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(Circulation Research. 2000;87:903.)
© 2000 American Heart Association, Inc.

Cellular Biology

G_i2 but Not G_i3 Is Required for Muscarinic Inhibition of Contractility and Calcium Currents in Adult Cardiomyocytes

Kohzo Nagata¹, Chianping Ye¹, Mohit Jain, David S. Milstone, Ronglih Liao, Richard M. Mortensen

From the Whitaker Cardiovascular Institute, Cardiac Muscle Research Laboratory (K.N., M.J., R.L.), Boston University School of Medicine, Boston, Mass; Department of Physiology and Medicine (Endocrine) (R.M.M.), University of Michigan Medical School, Ann Arbor, Mich; and Vascular Division, Departments of Pathology (D.S.M) and Medicine (C.Y.), Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass.

Correspondence to Richard M. Mortensen, Department of Physiology and Medicine (Endocrine), University of Michigan Medical School, 7726 Medical Science II, Ann Arbor, MI 48109-0622. E-mail rmort@umich.edu or rliao{at}bu.edu

	Abstract

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Abstract—Parasympathetic stimulation of the heart acts through M₂-muscarinic acetylcholine receptors to regulate ion channel activity and subsequent inotropic status. Although muscarinic signal transduction is mediated via pertussis toxin-sensitive G proteins G_i/o, the specific signal transduction requirements of G_i2 and G_i3 in mediating muscarinic regulated L-type calcium currents (I_{Ca, L}), intracellular calcium, and cell contractility remain to be determined. Adult ventricular myocytes were isolated from G_i2-null mice, G_i3-null mice, and their wild-type littermates. Cell shortening, intracellular calcium levels, and I_{Ca, L} were all measured in response to isoproterenol, a ß-adrenergic receptor agonist, and carbachol, a cholinergic receptor agonist. With isoproterenol stimulation, myocytes from all groups demonstrated a marked increase in calcium currents, correlating with augmented intracellular calcium transient amplitude and cell shortening. Carbachol significantly attenuated the isoproterenol response in wild-type and G_i3-null cells but had no effect in G_i2-null cells. This study demonstrates that G_i2, but not G_i3, is required for muscarinic inhibition of the ß-adrenergic response in adult murine ventricular myocytes.

Key Words: Gi proteins • muscarinic receptor • myocyte • contractility • intracellular calcium

	Introduction

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Parasympathetic stimulation of the heart acts through M₂-muscarinic acetylcholine receptors to regulate ion channel activity and subsequent cardiac chronotropic and inotropic status.^{1 2} In mammalian ventricular myocardium, M₂ activation is associated with little effect on basal cardiac function but a significant attenuation of the contractile response to ß-adrenergic (ßAR) stimulation, termed indirect inhibitory action.³ The stimulatory ßAR response is initiated via G_s protein activation of adenylyl cyclase and subsequent protein kinase A (PKA)-mediated phosphorylation of L-type calcium channels, troponin I, and phospholamban, resulting in increased calcium influx and augmented contractility (inotropy) as well as increased calcium reuptake and enhanced relaxation (lusitropy).⁴ The antiadrenergic response of the muscarinic receptor is mediated via pertussis toxin (PTX)-sensitive G proteins G_i/o.^{5 6 7} Although G_o, G_i2, and G_i3 are all known to couple the M₂ receptor, the mechanisms by which M₂-muscarinic activation influences L-type calcium channel (I_{Ca, L}) activity and corresponding intracellular calcium and myocyte contractility remain unclear.

Recently, G_o has been shown to mediate L-type calcium channel activity in response to M₂ stimulation.⁸ We have previously shown in spontaneously contracting nodal and atrial-like cells that, in addition to G_o, muscarinic regulation of I_{Ca, L} requires both G_i2 and G_i3 for normal calcium kinetics.⁹ Significant differences in the M₂ response and regulation exist between atrial and ventricular myocardium.³ The specific signal-transduction requirements of G_i2 and G_i3 in mediating muscarinic-regulated L-type calcium channel activity, intracellular calcium, and cell contractility in adult ventricular myocytes remain to be determined. Therefore, we examined the ability of the M₂ receptor agonist carbachol to inhibit isoproterenol-stimulated I_{Ca, L}, intracellular calcium, and cell contractility in ventricular myocytes from G_i2- and G_i3-null mice.

	Materials and Methods

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Animal Model
Using previously published constructs for gene targeting,^{10 11} the _i2 and _i3 genes were inactivated in J1 cells cultured on mouse embryo fibroblasts and injected into C57BL/6 blastocysts. Resulting chimeras passed the targeted mutation in the germline bred to C57Bl/6. Heterozygotes were mated to obtain littermates that were either wild type (WT) or homozygous for the gene inactivation. The inactivation of the targeted gene was confirmed by polymerase chain reaction. There was no difference between littermate controls for G_i2-null and G_i3-null mice; therefore, data were combined and presented as WT. G_i2-null and G_i3-null mice displayed no gross animal phenotype or cardiac morphological differences. All animal handling and procedures strictly conformed with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication number 85-23, 1996).

Adult Ventricular Myocyte Isolation
G_i2-null and G_i3-null mice and their WT littermates were intraperitoneally heparinized with 200 U heparin and anesthetized with ketamine (150 mg/kg) and xylazine (15 mg/kg). Left ventricular myocytes were dissociated using a modified protocol described previously.¹² Briefly, hearts were quickly excised, cannulated via the aorta, and perfused in the Langendorff mode with a constant perfusion pressure of 80 mm Hg. The hearts were first perfused for 5 minutes at 37°C with 1.8 mmol/L Ca²⁺ Tyrode (in mmol/L: NaCl 137, KCl 5.4, CaCl₂ 1.8, MgCl₂ 0.5, HEPES 10, and glucose 10, pH 7.4), followed by Ca²⁺-free Tyrode for an additional 5 minutes. They were then perfused with a digestion solution containing 0.06% collagenase D (Boehringer Mannheim) and 0.01% protease XIV (Sigma Chemical Co). After the hearts were palpably flaccid, the digestion solution was washed out with Ca²⁺-free Tyrode solution for 30 seconds. The left ventricle (including the septum) was cut into small pieces and gently agitated, allowing the myocytes to be dispersed in the KB solution (in mmol/L: KOH 85, KCl 30, KH₂PO₄ 30, MgSO₄ 3, EGTA 0.5, HEPES 10, 1-glutamic acid 50, taurine 20, and glucose 10, pH 7.4). After 60 minutes, the cells were resuspended in calcium-containing buffers, with Ca²⁺ concentrations gradually increasing from 0.2 to 0.6 and, finally, to 1.2 mmol/L Ca²⁺. Left ventricular myocytes were aliquot into 2 portions and resuspended in their appropriate buffers for electrophysiological and myocyte contractility measurements.

Myocytes included in the study met the following criteria: an overall rod-shape with a clear striation pattern (without granulation and without cauliflower-shaped cell edges); quiescent in the absence of electrical stimulation; and stable mechanical behavior at 5 Hz and 37°C for 5 to 10 minutes. No cells were included past 6 hours after isolation.

Myocyte Cell Shortening
Cells were electrically paced via platinum wires placed in the myocyte bath and connected to a commercially available stimulator. Cell length and contractile amplitude of myocytes were recorded in real time on a Pentium 120-MHz personal computer with a video edge detector and specialized data acquisition software (SoftEdge Acquisition System and IonWizard, IonOptix Inc), as previously described.¹²

Twitch amplitude was expressed as the difference between diastolic and peak systolic cell lengths. Percent cell shortening (%CS) was expressed as the ratio of absolute twitch amplitude to diastolic cell length. Maximum derivatives of both cell contraction and relaxation were measured as previously described.^{12 13 14}

Intracellular Calcium Measurement
Cytosolic calcium was measured by the fluorescent calcium indicator fura-2 (Molecular Probes) using a dual fluorescence, calcium ion sensing system (Ion Optix), as previously described.¹² Freshly dissociated ventricular myocytes were incubated in 1.2 mmol/L Ca²⁺ Tyrode solution containing 1 µmol/L of membrane permeate fura-2 for 15 minutes at room temperature. After washing out the fura-2 in the loading solution, an additional 40 minutes were allowed for the deesterification of the fura-2 ester in the cells. Throughout this procedure, 500 µmol/L probenecid was included to prevent the leakage of fura-2 from the cells. Fura-2–loaded myocytes were alternately excited with a xenon lamp at wavelengths of 360 and 380 nm. The emission fluorescence was collected by the objective and reflected through barrier filter (510±15 nm) to a photomultiplier tube. A subset of myocytes from each group was used to calibrate the fura-2 fluorescence ratio to intracellular calcium concentrations in situ, as previously described.¹²

Myocyte Protocol
Myocytes were stimulated at a physiological rate of 300 bpm at 37°C throughout entire protocol. After 15 minutes of baseline stabilization period, cell shortening was recorded as baseline data. A concentration of 1 µmol/L of isoproterenol was then superperfused, and data were collected when the reaction reached steady state after 3 minutes. Added to the isoproterenol was 10 µmol/L carbachol superperfusate, and measurements were recorded after another 3 minutes. Cell shortening and fura-2 fluorescence ratio were simultaneously recorded at each time point.

Electrophysiological Measurements
Isolated ventricular myocytes were attached to 1% gelatin-coated tissue culture dishes. Media was changed gradually to increase the calcium concentration to 1.8 mmol/L. All recordings were performed at ambient temperature (21°C to 23°C). Calcium currents (I_{Ca, L}) were recorded using a conventional whole-cell voltage clamp recording.¹⁵ The internal solution contained (in mmol/L) CsCl 120, EGTA 10, Mg-ATP 3, and HEPES 10, pH 7.3, adjusted by CsOH. The bath solution contained (in mmol/L) NaCl 137, CsCl 5.4, CaCl₂ 1.8, MgCl₂ 0.5, HEPES 10, and glucose 10, pH 7.4. Baseline and stimulated currents were measured in the presence and absence of 0.5 mmol/L GTP, which showed no effect of added GTP. In routine protocols, the cells were depolarized every 6 seconds from a holding potential of -80 to 0 mV for 200 ms after a 50-ms prepulse to -50 mV. This prepulse, together with the application of 30 µmol/L tetrodotoxin, was used to eliminate the fast sodium currents. Potassium currents were eliminated by substituting Cs⁺ for K⁺ in both pipette and bath solutions.

For the determination of current-voltage relations, currents were elicited between -50 and +50 mV from a holding potential of -80 mV. Voltage-clamp pulses of 200 ms after a prepulse at -50 mV (50 ms) were applied every 6 seconds. To measure the steady-state I_Ca inactivation, a double-pulse voltage-clamp protocol was used. A conditioning pulse from -80 mV to a voltage varying from -80 to +20 mV for 2 seconds was followed by a pulse at -50 mV for 20 ms and then a fixed test pulse at 0 mV for 200 ms. Current was obtained at the test potential of 0 mV by measuring the difference between peak inward current and the leak current at the end of the 2s pulse.

In the studies of I_{Ca, L} in response to isoproterenol or carbachol, 2 to 3 minutes after a stable whole-cell configuration was formed, the basal I_{Ca, L} was recorded for 3 minutes. Then isoproterenol (1 µmol/L) was added to the bath solution and recordings were made every minute for 4 minutes. Carbachol (10 µmol/L) was added, and the recordings continued for at least 7 minutes. In some experiments, isoproterenol and carbachol were washed out from the bath, with the current returning to baseline. Isoproterenol was then added, and the stimulated currents were recorded to assure that there was no significant current rundown.

Cell membrane capacitance (C_m) was measured by a voltage ramp with a slope (dV/dt) of 10 V/s from -80 to -60 mV. C_m was calculated as C_m=(I_ramp-I_ss)/(dV/dt), where I_ramp is the current at the end of the ramp pulse and I_ss is the steady state level of whole cell current at -80 mV.

Calculations
Current-voltage curve was plotted as current per unit membrane capacitance (pS/pF) versus the membrane potential (mV). Steady state inactivation data were plotted as I/I_max against voltage (mV), and the voltage shown is the conditioning potential. The voltage dependence of calcium current data was curve fitted by a Boltzmann distribution equation using Graphpad Prism software (Graphpad, Inc, San Diego, Calif) on a Macintosh G3 computer.

Statistical Analysis
Statistical differences between the mean values for 2 groups were evaluated by the Student’s unpaired t test. Measurements made sequentially were compared by 2- or 3-factor ANOVA, with repeated measures where appropriate, using standard statistical software. Data are expressed as mean±SEM. P<0.05 was considered statistically significant.

	Results

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Myocyte Contractility
The indirect inhibitory effect of M₂ stimulation on the ßAR response was investigated in isolated myocytes from adult WT (27 cells; 6 animals), G_i2-null (27 cells; 5 animals), and G_i3-null mice (27 cells; 4 animals). Cell contractility and intracellular calcium was simultaneously measured in the same batch of isolated myocytes in which electrophysiological parameters were determined. Representative recordings of cell shortening in response to isoproterenol and carbachol in adult ventricular myocytes isolated from WT, G_i2-null, and G_i3-null mice are shown in Figure 1A. Diastolic cell length was similar among WT, G_i2-null, and G_i3-null myocytes at baseline (120±3, 118±4, and 124±3 µm, respectively; P=NS). Baseline %CS (as seen in Figure 1) was 3.65±0.22%, 3.28±0.23%, and 3.37±0.22% for WT, G_i2-null, and G_i3-null myocytes, respectively, and suggests similar baseline contractile function among groups. With isoproterenol stimulation, myocytes had an 2.5-fold increase in %CS that was not significant among groups. The addition of carbachol in the presence of isoproterenol resulted in a significant attenuation of the ßAR response in WT and G_i3-null myocytes of 50% relative to isoproterenol alone. In G_i2-null cells, however, the addition of carbachol did not decrease %CS.

View larger version (28K): [in this window] [in a new window]   Figure 1. Figure 1. Effect of Gi gene inactivation on cell shortening in isolated adult cardiomyocytes. A, Representative recordings of cell shortening in response to isoproterenol and carbachol in myocytes isolated from WT, Gi2-null, and Gi3-null mice. B, Summary of %CS at baseline (black bar), after 3 minutes of 0.1 µmol/L isoproterenol infusion (white bar), and after 3 minutes of 0.1 µmol/L isoproterenol plus 10 µmol/L carbachol infusion (gray bar). All genotypes had a significant increase in %CS with isoproterenol (P<0.01). Isoproterenol plus carbachol decreased cell shortening relative to isoproterenol alone in WT and Gi3-null cells. *P<0.01 vs isoproterenol infusion in corresponding genotype.

Table 1 shows the maximum rate of contraction (-dL/dt_max) and the maximum rate of cell relengthening, or relaxation (+dL/dt_max) at baseline as well as during infusion of isoproterenol and isoproterenol plus carbachol. As with cell shortening, all groups had similar baseline maximum rate of contraction, which was markedly increased with isoproterenol. The addition of carbachol reduced -dL/dt_max in WT and G_i3-null myocytes but had no affect on maximum rate of contraction in G_i2-null cells. Both cell shortening and maximum rate of contraction suggest that the inhibitory effects of the muscarinic system on ßAR-stimulated contractility are mediated via G_i2 but not G_i3. In addition, to increased inotropy, ßAR stimulation also results in enhanced lusitropy, or relaxation, presumably via PKA-mediated phosphorylation of troponin I and phospholamban. In our isolated myocytes, isoproterenol stimulation resulted in a significant increase in maximum rate of relaxation in all groups. Carbachol inhibited the rise in +dL/dt_max in WT and G_i3-null cells but not in G_i2-null cells, suggesting that the inhibitory effects of M₂ stimulation on lusitropy are also mediated via G_i2.

View this table: [in this window] [in a new window]   Table 1. Cell Shortening and Relaxation

Intracellular Ca²⁺
The inhibitory inotropic and lusitropic effects of M₂ stimulation are believed to be directed by alterations in intracellular calcium influx and reuptake, and, therefore, we determined simultaneous intracellular calcium concentrations with calcium-sensitive probe fura-2. Representative recordings of intracellular calcium transients in response to isoproterenol and carbachol in adult ventricular myocytes isolated from WT, G_i2-null, and G_i3-null mice are shown in Figure 2A. WT, G_i2-null, and G_i3-null cells had similar diastolic intracellular calcium concentrations at baseline of 185±19, 204±25, and 190±23 nmol/L, respectively (P=NS). The amplitudes of the intracellular calcium transient at baseline, isoproterenol infusion, and isoproterenol plus carbachol infusion are shown in Figure 2B. At baseline, all groups had similar calcium transient amplitudes. With isoproterenol infusion, WT, G_i2-null, and G_i3-null cells had similar significant increases in intracellular calcium transients of 2.5-fold, consistent with the increase in contractility from ßAR stimulation. Carbachol attenuated the increase in calcium transient amplitude in WT and G_i3-null myocytes but had no effect on intracellular calcium in G_i2-null cells, resembling the contractile response to M₂ stimulation. The maximum rate of intracellular calcium influx and reuptake (+dCa/dt_max and -dCa/dt_max, respectively) were also determined as an assessment of intracellular calcium kinetics. As indicated in Table 2, isoproterenol caused an increase in the rate of calcium influx, consistent with augmented calcium transient amplitude and cell shortening in all groups. M₂ stimulation with carbachol reduced the max +dCa/dt_max in WT and G_i3 cells but had no effect on G_i2 cells. Intracellular calcium efflux was also increased with isoproterenol stimulation in all cells, in accordance with enhanced cellular relaxation. Carbachol treatment inhibited the lusitropic effect of ßAR stimulation in WT and G_i3 cells. Max -dCa/dt_max was unchanged in G_i2-null myocytes with carbachol, suggesting that in addition to the calcium transient amplitude, calcium influx and efflux kinetics were also unaffected by carbachol treatment in G_i2 cells.

View larger version (28K): [in this window] [in a new window]   Figure 2. Figure 2. Effect of Gi gene inactivation on the intracellular calcium transient amplitude as measured with fura-2 fluorescence. A, Representative recordings of intracellular calcium transients in response to isoproterenol and carbachol in myocytes isolated from WT, Gi2-null, and Gi3-null mice. B, Intracellular calcium transient at baseline (black bar), after 3 minutes of 0.1 µmol/L isoproterenol infusion (white bar), and after 3 minutes of 0.1 µmol/L isoproterenol plus 10 µmol/L carbachol infusion (gray bar). All genotypes had a significant increase in calcium transient amplitude with isoproterenol (P<0.01). Isoproterenol plus carbachol decreased calcium transient amplitude relative to isoproterenol alone in WT and Gi3-null cells. *P<0.01 vs isoproterenol infusion in corresponding genotype.

View this table: [in this window] [in a new window]   Table 2. Intracellular Calcium Kinetics

Characterization of I_{Ca, L}
Augmented intracellular calcium transients and subsequent increased cell contractility are believed to be activated by PKA-stimulated phosphorylation of the L-type calcium channel. Therefore, to additionally investigate the mechanism underlying altered M₂ response in G_i2-null myocytes, calcium currents were recorded in ventricular myocytes from WT, G_i2-null, and G_i3-null mice. The current-voltage relationship and inactivation showed no differences in the calcium channel characteristics (Figure 3). In whole-cell configuration, mean cell membrane capacitance (C_m) of these ventricular myocytes was 96.8±4.8 for WT (n=8), 92.6±6.7 for G_i2-null (n=7), and 93.7±8.4 for _i3-null (n=6) pF, with no significantly differences among the genotypes. The inactivation curve showed a half-maximal response (V_h) in the range of 23.1 to 24 mV and slope factors of -7.1 to -7.5 mV at baseline. These results are similar to previously reported characteristics of L-type calcium channels in cardiomyocytes.^{16 17} and showed that major characteristics of these channels were not altered by gene inactivation.

View larger version (16K): [in this window] [in a new window]   Figure 3. Figure 3. Characterization of calcium currents in isolated ventricular myocytes. A, Representative calcium current traces recorded at various testing potentials from -50 to +10 mV, indicated by the numbers next to the traces (in mV). The dashed line represents the steady current level at -50 mV. B, Current-voltage curves. C, Representative current traces recorded at 0 mV, with various conditioning potentials indicated by the numbers (in mV). D, Inactivation curves. In B and D, • indicates WT; , Gi2-null, and x, Gi3-null. n=4 cells for each group.

Representative recordings of I_{Ca, L} in response to isoproterenol and carbachol in adult ventricular myocytes isolated from WT, G_i2-null, and G_i3-null mice are shown in Figure 4A. As shown in Figure 4B, the time course and degree of I_{Ca, L} in response to isoproterenol stimulation was similar among groups. The inhibition of the response to isoproterenol stimulation exerted by carbachol was rapid and returned to baseline current within 3 minutes after the addition of carbachol in both WT and G_i3-null myocytes. However, the response to carbachol was severely blunted in G_i2–null myocytes, consistent with the effects on intracellular calcium transients and cell shortening.

View larger version (16K): [in this window] [in a new window]   Figure 4. Figure 4. Inhibitory effects of carbachol on ICa, L. A, Representative tracings. Arrows a through d indicate the time that traces were recorded: a indicates basal; b, 3 minutes after applying 1 µmol/L isoproterenol (ISO); c, 1 minute after addition of 10 µmol/L carbachol (CARB); and d, 3 minutes after addition of CARB. B, Time course of the ISO and CARB-mediated effects on ICa, L. After a stable basal current was recorded, the bath solution was perfused with ISO and stimulated currents were recorded for 4 minutes. CARB was added to the bath solution (indicated by the arrow), and recordings continued for at least 7 minutes. Whole-cell calcium currents were calculated a 1-minute intervals. Horizontal bar on top denotes period of ISO or CARB perfusion. • indicates WT; , Gi2-null; and , Gi3-null. *P<0.01 vs other groups. C, Summary of isoproterenol and carbachol: mediated effects on ICa, L. The currents, normalized to individual cell’s membrane capacitance (pA/pF), were measured at baseline (black bar), 3 minutes after addition of isoproterenol (gray bar), and 3 minutes after addition of isoproterenol plus carbachol (white bar). *P<0.01 vs isoproterenol in corresponding genotype. n=5 to 6 cells from each group.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The antagonistic effects of the muscarinic system on ßAR stimulation have long been recognized,¹⁸ although the mechanisms responsible for the antiadrenergic effect of the M₂ receptor remain unclear. This study demonstrates that G_i2 but not G_i3 is required for the indirect inhibitory action of muscarinic receptors on cell contractility and L-type calcium currents in adult ventricular myocytes.

G_i2 Required for Muscarinic Response
Mice with selective knockout of the G_i2 subunit exhibited a marked attenuation of the antiadrenergic M₂ effect on ßAR-stimulated increases in intracellular calcium and corresponding myocyte cell shortening as well as calcium influx kinetics and rate of contraction, in contrast to WT and G_i3-null mice. Furthermore, patch-clamp experiments determined that myocytes deficient in G_i2 had little or no M₂-mediated inhibition of L-type calcium currents. In addition to a reduction of the inotropic response, carbachol stimulation decreased the lusitropic response to isoproterenol, diminishing both calcium uptake and cellular relaxation in WT and G_i3-null cells but not in G_i2-null myocytes. Taken together, our data suggest that both the calcium and contractile inotropic and lusitropic responses to ßAR stimulation are inhibited by activation of the M₂ receptor via a G_i2 signal transduction pathway.

Mechanism of G_i2 Signal Transduction
Although G_i2 is essential for M₂-muscarinic response, the downstream signal transduction pathway still remains somewhat unclear. Because the calcium and contractile inotropic and lusitropic effects of ßAR activation are mediated though stimulation of adenylyl cyclase and subsequent PKA-mediated phosphorylation of intracellular proteins, factors that affect adenylyl cyclase activity, cAMP levels, or protein phosphorylation remain obvious candidates for M₂ activity. Stimulation of calcium currents through exogenous cAMP or nonhydrolyzable analogs of cAMP is not inhibited by muscarinic activity or through the use of nonhydrolyzable GTP analogs, suggesting that M₂ inhibition occurs at the level of cAMP.^{7 18 19} Importantly, we have previously shown that selective knockout of the G_i2 subunit does not alter protein levels of G_i3 or Gß in our mice,²⁰ suggesting that in adult ventricular myocytes, G_i2 rather than Gß regulates muscarinic response through the proposed inhibition of adenylyl cyclase activity.

Alternative G_o and G_i2 Pathways
Previously, G_o has been shown to be required for M₂-stimulated changes in L-type calcium channel activity in adult myocytes, and the level at which the G_o and G_i2 pathways converge is not presently known. Whereas G_i2 has been shown to mediate muscarinic inhibition of adenylyl cyclase,²¹ G_o inactivation does not affect adenylyl cyclase activity.²² Although NO production is reported to be required for muscarinic inhibition of I_{Ca, L} in nodal or nodal-like cells,^{9 23 24} there is still considerable controversy as to the role of NO in atrial cardiocytes and adult ventricular myocytes.^{25 26} In rabbit sino atrial and atrioventricular nodal cells, an inhibitor of NOS was shown to block muscarinic inhibition of L-type calcium channels and an NO producer to reproduce the effects.^{23 24} In murine atrial and nodal-like cardiocytes derived in vitro from embryonic stem cells, we have shown a similar dependence on NO generation.⁹ However, in adult atria and ventricular cells using NOS3 knockout animals, both a dependence²⁶ and an independence²⁵ of NOS have been reported. In addition, it is has been suggested that muscarinic-stimulated phosphatase activity,^{27 28 29} as well as cGMP phosphodiesterases,^{30 31} could potentially contribute to the inhibition of PKA phosphorylation, although the role of these enzymes in either the G_i2 or G_o muscarinic pathways remains unclear.

The muscarinic system and inhibitory G_i proteins play important roles in the pathophysiology of various cardiac phenotypes, from aging^{32 33 34 35} to cardiac failure,^{36 37 38} and have been proposed to primarily influence cardiac function through altered ßAR response. Characterization and understanding of the signal transduction pathway responsible for the muscarinic effect are crucial to influencing these phenotypes. In adult ventricular myocytes, our data demonstrate an independent pathway via the G_i2 subunit that is essential for functional signal transduction of the M₂-muscarinic receptor.

	Acknowledgments

 

This work was supported by the National Institutes of Health (National Research Service Award 1F32HL09531 to C.Y., K14H03377 to R.L., and R01 GM49122 and HL58606 to R.M.M.) and the American Heart Association (Established Investigator Award to R.M.M.).

	Footnotes

 
¹ Both authors contributed equally to this study.

Received July 25, 2000; revision received August 24, 2000; accepted September 14, 2000.

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