RGS6, a Modulator of Parasympathetic Activation in HeartNovelty and Significance
Rationale: Parasympathetic regulation of heart rate is mediated by acetylcholine binding to G protein–coupled muscarinic M2 receptors, which activate heterotrimeric Gi/o proteins to promote G protein–coupled inwardly rectifying K+ (GIRK) channel activation. Regulator of G protein signaling (RGS) proteins, which function to inactivate G proteins, are indispensable for normal parasympathetic control of the heart. However, it is unclear which of the more than 20 known RGS proteins function to negatively regulate and thereby ensure normal parasympathetic control of the heart.
Objective: To examine the specific contribution of RGS6 as an essential regulator of parasympathetic signaling in heart.
Methods and Results: We developed RGS6 knockout mice to determine the functional impact of loss of RGS6 on parasympathetic regulation of cardiac automaticity. RGS6 exhibited a uniquely robust expression in the heart, particularly in sinoatrial and atrioventricular nodal regions. Loss of RGS6 provoked dramatically exaggerated bradycardia in response to carbachol in mice and isolated perfused hearts and significantly enhanced the effect of carbachol on inhibition of spontaneous action potential firing in sinoatrial node cells. Consistent with a role of RGS6 in G protein inactivation, RGS6-deficient atrial myocytes exhibited a significant reduction in the time course of acetylcholine-activated potassium current (IKACh) activation and deactivation, as well as the extent of IKACh desensitization.
Conclusions: RGS6 is a previously unrecognized, but essential, regulator of parasympathetic activation in heart, functioning to prevent parasympathetic override and severe bradycardia. These effects likely result from actions of RGS6 as a negative regulator of G protein activation of GIRK channels.
Since the discovery that acetylcholine (ACh) release from the vagus produces bradycardia, key proteins and mechanisms underlying this action of ACh in heart have been identified. It is now known that ACh binds to muscarinic M2 receptors (M2Rs) that activate heterotrimeric G proteins (Gi/o) in key pacemaking regions of the heart. Activation of these G proteins causes release of Gβγ subunits that bind to and activate G protein–coupled inwardly rectifying K+ (GIRK) channels, which results in a large K+ current (acetylcholine-activated potassium current [IKACh]) and membrane hyperpolarization.1
RGS proteins function as GTPase-activating proteins (GAPs) for Gα subunits, accelerating their conversion to the inactive GDP-bound form.2 This results in their reassembly with Gβγ to form inactive G protein heterotrimers, thereby terminating signaling by both Gα and Gβγ proteins. Heterologous expression of various members of the RGS protein family with GIRK channels and M2Rs are required to reconstitute the normal activation and deactivation kinetics of native atrial GIRK channels.3 In vivo evidence for this key role of RGS proteins in controlling ACh-mediated bradycardia was provided using knock-in mice expressing mutant forms of Gi or Go that cannot be acted on by RGS proteins.4,5 Thus, endogenous RGS proteins are required to regulate parasympathetic signaling in heart.
Therefore, it is of considerable importance and interest to identify which specific RGS proteins are responsible for this activity in heart. Here, we show that RGS6 is expressed highly in the heart, including sinoatrial (SAN) and atrioventricular (AVN) nodal regions, leading us to assess its involvement in parasympathetic control of the heart and IKACh signaling.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
RGS6+/− mice in a 129/Sv×C57BL6 background, obtained from TIGM, were bred to generate RGS6 knockout (RGS6−/−) mice.
Robust Expression of RGS6 in Heart
We cloned RGS6 in 1998 (GenBank accession no. AF073920) and later discovered multiple splice forms of RGS6 in brain.6 Given evidence that RGS6 transcripts are expressed in heart,7 we examined RGS6 protein expression in heart. We found robust expression of a single immunoreactive form of RGS6 in heart, corresponding to that of RGS6L,6 with higher levels of expression in atria compared with ventricles (Figure 1A; Online Figure I, A). RGS4, which has been found to regulate parasympathetic signaling in the SAN,8 was barely detectable in atria and was absent in ventricles. Examination of mRNA levels of RGS6 revealed strong enrichment in the SAN and AVN and higher expression in atrium compared with ventricle (Figure 1B; Online Figure I, B). RGS4 mRNA levels also were enriched in the SAN and AVN and were about two-fold higher than those of RGS6 in these regions. The low or undetectable levels of RGS4 protein in atria and ventricles is consistent with our finding that RGS4 mRNA levels in these tissues are 3% to 6% of those in brain.
To examine the role of RGS6 in heart, we developed RGS6−/− mice (Figure 1C, top), which would generate a short truncated form of RGS6L lacking its RGS domain (responsible for GAP activity). A typical genotyping result and immunoblot of RGS6 in atria and ventricles of mice of each genotype is shown (Figure 1C, bottom). RGS6 expression is reduced in atria and ventricles of RGS6+/− mice compared with wild-type (WT) mice, illustrating a gene dosage effect in both of these tissues (Online Figure I, A) and is absent in RGS6−/− mice.
Gross morphological examination of WT and RGS6−/− mice failed to identify any pathology as a result of RGS6 loss (Figure 2A; Online Figure II). Immunohistochemistry confirmed our immunoblotting results demonstrating that RGS6 is expressed more highly in the atrium than the ventricle and is absent in RGS6−/− mice (Figure 2B). In agreement with the observed expression of RGS6 mRNA in the SAN and AVN, we found that RGS6 protein is expressed in the SAN and AVN, which was confirmed by using HCN4 as a marker (Figure 2C and 2D). These results demonstrate robust expression of RGS6 in mouse heart, particularly in the SAN and AVN regions, and validate the use of RGS6−/− mice to study the functional role of RGS6 in heart.
Loss of RGS6 Provokes Exaggerated Bradycardia and Altered SAN Action Potential Firing
We examined carbachol (CCh)-induced bradycardia in conscious unrestrained WT and RGS6−/− mice (Figure 3A and 3B). A dramatic enhancement in CCh-induced bradycardia was seen in RGS6−/− mice compared with WT mice, 66% versus 34%, respectively. Similar results were found in anesthetized mice (Online Figure III, A). Thus, RGS6−/− mice exhibit enhanced M2R-mediated bradycardia as found in mice expressing RGS-insensitive Gαi2.5 Perfused hearts from RGS6−/− mice showed a normal chronotropic response to isoproterenol, but an enhanced bradycardia (60% versus 34%, Figure 3C) and atrioventricular block (Online Figure III, B; Online Figure IV, D) in response to CCh compared with WT mice. Consistent with these findings, inhibition of spontaneous action potential firing rates in SAN cells by CCh was significantly enhanced by loss of RGS6 (Figure 3D; Online Figure V). These results show that genetic deletion of RGS6 in heart leads to increases in CCh-mediated bradycardia likely by actions on cardiac pacemaker cells in the SAN, demonstrating that RGS6 is essential for modulation of M2R signaling in heart.
Altered M2R Regulation of IKACh in RGS6−/− Atrial Myocytes
The role of IKACh in M2R-mediated bradycardia is well established. Knockout of GIRK1 or GIRK4 channel subunits in mice causes loss of IKACh in atrial myocytes and severe impairment in vagal-mediated bradycardia.9 RGS proteins reconstitute the rapid gating kinetics of GIRK channels in atrial myocytes.3 In view of our findings above, we examined whether atrial myocytes from RGS6−/− mice exhibited altered M2R regulation of GIRK channels. In atrial myocytes from WT mice, application of CCh elicited rapid IKACh that showed significant desensitization over time, followed by rapid deactivation on removal of CCh (Figure 4A). Atrial myocytes from RGS6−/− mice exhibited a significant reduction in the time course of activation and deactivation, as well as the extent of IKACh desensitization (Figure 4A, 4C, and 4D). In some RGS6−/− cells, we found a smaller amplitude of IKACh, but this effect was not significant (Figure 4B). Thus, RGS6 is required for the normal activation and deactivation kinetics, as well as the rate of desensitization of GIRK channels. These effects are likely mediated by the GAP activity of RGS6 on Gi/o proteins that activate GIRK channels by releasing Gβγ. Indeed, the GAP function of RGS proteins accelerates both the onset of G protein–coupled receptor signaling and rate of deactivation.10 Although RGS6 forms a complex with Gβ5 in atria, we found no evidence for direct interaction of RGS6 with GIRK1 or R7BP (Online Figure VI).
This study establishes RGS6 as an essential modulator of parasympathetic activation in heart, where it functions to prevent parasympathetic override and severe bradycardia. Loss of RGS6 was associated with severely exaggerated bradycardia in response to CCh in both mice and isolated perfused hearts, showing that this response was not dependent on effects of RGS6 in tissues beyond the heart. Indeed, RGS6 is expressed highly in heart, especially within the SAN and AVN regions. This is the first demonstration of endogenous expression of RGS6 protein in heart and the only member of the RGS protein family to be detected at the protein level in heart. Loss of RGS6 within SAN cardiac pacemaker cells, which dramatically enhanced CCh inhibition of spontaneous action potential firing, likely accounts for the observed effects on CCh-mediated bradycardia. Our findings support a role for RGS6 as a major negative modulator of M2R signaling in the SAN. It may play other important roles in the heart in view of its expression in both atrium and ventricle. Indeed, we found that RGS6 was expressed in the AVN, and we observed CCh-induced atrioventricular block in RGS6−/− mice.
We provide the first evidence that RGS6 is required for desensitization and rapid deactivation of GIRK-mediated IKACh in atrial myocytes, consistent with its role as a GAP for Gi/o.10,11 Delayed deactivation of IKACh in RGS6-deficient atrial pacemaker cells slows channel closing, prolonging membrane hyperpolarization. This effect would be expected to produce the dramatic increase in CCh-induced bradycardia in RGS6−/− mice and isolated hearts and enhanced inhibition of spontaneous action potential firing in SAN cells. In fact, the finding that loss of RGS6 has such dramatic effects on GIRK channel deactivation implies that other RGS proteins do not compensate for loss of RGS6 or have a role similar to that of RGS6. We speculate that this may result from lack of expression of other RGS proteins specifically in SAN regions because most RGS proteins are capable GAPs for Gi/o. However, Cifelli et al8 recently reported that RGS4 modulates parasympathetic activation and heart rate control in the SAN based on studies with RGS4−/− mice. In their study, RGS4 expression measured by β-galactosidase staining (LacZ in the knockout allele) was limited to the SAN region, in contrast to the expression of RGS6 protein shown here. Here, we showed enriched expression of RGS6 and RGS4 mRNA in SAN and AVN regions of the heart. We envision 2 possible ways to reconcile our findings with those of Cifelli et al.8 It is possible that RGS6 and RGS4 are both expressed in the same SAN cells and act on the same G proteins mediating GIRK channel opening as both proteins function as GAPs for Gi1–3 and Go. In this case, we might expect to see even greater exaggeration of parasympathetic signaling in mice with combined loss of RGS4 and RGS6. Alternatively, it is possible that RGS6 and RGS4 act in different cells or on different G proteins in vivo; in which case, they might not produce additive effects on their loss.
The present results provide new evidence for an important role of RGS6 in the heart. RGS6 is expressed highly in heart, and its loss promotes severe bradycardia during parasympathetic activation. These findings suggest that alterations in RGS6 expression or activity in heart could potentially contribute to diseases such as sick sinus syndrome or other maladies involving abnormal parasympathetic activation in heart. Thus, this work identifies RGS6 as a possible therapeutic target for treatment of such diseases.
Sources of Funding
This work was supported by NIH grants GM075033-02 and ARRA GM075033-03S1 (to R.A.F.); HL084583 and HL083422 (to P.J.M.); and HL007121 (Cardiovascular Interdisciplinary Research Fellowship) (to H.G.). R.A.F. and D.P.M. were also supported by The University of Iowa Carver Collaborative Pilot Grant.
We thank Dr Chantal Allamargot (University of Iowa Central Microscopy Core Facility) for assistance with histology/microscopy of hearts and Dr John Koland for careful reading of and useful suggestions for this manuscript.
In August 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.2 days.
Non-standard Abbreviations and Acronyms
- atrioventricular node
- GTPase-activating protein
- G protein–coupled inwardly rectifying K+ channel
- acetylcholine-activated potassium current
- muscarinic M2 receptor
- regulator of G protein signaling
- sinoatrial node
- wild type
- Received May 12, 2010.
- Revision received September 7, 2010.
- Accepted September 15, 2010.
- © 2010 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Parasympathetic stimulation of the heart is achieved through acetylcholine (ACh) release from the vagus, which binds to G protein–coupled muscarinic M2 receptors (M2Rs) located at pacemaking nodes and electrically conducting portions of the heart.
Stimulation of M2Rs by ACh results in release of the Gβγ component of the heterotrimeric G protein complex associated with the receptor which promotes activation of G protein–coupled inwardly rectifying K+ (GIRK) channels, hyperpolarization of the membrane, inhibition of cell firing, and a net decrease in heart rate.
Regulator of G protein signaling (RGS) proteins determine the magnitude and duration of the cellular response to G protein–coupled receptor stimulation through inactivation of G proteins and are essential for proper GIRK channel gating kinetics and normal parasympathetic control of the heart.
What New Information Does This Article Contribute?
RGS6 is expressed robustly in heart, particularly in the sinoatrial node (SAN) and atrioventricular node (AVN), which are known to control heart rate and cardiac contractility.
RGS6 is required for desensitization and rapid deactivation of M2R GIRK-mediated IKACh channel current and suppression of atrial myocyte membrane excitability.
Loss of RGS6 is associated with severely exaggerated bradycardia and atrioventricular block in response to parasympathetic stimulation, demonstrating that RGS6 is essential for modulating M2R signaling in heart to prevent parasympathetic override.
Nearly a century has elapsed since the discovery that vagal ACh release produces bradycardia and reduced cardiac contractility. Parasympathetic regulation of cardiac automaticity is now known to be achieved by ACh-stimulating M2Rs, causing G protein release and GIRK activation. RGS proteins negatively regulate this process; however, it is unclear which specific RGS protein(s) is critical. This work identifies RGS6 as an essential modulator of parasympathetic cardiac regulation. Our findings suggest that alteration of RGS6 expression or activity could potentially contribute to cardiac pathologies involving aberrant parasympathetic activity, establishing RGS6 as a potential therapeutic target for such conditions.