Introduction

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During regional ischemia, myocardial ATP-sensitive potassium (K_ATP) channels open (Noma, 1983) and extracellular potassium rises, leading to enhanced automaticity and a shortening of the refractory period. The arrhythmogenic potential is strongly enhanced by the local nature of myocardial ischemia, which translates into spatial heterogeneities in excitability, conduction, and refractoriness favoring reentrant arrhythmias. Regional ischemia also leads to typical electrocardiographic (ECG) changes. The shift of the S-T segment reflects heterogeneity in the plateau phase of action potentials as a consequence of the accelerated repolarization through opening of K_ATP channels. Intraventricular conduction is disturbed in both space and time (Gettes and Cascio, 1992), leading to an altered spectrum of the QRS complexes (Hatala et al., 1995) and an increase in Q-J time, which is mainly due to the slower, or even blocked, impulse conduction in the ischemic region. Several factors may be involved in the delay in ventricular activation, including an inexcitability of cells in the ischemic area, a decrease in diastolic membrane potential leading to a decreased availability of fast sodium channels with a lower conduction velocity, and an increase in the intercellular electrical resistance by uncoupling (Kleber et al., 1987). The ischemically induced increase in extracellular potassium concentration due to the opening of K_ATP channels is discussed as contributing to the deterioration of each of these changes (Hill and Gettes, 1980; Bekheit et al., 1990; Gettes and Cascio, 1992). Therefore, blocking the ischemia-induced opening of K_ATP channels seems to be a promising antiarrhythmic approach. In fact, the K_ATP channel blocker glibenclamide has been shown to inhibit ventricular fibrillation (VF) in various models of ischemia (Ballagi-Pordan et al., 1987; Kantor et al., 1987; Homburg et al., 1991; Gwilt et al., 1992; Billman et al., 1993; Wilde et al., 1993; Baczko et al., 1997), and more recently, the new compound HMR 1883, a cardioselective blocker of myocardial K_ATP channels (Gögelein et al., 1998), has been reported to inhibit sudden death due to VF in conscious dogs (Billman et al., 1998) and anesthetized rats (Linz et al., 1998a). In support of the profibrillatory action of sarcolemmal K_ATP channel opening as a consequence of myocardial ischemia, the K_ATP channel opener pinacidil was found to be profibrillatory in different animal models of cardiac ischemia (Chi et al., 1990, 1993; Tosaki et al., 1992).

HMR 1883 differs from the prototype glibenclamide by several properties, which could make it appropriate for the prevention of severe ischemic arrhythmias in patients. It is well tolerated, devoid of an insulin-releasing and vasoconstrictor effect at antifibrillatory doses, and does not inhibit the mitochondrial K_ATP channel (T. Sato, N. Sasaki, J. Seharaseyon, B. O'Rourke, E. Marbán, unpublished observations; Linz et al., 1998b) in contrast to the sarcolemmal K_ATP channel. The mitochondrial K_ATP channel is held responsible for the beneficial effect of ischemic preconditioning. Because myocardial ischemia is a major cause of sudden death due to VF (Gillum, 1989; Green, 1990), HMR 1883 has a life-saving potential against ischemically induced arrhythmias. Because K_ATP channels will be closed during normoxia, a K_ATP channel blocker would be expected to be electrophysiologically silent, having no effect on the ECG (particularly the Q-T interval) under baseline conditions. Therefore, it should be devoid of the typical proarrhythmic effects of class III potassium channel-blocking compounds, the occurrence of early afterdepolarizations and torsade de pointes, that arise from prolonged repolarization reflected by a longer Q-T interval in the ECG. If opening of K_ATP channels were significantly involved in shaping the ischemic ECG, K_ATP channel blockade should attenuate the typical effects of ischemia on the ECG.

In the current study, we investigated the antiarrhythmic effect of HMR 1883 in anesthetized pigs subject to left anterior descending coronary artery (LAD) occlusion and compared its ECG and hemodynamic effects during normoxia and ischemia.

	Materials and Methods

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Two separate studies were performed in anesthetized pigs of the German Landrace. In the first study, the effects of HMR 1883 on hemodynamics and ECG were investigated under baseline conditions. In the second study, we attempted to determine the effect of the K_ATP channel blocker on survival and ECG changes, S-T-segment deviation, and Q-J time. Detailed protocols for the two studies, including anesthesia, are given below.

The pigs were ventilated with room air and oxygen by a Bird Mark-7 respirator. Blood gas analysis (pO₂ and pCO₂) was performed at regular time intervals to control oxygen supply via the respirator to maintain pO₂ at >100 mm Hg and pCO₂ at <35 mm Hg.

Observations and Measurements

To measure hemodynamic parameters, tip catheters (Millar PC 350) were inserted into the left femoral artery ]systolic blood pressure (BP_s) and diastolic BP] and into the left ventricle via the right carotid artery [left ventricular pressure (LVP), left ventricular end-diastolic pressure (LVEDP), and heart rate (HR)]. The maximal rate of LVP increase (dp/dt) was derived by an analog differentiator. The LVP signal also triggered a cardiotachometer (HR).

Cardiac output (CO) was measured continuously by an ultrasound flow probe placed around the pulmonary artery and connected to an active redirection transit time flowmeter (model 206; ART2, Triton Technology Inc., San Diego, CA). Bipolar body surface ECGs were recorded using subcutaneous needle electrodes in the classic lead III arrangement. Hemodynamic parameters together with the ECG were recorded on an 8-channel polygraph (model TA 4000; Gould, Cleveland, OH) at a paper speed of either 5 mm/min (continuously) or 50 mm/s (intermittent for ECG evaluation). In some experiments, the ECG was also digitized at a sample rate of 1 kHz and periodically stored on a computer hard disk for later evaluation (Data Acquisition System MP 100WSW; AcqKnowledge Software, Harry Fein, World Precision Instruments, Berlin, Germany).

Effect of HMR 1883 on Hemodynamic and ECG under Baseline Conditions. Pigs of either sex (20-35 kg) were premedicated with 1.3 ml of a mixture of Tilest 500 and Rompun i.m. (75.6 mg of tiletamin HCl, 73.28 mg of zolacepam HCl, and 30.32 mg of xylacin HCl) and anesthetized with an intravenous bolus of 20 to 30 mg/kg pentobarbital sodium followed by a continuous infusion of 12 to 17 mg of pentobarbital/kg/h i.v. to maintain anesthesia. When stabile hemodynamic conditions and blood gas values were achieved for at least 20 min, the control values for the hemodynamic and ECG parameters were taken.

Calculated Parameters

Stroke volume (SV; ml/beat) is defined as SV = CO/HR. Total peripheral resistance (TPR; dyne * s * cm⁵) is defined as TPR = (BP_mean/CO)  * 79.9. Left ventricular stroke work (LVSW; J/beat) is defined as LVSW = (BP_mean  LVEDP)  * SV * 0.133 * 10³. Left ventricular minute work (J/min) is defined as LVSW * HR. Myocardial oxygen consumption (MO₂; ml O₂/min/100 g) is defined as MO₂ = K₁ * BP_s * HR + K₂[(0.8 BP_s + 0.2 diastolic BP) * HR * SV)/BW] + 1.43, where K₁ = 4.08 * 10⁴, K₂ = 3.25 * 10⁴, and BW = body weight (kg; Rooke and Feigl, 1982). Corrected Q-T (Q-T_c; ms) is defined as Q-T_c = Q-T/(R  R)^1/2 (Bazett, 1920).

Effect of HMR 1883 on Survival and ECG during Coronary Occlusion

Twenty-four pigs of the German Landrace (castrated males, 28-40 kg) were anesthetized with 10 ml of Ketavet (1 g of ketamine base) i.m. and 30 to 40 mg/kg pentobarbital sodium as i.v. bolus plus a continuous infusion of 12 to 17 mg pentobarbital/kg/h i.v. to maintain anesthesia.

A left thoracotomy was performed at the fifth intercostal space, the lung was retracted, the pericardium was incised, and the heart was suspended in a pericardial cradle. To induce ischemia, a small segment of the LAD, approximately 1 cm distal from its origin, was dissected from surrounding tissue and encircled loosely with a snare occluder. Occlusion was maintained for 20 min, and then the occluder was loosened to allow for reperfusion (20 min).

Experimental Protocol. When stabile hemodynamic conditions and blood gas values were achieved for at least 20 min, the control values were taken for the hemodynamic parameters and for the ECG. The animals were randomly assigned to one of two groups. In group 1, the animals received an i.v. bolus of physiological saline, whereas in group 2, HMR 1883 was administered at the dose 3.0 mg/kg i.v. Five minutes later, the LAD was occluded for 20 min, followed by a 20-min reperfusion period.

Evaluation of Area at Risk. A dye exclusion method (Evans blue) was applied to delineate the ischemic tissue from the nonischemic one. Briefly, at the end of the experiment, the LAD was occluded, the heart was quickly removed, and the aorta was cannulated. Via this cannula, 400 ml of a Evans blue solution (0.5% in physiological saline) were infused into the coronary vascular bed at a pressure of 80 mm Hg, which resulted in a heavy, dark-blue staining of the nonischemic area. After removal of the atria, the heart was cut into 8 to 10 sections at right angles to the long axis of the left ventricle. The nonstained ischemic areas and the blue-stained normal areas of the right ventricular free wall and of the left ventricle plus septum were dissected and weighed separately. The size of the ischemic zones were expressed as the percentage of the total left or right ventricular tissue.

Evaluation of S-T Segment Deviation and Q-J Time. Two parameters were analyzed from the ECG records: the ventricular activation time measured as the distance between the beginning of the Q wave and the J point (beginning of the S-T segment) and the S-T deviation. The latter was defined as the difference between the baseline of the ECG (T-Q segment) and the actual voltage 100 ms after the onset of the Q wave. This calculation procedure for the S-T segment was selected because the S-T segment was sometimes not parallel to the baseline but rather was downsloping or slow rising during ischemia.

Statistical Analysis. All data were expressed as mean ± S.E.. Differences in the mean values within the groups were analyzed with use of the paired Student's t test. To analyze the differences between the groups, either the unpaired Student's t test (hemodynamics, S-T segment deviation, ventricular activation time) or the Fisher's exact probability test (survival data) were used. A value of P < .05 was regarded as significant. In the control group of the LAD study, the animals that died early during ischemia were not included for the evaluation of the ECG parameters.

Preparation of Test Compound. HMR 1883 (1-[[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea) was synthesized in the Department of Medicinal Chemistry (Hoechst AG, Frankfurt/Main, Germany). For i.v. injection, HMR 1883 was dissolved immediately before use. Because of the poor solubility of HMR 1883, a special dissolution procedure had to be performed; 250 mg of the compound and 65 mg of Na₂CO₃ were added to 50 ml of distilled water. The suspension was stirred for approximately 30 min at 70-80°C until dissolution was completed.

	Results

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Effect of HMR 1883 on Hemodynamics and ECG under Baseline Conditions (First Study)

HMR 1883, given cumulatively at 1 and 3 mg/kg i.v. separated by an interval of 30 min, had no effect on hemodynamics and ECG (data not shown). A dose of 10 mg/kg was administered to a separate group (Table 1). Even at 10 mg/kg, HMR 1883 had no significant effects on hemodynamics, including BP, LVP, left ventricular contractility, CO, TPR, LVSW, and left ventricular oxygen consumption and ECG parameters, including R-R, P-Q (P-R), Q-T, Q-T_c interval (Table 1), S-T segment, and Q-J time. In addition, the shape of the ECG waves did not change significantly (Fig. 1).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Absence of significant effects of HMR 1883 (3 mg/kg i.v.) on ECG in an anesthetized pig before occlusion of the LAD.

Effect of HMR 1883 on Survival and ECG during Coronary Occlusion (Second Study)

Location and Size of Ischemic Area after Occlusion of LAD. In this separate study, occlusion of the LAD resulted in severe ventrolateral ischemia that affected significant parts of the left ventricle and the septum and, to a lesser extent, the right ventricle. Ischemia was transmural. A well-defined borderline was present between the ischemic zone and the adjacent nonischemic cardiac tissue.

Hemodynamic Effects of LAD Occlusion. In control pigs within 1 min, occlusion of the LAD resulted in a significant decrease in BP (BP_s, 18 mm Hg; diastolic BP, 13 mm Hg), LVP (17 mm Hg), and dp/dt (559 mm Hg/s; Table 2). HR showed a slight tendency to increase, whereas the LVEDP markedly increased. These effects persisted, with minor changes, throughout the entire occlusion period. The administration of 3 mg/kg HMR 1883 had no significant effect on the hemodynamic parameters before the coronary artery occlusion, as already shown in the previous experiment with 10 mg/kg.

ECG Effects, Arrhythmias, and Sudden Cardiac Death. Figure 2 (top) shows a representative example of the changes in the ECG observed during ischemia in a control pig. The first sign of ischemia was a depression of the S-T segment, which was detectable at 30 s of the occlusion. The S-T segment depression increased with time, was maximal at 5 min, and thereafter, showed a slight tendency to return toward baseline values (Figs. 2 and 3, top). Approximately 1 min after the beginning of the occlusion, the positive R wave started to decline. At 2 min, the shape of the QRS complex has completely changed into a Q-S pattern consisting of a large negative wave that was preceded by the more-or-less-marked initial Q wave.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   ECG effects of LAD occlusion in a control pig (top) and an HMR 1883-treated pig (bottom) at baseline (0 min) and 5, 10, and 15 min after occlusion. The untreated pig survived 19 min of occlusion (ischemic area, 35% of left ventricle and 7% of right ventricle). The HMR 1883-treated pig survived the entire experiment (ischemic area, 42% of left ventricle and 20% of right ventricle).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Effect of HMR 1883 on S-T-segment deviation (top) and Q-J time (bottom) during LAD occlusion. , control group. , HMR 1883. *p < .05. Values are mean ± S.E. (n = 8).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Effect of HMR 1883 (3 mg/kg i.v.) on survival during occlusion of the LAD and reperfusion. *p < .05 for the end of the occlusion period. Top curve, HMR 1883. Bottom curve, control animals.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Representative example of sudden death from ventricular fibrillation 17 min after LAD occlusion in a control pig.

	Discussion

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HMR 1883 significantly protected anesthetized pigs against ischemically induced arrhythmias and sudden cardiac death. The second major finding was the prevention of electrophysiological changes typically associated with myocardial ischemia. HMR 1883 reduced the ischemic S-T segment shift and the increase in Q-J time, the former reflecting heterogeneity in the plateau phase of action potentials and the latter reflecting irregular impulse propagation and delayed ventricular activation. In some animals, the ECG appeared quite normal despite large ischemic areas. During normoxia, however, HMR 1883 was silent, affecting neither the ECG nor hemodynamics. HMR 1883 had no effect on the delayed rectifier at 100 µM and no effect on action potential duration in vitro (Gögelein et al., 1998). Thus, in contrast to class III compounds, blockers of the delayed rectifier, the K_ATP channel blocker did not affect repolarization during normoxia reflected by an unchanged Q-T-interval, and hence, it should be devoid of the typical proarrhythmic effects of class III antiarrhythmic compounds. The protective effect of the K_ATP channel blocker HMR 1883 in our study indicates that opening of K_ATP channels during ischemia induces proarrhythmic mechanisms that are responsible for ischemic sudden cardiac death and for the typical ECG changes during ischemia.

Protection against sudden death by HMR 1883 in our study is consistent with results of Billman et al. (1993, 1998), who could demonstrate that pretreatment with either glibenclamide or HMR 1883 significantly reduced the incidence of VF induced by the combination of exercise and acute myocardial ischemia in conscious dogs susceptible to life-threatening arrhythmias. Moreover, HMR 1883 inhibited VF during ischemia and reperfusion in anesthetized rats (Linz et al., 1998a).

Effects of HMR 1883 on S-T Segment. One of the interesting findings of the present study was the fact that HMR 1883 could attenuate the S-T segment shift, although the LAD was completely occluded. This indicates a reduction in the heterogeneity of repolarization of the heart. A shift of the S-T segment is generally regarded as a clear indication for myocardial ischemia (Scher and Spach, 1979), and even a linear relationship between coronary flow reduction (stepwise down to 50%) and S-T segment deviation could be demonstrated in the anesthetized pig (Watanabe and Buffington, 1994). It is very likely, however, that this compound directly interfered with the electrophysiological mechanisms that occur as a consequence of reduced flow, the ischemically induced opening of K_ATP channels, and the ensuing electrophysiological derangements rather than with coronary flow itself for the following reasons. The pig, similar to healthy humans, has very few coronary collaterals (Weaver et al., 1986; Patterson and Kirk, 1983), and hence, residual flow is very low (6% of the normal blood flow; Muller et al., 1986), and the transition zone between normal flow and no-flow regions is less than 2 mm in width (Hearse et al., 1986). Although we cannot fully exclude that HMR 1883 may have somewhat improved the low residual flow in the ischemic area via collaterals, this is unlikely because the blockade of vascular K_ATP channels is a vasoconstrictor mechanism as shown with glibenclamide. HMR 1883 is devoid of a significant vasoconstrictor effect, however. In contrast to glibenclamide, it neither reduced coronary blood flow during normoxia (Gögelein et al., 1998) nor inhibited the reactive hyperemia occurring after the opening of an occluded coronary artery in dogs (Billman et al., 1998).

Effect of HMR 1883 on Q-J Time. Besides enhanced automaticity, ischemia also provides the substrate for reentry by increasing dispersion of excitability, reducing conduction velocities, and shortening refractory periods in ischemic areas (Coronel, 1994). Occlusion of the LAD resulted in a significant increase in Q-J time. The Q-J time reflects ventricular activation, the time needed for the electrical impulse to travel through the fast-conducting His-Purkinje system to the cardiomyocytes, and to activate the majority of these cells so that their action potential is in phase 2. Regional ischemic injury within the myocardium disturbs intraventricular conduction (Gettes and Cascio, 1992), in both space and time, leading to an altered spectrum of the QRS complex (Hatala et al., 1995) and an increase in Q-J time. The latter is mainly due to the slower or even blocked impulse conduction in the ischemic region. An altered spectrum of the QRS complex and an increase in the Q-J time have been demonstrated in the ischemic control group of our study. Several factors may have contributed to the observed delay in ventricular activation, including an inexcitability of cells in the ischemic area, a decrease in membrane potential leading to a decreased availability of fast sodium channels with a lower conduction velocity, and an increase in the intercellular electrical resistance via uncoupling (Kleber et al., 1987). Intercellular coupling with low-resistance pathways is essential for impulse propagation. Uncoupling starts after 10 min of coronary artery occlusion (Smith et al., 1995). Ischemically induced increase in extracellular potassium due to the opening of K_ATP channels is considered to contribute to the deterioration of each of these changes (Hill and Gettes, 1980; Bekheit et al., 1990; Gettes and Cascio, 1992).