RGS6, a Modulator of Parasympathetic Activation in Heart

Abstract

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 GIRK channel activation. 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 twenty 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 (SAN) and atrioventricular (AVN) 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 SAN cells. Consistent with a role of RGS6 in G protein inactivation, RGS6-deficient atrial myocytes exhibited a significant reduction in the time course of I_KAch activation and deactivation, as well as the extent of I_KAch 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.

Introduction

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⁺ channels (GIRKs), which results in a large K⁺ current (I_KAch) and membrane hyperpolarization¹.

RGS proteins function as GTPase-activating proteins (GAPs) for Gα subunits, accelerating their conversion to the inactive GDP bound form². 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 channels³. 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 upon 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 SAN and AVN regions, leading us to assess its involvement in parasympathetic control of the heart and I_KAch signaling.

Methods

An expanded Methods section is available as Online Supplementary Material.

Mice

RGS6^+/- mice in a 129/Sv × C57BL6 background obtained from TIGM were bred to generate RGS6 knockout (RGS6^-/-) mice.

Results

Robust Expression of RGS6 in Heart

We cloned RGS6 in 1998¹ and later discovered multiple splice forms of RGS6 in brain⁶. Given evidence that RGS6 transcripts are expressed in heart⁷, we examined RGS6 protein expression in heart. We found robust expression of a single immunoreactive form of RGS6 in heart, corresponding to that of RGS6L⁶, with higher levels of expression in atria compared to ventricles (Figs. 1A, IA). RGS4, which has been found to regulate parasympathetic signaling in the SAN⁸, was barely detectable in atria and absent in ventricles. Examination of mRNA levels of RGS6 revealed strong enrichment in the SAN and AVN and higher expression in atrium compared to ventricle (Figs. 1B, IB). 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 3-6% of those in brain.

Fig 1.

Cardiac expression of RGS6 and characterization of RGS6-/- mouse. Protein (A) and mRNA (B) levels of RGS4 and RGS6 in mouse heart tissues (n=3 mice). C, Generation of RGS6^-/- mice. Upper, illustration (in scale) of targeted region of RGS6 gene (black bars, exons; P1, P2, P3, PCR primers used for genotyping). Lower left, A typical genotyping result. Lower right, RGS6 protein expression in hearts of RGS6^+/+, RGS6^+/- and RGS6^-/- mice.

To examine the role of RGS6 in heart, we developed RGS6^-/^- mice (Fig. 1C, upper), that 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 (Fig. 1C, lower). RGS6 expression is reduced in atria and ventricles of RGS6⁺/^- mice compared to wild-type (wt) mice, illustrating a gene dosage effect in both of these tissues (Fig. IA), and is absent in RGS6^-/^- mice.

Gross morphologic examination of wt and RGS6^-/^- mice failed to identify any pathology as a result of RGS6 loss (Figs. 2A, 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 (Fig. 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, authenticated using HCN4 as a marker (Figs. 2C, 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.

Fig 2.

Histochemical staining of wt and RGS6-/- mouse hearts. A, H&E staining of hearts from wt and RGS6^-/- mice. B, Immunohistochemistry of RGS6 in hearts from wt and RGS6^-/- mice (yellow boxes, regions shown in atrium and ventricle panels). Expression of RGS6 in SAN and AVN regions in mouse heart (C) and SAN cell lysates (D).

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 (Figs. 3A, 3B). A dramatic enhancement in CCh-induced bradycardia was seen in RGS6^-/^- mice compared to wt mice, 66% vs 34%, respectively. Similar results were found in anesthetized mice (Fig. IIIA). Thus, RGS6^-/^- mice exhibit enhanced M2R-mediated bradycardia as found in mice expressing RGS-insensitive G_αi2⁵. Perfused hearts from RGS6^-/^- mice showed a normal chronotropic response to isoproterenol, but an enhanced bradycardia (60% vs 34%, Fig. 3C) and AV block (Figs. IIIB, IVD) in response to CCh compared to 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 (Figs. 3D, 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.

Fig 3.

Effects of loss of RGS6 on CCh-induced bradycardia and SAN cell action potential firing. A, Heart rates in conscious wt (n=3) and RGS6^-/- mice (n=5) at rest and following CCh (0.1 mg/kg i.p.). B, Average heart rates of conscious wt (n=3) and RGS6^-/- mice (n=5) in the first five minutes after administration of saline or CCh. *, p<0.05 vs wt. C, Heart rates of perfused hearts from wt and RGS6^-/- mice (n=3 each). *, p<0.01 vs wt. D, CCh inhibition of spontaneous action potential firing in SAN cells from wt and RGS6^-/- mice (n=5 each). *, p<0.01 vs wt.

Altered M2R Regulation of I_KAch in RGS6^-/^- Atrial Myocytes

The role of I_KAch in M2R-mediated bradycardia is well established. Knockout of GIRK1 or GIRK4 channel subunits in mice causes loss of I_KAch in atrial myocytes and severe impairment in vagal-mediated bradycardia⁹. RGS proteins reconstitute the rapid gating kinetics of GIRKs in atrial myocytes³. In view of our findings above, we examined whether atrial myocytes from RGS6^-/^- mice exhibited altered M2R regulation of GIRKs. In atrial myocytes from wt mice, application of CCh elicited rapid I_KAch that showed significant desensitization over time, followed by rapid deactivation upon removal of CCh (Fig. 4A). Atrial myocytes from RGS6^-/^- mice exhibited a significant reduction in the time course of activation and deactivation, as well as the extent of I_KAch desensitization (Figs. 4A, 4C, 4D). In some RGS6 ^-/- cells we found a smaller amplitude of I_KAch but this effect was not significant (Fig. 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 by releasing Gβγ. Indeed, the GAP function of RGS proteins accelerates both the onset of GPCR signaling and rate of deactivation¹⁰. Although RGS6 forms a complex with Gβ5 in atria, we found no evidence for direct interaction of RGS6 with GIRK1 or R7BP (Fig. VI).

Fig 4.

I_kAch properties in atrial myocytes from wt and RGS6^-/- mice. A, Representative I_kAch recordings from myocytes of wt and RGS6^-/- mice. Current density (peak current/cell capacitance; B), extent of desensitization (ratio of peak current to the current at the time of CCh removal; C), and half-maximal activation/deactivation time constant (t1/2; D) analysis of I_kAch obtained from wt (n=9) and RGS6^-/- (n=12) myocytes isolated from six pups of each genotype. *, p < 0.05 vs. wt.

Discussion

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 upon 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 AV block in RGS6^-/- mice.

We provide the first evidence that RGS6 is required for desensitization and rapid deactivation of GIRK-mediated I_KAch in atrial myocytes, consistent with its role as a GAP for Gi/o^{10, 11}. Delayed deactivation of I_KAch 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/Go. However, Cifelli et al ⁸ recently reported that RGS4 modulates parasympathetic activation and heart rate control in the SAN based upon 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 two 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 upon the same G proteins mediating GIRK channel opening as both proteins function as GAPs for Gi_1-3 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 upon different G proteins in vivo, in which case they might not produce additive effects upon 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.

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 G_βγ 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 (GPCR) 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 (SAN) and atrioventricular (AVN) nodes known to control heart rate and cardiac contractility.

• RGS6 is required for desensitization and rapid deactivation of M2R GIRK-mediated I_KACh channel current and suppression of atrial myocyte membrane excitability.

• Loss of RGS6 is associated with severely exaggerated bradycardia and AV block in response to parasympathetic stimulation, demonstrating that RGS6 is essential for modulating M2R signaling in heart to prevent parasympathetic override.

Summary

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/are 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.

Supplementary Material

Online Figure I. A, Quantification of RGS6 protein levels in hearts of RGS6 +/+, +/-, and -/- mice. Results are expressed as means ± S.E. of three hearts from mice of each RGS6 genotype. B, Expression of HCN4 in mouse tissues. To authenticate the SAN and AVN tissues used in Figure 1B, relative levels of HCN4 mRNA were determined in mouse brain and ventricle, atrium, SAN, and AVN of heart using real time PCR, and normalized to 18S rRNA mRNA level. The value of relative HCN4 mRNA level in brain was set as 1. Results are expressed as means ± S.E. of tissues from three wild-type mice, and the mean values are indicated in the figure.

Online Figure II. Loss of RGS6 does not influence heart size. Weights of atria and ventricles, expressed as percentage of body weight, were plotted from five wild-type and five RGS6^-/- mice. The size of hearts from wild-type and RGS6^-/- mice are not significantly different.

Online Figure III. Effects of loss of RGS6 on CCh-induced bradycardia. A, Heart rates in anesthetized wt and RGS6^-/- mice (n=5 each) at rest and following CCh (0.1 mg/kg i.p.). *, p<0.05 vs wt. B, Representative ECG traces of isolated perfused hearts from wild-type and RGS6^-/- mice at baseline, in the presence of 50 nM isoproterenol (Iso) or 50 nM + 0.5 μM carbachol (CCh). Severe bradycardia and AV block (prolonged PR interval) was noted in RGS6^-/- mice when 0.5 μM CCh was infused.

Online Figure IV. Effects of loss of RGS6 on QRS duration, PR, and QT intervals in isolated perfused mouse hearts. A, A hypothetical ECG trace is used to show the definition of QRS duration, PR, and QT intervals. ECGs of isolated mouse hearts (n=3) were recorded at basal condition (B), in the presence of 50 nM isoproterenol (C), or 50 nM isoproterenol plus 500 nM carbachol (D). Intervals of PR, QT, and QRS duration were calculated from these ECG traces. Results are means ± S.E. of three measurements. *, p<0.001.

Online Figure V. Representative recordings of spontaneous action potential firing in SA nodal myocytes isolated from wild-type (wt) and RGS6^-/- mice. Recording was done at control, 0.1μM carbachol (CCh)-treated, and washout conditions.

Online Figure VI. RGS6 co-immunoprecipitates with Gβ5 but not R7BP or GIRK1 in atrial lysates. Lysates (∼1 mg) from wild-type (wt) or RGS6^-/- mouse atria were immunoprecipitated using an anti-RGS6 antibody. The precipitates were then resolved using SDS-PAGE and blotted for Gβ5, R7BP, GIRK1, and RGS6. Brain lysate from a wild-type mouse (20 μg) was used as a positive control. NS, non-specific cross-reactive band.

Non-standard Abbreviations and Acronyms

AVN

atrioventricular node

CCh

carbachol

GAP

GTPase-activating proteins

GIRK

G protein-coupled inwardly rectifying K⁺ channels

I_kAch

Acetylcholine-activated potassium current

M2Rs

muscarinic M2 receptors

RGS4

regulator of G-protein signaling 4

RGS6

regulator of G-protein signaling 6

SAN

sinoatrial node