RGS6/Gβ5 complex accelerates I_KACh gating kinetics in atrial myocytes and modulates parasympathetic regulation of heart rate

Abstract

Rationale

The parasympathetic reduction in heart rate (HR) involves the sequential activation of m2 muscarinic cholinergic receptors (m₂R), pertussis toxin-sensitive (Gi/o) heterotrimeric G proteins, and the atrial potassium channel I_KACh. 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-I_KACh 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 electrocardiogram (ECG) telemetry. Loss of Rgs6 yielded profound delays in m₂R-I_KACh deactivation kinetics in both neonatal atrial myocytes and adult sino-atrial nodal cells. Rgs6^−/− mice exhibited mild resting bradycardia and altered heart rate responses to pharmacologic manipulations that were consistent with enhanced m₂R-I_KACh signaling.

Conclusions

The cardiac Rgs6/Gβ5 complex modulates the timing of parasympathetic influence on atrial myocytes and heart rate in mice.

Introduction

Cardiac output is shaped to a great extent by sympathetic and parasympathetic influences. Parasympathetic input tempers heart rate (HR) and counteracts the pro-arrhythmic effects of sympathetic activation, and is mediated by acetylcholine (ACh)¹. ACh is released from post-ganglionic parasympathetic neurons and binds to m₂ muscarinic receptors (m₂R) on pacemaker cells and atrial myocytes, triggering activation of pertussis toxin-sensitive (Gi/o) heterotrimeric G proteins². 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 I_KACh, a heterotetramer composed of Girk1 and Girk4 subunits³. Binding of Gβγ to I_KACh enhances its gating which leads to cell hyperpolarization and ultimately, decreased HR⁴.

The duration of G protein signaling is controlled by members of the Regulator of G protein Signaling (RGS) family⁵. RGS proteins stimulate inactivation of Gα-GTP, facilitating its re-assembly 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 I_KACh regulation by m₂R signaling, via both Gα_o and Gα_i2 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-I_KACh signaling.

Methods

Littermate mice were used for all experiments in this study. All procedures were carried out in accordance with the NIH guidelines and were approved by the Institutional Animal Care and Use Committee of the University of Minnesota. A detailed Methods section is available as Supplementary Material.

Results

Profiling Rgs6 protein expression across mouse tissues revealed its readily detectable levels in the heart in addition to abundant presence in the brain (Fig. 1A; Online Fig. I). Rgs6 protein was enriched in atria, where it was found predominantly in myocytes (Online Fig. II), consistent with a recent report⁹, and similar to the distribution of Girk1, an integral subunit of I_KACh (Fig. 1B). To begin exploring the role of Rgs6 in cardiac physiology, we obtained Rgs6^−/− mice where exons 5–7 encoding the critical N-terminal portion of the protein were eliminated (Fig. 1C, D). Immunoblotting verified the complete absence of Rgs6 protein in the hearts of Rgs6^−/− mice (Fig. 1E).

Figure 1. Rgs6 protein level and complex formation in the mouse heart.

A, Rgs6 protein levels across mouse tissues as analyzed by Western blotting. Equal amounts of total protein (20 μg) were loaded in each lane. Coomasie staining (CBB) was used as a loading control. B, Rgs6 is co-enriched with Girk1 in the atria. Atrial (ATR) and ventricular (VEN) lysates were analyzed by Western blotting (upper panels). CBB staining confirms equal protein loading. Graph: Quantification of Rgs6 and Girk1 band densities. C, Strategy for Rgs6 ablation by homologous recombination. D, Structural organization of Rgs6. Frames designate the deleted region (Δ) and recognition site of the RGS6 FL antibody (αRGS6 FL Ab) used throughout the study. Colored boxes designate known structural domains. E, Co-immunoprecipitation of Rgs6 with Gβ5 and R7BP from heart and brain tissues. F, Co-dependence of Rgs6 and Gβ5 expression in the heart and brain as analyzed by Western blotting.

Rgs6 interacts with the type 5 G protein β subunit (Gβ5) and the R7 Binding Protein (R7BP) in the CNS¹⁰ (Fig. 1F). In the mouse heart, however, only Gβ5 is available for the interaction with Rgs6 (Fig. 1E). 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 (Fig. 1G). 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 upon 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-I_KACh signaling⁶.

Given the co-enrichment of Rgs6 and I_KACh in atria and the role of R7 RGS/Gβ5 complexes in GPCR-GIRK signaling in the CNS¹¹, we next measured the impact of Rgs6 ablation on m₂R-I_KACh signaling in neonatal atrial myocytes, which exhibit robust inward current triggered by the non-selective muscarinic agonist carbachol (CCh). While 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 Fig. III). Current activation kinetics were also delayed in Rgs6^−/− myocytes, though only for the lower CCh concentrations tested.

We next compared CCh-induced currents in sino-atrial node (SAN) cells, the key anatomic substrate for parasympathetic control of heart rate (Fig. 2). While 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 (Fig. 2). Furthermore, deletion of the Gβ5 replicated prolonged deactivation kinetics seen in Rgs6^−/− myocytes (Fig. 2E), indicating that regulation of the m₂R-I_KACh signaling in heart atria is mediated by the Rgs6/Gβ5 complex rather than Rgs6 by itself.

Figure 2. Impact of Rgs6 ablation on m₂R-I_KACh signaling in atrial myocytes and SAN cells.

A, Inward currents evoked by CCh (10 μmol/L) in atrial myocytes from wild-type and Rgs6^−/− mice. Scale bars: 5 s/200 pA. B, Inward currents evoked by CCh (10 μmol/L) in SAN cells from wild-type and Rgs6^−/− mice. Scale bars: 5 s/400 pA. Inset: image of the wild-type SAN cell evaluated in the adjacent trace. Summary of steady-state CCh-induced current density (C), activation kinetics (D), and deactivation kinetics (E) in wild-type, Rgs6^−/−, and Gβ5^−/− atrial myocytes (n=5–11 per group), and in wild-type and Rgs6^−/− SAN cells (n=10–12 per genotype). Genotype did not impact current density (atrial myocytes: F_2,21=1.9, P=0.18; SAN cells: t(20)=0.13, P=0.90) or activation kinetics (atrial myocytes: F_2,21=1.0, P=0.38; SAN cells: t(20)=1.69, P=0.11), but did influence deactivation kinetics (atrial myocytes: F_2,21=24.8, P<0.001; SAN cells: t(20)=3.71, P<0.01). Symbols: **^, *** P<0.01 and 0.001, respectively, vs. wild-type (within cell type).

The striking impact of Rgs6 ablation on m₂R-I_KACh signaling kinetics in atrial myocytes and SAN cells, prompted us to test whether Rgs6/Gβ5 can physically associate with the I_KACh channel. In transfected HEK293 cells, we detected robust co-immunoprecipitation of the Rgs6/Gβ5 complex with Girk4 but not Girk1 by both forward and reverse precipitation strategies (Fig 3). Thus, the involvement of Rgs6/Gβ5 in m₂R-I_KACh signaling is likely aided by a direct protein-protein interaction mediated by the cardiac-specific Girk subunit, Girk4.

Figure 3. RGS6/Gβ5 forms a complex with Girk4.

Rgs6 and Gβ5 were co-expressed with either AU1-tagged Girk4 or AU5-tagged Girk1 in HEK293 cells. Forward and reciprocal co-immunoprecipitation assays were performed as described in the Methods using indicated antibodies. Eluates were analyzed using Western blotting.

The delay in I_KACh deactivation kinetics triggered by Rgs6/Gβ5 elimination is expected to enhance m₂R-I_KACh signaling as 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 pharmacologic manipulation. Analysis of ECG traces did not reveal gross abnormalities in cardiac physiology in Rgs6^−/− mice (Fig. 4A and Online Fig. IV). Rgs6^−/− mice did, however, display a mild resting bradycardia (511±13 vs. 476±4 bpm, P<0.05), consistent with the effect of Rgs6/Gβ5 ablation on m₂R-dependent signaling in atrial myocytes (Fig. 4B). While CCh administration (0.1 mg/kg, i.p.) triggered a rapid decrease in HR in wild-type and Rgs6^−/− animals, the effect was significantly larger and persisted longer in Rgs6^−/− mice (Fig. 4C). Similarly, parasympathetic blockade with atropine (1 mg/kg, i.p.) had a positive chronotropic effect in both groups, with a significantly larger effect seen in Rgs6^−/− mice (Fig. 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.

Figure 4. Effect of Rgs6 ablation on resting HR and muscarinic regulation.

A, Baseline ECG recorded during light phase from conscious, unrestrained wild-type (WT, n=5) and Rgs6^−/− mice (n=5). B, Average HR as determined from the analysis of the ECG recordings (6hr). Symbols: * P<0.05 vs. wild-type. C, Effect of CCh (0.1 mg/kg, i.p.) on HR, analyzed and plotted as simple moving average with a period of 10 min. HR at 0 min corresponds to the 30-min average baseline HR on the day of the experiment. Two-way ANOVA analysis (genotype and time) of the 120-min post-injection interval revealed main effects of genotype (F_1,104=21.6; P<0.001). D, Effect of atropine (1 mg/kg, i.p.) on HR, analyzed and plotted as simple moving averages with a period of 10 min. No significant difference in HR was observed during the 60-min post-injection interval (F_1,56=0.02; P=0.89).

Discussion

Here, we report that Rgs6/Gβ5 negatively regulates m₂R-I_KACh signaling in atrial myocytes by accelerating I_KACh deactivation kinetics. These observations, together with the effect of Rgs6 ablation on HR and responses to pharmacologic manipulation, indicate that Rgs6/Gβ5 represents a key node of regulation in the parasympathetic control of cardiac output. Since dysregulation of the parasympathetic tone by deficiencies in I_KACh function is increasingly accepted as a major factor in the pathogenesis of the atrial fibrillation¹, 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 locomotion¹⁰. 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 two proteins, Gβ5 and R7BP that specify its stability, subcellular distribution, and activity¹⁰. 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 co-expression, and the phenotypic similarities in m₂R-I_KACh 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 GABA_B receptor activation¹¹. Thus, the present work reveals the conservation of this compartmentalization mechanism by showing that Rgs6/Gβ5 can likewise regulate m₂R-I_KACh signaling.

Previous work has identified Rgs4 as a critical regulator of m₂R-I_KACh signaling in sinoatrial nodal cells⁶. Indeed, the deficiencies in m₂R-I_KACh signaling linked to Rgs6 ablation reported herein are reminiscent of those reported in Rgs4^−/− mice⁶. Therefore, murine sinoatrial nodal cells may employ parallel approaches involving Rgs4 and Rgs6/Gβ5 to regulate m₂R-I_KACh signaling. It is possible, for example, that Rgs4 and Rgs6/Gβ5 selectively regulate different G protein subtypes involved in I_KACh gating. Indeed, studies with knock-in mice expressing RGS-insensitive G proteins reveal a differential contribution of Gα_i2 and Gα_o to m₂R-dependent actions^7,8. Furthermore, Rgs6/Gβ5 shows selectivity towards Gα_o over Gα_i2 in vitro¹⁴. However, while the role of Gα_i2 in mediating m₂R-I_KACh coupling is well established⁸, the involvement of Gα_o in this process is less certain. Moreover, it remains possible that other proteins of 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-I_KACh signaling in the mouse models and their relevance to human physiology will serve as an exciting future research direction.

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 (m2R) on sinoatrial nodal cells and atrial myocytes, leading to the G protein-dependent activation of the potassium channel I_KACh.

• Modulating m2R-I_KACh signaling can impact heart rate.

What new information does this article contribute?

• The Rgs6/Gβ5 protein complex is an essential modulator of m2R-I_KACh 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.

Summary

m2R-I_KACh signaling plays 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-I_KACh signaling in cardiomyocytes and sinoatrial cells. We show that Rgs6/Gβ5 can physically associate with I_KACh 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.

Abbreviations

GIRK channel

G protein-gated inwardly-rectifying K⁺ channel

CCh

carbachol

RGS

Regulator of G protein Signaling

HR

heart rate

ECG

electrocardiogram

m₂R

type 2 muscarinic receptor