Contribution of KATP+ channels to coronary vasomotor tone regulation is enhanced in exercising swine with a recent myocardial infarction

Daphne Merkus, Birgit Houweling, Marion van Vliet, Dirk J. Duncker

Abstract

Previous studies demonstrated a decreased flow reserve in the hypertrophied myocardium early after myocardial infarction (MI). Previously, we reported that exacerbation of hemodynamic abnormalities and neurohumoral activation during exercise caused slight impairment of myocardial O2 supply in swine with a recent MI. We hypothesized that increased metabolic coronary vasodilation [via ATP-sensitive K+ (KATP+) channels and adenosine] may have partially compensated for the increased extravascular compressive forces and increased vasoconstrictor neurohormones, thereby preventing a more severe impairment of myocardial O2 balance. Chronically instrumented swine were exercised on a treadmill up to 85% of maximum heart rate. Under resting conditions, adenosine receptor blockade [8-phenyltheophylline (8-PT), 5 mg/kg iv] and KATP+ channel blockade (glibenclamide, 3 mg/kg iv) produced similar decreases in myocardial O2 supply in normal and MI swine. However, while glibenclamide's effect waned in normal swine during exercise (P < 0.05), it was maintained in MI swine. 8-PT's effect was maintained during exercise and was not different between normal and MI swine. Finally, in normal swine combined treatment with 8-PT and glibenclamide produced a vasoconstrictor response that equaled the sum of the responses to blockade of the individual pathways. In contrast, in MI swine the vasoconstrictor response to 8-PT and glibenclamide was similar to that produced by glibenclamide alone. In conclusion, despite significant hemodynamic abnormalities in swine with a recent MI, myocardial O2 supply and O2 consumption in remodeled myocardium are still closely matched during exercise. This close matching is supported by increased KATP+ channel-mediated coronary vasodilation. Although the net vasodilator influence of adenosine was unchanged in remodeled myocardium, it became exclusively dependent on KATP+ channel opening.

  • heart failure
  • hypertrophy
  • myocardial oxygen balance
  • adenosine

heart failure is currently the only major cardiovascular disorder of which the incidence is increasing (5). Myocardial infarction (MI) is becoming an increasingly important risk factor for heart failure because of the reduction in acute infarction-associated mortality (5). The role of myocardial blood flow abnormalities in the progression from left ventricular (LV) remodeling after MI to overt heart failure is incompletely understood, but in vivo studies in rats (23, 24) and pigs (32) indicate a reduction in flow reserve of up to 35% in the remodeled surviving LV myocardium, 3–8 wk after infarction. Recently, our laboratory (17) reported the presence of exacerbated hemodynamic (increased LV filling pressure and heart rate) and neurohumoral (increased catecholamines and endothelin) responses to exercise in swine with MI-induced LV remodeling compared with normal swine. Surprisingly, however, myocardial O2 supply was only slightly impaired in MI vs. normal swine, even during severe exercise levels. We hypothesized that increased metabolic coronary vasodilation [e.g., adenosine and ATP-sensitive K+ (KATP+) channel activity] may have acted to compensate for the increased intramyocardial extravascular compressive forces acting on the coronary bed as well as increased vasoconstrictor neurohormones, thereby preventing a more marked impairment of myocardial O2 balance. Consequently, in the present study, we tested the hypothesis that an increased vasodilator influence of KATP+ channels and adenosine helps to maintain coronary blood flow (CBF) and O2 supply in swine with remodeled LV myocardium early after MI.

METHODS

Animals

Studies were performed in accordance with the “Guiding Principles in the Care and Use of Laboratory Animals” as approved by the Council of the American Physiological Society and with approval of the Animal Care Committee of the Erasmus University Medical Center-Rotterdam. Thirty-eight 2- to 3-mo-old Yorkshire × Landrace pigs (23 ± 1 kg at the time of surgery) of either sex (19 male, 19 female) entered the study; 21 were assigned to the MI group and 17 to the normal group. Daily adaptation of animals to laboratory conditions started 1 wk before surgery.

Surgery

Pigs were sedated (ketamine, 20 mg/kg im), anesthetized (thiopental, 10 mg/kg iv), intubated, and ventilated with O2 and N2O to which 0.2–1.0% (vol/vol) isoflurane was added (10, 17, 27). Anesthesia was maintained with midazolam (2 mg/kg followed by 1 mg·kg−1·h−1 iv) and fentanyl (10 μg·kg−1·h−1 iv). Under sterile conditions, the chest was opened via the fourth left intercostal space, and a fluid-filled polyvinyl chloride catheter was inserted into the aortic arch for mean aortic pressure (MAP) measurement (Combitrans pressure transducers; Braun) and blood sampling for determination of blood gases (Acid-Base Laboratory model 505; Radiometer), O2 saturation and hemoglobin concentration (OSM2; Radiometer), and computation of O2 content, O2 supply, and O2 consumption (V̇o2) (10, 17, 18, 27). A microtipped pressure transducer (P4.5; Konigsberg Instruments) was inserted into the LV via the apex as well as a polyvinyl chloride catheter to calibrate the Konigsberg transducer LV pressure signal. Polyvinyl chloride catheters were also implanted into the left atrium to measure pressure and into the pulmonary artery to administer drugs. An angiocatheter was inserted into the anterior interventricular vein for blood sampling (911), and a transit-time flow probe (2.5–3.0 mm; Transonics Systems) was placed around the left anterior descending coronary artery (27, 28). The proximal left circumflex coronary artery (LCx) was permanently occluded in 21 MI swine (17, 18), which were monitored for 1 h and, if needed, internally defibrillated (10–30 J); three MI swine died because of recurrent fibrillation. Catheters were tunneled to the back, and animals were allowed to recover, receiving analgesia (0.3 mg buprenorphine im) for 2 days and antibiotic prophylaxis (25 mg/kg amoxicillin and 5 mg/kg gentamicin iv) for 5 days (10, 17, 18, 27). Three additional MI swine died during the first night after surgery.

Experimental Protocols

Studies were performed in 17 normal (27 ± 1 kg) and 15 MI (28 ± 1 kg) swine, at 15 ± 2 and 16 ± 2 days after surgery, respectively, with animals exercising on a motor-driven treadmill (1–4 km/h for MI swine and 1–5 km/h for normal swine). Three protocols were performed in random order, each on a different day. We have previously shown excellent reproducibility (<5% difference) of hemodynamic responses to consecutive exercise trials in both normal (911) and MI (18) swine.

KATP+ channels.

With swine (10 normal and 9 MI animals) lying quietly on the treadmill, resting hemodynamic measurements, consisting of LV pressure, rate of rise of LV pressure (LV dP/dt), MAP, left atrial pressure (LAP), and CBF were obtained and blood samples were collected. Hemodynamic measurements were repeated and rectal temperature was measured with animals standing on the treadmill, and the exercise trial was started. Hemodynamic variables were continuously recorded, and blood samples were collected during the last 30 s of each 3-min exercise stage. Blood samples were maintained in iced syringes until the conclusion of each exercise trial. Measurements of Po2 (Torr), Pco2 (Torr), and pH were then immediately performed with a blood gas analyzer (Acid-Base Laboratory model 505) while O2 saturation (%) and hemoglobin (g/100 ml) were measured with a hemoximeter (OSM2). After completing the control exercise trial, swine were allowed to rest on the treadmill for 90 min, after which animals received glibenclamide (3 mg/kg iv dissolved in 20 ml H2O + 2 drops 5 M NaOH; Refs. 9, 27), and the exercise trial was repeated.

Adenosine.

After baseline measurements had been obtained in resting swine (12 normal and 12 MI animals), the control exercise trial was started. After completing the trial, swine were allowed to rest on the treadmill for 90 min, after which animals received 8-phenyltheophylline (8-PT, 5 mg/kg iv dissolved in 18.7 ml saline + 1.3 ml propanediol + 2 drops 5 M NaOH; Refs. 11, 22, 27), and the exercise trial was repeated.

Adenosine and KATP+ channels.

A subgroup of swine (8 normal and 7 MI animals) that underwent the exercise trial in the presence of adenosine receptor blockade were used to study combined blockade of adenosine receptors and KATP+ channels. Ninety minutes after the second exercise trial, animals received 8-PT (2.5 mg/kg iv; Refs. 22, 27) and glibenclamide (3 mg/kg iv) and underwent a third exercise trial.

Data Analysis

Hemodynamic data were digitally recorded and analyzed offline (911). Blood O2 content (μmol/ml) was computed as (Hb·0.621·O2 saturation) + (0.00131Po2). Myocardial O2 consumption (MVo2) in the region of myocardium perfused by the left anterior descending coronary artery was calculated as the product of CBF and the difference in O2 content between arterial and coronary venous blood. Myocardial O2 extraction (MEo2) was computed as the ratio of the arteriovenous O2 content difference and the arterial O2 content. To correct for any alterations in MVo2, the parameters MEo2, coronary venous Po2 (PvO2), and coronary venous O2 saturation (SvO2) are all presented as a function of MVo2 (27).

Statistical analysis on hemodynamic data was performed with three-way (exercise level, drug treatment, and MI) ANOVA for repeated measures, followed by Dunnett's test (exercise effect), paired t-test (treatment effect), or unpaired t-test (MI vs. normal). Analysis of covariance (ANCOVA) was used to test the effect of drug treatment, MI, and their interaction on the relations between MVo2 (with MVo2 as covariate) and MEo2, coronary PvO2, and coronary SvO2. Because no differences were found between male and female swine, data from both sexes were pooled. Statistical significance was accepted when P ≤ 0.05. All data are presented as means ± SE.

RESULTS

Cardiac Anatomic Data

Despite the loss of viable LV myocardial tissue due to infarction, the LV weight-to-body weight ratio was 15% higher in MI (3.64 ± 0.14 g/kg) than in normal (3.17 ± 0.15 g/kg; P ≤ 0.05) swine, reflecting significant LV hypertrophy.

Systemic Hemodynamics

Under resting conditions, MI swine were characterized by a lower maximum LV dP/dt (LV dP/dtmax) and increased LAP, whereas the exercise-induced increases in LV dP/dtmax and LV systolic pressure were blunted in MI compared with normal animals (Fig. 1, top; Table 1). Arterial Po2 and hemoglobin saturation were similar in MI (101 ± 2 Torr, 94 ± 1%) and normal swine (105 ± 2 Torr, 95 ± 1%) at rest; no significant changes occurred in either group during exercise (data not shown).

Fig. 1.

Top: relations between heart rate and maximum rate of rise of left ventricular (LV) pressure (LV dP/dtmax; left), left atrial pressure (middle), and LV systolic pressure (right) in 16 normal swine and 15 swine with a recent myocardial infarction (MI) of the left circumflex coronary artery (LCx) area. bpm, Beats/min. Bottom: myocardial O2 balance in the LV anterior free wall of 14 normal swine and 11 swine with a recent MI of the LCx area. MVo2, myocardial O2 consumption; MEo2, myocardial O2 extraction; CVPo2, coronary venous Po2; CVSo2, coronary venous So2. Data are means ± SE. *P ≤ 0.05, MI vs. normal.

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Table 1.

Hemodynamic effects of KATP+ channel blockade with Glib in 10 normal swine and 9 swine with recent MI of LCx area

In both normal and MI swine, glibenclamide increased mean aortic and LV systolic blood pressure under resting conditions, which was accompanied by a (probably baroreflex mediated) decrease in heart rate (Table 1). In normal but not MI animals, the pressor effect of glibenclamide waned during exercise. Glibenclamide had no effect on arterial hemoglobin, Po2, or So2 levels.

Except for a small increase in heart rate, 8-PT had no effects on systemic hemodynamics in either group of animals at rest or during exercise (Table 2). Combined administration of 8-PT and glibenclamide resulted in hemodynamic changes that were similar to the alterations produced by glibenclamide alone (Table 3).

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Table 2.

Hemodynamic effects of adenosine receptor blockade with 8-PT in 12 normal swine and 12 swine with recent MI of LCx area

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Table 3.

Hemodynamic effects of combined administration of KATP+ channel blockade (Glib) and adenosine receptor blockade (8-PT) in 8 normal swine and 7 swine with recent MI of LCx area

Coronary Circulation

In MI swine the relation between MVo2 and MEo2 in the anterior LV free wall was shifted slightly upward, whereas the relations between MVo2 and both coronary PvO2 and coronary SvO2 shifted slightly downward, indicating a mild perturbation in O2 balance in remote surviving myocardium of swine with a recent MI compared with normal animals (Fig. 1, bottom).

In resting normal and MI swine, glibenclamide decreased CBF (Table 1) and myocardial O2 supply, necessitating an increase in MEo2, which led to a decrease of coronary PvO2 and coronary SvO2 (Fig. 2). In normal animals, the effect of glibenclamide was blunted at higher levels of exercise. In contrast, in MI swine the effects of glibenclamide were maintained during exercise.

Fig. 2.

Effect of the ATP-sensitive K+ (KATP+) channel blocker glibenclamide (Glib, 3 mg/kg iv) on myocardial O2 balance in the LV anterior free wall of 9 normal swine (top) and 8 swine with a recent MI (bottom). Data are means ± SE. *P ≤ 0.05 vs. corresponding control; †P ≤ 0.05, effect of Glib blunted at higher MVo2 levels (Glib × MVo2); ‡P ≤ 0.05, effect of Glib different after MI (Glib × MVo2 × MI).

Although 8-PT did not produce a statistically significant decrease in CBF at rest or during exercise (Table 2), 8-PT limited myocardial O2 supply at each level of MVo2, which was reflected in the increase in MEo2 and a decrease of coronary PvO2 and coronary SvO2 (Fig. 3). In normal swine, the effects of 8-PT during exercise were identical to its effects under resting conditions. The effects of 8-PT in MI swine were similar to those in normal swine, although a trend toward an increased effect of 8-PT with higher levels of exercise was observed in the relation between MVo2 and coronary PvO2 after MI compared with normal swine [MVo2 × MI × 8-PT; P = 0.08 (ANCOVA)]. However, such a trend was absent in the relation between MVo2 and coronary SvO2 or between MVo2 and MEo2 (both P > 0.50).

Fig. 3.

Effect of the adenosine receptor antagonist 8-phenyltheophylline (8-PT, 5 mg/kg iv) on myocardial O2 balance in the LV anterior free wall of 12 normal swine (top) and 12 swine with a recent MI (bottom). Data are means ± SE. The effect of 8-PT was maintained at higher MVo2 levels; the effect of 8-PT in MI swine was not different from that in normal animals. *P ≤ 0.05 vs. corresponding control.

In normal swine, the effect of combined administration of 8-PT and glibenclamide on myocardial O2 balance (Fig. 4) equaled the sum of effects of individual drug treatments (compare Figs. 24). In contrast, in MI swine the effect of 8-PT and glibenclamide was identical to the effect of glibenclamide alone (compare Figs. 2 and 4).

Fig. 4.

Effect of combined administration of the adenosine receptor antagonist 8-PT and the KATP+ channel blocker Glib on myocardial O2 balance in the LV anterior free wall of 8 normal swine (top) and 7 swine with a recent MI (bottom). Data are means ± SE. *P ≤ 0.05 vs. corresponding control; †P ≤ 0.05, effect of 8-PT+Glib greater than effect of 8-PT alone (Fig. 3); ‡P ≤ 0.05, effect of 8-PT+Glib greater than effect of Glib alone (Fig. 2).

DISCUSSION

The major findings in the present study are that 1) under resting conditions, KATP+ channels exert a similar vasodilator influence on the coronary circulation of swine with a normal LV and in remodeled myocardium of swine with a 2- to 3-wk-old MI; 2) during exercise, the coronary vasodilator influence of KATP+ channels is blunted in swine with normal hearts, whereas KATP+ channels maintain their vasodilator influence in remodeled myocardium; 3) the coronary vasodilator influence of adenosine is similar in normal swine and swine with a MI under resting conditions; 4) coronary vasodilator influence of adenosine in both normal swine and MI swine is maintained during exercise; and 5) in normal swine the vasodilator influences of adenosine and KATP+ channels are additive, whereas in MI swine adenosine-induced vasodilation appears to be mediated exclusively through KATP+ channels.

KATP+ Channels

In addition to the role for KATP+ channels in the regulation of CBF under physiological conditions (9, 12, 15), there is evidence for an increased KATP+ channel activity in the coronary circulation of remodeled hearts. For example, in anesthetized dogs with pacing-induced severe heart failure (31), glibenclamide resulted in an exaggerated vasoconstrictor response compared with normal dogs. Interestingly, in dogs subjected to only 1 wk of pacing (when LV function was still normal) KATP+ channel activity was not different from that in normal dogs (31), suggesting that KATP+ channel activity in the basal resting state was only enhanced in the presence of overt heart failure. Similarly, in awake dogs with compensated pressure overload-induced LV hypertrophy, the reduction in CBF produced by glibenclamide was similar to that in normal dogs (26). During exercise, however, glibenclamide produced a greater reduction in CBF in hypertrophied hearts, indicating increased KATP+ channel contribution to coronary vasodilation when O2 requirements of the hypertrophied heart were augmented (26).

In the present study in swine with MI-induced moderate LV remodeling and dysfunction, glibenclamide caused a marked decrease in coronary PvO2 in remodeled LV under resting conditions, which was similar to the decrease in coronary PvO2 in normal hearts. Although the vasoconstriction under resting conditions in response to KATP+ channel blockade with glibenclamide was similar in normal and post-MI remodeled hearts, the responses to exercise were different. Thus in normal swine the effects of KATP+ channel blockade waned during exercise, suggesting that other vasodilator systems, such as adenosine and NO, compensated for the loss of KATP+ channels during exercise (13, 14, 22, 27). In contrast, in the post-MI remodeled hearts the effects of glibenclamide were maintained during exercise. Our findings, which are consistent with the observations in dogs (26), support the hypothesis that KATP+ channel opening is of greater importance in resistance vessel dilation during exercise in hypertrophied than in normal hearts. It is likely that with the progression from LV dysfunction to overt heart failure, increased KATP+ channel activity may also become important under resting conditions (31).

Adenosine

Adenosine has been proposed to be a metabolic messenger by which tone in the coronary resistance vessels is regulated in response to changes in metabolic needs of the myocardium (2). However, adenosine receptor blockade with 8-PT and/or augmentation of adenosine catabolism with intracoronary adenosine deaminase had either no effect (1) or produced a small decrease (11, 27, 30) in basal coronary PvO2 but did not interfere with the normal exercise-induced increase in CBF (1, 11, 27, 30), indicating that adenosine is not critical for exercise-induced coronary vasodilation or that loss of adenosine-mediated vasodilation can be compensated for by increased contributions of other vasodilator pathways to maintain adequate metabolic vasodilation.

In contrast to the lack of evidence for an essential role for adenosine in regulation of CBF under physiological conditions, endogenously released adenosine does contribute to coronary vasodilation when there is an insufficient supply of O2 (25). Similarly, adenosine production could be increased in the remodeled myocardium after MI as a result of the, albeit mild, perturbations in the myocardial O2 balance (17, 32). However, we found no evidence for an increased contribution of adenosine to regulation of coronary resistance vessel tone in the present study, as adenosine receptor blockade with 8-PT caused a similar decrease in coronary PvO2 in postinfarct remodeled hearts compared with normal hearts both at rest and during exercise. These findings are in agreement with observations in dogs with pressure-overload LV hypertrophy, in which adenosine receptor blockade did not affect CBF either at rest or during exercise (26).

Several reasons could be put forward for the failure to observe a larger contribution of adenosine to regulation of coronary resistance vessel tone. First, it is possible that the perturbations in the myocardial O2 balance were too mild to increase adenosine production. Second, the activity of enzymes that regulate tissue adenosine levels (8) may have been altered. For example, the activity of adenosine deaminase, the enzyme responsible for breakdown of adenosine to inosine, was found to be elevated in LV hypertrophy (4, 6, 7). Furthermore, there is evidence that the activity of cytosolic 5′-nucleotidase, which converts 5′-AMP to adenosine, is lower in certain models of pressure overload (6)- and volume overload (4)-induced LV hypertrophy, which could be related to intermittent hypoperfusion of the hypertrophic myocardium (16). Together these enzymatic alterations, which act to decrease myocardial levels of adenosine, may have prevented a significant increase in myocardial adenosine levels in the post-MI remodeled hearts.

Finally, it is possible that an increased role of adenosine in hypertrophied myocardium is masked by an increased contribution of other vasodilator systems during adenosine receptor blockade, as the process of metabolic vasodilation is thought to be mediated through multiple parallel or redundant pathways (16, 22, 27). Thus KATP+ channel activity may have increased in response to adenosine receptor blockade to compensate for the loss of adenosine-mediated vasodilation. Hence, we evaluated the interactions between these vasodilator pathways.

Interaction Between KATP+ Channels and Adenosine

In contrast to the canine heart, in which adenosine can act as a back-up system (14, 29), adenosine and KATP+ channels appear to exert additive vasodilator influences on coronary vasomotor tone in the normal porcine heart (Ref. 27 and present study). Thus the coronary vasoconstriction that occurs in response to combined adenosine receptor blockade and KATP+ channel blockade equals the sum of the vasoconstriction induced by blockade of the individual pathways. Adenosine mediates its vasodilator effect on porcine coronary resistance vessels via KATP+, Ca2+-activated K+ (KCa+), and voltage-dependent K+ (Kv+) channels (3, 1921). It is therefore possible that after KATP+ channel blockade adenosine maintained its vasodilator influence via KCa+ and/or Kv+ channels.

In contrast to the normal porcine heart, the magnitude of the constriction in remodeled hearts induced by combined blockade of adenosine receptors and KATP+ channels was virtually identical to that produced by blockade of KATP+ channels alone (compare Figs. 2 and 4). These findings could be interpreted to suggest that in remodeled myocardium the vasodilator influence of endogenous adenosine was entirely mediated through opening of KATP+ channels, observations that are corroborated by findings in pressure-overload hypertrophied canine hearts (26). Together these observations in the porcine and canine coronary circulations suggest that, although the magnitude of the vasodilator influence exerted by endogenous adenosine was similar in normal and remodeled hearts, its effector pathway was different.

In conclusion, in post-MI remodeled LV myocardium, an increased KATP+ channel-mediated metabolic vasodilator influence helps to maintain myocardial O2 supply commensurate with MVo2 during exercise. In contrast, the net contribution of adenosine to regulation of coronary resistance vessel tone was not increased in remodeled myocardium. However, although in normal hearts the coronary vasodilator influence of adenosine did not depend on intact KATP+ channel activity, the endogenous adenosine-induced vasodilation in remodeled myocardium appeared exclusively mediated via KATP+ channels.

GRANTS

This study was supported by The Netherlands Heart Foundation Grants 2000T038 (to D. J. Duncker) and 2000T042 (to D. Merkus).

Acknowledgments

Reier Hoogendoorn and Rob van Bremen are gratefully acknowledged for technical assistance.

Footnotes

  • 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.

REFERENCES

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