreasonable, it remains speculative because vascular ecNOS was not measured directly in our study.
A second explanation that is not mutually exclusive to Etr-induced increases in blood flow and/or shear stress involves improved antioxidant defense mechanisms. Specifically, shear stress reportedly elevates Cu/Zn SOD mRNA in 1) cultured human aortic endothelial cells (12), 2) isolated porcine coronary arterioles perfused in vitro (38), and 3) coronary arterioles from exercise-trained pigs (27). Of particular note is the finding by Woodman et al. that Cu/Zn SOD mRNA is elevated in response to high flow and/or shear stress but not low flow and/or shear stress (38). Because Cu/Zn SOD is the principal scavenger for superoxide anion in vascular cells and because superoxide anion increases during myocardial ischemia and degrades NO to peroxynitrite, it follows that ischemia-induced vascular dysfunction may be preserved by elevating antioxidant defense mechanisms. Although this mechanism remains speculative because coronary vascular Cu/Zn SOD was not measured directly in our study, our findings suggest that Etr did not confer an overall improvement in antioxidant status or oxidant load. In this regard, general estimates of 1) lipid (i.e., plasma and liver thiobarbituric acid-reactive substances) and 2) protein (i.e., plasma and liver protein carbonyls) oxidation, and 3) overall antioxidant status (i.e., isoforms of liver superoxide dismutase) were similar among groups.
Our secondary hypothesis, that Etr improves myocardial contractile function and thereby attenuates ischemia-induced contractile dysfunction, was not supported. Instead, we observed that indexes of LV contractile function were similar in Sham animals regardless of their training status and that ischemia-induced reductions in global myocardial function (i.e., LVDP and LV dP/dt@DP40 in Isc rats) were similar regardless of Etr. Therefore, Etr-induced improvements in coronary vascular function (observed in both Sham and Isc animals) did not result in improved contractile function. The possibility exists, however, that Etr-induced coronary vascular protection benefits myocardial contractile function only during periods of elevated myocardial oxygen demand that require increases in blood flow. For example, Bowles et al. (1) evaluated myocardial contractile function in isolated hearts after global ischemia in response to a low workload (i.e., preload, 10 cmH2O; afterload, 80 cmH2O; heart rate, 300 beats/min) and high workload (i.e., preload, 20 cmH2O; afterload, 130 cmH2O; heart rate, 420 beats/min). The relative increase in cardiac output, systolic pressure, cardiac work (i.e., cardiac output x peak aortic systolic pressure), and stroke volume from the low to high workload was greatest in hearts from high-intensity trained rats vs. sedentary controls (1), demonstrating enhanced myocardial reserve. Future studies in trained and untrained animals that evaluate coronary vascular function together with myocardial contractile function at different workloads are required to address this possibility.
Our study is the first to report that high-intensity treadmill running improves coronary vascular function, and that this improvement persists in vessels exposed to ischemia and reperfusion. These results, taken together with our laboratory's previous finding that a more traditional exercise protocol was not effi-cacious in this regard (32), suggest that training intensity is important concerning vascular function in the absence and presence of myocardial ischemia. Although extrapolation of findings from experimental animals to humans should be done cautiously, our data suggest that repeated bouts of treadmill-running performed at high intensities improve coronary vascular function. Importantly, these improvements are maintained in the presence of ischemia and reperfusion.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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This work was funded, in part, by an American Heart Association, Western States Affiliate, Grant-In-Aid 98-201; National Affiliate, Scientist Development Grant 0130099N; and by a New Investigator Award from the University of California, Davis, Clinical Nutrition Research Unit (NIDDK 35747, Dr. Charles H. Halsted, Principal Investigator) to J. D. Symons.
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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We thank T. I. Musch and K. Sue Hageman for assistance with blood flow measurements. We thank Drs. R. M. McAllister and C. L. Stebbins for critically evaluating the manuscript.
Preliminary results were presented at Experimental Biology 2002 in New Orleans, LA and the 2003 Annual General Meeting of the American College of Sports Medicine in San Francisco, CA.
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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. Section 1734 solely to indicate this fact.
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) and (30 min later) presence (
) of nitric oxide synthase inhibition using NG-monomethyl-L-arginine (L-NMMA, 10-6 M) are shown. Values are means ± SE. *P < 0.05, KCl vs. KCl+ L-NMMA.
