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(Circulation Research. 2006;98:659.)
© 2006 American Heart Association, Inc.

Cellular Biology

Endogenous RGS Proteins and G Subtypes Differentially Control Muscarinic and Adenosine-Mediated Chronotropic Effects

Ying Fu, Xinyan Huang, Huailing Zhong, Richard M. Mortensen, Louis G. D’Alecy, Richard R. Neubig

From the Departments of Pharmacology (Y.F., X.H., H.Z., R.M.M., R.R.N.), Molecular and Integrative Physiology (R.M.M., L.G.D’A.), and Surgery (Vascular) (L.G.D’A.), University of Michigan, Ann Arbor; and the Department of Surgery (L.G.D’A.), William Beaumont Hospital, Royal Oak, Mich.

Correspondence to R. R. Neubig, Department of Pharmacology, 1301 MSRB III, Ann Arbor, MI 48109. E-mail rneubig{at}umich.edu

	Abstract

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Cardiac automaticity is controlled by G protein–coupled receptors, such as adrenergic, muscarinic, and adenosine receptors. The strength and duration of G protein signaling is attenuated by regulator of G protein signaling (RGS) proteins acting as GTPase-activating proteins for G subunits; however, little is known about the role of endogenous RGS proteins in cardiac function. We created point mutations in G subunits that disrupt G-RGS binding and introduced them into embryonic stem (ES) cells by homologous recombination. Spontaneously contacting cardiocytes derived from the ES cells were used to evaluate the role of endogenous RGS proteins in chronotropic regulation. The RGS-insensitive G_oG184S homozygous knock-in (G_oGS/GS) cells demonstrated enhanced adenosine A₁ and muscarinic M₂ receptor–mediated bradycardic responses. In contrast, G_i2GS/GS cells showed enhanced responses to M₂ but not A₁ receptors. Similarly M₂ but not A₁ bradycardic responses were dramatically enhanced in G_i2GS/GS mice. Blocking G protein–coupled inward rectifying K⁺ (GIRK) channels largely abolished the mutation-induced enhancement of the M₂ receptor–mediated response but had a minimal effect on A₁ responses. The G_s-dependent stimulation of beating rate by the ß₂ adrenergic receptor agonist procaterol was significantly attenuated in G_oGS/GS and nearly abolished in G_i2GS/GS cells because of enhanced signaling via a pertussis toxin sensitive mechanism. Thus, endogenous RGS proteins potently reduce the actions of G_i/o-linked receptors on cardiac automaticity. Furthermore, M₂ and A₁ receptors differentially use G_i2 and G_o and associated downstream effectors. Thus, alterations in RGS function may play a role in pathophysiological processes and RGS proteins could represent novel cardiovascular therapeutic targets.

Key Words: RGS • automaticity • adenosine receptor • ß₂ adrenergic receptor • muscarinic receptor

	Introduction

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G protein–coupled receptors (GPCRs) control many cardiovascular functions, such as automaticity and contractility and cell growth and survival.¹ Cardiac ion channels are key effectors, including activation of the G protein–coupled inward rectifying potassium (GIRK) channels by ß subunits released from G_i and inhibition of I_f and I_Ca,L by G_o, whereas I_Ca,L is also regulated by G_i and nitric oxide.^2–5 GIRK channels contribute approximately 50% of the in vivo bradycardic response to vagal stimulation,⁶ but the relative contribution of different G subunits to in vivo cardiac automaticity is less clear.

The recently identified regulators of G protein signaling (RGS) proteins reduce the strength and duration of G protein signaling by acting as GTPase accelerating proteins (GAPs) through their conserved RGS domain.⁷ Mammalian myocardium expresses more than 10 different RGS proteins (2, 3, 4, 5, 6, 7, 12, 14, 16, and 19), which have GAP activity toward G_i/o and G_q/11 family G proteins.⁸ Indeed, the first reported mammalian RGS effect was the rapid deactivation of GIRK currents in atrium⁹ and RGS proteins also contribute to agonist-, calcium-, and voltage-dependent relaxation of GIRK currents.^10–12 These results suggest an important role for RGS proteins in sinoatrial (SA) node function and potentially in atrial arrhythmias. More recently, hypertension in the RGS2 knockout mouse illustrates the importance of RGS proteins in cardiovascular regulation.^13,14

Little is known about the role of endogenous cardiac RGS proteins, but they are dynamically regulated in a variety of pathophysiological conditions, including cardiac hypertrophy¹⁵ and heart failure.^16,17 It was proposed recently that RGS proteins represent potential new therapeutic targets for cardiovascular diseases.¹⁸ However, it has been difficult to ascertain the full physiological role of endogenous RGS proteins in vivo in part because of their functional redundancy. Thus, we used RGS-insensitive (RGSi) mutant G subunits, which contain a G184S point mutation in their switch I region that prevents binding of RGS proteins and the subsequent G deactivation.¹⁹ These G mutations do not affect the intrinsic GTPase activity, Gß subunit binding, receptor stimulation, or the ability to signal to downstream effectors.²⁰ Expression of these RGSi G subunits significantly enhances G_i/o-mediated signaling.^21,22 However, overexpression of RGSi G mutants and the use of pertussis toxin (PTX)-insensitive mutants complicate data interpretation. Therefore, we introduced RGSi mutants of G_o and G_i2 into embryonic stem (ES) cells by homologous recombination so that expression of the mutant G is under the control of their endogenous promoters (GRGSi knock-ins), thus allowing us to assess the functional contribution of all RGS proteins involved in regulation of that specific G subunit.

Two questions have been addressed in this study. First, do endogenous RGS proteins modulate cardiac signaling by G_o and G_i2? Second, is there specificity in the signaling pathways from receptors to G protein subunits and their downstream effectors? We show that A₁ adenosine, M₂ muscarinic, and ß₂-adrenergic chronotropic signals are all strongly modulated by endogenous RGS proteins. Furthermore, A₁ receptor (A₁R) preferentially couples to G_o and leads to a largely GIRK-independent bradycardic response. In contrast, G_i2 plays a major role in the M₂ muscarinic response mediated through GIRK channel activation. Also, G_i2 couples better to ß₂ adrenergic receptor (ß₂AR) than G_o does. Thus inhibiting RGS proteins that reduce G_o or G_i2 function may permit selective enhancement of adenosine or muscarinic, ß₂-negative chronotropic, or cardioprotective actions.

	Materials and Methods

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Generation of RGS-Insensitive ES Cells and Mice and In Vitro Differentiation to Cardiocytes
We generated heterozygous and homozygous G_oRGSi²⁰ and G_i2RGSi knock-in ES cell lines and created G_i2 RGSi knock-in mice (see the online data supplement available at http://circres.ahajournals.org). Wild-type cells and mice are designated +/+, heterozygote G_o G184S are G_o+/GS, and homozygous G_oGS/GS with similar designations of G_i2+/GS and G_i2GS/GS. Two clones each from 2 independently derived knock-in cell lines were examined for each G to ensure consistency of the phenotype. ES-derived cardiocytes (ESDC) were differentiated by a hanging drop protocol²⁰ modified from Hescheler et al.²³ These cells have been described as having an "atrial-nodal" character and their differentiation was confirmed by mRNA expression of cardiac-specific genes, such as - and ß-myosin heavy chain, MLC2a, MLC2v, atrial natriuretic factor, and the cardiac transcriptional factors GATA4 and Nkx2.5 (data not shown). G_i2+/GS mice show minimal overt phenotypic changes, whereas G_i2GS/GS have reduced body weights and show reduced viability.

Beating Rate Measurements
ESDC beating rate measurements were performed 21 to 25 days after withdrawal of leukemia inhibitory factor. Basal beating rate was recorded after at least 30 minutes at room temperature, then beating rates were counted for 30 seconds, 5 minutes after each drug administration.²⁰ Isoproterenol (Iso), procaterol, R-PIA, carbachol, CPX (8-cyclopentyl, 1,3-dipropylxanthine), PTX, and ICI 118,551 were obtained from Sigma. rTertiapinQ was obtained from Alomone Labs.

Statistical Analysis
Data are reported as mean±SEM. Beating rates are compared by 2-way ANOVA with Bonferroni post test (GraphPad Prism 4.0). Nonlinear least squares analysis with global fitting (online data supplement) and F test was used to fit and compare dose-response data, with P<0.05 considered significant.

	Results

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Enhanced Adenosine and Muscarinic Bradycardic Responses in G_oRGSi Mutant Cells
In ESDC, adrenergic, cholinergic, and adenosine signaling pathways are functionally intact and G subunit expression is normal (Figures I and II in the online data supplement). The selective A₁R agonist R(-)N6-(2-phenylisopropyl)-adenosine (R-PIA) was significantly more potent in the G_oRGSi cells with 4- and 6.4-fold reductions in IC₅₀ for heterozygous (G_o+/GS) and homozygous (G_oGS/GS) cells, respectively (Figure 1A). Similar results were seen in 2 clones of heterozygous and homozygous cells (not shown). Moreover, maximal inhibition by R-PIA was significantly enhanced in both knock-in cell lines (+/GS, 88±3%; GS/GS, 82±3%) compared with wild-type cells (68±3%) (P<0.001). The increase in agonist sensitivity in heterozygous G_o+/GS cells is consistent with the expected semidominant phenotype because loss of RGS GAP activity at even half of the G subunits should lead to a substantial increase in active G protein. G_oGS/GS cells also showed enhanced sensitivity to the M₂ receptor (M₂R) agonist carbachol (7-fold decrease in IC₅₀) without a significant difference in maximal inhibition (Figure 1B).

View larger version (27K): [in this window] [in a new window]   Figure 1. Negative chronotropic responses are enhanced in G RGSi mutant cells. Iso-stimulated (50 nmol/L) beating rates were measured with increasing concentrations of the adenosine A1 agonist, R-PIA (A and C), or the muscarinic M2 agonist carbachol (Carb) (B and D). Experiments were conducted in parallel with beating foci from wild-type +/+ (solid line), Go+/GS (dashed line), and Go/i2GS/GS (dotted line) cell lines on the same day after differentiation. Beating colonies of +/GS and GS/GS knock-in cells were derived from 2 independent clones, which demonstrated similar results, so the combined results are shown. A preliminary version of the data in A has been reported previously.20 Data were normalized to the total beating rate after Iso stimulation. **P<0.001.

Enhanced Muscarinic but Not Adenosine Bradycardic Responses in Cells Expressing G_i2RGSi Mutant G Protein
G_i2 is the predominant isoform of inhibitory G proteins in mammalian myocardium and plays a largely parallel role with G_o in regulation of cardiac automaticity. We obtained targeted G_i2RGSi ES cells and Western blotting showed that the mutation does not alter the expression of G_i2 in either ES cells or in heart from the G_i2 mutant mice (Figure IC and ID in the online data supplement). Interestingly, G_i2 appears to play less of a role in adenosine-mediated bradycardia than does G_o, with only a nonsignificant 2.5-fold reduction in the R-PIA IC₅₀ for G_i2GS/GS cells compared with the 6.4-fold decrease for G_oGS/GS cells. Plus, there was no significant difference in maximal stimulation (Figure 1C). In contrast, G_i2GS/GS cells showed dramatically enhanced M₂R responses with 5.4-fold reduction in IC₅₀ and 71% increase in maximal inhibition (72% versus 42%) when compared with +/+ cells (Figure 1D), indicating that G_i2 preferentially couples to M₂R versus A₁R.

Pacemaker I_f and GIRK Currents Regulate Beating Rate in ESDC
To begin to address the ionic mechanisms of these receptor signals, we examined the role of I_f and GIRK in ESDC automaticity. The pacemaker current, I_f, has been shown to mediate ß-adrenergic stimulation and to contribute to cholinergic inhibition of heart rate²⁴ and is a major mechanism for pacemaker activity. Deletion of the gene underlying I_f, HCN4, results in severe bradycardia and chronotropic incompetence.²⁵ Similarly in ESDC, the selective I_f blocker ZD7288 nearly inhibits spontaneous beating by &60% and virtually eliminates ß-adrenergic stimulation (Figure 2A). Thus, it was difficult to assess the effects of inhibitory agonists on this channel as most of our other functional studies were in the presence of Iso.

View larger version (20K): [in this window] [in a new window]   Figure 2. Pacemaker If and GIRK currents are functional in ESDC. A, Spontaneously contracting embryoid bodies derived from wild-type D3 ES cells were treated with increasing concentrations of the pacemaker channel blocker ZD7288, which dose-dependently inhibited both basal () and Iso-stimulated () beating rates. B, Carbachol (30 µmol/L) and R-PIA 1 (µmol/L) inhibited the Iso-stimulated beating rate, and the effect of rTertiapinQ (100 nmol/L) on that inhibition is shown. Two-way ANOVA was used to determine statistical significance. **P<0.01.

In contrast, the GIRK blocker rTertiapinQ at 100 nmol/L did not affect basal or Iso-stimulated beating but decreased the carbachol-mediated reduction of Iso-stimulated beating rate from 40±3% to 22±2% (Figure 2B). This result is consistent with the 50% contribution of GIRK channel in negative chronotropic response mediated by M₂R reported in GIRK knockout animals.⁶ Interestingly, however, inhibition of beating by R-PIA was barely affected by rTertiapinQ (43±5% versus 36±6%), indicating that GIRK channel activation contributes minimally to regulation of automaticity by the A₁R despite its ability to activate GIRK currents.²⁶ Although we cannot completely rule out a role for GIRK currents, it is apparent that the M₂ and A₁ responses differ substantially in their dependence on GIRK currents.

Enhanced Bradycardia by M₂R but Not A₁R in G_o/i2RGSi Cells Is Primarily Mediated by Enhanced of GIRK Channel Activation
In presence of the specific GIRK channel blocker rTertiapinQ, carbachol produced a much smaller bradycardic response, but, more importantly, the difference in IC₅₀ and maximal inhibition between +/+ and G_i2GS/GS cells was largely abolished (Figure 3A). Thus, augmented GIRK channel activation caused by lack of RGS regulation is largely responsible for the enhanced bradycardic response on M₂R activation, whereas other pathways are less RGS regulated. Interestingly, the IC₅₀ shift for carbachol with the G_oGS/GS cells was also reduced (2.3-fold versus 6.8-fold) with rTertiapinQ (Table). On the other hand, rTertiapinQ has a minimal effect on R-PIA–induced inhibition of beating rate. In the presence of the GIRK blocker, G_oGS/GS cells still show a 5.4-fold lower IC₅₀ for R-PIA than do +/+ cells. This suggests that endogenous RGS proteins regulate pathways signaling to other downstream effectors such as I_Ca,L and I_f rather than GIRK channel on activation of G_o by A₁R.

View larger version (27K): [in this window] [in a new window]   Figure 3. Effects of GIRK channel blocker on the RGSi-enhanced bradycardic responses to A1R and M2R. rTertiapinQ (100 nmol/L) was used with increasing concentrations of carbachol (Carb) (A) or R-PIA (B) to examine whether GIRK channel activation contributes to beating rate in GoGS/GS or Gi2GS/GS cells. Gray dotted lines show data from Figure 1 for reference. Arrows indicate the carbachol and R-PIA IC50 values.

View this table: [in this window] [in a new window]   Table 1. Summary of Agonist Sensitivity in GoGS/GS or Gi2GS/GS Versus +/+ Cells and in the Presence of rTertiapinQ

Diminished Positive Chronotropic Response in G_o/i2RGSi Mutant Cells Following ß₂AR Activation
ßARs play a pivotal role in the control of cardiovascular function by mediating the actions of sympathetic nervous system activation and circulating catecholamines. Although the ß₁AR is the predominant subtype in the hearts of many species, both ß₁ and ß₂ subtypes can increase cardiac automaticity and contractility.²⁷ Although most of actions of the ß₂AR are mediated through G_s proteins and protein kinase A, ß₂AR can also couple to inhibitory G proteins, which have been suggested to account for their apparent cardioprotective action.^28,29 Because endogenous RGS proteins act on G_i/o family G proteins but not on G_s, it is plausible to hypothesize that RGS proteins could modulate the actions of the ß₂AR, including chronotropic regulation.

Procaterol, a selective ß₂AR agonist,³⁰ stimulates the ESDC beating rate. This tachycardia is mediated by ß₂ARs because it is blocked by 200 nmol/L ICI 118,551, a ß₂AR selective antagonist, and not by the selective ß₁AR antagonist CGP 20712A, which inhibits Iso simulation (Figure III in the online data supplement). The EC₅₀ for procaterol was not different between +/+ cells and 2 clones of G_oGS/GS cells (Figure 4A). However, maximal stimulation by procaterol is significantly attenuated in 2 independent G_oGS/GS cells lines (dashed and dotted lines, 131% and 135%, respectively) compared with +/+ cells (solid line, 178%). In contrast, Iso-stimulated beating rates did not differ among the 3 cell types. This suggests that endogenous RGS proteins control ß₂AR effects on beating rate by reducing the action of G_o. This is intriguing because ß₂AR coupling to G_o has not been reported previously. PTX was used to test whether the difference in procaterol response was caused by overactive inhibitory G proteins. The difference between G_oGS/GS and wild-type cells was abolished by PTX treatment (Figure IV in the online data supplement), confirming that G_i/o family coupling of the ß₂AR is under tonic control by endogenous RGS proteins.

View larger version (26K): [in this window] [in a new window]   Figure 4. ß2AR-mediated stimulation of beating rate was attenuated in Go/i2RGSi mutant cells. A, The beating rate of wild-type and GoGS/GS cells was measured after 20 minutes incubation with 50 nmol/L Iso at room temperature. Cells were then washed 3 times with drug-free medium and allowed to sit for 40 minutes to allow recovery to the basal beating rate. Increasing concentrations of the ß2AR selective agonist, procaterol, were added to obtain dose-response curves. The experiment was run in parallel with +/+ cells (solid square) and 2 independent clones of GoGS/GS cells (open square and open circle). Data are normalized to the basal beating rate which did not differ among the clones. The mean of the Iso response for +/+ (solid bar) and 2 GoGS/GS clones (hatched bars) are shown on the right. B, Wild-type CJ7 cells (solid line) and Gi2GS/GS cells (dashed line) derived from 2 separate clones were tested. Data from the wild-type cells was fit to a sigmoidal dose-response curve, whereas the Gi2GS/GS mutants showed a biphasic response. ***P<0.0001.

An even more striking difference was seen in G_i2GS/GS mutant cells. A slight negative chronotropic response to procaterol occurred at low doses but was overcome at higher doses. Thus endogenous RGS proteins dramatically regulate ß₂AR chronotropy such that the G_i2-mediated suppression overcomes G_s-mediated stimulation if the GAP activity of RGS is blocked. Similar to the striking left shifts of the carbachol and R-PIA dose-response curves in RGSi cells, the effect of procaterol on G_i2-mediated inhibition occurs at much lower concentrations, whereas G_s effects do not shift. Thus endogenous RGS proteins strongly suppress G_i2 effects in the heart.

M₂R-Mediated Response Is Selectively Enhanced in G_i2GS/GS Mice
Because ES cell studies may not fully replicate results in vivo, we developed G_i2RGSi knock-in mice to assess the role of RGS proteins in the M₂ receptor (M₂R)/A₁R specificity of chronotropic regulation in the intact animal. Heart rate was continuously monitored in unconstrained mice by telemetry. Baseline heart rates in the G_i2GS/GS mice were slightly higher than wild type (588±11 versus 571±8 bpm). Administration of the A₁R agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA, 0.1 mg/kg, IP) led to a significant reduction in heart rate in both +/+ and G_i2GS/GS mice but showed no difference between genotypes.

In contrast, IP injection of the standard dose of 0.5 mg/kg carbachol resulted in profound bradycardia and death in 1 homozygous G_i2 GS/GS mouse, whereas the wild-type mice showed a 293±44 bpm decrease (n=2). A much smaller dose of 0.2 mg/kg carbachol (Figure 5) induced very little effect in +/+ animals but produced a dramatic reduction in heart rate of G_i2GS/GS mice (–48 versus –295 bpm, P<0.001). This confirmation of the muscarinic selectivity of the G_i2 RGSi effect strongly supports the validity of our ESDC model of chronotropic regulation (Figure 6) and also illustrates, in vivo, the strength and specificity of RGS effects in modulating GPCR signals despite similar signaling pathways.

View larger version (31K): [in this window] [in a new window]   Figure 5. Muscarinic M2-mediated bradycardic responses were dramatically enhanced in Gi2GS/GS mice compared with littermate controls. Heart rates (HR) of wild-type +/+ (squares) and Gi2GS/GS (circles) mice were recorded telemetrically in unconstrained animals. Ninety minutes after a saline injection as control (dashed lines), 0.1 mg/kg CCPA (A) or 0.2 mg/kg carbachol (B) was administrated IP and the heart rate change from basal was calculated. Statistical significance was determined by 2-way ANOVA with Bonferroni post test at each time point. ***P<0.001.

View larger version (16K): [in this window] [in a new window]   Figure 6. Proposed roles of G and RGS proteins in chronotropic regulation of the heart. Some GPCRs can couple to multiple isoforms of G proteins from same family (ie, M2R), and some couple to G proteins from different families exerting counteracting effects (ie, ß2AR). Vagal stimulation activates M2R, which couples well to both Gi2 and Go. The ß subunits from Gi2 but not Go directly activate GIRK currents, leading to negative chronotropic effects. This pathway is highly regulated by endogenous RGS proteins. In contrast, ß released from Go does not normally activate GIRK currents, but, in the absence of RGS control (GoGS/GS), there is modest activation of GIRK. Adenosine A1Rs preferentially couple to Go rather than Gi2 to inhibit heart rate, but they only minimally use GIRK currents for this response rather ICa,L or If are likely effectors. Also, ß2AR can couple to multiple isoforms in the Gi family with preference for Gi2 over Go. Furthermore, these effects are normally kept in check by endogenous RGS proteins. Thus, RGS proteins play a central role in chronotropic regulation and are likely to modulate cardioprotective functions of Gi/o signaling.

	Discussion

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We demonstrate that endogenous RGS proteins potently regulate chronotropic responses to muscarinic, adenosine, and ß₂ adrenergic receptors. Furthermore, the RGS-insensitive G subunits reveal dramatic differences in the signaling pathways and channel mechanisms used in cardiac chronotropic control by A₁ adenosine and M₂ muscarinic receptors. The remarkable, and even lethal, increase in carbachol responsiveness in vivo illustrates the potential importance of RGS protein modulation in both pathological and pharmacological situations.

Role of RGS Proteins in the Heart
Most information on mammalian RGS proteins derives from transfection experiments but overexpressed proteins may behave differently from their endogenous counterpart both in cellular localization and relative abundance to other family members.³¹ In contrast, we used homologous recombination in ES cells in which the mutant protein is under the control of its native promoter and expressed at normal levels. More importantly, the RGSiG subunits reveal the contribution from all endogenous RGS proteins, avoiding complications from redundancy caused by multiple RGS proteins. Thus, we provide the first demonstration of a role of endogenous RGS proteins in the normal physiological regulation of cardiac function.

G Protein Pathways Used by A₁R and M₂R
The enhanced activation of G_o and G_i2 subunits by incorporation of the RGSi mutation reveals the broad coupling of muscarinic M₂R to G_o and G_i2 but preferential coupling of adenosine A₁ receptor to G_o. This is despite the quite similar actions of A₁R and M₂R in regulating chronotropy and inotropy. The physiological significance of the preferential M₂R-G_i2 coupling is strongly supported by our in vivo data, whereas G_i2-RGSi mice had unchanged responses after CCPA administration.

Previous data have suggested some G_i and G_o selectivity of these receptors, but the degree to which this is functionally relevant has not been clear. In human myocardium, M₂R but not A₁R is able to maximally activate [³⁵S]GTPS binding to G subunits.³² In cardiac membranes, M₂R coimmunoprecipitated with both G_o and G_i proteins,³³ whereas purified G_o was found to efficiently reconstitute A₁R.³⁴ Coimmunoprecipitation of activated A₁R with G_o and G_i3 but not G_i2 was also seen in rat ventricle.³⁵

There may, however, be factors beyond just G protein that determine RGS control of the functional responses to M₂ and A₁ receptors. Unlike the M₂R, which is dynamically targeted to caveolae on agonist binding,³⁶ A₁R localizes in caveolae in unstimulated ventricular myocytes and translocates out after receptor occupancy.³⁷ It is possible that the more pronounced effect of the GIRK channel blocker on the enhanced M₂ responses in the G_o GS/GS cells versus the A₁ response in the same cells (Table) may be related to localization rather than just G protein specificity. Indeed, we predicted³⁸ that RGS proteins may enhance specificity of G protein signaling by a "kinetic scaffolding" mechanism that limits the diffusion of active G and ß subunits to very short (nanometer) distances from the receptor. Thus, the modest but significant difference in maximal stimulation by R-PIA and 7-fold EC₅₀ shift for carbachol between +/+ and G_oGS/GS cells that were lost in presence of rTertiapinQ suggests potential GIRK coupling by the mutant G_o.

Role of GIRK Currents in A₁R- and M₂R-Mediated Negative Chronotropy
The contribution of GIRK currents to M₂R- versus A₁R-mediated bradycardia appears to be quite different. Despite the ability of both M₂R and A₁R to activate GIRK currents,²⁶ the A₁R responses in ESDC are largely untouched by the GIRK blocker rTertiapinQ, whereas M₂R-induced negative chronotropy is reduced by 45% (Figure 2B). Furthermore, rTertiapinQ almost completely abolished the increase in maximal inhibition and reduction in IC₅₀ to carbachol in G_i2GS/GS mutant. This suggests that M₂-G_i2-GIRK is a primary pathway mediating the M₂R chronotropic response, and it is tightly regulated by endogenous RGS proteins.

In contrast, the enhanced sensitivity to R-PIA in G_oGS/GS mutant cells is largely maintained in presence of rTertiapinQ, indicating minimal contribution of GIRK activation in A₁R-mediated negative chronotropy. Given that A₁R preferentially couple to G_o, the minimal role of GIRK is perhaps not surprising because specificity of G_i2 over G_o in activating GIRK currents is well established. Sowell et al² demonstrated that the expression of G_i2 and G_i3 is required for normal agonist-dependent activation of GIRK current, whereas G_o knockout cells show normal GIRK responses but altered I_Ca,L regulation.³ Furthermore, the A₁R has an 11-fold lower EC₅₀ for suppression of Iso-stimulated I_Ca,L compared with its action on GIRK current.³⁹ Altogether, these prior data suggest that A₁R ought to use a non-GIRK pathway (eg, I_Ca,L) in mediating its bradycardic effects. Our results illustrate in vitro and in vivo the dramatic difference in signaling pathways (G_i2-GIRK and G_o-non-GIRK) in negative chronotropic regulation by M₂R and A₁R, respectively.

There is a discrepancy, however, between our conclusion and that of Wickman et al⁶ where disruption of GIRK4 in vivo results in a 50% loss of the A₁R-mediated negative chronotropic response, an effect similar to that for vagal bradycardia.⁶ They used a relatively high dose of CCPA (0.3 mg/kg), which may lead to more "spill-over" of A₁ responses to a G_i2 pathway or to high degrees of G_o activation that could lead to GIRK coupling. Previous studies showed that the A₁R in rat myocytes can activate only a fraction of the magnitude of GIRK current that is activated via the M₂R.^40,41 A₁R density was suggested to limit responsiveness because the receptor overexpression does enhance GIRK current activation.⁴¹ It is also possible that the discrepancy between our results and those Wickman et al, could derive from the ESDC versus animal models, but we also see a dramatic difference between carbachol and CCPA in the G_i2 RGSi mice. Interestingly, the effect of the G_i2 RGSi on vagal bradycardia induced by methoxamine was significant but much less than with direct activation by carbachol (data not shown); therefore, perhaps CNS control of vagal reflexes (which are involved for methoxamine but less so for carbachol responses) is altered in GIRK4 knockout.

This strong regulation by endogenous RGS proteins on GIRK currents suggests potential involvement in pathophysiology of arrhythmias. GIRK4 knockout mice and mice with reduced expression of membrane-associated G protein ß subunits exhibited significantly reduced beat-to-beat fluctuation in heart rate and had a reduced propensity for atrial fibrillation.^42,43 Therefore, loss of RGS function, either by G mutations or loss of individual RGS proteins, might be expected to increase GIRK activation and enhanced propensity for atrial fibrillation. Furthermore, G_i2 plays a clear role in ß₂AR signaling and G_i2 enhances survival in the face of ß₂AR overexpression, so inhibiting cardiac RGS may provide a novel approach to cardioprotection. The blunted maximal stimulation by ß₂AR in G_oGS/GS mutants is the first convincing evidence for ß₂AR-G_o coupling. Taken together, these data suggest that ß₂AR-mediated action may also involve multiple G_i/o subunits.

In summary, we show that endogenous RGS proteins potently modulate the G_i/o-mediated regulation of cardiac automaticity, leading to enhanced bradycardic responses on A₁R and M₂R activation. Moreover, there is clear specificity between G_i2 and G_o in this chronotropic control in vivo and in vitro. A₁R-mediated negative chronotropic actions are through preferential coupling to G_o and minimally involve GIRK currents, whereas M₂R coupling uses both G_i2 and G_o and their appropriate effectors. Thus, the GRGSi knock-in mice represent a valuable model for studying the both G subunit roles in signaling by different receptors as well as in understanding the function of endogenous RGS proteins in vivo. Our results suggest that RGS inhibitors targeting the G_o-selective RGS proteins, such as RGS6, should potentiate adenosine responses more than M₂ effects. The striking enhancement of carbachol-induced bradycardia with the RGSi G subunits shows that pathophysiological alterations in RGS control of G_i/o could contribute to sinus node dysfunction and/or atrial arrhythmias. Furthermore, ß₂AR action via both G_i2 and G_o subunits is under tonic suppression by endogenous RGS proteins. Future studies to determine whether cardioprotective actions of ß₂AR and other G_i/o-coupled receptors can be enhanced by blocking RGS proteins should be of great interest, as should be identification of the individual RGS protein(s) responsible for these regulatory processes.

	Acknowledgments

 
This work was supported by an American Heart Association Predoctoral Fellowship (to Y.F.), NIH grant R01 GM039561 (to R.R.N.), NIH grant R01 HL070902 (to R.M.M.), and NIH grant T32HL007853 (to X.H.). This work was also supported, in part, by the Michigan Diabetes, Research, and Training Center (NIH grant P60 DK20572) and the University of Michigan Cancer Center (NIH grant P30 CA046592). We appreciate the assistance of Steven Whitesall with the ECG transmitter studies. We also thank Raelene Charbeneau and Min Liu for help in managing the mouse colony and Dr Susanne Mumby (University of Texas, Southwestern Medical Center) for the G_i2 antiserum.

	Footnotes

 
Original received February 4, 2005; resubmission received October 20, 2005; revised resubmission received January 10, 2006; accepted January 19, 2006.

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