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(Circulation. 1997;96:1914-1922.)
© 1997 American Heart Association, Inc.

Articles

ß₂-Adrenergic Receptor Antagonists Protect Against Ventricular Fibrillation

In Vivo and In Vitro Evidence for Enhanced Sensitivity to ß₂-Adrenergic Stimulation in Animals Susceptible to Sudden Death

George E. Billman, PhD; Lourdes C. Castillo, BS; James Hensley, BS; Charlene M. Hohl, PhD; ; Ruth A. Altschuld, PhD

From the Departments of Physiology and Medical Biochemistry, Ohio State University, Columbus, Ohio.

Correspondence to George E. Billman, PhD, Department of Physiology, The Ohio State University, 302 Hamilton Hall, 1645 Neil Ave, Columbus OH 43210-1218. E-mail billman.1{at}postbox.acs.ohio-state.edu

	Abstract

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Background The ventricular myocardium contains functional ß₂-adrenergic receptors that when activated increase intracellular Ca²⁺ transients. Because elevated Ca²⁺ has been implicated in the induction of ventricular fibrillation (VF), it is possible that the activation of these receptors may also provoke malignant arrhythmias.

Methods and Results To test this hypothesis, a 2-minute occlusion of the left circumflex coronary artery was made during the last minute of exercise in 28 dogs with healed anterior myocardial infarctions: 17 had VF (susceptible) and 11 did not (resistant). On a subsequent day, this test was repeated after administration of the ß₂-adrenergic receptor antagonist ICI 118,551 (0.2 mg/kg). This drug did not alter the hemodynamic response to the coronary occlusion, yet it prevented VF in 10 of 11 animals tested (P<.001). However, heart rate was reduced in 6 animals. Therefore, the ICI 118,551 exercise-plus-ischemia test was repeated with heart rate held constant by ventricular pacing (n=3). ICI 118,551 still prevented VF when heart rate was maintained. Next, the effects of increasing doses of the ß₂-adrenergic receptor agonist zinterol on Ca²⁺ transient amplitudes were examined in ventricular myocytes. Zinterol elicited significantly greater increases in Ca²⁺ transient amplitudes at all doses tested (10^-9 to 10^-6 mol/L) in myocytes prepared from susceptible versus resistant animals. The cardiomyocyte response to isoproterenol (10^-7 mol/L) in the presence or absence of the selective ß₁- (CGP-20712A, 300 nmol/L) or ß₂- (ICI 118,551, 100 nmol/L) adrenergic receptor antagonist was also examined. Isoproterenol elicited larger Ca²⁺ transient increases in the susceptible myocytes, which were eliminated by ICI but not by CGP.

Conclusions When considered together, these data demonstrate that canine myocytes contain functional ß₂-adrenergic receptors that are activated to a greater extent in the susceptible animals. The resulting cytosolic Ca²⁺ transient increases may lead to afterpotentials that ultimately trigger VF in these animals.

Key Words: cells • death, sudden • myocardial ischemia • receptors, adrenergic, beta

	Introduction

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Disturbances in the autonomic control of the heart, particularly in response to myocardial ischemia, increase the propensity for malignant cardiac arrhythmias.^{1 2} Specifically, enhanced sympathetic activity can reduce cardiac electrical stability and induce ventricular fibrillation (VF) during ischemia.^{1 2} Presumably, the activation of myocardial adrenergic receptors mediates the arrhythmogenic effects of catecholamines released from sympathetic nerve terminals. It is widely accepted that the cardiac response to catecholamines is mediated primarily by ß-adrenergic receptors. Indeed, ß-adrenergic antagonists decrease the incidence of sudden cardiac death in post–myocardial infarction patients (for reviews see References 3 and 4^{3 4} ).

Until recently, it has been thought that myocardial ß-adrenergic receptors were primarily of the ß₁-subtype. However, evidence has accumulated that ventricular muscle also contains functional ß₂-adrenergic receptors (for review see Reference 5⁵ ). Furthermore, this subtype may become particularly important in certain pathological conditions.^{6 7 8} For example, ß₁-adrenergic receptor sensitivity decreases substantially during chronic heart failure, whereas the number of ß₂-adrenergic receptors remains relatively constant.^{6 7} As a consequence, the failing heart becomes more dependent on ß₂-adrenergic receptors for inotropic support. Under these conditions, it is possible that enhanced activation of ß₂-adrenergic receptors may increase the propensity for VF. Indeed, it is well established that post–myocardial infarction patients with poor cardiac pump function exhibit a much higher incidence of sudden cardiac death than patients in whom cardiac function is preserved.⁹ The role that ß₂-adrenergic receptors play in the induction of malignant arrhythmias, however, remains largely to be determined.

It was the purpose of this series of experiments to investigate the role that ß₂-adrenergic receptors play in the formation of malignant arrhythmias. Specifically, the hypothesis that the ß₂-adrenergic receptor antagonist ICI 118,551 would protect against VF during myocardial ischemia was tested in an intact, conscious animal model of sudden death. In addition, the effects of zinterol, a highly selective ß₂-adrenergic receptor agonist, were evaluated in myocytes isolated from the hearts of animals identified as being susceptible or resistant to the formation of VF during an exercise-plus-ischemia test. In particular, the hypothesis that ß₂-adrenergic receptor stimulation would elicit larger increases in Ca²⁺ transients in myocytes prepared from arrhythmia-prone hearts compared with arrhythmia-resistant hearts was tested.

	Methods

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The principles governing the care and use of animals, as expressed by the Declaration of Helsinki, adapted 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 Laboratory Animal Care and Use Committee.

Surgical Preparation
Heartworm-free mongrel dogs (n=41) weighing 15.4 to 20.9 kg were used in this study. The animals were anesthetized and instrumented to measure left circumflex coronary blood flow, left ventricular pressure, and the ventricular electrogram, as previously described.^{2 10} A hydraulic occluder was also placed around the left circumflex artery. 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 minutes and then tied off.^{2 10} All leads from the cardiovascular instrumentation were tunneled under the skin to exit on the back of the animal's neck. Finally, the animals were placed in an "intensive care" setting for the first 24 hours and placed on antiarrhythmic therapy as previously described.¹¹ Eleven animals died within the first 72 hours after myocardial infarction. Two additional animals could not be classified (see below) because of rupture of the coronary occluder. Thus, studies were completed on 28 of the original 41 animals.

Exercise-Plus-Ischemia Test
The studies began 3 to 4 weeks after 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.^{2 10} Briefly, the animals ran on a motor-driven treadmill while workload was increased every 3 minutes for a total of 18 minutes. The protocol began with a 3-minute warm-up period during which the animals ran at 4.8 km/h at a 0% grade. The speed was increased to 6.4 km/h and the grade was increased every 3 minutes 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 more minute (total occlusion time, 2 minutes). 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 to 20 seconds after VF began). The occlusion was immediately released if VF occurred. Seventeen animals developed VF (susceptible), and the remaining 11 did not (resistant).

One week later, the exercise-plus-ischemia test was repeated in 11 randomly selected susceptible animals after pretreatment with the selective ß₂-adrenergic receptor antagonist ICI 118,551¹² (0.2 mg/kg IV, Research Biochemical International). In a similar manner, the hemodynamic response to exercise was evaluated after pretreatment with ICI 118,551 in 7 randomly selected resistant animals. The hearts of the remaining susceptible (n=6) and resistant (n=4) animals were used for the preparation of myocytes (see below). The ß₁-adrenergic receptor agonist dobutamine HCl¹³ (Research Biochemical International) was injected as a bolus (250 µg IV) before and 3 to 5 minutes after the ICI 118,551 injection. In addition, the nonselective ß-adrenergic receptor agonist isoproterenol HCl (Isuprel, Winthrop Pharmaceuticals) was injected (1.0 µg/kg IV) before and after ICI 118,551. ICI 118,551 did not alter the inotropic (dP/dt_max) response to dobutamine (Fig 1; no drug, 3662±360; dobutamine, 6855±612; ICI, 3386±298; ICI plus dobutamine, 6993±463 mm Hg/s). In contrast, ICI 118,551 significantly (by 23.4%; P<.01) reduced the inotropic response to isoproterenol HCl (Fig 1; no drug, 3023±146; isoproterenol, 7686±1093; ICI, 2721±265; ICI plus isoproterenol, 6432±704 mm Hg/s). Finally, a second control (saline injection) exercise-plus-ischemia test was repeated 1 week after the ICI 118,551 treatment (susceptible only, n=7).

View larger version (0K): [in this window] [in a new window]   Figure 1. Representative recordings from same animal before and after pretreatment with ß2-adrenergic receptor antagonist ICI 118,551 (0.2 mg/kg). ICI 118,551 reduced inotropic response to isoproterenol (1.0 µg/kg, A) but did not alter response to ß1-adrenergic agonist dobutamine (250 µg, B). LVP indicates left ventricular pressure; HR, heart rate.

Because ICI 118,551 treatment produced a small reduction in heart rate (see below), the ICI 118,551 exercise-plus-ischemia test was repeated in 3 susceptible dogs with heart rate held constant by ventricular pacing with the epicardial leads. Heart rate was paced 20 beats above the highest heart rate noted during the control exercise-plus-ischemia test beginning 30 seconds before the occlusion and maintained until 30 seconds after the occlusion release.

Myocyte Isolation
On a subsequent day, the animals were anesthetized with sodium pentobarbital (10 mg/kg IV) and the heart was rapidly removed for the isolation of ventricular cardiomyocytes. Ventricular cardiomyocytes were isolated from 11 susceptible and 9 resistant animals by use of a collagenase perfusion as previously described.¹⁴ This procedure yields cells from primarily the midmyocardial wall, with very few cells obtained from either the epicardial or endocardial layers. The cells were obtained from the area perfused by the left circumflex coronary artery, and the ischemic "border" zone was avoided. Approximately half of the hearts (5 susceptible and 5 resistant animals) had been previously treated with ICI 118,551 (see above) 1 week before myocyte preparation. Thus, myocytes were obtained from 6 susceptible and 4 resistant animals that had not received prior treatment with ICI 118,551. The cells were suspended in a modified Krebs-Henseleit buffer, pH 7.4, containing (in mmol/L) NaCl 118, KCl 4.8, MgSO₄ 1.2, KH₂PO₄ 1.2, CaCl₂ 1.0, HEPES 20, Na pyruvate 5, glucose 4, insulin 0.001, glutamine 0.68, and NaHCO₃ 5, as well as penicillin-streptomycin, a complete mixture of amino acids (basal medium Eagle), vitamins, and 2% BSA.

Ca²⁺ Transient Measurement
The isolated cells were loaded with 2 µmol/L fura 2-AM for 3 minutes, followed by a 45-minute postincubation at room temperature to ensure complete hydrolysis of the ester groups. Cells were loaded into a Plexiglas superfusion chamber and allowed to attach to the bottom glass coverslip. After 5 minutes, superfusion at 2 mL/min, 37°C, pH 7.4 was begun with a 95% oxygen/5% carbon dioxide saturated Krebs-Henseleit buffer. The cells were field-stimulated with parallel platinum electrodes at 0.2 Hz. Fluorescence measurements, with excitation alternating between 340 and 380 nm, were collected at 30 points per second with a PTI filterscan. Sixteen consecutive transients were signal-averaged; the data were not smoothed. Cumulative dose-response curves were obtained for the ß₂-adrenergic agonist zinterol⁸ (Bristol-Myers) for cells obtained from 5 susceptible and 5 resistant animals (5 cells per animal). In a similar manner, the response to the mixed ß₂-adrenergic receptor agonist isoproterenol (100 nmol/L) was obtained before and after treatment with the ß₂-adrenergic receptor antagonist ICI 118,551 (100 nmol/L) or the ß₁-adrenergic receptor antagonist CGP-20712A⁸ (300 nmol/L, CIBA-Geigy). All three drug treatments (isoproterenol, isoproterenol plus ICI 118,551, and isoproterenol plus CGP-20712A) were given in a random order with a 10-minute washout period between treatments. Cells were obtained from 4 susceptible and 4 resistant animals for the study of isoproterenol effects.

Because mitochondria also accumulate and hydrolyze fura 2-AM, accurate calibration of the cytosolic dye signal becomes problematic.¹⁵ Data are therefore presented as the ratio between fluorescence intensity at 340 and 380 nm excitation. However, the amplitude and configuration of the fura 2–ratio transients are thought to reflect accurately the magnitude and time course of changes in cytosolic free Ca²⁺.⁸

Measurement of cAMP in Suspensions of Intact Canine Myocytes
Suspensions of canine myocytes (1.5 mg protein/mL) were incubated for 5 minutes at 37°C with increasing concentrations of zinterol or isoproterenol. Cells were then separated from the suspending medium and simultaneously extracted by rapid centrifugation through a layer of bromododecane into 2N perchloric acid. The acid extracts were neutralized with 1,2,2-trichlorotrifluoroethane-trioctylamine, and cAMP content was analyzed by radioimmunoassay as previously described.¹⁴

Data Analysis
All hemodynamic data were recorded on a Gould model 2800S eight-channel recorder and a Teac model MR-30 FM tape recorder. Coronary blood flow was measured with a University of Iowa Bioengineering flowmeter model 545 C-4. 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 last 5 seconds of each exercise level. The coronary occlusion data were averaged over the last 5 seconds before and at the 60-second line point (or VF onset) after occlusion onset. The in vivo and in vitro data were analyzed by ANOVA for repeated measures. When the F ratio was found to exceed a critical value (P<.05), Scheffé's test was used to compare the means. The effects of the drug intervention on arrhythmia formation were determined with a ² test with Yates' correction for continuity. All data are reported as mean±SEM. Cardiac arrhythmias were evaluated at a paper speed of 100 mm/s. Myocardial infarct size was not determined in the present study, because fresh tissue was needed to prepare the myocytes. However, previous studies demonstrated that the myocardial infarction was significantly larger (approximately twice as large) in the susceptible than in the resistant animals.^{11 16}

	Results

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In agreement with previous studies,^{2 10 11} the animals could be divided into two groups based on the response to the exercise-plus-ischemia test. Seventeen dogs developed VF (susceptible) and 11 did not (resistant). VF was reproducibly induced in the susceptible animals with each presentation of the control exercise-plus-ischemia test both before and 1 week after the ICI 118,551 treatment. The average time to VF onset was similar for both the first (76±13 seconds; range, 35 to 121 seconds) and the second (60±9 seconds; range, 41 to 101 seconds) control occlusions. The heart rate response to each control occlusion was also similar (neither the trial, F=0.97, nor the trialxocclusion interaction, F=3.58, was significantly different; first occlusion: control, 190±12; occlusion, 229±14 bpm; second occlusion: control, 170±15; occlusion, 225±12 bpm).

Effects of ß₂-Adrenergic Receptor Blockade
The effects of the selective ß₂-adrenergic receptor antagonist ICI 118,551 were evaluated in 11 susceptible animals. Representative recordings from the same susceptible animal before and after pretreatment with ICI 118,551 are displayed in Fig 2. ICI 118,551 significantly reduced the incidence of VF, suppressing life-threatening arrhythmias in 10 of 11 animals (², 14.85; P<.001). The hemodynamic responses to the coronary occlusion before and after ICI 118,551 are shown in the Table. ICI 118,551 did not alter these parameters either before or during the coronary occlusion. The coronary occlusion elicited significant (P<.01) increases in heart rate and left ventricular diastolic pressure, whereas left ventricular systolic pressure and left ventricular dP/dt decreased both before and after ß₂-adrenergic receptor blockade. ICI 118,551 significantly (P<.05) reduced heart rate both before and during the coronary occlusion. The change in heart rate elicited by the coronary occlusion was not affected by this intervention. In addition, the maximum heart rate noted during the occlusion was not changed in 5 animals but was reduced in 6 animals. Interestingly, the 1 animal that was not protected by ICI 118,551 exhibited a large reduction in heart rate. Nevertheless, because reductions in heart rate could contribute to the protection noted for ß₂-adrenergic receptor blockade, the ICI 118,551 exercise-plus-ischemia test was repeated with heart rate held constant by ventricular pacing. Three animals in which ICI 118,551 produced a reduction in heart rate were used in this study (control, 188±19; occlusion, 204±18; ICI 118,551, 173±27; occlusion, 194±17 bpm). Even when heart rate was maintained (220±20 bpm), ICI 118,551 prevented VF in all 3 animals. It therefore seems unlikely that changes in heart rate alone were responsible for the protection noted for the ß₂-adrenergic antagonist.

View larger version (0K): [in this window] [in a new window]   Figure 2. Representative recordings from same susceptible animal before and after pretreatment with ß2-adrenergic receptor antagonist ICI 118,551 (0.2 mg/kg). Note absence of ventricular arrhythmias. LVP indicates left ventricular pressure; CBF, left circumflex coronary blood flow; and HR, heart rate.

View this table: [in this window] [in a new window]   Table 1. Hemodynamic Effects of Coronary Artery Occlusion

ICI 118,551 produced similar effects on the hemodynamic response to exercise in both the susceptible and resistant animals; these data have therefore been combined. ICI 118,551 did not alter the left ventricular systolic pressure (Fig 3C) response to exercise. In contrast, ICI 118,551 elicited significant (P<.01) reductions in left ventricular dP/dt_max (Fig 3B) during the early lower-intensity exercise levels (ie, the first three levels of exercise). A small but significant (P<.05) reduction in heart rate was also noted during exercise (Fig 3A). These data suggest that ß₂-adrenergic stimulation may contribute to the positive inotropic and chronotropic responses to exercise, whereas other factors (presumably the activation of ß₁-adrenergic receptors) become more important as exercise progresses to more intense levels of effort.

View larger version (0K): [in this window] [in a new window]   Figure 3. Effects of ß2-adrenergic receptor antagonist ICI 118,551 (0.2 mg/kg) on hemodynamic response to submaximal exercise. Left ventricular (LV) pressure maximum (B) and heart rate (A) were reduced by ICI 118,551, while LV systolic pressure (LVSP, C) was not affected by this treatment. *P<.05, **P<.01 ICI 118,551 vs control (no drug).

ß₂-Adrenergic Receptor Agonist Effects on Isolated Myocytes
The data described above suggest that the activation of ß₂-adrenergic receptors may contribute to the development of malignant arrhythmias in susceptible animals. Because ß₂-adrenergic receptor agonists promote the release of catecholamines from nerve terminals,¹⁷ the effects of ß₂-adrenergic receptor agonists were further evaluated in isolated myocytes in which the direct cardiac effects could be evaluated free of the interference of neural actions. Representative examples of Ca²⁺ transients recorded in myocytes from 1 susceptible and 1 resistant animal before and after the ß₂-adrenergic receptor agonist zinterol are displayed in Fig 4. The same dose of zinterol elicited much larger increases in Ca²⁺ transient amplitudes in myocytes from susceptible versus resistant animals. The pooled data from 5 susceptible and 5 resistant animals (average, 5 cells per dog) are shown in Fig 5. Zinterol elicited significantly greater increases in Ca²⁺ transient amplitudes in myocytes from susceptible animals at the doses used in this study (10^-9 to 10^-6 mol/L).

View larger version (0K): [in this window] [in a new window]   Figure 4. Representative examples of Ca2+ transients recorded in myocytes obtained from one susceptible and one resistant animal before and after ß2-adrenergic receptor agonist zinterol (ZIN). Same dose of zinterol elicited a much larger increase in transient amplitude in susceptible vs resistant animal.

View larger version (0K): [in this window] [in a new window]   Figure 5. Effects of increasing doses of ß2-adrenergic receptor agonist zinterol on amplitude of Ca2+ transient recorded in cardiomyocytes obtained from hearts of susceptible (open bars, n=5) and resistant (solid bars, n=5) animals. Zinterol induced much larger increases in Ca2+ transient in susceptible vs resistant animals. **P<.01 susceptible vs resistant.

The contribution of the ß₁- and ß₂-adrenergic receptors to the elevation of Ca²⁺ transient amplitudes was further evaluated with the nonselective ß-adrenergic agonist isoproterenol in the presence or absence of the selective ß₁-adrenergic receptor antagonist CGP-20712A or the ß₂-adrenergic receptor antagonist ICI 118,551. Typical examples for 1 susceptible and 1 resistant animal are shown in Figs 6 and 7, respectively. The composite data from cells obtained from 4 susceptible and 4 resistant animals are presented in Fig 8. Isoproterenol elicited a significantly greater increase in the Ca²⁺ transient amplitude in the susceptible myocytes than in cells from the resistant animals. Ca²⁺ transient amplitudes were significantly reduced by either CGP-20712A or ICI 118,551 in both groups of cells. However, ICI 118,551 elicited a greater reduction in the Ca²⁺ transient amplitude than did CGP-20712A in susceptible myocytes. In contrast, the pattern was reversed in cells from the resistant animals. The ß₁-adrenergic receptor CGP-20712A elicited a much greater reduction in Ca²⁺ transient amplitude than was noted for the ß₂-adrenergic receptor antagonist ICI 118,551 in cells from the resistant animals. It is interesting to note that the Ca²⁺ transient amplitude after ICI 118,551 pretreatment was similar in both groups of cells; that is, ß₂-adrenergic receptor blockade eliminated the excess increase in peak Ca²⁺ induced by isoproterenol in cells from susceptible animals. These data suggest that ß₂-adrenergic receptor–mediated increases in cytosolic Ca²⁺ may contribute to the increased susceptibility to VF. In fact, isoproterenol induced aftertransients in myocytes from two additional susceptible animals. These aftertransients were eliminated by ICI 118,551 but not by CGP-20712A (Fig 9).

View larger version (0K): [in this window] [in a new window]   Figure 6. Representative examples of Ca2+ transients recorded in myocytes from one susceptible animal in response to ß-adrenergic receptor agonist isoproterenol (ISO, 100 nmol/L) before and after treatment with ß2-adrenergic receptor antagonist ICI 118,551 (ICI, 100 nmol/L) or ß1-adrenergic receptor antagonist CGP-20712A (CGP, 300 nmol/L).

View larger version (0K): [in this window] [in a new window]   Figure 7. Representative examples of Ca2+ transients recorded in myocytes from one resistant animal in response to ß-adrenergic receptor agonist isoproterenol (ISO, 100 nmol/L) before and after treatment with ß2-adrenergic receptor antagonist ICI 118,551 (ICI, 100 nmol/L) or ß1-adrenergic receptor antagonist CGP-20712A (CGP, 300 nmol/L).

View larger version (0K): [in this window] [in a new window]   Figure 8. Effects of ß2-adrenergic receptor antagonist ICI 118,551 (ICI, 100 nmol/L) and ß1-adrenergic receptor antagonist CGP-20712A (CGP, 300 nmol/L) on change in Ca2+ transient amplitude induced by isoproterenol (ISO, 100 nmol/L). Average increase (% from control) is presented for susceptible (n=4 hearts) and resistant (n=4 hearts) animals. **P<.01 susceptible vs resistant; P<.01 isoproterenol vs other treatments.

View larger version (0K): [in this window] [in a new window]   Figure 9. Ca2+ transients recorded in myocytes from one susceptible animal in which ß-adrenergic receptor agonist isoproterenol (ICI, 100 nmol/L) induced aftertransients. Treatment with ß2-adrenergic receptor antagonist ICI 118,551 (ICI, 100 nmol/L) but not ß1-adrenergic receptor antagonist CGP-20712A (CGP, 300 nmol/L) completely suppressed these aftertransients.

Myocyte cAMP Production
To characterize further the effects of ß₂-adrenergic receptor stimulation, the effects of isoproterenol and zinterol on cAMP production were examined in suspensions of myocytes obtained from susceptible and resistant animals. The enhanced effect of isoproterenol on Ca²⁺ transient amplitudes in myocytes from susceptible dogs was not associated with increased cAMP production. As shown in Fig 10, cAMP content of myocytes incubated with 10^-7 or 10^-6 mol/L isoproterenol did not differ between the two groups of cells. It was also found that these isoproterenol-induced increases in cAMP were not significantly reduced by blockade of the ß₂-adrenergic receptors with 100 nmol/L ICI 118,551 in either the susceptible or resistant dog myocytes. Conversely, the isoproterenol effect on cAMP production was reduced >80% by the addition of 300 nmol/L CGP-20712A to block the ß₁-adrenergic receptors. The greater zinterol response in myocytes from susceptible dogs was also unrelated to cAMP production: 10 µmol/L zinterol increased cAMP by an average of only 1.1 and 1.9 pmol/mg in cells from susceptible and resistant dogs, respectively. The slight increase in cAMP for both groups of cells may have resulted from activation of ß₁-receptors by the high zinterol concentration.

View larger version (0K): [in this window] [in a new window]   Figure 10. Effect of ß-adrenergic receptor agonist isoproterenol on myocyte cAMP levels. Open bars indicate susceptible animals; solid bars, resistant.

	Discussion

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Enhanced cardiac sympathetic activity elicited during myocardial ischemia can profoundly affect electrical stability¹ and has been linked to an increased risk for cardiac mortality in patients with ischemic heart disease.¹⁸ It therefore is not surprising that treatment with ß-adrenergic receptor antagonists significantly reduces cardiac mortality after myocardial infarction.^{3 4} The results of the present study suggest that cardiac ß₂- rather than ß₁-adrenergic receptors contribute substantially to the formation of malignant arrhythmias. The resulting Ca²⁺ overload could ultimately provoke the increased vulnerability to life-threatening ventricular arrhythmias.

ß-Adrenergic Receptors and Susceptibility to VF
In agreement with the present study, the nonselective ß-adrenergic receptor antagonist propranolol has been reported to prevent VF in this animal model.¹⁹ However, the effects of ß₂-adrenergic receptors were not evaluated in these studies. There have been at least 32 trials involving 29 000 patients in which ß-adrenergic receptor blockers have been initiated within the first few hours of either confirmed or suspected myocardial infarction.^{3 4} The pooled data from these studies suggest that early treatment with ß-adrenergic receptor blockers results in a 20% to 30% reduction of infarct size and a 15% decrease in cardiac mortality, probably as the result of a corresponding reduction in the incidence of VF.³ In contrast, we are not aware of any studies in which either the long- or short-term effects of ß₂-adrenergic receptor agonists or antagonists in arrhythmia formation during myocardial ischemia have been systematically investigated. However, when one carefully examines the clinical studies cited above, it becomes apparent that not all ß-adrenergic antagonists offer the same level of protection, particularly during acute myocardial infarction. For example, it is interesting to note that many but by no means all²⁰ of the studies using the ß₁-adrenergic receptor antagonist metoprolol failed to report significant reductions in the incidence of VF during acute myocardial infarction.^{21 22 23} Furthermore, although atenolol did reduce overall mortality by 15%, the number of patients who died as the result of malignant arrhythmias was not altered.²⁴ In contrast, propranolol almost completely eliminated death due to VF.^{25 26} These data suggest that a better antiarrhythmic protection can be achieved with complete (ie, ß₁- and ß₂-) rather than selective (ie, ß₁-) ß-adrenergic receptor blockade. Indeed, Newton and Parker²⁷ found that the nonselective ß-adrenergic antagonist propranolol afforded a more favorable inhibition of sympathetic outflow (decreased norepinephrine spillover) than did metoprolol (which markedly increased norepinephrine levels) in patients with heart failure. In agreement with the present results, these studies indicate that the activation of myocardial ß₂-adrenergic receptors may play a particularly important role in the induction of malignant arrhythmias during acute myocardial infarction. In this regard, it is interesting to note that there are isolated clinical reports in which ß₂-adrenergic receptor agonists have precipitated sudden death as a consequence of cardiac actions in asthmatic patients.^{28 29}

Speculation on ß₂-Adrenergic Receptor Mechanisms for VF
The mechanisms mediating the effects of ß₂-adrenergic receptor stimulation on cardiac muscle remain to be determined. However, as a consequence of chronic heart failure, there is a substantial loss of ß₁-adrenergic receptors with little or no loss in ß₂-adrenergic receptors.^{6 7} The failing heart therefore may become more dependent on the activation of ß₂-adrenergic receptors for inotropic support during sympathetic stimulation. Indeed, recent studies demonstrated that the ß₂-adrenergic receptor agonist zinterol elicited significantly larger Ca²⁺ transients in ventricular myocytes prepared from failing hearts compared with normal tissue.⁸ These ß₂-adrenergic receptor–mediated changes in cytosolic Ca²⁺ could also contribute significantly to the induction of VF. Elevations in intracellular Ca²⁺ can provoke oscillations in membrane potential^{30 31 32} that, if sufficient to reach threshold, can trigger repetitively sustained action potentials. These afterdepolarizations have been recorded in isolated tissue in response to interventions that favor Ca²⁺ loading (hypoxia, catecholamines, digitalis toxicity) and can be suppressed by Ca²⁺ antagonists (for reviews see References 31 and 32^{31 32} ). In fact, recent studies in isolated rabbit hearts demonstrated that a slow inward Ca²⁺ current was required for the initiation and maintenance of VF.³³ In related studies using the same animal model as the present study, Billman^{10 11 34 35} demonstrated that several organic and inorganic Ca²⁺ channel antagonists could prevent VF induced by an exercise-plus-ischemia test. Conversely, Bay K8644, a Ca²⁺ channel agonist, induced malignant arrhythmias in animals resistant to the development of VF.¹⁰ Ryanodine failed to prevent malignant arrhythmias in this model despite large reductions in cytosolic [Ca²⁺], as indicated by large reductions in contractile function.³⁶ When considered together, these studies strongly suggest that Ca²⁺ influx across the sarcolemma is critical for the induction of VF. Thus, ß₂-adrenergic receptor–mediated changes in Ca²⁺ influx could lead to the formation of arrhythmias and sudden death. In this regard, it is interesting to note that heart failure patients and patients with poor left ventricular contractile function are at a substantially greater risk of sudden death than are patients with more normal pump function.⁹ One might speculate that changes in Ca²⁺ influx, mediated by a greater activation of myocardial ß₂-adrenergic receptors, may provoke lethal cardiac arrhythmias in these patients.

In the present study, the selective ß₂-adrenergic receptor agonist zinterol elicited significantly larger increases in Ca²⁺ transient amplitudes in myocytes obtained from the hearts of susceptible compared with resistant animals. Furthermore, isoproterenol provoked larger Ca²⁺ transients in susceptible myocytes, an effect that was eliminated by ß₂-adrenergic receptor but not ß₁-adrenergic receptor antagonists. These data suggest that ß₂-adrenergic receptor activation does, in fact, provoke larger changes in cytosolic Ca²⁺ in the hearts of susceptible animals. Any resulting oscillations in membrane potential could provoke arrhythmia formation, particularly during myocardial ischemia. Indeed, aftertransients, which are generally accepted to trigger these oscillations in membrane potential,^{30 37} were recorded in cells obtained from the hearts of two susceptible dogs during stimulation with isoproterenol. These aftertransients were eliminated by the ß₂-adrenergic receptor antagonist ICI 118,551 but were not affected by the ß₁-adrenergic receptor antagonist CGP-20712A. Thus, these data suggest that an enhanced sensitivity to ß₂-adrenergic receptor stimulation can trigger abnormalities in intracellular Ca²⁺ that in turn may give rise to fluctuations in membrane potential that culminate in VF.

ß₂-Adrenergic Receptor Effects on cAMP Production
In contrast to isoproterenol, zinterol failed to increase cAMP levels in myocytes obtained from susceptible animals. These data suggest that alterations in cytosolic Ca²⁺ levels induced by ß₂-adrenergic receptor stimulation are largely cAMP independent. Consistent with these findings, previous studies demonstrated that interventions that increased cAMP levels (forskolin, IBMX, and 8-bromo-cAMP) failed to induce arrhythmias in resistant animals.³⁸ Other, as yet unidentified, mechanisms must be responsible for the Ca²⁺ transient changes noted as a consequence of ß₂-adrenergic receptor stimulation. For example, it is possible that ß₂-adrenergic receptor stimulation may either directly or via actions of a G protein alter cardiac ion channels (ie, increase the open probability of L-type Ca²⁺ channels). Indeed, direct G-protein effects have been reported for L-type Ca²⁺ channel activation.³⁹ These findings agree with results obtained from myocytes from failing hearts⁸ and normal hearts.⁴⁰ However, unlike the response noted in the failing heart, isoproterenol induced large increases in cAMP in the myocytes obtained from susceptible animals. Thus, ß₁-adrenergic receptor activity may be relatively normal in the susceptible myocardium, at least compared with the failing heart.

In summary, a ß₂-adrenergic receptor antagonist (ICI 118,551) significantly protected against VF in animals shown to be susceptible to malignant arrhythmias. In a similar manner, ß₂-adrenergic receptor agonists elicited significantly greater increases in the amplitude of Ca²⁺ transients in myocytes obtained from susceptible animals compared with those obtained from hearts of resistant animals. These increases in cytosolic Ca²⁺ occurred independently of changes in cAMP levels. When considered together, these data suggest that the activation of ß₂-adrenergic receptors may precipitate VF during myocardial ischemia via cAMP-independent increases in cytosolic Ca²⁺. It therefore seems likely that the inhibition of specific ß₂-adrenergic receptor pathways may represent a novel approach in the management of arrhythmias in patients with ischemic heart disease. The data further indicate that the recent suggestion that cardiac function may be improved by the enhanced activation of ß₂-adrenergic receptors in patients in cardiac failure without adverse cardiac actions⁴¹ needs to be reexamined. The activation of ventricular ß₂-adrenergic receptors could provoke life-threatening arrhythmias, particularly during myocardial ischemia in those patients with compromised cardiac function.

	Acknowledgments

 
These studies were supported by grants from the American Heart Association, Ohio Affiliate, and by NIH grants HL-36240 and HL-48835. Zinterol and CGP-20712A were generously provided by Bristol-Myers, Evansville, Ind, and CIBA-Geigy, Summit, NJ, respectively.

Received December 26, 1996; revision received April 2, 1997; accepted April 4, 1997.

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