Introduction

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Ventricular fibrillation (VF) has been identified as a leading cause of sudden death during myocardial ischemia in humans (Gillum, 1989). It has been proposed that alterations in cellular electrophysiological properties that culminate in the formation of malignant arrhythmias may result from abnormalities in the biochemical homeostasis of the cardiac cells (Opie et al., 1979). Alterations in extracellular potassium, in particular, have been linked to an increased propensity for malignant arrhythmias as a consequence of the interruption in CBF (for reviews, see Billman, 1994; Coronel, 1994). Extracellular potassium concentration rises rapidly during myocardial ischemia (Coronel et al., 1988; Harris, 1966; Kléber, 1984). The resulting depolarization of the surrounding tissue, decreases in action potential duration and nonuniformities of repolarization (refractory period) could all contribute to the induction of malignant arrhythmias (Janse and Wit, 1989).

Since the first characterization of K_ATP in cardiac tissue (Noma, 1983), an increasing body of evidence has implicated the activation of the channel in many of the electrical consequences of myocardial ischemia (for review, see Billman, 1994). For example, glibenclamide, a sulfonylurea drug that selectively blocks the K_ATP (Sturgess et al., 1985), has recently been shown to reduce the extracellular accumulation of potassium and reverse the shortening of the action potential duration provoked by hypoxia or myocardial ischemia (Nakaya et al., 1991; Nichols et al., 1991; Venkatesh et al., 1991, 1992). If the extracellular potassium accumulation contributes to the development of VF, then drugs that selectively block the K_ATP should also protect against these malignant arrhythmias. A limited number of experimental (Billman et al., 1993; Wollenben et al., 1989) and clinical (Cacciapuoti et al., 1991; Davis et al., 1996; Lomuscio and Fiorentini, 1996; Lomuscio et al., 1994) studies, in fact, provide preliminary support for this hypothesis. Glibenclamide, for example, has been shown to prevent VF in isolated ischemic rat hearts (Wollenben et al., 1989), as well as reduce the number and severity of arrhythmias during transient ischemia in diabetic patients with coronary artery disease (Cacciapuoti et al., 1991). In a similar manner, glibenclamide significantly reduced the incidence of VF in non-insulin-dependent diabetic patients with acute myocardial infarction (Lomuscio et al., 1994). Recently, glibenclamide also significantly reduced the incidence of VF induced by the combination of acute ischemia during exercise in dogs with previously healed myocardial infarctions (Billman et al., 1993). This drug also induced large reductions in left ventricular dP/dt maximum (an index of inotropic state) and mean CBF (Billman et al., 1993). Glibenclamide, however, is not selective for cardiac tissue. For example, this drug also blocks pancreatic K_ATPs, which promotes insulin release and often results in profound hypoglycemia. In addition, the activation of this channel may play an important role in the regulation of CBF (Aversano et al., 1991; Daut et al., 1990), and as such, glibenclamide has been shown to reduce coronary perfusion (Billman et al., 1993). Therefore, one would predict that compounds that selectively block cardiac K_ATPs should protect against arrhythmias induced by myocardial ischemia without compromising either CBF or blood glucose levels. Recently, a number of K_ATP subtypes have, in fact, been isolated from different tissues (Inagaki et al., 1996; Isomoto et al., 1996; Garlid et al., 1997). In addition, a novel sulfonylthiourea compound, HMR 1883, 1-[[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea (fig. 1) has been shown to block K_ATP channels in cardiac muscle cells with a much higher potency than in pancreatic  cells (Gögelein et al., 1998). However, the effects of this drug in intact preparations have not been fully characterized. Therefore, it was the purpose of this series of experiments to evaluate the effects of a novel cardioselective K_ATP antagonist, HMR 1883, on the susceptibility to ventricular fibrillation using an unanesthetized canine model of sudden death.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   The chemical structures of HMR 1883 and glibenclamide.

	Methods

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The principles governing the care and use of animals, as expressed by the Declaration of Helsinki and as adopted by the American Physiological Society, were followed at all times during this study. In addition, all procedures were approved by the Ohio State University Institutional Animal Care and Use Committee.

Surgical preparation. Fifty heartworm-free mongrel dogs (Kaiser Lake Kennels, Kaiser Lake, OH), weighing 15.4 to 19.1 kg, were used in this study. The animals were anesthetized and instrumented to measure left circumflex CBF, left ventricular pressure and ventricular electrogram, as previously described (Billman and Hamlin, 1996; Billman et al., 1993, 1997; Schwartz et al., 1984). Briefly, the animals were given Innovar Vet (0.02 mg/kg fentanyl citrate and 1 mg/kg hydroperidol i.v.; Pittman-Moore, Washington Crossing, NH) as a preanesthetic, whereas a surgical plane of anesthesia was induced with sodium pentobarbital (10 mg/kg i.v.; Ampro Pharmaceutical, Arcadia, CA). A left thoracotomy was made in the fourth intercostal space, and the heart was exposed and supported by a pericardial cradle. A 20-MHz pulsed Doppler flow transducer and a hydraulic occluder were placed around the left circumflex artery. A pair of insulated silver-coated wires were sutured to the epicardial surface of both the left and right ventricles. These electrodes were used for ventricular pacing (see below) or to record a ventricular electrogram from which HR was determined using a Gould Biotachometer (Gould Instruments, Cleveland, OH). A precalibrated solid-state pressure transducer (Konigsberg Instruments, Pasadena, CA) was inserted into the left ventricle via a stab wound in the apical dimple. Finally, a two-stage occlusion of the left anterior descending coronary artery was performed approximately one third the distance from the origin to induce an anterior wall myocardial infarction. This vessel was partially occluded for 20 min and then tied off. All leads from the cardiovascular instrumentation were tunneled under the skin to exit on the back of the animal's neck.

Exercise-plus-ischemia test. The studies began 3 to 4 weeks after the production of the myocardial infarction. The animals were walked on a motor-driven treadmill and trained to lie quietly without restraint on a laboratory table during this recovery period. Susceptibility to VF was then tested, as previously described (Billman and Hamlin, 1996; Billman et al., 1993, 1997; Schwartz et al., 1984). Briefly, the animals ran on a motor-driven treadmill while workload was increased every 3 min for a total of 18 min. The protocol began with a 3-min warm-up period, during which the animals ran at 4.8 km/hr at 0% grade. The speed was increased to 6.4 km/hr, and the grade was increased every 3 min as follows: 0%, 4%, 8%, 12% and 16%. During the last minute of exercise, the left circumflex coronary artery was occluded, the treadmill was stopped and the occlusion was maintained for 1 additional min (total occlusion time, 2 min). Large metal plates (diameter, 11 cm) were placed across the animal's chest so that electrical defibrillation could be achieved with minimal delay but only after the animal was unconscious (10-20 sec after VF began). The occlusion was immediately released if VF occurred. Eighteen animals developed VF (susceptible; 5 were not successfully defibrillated), and the remaining 17 did not (resistant).

Refractory period determination. On a subsequent day, the effective refractory period was determined as previously described (Billman and Hamlin, 1996), using a Medtronic model 5325 programmable stimulator, both at rest (n = 20) and during myocardial ischemia (n = 10). Briefly, the heart was paced for 8 beats (S₁; intrastimulus interval, 300 msec; pulse duration, 1.8 msec at twice-diastolic threshold of ~6 mA). The intrastimulus interval was progressively shortened between the last paced beat and a single extrastimulus (S₂). The refractory period represented the shortest interval capable of generating a cardiac response and was measured using either the left or right ventricular electrodes. This procedure was completed within 30 sec. No differences were noted between either pair of electrodes. The data therefore were combined.

Reactive hyperemia studies. The K_ATP has been implicated in vascular regulation, particularly CBF (Aversano et al., 1991; Belloni and Hintze, 1991; Daut et al., 1990). Therefore, the effects of HMR 1883 and glibenclamide on the response to brief interruptions in CBF were also evaluated. Animals (n = 5) were placed on a laboratory table, and the left circumflex coronary was occluded three or four times for 15 sec. At least 2 min (or until CBF had returned to preocclusion base line) elapsed between occlusions. The occlusions were then repeated 5 min after either HMR 1883 (3.0 mg/kg i.v.) or glibenclamide (1.0 mg/kg i.v.). On the subsequent day, the studies were repeated with the drug that had not been given the previous day.

Data analysis. All hemodynamic data were recorded on a Gould model 2800S eight-channel recorder (Cleveland, OH) and a Teac model MR-30 FM tape recorder (Tokyo, Japan). Coronary blood flow was measured with a University of Iowa Bioengineering flowmeter model 545 C-4 (Iowa City, IA). The rate of change of left ventricular pressure [d(LVP)/dt] was obtained by passing the left ventricular pressure through a Gould differentiator that has a frequency response linear to >300 Hz. The data were averaged over the past 5 sec of each exercise level. The coronary occlusion data were averaged over the last 5 sec before and at the 60-sec line point (or VF onset) after occlusion onset. The total area between the peak CBF and return to base line was measured for each 15-sec occlusion, and the percent repayment was calculated. The reactive hyperemia response to each occlusion was then averaged to obtain one value for each animal. The data were then analyzed using analysis of variance for repeated measures. When the F ratio was found to exceed a critical value (P < .05), Scheffé's test was used to compare the mean values. The effects of the drug intervention on arrhythmia formation were determined using a ² test with Yates' correction for continuity. All data are reported as mean ± S.E.M. Cardiac arrhythmias, PR interval and QT interval were evaluated at a paper speed of 100 mm/sec. QT interval was corrected for HR using Bazett's method.

	Results

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Effects on susceptibility to VF. The exercise-plus-ischemia test induced VF in 18 animals (5 were not successfully defibrillated). In agreement with previous studies (Billman and Hamlin, 1996; Billman et al., 1993, 1997; Schwartz et al., 1984), VF was reproducibly induced in the susceptible animals with each presentation of both control exercise-plus-ischemia tests. The average time to the onset of VF was 62 ± 4 sec (range, 35-90 sec). The control exercise-plus-ischemia test elicited similar hemodynamic changes (e.g., HR: first occlusion control, 198.7 ± 8.6; occlusion, 224.2 ± 8 beats/min; second occlusion control, 181.6 ± 11.0; occlusion, 232.2 ± 8.1 beats/min) with a similar time to VF onset (59 ± 5.4 sec; range, 37.5-101 sec) as the first test. The exercise-plus-ischemia test was repeated after the following treatments: HMR 1883 (3.0 mg/kg i.v., n = 8), HMR 1883 (3.0 mg/kg i.v., 1 hr before the exercise-plus-ischemia test, n = 5) and glibenclamide (1.0 mg/kg i.v., n = 7). Representative examples for the same animal before and after pretreatment with HMR 1883 are shown in figure 2. HMR 1883 given 3 min before the onset of exercise prevented VF in 7 of 8 animals (² = 9.1, P < .005). This drug completely suppressed ventricular arrhythmia formation in 6 animals. One animal exhibited a brief run of ventricular tachycardia (a run of four extra beats). In a similar manner, HMR 1883, 3.0 mg/kg i.v., given 1 hr before the exercise-plus-ischemia test, prevented ventricular fibrillation in 4 of 5 additional animals (² = 3.8, P < .05). Thus, 3.0 mg/kg i.v. HMR 1883 prevented malignant arrhythmias in a total of 11 of 13 animals (² = 15.8, P < .001, figure 3). Finally, 1.0 mg/kg i.v. glibenclamide elicited a similar protection, protecting 6 of 7 animals (² = 7.3, P < .01).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Representative recordings from the same animal before and after pretreatment with the cardioselective ATP-sensitive potassium channel antagonist HMR 1883 (3.0 mg/kg i.v.). Note that this drug prevented malignant arrhythmias despite a similar HR change induced by the ischemia. Note further the similar ischemic ECG changes (ST depression) both before and after HMR 1883 treatment.

View larger version (8K): [in this window] [in a new window]   Fig. 3.   The effect of HMR 1883 on the incidence of VF. During the control (no drug) test, VF was induced in 13 of 13 (100%) animals; after HMR 1883 (3.0 mg/kg i.v.), VF was induced in 2 of 13 (15%). * P < .001 (no drug vs. HMR 1883).

View larger version (31K): [in this window] [in a new window]   Fig. 4.   The effect of HMR 1883 (3.0 mg/kg i.v.) on the hemodynamic response to exercise. Note that this drug did not alter the response of any variable to exercise. , Control (i.e., no drug). , HMR 1883. HR, LVSP and LV dp/dt maximum, n = 10; mean CBF, n = 7. Exercise levels: 1, 0% grade/0 kph; 2, 0%/4.8 kph; 3, 0%/6.4 kph; 4, 4%/6.4 kph; 5, 8%/6.4 kph; 6, 12%/6.4 kph; 7, 16%/6.4 kph.

View larger version (33K): [in this window] [in a new window]   Fig. 5.   The effect of glibenclamide (1.0 mg/kg i.v.) on the hemodynamic response to exercise. Note that this drug significantly reduced both mean CBF and left ventricular dp/dt maximum. , control (i.e., no drug). , HMR 1883. * P < .01 glibenclamide vs. corresponding control (i.e., no drug exercise level). HR n = 7; LVSP, LV dp/dt maximum and mean CBF, n = 5. Exercise levels: 1, 0% grade/0 kph; 2, 0%/4.8 kph; 3, 0%/6.4 kph; 4, 4%/6.4 kph; 5, 8%/6.4 kph; 6, 12%/6.4 kph; and 7, 16%/6.4 kph.

Electrophysiological and hemodynamic effects of K_ATP agonists and antagonists before and during myocardial ischemia. The electrophysiological and hemodynamic effects of the K_ATP-modulating drugs before myocardial ischemia are displayed in table 2. HMR 1883 did not alter the resting values of any these of variables. Glibenclamide, however, provoked significant reductions in both left ventricular dP/dt maximum and mean CBF (table 2). In contrast, pinacidil significantly increased HR and mean CBF, which was accompanied by significant reductions in LVSP, PR interval and refractory period (table 2). Myocardial ischemia (n = 10) elicited significant increases in HR (control, 131 ± 7; occlusion, 151 ± 9 beats/min), which, in agreement with previous studies (Li and Ferrier, 1991; Billman and Hamlin, 1996), was accompanied by small but significant reductions in refractory period (fig. 6). Pretreatment with either glibenclamide (n = 7) or HMR 1883 (n = 7) prevented the ischemia-induced reductions in refractory period. Pinacidil, as noted above, significantly reduced refractory period, which was not further reduced by ischemia.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   The effects of coronary artery occlusion on refractory period. Note that coronary occlusion significantly reduced refractory period in the control (no drug, n = 10) condition. Pinacidil (n = 8, 0.5 mg/kg i.v.) significantly reduced refractory period, whereas both glibenclamide (glib, n = 7, 1.0 mg/kg i.v.) and HMR 1883 (n = 7, 3.0 mg/kg i.v.) prevented the reduction in refractory period induced by ischemia. * P < .01 control vs. occlusion, , P < .01 no drug vs. the corresponding drug treatment.

Effects on reactive hyperemia. Representative examples of CBF response to a 15-sec coronary artery occlusion before and after either glibenclamide or HMR 1883 are shown in figure 7. The release of 15-sec coronary occlusion was accompanied by a large repayment of the flow deficit (control, 426.8 ± 102%), which was not significantly altered by HMR 1883 pretreatment (414.6 ± 133.3%, 3.0 ± 4.7% compared with control values). In contrast, glibenclamide provoked a large and significant reduction (30.0 ± 11% compared with control values) in the flow repayment after occlusion release (310.6 ± 72.6%). Similar reductions in CBF have been reported after either glibenclamide (Aversano et al., 1991) or the administration of the adenosine antagonists aminophylline (Billman, 1987) and adenosine deaminase (Saito et al., 1981). It should be noted that it has been proposed that adenosine elicits coronary vasodilation via the activation of the K_ATP channel (Daut et al., 1990). Finally, glibenclamide provoked significant decreases in plasma insulin accompanied by large increases in plasma glucose (fig. 8). When considered together, these data demonstrate that HMR 1883, in contrast to glibenclamide, does not inhibit the activation of either vascular or pancreatic K_ATPs.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Representative recordings from the same animal of the reactive hyperemia response to a 15-sec coronary artery occlusion before and after treatment with either HMR 1883 (3.0 mg/kg i.v.) or glibenclamide (1.0 mg/kg i.v.). The drugs were given 24 hr apart. Note that HMR 1883 did not alter the hyperemia, whereas glibenclamide reduced the duration of the hyperemia.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   The effect of HMR 1883 (3.0 mg/kg i.v.) and glibenclamide (1.0 mg/kg i.v.) on plasma insulin concentration (A) and blood glucose levels (B). Note that glibenclamide, but not HMR 1883, elicited significant changes in both glucose and insulin levels. * P < .01 control vs. drug.

	Discussion

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In the present study, both glibenclamide and a novel K_ATP antagonist, HMR 1883, prevented ischemically induced VF without altering the hemodynamic response to the coronary occlusion. Furthermore, both compounds prevented ischemically induced reductions in refractory period. Conversely, the activation of the K_ATP with pinacidil provoked reductions in refractory period similar to those noted during myocardial ischemia. Glibenclamide, in contrast to HMR 1883, significantly attenuated the reactive hyperemia associated with coronary occlusion release, reduced both mean CBF and left ventricular dP/dt maximum, increased plasma insulin concentration and promoted hypoglycemia. When considered together, these data suggest that activation of cardiac K_ATPs during myocardial ischemia may play an important role in both ischemically induced reductions in refractory period and the formation of malignant ventricular arrhythmias. The data further demonstrate that HMR 1883 preferentially blocks cardiac K_ATPs without adversely affecting either vascular or pancreatic K_ATPs.

HMR 1883 and selectivity for cardiac K_ATPs. In the present study, HMR 1883 could prevent ischemically induced reductions in ventricular refractory period without altering either plasma insulin, blood glucose or coronary reactive hyperemia. In agreement with these findings, Gögelein et al. (1998) demonstrated that in isolated guinea pig papillary muscle or single cardiomyocytes, HMR 1883 could attenuate the reductions in action potential duration induced by either the potent K_ATP opener rilmakalim hypoxia or metabolic inhibition. A similar inhibition has been reported for glibenclamide (Gögelein et al., 1998; Krause et al., 1995; Nakaya et al., 1991; Takizawa et al., 1996). However, HMR 1883 was >3 orders of magnitude less potent than glibenclamide in blocking the K_ATP activation in pancreatic  cells (Gögelein et al., 1998). The authors concluded that at therapeutic concentrations, HMR 1883 would preferentially block the activation of cardiac K_ATPs.

HMR 1883 and susceptibility to VF. In agreement with the present study, glibenclamide has been shown to prevent ventricular arrhythmias induced by myocardial ischemia in both isolated hearts (Bellemin-Baurreau et al., 1994; Chi et al., 1993; Gwilt et al., 1992; Tosaki and Hellegouarch, 1994) and intact preparations (Billman et al., 1993). For example, Wolleben et al. (1989) demonstrated that ischemia-induced VF in isolated rat heart was prevented by the sulfonylurea drugs glibenclamide and tolbutamide. In contrast, potassium channel agonists decreased the time to fibrillation (Chi et al., 1990, 1993; Wolleben et al., 1989). In a similar manner, Billman et al. (1993) further demonstrated that glibenclamide significantly reduced the incidence of VF induced by the combination of exercise and acute myocardial ischemia in animals previously shown to be susceptible to life-threatening arrhythmias. This drug suppressed ventricular fibrillation in 13 of 15 animals. However, in agreement with the present study and in contrast to HMR 1883 (compare figs. 4 and 5), glibenclamide provoked large reductions in both mean CBF and myocardial contractility as measured by left ventricular dP/dt maximum.

Cardiac K_ATP and ischemically induced VF: Possible mechanisms. Extracellular potassium has been known for some time to increase rapidly during ischemia (for reviews, see Billman, 1994; Coronel, 1994). The increased extracellular potassium promotes the depolarization of the tissue surrounding the ischemic regions, as well as reductions in action potential duration, which can provoke abnormalities of impulse conduction (Hicks and Cobbe, 1990; Nakaya et al., 1991; Venkatesh et al., 1991; Wilde et al., 1990). A major factor contributing to VF, particularly during myocardial ischemia, is a dispersion or inhomogeneity of refractory period resulting from regional differences in action potential duration (Janse and Wit, 1989). This allows for the fragmentation of impulse conduction during ensuing beats. The activation of the K_ATP produces large reductions in action potential duration, which are inhibited by glibenclamide (Bekheit et al., 1990; Kantor et al., 1990; Ruiz-Petrich et al., 1992) but exacerbated by pinacidil (Vanheel and de Hemptinne, 1992; Venkatesh et al., 1992). Glibenclamide has been reported to attenuate ischemically induced regional differences in refractory period (DiDiego and Antzelevitch, 1993; Kubota et al., 1993; Tweedie et al., 1993). In contrast, the K_ATP agonist pinacidil elicited a marked dispersion of repolarization and refractory period between the epicardium and endocardium, leading to the formation of extrasystoles (DiDiego and Antzelevitch, 1993). Therefore, HMR 1883, by preferentially blocking cardiac K_ATPs, could prevent potassium efflux and the resulting nonuniformities in refractory period between the ischemic and nonischemic tissue. As such, HMR 1883 could prevent VF by abolishing this substrate for reentrant arrhythmias induced by ischemia.