(Circulation. 1996;94:102-107.)
© 1996 American Heart Association, Inc.
Articles |
Physiological and Biochemical Evidence for Coordinate Increases in Muscarinic Receptors and Gi During Pacing-Induced Heart Failure
From the Departments of Medicine, Harvard Medical School, Brigham and Women's Hospital, Boston, and Massachusetts General Hospital, Boston, and the New England Regional Primate Research Center, Southborough, Mass.
Correspondence to Dorothy E. Vatner, MD, New England Regional Primate Research Center, 1 Pine Hill Dr, PO Box 9102, Southborough, MA 01772-9102.
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Abstract |
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Background It is not clear whether the increase in the myocardial guanylyl nucleotide inhibitory protein (Gi), frequently observed in heart failure, is associated with any functional effects.
Methods and Results Eight sham-operated dogs and 10
dogs were studied with pacing-induced heart failure (240 bpm for 4
to 7 weeks), characterized by reduced (P<.05) left
ventricular dP/dt (from 2926±99 to 1303±126 mm Hg/s). The
muscarinic agonist acetylcholine (10 µg/kg IV) in the presence of
ganglionic blockade reduced left ventricular dP/dt more
(P<.05) in heart failure (-23±2%) than before heart
failure (-8±2%), despite lesser reductions in
arterial pressure.
Gi
2 was increased by 55% in
heart failure. Dose-response curves for carbachol
(10-8 to
10-3 mol/L) inhibition of
isoproterenol-stimulated adenylyl cyclase demonstrated
significantly greater (P<.05) inhibition in heart failure
compared with sham-operated dogs. These changes were associated
with a coordinate increase in muscarinic receptor density, determined
by antagonist binding with 3H-quinuclidinyl
benzilate, in heart failure (153±6.2 fmol/mg protein) compared with
sham-operated dogs (124±7.4 fmol/mg protein). Agonist binding with
carbachol also revealed an increase in total muscarinic receptors in
heart failure without a change in fraction of high- and
low-affinity receptors.
Conclusions These data, in the aggregate, provide physiological and biochemical evidence to support the concept that the coordinate increases in muscarinic receptor number and Gi levels in heart failure are coupled to increased inhibition of adenylyl cyclase activity and an increased inhibition of myocardial contractility.
Key Words: receptors • proteins • heart failure • nervous system, autonomic • signal transduction
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Altered ß-adrenergic receptor signaling and decreased adenylyl cyclase activity are hallmarks of heart failure.1 2 3 4 5 6 7 8 In most studies, the failing heart also is characterized by increased Gi levels, quantified by immunoblotting and pertussis toxin labeling.1 2 9 10 11 12 Other studies, including a previous one from our laboratory on aortic banding–induced heart failure,13 14 15 16 failed to demonstrate an increase in Gi. More importantly, prior studies that did demonstrate an increase in Gi in heart failure have not been able to demonstrate a physiological correlate of the enhanced Gi, eg, a decrease in isoproterenol-stimulated adenylyl cyclase activity.17
Accordingly, the primary goal of the present investigation was to
determine whether muscarinic inhibition of isoproterenol-stimulated
adenylyl cyclase is enhanced in the rapid ventricular
pacing model of CHF. Because this was the primary goal of the
present investigation, it was important to conduct the
investigation in the pacing-induced heart failure model in which a
significant increase in Gi can be
demonstrated.1 Because the inhibition of adenylyl cyclase
activity by muscarinic cholinergic stimulation involves the interaction
of the muscarinic receptor with
Gi,18 19 20 21 22 23 it also was considered
important to examine muscarinic receptor density by use of both agonist
and antagonist ligand binding studies, which allowed us to
determine whether the results could be attributed to coordinate
increases in levels of muscarinic receptors and
Gi2. A final goal was to
determine whether the changes in Gi and muscarinic
receptors in vitro could be associated with increased muscarinic
inhibition of myocardial contractility in vivo in dogs
with heart failure. These latter experiments were conducted in the
presence of ganglionic blockade to minimize complications of
reflex-induced increases in myocardial
contractility, elicited by fall in arterial
pressure after intravenous acetylcholine.
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Methods |
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Eighteen adult mongrel dogs were anesthetized with thiamylal sodium (10 to 15 mg/kg IV) and then with halothane (0.5 to 1.5 vol/100 mL in oxygen). By use of a sterile technique, a left thoracotomy was performed through the fifth intercostal space. Stainless steel pacing wires were placed on the right ventricle. To measure LV and aortic pressures, a solid-state miniature pressure transducer (Konigsberg Instruments) was implanted in the LV through the apex, and a Tygon catheter was placed in the descending thoracic aorta. Hemodynamic measurements were made 2 to 3 weeks after surgical instrumentation in fully awake dogs before the initiation of rapid ventricular pacing and periodically during the pacing protocol but only after the pacemakers were off for 30 minutes. Rapid ventricular pacing was controlled with a programmable pacemaker set at 240 bpm in 10 dogs until heart failure developed, which required 3 to 5 weeks. At that time, final hemodynamic measurements were made. Eight sham-operated dogs had the instrumentation surgically implanted and remained in sinus rhythm without pacing for hemodynamic studies. In 5 conscious dogs, the effects of acetylcholine (10 µg/kg IV) were examined before pacing and after pacing-induced heart failure. These experiments were conducted in the presence of ganglionic blockade with hexamethonium bromide (30 mg/kg IV), which eliminated reflex tachycardia to both nitroglycerin (15 µg/kg) and acetylcholine (10 µg/kg). No atropine was administered. Dogs used in this study were maintained according to the guidelines of the Committee on Animals of the Harvard Medical School and the Guidelines for the Care and Use of Laboratory Animals (Department of Health and Human Services Publication NIH 83-23, revised 1985).
Membrane Preparation
After the dogs were anesthetized with sodium
pentobarbital (20 to 30 mg/kg), the hearts were removed and placed in
iced saline. The LV and septum were weighed, trimmed of fat and
connective tissue, and homogenized in 4 mL/g tissue of
buffer 1 (0.75 mol/L NaCl and 10 mmol/L histidine, pH 7.5) with a
Polytron S-20 (Brinkmann Instruments, Inc) for 5 seconds at low speed.
All buffers contained protease inhibitors, including
soybean trypsin inhibitor (5 mg/L), benzamidine (10
µmol/L), and phenylmethylsufonyl fluoride (10 µmol/L). The
homogenate was centrifuged at 14 000g
for 20 minutes. The pellet was resuspended in buffer 1,
homogenized, and centrifuged twice more as
described above. The pellet was resuspended in buffer 2 (10 mmol/L
NaHCO3 and 5 mmol/L histidine, pH 8.0),
homogenized, and centrifuged at 14 000g
for 20 minutes. The pellet was resuspended in buffer 3 (100 mmol/L
Tris-HCl, pH 7.2; 1 mmol/L EGTA; and 5 mmol/L MgCl2). The
suspension was filtered through a silk screen (size 12) and stored at
-70°C.
Muscarinic Cholinergic Receptor Binding
Studies were performed with 100 µL of 0.1 to 50 nmol/L
3H-QNB (36 Ci/mmol), 100 µL atropine (1 µmol/L) or
buffer 3, and 800 µL membrane protein (100 µg per tube). Assays
were performed in triplicate, incubated at 37°C for 60 minutes,
terminated by rapid filtration on Whatman GF/C filters, and counted in
5-mL Ecoscint in a beta counter (TM Analytic) for 5 minutes.
Nonspecific binding was defined as that measured in the presence of 1
µmol/L atropine. Specific binding (90% of total binding) was
determined by subtracting nonspecific from total binding.
Agonist binding studies were performed by studying competition binding
curves with 100 µL 3H-QNB (0.3 nmol/L), 800 µL
sarcolemmal membranes (100 µg per assay), and 100 µL carbachol (10
nmol/L to 1 mmol/L) in 22 different concentrations. Assays were
performed in duplicate. Incubation, filtration, and counting were
carried out as described above. Linear regression was performed on
antagonist binding studies on the amount bound versus bound
to free ligand. An r2 value of 
.85 was
the criterion for acceptability of the data. Accordingly, 2 of 10 data
points in each graph did not qualify for antagonist
binding. With agonist binding, data from one dog in each group
contained unacceptable scatter. All binding data were analyzed
by the Ligand program of Munson and Rodbard.24 In the
computer analysis of the binding data, the F test was used to
compare the best fit for the ligand competition binding data. The best
fit (two sites versus one site) was determined by the probability value
for the F test and by the change in the residual sum of squares for the
various fits. One- and two-site models were tested, and the model
yielding the least residual sum of squares was used to describe the
data.24 Nonspecific binding was similar in both shams and
dogs with heart failure.
Adenylyl Cyclase Activity
Adenylyl cyclase activity was assayed as previously
described.25 The reaction volume was 150 µL, including 1
mmol/L ATP (106 cpm [-32P]ATP), 1
IU creatine phosphokinase, 20 mmol/L creatine phosphate, 1 mmol/L cAMP
(1 to 2000 cpm [3H]cAMP per 0.15 mL), 25 mmol/L Tris HCl,
pH 7.2, 5 mmol/L MgCl2, 1 mmol/L EDTA, 25 µg
sarcolemmal membranes, and the following stimulators added to measure
maximal adenylyl cyclase activity: 0.1 mmol/L GTP, 0.1 mmol/L
isoproterenol, and 0.1 mmol/L 5'-guanylylimidodiphosphate [Gpp(NH)p].
To terminate the reaction, 10 µL stopping solution (20 mmol/L ATP, 10
mmol/L cAMP, and 2% SDS) was added. The reaction tubes were heated on
a dry bath (100°C), and cAMP was separated. Recovery of cAMP was 60%
to 80%. To study optimal conditions for carbachol inhibition of
isoproterenol- (0.1 mmol/L) and GTP- (0.1 mmol/L) stimulated adenylyl
cyclase activity, the assay was tried with several different
temperatures, ie, 37°C or 30°C, and several 5- or 15-minute
preincubation times were assayed. During the preincubation, all
reagents except for 32P-ATP were added to each assay tube.
At 37°C and with no preincubation period, inhibition of
isoproterenol-stimulated adenylyl cyclase activity with carbachol
was found to be optimal. In addition, the effects of various
concentrations of MgCl2 were studied (0.5, 1.0, 1.5, 2.0,
and 2.5 mmol/L) on muscarinic inhibition of isoproterenol- (0.1 mmol/L)
and GTP- (0.1 mmol/L) stimulated adenylyl cyclase activity. Carbachol
inhibition was optimal in the presence of 2.5 mmol/L
MgCl2, and this MgCl2 concentration was
used with the carbachol inhibition studies.
Because both adenosine and acetylcholine inhibit adenylyl cyclase activity, the presence of endogenously released adenosine would decrease basal adenylyl cyclase activity and interfere with carbachol inhibition of adenylyl cyclase. Adenosine deaminase (0.3 U) was added before incubation of myocardial membranes with carbachol and isoproterenol. A series of experiments with and without adenosine deaminase was conducted in the presence of various magnesium concentrations (0.5, 1.0, 1.5, 2.0, and 2.5 mmol/L). In the presence of adenosine deaminase, basal adenylyl cyclase levels were decreased by 36%, and isoproterenol-stimulated activity was decreased by 8%; however, adenosine deaminase did not alter carbachol inhibition of adenylyl cyclase (data not shown).
Immunoblotting
Gi2 protein content was
quantified by immunoblotting by use of rabbit antisera
against synthetic peptides that correspond to defined regions of G
proteins as previously described.1 26
Statistical Analysis
Data are expressed as mean±SE and were analyzed with
the SAS program on an IBM-PC 486 (IBM Instruments). Statistical
evaluation was performed with Student's t test for grouped
data. When more than one data point was involved, a one-way ANOVA
for repeated measures with analysis of contrast to highlight
the individual differences (Fig 3
) was used. Differences of
P<.05 were considered significant.
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) compared with four sham-operated
control hearts (
). Assays were incubated at 37°C for 10
minutes. Isoproterenol-stimulated (0.1 mmol/L) adenylyl cyclase
activity with 2.5 mmol/L MgCl2 in the absence of carbachol
was 15.8±1.6 and 15.0±1.4
pmol · min-1 · mg-1
for the sham-operated control and the failing hearts, respectively.
The entire data from all points on the curves derived from four dogs in
each group were analyzed by ANOVA with repeated measures and
found to be significantly different (*P<.05) in dogs with
heart failure compared with controls. The inset examines a single point
and shows a significant increase in percent inhibition of 1 mmol/L
carbachol in four failing hearts (cross-hatched bar) compared with
four sham-operated controls (open bar). |
Results |
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Baseline Hemodynamics
After heart failure developed, heart rate rose (P<.05) from 88±3 to 117±11 bpm and LV end-diastolic pressure rose from 5.7±0.5 to 24±2.8 mm Hg, while mean arterial pressure decreased (P<.05) from 92±2 to 72±7 mm Hg and LV dP/dt decreased (P<.05) from 2926±99 to 1303±126 mm Hg/s (Table 1
). Heart failure was also characterized by
ascites and exercise intolerance.
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Effects of Acetylcholine Infusion
These experiments were conducted in the presence of ganglionic
blockade to minimize reflex effects induced by the decrease in
arterial pressure. In contrast to the baseline
hemodynamics in the intact state, in the presence of
ganglionic blockade, heart rate tended to be lower after heart failure
(110±7 versus 124±7 bpm) rather than higher, although this difference
was not significant (Table 2). LV dP/dt was still lower
(P<.05) in dogs with heart failure (1256±30 mm Hg/s)
compared with before pacing (2052±142 mm Hg/s). Heart failure induced
two major differences in the responses to acetylcholine (Table 2): mean
arterial pressure decreased less (27±3% versus 50±2%,
P<.05) and LV dP/dt decreased more (-23±3% versus
-8±2%, P<.05) with acetylcholine after heart
failure.
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Muscarinic Cholinergic Receptor Density
Muscarinic cholinergic receptor density was significantly higher
in the group with pacing-induced heart failure (153±6.2 versus
124±7.4 fmol/mg) compared with the sham-operated dogs. However,
3H-QNB receptor affinity was not different between the two
groups (54±7 versus 55±6 pmol/L; Fig 1). Specific
binding near the Kd was 
90% of total binding
of the ligand to the membranes in the absence of atropine. The binding
curves reached saturation.
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Muscarinic Receptor Agonist Binding
Competition curves for muscarinic agonist binding with increasing
concentrations of carbachol (10 nmol/L to 1 mmol/L) were performed (Fig 2). High- and low-affinity dissociation constants
(KH and KL) were similar
for sham-operated dogs and dogs with pacing-induced heart
failure (KH=0.23±0.07 versus 0.29±0.07
µmol/L; KL=12±3.8 versus 20±5.9
µmol/L; Table 3). The percentage of high- and
low-affinity receptors (%RH and %RL) as
modeled by the Ligand program24 were similar for
sham-operated and heart failure dogs (%RH=54±4.1%
versus 55±1.9%; %RL=46±4.1% versus 45±1.9%).
However, there was a significant increase (P<.05) in both
high- and low-affinity muscarinic receptor densities in the failing
hearts compared with normal hearts as calculated from agonist binding
curves, corroborating the antagonist binding data (Table 3).
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Muscarinic Inhibition of Adenylyl Cylase Activity
To study muscarinic inhibition of adenylyl cyclase activity, the
effect of increasing concentrations of carbachol (0.01 µmol/L to 1
mmol/L) was studied in sham and heart failure dogs. Using optimized
assay conditions for muscarinic inhibition of
isoproterenol-stimulated adenylyl cyclase, we compared the effect
of increasing concentrations (0.01 µmol/L to 1 mmol/L) of the
muscarinic agonist carbachol on isoproterenol-stimulated adenylyl
cyclase activity in failing and sham hearts. The percent inhibition of
adenylyl cyclase activity is modestly but significantly
(P<.05) increased in the failing heart at each
concentration studied (Fig 3
). Furthermore, we have
previously demonstrated that Gi2
as measured by immunoblotting is increased in heart
failure.1 In the present study, this measurement was
verified and found to be increased in heart failure (1.58±0.09)
compared with sham hearts (1.02±0.02 arbitrary units). Thus, a
functional measure of muscarinic activity, ie, increased inhibition of
isoproterenol-stimulated adenylyl cyclase activity in the failing
heart, also is observed with the coordinate increase in muscarinic
receptor number and increased
Gi2 levels.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The underlying hypothesis of the present investigation was that coordinate increases in muscarinic receptors and Gi coupled with altered activity of the catalytic subunit of adenylyl cyclase result in an increased level of inhibition of adenylyl cyclase activity. Accordingly, the aims of the present investigation were to provide physiological and biochemical evidence for a functional role of increased Gi and possible changes in muscarinic receptors in heart failure. The pacing-induced heart failure model was used, which already has been characterized by reductions in adenylyl cyclase1 2 6 27 and increased Gi.1
Acetylcholine, which stimulates muscarinic receptors, was used to determine the extent to which LV contractile activity could be impaired in vivo by use of an agonist that affects the Gi pathway. Acetylcholine acts directly to reduce arterial pressure and LV dP/dt, an index of myocardial contractility. In the intact conscious animal, however, the effects on LV dP/dt are masked by arterial baroreflex-mediated increases in heart rate and LV dP/dt, which parenthetically are altered in heart failure. Accordingly, the present experiments were conducted in the presence of ganglionic blockade, in which secondary reflex effects were minimized, as reflected by the lack of reflex tachycardia in response to the acetylcholine-induced hypotension. In the presence of ganglionic blockade, the same dose of acetylcholine reduced LV dP/dt more in the presence of heart failure than in the same dogs studied before pacing, suggesting a greater muscarinic inhibitory action on contractility in heart failure. Because the LV dP/dt measurement is sensitive to changes in afterload, a greater decrease in LV and arterial pressures induced by acetylcholine could have caused the greater reduction in LV dP/dt. However, the reverse was noted; ie, acetylcholine induced greater decreases in LV and arterial pressures in the dogs before pacing-induced heart failure. This indicates that the greater decrease in LV dP/dt in dogs with heart failure induced by acetylcholine was not related to load-dependent mechanisms but more likely reflected its action to inhibit myocardial contractility directly.28 29 That acetylcholine caused a greater decrease in arterial pressure in the dogs before pacing was expected because one part of the action of acetylcholine is to elaborate endothelium-derived relaxing factor.30 This mechanism is altered in heart failure,31 32 33 34 35 36 thereby explaining blunted hypotension induced by acetylcholine in heart failure.
An interesting incidental finding was the paradoxical directional change in heart rate in heart failure in the presence and absence of ganglionic blockade. As expected, in dogs with reflexes intact, heart rate rose in heart failure (from 88±3 to 117±11 bpm). Interestingly, in the presence of ganglionic blockade, heart rate fell in heart failure (124±7 to 110±7 bpm). These data most likely are explained by the preponderance of vagal tone in normal animals and the preponderance of sympathetic tone in animals with heart failure. It is under these conditions of increased sympathetic tone that any potential role of increased Gi and muscarinic receptors may be augmented.
Prior studies in human tissue from failing hearts have shown increased Gi without increased inhibition of adenylyl cyclase in heart failure.17 Studies in rats with chronic ischemia-induced heart failure demonstrated enhanced cyclase inhibition by muscarinic agonists but no increase in Gi.13 A prior study from our laboratory in dogs with pressure-overload hypertrophy and heart failure also failed to show increases in either Gi or enhanced muscarinic inhibition of cyclase in that model of heart failure.16 There also has been a study of pacing-induced heart failure in pigs, which found a decrease in Gi.37 There are two possible explanations for the differences observed between the present investigation and prior studies. First, the results in the field clearly have been model dependent. The pressure-overload model used previously in our own laboratory16 is characterized by complicating factors, eg, severe hypertrophy and subendocardial ischemia and fibrosis, when heart failure develops.38 That model also is not characterized by an increase in Gi.16 We hypothesize that the pacing-induced heart failure model in the dog, used in the present investigation, is more closely akin to human cardiomyopathy, particularly in view of the increase in Gi observed in both cases, with one difference being the ability to demonstrate enhanced muscarinic inhibition of cyclase. Second, an important difference between the present results and clinical studies is that the changes observed in this study of pacing-induced heart failure were significant but not so great that they might easily be demonstrated in a more heterogeneous population of diseased human hearts. It also has been shown that halothane affects inhibitory G proteins in the failing human myocardium39 and measurements of cardiac function in heart failure,40 as has been demonstrated repeatedly in animals.41 Our physiological studies were conducted in awake dogs, and final tissue samples were collected after bolus barbiturate anesthesia, thereby obviating these problems.
The present study examined muscarinic receptor binding by use of both antagonist and agonist binding techniques. Both of these techniques demonstrated increased receptor binding in heart failure without an alteration in affinity or fraction of receptors in the high-affinity state. This result was unexpected because an increase in Gi and receptor number should result in an increase of high-affinity receptor sites. However, at least half the increased receptors are in the high-affinity form and may contribute to the enhanced ability of muscarinic agonists to inhibit adenylyl cyclase in heart failure. Although one might predict that muscarinic receptor density would increase in heart failure because of the chronic reduction in vagal tone, prior studies have not found an increase in muscarinic receptors in heart failure.10 13 14 15 42 More importantly, we used two techniques, ie, antagonist and agonist binding, to demonstrate the increase in muscarinic receptor density. Again, the differences between the current results and prior experimental studies are most likely model dependent. As noted above, in our prior study, aortic banding–induced heart failure also is associated with significant subendocardial hypoperfusion and fibrosis.38 In contrast, the complicating influences of hypertrophy and fibrosis are not of major significance in this model of pacing-induced heart failure.43 As with the differences with Gi inhibition of adenylyl cyclase noted above, failure to demonstrate increases in muscarinic receptor density are most likely caused by the difficulty in demonstrating modest changes in a patient population characterized by different origins of heart failure and therapeutic regimens.
In summary, our study is the first to provide both
physiological and biochemical evidence for a
functional role of the coordinate increase in muscarinic receptors and
Gi in heart failure. It is possible that the modest
increase in muscarinic density observed in dogs with heart failure
allowed the functional inhibitory role in adenylyl cyclase
to be manifested. In the pacing-induced model of heart failure in
which both Gi2 and muscarinic
receptors increase, significant biochemical and
physiological evidence of
isoproterenol-stimulated adenylyl cyclase inhibition is
demonstrated.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported in part by US Public Health Service grants HL-38070, HL-37404, HL-45332, and RR00168. Dr Sato is the recipient of the Samuel A. Levine Fellowship grant from the American Heart Association, Massachusetts Affiliate.
Received October 30, 1995; revision received December 18, 1995; accepted December 21, 1995.
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