G protein-coupled receptors play a pivotal role in regulating cardiac automaticity. Their function is controlled by regulator of G protein signaling (RGS) proteins acting as GTPase-activating proteins for Gα subunits to suppress Gαi and Gαq signaling. Using knock-in mice in which Gαi2-RGS binding and negative regulation are disrupted by a genomic Gαi2G184S (GS) point mutation, we recently (Fu Y, Huang X, Zhong H, Mortensen RM, D'Alecy LG, Neubig RR. Circ Res 98: 659–666, 2006) showed that endogenous RGS proteins suppress muscarinic receptor-mediated bradycardia. To determine whether this was due to direct regulation of cardiac pacemakers or to alterations in the central nervous system or vascular responses, we examined isolated, perfused hearts. Isoproterenol-stimulated beating rates of heterozygote (+/GS) and homozygote (GS/GS) hearts were significantly more sensitive to inhibition by carbachol than were those of wild type (+/+). Even greater effects were seen in the absence of isoproterenol; the potency of muscarinic-mediated bradycardia was enhanced fivefold in GS/GS and twofold in +/GS hearts compared with +/+. A1-adenosine receptor-mediated bradycardia was unaffected. In addition to effects on the sinoatrial node, +/GS and GS/GS hearts show significantly increased carbachol-induced third-degree atrioventricular (AV) block. Atrial pacing studies demonstrated an increased PR interval and AV effective refractory period in GS/GS hearts compared with +/+. Thus loss of the inhibitory action of endogenous RGS proteins on Gαi2 potentiates muscarinic inhibition of cardiac automaticity and conduction. The severe carbachol-induced sinus bradycardia in Gαi2G184S mice suggests a possible role for alterations of Gαi2 or RGS proteins in sick sinus syndrome and pathological AV block.
- G protein signaling
- heart rate
- muscarinic receptors
- adenosine receptors
cardiac automaticity is highly regulated by the autonomic nervous system through G protein-coupled receptors, including β-adrenergic receptors (β-AR) and muscarinic M2 receptors (M2R), which are located in specialized conducting tissues, i.e., sinoatrial node (SAN) and atrioventricular (AV) node (AVN). Stimulation of the sympathetic nervous system in response to exercise or stress results in a rapid increase in heart rate, which is mediated by activation of the Gαs-coupled β-AR. Subsequently, adenylyl cyclase is activated, which leads to an increase in cAMP level and activation of protein kinase A. A wide spectrum of ion channels [i.e., pacemaker current (If), delayed rectifier current (IKs), L-type Ca2+ current (ICa,L)] can be regulated by phosphorylation or by direct binding of cyclic nucleotides, leading to increased pacemaker rates (2, 28, 29). On the other hand, vagal stimulation suppresses activation of adenylyl cyclase through M2R, which are coupled to inhibitory G proteins (Gαi/o), thereby counteracting the effect of sympathetic activation. Meanwhile, Gβγ subunits released from Gαi/o can directly activate G protein-coupled inward rectifying K+ channels (GIRK), causing a decrease in heart rate. The generation of GIRK4 knockout mice (38) clearly demonstrated that this second-messenger-independent pathway contributes ∼50% of the bradycardic response mediated by M2R without affecting basal heart rate.
Besides being a major effector mechanism for negative chronotropic regulation upon vagal stimulation, GIRK currents are highly regulated by endogenous regulators of G protein signaling (RGS) proteins (4), which function primarily as GTPase-accelerating proteins through their conserved RGS domains (11, 17). RGS proteins enhance the intrinsic GTPase activity of G proteins and accelerate the turn-off step of the G protein cycle, thereby serving as negative RGS. More than 10 mammalian RGS proteins have been found in myocardium (5, 21, 39), with minimal distinction in their expression pattern between atria and ventricles (39). In addition, the specificity of different RGS proteins toward G protein isoforms is rather loose. The majority of these RGS proteins can act as a GTPase-activating protein for most members of the Gαi/o and Gαq families (17). Transgenic models with overexpression or elimination of a specific RGS protein have provided essential information to our understanding of the physiological role of this family of proteins in the cardiovascular system. Elevated levels of RGS4 in heart were associated with higher mortality after transverse aortic constriction in mice (30), whereas cardiomyopathy from Gq overexpression or diabetes was ameliorated by RGS4 overexpression (15, 31). These studies suggest that upregulation of RGS4 might be causal or compensatory, depending on the physiological context. Furthermore, RGS2 knockout mice develop severe hypertension, indicating a critical role of RGS2 as a modulator of Gαq signaling in the vasculature (16, 37). Despite the knowledge gained from these models, it is clear that generation of knockout or transgenic models for individual RGS proteins expressed in heart is a daunting task. Furthermore, functional redundancy among family members may well mask some significant phenotypes, which might explain the minimal phenotype observed in RGS4 knockout mice (13). As described in our laboratory's previous studies (8, 9), we employed RGS-insensitive (RGSi) mutant Gα subunits, which contain a G184S point mutation in their switch I region to keep the Gα subunit from binding to RGS proteins without altering other properties of the G protein, such as its intrinsic GTPase activity, effector binding, etc. (22). Generation of knock-in mice with the RGSi mutation allows us to examine the overall physiological role of endogenous RGS proteins as a group on a particular Gα subunit.
Indeed, our previous work in embryonic stem cell-derived cardiocytes (ESDC) (8, 9) demonstrated that endogenous RGS proteins potently modulate Gαi/o-coupled receptor [i.e., M2R, adenosine receptor (A1R), and β2-AR] signaling in control of cardiac automaticity in a Gα isoform-specific manner. In intact animals with the Gαi2RGSi mutation, we have also confirmed a profound increase in sensitivity to carbachol-induced bradycardia (8), which may largely be due to enhanced GIRK activation. Loss of regulation by RGS proteins on Gαi2, however, revealed a wide spectrum of phenotypic changes in these mice (19), including behavioral hyperactivity, elevated daytime baseline heart rate, and mild cardiac hypertrophy, which may point to alterations in the central nervous system (CNS). It is possible that vascular responses to different agonists are altered as well. Therefore, it remains unclear whether the enhanced carbachol-induced bradycardia is due to changes in the CNS or peripheral vasculature, or if it is due to intrinsic alterations in cardiac pacemaker activity.
To investigate the role of endogenous RGS proteins in heart rate regulation, we used Gαi2 RGSi knock-in mice, where expression of the mutant protein is under control of its own promoter. An isolated heart perfusion system was used to further dissect the role of RGS proteins on pacemaker activity of the heart, independent of potential CNS effects or vascular influences. Here we demonstrate that endogenous RGS proteins not only modulate muscarinic control of pacemaking activity of SAN, but also profoundly influence AVN conduction, suggesting a potential role in the etiology of AV block and sick sinus syndrome.
MATERIALS AND METHODS
Gαi2RGSi knock-in mice.
Generation of the Gαi2RGSi mouse colony was described previously (19). All animals used in these studies had been backcrossed for more than five generations (N5–N9) onto a C57Bl/6 background. Control animals used in the experiments were wild-type littermates (+/+) of the Gαi2G184S RGSi heterozygous (+/GS) or homozygous (GS/GS) mice. Animals were maintained under a standard 12:12-h light-dark cycle regime and had ad libitum access to standard chow and water. Male or female mice used in these studies were 4–7 mo of age. The study was performed with approval of the University Committee on Use and Care of Animals (UCUCA) at University of Michigan.
Isolated heart perfusion.
Mice were anesthetized with 2.5% Avertin (20 μl/g ip) with 1 mg/ml of heparin (Sigma; 187 USP units/mg) (25). Hearts were rapidly excised and rinsed in ice-cold modified Tyrode solution [consisting of (in mM) 137 NaC1, 5.4 KCl, 0.5 MgCl2, 0.16 NaH2PO4, 3 NaHCO3, 5 HEPES, and 5 glucose; pH 7.35] supplemented with 2.5 mM CaCl2 solution and immediately perfused with the same solution for <1 min at room temperature. Hearts were then mounted on a perfusion apparatus (Kent Scientific) for retrograde aortic perfusion with oxygenated Krebs-Henseleit solution [consisting of (in mM) 25.0 NaHCO3, 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 2.5 CaCl2, 0.5 Na-EDTA, and 15 glucose, oxygenated with 95% O2-5% CO2, pH 7.3–7.4 at 37°C] at a constant pressure of 75–85 cmH2O (37°C) and allowed to stabilize for 25 min. Hearts with signs of ischemia, physical damage, persistent arrhythmia over 5 min after the start of perfusion, or slow flow rate (<1 ml/min) were discarded.
ECG recording and beating rate measurement.
ECG was recorded using a DP-304 differential amplifier (Axon Instruments), digitized at 300 μs with Digidata 1322A and pCLAMP 8 software. Beating rate (BR) was calculated from RR intervals extracted using custom-written software. Baseline BRs were recorded for 5 min after initial stabilization, and increasing concentrations of agonist were given every 5 min. RR intervals of the last minute of each 5-min segment were used as the steady-state BR to obtain concentration-response curves. Studies conducted on baseline inhibition of BR by the adenosine receptor agonist (R)-phenylisopropyladenosine (R-PIA) were subsequent to carbachol inhibition and atropine reversal; thus the BR after atropine was used as baseline for R-PIA inhibition. Isoproterenol (Iso), R-PIA, carbachol, and atropine were obtained from Sigma.
Overdrive atrial pacing and effective refractory period recordings.
Bipolar pacing electrodes were placed on the right atrium, and pacing was usually conducted at cycle lengths of 150, 120, and 100 ms. Each pacing episode lasted for 10 s and was repeated three times to obtain average PR intervals, which is defined from the beginning of P wave to the peak of R wave, instead of the beginning of R wave. This modification was made for more precise measurement. Programmed stimulation was also performed by delivering a train of pulses at a basic cycle length of 100 ms (S1) followed by a single premature pulse (S2). S2 was decremented by 2 ms in multiple runs to determine the effective refractory period (ERP) for atrial-ventricular conduction.
All of the data are reported as means ± SE. Two-way ANOVA with a Bonferroni posttest (GraphPad Prism 4.0) was used to compare averages from multiple groups. A nonlinear least squares method with global fitting and F-test comparisons of curves (Graphpad Prism 4.0) was used to fit dose-response data, and P < 0.05 was considered significant. Global fitting performs nonlinear least squares fits of the whole family of data sets (e.g., +/+, +/GS, GS/GS) at once with sharing of selected parameters (i.e., IC50 or maximal response) to obtain one value that applies to the entire family. A second fit is done with that parameter floating. When the best fitting model from the entire family requires different values for a parameter, a statistical difference is reported.
Since the occurrence of AV block is a binary response, the outcome was considered as dichotomous values (case and control) and treated as categorical data. Three genotypes (+/+, +/GS, and GS/GS) were dummy coded. The number of cases was counted, and a logistic regression model (Stata 8.0, Statacorp) was used to detect statistical differences.
Enhanced M2R-mediated sinus bradycardia and AV block in Gαi2RGSi mutants subsequent to β-adrenergic stimulation by Iso.
Hearts from male mice 4–7 mo old were used in this study. Increasing concentrations of the β-AR agonist Iso (1–100 nM) were added to the perfusion buffer to obtain the concentration-response relationship. Baseline BR (beats/min) among wild-type and Gαi2RGSi mutants were similar (316 ± 13, 320 ± 14, and 313 ± 25 for +/+, +/GS, and GS/GS mice, respectively, not significant) (Fig. 1A). Moreover, there was no significant difference in maximal Iso-stimulated BR. This is consistent with our laboratory's previous findings (8) in ESDC, indicating that mutation of the Gαi2 subunit does not affect Gαs-mediated responses, and all animals respond to Iso-mediated β-adrenergic stimulation in a similar manner. After the maximal stimulation was achieved, the Iso concentration was reduced to 50 nM and used as a baseline for inhibition by carbachol. Both +/GS and GS/GS mice show increased sensitivity to carbachol compared with their counterparts (IC50 as μM: 0.33 and 0.36 vs. 0.51, P < 0.0001), and maximal inhibition by carbachol was also slightly increased in GS/GS mice compared with wild type (88 vs. 82%, P < 0.05) (Fig. 1B). This result indicates that loss of RGS regulation on Gαi2 significantly enhanced both the potency and magnitude of carbachol-induced bradycardia, although the enhancement was modest.
In the presence of Iso, we observed that carbachol not only inhibited the BR, but also induced third-degree AV block at higher concentrations. On the ECG, the normal relationship between the P waves and the QRS complexes is lost for the GS/GS hearts (Fig. 2A). Interestingly, both +/GS and GS/GS mice showed a higher frequency of AV block at lower concentration of carbachol. For example, none of 12 wild-type hearts developed AV block at 1 μM carbachol, whereas three out of five GS/GS mutants showed AV block, indicating significantly increased susceptibility compared with wild type. There was no third-degree AV block observed during perfusion with Iso alone.
Pronounced sinus bradycardia in response to carbachol in Gαi2RGSi mutants and increased AV block only in +/GS mutants in the absence of β-adrenergic stimulation.
M2R-mediated bradycardia was further examined in the absence of Iso. Similar to results in Fig. 1, basal BR among three genotypes are not significantly different (BR as beats/min: 312 ± 10, 343 ± 20, 315 ± 8 for +/+, +/GS, and GS/GS mice, respectively). However, both +/GS and GS/GS mutants demonstrated substantial increases in sensitivity to carbachol with 2.4- and 4.7-fold reductions in IC50 compared with +/+ mice (Fig. 3A). In these experiments, the BR of all hearts was suppressed to zero by carbachol before the addition of atropine. Furthermore, female wild-type and GS/GS mutant mice demonstrated virtually the same phenomenon with more than a sixfold reduction in IC50 for carbachol (Fig. 3B). Therefore, in the absence of β-adrenergic stimulation, loss of RGS control on Gαi2 dramatically enhanced sinus bradycardia in response to carbachol.
Interestingly, a different type of heart block, second-degree AV block, was observed with carbachol in the absence of Iso. On the ECG, this is demonstrated by P waves that are not followed by a QRS complex (Fig. 4A). Similar to what is observed with third-degree AV block in the presence of Iso, the +/GS hearts showed an increased occurrence of AV block at lower concentrations of carbachol. Surprisingly, none of the GS/GS mutant hearts showed second-degree AV block (Fig. 4B), which seems to contradict our hypothesis of enhanced Gαi2 signaling in the AVN. This discrepancy could be due to the severe sinus bradycardia in GS/GS mutants that limits atrial inputs to the AVN below the blocking threshold. To test this hypothesis, we conducted atrial overdrive pacing studies to focus on AV conduction properties while holding atrial rate constant.
Prolonged AV conduction in Gαi2RGSi mutants.
Overdrive pacing is commonly used clinically or experimentally while obtaining intracavitary ECGs to unmask sinus node dysfunction and AV conduction abnormalities (20, 23). Hearts were paced at cycle lengths of 150, 120, and 100 ms, and the PR interval was measured. The PR intervals at all cycle lengths from GS/GS mutant mice were significantly longer than for their wild-type counterparts (51.8 ± 0.9 vs. 48.6 ± 0.6 ms; 54.4 ± 0.9 vs. 50.5 ± 0.6 ms; 60.2 ± 1.3 vs. 54.6 ± 1.0 ms for cycle length of 150, 120, and 100 ms, respectively), indicating slower AV conduction (Fig. 5A). Interestingly, this is true even without exogenously applied muscarinic agonist.
Furthermore, the AV ERP was measured by introducing a single extra stimulus after a drive train of impulses at a fixed rate (all S1), at which time the S1–S2 interval is decreased until the S2 impulse does not conduct to the ventricle. This interval defines the AV nodal ERP. Using the S1–S2 protocol, ERP measured in Gαi2 GS/GS mutants was significantly longer than that of wild type (91.0 ± 1.4 vs. 86.7 ± 1.0 ms, P < 0.05). In summary, genetically blocking the action of RGS proteins on Gαi2 leads to delayed AV conduction, which may lead to increased risk for AV block.
A1R-mediated bradycardia remains unaltered in Gαi2RGSi mutant mice.
As previously reported in intact animals and ESDC (8), augmented Gαi2 signaling due to loss of RGS regulation preferentially amplifies M2R-mediated responses, but not those of the A1R. In the present study, we further confirmed this selectivity in isolated hearts. Hearts from male and female GS/GS mutants and their littermate controls were subjected to increasing concentrations of R-PIA (Fig. 6). Comparable responses (IC50 and maximal inhibition) were observed in +/+ and GS/GS mutant mice. No specific types of arrhythmia were associated with R-PIA administration for either genotype.
In the present study, we demonstrate that endogenous RGS proteins potently modulate muscarinic-mediated negative chronotropic responses through Gαi2 in cardiac pacemaker cells, independent of CNS input or peripheral vascular changes. Furthermore, in the mutant mice, two distinct types of conduction defects were associated with carbachol, depending on the presence or absence of β-adrenergic stimulation. Loss of RGS regulation on Gαi2 significantly increased the occurrence of these abnormalities, suggesting a potential role for etiological involvement of either RGS proteins or Gαi2.
Enhanced Gαi2 signaling and sinus bradycardia.
Despite predictions of enhanced vagal bradycardia on loss of RGS actions at Gαi2, our previous study of unrestrained Gαi2 GS/GS mice in vivo showed a slightly higher daytime baseline heart rate during daytime than their littermate controls, suggesting elevated sympathetic tone (19). Other phenotypes, including behavioral hyperactivity and increased heart-to-body weight ratio, also suggested CNS alterations in these mutant mice. Furthermore, bradycardia induced by the α1-AR receptor agonist methoxamine was only moderately enhanced in Gαi2GS/GS mutant mice compared with their littermate controls (7). In contrast, direct activation of the M2R by carbachol showed a dramatic difference between GS/GS and +/+ mice (8), suggesting the possibility of changes in the peripheral vasculature or in baroreflex modulation of heart rate. CNS control or peripheral input through baroreflexes may complicate the interpretation of these prior data. Therefore, in this study, we employed an isolated heart perfusion system, which allows us to dissect cardiac changes from the complex interactions in intact animals.
Unlike humans, the heart rate in rodents at rest is dominated by sympathetic stimulation, which gives an average HR around 600 beats/min in mice. Upon removal and perfusion ex vivo, heart rates drop to ∼300 beats/min, which is determined by the intrinsic activity of pacemakers in the SAN. To examine the cholinergic inhibition of pacemaker activity, we applied carbachol in the absence of Iso, as well as in the presence of Iso, which better mimics the in vivo situation. Our data demonstrate that the Gαi2RGSi mutation significantly enhances sensitivity to carbachol, both under basal conditions and following Iso stimulation. These results are consistent with what we observed previously with carbachol in vivo. In the presence of 50 nM Iso, however, the difference in IC50 for carbachol inhibition between Gαi2RGSi mutants and +/+ mice was less than twofold, which seems unlikely to account for the dramatic difference observed in vivo (8). It might be partially due to the high concentration of Iso used in this study, which activates β-AR more strongly than in vivo and may mask the difference in carbachol inhibition. Nonetheless, 300 nM carbachol still distinguished Gαi2RGSi mutant hearts (both +/GS and GS/GS) from wild type by 65–100 beats/min, which is a physiologically relevant difference. Similarly, once the β-AR activation is eliminated, Gαi2RGSi mutants demonstrated a dramatic increase in sensitivity to carbachol, with the homozygous hearts showing much more pronounced bradycardia. Some transgenic models, such as overexpression of RhoA (32) and angiotensin receptor AT1 (12), have shown abnormalities in AV conduction, but probably by a different mechanism. Redfern et al. (27) used transgenic mice with overexpression of a modified Gαi/o-coupled receptor (Ro1) that can only be activated by a synthetic ligand and demonstrated that increased Gαi/o signaling caused dramatic sinus bradycardia and delayed ventricular conduction, as well as cardiomyopathy on chronic exposure. The authors suggest that chronic activation of the Gαi/o pathway in other models would result in a phenotype similar to that in the Ro1 model. Interestingly, the cardiac phenotype of our Gαi2RGSi model is similar to that in the Ro1 model, in which bradycardia and cardiac hypertrophy have been observed. However, there is no overexpression of mutant protein in Gαi2RGSi mice (8), and enhancement of Gαi2 signaling is probably intermittent and milder compared with their Ro1 model. Furthermore, the mutation in our model is specific for the Gαi2 subunit, which may explain the more limited cardiac phenotype in our model.
In addition, our data are consistent with the classic observation that parasympathetic inhibition is much stronger when sympathetic drive is elevated. Comparing the IC50 values for wild-type hearts for carbachol inhibition in the presence or absence of Iso, approximately sixfold lower values are seen when Iso is present (0.5 vs. 3.3 μM). Interestingly, a smaller difference in the IC50 of Gαi2RGSi mutants and wild type (1.4-fold vs. 4.7-fold) was observed even in presence of Iso. As our laboratory's previous study (8) suggested, enhanced GIRK activation is primarily responsible for the severe carbachol-induced bradycardia in Gαi2RGSi mutant hearts. In the presence of β-adrenergic stimulation by Iso, however, other mechanisms mediated by different members of Gαi family, i.e., Gαo, or G protein-independent mechanisms may lead to differential participation of other ion channels, such as If or ICa,L currents (33), thereby affecting the overall contribution of GIRK enhancement in heart rate inhibition. Taken together, our data clearly show that endogenous RGS proteins potently regulate the pacemaker activity of SAN in heart.
RGS proteins and atrial arrhythmias.
It is well established that expression of and signaling by inhibitory G proteins are altered in some cardiac diseases, such as dilated cardiomyopathy (18, 35), which is often accompanied by different kinds of arrhythmias. Little is known about RGS proteins in cardiac diseases; however, as a modulator of G protein signaling, changes or defects in RGS proteins could well result in similar alterations and may be implicated in disease states. For example, RGS2 knockout mice developed hypertension (16), and a recent study demonstrated that hypertensive patients have a lower RGS2 expression (36). Similarly, we have recently shown that a SNP in RGS2 identified in a Japanese hypertensive cohort leads to reduced RGS2 expression (1). Thus RGS proteins may be new candidate genes in various cardiovascular diseases, where dysfunction of G protein-coupled receptors might be involved.
As an example, the present study demonstrates that loss of RGS function may lead to heart block upon vagal stimulation. In the presence of Iso, strong M2R activation by carbachol leads to complete heart block in vitro, which is much more pronounced in the Gαi2RGSi mutant hearts. Complete heart block may lead to serious complications in patients, such as stroke or syncope, and often requires pacemaker implantation. In the absence of Iso stimulation, the +/GS mutant shows increased second-degree AV block compared with wild type. However, no AV block was observed in GS/GS mutant without Iso. This appears to be explained by the severe bradycardia in GS/GS mutant where the sinus rate is too slow to reveal a block in the AVN. To support our hypothesis, we further evaluated whether AV conduction is slowed in GS/GS mutant using atrial pacing protocols. As we predicted, the Gαi2RGSi mutant showed delayed AV conduction, consistent with increased susceptibility to AV block. Interestingly, this prolonged AV conduction was observed in the absence of exogenous agonist. However, the pacing electrodes may stimulate the release of acetylcholine from the nerve endings or release of other endogenous agonists. Therefore, this AV conduction delay may still be mediated by activation of muscarinic receptors and enhanced Gαi2 activation.
There are a number of mutations in ion channels that have been linked to AV block. For example, a mutation in SCN5A, the gene encoding the cardiac sodium channel, was reported in patients with long QT syndrome and AV block (24, 26). Furthermore, inhibition of ICa,L by a maternal auto-antibody in infants also causes congenital heart block (10). In animal models, infusion of acetylcholine causes AV block in mice, indicating that profound hypervagotonicity can lead to this conduction abnormality (14). This was further confirmed in guinea pigs (6), and, interestingly, GIRK channel blockers can dose-dependently reverse AV block induced by acetylcholine, indicating that GIRK current activation is the major mechanism for AV block associated with abnormal vagal stimulation. Our previous study (8) demonstrated that the enhanced bradycardia in Gαi2RGSi cardiocytes is primarily due to increased GIRK currents upon M2R activation. Therefore, it is plausible to propose that enhanced GIRK activation is largely responsible for the increased occurrence of AV block in response to carbachol and prolonged AV conduction possible in response to acetylcholine. On the other hand, we also found that A1R-mediated bradycardia was not altered in the Gαi2RGSi mutants and minimally involves GIRK activation. In the present isolated heart perfusion system, we were able to confirm that the bradycardic response to R-PIA is comparable among all animals, and, interestingly, no AV block was associated with R-PIA administration, thus supporting the concept that A1R-mediated bradycardia uses other channels as effector. It is important to mention that there is no sinus bradycardia in vivo in Gαi2GS/GS mice under basal conditions, which can be explained by low level of parasympathetic activation at rest. This is opposite to humans, where parasympathetic tone is dominant at rest. Therefore, it is plausible that sinus bradycardia or AV block can be induced by enhanced Gαi2 activation when combined with the dominant parasympathetic tone in humans.
As an important effector for parasympathetic control of heart rate, GIRK currents have also been proposed to be implicated in the development of atrial fibrillation (AF), which affects more than two million Americans. Several lines of evidence have suggested that abnormal activation of GIRK currents is associated with AF. Dobrev et al. (3) showed that GIRK currents are constitutively active in the heart of chronic AF patients. Furthermore, a polymorphism in the Gβ3 subunit that leads to reduced GIRK current has been linked to decreased risk of AF in humans (34). Since the GIRK current is preferentially activated upon M2R activation when actions of RGS proteins on Gαi2 are eliminated, it is conceivable that Gαi2RGSi mutant mice or humans may have increased risk for AF as well.
In summary, our study demonstrates that endogenous RGS proteins are involved in regulation of cardiac automaticity at both the SAN and AVN, and they play an important role in modulating M2R-mediated negative chronotropy. Loss of function in RGS proteins in mouse hearts was also implicated in the development of atrial arrhythmias, such as AV block. Therefore, changes in RGS expression, mutations, or polymorphisms in RGS proteins, as well as in G proteins, may lead to various cardiovascular diseases.
This study was supported by an American Heart Association Predoctoral Fellowship (Y. Fu), and National Institutes of Health Grants NIH R01 GM039561 (R. R. Neubig), RO1 HL69052 (A. N. Lopatin), and T32HL007853 (X. Huang).
We thank Raelene Charbeneau for help managing the mouse colony.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 by the American Physiological Society