INTRODUCTION

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IN THE HEART, ATP-sensitive potassium (K_ATP) channels are found on the sarcolemma of coronary vascular smooth muscle cells and myocardial myocytes as well as the inner mitochondrial membrane (25). In each of these locations, K_ATP channels have the potential to cause alterations of energy metabolism. K_ATP channels on coronary arteriolar smooth muscle cells participate in autoregulation and metabolic vasoregulation, thereby regulating the oxygen and metabolic substrate available to the myocardium (13). Mitochondrial K_ATP channels are important regulators of matrix volume; opening of the channels allows influx of potassium with osmotically obligated water to increase mitochondrial volume (5, 6, 8). During high rates of ATP synthesis, a decrease of mitochondrial membrane potential would reduce the driving force for potassium uptake, thereby tending to decrease matrix volume. Kowaltowski et al. (14) proposed that during high rates of ATP synthesis, opening of K_ATP channels may act to prevent respiratory inhibition secondary to matrix contraction. In isolated cardiac mitochondria, Holmuhamedov et al. (9) reported that pharmacological K_ATP channel openers accelerated respiration but slowed ATP production. Thus the findings in isolated mitochondria suggest that K_ATP channel activity may have the potential to influence respiration, especially during the high cardiac workloads associated with exercise.

In normal resting dogs, we (2) found that K_ATP channel blockade with glibenclamide caused a 18 ± 5% decrease of coronary blood flow with a parallel reduction of myocardial oxygen consumption (MO₂) and a slight but significant decrease of regional systolic wall thickening. These data suggested that glibenclamide caused coronary vasoconstriction and a decrease in myocardial blood flow sufficient to decrease contractile performance secondary to reduced oxygen availability. Alternatively, because glibenclamide acts as a nonselective blocker of K_ATP channels, the observed effects might have resulted from blocking of mitochondrial K_ATP channels (11, 15). Consequently, the present study was performed to determine whether the decrease in MO₂ observed after nonselective K_ATP channel blockade with glibenclamide resulted, at least in part, from blocking the opening of mitochondrial K_ATP channels.

5-Hydroxydecanoate (5-HD) was used to produce selective mitochondrial K_ATP channel blockade, because this agent has little effect on sarcolemmal K_ATP channel activity (15) or on vascular smooth muscle K_ATP channels (it did not block the diazoxide-induced increases in coronary flow in perfused rabbit hearts) (23). The effects on MO₂ at rest and during exercise were compared with the response to nonselective K_ATP channel blockade with glibenclamide.

	METHODS

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Studies were performed in 10 adult mongrel dogs weighing 25-30 kg trained to run on a motor-driven treadmill. All experiments were performed in accordance with the "Guiding Principles in the Care and Use of Laboratory Animals" approved by the council of the American Physiological Society and with the prior approval of the Animal Care Committee of the University of Minnesota.

Surgical preparation. The animals were anesthetized with pentobarbital sodium (35 mg/kg with supplemental doses to maintain surgical anesthesia), intubated, and ventilated with a mixture of oxygen (30%) and room air (70%). Respiratory rate and tidal volume were set to keep arterial blood gases within the physiological range. A left thoracotomy was performed in the fifth intercostal space, and the heart was suspended in a pericardial cradle. A polyvinyl chloride catheter (3.0 mm outer diameter) filled with heparinized saline was inserted into the internal thoracic artery and advanced into the ascending aorta. A similar catheter was introduced into the left ventricle (LV) through the apex and secured in place. A solid-state micromanometer (model P5, Konigsberg Instruments; Pasadena, CA) was also introduced into the LV at the apex. A final catheter was introduced into the right atrial appendage, manipulated into the coronary sinus ostium, and advanced into the great cardiac vein until the tip could be palpitated within 1 cm of the interventricular sulcus to allow selective sampling of coronary venous blood draining the myocardium perfused by the left anterior descending (LAD) coronary artery. Approximately 1.5 cm of the proximal LAD was dissected free, and a Doppler velocity probe (Craig Hartley; Houston, TX) was positioned around the artery. Immediately distal to the velocity probe, a hydraulic occluder was placed around the vessel. A silicone catheter (0.3 mm inner diameter) bonded to a larger silicone catheter (1.6 mm inner diameter) was introduced into the LAD immediately distal to the hydraulic occluder to allow drug infusion. The pericardium was then loosely closed, and the catheters and electrical leads were tunneled subcutaneously to exit at the base of the neck. The chest was closed in layers, and the pneumothorax was evacuated. Postoperative analgesia was provided with butorphanol (0.4 mg/kg sq) every 4-6 h. Catheters were flushed daily with heparinized saline. The catheters, electrical leads, and occluder tubing were protected with a nylon vest.

Hemodynamic measurements. Aortic pressure was measured with a Gould P23XL pressure transducer positioned at midchest level. LV pressure was measured with the micromanometer calibrated with the fluid-filled LV catheter. The first time derivative of LV pressure (dP/dt) was obtained via electrical differentiation of the LV pressure signal. Coronary blood velocity was measured with a Doppler flowmeter system (Craig Hartley). Data were recorded on an eight-channel direct-writing oscillograph (Coulbourn Instruments; Lehigh Valley, PA).

Myocardial oxygen consumption. Blood specimens were maintained in iced syringes until the completion of each exercise trial. Measurements of PO₂, PCO₂, and pH were then immediately performed with an Instrumentation Laboratory model 113 blood gas analyzer (Lexington, MA). Hemoglobin content was determined by the cyanmethemoglobin method. Hemoglobin oxygen saturation was calculated from the blood PO₂, pH, and temperature using the oxygen dissociation curve for canine blood. Blood oxygen content was computed as follows: (hemoglobin × 1.34 × percent oxygen saturation) + (0.0031 × PO₂). Oxygen consumption in the region of myocardium perfused by the LAD was calculated as the product of blood flow measured with the Doppler flow probe and the difference in oxygen content between aortic and coronary venous blood.

Experimental protocol. Studies were performed 10-14 days after surgical preparation. With the dog standing quietly on the treadmill, resting hemodynamics and coronary blood flow were recorded, and 2 ml of blood were withdrawn anaerobically from the aortic and coronary venous catheters and maintained on ice until blood gas analysis could be performed (within 20 min after sample collection). Subsequently, a three-stage graded treadmill exercise began (stage 1: 6.4 km/h at 0% grade, stage 2: 6.4 km/h at 5% grade, and stage 3: 6.4 km/h at 10% grade). Each exercise stage was 3 min in duration. LV and aortic pressures and coronary blood flow were measured continuously. Aortic and coronary venous blood samples were withdrawn during the last 30 s of each exercise stage when hemodynamics had reached a steady state. In 7 dogs, after a 2-h rest period, the selective mitochondrial K_ATP channel blocker 5-HD was administered at a dose of 0.7 mg · kg¹ · min¹ by intracoronary infusion over 10 min. This dose has been previously demonstrated to block the myocardial protective effects produced by ischemic preconditioning in the dog (1). Beginning 15 min after drug administration, all measurements were repeated at rest and during the three-stage treadmill exercise protocol. Animals were subsequently allowed to rest for 2 h. Nonselective K_ATP channel blockade was then produced by intracoronary infusion of glibenclamide (50 µg · kg¹ · min¹). Ten minutes after the beginning of the infusion, all measurements were repeated at rest and during treadmill exercise. In three additional dogs, an infusion of 5-HD was continued throughout the entire protocol to ensure that an adequate drug dose was present at the time that exercise measurements were performed. In these animals, 5-HD was infused at a dose of 0.7 mg · kg¹ · min¹ beginning 10 min before exercise. After 10 min of infusion of 5-HD, the exercise protocol began while the 5-HD infusion continued. The infusion of 5-HD was stopped after the conclusion of the exercise measurements.

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Data analysis. Heart rate, LV and aortic pressures, and coronary velocity were measured from the strip-chart recordings. Coronary blood flow was computed from the Doppler shift as previously described using the equation Q = 2.5 × f × D², where Q is coronary blood flow (in ml/min), f is the Doppler shift (in kHz), and D is the internal diameter of the coronary artery within the velocity probe (2). Statistical analysis was performed using two-way (exercise level and treatment) ANOVA for repeated measures. When a significant effect of exercise was observed, comparisons within treatment groups were made using one-way ANOVA followed by Scheffe's post hoc test. When a significant difference between treatments was observed, comparisons between groups were made using the Student's t-test with the Bonferroni correction. The effects of treatment on the relationship between two variables were analyzed by analysis of covariance (ANCOVA). Data for the three animals in which 5-HD was infused continuously beginning 10 min before exercise and continuing until the end of the exercise were initially analyzed separately from the seven animals in which a 10-min infusion of 5-HD was performed 15 min before beginning exercise. Because no differences were found between the two groups, the data from all 10 animal were pooled for the final analysis. Statistical significance was accepted at P < 0.05. All data are presented as means ± SE.

	RESULTS

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Hemodynamic measurements. Hemodynamic data obtained at rest and during treadmill exercise are shown in Table 1. Heart rate increased from 141 ± 7 beats/min during resting conditions to 262 ± 10.3 beats/min during peak exercise. Mean aortic pressure and LV systolic pressure increased progressively during exercise, whereas LV end-diastolic pressure was significantly increased during exercise stages 2 and 3 compared with resting measurements. Maximal LV dP/dt increased progressively during exercise. Selective mitochondrial K_ATP channel blockade with 5-HD did not significantly change hemodynamic measurements obtained at rest or during exercise. Nonselective K_ATP channel blockade with glibenclamide did not significantly alter heart rate, mean aortic pressure, or LV systolic pressure at rest or during exercise. However, LV end-diastolic pressure was significantly higher during exercise stage 3 than during control exercise or exercise with 5-HD. Maximal LV dP/dt was significantly lower after glibenclamide during all three exercise stages compared with control measurements and measurements obtained after 5-HD.

Coronary hemodynamics. During control conditions, coronary blood flow increased from 40.5 ± 3.8 ml/min during resting conditions to a maximum of 65.0 ± 6.0 ml/min during the heaviest level of exercise (Table 2). Exercise resulted in a significant increase of hemoglobin from 13.7 ± 0.04 g/dl at rest to 14.4 ± 0.4 g/dl during the heaviest level of exercise, with a corresponding increase of arterial oxygen content (P < 0.05). Compared with resting measurements, coronary vein oxygen tension decreased significantly during exercise stages 2 and 3. As a result of these alterations, MO₂ in the LAD region increased from 6.1 ± 0.8 ml/min at rest to 11.0 ± 1.2 ml/min during the heaviest level of exercise.

View this table: [in this window] [in a new window]   Table 2.   Effect of 5-HD and Glib on LAD coronary artery blood flow, coronary venous PO2, and MO2 at rest and during a three-stage graded treadmill exercise protocol

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Relationship between coronary blood flow and the heart rate times systolic blood pressure product (rate-pressure product × 103) at rest and during graded treadmill exercise. Data were obtained during control conditions, after selective mitochondrial ATP-sensitive K+ (KATP) channel blockade with 5-hydroxydecanoate (5-HD; 0.7 mg · kg1 · min1 ic), and during nonselective KATP channel blockade with glibenclamide (Glib; 50 µg · kg1 · min1 ic). Results are means ± SE; n = 7.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Relationship between coronary venous oxygen tension and myocardial oxygen consumption at rest and during graded treadmill exercise. Data were obtained during control conditions, after selective mitochondrial KATP channel blockade with 5-HD (0.7 mg · kg1 · min1 ic), and after nonselective KATP channel blockade with Glib (50 µg · kg1 · min1 ic). Results are means ± SE; n = 7.

Magnitude and selectivity of K_ATP channel blockade. The effects of glibenclamide and 5-HD on the increases in coronary blood flow caused by pinacidil are shown in Fig. 3. Glibenclamide caused dose-dependent inhibition of the coronary vasodilation produced by pinacidil, with >90% inhibition at all doses of pinacidil tested. As shown in Fig. 3, 5-HD did not significantly inhibit the coronary vasodilation produced by pinacidil at any dose tested. These findings indicate that the dose of glibenclamide used produced a high degree of blockade of K_ATP channels on coronary resistance vessel smooth muscle. In contrast, 5-HD did not significantly inhibit the effect of pinacidil on vascular smooth muscle K_ATP channels.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effect of nonselective KATP channel blockade with Glib (50 µg · kg1 · min1 ic; A) and selective mitochondrial KATP channel blockade with 5-HD (0.7 mg · kg1 · min1 ic; B) on the increase in coronary blood flow produced by intracoronary infusions of pinacidil in awake resting dogs. Data are means ± SE.

	DISCUSSION

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This study addressed the question of whether glibenclamide caused a primary decrease of MO₂ by blocking mitochondrial K_ATP channels or whether the decreased oxygen consumption resulted from vasoconstriction of coronary resistance vessels with a decrease of oxygen availability. The finding that selective mitochondrial K_ATP channel blockade with 5-HD had no effect on MO₂ at rest or during exercise supports the conclusion that the reduction of MO₂ produced by glibenclamide resulted from inhibition of K_ATP channels on the sarcolemma of the coronary smooth muscle cells, which resulted in vasoconstriction and a decrease of coronary blood flow. This suggests that glibenclamide caused a decrease of oxygen availability to the myocardium rather than a primary reduction of mitochondrial respiration.

K_ATP channels exist on the sarcolemma of coronary vascular smooth muscle cells and myocardial myocytes as well as on the inner mitochondrial membrane of cardiac myocytes (11). Channels open in response to low levels of ATP or a decrease of the ATP-to-ADP ratio as well as to decreases of redox potential and pH (7, 12). Sarcolemmal K_ATP channels exist as octomeres of four regulatory proteins containing sulfonylurea receptors associated with four inward rectifying potassium channels, which serve as pore-forming subunits (10, 21). K_ATP channels at different sites within the heart appear to be distinct, although the molecular identify of the channel associated with the inner mitochondrial membrane has not yet been characterized. The structural differences of K_ATP channels are of importance, because they have allowed development of agonists and antagonists that are selective for K_ATP channels at different sites within the heart. K_ATP channels in vascular smooth muscle cells of coronary arterioles appear to participate in metabolic vasoregulation (13). Opening of these channels results in an outward flux of potassium, which increases the sarcolemmal membrane potential; the resultant hyperpolarization closes voltage-dependent calcium channels, leading to decreased influx of calcium, thereby causing vasodilation (13, 19). Conversely, closing of K_ATP channels decreases the membrane potential, thereby opening voltage-dependent calcium channels to cause vasoconstriction.

The discovery of K_ATP channels on the inner mitochondrial membrane suggested a strategic locus to influence energy metabolism in the myocardial myocytes (11). In vitro studies have demonstrated that mitochondrial K_ATP channels participate in regulation of mitochondrial matrix volume. Opening of mitochondrial K_ATP channels causes a net uptake of potassium that is accompanied by an electroneutral influx of anions and osmotically obligated water, resulting in an increase of mitochondrial matrix volume (4, 6, 8). Evidence has been presented that expansion of mitochondrial matrix volume can activate electron transport and stimulate mitochondrial respiration (8, 9). Kowaltowski et al. (14) observed that the decreased mitochondrial membrane potential during state 3 respiration (to mimic a high workload state of the cardiomyocyte) caused decreased electrogenic potassium uptake with consequent matrix contraction. They hypothesized that opening of mitochondrial K_ATP channels during high rates of ATP synthesis can act to maintain constant matrix volume by providing additional potassium conductance to compensate for the lower driving force for electrogenic potassium uptake. They proposed that in this way K_ATP channel opening can act to prevent respiratory inhibition due to matrix contraction during high rates of ATP synthesis.

A physiological role for mitochondrial K_ATP channels in the intact heart was demonstrated by their participation in the preconditioning response. In myocardial preconditioning, an initial brief period of ischemia protects the heart during a subsequent more prolonged ischemic episode (18). Preconditioning is, at least in part, dependent on activation of mitochondrial K_ATP channels, because selective inhibition of mitochondrial K_ATP channel activity has been found to interrupt the preconditioning response (1, 3, 4). Thus diazoxide, in concentrations that caused opening of K_ATP channels on mitochondria and vascular smooth muscle but did not open sarcolemmal K_ATP channels on cardiac myocytes, exerted a cardioprotective effect equivalent to that produced by cromokalim or pinacidil, which opened both sarcolemmal and mitochondrial K_ATP channels (4, 15). Conversely, 5-HD, which selectively inhibits mitochondrial K_ATP channels, was found to block the protective effect of preconditioning (1, 3).

The decreased MO₂ during glibenclamide administration in the present study was likely the result of effects on coronary vascular smooth muscle. The decrease of coronary blood flow produced by glibenclamide resulted in increased myocardial oxygen extraction and decreased coronary venous oxygen tension, indicating that myocardial oxygen delivery was decreased relative to oxygen demands. This interpretation is supported by a previous study (2) in which regional LV systolic wall thickening was measured as an index of contractile performance. In that study (2), the decreased coronary blood flow produced by intracoronary glibenclamide resulted in hypokinesis of the dependent myocardial region. When blood flow was returned to the preglibenclamide control level by intracoronary infusion of nitroprusside, myocardial contractile performance improved. This finding suggested that the decrease in coronary blood flow resulted in oxygen insufficiency in the dependent myocardium with impaired contractile performance. In support of this, Samaha et al. (19), using ³¹P nuclear magnetic resonance spectroscopy to assess myocardial high-energy phosphate levels, observed that the decreased coronary artery blood flow produced by glibenclamide was associated with a decrease of phosphocreatine and a reduction of the phosphorylation potential similar to that observed during ischemia. The concept that the primary effect of glibenclamide on MO₂ resulted from coronary vasoconstriction with a decrease in oxygen delivery to the heart is supported by the finding that K_ATP channel blockade with glyburide in isolated rat hearts perfused at constant flow did not alter oxygen consumption (20). These findings suggest that the primary effect of glibenclamide was to cause vasoconstriction of the coronary resistance vessels with a decrease in oxygen delivery to the heart.

If the decrease in MO₂ resulted from the decrease in coronary blood flow produced by glibenclamide, then one might have expected a greater compensatory increase in oxygen extraction. As seen in Table 2, during resting conditions, glibenclamide caused a significant decrease of MO₂ even though coronary venous oxygen tension fell to only 14.4 ± 1.0 Torr. Clearly, the heart was able to extract oxygen to a greater degree than this, because during control conditions coronary venous oxygen tension fell to 11.9 ± 1.1 Torr during the heaviest level of exercise. The failure of the heart to achieve a greater average level of oxygen extraction may be related to an effect of glibenclamide on the distribution of blood flow at the microvascular level. In studies of the response to progressive hypoperfusion in the pig hindlimb, Vallet et al. (23) observed that glibenclamide caused reductions of oxygen uptake and the cytochrome aa₃ oxidation level during moderate decreases of blood flow that did not cause these changes in control limbs. The investigators suggested that this occurred because glibenclamide interfered with capillary recruitment as blood flow and perfusion pressure were decreased, resulting in a local perfusion-metabolism mismatch. The resultant microheterogeneity of blood flow could result in small areas of ischemia in parallel with other areas that were adequately perfused, so that metabolic evidence of ischemia and contractile dysfunction could occur at levels of coronary venous oxygen tension greater than those observed during high levels of oxygen utilization. The finding in the present study that glibenclamide resulted in a decrease of MO₂ during resting conditions with only a modest (not significant) decrease of venous PO₂ similarly suggests that the decreased oxygen consumption may have been, at least in part, the result of microheterogeneity of perfusion.

Limitations. Because selective mitochondrial K_ATP channel blockade failed to decrease MO₂, it is important to establish that an adequate dose of 5-HD was administered. Because there is no methodology to directly assess the state of mitochondrial K_ATP channels in vivo, we relied on previous reports as well as calculations of coronary blood levels of drug. Several investigators have demonstrated that doses of 5-HD of 3 mg/kg (1) to 7 mg/kg (22) administered by the intracoronary route abolished the protective effect of myocardial preconditioning in the dog. Because myocardial preconditioning appears to involve the opening of mitochondrial K_ATP channels, the ability of 5-HD to block preconditioning implies that the dose used was sufficient to achieve intracellular concentrations needed to inhibit the mitochondrial K_ATP channels. We used a similar infusion protocol with a dose of 5-HD at the higher end (7 mg/kg over 10 min). During our initial studies, measurements began 15 min after completion of the 5-HD infusion. However, because of the possibility that 5-HD might have a very short biological half-life in vivo, in three additional animals, the infusion of 5-HD was continued during the time that measurements were obtained. In both groups of animals, 5-HD did not decrease MO₂ either at rest or during exercise. With the use of the measured coronary blood flow rates in the LAD coronary artery, and neglecting any contribution from recirculation, this infusion rate produced a calculated coronary artery blood concentration of ~2 mM at rest and 1.2 mM during the heaviest level of exercise, far higher than the perfusate concentrations of 5-HD that have been found to block preconditioning in isolated hearts (200-300 µM) (16, 24) or to block the increase in matrix volume of isolated cardiac mitochondria (300 µM) by diazoxide (14). These lines of evidence support the contention that a concentration of 5-HD sufficient to inhibit mitochondrial K_ATP channels in the myocardium was present at the time the experimental measurements were obtained. Although 5-HD did not decrease MO₂ in the range of cardiac workloads observed in the present study, it is possible that different results would be found during heavier levels of exercise, which result in higher cardiac workloads.