RGS6/Gβ5 Complex Accelerates IKACh Gating Kinetics in Atrial Myocytes and Modulates Parasympathetic Regulation of Heart RateNovelty and Significance
Rationale: The parasympathetic reduction in heart rate involves the sequential activation of m2 muscarinic cholinergic receptors (m2Rs), pertussis toxin–sensitive (Gi/o) heterotrimeric G proteins, and the atrial potassium channel IKACh. Molecular mechanisms regulating this critical signal transduction pathway are not fully understood.
Objective: To determine whether the G protein signaling regulator Rgs6/Gβ5 modulates m2R-IKACh signaling and cardiac physiology.
Methods and Results: Cardiac expression of Rgs6, and its interaction with Gβ5, was demonstrated by immunoblotting and immunoprecipitation. Rgs6−/− mice were generated by gene targeting, and the cardiac effects of Rgs6 ablation were analyzed by whole-cell recordings in isolated cardiomyocytes and ECG telemetry. Loss of Rgs6 yielded profound delays in m2R-IKACh deactivation kinetics in both neonatal atrial myocytes and adult sinoatrial nodal cells. Rgs6−/− mice exhibited mild resting bradycardia and altered heart rate responses to pharmacological manipulations that were consistent with enhanced m2R-IKACh signaling.
Conclusions: The cardiac Rgs6/Gβ5 complex modulates the timing of parasympathetic influence on atrial myocytes and heart rate in mice.
Cardiac output is shaped to a great extent by sympathetic and parasympathetic influences. Parasympathetic input tempers heart rate (HR), counteracts the proarrhythmic effects of sympathetic activation, and is mediated by acetylcholine (ACh).1 ACh is released from postganglionic parasympathetic neurons and binds to m2 muscarinic receptors (m2Rs) on pacemaker cells and atrial myocytes, triggering activation of pertussis toxin–sensitive (Gi/o) heterotrimeric G proteins.2 Once activated, G proteins dissociate into Gα-GTP and Gβγ subunits, leading to modulation of adenylyl cyclase and multiple ion channels. Central among these reactions is the binding of Gβγ to the atrial potassium channel IKACh, a heterotetramer composed of G protein–gated inwardly rectifying K+ (Girk)1 and Girk4 channel subunits.3 Binding of Gβγ to IKACh enhances its gating, which leads to cell hyperpolarization and, ultimately, decreased HR.4
The duration of G protein signaling is controlled by members of the RGS (regulator of G protein signaling) family.5 RGS proteins stimulate inactivation of Gα-GTP, facilitating its reassembly with Gβγ. RGS proteins play a critical role in shaping bradycardic effects of m2R receptor activation.6,–,8 Indeed, eliminating RGS influence by expressing Gα subunits insensitive to RGS action results in a substantial enhancement of IKACh regulation by m2R signaling, via both Gαo and Gαi2 pathways.7,8 Although more than 30 RGS proteins have been identified, the involvement of specific RGS proteins in the regulation of parasympathetic input is not fully understood. Here, we report an unexpected role of the Rgs6/Gβ5 complex, previously thought to be neuron-specific regulator, in the temporal regulation of m2R-IKACh signaling.
Littermate mice were used for all experiments in this study. All procedures were carried out in accordance with NIH guidelines and were approved by the Institutional Animal Care and Use Committee of the University of Minnesota.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
Profiling Rgs6 protein expression across mouse tissues revealed its readily detectable levels in the heart in addition to abundant presence in the brain (Figure 1 A and Online Figure I). Rgs6 protein was enriched in atria, where it was found predominantly in myocytes (Online Figure II), consistent with a recent report,9 and similar to the distribution of Girk1, an integral subunit of IKACh (Figure 1 B). To begin exploring the role of Rgs6 in cardiac physiology, we obtained Rgs6−/− mice where exons 5 to 7 encoding the critical N-terminal portion of the protein were eliminated (Figure 1 C and Figure 1 D). Immunoblotting verified the complete absence of Rgs6 protein in the hearts of Rgs6−/− mice (Figure 1 E).
Rgs6 interacts with the type 5 G protein β subunit (Gβ5) and the R7 binding protein (R7BP) in the CNS (Figure 1 F).10 In the mouse heart, however, only Gβ5 is available for the interaction with Rgs6 (Figure 1 E). Rgs6 was undetectable in hearts from Gβ5−/− mice, indicating that the physical association with Gβ5 is critical for the expression and/or stability of Rgs6 (Figure 1 G). Similarly, Gβ5 levels were dramatically reduced in the Rgs6−/− heart but not brain, indicating that in the heart Rgs6 is the predominant RGS bound to Gβ5. No effect on Rgs6 or Gβ5 levels was observed on elimination of R7BP or Girk4. Notably, we detected no compensatory changes in either Gαi/o proteins or RGS4, a protein previously implicated in regulation of the m2R-IKACh signaling.6
Given the coenrichment of Rgs6 and IKACh in atria and the role of R7 RGS/Gβ5 complexes in G protein–coupled receptor–GIRK signaling in the CNS,11 we next measured the impact of Rgs6 ablation on m2R-IKACh signaling in neonatal atrial myocytes, which exhibit robust inward current triggered by the nonselective muscarinic agonist carbachol (CCh). Whereas CCh evoked currents with comparable potency in atrial myocytes from wild-type mice, current deactivation kinetics were notably slower across all CCh concentrations tested in myocytes from Rgs6−/− mice (Online Figure III). Current activation kinetics were also delayed in Rgs6−/− myocytes, although only for the lower CCh concentrations tested.
We next compared CCh-induced currents in sinoatrial node (SAN) cells, the key anatomic substrate for parasympathetic control of heart rate (Figure 2). Although some differences in the density and kinetics of CCh-induced responses between adult SAN cells and neonatal atrial myocytes were evident, Rgs6 ablation correlated with significantly delayed deactivation rates in both cell types. Under the same conditions, no differences in CCh-induced steady-state current density or activation kinetics were observed between genotypes in either atrial myocytes or SAN cells (Figure 2). Furthermore, deletion of the Gβ5 replicated prolonged deactivation kinetics seen in Rgs6−/− myocytes (Figure 2 E), indicating that regulation of the m2R-IKACh signaling in heart atria is mediated by the Rgs6/Gβ5 complex rather than Rgs6 by itself.
The striking impact of Rgs6 ablation on m2R-IKACh signaling kinetics in atrial myocytes and SAN cells prompted us to test whether Rgs6/Gβ5 can physically associate with the IKACh channel. In transfected HEK293 cells, we detected robust coimmunoprecipitation of the Rgs6/Gβ5 complex with Girk4 but not Girk1 by both forward and reverse precipitation strategies (Figure 3). Thus, the involvement of Rgs6/Gβ5 in m2R-IKACh signaling is likely aided by a direct protein–protein interaction mediated by the cardiac-specific Girk subunit Girk4.
The delay in IKACh deactivation kinetics triggered by Rgs6/Gβ5 elimination is expected to enhance m2R-IKACh signaling because the channel would stay open longer, which would potentiate the parasympathetic regulation of HR. We addressed this possibility by analyzing cardiac function in mice using ECG telemetry, at baseline and following pharmacological manipulation. Analysis of ECG traces did not reveal gross abnormalities in cardiac physiology in Rgs6−/− mice (Figure 4A and Online Figure IV). Rgs6−/− mice did, however, display a mild resting bradycardia (511±13 versus 476±4 bpm, P<0.05), consistent with the effect of Rgs6/Gβ5 ablation on m2R-dependent signaling in atrial myocytes (Figure 4B). Although CCh administration (0.1 mg/kg, IP) triggered a rapid decrease in HR in wild-type and Rgs6−/− animals, the effect was significantly larger and persisted longer in Rgs6−/− mice (Figure 4C). Similarly, parasympathetic blockade with atropine (1 mg/kg, IP) had a positive chronotropic effect in both groups, with a significantly larger effect seen in Rgs6−/− mice (Figure 4D). Importantly, there was no difference in HR immediately following atropine administration, indicating that the bradycardia seen in Rgs6−/− mice results from enhanced intrinsic m2R signaling.
Here, we report that Rgs6/Gβ5 negatively regulates m2R-IKACh signaling in atrial myocytes by accelerating IKACh deactivation kinetics. These observations, together with the effect of Rgs6 ablation on HR and responses to pharmacological manipulation, indicate that Rgs6/Gβ5 represents a key node of regulation in the parasympathetic control of cardiac output. Because dysregulation of the parasympathetic tone by deficiencies in IKACh function is increasingly accepted as a major factor in the pathogenesis of the atrial fibrillation,1 our study introduces Rgs6/Gβ5 complex as an attractive candidate for better understanding of cardiac pathophysiology and development of corrective therapies.
Rgs6 belongs to the R7 family of RGS proteins, members of which were thought to be expressed exclusively in the nervous system, where they play roles in nociception, vision, reward behavior and locomotion.10 Although Rgs6 expression was reported previously in the heart,9,12,13 our study documents for the first time the functional relevance of Rgs6 to cardiac physiology. In the CNS, Rgs6 forms complexes with 2 proteins, Gβ5 and R7BP, that specify its stability, subcellular distribution, and activity.10 Here, we show that cardiac Rgs6 forms a complex with Gβ5, but not with R7BP which is undetectable in the heart. The obligate and functionally relevant nature of the Rgs6/Gβ5 interaction was underscored by the mutual dependence of Rgs6 and Gβ5 levels on their coexpression and the phenotypic similarities in m2R-IKACh signaling in myocytes from Rgs6−/− and Gβ5−/− mice. In neurons, Gβ5 recruits R7 RGS proteins to GIRK channels, resulting in accelerated channel kinetics associated with GABAB receptor activation.11 Thus, the present work reveals the conservation of this compartmentalization mechanism by showing that Rgs6/Gβ5 can likewise regulate m2R-IKACh signaling.
Previous work has identified Rgs4 as a critical regulator of m2R-IKACh signaling in sinoatrial nodal cells.6 Indeed, the deficiencies in m2R-IKACh signaling linked to Rgs6 ablation reported herein are reminiscent of those reported in Rgs4−/− mice.6 Therefore, murine sinoatrial nodal cells may use parallel approaches involving Rgs4 and Rgs6/Gβ5 to regulate m2R-IKACh signaling. It is possible, for example, that Rgs4 and Rgs6/Gβ5 selectively regulate different G protein subtypes involved in IKACh gating. Indeed, studies with knock-in mice expressing RGS-insensitive G proteins reveal a differential contribution of Gαi2 and Gαo to m2R-dependent actions.7,8 Furthermore, Rgs6/Gβ5 shows selectivity toward Gαo over Gαi2 in vitro.14 However, whereas the role of Gαi2 in mediating m2R-IKACh coupling is well established,8 the involvement of Gαo in this process is less certain. Moreover, it remains possible that other proteins of the more than 30-member RGS family also play roles in this regulation. Delineating the mechanisms of the functional involvement of RGS proteins in controlling m2R-IKACh signaling in the mouse models and their relevance to human physiology will serve as an exciting direction for future research.
Sources of Funding
This work was supported by NIH grants DA026405 (to K.A.M.), MH061933 (to K.W.), DA011806 (to K.W.), and T32 DA07234 (to K.L.A.), and a McKnight Land–Grant Award (to K.A.M.).
The authors thank Dr. Simonds (NIH/NIDDK) for the gift of the anti-Gb5 and anti-R7BP antibodies and Texas Institute for Genomic Medicine (TIGM) for providing RGS6−/− mouse line.
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
- G protein–gated inwardly rectifying K+
- heart rate
- type 2 muscarinic receptor
- regulator of G protein signaling
- sinoatrial node
- Received May 12, 2010.
- Revision received September 16, 2010.
- Accepted September 20, 2010.
- © 2010 American Heart Association, Inc.
- Hollinger S,
- Hepler JR
- Cifelli C,
- Rose RA,
- Zhang H,
- Voigtlaender-Bolz J,
- Bolz SS,
- Backx PH,
- Heximer SP
- Fu Y,
- Huang X,
- Piao L,
- Lopatin AN,
- Neubig RR
- Fu Y,
- Huang X,
- Zhong H,
- Mortensen RM,
- D'Alecy LG,
- Neubig RR
- Snow BE,
- Betts L,
- Mangion J,
- Sondek J,
- Siderovski DP
- Hooks SB,
- Waldo GL,
- Corbitt J,
- Bodor ET,
- Krumins AM,
- Harden TK
Novelty and Significance
What Is Known?
Activation of the parasympathetic branch of the autonomic nervous system decreases heart rate via the neurotransmitter acetylcholine.
Acetylcholine stimulates m2 muscarinic receptors (m2Rs) on sinoatrial nodal cells and atrial myocytes, leading to the G protein–dependent activation of the potassium channel IKACh.
Modulating m2R-IKACh signaling can impact heart rate.
What New Information Does This Article Contribute?
The Rgs6/Gβ5 protein complex is an essential modulator of m2R-IKACh signaling in cardiac myocytes and sinoatrial cells.
Inactivation of the Rgs6 gene in mice results in a mild bradycardia and an enhanced effect of drug-induced parasympathetic stimulation.
m2R-IKACh signaling plays a critical role in controlling heart rate and has been implicated in the pathogenesis of atrial fibrillation. The molecular mechanisms regulating this process, however, are not fully understood. Here, we identify the Rgs6/Gβ5 protein complex as a key regulator of m2R-IKACh signaling in cardiomyocytes and sinoatrial cells. We show that Rgs6/Gβ5 can physically associate with IKACh and that deletion of the Rgs6 gene in mice significantly enhances the parasympathetic regulation of heart rate. Thus, Rgs6/Gβ5 may contribute to, or represent a novel therapeutic target for, pathological conditions characterized by abnormal regulation of cardiac output.