Endogenous RGS Proteins and G Subtypes Differentially Control Muscarinic and Adenosine-Mediated Chronotropic Effects
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Ying Fu, Xinyan Huang, Huailing Zhong, R
参见附件。
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.)
Surgery (Vascular) (L.G.D’A.), University of Michigan, Ann Arbor
the Department of Surgery (L.G.D’A.), William Beaumont Hospital, Royal Oak, Mich.
Abstract
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 GoG184S homozygous knock-in (GoGS/GS) cells demonstrated enhanced adenosine A1 and muscarinic M2 receptor–mediated bradycardic responses. In contrast, Gi2GS/GS cells showed enhanced responses to M2 but not A1 receptors. Similarly M2 but not A1 bradycardic responses were dramatically enhanced in Gi2GS/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 Gs-dependent stimulation of beating rate by the 2 adrenergic receptor agonist procaterol was significantly attenuated in GoGS/GS and nearly abolished in Gi2GS/GS cells because of enhanced signaling via a pertussis toxin sensitive mechanism. Thus, endogenous RGS proteins potently reduce the actions of Gi/o-linked receptors on cardiac automaticity. Furthermore, M2 and A1 receptors differentially use Gi2 and Go 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 2 adrenergic receptor muscarinic receptor
Introduction
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 subunits released from Gi and inhibition of If and ICa,L by Go, whereas ICa,L is also regulated by Gi 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 Gi/o and Gq/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, G subunit binding, receptor stimulation, or the ability to signal to downstream effectors.20 Expression of these RGSi G subunits significantly enhances Gi/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 Go and Gi2 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 Go and Gi2 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 2-adrenergic chronotropic signals are all strongly modulated by endogenous RGS proteins. Furthermore, A1 receptor (A1R) preferentially couples to Go and leads to a largely GIRK-independent bradycardic response. In contrast, Gi2 plays a major role in the M2 muscarinic response mediated through GIRK channel activation. Also, Gi2 couples better to 2 adrenergic receptor (2AR) than Go does. Thus inhibiting RGS proteins that reduce Go or Gi2 function may permit selective enhancement of adenosine or muscarinic, 2-negative chronotropic, or cardioprotective actions.
Materials and Methods
Generation of RGS-Insensitive ES Cells and Mice and In Vitro Differentiation to Cardiocytes
We generated heterozygous and homozygous GoRGSi20 and Gi2RGSi knock-in ES cell lines and created Gi2 RGSi knock-in mice (see the online data supplement available at http://circres.ahajournals.org). Wild-type cells and mice are designated +/+, heterozygote Go G184S are Go+/GS, and homozygous GoGS/GS with similar designations of Gi2+/GS and Gi2GS/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 -myosin heavy chain, MLC2a, MLC2v, atrial natriuretic factor, and the cardiac transcriptional factors GATA4 and Nkx2.5 (data not shown). Gi2+/GS mice show minimal overt phenotypic changes, whereas Gi2GS/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
Enhanced Adenosine and Muscarinic Bradycardic Responses in GoRGSi 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 GoRGSi cells with 4- and 6.4-fold reductions in IC50 for heterozygous (Go+/GS) and homozygous (GoGS/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 Go+/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. GoGS/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).
Enhanced Muscarinic but Not Adenosine Bradycardic Responses in Cells Expressing Gi2RGSi Mutant G Protein
Gi2 is the predominant isoform of inhibitory G proteins in mammalian myocardium and plays a largely parallel role with Go in regulation of cardiac automaticity. We obtained targeted Gi2RGSi ES cells and Western blotting showed that the mutation does not alter the expression of Gi2 in either ES cells or in heart from the Gi2 mutant mice (Figure IC and ID in the online data supplement). Interestingly, Gi2 appears to play less of a role in adenosine-mediated bradycardia than does Go, with only a nonsignificant 2.5-fold reduction in the R-PIA IC50 for Gi2GS/GS cells compared with the 6.4-fold decrease for GoGS/GS cells. Plus, there was no significant difference in maximal stimulation (Figure 1C). In contrast, Gi2GS/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 Gi2 preferentially couples to M2R versus A1R.
Pacemaker If and GIRK Currents Regulate Beating Rate in ESDC
To begin to address the ionic mechanisms of these receptor signals, we examined the role of If and GIRK in ESDC automaticity. The pacemaker current, If, has been shown to mediate -adrenergic stimulation and to contribute to cholinergic inhibition of heart rate24 and is a major mechanism for pacemaker activity. Deletion of the gene underlying If, HCN4, results in severe bradycardia and chronotropic incompetence.25 Similarly in ESDC, the selective If blocker ZD7288 nearly inhibits spontaneous beating by &60% and virtually eliminates -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.
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 Go/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 Gi2GS/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 GoGS/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, GoGS/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 ICa,L and If rather than GIRK channel on activation of Go by A1R.
Diminished Positive Chronotropic Response in Go/i2RGSi Mutant Cells Following 2AR Activation
ARs play a pivotal role in the control of cardiovascular function by mediating the actions of sympathetic nervous system activation and circulating catecholamines. Although the 1AR is the predominant subtype in the hearts of many species, both 1 and 2 subtypes can increase cardiac automaticity and contractility.27 Although most of actions of the 2AR are mediated through Gs proteins and protein kinase A, 2AR 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 Gi/o family G proteins but not on Gs, it is plausible to hypothesize that RGS proteins could modulate the actions of the 2AR, including chronotropic regulation.
Procaterol, a selective 2AR agonist,30 stimulates the ESDC beating rate. This tachycardia is mediated by 2ARs because it is blocked by 200 nmol/L ICI 118,551, a 2AR selective antagonist, and not by the selective 1AR 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 GoGS/GS cells (Figure 4A). However, maximal stimulation by procaterol is significantly attenuated in 2 independent GoGS/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 2AR effects on beating rate by reducing the action of Go. This is intriguing because 2AR coupling to Go 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 GoGS/GS and wild-type cells was abolished by PTX treatment (Figure IV in the online data supplement), confirming that Gi/o family coupling of the 2AR is under tonic control by endogenous RGS proteins.
An even more striking difference was seen in Gi2GS/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 2AR chronotropy such that the Gi2-mediated suppression overcomes Gs-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 Gi2-mediated inhibition occurs at much lower concentrations, whereas Gs effects do not shift. Thus endogenous RGS proteins strongly suppress Gi2 effects in the heart.
M2R-Mediated Response Is Selectively Enhanced in Gi2GS/GS Mice
Because ES cell studies may not fully replicate results in vivo, we developed Gi2RGSi 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 Gi2GS/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 Gi2GS/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 Gi2 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 Gi2GS/GS mice (–48 versus –295 bpm, P<0.001). This confirmation of the muscarinic selectivity of the Gi2 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.
Discussion
We demonstrate that endogenous RGS proteins potently regulate chronotropic responses to muscarinic, adenosine, and 2 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 Go and Gi2 subunits by incorporation of the RGSi mutation reveals the broad coupling of muscarinic M2R to Go and Gi2 but preferential coupling of adenosine A1 receptor to Go. This is despite the quite similar actions of A1R and M2R in regulating chronotropy and inotropy. The physiological significance of the preferential M2R-Gi2 coupling is strongly supported by our in vivo data, whereas Gi2-RGSi mice had unchanged responses after CCPA administration.
Previous data have suggested some Gi and Go 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 Go and Gi proteins,33 whereas purified Go was found to efficiently reconstitute A1R.34 Coimmunoprecipitation of activated A1R with Go and Gi3 but not Gi2 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 Go 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 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 GoGS/GS cells that were lost in presence of rTertiapinQ suggests potential GIRK coupling by the mutant Go.
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 Gi2GS/GS mutant. This suggests that M2-Gi2-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 GoGS/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 Go, the minimal role of GIRK is perhaps not surprising because specificity of Gi2 over Go in activating GIRK currents is well established. Sowell et al2 demonstrated that the expression of Gi2 and Gi3 is required for normal agonist-dependent activation of GIRK current, whereas Go knockout cells show normal GIRK responses but altered ICa,L regulation.3 Furthermore, the A1R has an 11-fold lower EC50 for suppression of Iso-stimulated ICa,L compared with its action on GIRK current.39 Altogether, these prior data suggest that A1R ought to use a non-GIRK pathway (eg, ICa,L) in mediating its bradycardic effects. Our results illustrate in vitro and in vivo the dramatic difference in signaling pathways (Gi2-GIRK and Go-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 Gi2 pathway or to high degrees of Go 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 Gi2 RGSi mice. Interestingly, the effect of the Gi2 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 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, Gi2 plays a clear role in 2AR signaling and Gi2 enhances survival in the face of 2AR overexpression, so inhibiting cardiac RGS may provide a novel approach to cardioprotection. The blunted maximal stimulation by 2AR in GoGS/GS mutants is the first convincing evidence for 2AR-Go coupling. Taken together, these data suggest that 2AR-mediated action may also involve multiple Gi/o subunits.
In summary, we show that endogenous RGS proteins potently modulate the Gi/o-mediated regulation of cardiac automaticity, leading to enhanced bradycardic responses on A1R and M2R activation. Moreover, there is clear specificity between Gi2 and Go in this chronotropic control in vivo and in vitro. A1R-mediated negative chronotropic actions are through preferential coupling to Go and minimally involve GIRK currents, whereas M2R coupling uses both Gi2 and Go 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 Go-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 Gi/o could contribute to sinus node dysfunction and/or atrial arrhythmias. Furthermore, 2AR action via both Gi2 and Go subunits is under tonic suppression by endogenous RGS proteins. Future studies to determine whether cardioprotective actions of 2AR and other Gi/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 Gi2 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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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.)
Surgery (Vascular) (L.G.D’A.), University of Michigan, Ann Arbor
the Department of Surgery (L.G.D’A.), William Beaumont Hospital, Royal Oak, Mich.
Abstract
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 GoG184S homozygous knock-in (GoGS/GS) cells demonstrated enhanced adenosine A1 and muscarinic M2 receptor–mediated bradycardic responses. In contrast, Gi2GS/GS cells showed enhanced responses to M2 but not A1 receptors. Similarly M2 but not A1 bradycardic responses were dramatically enhanced in Gi2GS/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 Gs-dependent stimulation of beating rate by the 2 adrenergic receptor agonist procaterol was significantly attenuated in GoGS/GS and nearly abolished in Gi2GS/GS cells because of enhanced signaling via a pertussis toxin sensitive mechanism. Thus, endogenous RGS proteins potently reduce the actions of Gi/o-linked receptors on cardiac automaticity. Furthermore, M2 and A1 receptors differentially use Gi2 and Go 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 2 adrenergic receptor muscarinic receptor
Introduction
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 subunits released from Gi and inhibition of If and ICa,L by Go, whereas ICa,L is also regulated by Gi 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 Gi/o and Gq/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, G subunit binding, receptor stimulation, or the ability to signal to downstream effectors.20 Expression of these RGSi G subunits significantly enhances Gi/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 Go and Gi2 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 Go and Gi2 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 2-adrenergic chronotropic signals are all strongly modulated by endogenous RGS proteins. Furthermore, A1 receptor (A1R) preferentially couples to Go and leads to a largely GIRK-independent bradycardic response. In contrast, Gi2 plays a major role in the M2 muscarinic response mediated through GIRK channel activation. Also, Gi2 couples better to 2 adrenergic receptor (2AR) than Go does. Thus inhibiting RGS proteins that reduce Go or Gi2 function may permit selective enhancement of adenosine or muscarinic, 2-negative chronotropic, or cardioprotective actions.
Materials and Methods
Generation of RGS-Insensitive ES Cells and Mice and In Vitro Differentiation to Cardiocytes
We generated heterozygous and homozygous GoRGSi20 and Gi2RGSi knock-in ES cell lines and created Gi2 RGSi knock-in mice (see the online data supplement available at http://circres.ahajournals.org). Wild-type cells and mice are designated +/+, heterozygote Go G184S are Go+/GS, and homozygous GoGS/GS with similar designations of Gi2+/GS and Gi2GS/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 -myosin heavy chain, MLC2a, MLC2v, atrial natriuretic factor, and the cardiac transcriptional factors GATA4 and Nkx2.5 (data not shown). Gi2+/GS mice show minimal overt phenotypic changes, whereas Gi2GS/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
Enhanced Adenosine and Muscarinic Bradycardic Responses in GoRGSi 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 GoRGSi cells with 4- and 6.4-fold reductions in IC50 for heterozygous (Go+/GS) and homozygous (GoGS/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 Go+/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. GoGS/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).
Enhanced Muscarinic but Not Adenosine Bradycardic Responses in Cells Expressing Gi2RGSi Mutant G Protein
Gi2 is the predominant isoform of inhibitory G proteins in mammalian myocardium and plays a largely parallel role with Go in regulation of cardiac automaticity. We obtained targeted Gi2RGSi ES cells and Western blotting showed that the mutation does not alter the expression of Gi2 in either ES cells or in heart from the Gi2 mutant mice (Figure IC and ID in the online data supplement). Interestingly, Gi2 appears to play less of a role in adenosine-mediated bradycardia than does Go, with only a nonsignificant 2.5-fold reduction in the R-PIA IC50 for Gi2GS/GS cells compared with the 6.4-fold decrease for GoGS/GS cells. Plus, there was no significant difference in maximal stimulation (Figure 1C). In contrast, Gi2GS/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 Gi2 preferentially couples to M2R versus A1R.
Pacemaker If and GIRK Currents Regulate Beating Rate in ESDC
To begin to address the ionic mechanisms of these receptor signals, we examined the role of If and GIRK in ESDC automaticity. The pacemaker current, If, has been shown to mediate -adrenergic stimulation and to contribute to cholinergic inhibition of heart rate24 and is a major mechanism for pacemaker activity. Deletion of the gene underlying If, HCN4, results in severe bradycardia and chronotropic incompetence.25 Similarly in ESDC, the selective If blocker ZD7288 nearly inhibits spontaneous beating by &60% and virtually eliminates -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.
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 Go/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 Gi2GS/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 GoGS/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, GoGS/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 ICa,L and If rather than GIRK channel on activation of Go by A1R.
Diminished Positive Chronotropic Response in Go/i2RGSi Mutant Cells Following 2AR Activation
ARs play a pivotal role in the control of cardiovascular function by mediating the actions of sympathetic nervous system activation and circulating catecholamines. Although the 1AR is the predominant subtype in the hearts of many species, both 1 and 2 subtypes can increase cardiac automaticity and contractility.27 Although most of actions of the 2AR are mediated through Gs proteins and protein kinase A, 2AR 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 Gi/o family G proteins but not on Gs, it is plausible to hypothesize that RGS proteins could modulate the actions of the 2AR, including chronotropic regulation.
Procaterol, a selective 2AR agonist,30 stimulates the ESDC beating rate. This tachycardia is mediated by 2ARs because it is blocked by 200 nmol/L ICI 118,551, a 2AR selective antagonist, and not by the selective 1AR 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 GoGS/GS cells (Figure 4A). However, maximal stimulation by procaterol is significantly attenuated in 2 independent GoGS/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 2AR effects on beating rate by reducing the action of Go. This is intriguing because 2AR coupling to Go 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 GoGS/GS and wild-type cells was abolished by PTX treatment (Figure IV in the online data supplement), confirming that Gi/o family coupling of the 2AR is under tonic control by endogenous RGS proteins.
An even more striking difference was seen in Gi2GS/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 2AR chronotropy such that the Gi2-mediated suppression overcomes Gs-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 Gi2-mediated inhibition occurs at much lower concentrations, whereas Gs effects do not shift. Thus endogenous RGS proteins strongly suppress Gi2 effects in the heart.
M2R-Mediated Response Is Selectively Enhanced in Gi2GS/GS Mice
Because ES cell studies may not fully replicate results in vivo, we developed Gi2RGSi 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 Gi2GS/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 Gi2GS/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 Gi2 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 Gi2GS/GS mice (–48 versus –295 bpm, P<0.001). This confirmation of the muscarinic selectivity of the Gi2 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.
Discussion
We demonstrate that endogenous RGS proteins potently regulate chronotropic responses to muscarinic, adenosine, and 2 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 Go and Gi2 subunits by incorporation of the RGSi mutation reveals the broad coupling of muscarinic M2R to Go and Gi2 but preferential coupling of adenosine A1 receptor to Go. This is despite the quite similar actions of A1R and M2R in regulating chronotropy and inotropy. The physiological significance of the preferential M2R-Gi2 coupling is strongly supported by our in vivo data, whereas Gi2-RGSi mice had unchanged responses after CCPA administration.
Previous data have suggested some Gi and Go 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 Go and Gi proteins,33 whereas purified Go was found to efficiently reconstitute A1R.34 Coimmunoprecipitation of activated A1R with Go and Gi3 but not Gi2 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 Go 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 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 GoGS/GS cells that were lost in presence of rTertiapinQ suggests potential GIRK coupling by the mutant Go.
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 Gi2GS/GS mutant. This suggests that M2-Gi2-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 GoGS/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 Go, the minimal role of GIRK is perhaps not surprising because specificity of Gi2 over Go in activating GIRK currents is well established. Sowell et al2 demonstrated that the expression of Gi2 and Gi3 is required for normal agonist-dependent activation of GIRK current, whereas Go knockout cells show normal GIRK responses but altered ICa,L regulation.3 Furthermore, the A1R has an 11-fold lower EC50 for suppression of Iso-stimulated ICa,L compared with its action on GIRK current.39 Altogether, these prior data suggest that A1R ought to use a non-GIRK pathway (eg, ICa,L) in mediating its bradycardic effects. Our results illustrate in vitro and in vivo the dramatic difference in signaling pathways (Gi2-GIRK and Go-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 Gi2 pathway or to high degrees of Go 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 Gi2 RGSi mice. Interestingly, the effect of the Gi2 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 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, Gi2 plays a clear role in 2AR signaling and Gi2 enhances survival in the face of 2AR overexpression, so inhibiting cardiac RGS may provide a novel approach to cardioprotection. The blunted maximal stimulation by 2AR in GoGS/GS mutants is the first convincing evidence for 2AR-Go coupling. Taken together, these data suggest that 2AR-mediated action may also involve multiple Gi/o subunits.
In summary, we show that endogenous RGS proteins potently modulate the Gi/o-mediated regulation of cardiac automaticity, leading to enhanced bradycardic responses on A1R and M2R activation. Moreover, there is clear specificity between Gi2 and Go in this chronotropic control in vivo and in vitro. A1R-mediated negative chronotropic actions are through preferential coupling to Go and minimally involve GIRK currents, whereas M2R coupling uses both Gi2 and Go 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 Go-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 Gi/o could contribute to sinus node dysfunction and/or atrial arrhythmias. Furthermore, 2AR action via both Gi2 and Go subunits is under tonic suppression by endogenous RGS proteins. Future studies to determine whether cardioprotective actions of 2AR and other Gi/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 Gi2 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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