Vol. 91, Issue 4, 1627-1637, October 2001
Enhanced in vivo and in vitro contractile responses to
2-adrenergic receptor stimulation in dogs
susceptible to lethal arrhythmias
Melanie S.
Houle1,
Ruth
A.
Altschuld2, and
George E.
Billman1
Departments of 1 Physiology and Cell Biology and
2 Molecular and Cellular Biochemistry, The Ohio State
University, Columbus, Ohio 43210
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ABSTRACT |
The response to 
-adrenergic
receptor (-AR) stimulation was evaluated in both isolated
cardiomyocytes (video edge detection) and the intact animal
(echocardiography) in dogs either susceptible (S) or resistant (R) to
ventricular fibrillation induced by a 2-min coronary occlusion during
the last minute of exercise. In the intact animal, velocity of
circumferential fiber shortening (Vcf) was evaluated both before
(n = 27, S = 12 and R = 15) and after
myocardial infarction. Before infarction, increasing doses of
isoproterenol provoked similar contractile and heart rate responses in
each group of dogs. Either 1-AR (bisoprolol) or
2-AR (ICI-118551) antagonists reduced the isoproterenol
response, with a larger reduction noted after the 1-AR
blockade. In contrast, after infarction, isoproterenol induced a
significantly larger Vcf and heart rate response in the susceptible
animals that was eliminated by 2-AR blockade. The
single-cell isotonic shortening response to isoproterenol (100 nM) was
also larger in cells obtained from susceptible compared with resistant
dogs and was reduced to a greater extent by 2-AR blockade in the susceptible dog myocytes (S, 
48%, n = 6; R, 15%, n = 9). When considered together, these
data suggest that myocardial infarction provoked an enhanced
2-AR response in susceptible, but not resistant, animals.
sudden cardiac death; -adrenergic receptors; myocardial
ischemia; inotropy; contractility; ventricular fibrillation
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INTRODUCTION |
DISTURBANCES IN THE
AUTONOMIC control of the heart associated with cardiovascular
disease increase the risk for malignant cardiac arrhythmias (for
reviews, see Refs. 13, 24). Specifically, enhanced sympathetic activity can reduce cardiac electrical stability, culminating in ventricular fibrillation (VF). The activation of myocardial adrenergic receptors presumably mediates the arrhythmogenic effects of the catecholamines released from the sympathetic nerve terminals. Until recently, the myocardial -adrenergic receptors (-AR) were thought to be primarily of the
1-adrenergic subtype. However, it is now known that
ventricular myocytes also contain functional 2-AR that
may become particularly important under certain pathological conditions
(1, 12). For example, 1-AR sensitivity
decreases substantially as the result of heart failure, whereas
2-AR number remains relatively constant (1, 9, 12). As a consequence, the failing heart becomes more dependent on the 2-AR for inotropic support. However, the
activation of these receptors may also alter the cardiac electrical
stability, increasing the propensity for the formation of malignant
arrhythmias in the diseased heart.
We recently demonstrated that the selective 2-AR
antagonist ICI-118551 almost completely suppressed VF induced by acute
myocardial ischemia in animals with healed myocardial
infarctions (4). Furthermore, the selective
2-AR agonist zinterol elicited significantly greater
increases in calcium transient amplitude during electrical stimulation
of ventricular myocytes isolated from dogs susceptible to VF compared
with cells from hearts of animals resistant to the formation of
malignant arrhythmias (4). One would predict that this
increased intracellular calcium should also produce an augmentation of
the contractile function in the isolated single cells and the heart in
the intact animal. The effects of 2-AR stimulation on
contractile function were not investigated and remain to be determined.
Furthermore, it is not known whether the altered 2-AR
response noted in the susceptible animals resulted from the myocardial
infarction or was present in these animals before the ischemic injury.
Therefore, it was the purpose of this study to examine the role that
cardiac 2-AR activation plays in the regulation of the contractile response both in single cardiomyocytes and in the intact
heart of animals known to be either susceptible or resistant to VF.
Specifically, the hypothesis that 2-AR stimulation
elicits a greater response in animals susceptible to malignant
arrhythmias than in those animals resistant to VF was investigated with
echocardiography in an intact canine model of sudden death. In a
similar manner, the single-cell shortening response to -AR
stimulation was used to evaluate the contractile response in cells
isolated from both groups of animals. Furthermore, to determine the
effects of myocardial infarction on the 2-AR
responsiveness, the echocardiography studies were performed both before
and after the permanent ligation of the left anterior descending artery.
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MATERIALS AND METHODS |
The principles governing the care and use of animals, as
expressed by the Declaration of Helsinki and adapted by the American Physiological Society, were followed at all times during this study. In
addition, the Ohio State University Institutional Laboratory Animals
Care and Use Committee approved all the procedures.
Surgical preparation of the canine model of sudden death.
A total of 45 heartworm-free purpose-bred mongrel dogs (Covance
Research Products, Cumberland, VA), weighing 14.3-20.4 kg, was
used in this study. Twenty-four hours before surgery, a transdermal fentanyl patch that delivers 75 µg/h for 72 h (Duragesic, Jansen Pharmaceutica, Titusville, NJ) was placed on the left side of the
animal's neck and secured with tape. On the day of surgery, the dogs
received 15 mg (volume 1 ml im) morphine (Elkins-Sinn, Cherry Hill, NJ)
and thiopental sodium (Baxter Healthcare, Glendale, CA; 20 mg/ml iv) to
induce anesthesia. Each dog was given between 17 and 20 mg/kg of the
thiopental sodium depending on the individual response. The dogs were
then intubated, and a surgical plane of anesthesia was maintained by
inhalation of isoflurane (1-1.5%, Baxter Healthcare, Glendale,
CA). With the use of strict aseptic techniques, a left thoracotomy was
made in the fourth intercostal space. The heart was exposed and
supported by a pericardial cradle. The left circumflex coronary artery
was dissected free of the surrounding tissue. A 20-MHz pulsed Doppler
flow transducer and a hydraulic occluder were placed around the vessel.
Insulated silver-copper wires were sutured to the epicardial surface of the left and right ventricles for the later recordings of a ventricular electrogram. A two-stage occlusion of the left anterior descending coronary artery was performed approximately one-third the distance from
the vessel's origin to produce an anterior wall myocardial infarction
(4, 6, 23). The vessel was partially occluded for 20 min
and then tied off. All of the leads to the cardiovascular instruments
were tunneled under the skin to exit on the back of the animal's neck.
In addition to the fentanyl patch described above, morphine sulfate
(1.0 mg/kg sc) was given as needed to control any postoperative pain.
The long-lasting local anesthetic 0.25% bupivacaine hydrochloride (Abbott Laboratories, North Chicago, IL) was also injected in each of
three sites (0.5 ml) to block the intercostal nerves in the area of the
incision to minimize discomfort to the animal. Each animal was placed
on antibiotic therapy (amoxicillin 500 mg per os, Teva Pharmaceuticals,
Sellersville, PA) twice daily for 7 days.
The animals were then placed in an "intensive care setting" for the
first 24 h. To minimize the incidence of arrhythmias, the dogs
received 100 mg lidocaine hydrochloride (im) (Elkins-Sinn) before
surgery, which was supplemented (60 mg iv) before each stage of the
two-stage occlusion. The dogs also received 500 mg of procainamide
hydrochloride (im; Abbott Laboratories) before the surgery.
Exercise-plus-ischemia test: classification of the dogs.
An exercise-plus-ischemia test was used only to classify the
animals as either susceptible or resistant to VF. Approximately 3-4 wk after the production of the myocardial infarction, the animals were trained to walk on a motor-driven treadmill. For several
days before the classification, the animals were brought to the
laboratory to familiarize them with the setting. After this adaptation
period, the exercise-plus-ischemia test was performed as
previously described (4, 6, 23). Briefly, the exercise level was increased every 3 min until a critical heart rate of 70% of
the maximum (~210 beats/min) had been achieved. During the last
minute of exercise, the left circumflex coronary artery was occluded.
The treadmill was then stopped, and the occlusion was maintained for an
additional minute. The total occlusion time was 2 min. Large metal
plates (11-cm diameter) were placed across the animal's chest so that
electrical defibrillation (Zoll M series defibrillator, Zoll Medical,
Burlington, MA) could be achieved with minimal delay but only after the
animal was unconscious. Of the 45 animals that underwent surgery, 11 died acutely within 48 h of surgery, 2 were unable to be
classified due to loss of the occluder, and there was 1 additional
animal in which the electrocardiogram could not be obtained. Of the 31 remaining dogs that were classified, 12 were susceptible and 19 were
resistant to VF. Electrocardiogram, heart rate, and left circumflex
coronary blood flow were recorded throughout the
exercise-plus-ischemia test. Left circumflex coronary blood
flow was measured to confirm that the coronary occlusion was complete.
Echocardiography studies.
The echocardiography studies described below were performed ~1 wk
before surgery. Of the 45 animals that eventually underwent surgery, 36 dogs were tested before surgery. One dog died acutely during surgery
due to anesthesia overdose, seven animals died within the first 48 h postsurgery, and one dog's heart adhered to the chest wall and as
such a reliable echocardiogram could not be obtained. Therefore, 27 of
the 36 dogs were eventually classified (susceptible n = 12 and resistant n = 15). The studies were again
performed 2-3 wk postsurgery on the animals surviving surgery
(n = 27 susceptible; n = 12 resistant
n = 15). All of the studies were performed, and the
data were analyzed before the classification test. The dogs were
lightly sedated with acepromazine maleate (0.5 mg/kg im; Ft. Dodge
Animal Health, Ft. Dodge, IA) before the studies. A conventional M-mode
echocardiogram (Fig. 1) was obtained by
using a Sonos 1000 system (Hewlett-Packard, Palo Alto, CA) with a
5.5-MHz transducer. The velocity of circumferential fiber shortening
(Vcf) of the left ventricle was determined to provide a
load-independent measure of contractility incorporating both the extent
and the velocity of fiber shortening (7). The Vcf was
calculated from M-mode echocardiograms by use of the
formula
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where LVIDd is the short axis of the left ventricle in
diastole (cm), LVIDs is the short axis of the left ventricle in systole (cm), and ET is the ejection time (s). Dividing by the square root of
the R wave-R wave interval from a simultaneously collected electrocardiograph corrected for changes in heart rate
(10).
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Fig. 1.
Representative left ventricular M-mode echocardiogram of
the left ventricle. LVIDd, left ventricular internal diameter during
diastole; LVIDs, left ventricular internal diameter during systole.
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The total -AR response was quantified by infusing increasing doses
of isoproterenol (Sigma Chemical, St. Louis, MO) (0.006, 0.017, 0.06, 0.18, and 0.66 µg · min
1 · kg1). The
echocardiogram was obtained after a steady-state heart rate response
had been achieved at each dose. Some of the dogs (presurgery,
n = 6 for susceptible and n = 2 for
resistant; postsurgery, n = 6 for susceptible and
n = 5 for resistant) did not receive the highest dose
of isoproterenol if the maximum response was reached at a lower dose.
After the mixed 1/2 in vivo response was
obtained, a bolus injection of either the 1-AR
antagonist (bisoprolol 0.6 mg/kg) (Merck, Darmstadt, Germany)
(21) or the 2-AR antagonist (ICI-118551
0.2 mg/kg) (RBI, Natick, MA) was given (4, 21). The
-AR blockers were given at a dose previously determined to be
selective for the specific -AR yet achieved an effective receptor
antagonism (4, 21). The isoproterenol dose-response
infusion was then repeated. The study was repeated 1 wk later with the
alternate -AR antagonist. Approximately half of the dogs received
the bisoprolol first whereas the other half received the ICI-118551 first.
To test the hypothesis that the decrease in isoproterenol response was
due to the presence of a -AR antagonist and not to desensitization
and/or downregulation of the receptor, the echocardiogram studies were
repeated in 6 animals ~2-3 wk after surgery. In this set of
experiments, the isoproterenol dose response was performed as described
above and then repeated a few minutes later (i.e., without the addition
of the -AR antagonist).
Isolation of ventricular myocytes.
On a subsequent day, the dogs (susceptible n = 6 and
resistant n = 9) were anesthetized with pentobarbital
sodium (10 mg/kg iv, Nembutal, Abbott Laboratories), and the heart was
rapidly excised (approximate removal time 3-5 min) for the
isolation of ventricular myocytes. Myocytes were isolated from these
animals by use of a collagenase perfusion procedure as previously
described (2, 4, 19). This procedure yields cells
primarily from the midmyocardial wall, with very few cells obtained
from either the epicardial or endocardial layers. Preliminary studies
demonstrated that the isoproterenol response was similar in cells
obtained from either the left or right ventricle. Because of the
implantation of the Doppler flow transducer around the left circumflex
artery and the ligation of the left anterior descending artery, it
proved to be easier technically to cannulate the right coronary artery. Therefore, the cells were obtained from a noninfarcted section of the
right ventricular free wall. The cells were suspended in a modified
Krebs-Henseleit buffer, pH 7.4, containing (in mM): 118 NaCl, 4.8 KCl,
1.2 MgSO4, 1.2 KH2PO4, 1.0 CaCl2, 20 HEPES, 5 sodium pyruvate, 4 glucose, 0.001 insulin, 0.68 glutamine, and 5 NaHCO3, as well
as penicillin-streptomycin, a complete mixture of amino acids (basal
medium Eagle), vitamins, and 2% bovine serum albumin. Cell
viability (exclusion of 0.3% trypan blue) was assessed by standard
cell-counting techniques using bright field light microscopy and a hemocytometer.
Edge-detection protocol.
Single-cell isotonic shortening was recorded in the isolated myocytes
(susceptible n = 6, resistant n = 9).
The visible motion of a single myocyte was collected by using a
low-light video camera (Diagnostics Instruments, Sterling Heights, MI)
and analyzed by using a video edge detector (Crescent Electronics,
Sandy, UT). The images were stored by use of a standard VCR.
Edge-detection software (Felix Software, Photon Technology
International, Monmouth Junction, NJ) was used for the quantitative
analysis of the digitized images.
The cells were placed in a flow-through chamber and were then allowed
to adhere to chamber surface (~10 min). The myocytes were stimulated
at the rate of 0.5 Hz (Crescent Electronics Stimulator) and superfused
with gassed (95% O2-5% CO2) 25 mM bicarbonate
buffer (pH 7.3-7.5) at 37° C containing (in mM): 121 NaCl, 4.8 KCl, 1.2 MgSO4, 1.2 KH2PO4, 1.8 CaCl2, and 5 Na pyruvate. The buffer with and without the
drug of interest was pumped across the flow-through chamber containing
the cells at the rate of 2 ml/min. The changes in the single-cell
shortening were evaluated in the presence of the control bicarbonate
buffer, 100 nM isoproterenol, and 100 nM isoproterenol combined with
either the 1- or 2-AR antagonists, ICI-118551 (100 nM) or bisoprolol (200 nM) (2, 4). The
isoproterenol perfusion was repeated a total of three time on cells
obtained from one susceptible and four resistant animals. This test
served as a control to confirm that the decreased response noted for a
given antagonist resulted from the actions of the antagonist and not as
the result of receptor desensitization or downregulation due to
repeated exposure to the isoproterenol.
Statistical and data analysis.
Results are expressed as means ± SE. Statistical significance of
the echocardiogram Vcf and heart rate data was determined by performing
a three-way ANOVA [group (2 levels) × drug (3 levels) × dose (6 levels)] with repeated measures on two factors (drug and
dose). Similar comparisons were made before and after myocardial infarction. For the single-cell unloaded shortening data, cells from a
given animal were averaged, and the mean values for each animal were
then used in the ANOVA. Statistical analysis of these data was
performed using a two-way ANOVA [group (2 levels) × drug (3 levels) with repeated measures on one factor (drug)]. If the F ratio was found to exceed a critical value
(P < 0.05), then the significance of the differences
between the group means were determined by using Scheffé's test.
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RESULTS |
Analysis of preinfarction echocardiograms.
Preinfarction echocardiograms were obtained from 36 of the 45 dogs used
in this study. Of the 36 dogs, 9 were unable to be subsequently
classified and therefore were excluded from the study. Of the 27 remaining dogs, 12 were eventually found to be susceptible to VF
whereas 15 were resistant to malignant arrhythmias.
Representative echocardiograms from one preinfarction susceptible and
one resistant dog are shown in Fig. 2.
The composite Vcf data obtained from the echocardiograms from all the
animals are displayed in Figs. 3-4. The susceptible and resistant
animals had similar Vcf values when challenged with increasing doses of isoproterenol (Fig. 3). In dogs given the
1-AR antagonist bisoprolol, the Vcf value was
significantly and equally decreased in both the resistant and the
susceptible animals; that is, bisoprolol elicited similar changes in
both groups of animals (Fig. 4).
Likewise, the 2-AR antagonist ICI-118551 significantly
decreased the Vcf value to the same extent in both groups of animals
(Fig. 4). The attenuation caused by bisoprolol, however, was
significantly greater at the highest dose of isoproterenol than that
achieved by ICI-118551 in both groups of animals, indicating that a
larger percentage of the isoproterenol response was due to
1-AR stimulation (Fig. 4).
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Fig. 2.
Representative left ventricular M-mode echocardiograms recorded
in 1 resistant (A) and 1 susceptible (B) dog
before myocardial infarction. a: Baseline before the
infusion of drug. b: During the infusion of 0.66 µg · min1 · kg1
isoproterenol (Iso). c: During the infusion of 0.66 µg · min1 · kg1 Iso
in the presence of 0.2 mg/kg of ICI-118551 (ICI). d: During
the infusion of 0.66 µg · min1 · kg1 Iso in
the presence of 0.6 mg/kg of bisoprolol (Bis).
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Fig. 3.
Effects of increasing doses of Iso on the velocity of
circumferential fiber shortening (Vcf) before myocardial infarction.
Note that Iso elicited similar increases in both susceptible (Sus,
n = 12) and resistant (Res, n = 15)
animals. Dose 0 is the baseline Vcf value before the
addition of drug, and doses 1-5 are 0.006, 0.017, 0.06, 0.18, and 0.66 µg · min1 · kg1 Iso,
respectively.
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Fig. 4.
Effects of selective -adrenergic receptor (-AR)
antagonists on the maximum Iso-induced contractile (Vcf) response
before myocardial infarction. Note the similar response in both
resistant (n = 15) and susceptible (n = 12) animals both before and after each -AR antagonist.
1-AR antagonist, Bis = 0.6 mg/kg;
2-AR antagonist, ICI = 0.2 mg/kg.
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Both the susceptible and the resistant animals experienced a
significant but equal increase in heart rate with increasing doses of
isoproterenol (Fig. 5). As with the Vcf
data, bisoprolol and ICI-118551 significantly decreased the heart rate
response to the same extent in both groups of animals, with bisoprolol having the greater effect (Fig. 6).
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Fig. 5.
Effects of increasing doses of Iso on heart rate before
myocardial infarction. Note that Iso elicited similar increases in both
susceptible (n = 12) and resistant (n = 15) animals. Dose 0 is the baseline heart rate value before
the addition of drug, and doses 1-5 are 0.006, 0.017, 0.06, 0.18, and 0.66 µg · min1 · kg1 Iso,
respectively.
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Fig. 6.
Effects of selective -AR antagonists on the maximum
Iso-induced heart rate response before myocardial infarction. Note the
similar response in both resistant (n = 15) and
susceptible (n = 12) animals both before and after each
-AR antagonist. Bis = 0.6 mg/kg; ICI = 0.2 mg/kg.
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Analysis of the postinfarction echocardiograms.
Of the 45 dogs that underwent surgery, postinfarction echocardiograms
were obtained from 27 dogs. Of these 27 dogs, 12 were subsequently
found to be susceptible and 15 were resistant to VF. These
echocardiograms were obtained ~2-3 wk after the surgery. Representative echocardiograms obtained from one postinfarction susceptible and one postinfarction resistant dog are shown in Fig.
7.
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Fig. 7.
Representative left ventricular M-mode echocardiograms recorded in
1 resistant (A) and a susceptible (B) dog after
myocardial infarction. a: Baseline before the infusion of
drug. b: During the infusion of 0.66 µg · min1 · kg1 Iso.
c: During the infusion of 0.66 µg · min1 · kg1 Iso in
the presence of 0.2 mg/kg of ICI. d: During the infusion of
0.66 µg · min1 · kg1 Iso
in the presence of 0.6 mg/kg of Bis.
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Myocardial infarction elicited significant (P < 0.01)
increases in baseline (i.e., predrug) Vcf values in the susceptible dogs (preinfarct 3.4 ± 0.2 vs. postinfarct 4.3 ± 0.2) but
not the resistant animals (preinfarct 3.4 ± 0.2 vs. postinfarct
3.9 ± 0.4). Increasing doses of isoproterenol caused a
significant increase in the Vcf value compared with the baseline
reading in both the susceptible and the resistant animals (Fig.
8). However, the susceptible animals
showed a much larger Vcf than the resistant animals at almost every
dose (Fig. 9). In comparing the
preinfarct isoproterenol increases in Vcf to those seen postinfarct, we
found that the resistant animals showed the same level of increase, whereas the susceptible animals had significantly larger increases at
doses 1-4 (compare Figs. 3 and 8). It should be
emphasized that, despite the elevated baseline Vcf values,
isoproterenol provoked greater increases in the susceptible animals
(e.g., change from baseline at drug dose 4, susceptible
6.4 ± 0.7 vs. resistant 4.6 ± 1.1).
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Fig. 8.
Effects of increasing doses of Iso on Vcf after
myocardial infarction. Note that Iso elicited significantly greater
increases in the Vcf response in the susceptible (n = 12) animals compared with the resistant (n = 15).
Dose 0 is the baseline Vcf value before the addition of
drug, and doses 1-5 are 0.006, 0.017, 0.06, 0.18, and
0.66 µg · min1 · kg1 Iso,
respectively. *P < 0.01 susceptible vs. resistant
dogs.
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Fig. 9.
Effects of selective -AR antagonists on the maximum
Iso-induced Vcf response after myocardial infarction in both resistant
(n = 15) and susceptible (n = 12)
animals. Note the increased Iso response in the susceptible animals.
Bis = 0.6 mg/kg; ICI = 0.2 mg/kg. *P < 0.01 susceptible vs. resistant animals at a given treatment.
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After myocardial infarction, both bisoprolol and ICI-118551 attenuated
the isoproterenol-induced increases in Vcf in each group of animals
(Fig. 9). In the resistant animals, bisoprolol caused a greater
attenuation of the isoproterenol Vcf response than ICI-118551 (Fig. 9).
These data indicate that most of the isoproterenol response observed in
the resistant dogs was due to 1-AR stimulation.
Susceptible animals, however, showed nearly equal decreases with either
-AR blocker. These data suggest that the isoproterenol response in
the susceptible animals resulted from equal contributions of the
1-AR and the 2-AR (Fig. 9). After
pretreatment with the 2-AR antagonist ICI-118551, both groups of animals responded similarly to isoproterenol. However, after pretreatment with 1-AR blocker bisoprolol, the
susceptible animals still exhibited significantly greater increases in
the Vcf response to isoproterenol. These data once again suggest
that both groups of animals had large and nearly equal
1-AR responses to isoproterenol. However, in contrast to
the resistant animals, the susceptible animals displayed an increased
2-AR response.
The first and second dose of isoproterenol (i.e., a second control
infusion given without the presence of a receptor antagonist) provoked
similar changes in both Vcf and heart rate (n = 6, data not shown). As such, the attenuated response to isoproterenol noted
after the pretreatment with a given -AR antagonist almost certainly
resulted from the actions of the antagonists on the target receptors
and not as the result of -AR desensitization or downregulation.
Myocardial infarction also provoked significant (P < 0.01) increases in the baseline (i.e., predrug) heart rate in both
susceptible (preinfarct 68 ± 5 vs. postinfarct 95 ± 6 beats/min) and resistant (preinfarct 73 ± 6 vs. postinfarct
92 ± 6 beats/min) animals. The heart rate response to increasing
doses of isoproterenol was also significantly (P < 0.01) elevated after myocardial infarction in both susceptible
(drug dose 4, preinfarct 108 ± 12.6 vs. postinfarct 141 ± 9 beats/min) and resistant (preinfarct 107 ± 9 vs.
postinfarct 126 ± 8 beats/min) animals. As observed with the
postinfarction Vcf values, increasing doses of isoproterenol elicited a
significantly greater increase in the heart rate of the susceptible
animals compared with the resistant animals (Fig.
10). Thus, despite an increased
baseline heart rate, isoproterenol provoked greater increases in the
susceptible animals (drug dose 4, change in heart rate
47 ± 8 beats/min) compared with the resistant dogs (drug dose 4, change in heart rate 34 ± 9 beats/min). In both the
susceptible and the resistant animals, bisoprolol caused a significant
decrease in the heart rate response at all of the doses of
isoproterenol, indicating a large 1-AR component to the
heart rate response (Fig. 11).
ICI-118551, however, caused a significantly greater decrease in
heart rate in the susceptible dogs compared with the resistant dogs
(Fig. 11).
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Fig. 10.
Effects of increasing doses of Iso on heart rate after
myocardial infarction. Note that Iso elicited significantly greater
increases in heart rate in the susceptible (n = 12)
animals compared with the resistant animals (n = 15).
Dose 0 is the baseline heart rate value before the addition
of drug, and doses 1-5 are 0.006, 0.017, 0.06, 0.18, and 0.66 µg · min1 · kg1
Iso, respectively. *P < 0.01 susceptible vs. resistant
dogs.
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Fig. 11.
Effects of selective -AR antagonists on the maximum
Iso-induced heart rate response after myocardial infarction in both
resistant (n = 15) and susceptible (n = 12) animals. Note the increased response to Iso in the susceptible
animals. Bis = 0.6 mg/kg; ICI = 0.2 mg/kg. *P < 0.01 susceptible vs. resistant animals for a given treatment.
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Analysis of single-cell unloaded shortening.
The unloaded single-cell shortening response to isoproterenol with and
without 1- or 2-AR antagonist was
obtained in cells from six susceptible and nine resistant animals.
Representative tracings from a single myocyte for both a resistant and
a susceptible animal are displayed in Fig.
12. The composite percent change in shortening data for cells from susceptible and resistant animals are
shown in Fig. 13. As would be expected,
isoproterenol elicited significant increases in the shortening in both
groups of animals. However, a significantly greater response was noted
in the susceptible animal myocytes. ICI-118551 elicited a greater
attenuation of the response in the susceptible group with only small
effects noted in cells from the resistant animals. In contrast,
bisoprolol produced significant reductions in both groups with a larger
decrease noted in cells from the resistant animals.
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Fig. 12.
Representative tracings of the unloaded single-cell
shortening of 1 ventricular cardiomyocyte obtained from a resistant
(A) and susceptible (B) animal. Tracings
represent the shortening response to electrical field stimulation at
0.5 Hz with bicarbonate buffer (control), 100 nM Iso, and Iso in the
presence of either 200 nM Bis or 100 nM ICI. Note the large effects of
the 2-AR antagonist ICI on the Iso response on the
susceptible, but not resistant, animal cardiomyocyte. Note further that
the 1-AR antagonist Bis produced similar reductions in
both cell types. A single tracing represents the average of 16 individual tracings taken from a single myocyte.
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Fig. 13.
Percent change from control for the unloaded single-cell
shortening. Note that Iso (100 nM) elicited a larger increase in cell
shortening in the cells obtained in susceptible animals compared with
those obtained from control animals. Note further the greater reduction
(change from peak response) in susceptible cells after ICI (100 nM)
treatment. Bis (200 nM) exhibited similar reductions from peak response
in both sets of cells. *P < 0.01 susceptible vs.
resistant animals for a given treatment.
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Isoproterenol also elicited significant increases in the rate of
contraction as measured by the rate of change in the cell length
(dL/dt) maximum (expressed as percent
change from control) in cells from both resistant and susceptible
animals with the largest increase noted in the susceptible animal
myocytes (Fig. 14). Once again, the
2-AR antagonist, ICI-118551, attenuated the isoproterenol response to a greater extent in the susceptible animals,
whereas the 1-AR antagonist bisoprolol exhibited greater effects on the cells from resistant animals compared with myocytes obtained from susceptible animals. As in the studies done on the intact
animals, single-cell isotonic shortening was not altered by repeated
doses of isoproterenol; that is, each dose of isoproterenol provoked
similarly large increases in both the cell shortening and the velocity
of cell shortening (dL/dt maximum).
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Fig. 14.
Effects of isoproterenol on the rate of change
(dL/dt maximum) in unloaded single-cell
shortening in cardiomyocytes obtained from susceptible and resistant
animals. Note that Iso (100 nM) elicited a larger increase in the
velocity of cell shortening in the cells obtained in susceptible
animals compared with those obtained from control animals. Note further
the greater reduction in susceptible cells after ICI (100 nM)
treatment. In contrast, Bis (200 nM) produced larger reductions from
peak response in the cells obtained from resistant animals.
*P < 0.01 susceptible vs. resistant animals for a
given treatment.
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DISCUSSION |
An enhanced cardiac sympathetic activity often accompanies
cardiovascular disease (13, 24) and has been linked to an
increased risk for sudden cardiac death (3, 6). Enhanced
2-AR activity may be particularly important in the
development of malignant arrhythmias (1, 4, 16, 17). We
recently demonstrated that the selective 2-AR antagonist
ICI-118551 almost completely suppressed VF induced by myocardial
ischemia in dogs with healed anterior wall myocardial
infarctions (4). Furthermore, the 2-AR
agonist zinterol provoked significantly greater increases in calcium
transient amplitude in cardiomyocytes isolated from the hearts of
susceptible dogs compared with cells obtained from the hearts of
resistant animals. One would predict that this enhanced 2-AR-induced increase in intracellular calcium should
also result in an augmented contractile response to catecholamines in
the susceptible animals. The results of the present study, in fact, confirm this hypothesis. The nonspecific -AR agonist, isoproterenol, elicited significantly greater contractile responses in both the intact
hearts (Fig. 8) and isolated cardiomyocytes (Figs. 13 and 14) obtained
from animals shown to be susceptible to VF compared with animals
resistant to malignant arrhythmias. The heart rate response to
isoproterenol was also elevated in the susceptible animals (Fig. 10).
Furthermore, ICI-118551, a specific 2-AR antagonist, attenuated the enhanced isoproterenol response in susceptible animals;
that is, the 2-AR antagonist elicited much greater
reductions in this response in susceptible dogs (Figs. 9 and 11). In
contrast, after pretreatment with bisoprolol, a 1-AR
antagonist, the susceptible animals still exhibited a larger Vcf value
in response to isoproterenol, a response that once again is consistent
with an accentuated 2-AR response in the susceptible
animals (Fig. 9). In agreement with these findings in the intact heart,
similar results were also noted in studies performed with isolated
cardiomyocytes. Isoproterenol elicited a greater single-cell isotonic
shortening (i.e., contraction to a greater extent, percent change in
cell length) and shortening velocity (dL/dt
maximum) in cells obtained from susceptible compared with resistant
dogs (Figs. 12-14). Once again, the accentuated contractile response was eliminated in the susceptible animal myocytes with the
2-AR antagonist ICI-118551 (Fig. 13 and 14). These data
support the conclusion that susceptible animals exhibit an enhanced
2-AR-mediated contractile response. Finally, the
enhanced 2-AR contractile response was noted only after,
but not before, myocardial infarction (compare Figs. 3 and 5 with Figs.
8 and 10), suggesting that the ischemic injury was most likely
responsible for the changes in the apparent receptor sensitivity noted
in the susceptible animals.
When these results are considered together, three important conclusions
can be drawn: 1) the two groups of animals responded similarly to both -AR stimulation and blockade before infarction yet
exhibited markedly different responses postinfarction; 2) the susceptible dogs had an increase in 2-AR activity in
addition to an apparently normal 1-AR response; and
3) the increase in the 2-AR response in the
susceptible dogs appears to affect all chambers of the heart as an
enhanced response was observed in both ventricles (increased left
ventricular Vcf in vivo, increased right ventricular myocyte
shortening) and the sinoatrial node (increase in heart rate).
Ventricular 2-ARs.
It is well established that -ARs modulate myocardial contractility,
heart rate, and peripheral vascular resistance. The classical view has
been that cardiac -ARs are of the 1-subtype and that 1-ARs are solely responsible for the positive inotropic
and chronotropic effects of -AR stimulation (22).
However, cardiac preparations from a number of species have been found
to contain 2-ARs and to respond, albeit to a variable
extent, to 2-AR agonists (1, 9, 11, 14).
The functional significance of mammalian ventricular 2-ARs is controversial. For most normal hearts or
myocytes, physiologically or therapeutically relevant concentrations of
2-AR agonists have surprisingly little effect on
contractility (14, 25). For example, Cui et al.
(14) demonstrated a marked difference between in vitro
-AR density and the ventricular response to an adrenergic agonist in
vivo. The 1/2-AR ratio was found to be
59/41% in myocytes isolated from baboon hearts, yet the ventricular
response to isoproterenol in intact, unanesthetized animals was reduced by ~85% by the selective 1-AR antagonist metropolol.
They concluded that the inotropic response to -adrenergic
stimulation was predominately mediated by 1-AR in the
nonhuman primate and, further, that a significant fraction of the
2-AR did not appear to be functional in vivo
(14).
Cardiac hypertrophy and failure are characterized by an overall loss of
sensitivity to -AR stimulation (1, 10, 12, 18). The
number of 1-AR declines, adenylyl cyclase activity decreases, and the amount of the adenylyl cyclase inhibitory G protein
Gi increases (8, 20). As a consequence, the
failing heart is unable to modulate contractility adequately. In
contrast to 1-AR, however, the number of
2-AR remain unchanged in the failing heart (10,
18). An elegant study of trabeculae carnae from explanted normal
and failing human hearts by Bristow et al. (10)
established that the positive inotropic effects of the highly selective
2-AR agonist zinterol were comparable in normal and
failing trabeculae, despite the fact that failure was associated with a
pronounced depression in the response to isoproterenol, a mixed
1/2-AR agonist. Consistent with these
observations, the density of 2-AR in crude membrane
preparations was not reduced in failing hearts, but total -AR
density was decreased by >50% (10). In agreement with
these finding, the whole cell -AR number was similar in both
susceptible and resistant animals (data not shown), yet, as has been
previously noted, isoproterenol elicited a significantly greater
inotropic response in the susceptible animals. The basis for this
upregulation of the 2-adrenergic response in hearts and
cardiomyocytes from susceptible dogs postinfarction is unknown and
currently under investigation.
A study of isolated human ventricular cardiomyocytes by Del Monte et
al. (15) demonstrated that individual cells contain both
1- and 2-ARs and further that a
surprisingly large proportion of the shortening response to
isoproterenol in electrically field-stimulated failing myocytes
resulted from 2-AR activation. In agreement with these
findings, we demonstrated that most of the stimulatory effects of
isoproterenol on myocytes prepared from failing human hearts
resulted from 2-AR rather than 1-AR
activation (2). We further demonstrated that the
2-AR agonist zinterol elicited an augmented
intracellular calcium transient amplitude response in myocytes obtained
from failing canine hearts (induced by tachypacing) (2). In contrast, cells from normal control animals
exhibited little response to 2-AR stimulation.
The data cited above strongly suggest that the mammalian ventricular
myocardium contains "latent" 2-AR that may provide
important inotropic support in the diseased heart. It is likely that
the cellular events that lead to an apparent increased dependence on
2-AR activation for inotropic support during heart
failure are similar to the mechanisms that trigger the enhanced
2-AR response in the postinfarction susceptible dogs in
the present study. In this regard, it is interesting to note that we
previously demonstrated that susceptible animals exhibit a
significantly greater impairment in cardiac function during exercise
than was noted in the resistant animals (5). One may
speculate that the susceptible animals may reflect an early or
transitional phase of heart failure. During this transition to heart
failure, the 2-AR coupling may change such that these
receptors become functional with regard to the control of ventricular
contractility before 1-AR sensitivity declines. As a
result, -adrenergic stimulation would provoke large increases in
intracellular calcium and reduce the electrical stability of the heart
increasing the propensity for the formation of malignant arrhythmias.
In summary, the animals susceptible to VF exhibited an enhanced in vivo
and in vitro contractile response to -AR stimulation that resulted
from an accentuated activation of 2-ARs. In vivo, the
nonselective -AR agonist isoproterenol elicited significantly greater increases in the Vcf in animals subsequently shown to be
susceptible to VF than in those animals that were resistant to the
formation of malignant arrhythmias. The selective 2-AR antagonist ICI-118551, but not the selective 1-AR
agonist bisoprolol, abolished the enhanced contractile response noted
in the susceptible animals. In a similar manner, isoproterenol provoked
significantly greater contractile increases in cardiomyocytes obtained
from the hearts of dogs susceptible to, as compared with cells
prepared from the hearts of animals resistant to, VF. Finally, this
increase in 2-AR responsiveness developed only after
myocardial infarction as there were no differences in the isoproterenol
response noted between the two groups of animals before the
ischemic injury. These data suggest that myocardial
infarction most likely produced changes in the
2-AR receptor coupling such that adrenergic stimulation provoked greater changes in cytosolic calcium in the susceptible animals that, in turn, led to an increased risk for the development of
lethal arrhythmias.
| |
ACKNOWLEDGEMENTS |
We thank Lou Castillo and Robert Kelly for technical assistance.
| |
FOOTNOTES |
These studies were supported by Aventis-Pharma, Frankfurt, Germany, and
by National Heart, Lung, and Blood Institute Grants HL-36240 and
HL-48835.
Address for reprint requests and other correspondence: G. E. Billman, Dept. of Physiology and Cell Biology, The Ohio State Univ.,
302 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210-1218 (E-mail:
billman.1{at}osu.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 20 March 2001; accepted in final form 24 May 2001.
| |
REFERENCES |
1.
Altschuld, RA,
and
Billman GE.
2-Adrenoceptors and ventricular fibrillation.
Pharmacol Ther
88:
1-14,
2000[Web of Science][Medline].
2.
Altschuld, RA,
Starling RC,
Hamilin RL,
Billman GE,
Hensley J,
Castillo L,
Fertel RH,
Hohl CM,
Robitaille PM,
Jones LR,
Xiao RP,
and
Lakatta EG.
Response of failing canine and human heart cells to 2-adrenergic stimulation.
Circulation
92:
1612-1618,
1995[Abstract/Free Full Text].
3.
Bigger, JT, Jr.
The predictive value of RR variability and baroreflex sensitivity in coronary heart disease.
Cardiac Electrophysiol Rev
1/2:
198-204,
1997.
4.
Billman, GE,
Castillo LC,
Hensley J,
Hohl CM,
and
Altschuld RA.
2-Adrenergic receptor antagonists protect against ventricular fibrillation: in vivo and in vitro evidence for enhanced sensitivity to 2-adrenergic stimulation in animals susceptible to ventricular fibrillation.
Circulation
96:
1914-1922,
1997[Abstract/Free Full Text].
5.
Billman, GE,
Schwartz PJ,
Gagnol JP,
and
Stone HL.
The cardiac response to submaximal exercise in dogs susceptible to sudden cardiac death.
J Appl Physiol
59:
890-897,
1985[Abstract/Free Full Text].
6.
Billman, GE,
Schwartz PJ,
and
Stone HL.
Baroreceptor reflex control of heart rate: a predictor of sudden death.
Circulation
66:
874-880,
1982[Free Full Text].
7.
Binkley, PF,
and
Boudoulas H.
Measurement of myocardial inotropy.
In: Cardiotonic Drugs: A Clinical Review, edited by Leier CV.. New York: Marcel Dekker, 1991, p. 24-25.
8.
Bristow, MR,
and
Feldman AM.
Changes in the receptor-G protein-adenylyl cyclase system in heart failure from various types of heart muscle disease.
In: Cellular and Molecular Alterations in the Failing Human Heart, edited by Hasenfuss G,
Holubarsch C,
Just H,
and Alpert NR.. New York: Springer, 1992, p. 15-35.
9.
Bristow, MR,
and
Ginsburg R.
2-Receptors on myocardial cells in human ventricular myocardium.
Am J Cardiol
57:
3F-6F,
1986[Medline].
10.
Bristow, MR,
Ginsburg R,
Umans V,
Fowler M,
Minobe W,
Rasmussen R,
Zera P,
Menlove R,
Shah P,
Jamieson R,
and
Stinson EB.
1- and 2-adrenergic receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective 1-receptor down-regulation in heart failure.
Circ Res
59:
297-309,
1986[Abstract/Free Full Text].
11.
Bristow, MR,
Hershberger RE,
Port JD,
Gilbert EM,
Sandoval A,
Rasmussen R,
Cates AE,
and
Feldman AM.
-Adrenergic pathways in the nonfailing and failing human ventricular myocardium.
Circulation
82:
112-125,
1990.
12.
Brodde, OE.
1- and 2-adrenoceptors in the human heart: properties, function, and alterations in chronic heart failure.
Pharmacol Rev
43:
203-242,
1992[Web of Science][Medline].
13.
Corr, PB,
Yamada K,
and
Witkowski FX.
Mechanisms controlling cardiac autonomic function and their relationships to arrhythmogenesis.
In: The Heart and Cardiovascular System: Scientific Foundations, edited by Fozzard HA,
Haber E,
Jennings RB,
Katz AM,
and Morgan HE.. New York: Raven, 1986, p. 1343-1404.
14.
Cui, YN,
Shen YT,
Kalthof B,
Iwase M,
Sato N,
Uechi M,
Vatner SF,
and
Vatner DE.
Identification and functional role of -adrenergic receptor subtypes in primate and rodent: in vivo vs. isolated myocytes.
J Mol Cell Cardiol
28:
1307-1317,
1996[Web of Science][Medline].
15.
Del Monte, F,
Kaumann AJ,
Poole-Wilson PA,
Wynne DG,
Pepper J,
and
Harding SE.
Coexistence of functioning 1- and 2-adrenoceptors in single myocytes from human ventricle.
Circulation
88:
854-863,
1993[Abstract/Free Full Text].
16.
Du, XJ,
Cox HS,
Dart AM,
and
Esler MD.
Sympathetic activation triggers ventricular arrhythmias in rat heart with chronic infarction and failure.
Cardiovasc Res
43:
919-929,
1999[Abstract/Free Full Text].
17.
Du, XJ,
and
Dart AM.
Role of sympathoadrenergic mechanisms in arrhythmogenesis.
Cardiovasc Res
43:
832-834,
1999[Free Full Text].
18.
Fowler, MB,
Laser JA,
Hopkins GL,
Minobe W,
and
Bristow MR.
Assessment of the -adrenergic receptor pathway in the intact failing human heart: progressive receptor down-regulation and subsensitivity to agonist response.
Circulation
74:
1290-1302,
1986[Abstract/Free Full Text].
19.
Hohl, CM,
and
Li Q.
Compartmentation of cAMP in adult canine ventricular myocytes: relation to single-cell free Ca2+ transients.
Circ Res
69:
1369-1379,
1991[Abstract/Free Full Text].
20.
Kiuchi, K,
Shannon RP,
Komamura K,
Cohen DJ,
Bianchi C,
Homcy CJ,
Vatner SF,
and
Vatner DE.
Myocardial -adrenergic receptor function during the development of pacing-induced heart failure.
J Clin Invest
91:
907-914,
1993.
21.
Kuschel, M,
Zhou YY,
Spurgeon HA,
Bartel S,
Karczewski P,
Zhang SJ,
Krause EG,
Lakatta EG,
and
Xiao RP.
2-Adrenergic cAMP signalling is uncoupled from phosphorylation of cytoplasmic proteins in canine heart.
Circulation
99:
2458-2465,
1999[Abstract/Free Full Text].
22.
Kuznetsov, V,
Pak E,
Robinson RB,
and
Steinberg SF.
2-Adrenergic receptor actions in neonatal and adult rat ventricular myocytes.
Circ Res
76:
40-52,
1995[Abstract/Free Full Text].
23.
Schwartz, PJ,
Billman GE,
and
Stone HL.
Autonomic mechanisms in ventricular fibrillation induced by myocardial ischemia during exercise in dogs with healed myocardial infarction: an experimental preparation for sudden cardiac death.
Circulation
69:
790-800,
1984[Abstract/Free Full Text].
24.
Schwartz, PJ,
and
Zipes DP.
Autonomic modulation of cardiac arrhythmias.
In: Cardiac Electrophysiology: From Cell to Bedside, edited by Zipes DP,
and Jalife J.. Philadelphia, PA: Saunders, 2000, p. 300-314.
25.
Xiao, RP,
and
Lakatta EG.
1-Adrenoceptor stimulation and 2-adrenoceptor stimulation differ in their effects on contraction, cytosolic Ca2+, and Ca2+ current in single rat ventricular cells.
Circ Res
73:
286-300,
1993[Abstract/Free Full Text].
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ABSTRACT
INTRODUCTION
