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(Circulation. 1996;93:763-771.)
© 1996 American Heart Association, Inc.
Articles |
Chronic Treatment With Carbachol Sensitizes the Myocardium to cAMP-Induced Arrhythmia
From Abteilung Allgemeine Pharmakologie, Universitäts-Krankenhaus Eppendorf, Hamburg (T.E., U.M., M.D., B.H., C.M., A.P., W.S., H.S., M.S.); Klinik III für Innere Medizin, Universität zu Köln (M.B.); Biochemisches Forschungslabor, Medizinische Klinik und Poliklinik, Universitätsklinikum Essen (M.C.M., O.-E.B.); and Beiersdorf-Lilly GmbH, Hamburg (A.R.), Germany.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background The present study investigated biochemical and functional consequences of chronic activation of the inhibitory Gi
-coupled adenylyl
cyclase pathway in the heart.
Methods and Results Rats (220 to 260 g) were treated with
4-day infusions of the M-cholinoceptor agonist carbachol (9.6 mg/kg per
day) or vehicle. An additional group that received the
ß-adrenoceptor agonist isoprenaline (2.4 mg/kg per day) served as
control. The main finding was that chronic infusion of carbachol led to
a marked increase in isoprenaline- or forskolin-induced
arrhythmia in electrically driven papillary muscles (in vitro).
Compared with control, the potency of isoprenaline and forskolin to
induce arrhythmia in cardiac preparations from
carbachol-treated rats was increased 36- and 2.2-fold and the
efficacy was increased 7.3- and 2.3-fold, respectively. The potency of
carbachol to antagonize the isoprenaline- and forskolin-induced
arrhythmia was decreased 30-fold. These changes were
accompanied by a decrease in left ventricular
M-cholinoceptor density by 15% (P<.05) and a decrease in
pertussis toxin–sensitive G proteins
(Gi) by 26% (P<.05)
without a decrease in the corresponding mRNAs. ß-Adrenoceptor density
and basal and stimulated adenylyl cyclase activity remained unchanged.
In contrast, isoprenaline infusion induced a decrease in arrhythmogenic
potency of forskolin (P=NS), which was accompanied by a
decrease in ß-adrenoceptor density, an increase in
Gi protein and mRNA levels, and a decrease
in basal and stimulated adenylyl cyclase activity.
Conclusions Chronic parasympathetic activation sensitizes
the myocardium to cAMP-induced arrhythmia. These
changes may be due to quantitative alterations in functional
Gi.
Key Words: acetylcholine • signal transduction • receptors, adrenergic, beta • arrhythmia
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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The hormone-sensitive adenylyl cyclase is under dual control of stimulatory and inhibitory receptors acting through stimulatory (Gs) and inhibitory (Gi) G proteins.1 Changes in the amount and/or function of components of this signaling pathway serve as a basic mechanism of modulating the response of cells when they are exposed to high concentrations of hormones or when hormonal stimuli have been withdrawn.2 3 Both stimulatory and inhibitory receptors and G-protein
-subunits participate in the
regulation.2 3 4 5 6 7
Most of these findings have been described in cultured noncardiac
cells under high agonist concentrations, and it is questionable whether
they also apply for the heart at more physiological
conditions. Furthermore, changes observed thus far are relatively
modest (
20% to 50%), and their functional consequences are not
well understood. The relative contribution of changes in receptor
number and G proteins to the observed overall changes in the
sensitivity of adenylyl cyclase pathways is especially unclear.
Increased expression of myocardial Gi
proteins has been demonstrated in several animal models of cardiac
failure or hypertrophy (see the review in Reference 8) and
in human heart
failure.8 9 10 11 Although
these findings
indicate a pathophysiological relevance, the
exact role of the increase in Gi in heart
failure remains controversial. One of the key questions is whether the
increase of Gi in heart failure
represents an element of a vicious cycle, contributing to the
blunted positive inotropic support of the heart by
catecholamines, or whether it protects the heart from the
fatal consequences of adrenergic overstimulation, such as proarrhythmic
effects, and may therefore be regarded as a beneficial mechanism.
The present study in rats was designed to study the following
questions: Does persistent activation of the
Gi-coupled inhibitory adenylyl cyclase pathway
induce changes in Gi proteins opposite to that observed
after stimulation of the Gs-coupled stimulatory pathway in
the heart? What are the molecular mechanisms of regulation? What are
the functional consequences? The primary result is that infusion of the
M-cholinoceptor agonist carbachol leads to a decrease in muscarinic
receptor density and a decrease in
Gi, which go along with marked
sensitization of the myocardium to cAMP-induced
arrhythmia. This sheds light on a new and
as-yet-unnoticed role of Gi in the
heart.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Animal Model
Male Wistar rats (220 to 260 g) were treated by 4-day subcutaneous infusions with osmotic minipumps (Alzet ML2) as described previously.5 12 Mean rate of infusion was 5 µL/h (varying between 4.4 and 5.6 µL/h). Three groups of animals (n=18 to 20) were treated with NaCl 0.9%, carbachol (9.6 mg/kg per day; Sigma Chemical Co), or (±)-isoprenaline-HCl (dissolved in 0.002N HCl, 2.4 mg/kg per day; Boehringer Ingelheim). The concentration of carbachol was chosen in initial dose-response experiments as a dose that produced maximal decreases in heart rate without a significant loss of animals after administration of a single injection of atropine (0.2 mg/kg IP). The dose of isoprenaline was taken from our previous studies. Heart rate was measured daily by recording surface ECG in conscious rats 3 days before and during treatment. Body weight was measured daily. At day 4, rats were stunned by a blow on the neck and bled through the carotid arteries. Hearts were removed rapidly and placed in ice-cold gassed Tyrode's solution (see below for composition), and one to two papillary muscles were excised from left ventricles. Contraction experiments and preparation of RNA were started immediately thereafter. Tissue samples were quickly frozen in liquid nitrogen and stored at -80°C for determination of the other biochemical parameters. All parameters were determined in left ventricles. For determination of blood pressure, an extra series of rats (n=8 to 11 per group) was anesthetized with 100 mg/kg IP hexobarbital. A 50-pp polypropylene catheter (Schulz und Sohn) was implanted into the left carotid artery and connected to a one-way Statham pressure transducer (courtesy of Peter van Berg, Kirchseeon/Egelharting). At the same time, the osmotic minipumps were freshly filled, rinsed with 0.9% NaCl, and immediately implanted subcutaneously. This variation of the normal procedure was chosen to delay the onset of 100% pumping rate and obtain "predrug" values. According to product information, the pumping rate of the 2ML2 minipump reaches 100% after 4 hours. The carbachol group received a single injection of atropine (0.2 mg/kg IP) before pump implantation. To control for possible effects of atropine alone, an additional group (n=5) received only the injection of atropine and a minipump filled with 0.9% NaCl. Blood pressure was measured every 15 minutes during the first 5 hours and every hour until the end of the experiment. The mean of the first four measurements (1 hour) was defined as predrug (time 0).
Contraction Experiments
The experiments were performed on
electrically driven (1 Hz;
duration, 5 ms; intensity, 20% above threshhold; stretched to
Lmax) papillary muscles in a modified Tyrode's
solution containing (mmol/L) NaCl 119.8, KCl 5.4, CaCl2
1.8, MgCl2 1.05, NaH2PO4 0.42,
NaHCO3 22.6, Na2EDTA 0.05, ascorbic acid 0.28,
and glucose 5.0, continuously gassed with 95%
O2/5% CO2 and maintained at 35°C as
described previously.5 In all muscles,
concentration-response curves (CRCs) were constructed for the
positive inotropic effect of ionic calcium (1.8 to 6.3 mmol/L). The
effect of calcium did not differ between different treatment groups and
therefore is not presented. There was no arrhythmia
during calcium CRC. After extensive washing and equilibration, one
group of muscles was exposed to isoprenaline; the other, to forskolin.
After extensive washing, a single concentration of isoprenaline (0.1
µmol/L) or forskolin (3 µmol/L) was applied, and then cumulative
CRCs were obtained for the negative inotropic effect of carbachol in
the presence of isoprenaline or forskolin. Isoprenaline and carbachol
were applied for 5 minutes each; forskolin, for 20 minutes until
equilibrium was reached. The length of the contraction experiments was
about 60 minutes for equilibration plus 30 minutes for calcium CRC plus
30 minutes for washout plus 45 or 90 minutes for isoprenaline or
forskolin CRC plus 30 or 60 minutes for washout plus 60 or 70 minutes
for carbachol CRC in the presence of isoprenaline or forskolin, giving
an average total of 4.5 or 6 hours. In that time, the basal force of
contraction decreased by a mean of 47% in all groups without
significant differences. Contractile response was expressed as an
increase in the force of contraction above the respective predrug value
(in milliNewtons per millimeter squared). For each individual
concentration, the occurrence of arrhythmia was registered. The
arrhythmia was of high frequency, typically of 5 Hz and
regular rhythm, sometimes in the form of postcontractions immediately
after the paced twitch. Only arrhythmia that prohibited valid
measurement of force of contraction at this concentration was counted.
Incidence of arrhythmia was expressed as percentage of all
papillary muscles investigated in the respective treatment groups.
Adenylyl Cyclase Activity
Adenylyl cyclase activity was
determined in crude
homogenates according to Salomon13 as
described previously.14 Homogenate protein (10
to 30 µg) and an incubation time of 10 minutes were used. Final assay
conditions were (mmol/L) HEPES 50 (pH 7.4), DTT 0.1, cAMP 0.1, EGTA
0.1, NaCl 50, papaverine-HCl 0.3, creatine phosphate 10,
MgCl2 0.5, and ATP 0.2, combined with 0.4 mg/mL creatine
kinase, 0.2% BSA, 1 µg/mL adenosine deaminase, and 0.2
µmol/L 32P-ATP (6 µCi/mL). The assay was linear between
10 and 100 µg protein and incubation between 5 and 30 minutes (not
shown). Stimulators were (mmol/L) GTP 0.1, GMPPNP
(guanylyl-imidodiphosphate) 0.01, isoprenaline 0.001, NaF 10, and
MnCl2 3, with forskolin 0.01 to 100 µmol/L. Except for
one set of experiments with basal, GTP, and isoprenaline stimulation,
samples contained 10 µmol/L propranolol to exclude
influences of remaining isoprenaline. Protein was determined according
to the technique used by Bradford15 with bovine IgG as
standard.
Determination of Myocardial
Catecholamines
Myocardial catecholamines were determined with
high-performance liquid chromatography with
electrochemical detection according to Weicker et al16 as
described previously.14
Radioligand Binding Experiments
Total ß-adrenoceptor
density and the ratio of
ß1 to ß2 adrenoceptors were
determined in a crude membrane fraction as previously
described.17 We incubated 50 µg membrane protein per
assay with six concentrations of
(-)-125I-iodocyanopindolol (5 to 200 pmol/L) for 1
hour at 37°C. Nonspecific binding was determined with 1
µmol/L of the nonselective ß-adrenoceptor
antagonist (±)-CGP 12177
(4-[3-tertiarybutylamino-2-hydroxypropoxy]-benzimidazole-2-on) and
amounted to 20% to 30% at 50 pmol/L of
125I-iodocyanopindolol. The relative amounts of
ß1 and ß2 adrenoceptors were determined
with a single concentration (300 nmol/L) of the
ß1-selective antagonist (±)-CGP 20712A
(1-[2-(3-carbamoyl-4-hydroxy)phenoxyethylamino]-3-[4-(1-methyl-4-tri-fluoromethyl-2-imidazolyl)phenoxyl]-2-propanol
methane sulfonate). Protein was determined according to the method of
Lowry et al18 with BSA as standard.
M-cholinoceptors were measured in a crude membrane fraction as described previously.19 We incubated 300 to 400 µg protein with eight concentrations of N-methyl-3H-scopolamine (0.1 to 10 nmol/L) for 120 minutes at 25°C. Nonspecific binding was determined with atropine (1 µmol/L) and amounted to 20% at 5 nmol/L N-methyl-3H-scopolamine. Protein was determined according to Bradford.15
Pertussis Toxin–Catalyzed ADP-Ribosylation
Pertussis
toxin–catalyzed ADP-ribosylation in the presence
of 32P-NAD was performed in crude homogenates
as previously described.5 The in vitro incorporation of
32P-NAD is considered to be a valid measure of the amount
of functional Gi.1 8
About 100 mg ventricular tissue was homogenized
with a Polytron homogenizer. We incubated 30 µg
protein for 1 hour at 30°C in a final volume of 60 µL containing
1.67 µmol/L 32P-NAD (30 Ci/mmol; NEN-DuPont) and 1 µg
activated pertussis toxin (List Biological Laboratories).
Proteins were subjected to SDS-PAGE (running gel, 9.6%
acrylamide 4 mol/L urea). Gels were stained with Coomassie
blue G250 and dried before exposure to x-ray film. For
quantification, the intensity of autoradiographic
signals in the 40/41-kD region was measured by two-dimensional
densitometry (TLC II, CAMAG) and expressed as arbitrary density units.
Protein was determined according to the method of
Bradford.15
Immunologic Determination of Gs and
Gß
Gs- and Gß-protein
levels were determined by quantitative Western blotting exactly as
described previously.20 We subjected 40 000 g membranes
(200 µg protein) to SDS-PAGE (4% and 10% acrylamide in
the stacking and running gel) and blotted the protein to nitrocellulose
membranes (Hybond ECL, Amersham Buchler) overnight. Blots were
incubated overnight at 4°C in 15 mL TTBS (10 mmol/L Tris, pH 7.4, 154
mmol NaCl, 0.125% Tween 20) containing 1% nonfat dry milk and a 1:500
dilution of antisera. The antisera were RM/1
anti-Gs and SW/1 anti-Gß (both
from NEN-DuPont). Antibodies were visualized by
125I-protein A and autoradiography and
counted in a scintillation counter. Protein dependency was established
in each tissue for each antibody by construction of standard curves
with 30 to 400 µg membrane protein. Protein amounts corresponding to
the middle part of those curves were used in all further experiments to
allow detection of possible changes in immunodetectable G proteins.
Steady State G-Protein mRNA Levels
Total RNA preparation,
slot blot analysis, cDNA and cRNA
probes, standardization by sense cRNAs, and hybridization were
performed exactly as described previously.21 The mean
OD260/OD280 of total RNA was 2.09±0.03
(n=36). A plasmid (pGEM-3Zf-) with a truncated cDNA
for human Gß was a kind gift from Dr P. Gierschik
(Deutsches Krebsforschungsinstitut, Heidelberg, FRG). Sense cRNAs used
as standards for slot blots were quantified by tracing the in vitro
transcription assay with 10 µCi fresh 32P-UTP (800
Ci/mmol) and determining the percent incorporation into cRNA after gel
filtration and precipitation. For each slot blot, a six-point
standard curve was established in duplicate.
Autoradiographic density of hybridization signals was
plotted versus the applied amount of cRNA standard and revealed a
linear relationship.
Protein Determination
Heart tissue total protein
concentration was measured to
determine whether changes in heart weight were accompanied by changes
in the absolute amount of protein or were caused by edema. Protein
concentration was measured in tissue homogenates in
solution after overnight incubation (see RNA preparation) according to
the technique of Bradford.15 The 4 mol/L LiCl/8 mol/L did
not interfere with the assay.
Statistical Analysis
All values presented are arithmetic
mean±SEM or
geometric mean with 95% confidence limits (EC50). The
equilibrium dissociation constant (Kd) and the
maximal number of binding sites (Bmax) were
calculated from plots according to Scatchard analysis.
Statistical significance between more than two groups was estimated
with ANOVA F test and Dunnett's test. Student's
t test for paired observations was used for heart rate
before and after treatment; Student's t test for unpaired
observations was used for comparing contractile responses between two
groups (NaCl and isoprenaline). A value of P<.05 was
considered significant.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Physiological Parameters and Norepinephrine Concentrations
Carbachol induced a drastic decrease in heart rate by about 280 beats per minute (range, 432 to 151 beats per minute) 5 minutes after implantation of the minipump, which was lethal for 3 of 5 rats (data not shown). After application of atropine (0.2 mg/kg IP) 5 minutes before implantation of carbachol-filled minipumps, the heart rate–lowering effect of carbachol was significantly reduced. The average heart rate showed a tendency to increase during the 4-day treatment of carbachol (Table 1
). This cannot be
explained by a decay of carbachol or other technical problems because
implanting the pump after 4 days into another animal resulted in the
same decrease in heart rate as after initial implanting. It may be
argued that atropine alone affects some of the parameters
investigated. We cannot completely exclude this possibility, but given
the short half-life (4 hours), a prolonged action of atropine
seems unlikely. In addition, atropine had no significant effect on
blood pressure (Table 2). The lack of a sustained effect
of carbachol on heart rate probably reflects the balance of the direct
negative chronotropic action and indirect baroreceptor-mediated
reflex tachycardia. Similarly, unchanged norepinephrine
levels in the carbachol group (Table 1) may result from direct
inhibition of norepinephrine release from sympathetic nerve endings and
central increases in sympathetic tone through the baroreceptor reflex.
In agreement with our previous5 12 21 and
other
studies,22 23 24 25 26
isoprenaline induced cardiac
hypertrophy, an increase in total cardiac protein and RNA,
and a decrease in tissue concentration of norepinephrine by 46%.
To further characterize the model, blood pressure was determined. As
expected, isoprenaline induced a slight and only transient
decrease in systolic pressure and a more pronounced and lasting
decrease in diastolic pressure (Table 2). In contrast,
neither carbachol (plus atropine) nor atropine alone significantly
altered blood pressure.
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Force of Contraction
Isometric force of contraction was
determined in isolated
electrically driven papillary muscles as a global parameter
of myocardial function and the sensitivity of stimulatory and
inhibitory adenylyl cyclase pathways. Muscles from
carbachol-treated rats consistently showed isoprenaline- or
forskolin-induced arrhythmia, which did not allow valid
measurement of inotropic effects in the majority of muscles (26 of 31
for isoprenaline and 15 of 21 for forskolin). In the remaining muscles,
the efficacy of isoprenaline and forskolin tended to be higher by 48%
and 53%, respectively (P=NS, not shown). In agreement with
earlier studies,5 25 papillary muscles from
isoprenaline-treated rats showed decreased maximal inotropic
responses to isoprenaline (not shown), which may result from changes in
both ß-adrenoceptor number and
Gi. As an extension, we now
demonstrate a 3.5-fold reduced positive inotropic potency of the
ß-adrenoceptor–independent stimulator of adenylyl cyclase
forskolin (Fig 1A). Because this alteration is unlikely
to result from changes in ß-adrenoceptor density, it adds to the
assumption that the increase in Gi
cross-regulates stimulatory adenylyl cyclase
pathways.4
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As shown previously,5 the negative
inotropic potency of
carbachol in the presence of isoprenaline was increased fourfold in
muscles from isoprenaline-treated rats (not shown). The present
experiments now show a similar sixfold increase in the potency of
carbachol when added in the presence of forskolin (Fig 1B
). In
addition, the efficacy of carbachol in reducing contractile force was
increased. Whereas carbachol reduced contractile force from 335% of
the predrug value (forskolin alone) to 139% in control muscles, it
reduced it from 271% to 77% in muscles from isoprenaline-treated
rats (data not shown). These findings again strongly suggest that the
increase in Gi is functionally important in
the heart under physiological conditions.
In cardiac preparations from carbachol-treated rats, force of contraction could not be evaluated in most (13 of 29 for isoprenaline, 13 of 21 for forskolin) of muscles because of arrhythmia. In the remaining muscles, however, the negative inotropic potency of carbachol in the presence of isoprenaline was decreased threefold compared with control (P<.05; not shown).
Incidence of Arrhythmia
Surprisingly, construction of CRCs
for the positive inotropic
effect of isoprenaline or forskolin was almost impossible in muscles
from carbachol-treated rats because of the occurrence of
spontaneous contractions of high frequency (Fig 2). The
arrhythmia could not be terminated by short periods of
overpacing. High concentrations of isoprenaline and forskolin (>0.3
and 3 µmol/L, respectively) also induced arrhythmia in
control muscles (12% and 31%, Fig 3A and 3B).
However,
the "arrhythmogenic" effect of isoprenaline and forskolin in
muscles from carbachol-treated rats was significantly greater (87%
and 71%, respectively) and started earlier (0.003 and 0.3 µmol/L,
respectively; Fig 3A and 3B). EC50
values (determined
geometrically) were 0.025 and 0.45 µmol/L for isoprenaline and
forskolin, respectively, compared with 0.9 and 1 µmol/L in the
control group. Thus, the apparent arrhythmogenic potency of
isoprenaline and forskolin was about 36 and 2.2 times higher,
respectively, in preparations from carbachol-treated rats.
In addition, the arrhythmogenic efficacy of isoprenaline and
forskolin was 7.3 and 2.3 times higher, respectively, than in control
muscles. In contrast, isoprenaline infusion induced a clear, albeit not
statistically significant, trend toward a reduced incidence of
forskolin-induced arrhythmia (Fig 3B).
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Cumulative
addition of carbachol abolished the isoprenaline- (Fig 4A) or
forskolin-induced arrhythmia (Fig 4B). In muscles from control
or isoprenaline-treated rats, the
initial frequency of arrhythmia was 0% (isoprenaline) or
20% (forskolin). In the latter, 0.1 µmol/L carbachol was
sufficient to almost completely suppress the arrhythmia. In
contrast, 0.1 µmol/L carbachol did not even reduce the 42% to 60%
incidence in muscles from carbachol-treated rats; 3 µmol/L
carbachol was necessary to fully suppress the arrhythmia in
these muscles (Fig 4A and 4B). Thus, the potency
of carbachol to
suppress cAMP-induced arrhythmia was about 30 times lower than
in control rats.
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Adenylyl Cyclase Activity
Adenylyl cyclase activity was
determined under conditions that
favor the influence of Gi proteins, namely high GTP, low
magnesium, and high NaCl.25 Isoprenaline infusion led to a
33% (no propranolol; Fig. 5A) or 44% (with
propranolol; Fig 5B) reduction in basal adenylyl cyclase
activity. Stimulation by GTP, isoprenaline, GMPPNP, and NaF was
decreased by 28%, 53%, 44.5%, and 49%, respectively (Fig 5B).
In
contrast, stimulation by MnCl2 and forskolin was unchanged
(Fig 5C). Infusion of carbachol had no significant influence on
basal
or stimulated adenylyl cyclase activity but was accompanied by a
tendency to increased values in all groups except stimulation by
MnCl2.
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ß-Adrenoceptors and M-Cholinoceptors
Isoprenaline induced
a decrease in total ß-adrenoceptor
density by 43% and shifted the ratio of ß1- to
ß2-adrenoceptor from 62%/38% to 76%/24% (Table
3). The reason and physiological
significance of this well-known shift are not clear (for
discussion, see Reference 5). Carbachol treatment did not alter
ß-adrenoceptor density. Kd values
for 125I-iodocyanopindolol did not differ
significantly between the groups
(Kd, 21±5, 16±3, and 23±3
[n=6] for
NaCl, isoprenaline, and carbachol, respectively).
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Carbachol treatment
induced a reduction in M-cholinoceptor density by
15% (Table 3). Isoprenaline did not affect M-cholinoceptor
density.
Kd values for
N-methyl-3H-scopolamine were not significantly
different in all groups studied
(Kd, 300±70, 367±60, and 353±50
pmol/L
[n=7 to 10] for NaCl, isoprenaline, and carbachol,
respectively).
Pertussis Toxin Substrates
Isoprenaline induced a 25%
increase in the 40/41-kD pertussis
toxin substrates, whereas treatment with carbachol induced a 26%
decrease (Fig 6). Similar but not identical results
(18% decrease, P=NS) were obtained by determining the
amount of Gi proteins by
radioimmunoassay26 (not shown).
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Quantitative Western Blotting of Gs and
Gß
The Gs antiserum detected two bands
with an apparent molecular weight of 42 and 49/50 kD. Both bands were
cut out and counted separately. The Gß antiserum detected
three bands, a doublet at about 37/38 kD and a single band of an
apparent molecular weight of 34 kD. The radioactive incorporation in
the doublet was approximately 10 to 15 times higher than in the 34 kD
band. Neither treatment induced any changes in
Gs or Gß level (Table 3).
G-Protein mRNA Levels
In agreement with our previous studies,
isoprenaline treatment
induced an increase in the steady state levels of
Gi-2 and
Gi-3 mRNA by 42%
and 63%, respectively (Table 3). Carbachol did not change mRNA
levels
of Gi-2 and
Gi-3 mRNA. Steady
state levels of Gs and Gß mRNA
were not altered by any treatment (Table 3).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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The aim of the present study was to investigate in an in vivo setting the mechanisms and functional consequences of chronic regulation of the cardiac adenylyl cyclase signaling pathway. The primary new finding was that long-term activation of the inhibitory Gi
-mediated pathway
by infusion of carbachol leads to a marked sensitization of the
myocardium to cAMP-induced arrhythmia.
Several experiments were performed to characterize the specificity and
possible underlying biochemical mechanisms of this effect. They
included determination of heart rate; blood pressure; heart weight;
protein and RNA content of the ventricular
myocardium; myocardial norepinephrine concentration;
ß-adrenoceptor and muscarinic receptor density;
Gs,
Gi, and Gß protein and
mRNA content; adenylyl cyclase activity; and inotropic responses of
isolated papillary muscles to calcium, isoprenaline, or forskolin.
Compared with control, the primary abnormalities that accompanied the
increase in arrhythmogenicity in carbachol-treated animals were a
15% decrease in muscarinic receptor density and a 26% decrease in
pertussis toxin–sensitive Gi
proteins.
The decreases in muscarinic receptor density and in
Gi proteins are in line with current
concepts of modulatory changes of components of the cardiac adenylyl
cyclase under long-term activation of inhibitory
pathways. Thus, several authors have shown that prolonged exposure to
adenosine receptor
agonists3 7 27 28 induces
downregulation of the respective receptor and immunodetectable amount
of Gi. Similarly, treatment of
adipocytes with prostaglandin E1, which
inhibits adenylyl cyclase in adipocytes, leads to a reduction in
Gi.29 The present
study extends these findings in showing that a 4-day infusion of the
muscarinic agonist carbachol decreases myocardial
Gi content. This effect was achieved by
application of a highly effective dose of carbachol that without
injection of a single dose of atropine, led to several dropouts in the
initial experiments. Infusions of carbachol and isoprenaline obviously
will have extracardial effects that by themselves may alter some of the
parameters determined in the present study. Infusion of
carbachol after a single injection of atropine, however, neither
changed tissue norepinephrine content nor significantly altered blood
pressure (Tables 1 and 2). Because identical
changes in
Gi were also seen in cultured cells after
incubation with adenosine analogues,6 27 it seems
justified to assume a direct cardiac effect.
The most prominent finding in carbachol-treated animals was the
marked increase in the potency (36- and 2.2-fold) and efficacy
(7.3- and 2.3-fold) of isoprenaline and forskolin, respectively, in
inducing spontaneous contractions in isolated papillary muscles.
This was accompanied by an 30-fold decrease in potency of carbachol
to suppress arrhythmia. Thus, carbachol infusion markedly
sensitizes the myocardium to cAMP-induced
arrhythmia ("cross-regulation") and compromises the
ability of carbachol to suppress them ("homologous
desensitization"). Similar effects seemed to apply to the
contractile responses, but the arrhythmia disturbed the
mechanograms too much to allow a clear statement.
Whereas the decreased potency of carbachol in carbachol-treated
rats may be due to a reduction in both muscarinic receptor density and
Gi, the increased susceptibility to
isoprenaline- and forskolin-induced arrhythmia cannot be
attributed to changes in receptors or any other measured
parameter. We cannot exclude the contribution of further
downstream elements such as changes in Ca2+-handling
proteins of the sarcoplasmic reticulum. However, our own recent
experiments did not reveal significant changes in the expression of
phospholamban, sarcoplasmic reticulum
Ca2+-ATPase,
Na+-Ca2+ exchanger, or ryanodine
receptors.30 Thus, the increase in arrhythmogenicity seems
to be due, at least in part, to the decrease in
Gi. This conclusion is
consistent with a recent study that shows that
ß1-adrenoceptor–mediated arrhythmia was
significantly more frequent in pertussis toxin–treated rat
cardiomyocytes.31 Thus, the present
experiments point to a so-far-unnoticed role of
Gi in controlling electric activity in the
heart.
This assumption is further supported by the fact that the incidence of
forskolin-induced arrhythmia was lower in
isoprenaline-pretreated than control rats. Even though the
difference did not reach statistical significance, it was a clear trend
and may indicate that an increase in
Gi, together with a decrease in
ß-adrenoceptor density, may protect the heart against
cAMP-induced arrhythmia. It is interesting in this respect that
pacing-induced heart failure in dogs is accompanied by a reduction
in ß-adrenoceptor density, an increase in
Gi,32 and a reduced
arrhythmogenic effect of catecholamines,33
suggesting a causative relation. Furthermore, we have shown by in situ
hybridization that Gs,
Gi-2, and
Go mRNAs are approximately twofold
concentrated in the AV conduction system.34 It may be
argued that the small (±25%) changes in the content of
Gi are not likely to account for the marked
changes in arrhythmogenicity. However, it has been estimated that one
adenosine or muscarinic receptor activates about 50 to
80 Gi molecules in human
myocardium.35 The effect is significant signal
amplification. If there is no spare
Gi, one can assume that even small
changes in the amplifier will significantly modulate the response. This
hypothesis, however, remains to be proved more directly.
In human heart failure, the concentration of pertussis
toxin–sensitive G proteins (Gi and
Go) is increased.9 10 11
Recent evidence from clinical studies on patients with heart failure
suggests that those with mild to moderate heart failure (New York Heart
Association class II to III) most often die from a sudden,
arrhythmogenic event, whereas patients with end-stage disease
(class IV) most often die from progressive heart
failure36 37 38 and bradyarrhythmic
events.39
The latter are patients with a high degree of ß-adrenoceptor
downregulation and increase in Gi and
Go. In view of the present
findings, it is tempting to speculate that these alterations in
end-stage human heart failure not only compromise contractile
performance but serve as a protective antiarrhythmic mechanism.
The importance of alterations in the ß-adrenoceptor adenylyl
cyclase pathway for the incidence of cardiac arrhythmia is
underlined by a recent study by Kaumann and colleagues.40
It reported an increased sensitivity toward
catecholamine-evoked arrhythmia in atrial
preparations of patients treated with ß1-blocking agents.
Such treatment induces an increase in ß1- but not in
ß2-adrenoceptor numbers and a reduction in
M-cholinoceptors.41 Both alterations could contribute to
the sensitization to cAMP-induced arrhythmia.
The underlying mechanisms of the isoprenaline- or forskolin-induced
arrhythmia in electrically driven papillary muscles are not
known in detail but probably involve spontaneous calcium release by the
sarcoplasmic reticulum and/or alterations in ion channel function.
Gi and Go may
interfere with protein kinase–phosphorylated
channels by inhibition of adenylyl cyclase and have been shown to
mediate the 1-adrenoceptor–induced slowing of
automaticity in the adult heart by activating the
Na+,K+-ATPase.44 It remains
to be elucidated what mechanisms may be relevant in the context of the
present experiments.
In contrast to the mechanism of upregulation of pertussis toxin
substrates after isoprenaline infusion that involves alterations in
gene transcription and mRNA21 45 the decrease in
pertussis
toxin substrates was not accompanied by changes in
Gi-2 or
Gi-3 mRNAs. This
finding is in accord with other in vivo7 and in vitro
studies3 and indicates alterations in protein turnover
rate.3
In conclusion, the present study shows that chronic muscarinic
stimulation markedly sensitizes the myocardium to
cAMP-induced arrhythmia. The results indicate that such
sensitization is probably caused by a decrease in
Gi, shedding light on a new and
so-far-unnoticed role of changes in G-protein content in the
heart.
|
Acknowledgments |
|---|
The study was supported by the Deutsche Forschungsgemeinschaft. We thank Dr R.R. Reed for providing the cDNA clones of Gi
-2,
Gi-3, and
Gs and Dr P. Gierschik for the gift of
Gß-cDNA. We thank Birgit Geertz, Monika Nose, and Ellen
Schäfer for excellent technical assistance. We thank Jochen
Scheel, Beiersdorf-Lilly GmbH, for his help in blood pressure
determination. |
Footnotes |
|---|
Reprint requests to Thomas Eschenhagen, MD, Abteilung Allgemeine Pharmakologie, Universitäts-Krankenhaus Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
Received January 23, 1995; revision received September 27, 1995; accepted October 4, 1995.
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