(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
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
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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
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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 43 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.
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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/dtmax) 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).
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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
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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.
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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/dtmax (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.
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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).
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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).
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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.
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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 potential30 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 3231 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,
Billman10 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.
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Acknowledgments |
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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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