Published online before print July 24, 2008, doi: 10.1161/CIRCRESAHA.108.180984
© 2008 American Heart Association, Inc.
Cellular Biology |
RGS4 Regulates Parasympathetic Signaling and Heart Rate Control in the Sinoatrial Node
From the Department of Physiology (C.C., R.A.R., H.Z., J.V.-B., S.-S.B., P.H.B., S.P.H.), Heart and Stroke/Richard Lewar Centre of Excellence in Cardiovascular Research, University of Toronto; and Department of Medicine (R.A.R., P.H.B.), University of Toronto, and Division of Cardiology, University Health Network, Toronto, Ontario, Canada.
Correspondence to Scott P. Heximer, Canada Research Chair in Cardiovascular Physiology, Heart and Stroke/Richard Lewar Centre of Excellence in Cardiovascular Research, University of Toronto, 1 King’s College Circle, Toronto, Ontario M5S 1A8, Canada. E-mail scott.heximer{at}utoronto.ca
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Abstract |
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Heart rate is controlled by the opposing activities of sympathetic and parasympathetic inputs to pacemaker myocytes in the sinoatrial node (SAN). Parasympathetic activity on nodal myocytes is mediated by acetylcholine-dependent stimulation of M2 muscarinic receptors and activation of G
i/o signaling. Although regulators of G protein signaling (RGS) proteins are potent inhibitors of Gi/o signaling in many tissues, the RGS protein(s) that regulate parasympathetic tone in the SAN are unknown. Our results demonstrate that RGS4 mRNA levels are higher in the SAN compared to right atrium. Conscious freely moving RGS4-null mice showed increased bradycardic responses to parasympathetic agonists compared to wild-type animals. Moreover, anesthetized RGS4-null mice had lower baseline heart rates and greater heart rate increases following atropine administration. Retrograde-perfused hearts from RGS4-null mice showed enhanced negative chronotropic responses to carbachol, whereas SAN myocytes showed greater sensitivity to carbachol-mediated reduction in the action potential firing rate. Finally, RGS4-null SAN cells showed decreased levels of G protein–coupled inward rectifying potassium (GIRK) channel desensitization and altered modulation of acetylcholine-sensitive potassium current (IKACh) kinetics following carbachol stimulation. Taken together, our studies establish that RGS4 plays an important role in regulating sinus rhythm by inhibiting parasympathetic signaling and IKACh activity.
Key Words: RGS proteins • sinoatrial node • parasympathetic • GIRK channels
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Introduction |
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Heart rate (HR) regulation by the autonomic nervous system is integrated by specialized autorhythmic (pacemaker) cells located within the sinoatrial node (SAN). Sympathetic neurotransmitters work via Gs-coupled β-adrenergic receptors to increase adenylyl cyclase activity, intracellular cAMP concentration and protein kinase A activity. As a result, cAMP-regulated effectors such as hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels, delayed rectifier, and voltage-gated Ca2+ channels are enlisted by sympathetic activity to increase pacemaker cell firing rate.1,2 By contrast, vagal parasympathetic activity decreases HR via G
i/o-coupled cholinergic M2 muscarinic receptors (M2Rs). Several effects, mediated by both Gi/o and Gβ
subunits, may contribute to this reduction in HR. Gβ heterodimers directly activate G protein–coupled inward rectifying potassium (GIRK) channels, resulting in membrane hyperpolarization. By contrast, Gi/o can both modulate phosphodiesterase activity and inhibit adenylyl cyclase activity, reduce both intracellular cAMP levels and protein kinase A activity, leading to decreased depolarizing currents carried by HCN and L-type calcium channels.2–5 Because dysregulation of parasympathetic activity occurs in heart failure, sick sinus syndrome, and selected cardiac arrhythmias,6 it is of clinical interest to identify key molecular regulators of parasympathetic signaling.
Regulators of G protein signaling (RGS) function as GTPase-activating proteins (GAPs) for G subunits via a conserved 
110 kDa RGS box domain.7,8 Accordingly, RGS proteins induce faster termination of signaling following removal of G protein–coupled receptor (GPCR) agonists. These proteins have recently emerged as inhibitory candidates of parasympathetic signaling in autorhythmic cells of the SAN because expression of RGS-resistant Gi2 or Go in mice reduced pacemaker cell automaticity.9,10 However, the pan-specific RGS protein inhibition in these models precluded identification of the specific RGS proteins involved. Although a large number of mammalian RGS proteins are expressed in the heart,11–13 their specific roles as regulators of parasympathetic pathway effectors are not well understood. Because RGS4 interacts with both M2R14 and GIRK channels,15 and because it can also modulate GIRK channel activation,16,17 we investigated its role as a regulator of parasympathetic signaling in the SAN. RGS4 was originally believed to be brain-specific with its high expression in the cerebral cortex and thalamus.18 Indeed motor memory defects have been identified in RGS4-deficient mice.19 This study investigates a role for RGS4 in the regulation of the SAN by the parasympathetic system.
Here, we use a RGS4 knockout mouse expressing LacZ behind the Rgs4 promoter to show that Rgs4 is highly expressed in the SAN. The discovery of increased parasympathetic-mediated signaling in RGS4-deficient animals, isolated hearts, and isolated SAN myocytes demonstrates that RGS4 is normally required for attenuation of parasympathetic-dependent G protein signaling in the SAN and intrinsic conduction system.
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Materials and Methods |
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An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Animals
The Rgs4tm1Dgen/J mouse strain was obtained from The Jackson Laboratory (Bar Harbor, Maine, http://jaxmice.jax.org/strain/005833.html). Mice were backcrossed > six generations into a C57Bl/6 background.
Statistical Analysis
Data are reported as means±SE. Data were analyzed using 1-way and 2-way ANOVA with Tukey’s or Dunn’s post hoc analysis and Student’s t tests, as appropriate. In all instances, P<0.05 was considered significant.
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Results |
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RGS4 Is Highly Expressed in the SAN
To characterize the role of RGS4 as a regulator of parasympathetic signaling in the SAN, we used RGS4-null mice expressing LacZ from the endogenous Rgs4 promoter. The targeted locus produces a truncated, nonfunctional RGS4 protein lacking its RGS domain (Figure 1A). As shown in Figure 1B, LacZ expression patterns in the brains of RGS4 heterozygous mice matched those previously reported for the endogenous RGS4 mRNA.18,19 Real-time RT-PCR analysis confirmed the loss of RGS4 mRNA in brains from knockout mice (Figure 1C).
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CT values were normalized to 18S and compared with RGS3 mRNA of +/+ hearts (as the calibrator sample), as described in the online data supplement. Data are representative of at least 3 independent experiments.Hearts from heterozygous animals showed an intense crescent-shaped pattern of LacZ staining on the exterior surface of the heart at the junction of the superior vena cava (SVC) and right atrium (Figure 2A, arrowheads), which is the region containing the SAN.20,21 To more precisely identify the location of RGS4 expression, the atria were dissected off the heart and mounted to visualize the endocardial SAN region.22,23 Low-magnification images revealed significant levels of RGS4 expression in the SAN and vasculature (Figure 2B). High magnification of this region showed the compact intense LacZ staining within the SVC-proximal region of the SAN extending toward the base of the right atrium (Figure 2C). LacZ expression was also observed, albeit at much lower levels in vascular smooth muscle cells and pericytes of the coronary vasculature. Notably, no LacZ expression was observed in working atrial myocardium, including the crista terminalis, and the right atrial appendage (RAA), Purkinje fibers, or in the working ventricular myocardium (data not shown). RT-PCR analysis of wild-type mice confirmed higher relative expression levels in SAN compared to RAAs (Figure 2D). To ensure our LacZ-stained regions corresponded to the SAN, immunohistochemistry for HCN4 (Figure 2F and Figure I in the online data supplement) was performed in parallel with LacZ staining (Figure 2E). These results revealed that Rgs4 is highly and selectively coexpressed with pacemaker channels in the SAN.
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Enhanced Parasympathetic-Mediated HR Regulation in RGS4-Deficient Mice
Despite high levels of RGS4 expression in the SAN, basal heart rates (Figure 3A) and blood pressures (data not shown) in conscious RGS4-null mice were not different from wild-type mice (controls). However, consistent with a role for RGS4 in the regulation of M2R signaling in the SAN, RGS4-null animals showed markedly enhanced heart rate responses to systemically administered carbachol (carbamylcholine chloride [CCh]) (0.1 mg/kg IP), suggesting that RGS4 function is important when parasympathetic tone is increased. Notably, under anesthetized conditions, in which increased parasympathetic tone is expected, RGS4-null mice showed reduced basal HR (Figure 4A) and reduced mean arterial pressures (data not shown) compared to wild-type controls. These differences in HR were abolished by the IV administration of atropine, a potent M2R antagonist. Specifically, atropine (0.3 mg/kg) had minimal effects on HR in wild-type animals, as expected from previous reports,24–26 while inducing large HR increases in RGS4-null mice (Figure 4A and 4B). Importantly, atropine eliminated the HR differences between the groups. These data suggest that autonomic control of SAN activity is biased toward greater parasympathetic activity because of reduced inhibition of M2R-coupled G protein signaling in RGS4-null mice relative to controls.
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; n=6) and null (KO) (
; n=6) mice were recorded in unrestrained waking mice using a radiotelemetric aortic blood pressure catheter inserted via the right common carotid artery. Forty-eight-hour (periodic sampling) (A) or 20-minute acute (continuous) CCh-stimulated (0.1 mg/kg IP) (B) traces were recorded. Two-way ANOVA with a Tukey’s honestly significant difference (HSD) post hoc test was used to determine statistical significance of baselines and change in HR with CCh between groups. *P<0.05.
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Increased Bradycardia in CCh-Treated RGS4-Deficient Hearts
It is conceivable that the results described above could be explained by differences in central nervous system activity or other neurohumoral factors between the groups of mice. To eliminate these differences, we studied isolated retrograde-perfused hearts. Although baseline HRs (430 bpm) and ECG patterns of isolated hearts did not differ between genotypes (Figure 5A), CCh application dose-dependently reduced heart rates (estimated from R-R intervals) to a greater extent in RGS4-null hearts. In fact, at the highest dose of CCh tested (10 µmol/L), all RGS4-null hearts showed SAN standstill, whereas wild-type and heterozygous hearts were merely slowed but continued to beat (Figure 5A and 5B). All hearts showed complete recovery of the ECG trace following CCh washout (data not shown). Thus, the loss of RGS4 renders the SAN more sensitive to the bradycardic effects of the parasympathetic agonist CCh.
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A separate group of isolated hearts was treated with 50 nmol/L isoproterenol (Iso) to mimic the in vivo conditions of high sympathetic tone before application of CCh. The response to sympathetic stimuli was not different between the groups, because wild-type and RGS4-deficient hearts had similar HRs ( 590 bpm) after Iso treatment, with all hearts showing normal ECG patterns (supplemental Figure IIA). As with non-Iso–treated hearts, CCh application slowed HR in RGS4-null far more than in wild-type hearts (supplemental Figure IIB). Interestingly, however, CCh effects were more pronounced in both genotypes following pretreatment with Iso. For example, discernable P or QRS waves could not be detected in RGS4-null hearts (ie, the hearts were in SAN standstill) at CCh concentrations above 3 µmol/L. Furthermore, RGS4-null hearts treated with Iso were more susceptible to atrioventricular node conduction block (ie, uncoupling of the P and QRS waves) and SAN standstill compared to nontreated hearts (supplemental Figure III and supplemental Table I). Together, these data suggest that RGS4 may modulate the previously reported crosstalk between β-adrenergic and M2R signaling.27
Enhanced Effect of CCh on Spontaneous Action Potential Firing in RGS4-Deficient Mice
Next, we evaluated the role of RGS4 as a regulator of spontaneous action potential (AP) firing rates in cardiac pacemaker cells (Figure 6). AP frequency was not different between genotypes under basal conditions (176±12.6 bpm in wild-type myocytes versus 164.7±9.2 bpm in RGS4-null myocytes; P=0.93). Superfusion of CCh (100 nmol/L) reduced AP frequency to 136±14.3 bpm in wild-type SAN myocytes, whereas in RGS-null myocytes, AP frequency was profoundly reduced to 14.2±11.1 bpm (Figure 6A and 6B). Strikingly, AP firing was completely suppressed at this dose of CCh in 9 of 11 RGS4-null SAN myocytes. In contrast, no wild-type myocytes stopped firing spontaneous APs.
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P<0.05 CCh response within wild-type, *P<0.05 between RGS4 KO and wild-type response to CCh.These changes in AP firing correlated with changes in the maximum diastolic potential (MDP) of the cells. Under basal conditions, MDP was –64.4±1.6 mV in wild-type myocytes and –65.1±0.9 mV in RGS4-null myocytes. CCh (100 nmol/L) hyperpolarized the MDP of wild-type cells to –67.1±1.6 mV, whereas RGS4-null SAN myocytes were significantly more hyperpolarized to –73.6±0.9 mV (Figure 6A and 6C). Although these effects of CCh on AP frequency and MDP were completely reversible on washout (Figure 6A), the time course of washout was significantly prolonged in RGS4-null SAN myocytes (151.0±6.6 seconds) compared to wild-type myocytes (90.6±7.8 seconds).
IKACh Is Altered in Isolated Sinoatrial Myocytes From RGS4-Deficient Mice
The results above suggest that RGS4 regulates heart function by modulating parasympathetic-dependent signaling. The correlation between changes in AP frequency and MDP following application of CCh suggest that acetylcholine-activated K+ currents (IKACh),28 produced by GIRK channels, are responsible for the altered parasympathetic signaling in RGS4-null mice. Thus, we measured IKACh in isolated SAN myocytes. Figure 7A shows that application of CCh (10 µmol/L) induces an increase in IKACh (recorded at –100 mV), which declines thereafter via a process referred to as desensitization.28 Whereas peak IKACh levels were similar between the groups, the time-dependent decline of IKACh is reduced in RGS4-null myocytes relative to wild-type. Indeed, the extent of IKACh desensitization (at –100 mV) is less in RGS4-deficient (13.8±1.4%) compared to wild-type (30.4±2.1%) SAN myocytes (Figure 7B). The reduced desensitization in RGS4-null myocytes is also evident from the current/voltage relationship measured 2 minutes after CCh application (Figure 7D), which shows increased IKACh in RGS4-null myocytes compared to control, without differences in peak IKACh between the groups (Figure 7C). Consistent with the AP studies above, the decay kinetics of IKACh following CCh removal (ie, the deactivation kinetics) were markedly delayed for RGS4-null myocytes during either CCh washout or the application of atropine to block M2Rs (Figure 7E). Together, these data indicate prolonged Gi/o signaling in SAN myocytes following CCh removal, consistent with the known function of RGS4 as a GAP for Gi/o.8 The delay in deactivation kinetics of IKACh coincided with a corresponding slowing (P<0.05) of activation kinetics in RGS4-deficient SAN myocytes compared to wild-type SAN myocytes (Figure 7F). Kinetic slowing of the actions of CCh in the absence of RGS4 is also consistent with the previously identified role of RGS4 in accelerating the kinetics of the response of GIRK channels to Gi/o stimulation.16 Thus, SAN myocytes from RGS4-null mice show markedly altered M2R-dependent signaling characteristics and regulation of GIRK channel kinetics.
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Finally, we evaluated the effects of 2 additional doses of CCh (1 µmol/L and 100 nmol/L) on IKACh to determine whether a component of the enhanced effect of parasympathetic signaling on HR regulation in RGS4-deficient hearts could be explained by a shift in the IKACh dose–response curve. Figure 8A illustrates peak IKACh current density at –100 and +40 mV. Peak currents were measured so that the maximal CCh response could be attained with minimal contribution from the desensitization effect. Despite a dose-dependent increase in peak IKACh density in both genotypes, no differences in peak IKACh density were observed between wild-type and RGS4-null SAN myocytes at any of the CCh doses tested. By contrast, the extent of IKACh desensitization following a 2-minute application of CCh at concentrations of 10 µmol/L (Figure 7B) and 1 µmol/L and 100 nmol/L (Figure 8B) was less for RGS4-deficient compared to wild-type myocytes at all concentrations tested. Notably, the greatest relative difference in the percentage of desensitization occurred at 100 nmol/L (Figure 8C), consistent with a role for potent regulation of GIRK activity by RGS4 at physiological M2R agonist concentrations.22 This may explain the dose-dependent differences in HR in the response to CCh between the genotypes. Together, these data suggest that enhanced CCh signaling in the SAN of RGS4-null mice is the result of altered IKACh desensitization and kinetics rather than a shift in the dose response.
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Discussion |
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Previous studies showed that RGS4 mRNA is expressed in atrial11,12,27 and ventricular12,29 myocytes; however, its expression in the murine SAN has not been examined previously. Using 2 approaches (ie, LacZ reporter/HCN4 immunostaining colocalization and real-time RT-PCR), our results demonstrate that RGS4 is more highly expressed in SAN myocytes compared to myocytes in the RAA. Because RGS4 is known to attenuate G
i/o but not Gs activation, we anticipated that its loss would cause selective increases in the response to parasympathetic stimulation in the SAN. Basal HRs in conscious RGS4-deficient mice were not different from wild-type controls, potentially because of the dominant effect of sympathetic versus parasympathetic tone on HR control in mice. However, consistent with a role for RGS4 in the regulation of HR under conditions of increased parasympathetic tone, conscious RGS4-deficient mice showed enhanced bradycardic responses to CCh and anesthetized RGS4-deficient animals showed lower basal HR levels. Atropine-mediated normalization of HR levels in the latter model supported the notion that increased parasympathetic activity could regulate RGS4-null hearts to a greater extent than wild-type. Although it is conceivable that the altered HR regulation in RGS4-null mice is partially attributable to the loss of RGS4 in the central nervous system and/or coronary vasculature, it seems likely that an enhanced intrinsic responsiveness of SAN myocytes to vagal stimulation plays a significant role. Enhanced intrinsic sensitivity of the SAN to vagal stimulation is also supported by the observation that isolated RGS4-deficient hearts showed enhanced bradycardia in response to the M2R agonist CCh. In fact, the RGS4-deficient hearts were so sensitive to the application of CCh that they experienced SAN standstill at doses of 3 µmol/L. Thus, it seems likely that loss of RGS4 in the SAN dramatically sensitizes these hearts to parasympathetic activity at the level of HR depression.
Because M2R is selectively coupled to the Gi/o subclass of heterotrimeric G proteins, it is likely that the majority of M2R-mediated responses in the SAN are mediated by signaling through Gi/o and its effectors. Additionally, because RGS proteins are potent inhibitors of Gi/o function at the plasma membrane, we expect that the loss of RGS proteins will increase Gi/o signaling in SAN myocytes. Although a number of end effectors in SAN myocytes could transduce the Gi/o-mediated signals to produce the lower HRs observed in RGS4-deficient mice, we focused on comparing IKACh between the different mouse groups because of the observed changes in MDP during spontaneous AP firing (Figure 6) and the prominent role that this current plays in mediating HR slowing in response to vagal stimulation.30 Moreover, RGS4 is known to modulate GIRK channel function in heterologous expression systems.17,31,32 Consistent with an increased level of Gi/o signaling, CCh-treated SAN myocytes from RGS4-deficient mice showed increased IKACh as a result of reduced desensitization and altered GIRK gating kinetics. However, because RGS4 functions at the receptor level to inhibit all Gi/o-mediated signaling, it is possible that other pathways regulated by parasympathetic stimuli, including adenylyl cyclase activity, phosphodiesterase activity, intracellular cyclic nucleotide levels, protein kinase A activity, HCN, and L-type calcium channels,2–4 may also be altered in RGS4-null hearts.
This is the first demonstration that RGS4 is required for rapid desensitization of GIRK-mediated IKACh in the SAN. These data suggest that RGS4 is part of a negative feedback regulatory mechanism for activated M2R-Gi/o-GIRK complexes. Previous work to define the mechanisms of GIRK desensitization suggested desensitization was resolved into fast and slow phases, where the fast phase is explained by IKACh channel dephosphorylation33 and RGS protein GAP activity34 and the slow phase involves G protein receptor kinase activity.35,36 Because RGS4 has not been implicated in the regulation of kinases or phosphatases in the cell, the loss of rapid phase desensitization in RGS4-deficient SAN myocytes likely indicates the loss of a GAP-dependent desensitization mechanism. It has been shown that RGS4 forms stable protein–protein interactions with both M2R14 and GIRK315 channels, and, thus, it is proposed to be a component of an integrated kinetic scaffolding complex that promotes efficient coordinated regulation of both G protein and GIRK activation.37 Consistent with the reported effects of RGS4 on the kinetic regulation of Gi/o-mediated modulation of GIRK channels, IKACh measured in SAN myocytes lacking RGS4 showed slower activation and deactivation compared to wild-type cells. Taken together, these data provide strong evidence for defective Gi/o signaling and GIRK regulation in SAN myocytes lacking a selective Gi/o GAP and point to the possibility that RGS4 plays a role in parasympathetic regulation of beat-to-beat changes in intact animals.
These data raise the possibility that reduction of RGS4 expression or function in a pathophysiological setting could increase susceptibility to bradycardia and arrhythmia. It is interesting that, like the RGS-resistant Gi- or Go-expressing mice, RGS4-null mice do not show increased vagal-mediated effects on baseline HR in vivo,9 perhaps reflecting an increased level of sympathetic drive in the murine models. Future studies will determine whether RGS4 is critical for SAN regulation under basal conditions in humans, who normally have higher intrinsic levels of parasympathetic tone.
In addition to showing a chronotropic phenotype, RGS-resistant Gi2 mice showed slowed conduction through the atrioventricular node and susceptibility to atrioventricular block and other conduction defects.10 Similarly, Iso-treated hearts from mice lacking RGS4 show M2R-dependent atrioventricular node block and cardiac arrest, implicating its potential role as a regulator of the crosstalk mechanisms between β-adrenergic receptors and M2R.27 However, it remains to be determined whether atrioventricular node conduction is altered in RGS4-deficient mice and whether loss of RGS4 in regions of the heart outside of the SAN significantly increases the susceptibility of hearts to conduction defects and arrhythmogenesis in vivo.
In summary, we show that RGS4 modulates the Gi/o-mediated regulation of cardiac automaticity, leading to enhanced bradycardic responses following M2R activation in RGS4-deficient mice. Moreover, the conduction defects associated with dysregulation of Gi/o-mediated activation of GIRK channels and other parasympathetic effectors suggest that RGS4 may normally provide protection from arrhythmogenic stimuli. Indeed, it has been shown that GIRK4 knockout mice and mice with altered expression of Gβ subunits exhibited significantly reduced HR variability and a reduced propensity for atrial fibrillation.38,39 Because increases in parasympathetic activity is associated with susceptibility to cardiac arrhythmias,6 conditions that lead to loss of RGS4 function might be expected to increase the probability of arrhythmia and atrial fibrillation. Accordingly, it will be of interest to characterize the expression and function of RGS4 in sick sinus syndrome and heart failure in humans. In the future, the search for compounds that increase both the expression and function of RGS4 may provide a valuable therapeutic strategy for the treatment and prevention of heart disease.
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Acknowledgments |
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We acknowledge technical support from the Cell Biology of Atherosclerosis Group at the University of Toronto, with special thanks to Drs Philip Marsden and Michelle Bendeck for assistance with real-time RT-PCR and histological analysis.
Sources of Funding
Technical and financial assistance for this work was provided by the Heart & Stroke/Richard Lewar Centre of Excellence (HSRLCE) in Cardiovascular Research. This work was supported by Heart and Stroke Foundation of Ontario Grant-in-Aid Program grants NA5921/T5835 (to S.P.H.) and operating grants from the Canadian Institutes of Health Research (MOP-68965 to P.H.B.). Career support came from the Canada Research Chairs Program (S.P.H.), the Heart & Stroke Foundation of Ontario Career Investigator Program (P.H.B.), the Canadian Institutes of Health Research–Tailored Advanced Collaborative Training in Cardiovascular Science program (R.A.R.) and Canadian Institutes of Health Research–Canada Graduate Scholarship Program (C.C.), and the Alberta Heritage Foundation for Medical Research (R.A.R.).
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this work.
Original received February 12, 2008; resubmission received June 9, 2008; revised resubmission received July 9, 2008; accepted July 10, 2008.
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