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Circulation. 1996;93:763-771

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*CARBACHOL CHLORIDE

(Circulation. 1996;93:763-771.)
© 1996 American Heart Association, Inc.

Articles

Chronic Treatment With Carbachol Sensitizes the Myocardium to cAMP-Induced Arrhythmia

Thomas Eschenhagen, MD; Ulrike Mende, MD; Matthias Diederich, MD; Boris Hertle, MD; Christian Memmesheimer, MD; Andreas Pohl, MD; Wilhelm Schmitz, MD; Hasso Scholz, MD; Markus Steinfath, MD; Michael Böhm, MD; Martin C. Michel, MD; Otto-Erich Brodde, PhD; Achim Raap, MD

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.


*    Abstract
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 *Abstract
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Background The present study investigated biochemical and functional consequences of chronic activation of the inhibitory Gi{alpha}-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{alpha}) 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{alpha} 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{alpha}.


Key Words: acetylcholine • signal transduction • receptors, adrenergic, beta • arrhythmia


*    Introduction
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 up arrowAbstract
 *Introduction
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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 {alpha}-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 ({approx}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{alpha} 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{alpha} in heart failure remains controversial. One of the key questions is whether the increase of Gi{alpha} 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{alpha}, 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{alpha} in the heart.


*    Methods
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 *Methods
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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 {approx}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{alpha}.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{alpha} and Gß
Gs{alpha}- 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{alpha} 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.


*    Results
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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 1Down). 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 ({approx}4 hours), a prolonged action of atropine seems unlikely. In addition, atropine had no significant effect on blood pressure (Table 2Down). 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 1Down) 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 2Down). In contrast, neither carbachol (plus atropine) nor atropine alone significantly altered blood pressure.


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  		Table 1. Physiological Parameters and Tissue Norepinephrine Content


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  		Table 2. Blood Pressure

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{alpha}. 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 1ADown). Because this alteration is unlikely to result from changes in ß-adrenoceptor density, it adds to the assumption that the increase in Gi{alpha} cross-regulates stimulatory adenylyl cyclase pathways.4



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  		Figure 1. Plots showing that the infusion of isoprenaline (Iso) desensitizes the myocardium to forskolin (Forsk) and sensitizes it to carbachol. Influence of 4-day infusions of isoprenaline (Iso; 2.4 mg/kg per day) compared with 0.9% NaCl on the positive inotropic effect of forskolin (A) and the negative inotropic effect of carbachol in the presence of forskolin (B) in electrically driven papillary muscles. A, Predrug value (mN/mm2): 1.1±0.2, Iso 1.7±0.3 (P=NS vs NaCl); maximal increase in force of contraction: NaCl 172% (1.61 mN/mm2), Iso 121% (2.0 mN/mm2; P=NS vs NaCl); and EC50 (µmol/L): 0.39 (0.24 to 0.63), Iso 1.39 (0.96 to 2.04; P<.05 vs NaCl). B, Predrug value (mN/mm2): NaCl 1.0±0.2, Iso 1.1±0.1; forskolin increased force of contraction by NaCl 235±40% (1.8 mN/mm2), Iso 171±35% (1.72 mN/mm2; P=NS vs NaCl); IC50 for carbachol (µmol/L): NaCl 0.50 (0.32 to 0.77); Iso 0.08 (0.05 to 0.14; P<.05 vs NaCl). Numbers in brackets give numbers of papillary muscles.

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 1BUp). 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{alpha} 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 2Down). 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 3ADown and 3BDown). 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 3ADown and 3BDown). 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 3BDown).



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  		Figure 2. Representative original tracings of papillary muscles from rats treated by 4-day infusions of 0.9% NaCl (A) or carbachol (9.6 mg/kg per day; B). Shown is the response to application of 3 µmol/L forskolin and the cumulative concentration-response curve for carbachol (0.01 to 0.03 to 0.1 . . . 1000 µmol/L, 5 minutes each).



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  		Figure 3. Plots showing that the infusion of carbachol sensitizes the myocardium to cAMP-induced arrhythmia. Influence of 4-day infusions of isoprenaline (Iso; 2.4 mg/kg per day) or carbachol (Carb; 9.6 mg/kg per day) compared with 0.9% NaCl on the incidence of arrhythmia in electrically driven papillary muscles after addition of isoprenaline (A) or forskolin (B). Ordinates give the incidence of arrhythmia in percentage of all papillary muscles investigated at the respective drug concentrations. Numbers in brackets are numbers of all papillary muscles investigated.

Cumulative addition of carbachol abolished the isoprenaline- (Fig 4ADown) or forskolin-induced arrhythmia (Fig 4BDown). 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 4ADown and 4BDown). Thus, the potency of carbachol to suppress cAMP-induced arrhythmia was about 30 times lower than in control rats.



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  		Figure 4. Plots showing that the infusion of carbachol desensitizes the myocardium to the arrhythmia-suppressing effect of carbachol. Influence of 4-day subcutaneous infusions of isoprenaline (Iso; 2.4 mg/kg per day) or carbachol (Carb; 9.6 mg/kg per day) compared with 0.9% NaCl on the ability of carbachol to suppress the isoprenaline (0.1 µmol/L; A)– or forskolin (Forsk; 3 µmol/L; B)–induced arrhythmia. Ordinates are identical to Fig 3Up.

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. 5ADown) or 44% (with propranolol; Fig 5BDown) reduction in basal adenylyl cyclase activity. Stimulation by GTP, isoprenaline, GMPPNP, and NaF was decreased by 28%, 53%, 44.5%, and 49%, respectively (Fig 5BDown). In contrast, stimulation by MnCl2 and forskolin was unchanged (Fig 5CDown). 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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  		Figure 5. Bar graphs showing that the infusion of isoprenaline leads to desensitization of myocardial adenylyl cyclase. Influence of 4-day infusions of isoprenaline (Iso; 2.4 mg/kg per day) or carbachol (Carb; 9.6 mg/kg per day SC) compared with 0.9% NaCl on basal and stimulated adenylyl cyclase activity. A, Stimulation with GTP (100 µmol/L) and isoprenaline (1 µmol/L) plus GTP. B, Stimulation with GTP (100 µmol/L), GMPPNP (10 µmol/L), NaF (10 mmol/L) plus GTP, and MnCl2 (3 mmol/L) plus GTP. The assay contained 10 µmol/L propranolol. C, Concentration-dependent effect of forskolin in the presence of GTP (100 µmol/L).

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 3Down). 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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  		Table 3. ß-Adrenoceptors; M-Cholinoceptors; Gs{alpha} and Gß Protein; and Gi{alpha}, Gs{alpha}, and Gß mRNA

Carbachol treatment induced a reduction in M-cholinoceptor density by 15% (Table 3Up). 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 6Down). Similar but not identical results (18% decrease, P=NS) were obtained by determining the amount of Gi{alpha} proteins by radioimmunoassay26 (not shown).



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  		Figure 6. Infusion of isoprenaline increases and infusion of carbachol decreases Gi{alpha}. Influence of 4-day infusions of isoprenaline (Iso; 2.4 mg/kg per day SC) or carbachol (Carb; 9.6 mg/kg per day SC) compared with 0.9% NaCl on Gi{alpha} protein content determined by pertussis toxin–catalyzed 32P-ADP–ribosylation. A, Representative autoradiography of the region of interest. Exposure time of the autoradiography was 72 hours. B, Bar graph showing densitometric quantification of the incorporation of 32P-ADP–ribose into the 40/41-kD pertussis toxin substrates (in arbitrary units). Numbers in columns are numbers of hearts.

Quantitative Western Blotting of Gs{alpha} and Gß
The Gs{alpha} 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{alpha} or Gß level (Table 3Up).

G-Protein mRNA Levels
In agreement with our previous studies, isoprenaline treatment induced an increase in the steady state levels of Gi{alpha}-2 and Gi{alpha}-3 mRNA by 42% and 63%, respectively (Table 3Up). Carbachol did not change mRNA levels of Gi{alpha}-2 and Gi{alpha}-3 mRNA. Steady state levels of Gs{alpha} and Gß mRNA were not altered by any treatment (Table 3Up).


*    Discussion
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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{alpha}-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{alpha}, Gi{alpha}, 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{alpha} proteins.

The decreases in muscarinic receptor density and in Gi{alpha} 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{alpha}. Similarly, treatment of adipocytes with prostaglandin E1, which inhibits adenylyl cyclase in adipocytes, leads to a reduction in Gi{alpha}.29 The present study extends these findings in showing that a 4-day infusion of the muscarinic agonist carbachol decreases myocardial Gi{alpha} 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 1Up and 2Up). Because identical changes in Gi{alpha} 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 ({approx}36- and {approx}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 {approx}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{alpha}, 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{alpha}. 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{alpha} 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{alpha}, 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{alpha},32 and a reduced arrhythmogenic effect of catecholamines,33 suggesting a causative relation. Furthermore, we have shown by in situ hybridization that Gs{alpha}, Gi{alpha}-2, and Go{alpha} 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{alpha} 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{alpha} molecules in human myocardium.35 The effect is significant signal amplification. If there is no spare Gi{alpha}, 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{alpha} and Go{alpha}) 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{alpha} and Go{alpha}. 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{alpha} and Go{alpha} may interfere with protein kinase–phosphorylated channels by inhibition of adenylyl cyclase and have been shown to mediate the {alpha}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{alpha}-2 or Gi{alpha}-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{alpha}, 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{alpha}-2, Gi{alpha}-3, and Gs{alpha} 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.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
up arrowDiscussion
*References
 

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