doi: 10.1161/CIRCRESAHA.108.183384
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© 2008 American Heart Association, Inc.
Editorials |
And the Winner Is ... RGS4!
From the Departments of Pharmacology and Internal Medicine, University of Michigan Medical School, Ann Arbor.
Correspondence to Richard R. Neubig, MD, PhD, Department of Pharmacology, University of Michigan Medical School, 1150 W Medical Center Dr, 1303 MSRB III, Ann Arbor, MI 48109. E-mail RNeubig{at}umich.edu
See related article, pages 527–535
Key Words: arrhythmia • automaticity • mouse mutants • potassium channels • G proteins
RGS proteins play a major role in controlling G protein signaling. Cifelli et al1 show very high expression of RGS4 in sinoatrial (SA) node in the mouse. Of the 10 RGS proteins expressed supraventricularly, genetic disruption of RGS4 alone produces a profound slowing of IK,Ach turnoff kinetics in SA node and enhanced carbachol-induced bradycardia in vivo. This is accompanied by a loss of rapid desensitization of the IK,Ach current, suggesting a selective action of RGS4 in controlling prolonged vagal responses. This has implications for clinical conditions dependent on cholinergic signals such as atrial fibrillation (AF), vasovagal syncope, and possibly torsades de point. These results make RGS4 an intriguing candidate gene in certain arrhythmias.
Approximately 12 years ago, the G protein and G protein–coupled receptor fields were rocked by the discovery of the RGS (regulators of G protein signaling) proteins.2,3 Here was a key regulatory protein family that inhibited G protein signaling but was virtually unknown a year earlier. Very rapidly, the core molecular function of RGS proteins was defined as GTPase accelerator protein activity, which sped the deactivating GTP hydrolysis step,4 which, for G proteins, was notoriously slow. The crystal structure of a complex of G
i1 and RGS4 "caught in the act" of GTP hydrolysis5 followed shortly thereafter.
Cardiac atrial function figured prominently in early studies of RGS proteins. The negative chronotropic actions of acetylcholine via the M2 muscarinic receptor involved activation of the IK,Ach current, which is carried by the G protein–coupled inwardly rectifying K+ (GIRK) channel, composed of a heterotetramer of subunits Kir3.1/Kir3.4. GIRK channels are activated by Gβ
subunits released from Gi/o family proteins. Vagal control of heart rate occurs on a very rapid times scale, with slowing and recovery within single cardiac cycles. This very rapid time course was inconsistent with the slow biochemical GTPase activity of Gi subunits. In a landmark report in 1997, Doupnik et al6 showed that the onset and recovery of IK,Ach currents in atrial myocytes was very fast (t
, <1 second), whereas M2 receptor control of expressed GIRK channels in CHO cells was much slower (t for turnoff, 
10 seconds). Coexpression of RGS4 reconstituted the fast, physiological on-and-off kinetics of IK,Ach in the CHO cells, suggesting that an RGS protein was necessary for the normal control of atrial GIRK channel function. Although RGS4 was used in that study, 10 different RGS proteins are expressed in atrial myocytes,7,8 and many of them can control IK,Ach kinetics.6 Showing that this regulatory mechanism was relevant in vivo, mice with a mutant Gi2 protein that is insensitive to RGS–GTPase accelerator protein activity had a marked enhancement of muscarinic agonist-mediated bradycardia9 and slowing of conduction through the atrioventricular (AV) node.10 These studies left open the question of which of the 10 atrial RGS proteins was responsible for control of acetylcholine responses in vivo.
The study by Cifelli et al1 in this issue of Circulation Research provides an answer to that question. RGS4, the original choice of Doupnik et al6 for their in vitro study, appears to play the major role in the control of chronotropic actions of Ach. A knockout mouse expressing lacZ from the RGS4 promoter has very high expression in the SA node relative to other cardiac tissues.1 This was confirmed by RT-PCR. Physiologically, the RGS4–/– mice had markedly enhanced sensitivity to carbachol-mediated bradycardia despite normal resting heart rates. However, anesthetized RGS4–/– mice showed a significantly lower heart rate, which was reversed by atropine, indicating increased vagal tone. Similar enhancements of muscarinic signaling were also shown in isolated perfused hearts and in single SA node cells. The latter showed lower rates of spontaneous action potentials and a dramatically increased sensitivity to carbachol-induced arrest.
GIRK currents appear to contribute to these effects. The maximal negative diastolic potential in the presence of carbachol was increased in the knockout SA node cells, and the kinetics of IK,Ach were dramatically slowed, reminiscent of the earlier studies in expression systems.6,11 In a result that also replicates in vitro findings, the initial peak IK,Ach current is not decreased in the RGS4–/– SA node cells. This paradox of a markedly faster channel deactivation with little change in current amplitude is only partially explained by an increase in channel activation rate.
Perhaps the most intriguing result is the prominent effect on rapid channel desensitization in the RGS4–/–. After the initial peak in GIRK current activation, there is a rapid (ca. 10–20 seconds) decrease in current. There are several potential mechanisms for rapid desensitization including RGS proteins.12 The effect seen by Cifelli et al1 is quite striking at the lowest concentration of carbachol (Figure). There was a 40% decrease in IK,Ach over 2 minutes in the wild-type SA node cells but only a 7% decrease in the RGS4–/– SA node cells. This may have important implications for the pathophysiology of vagal actions. Under normal conditions, it is likely that vagal efferent activity occurs in short bursts (the high-frequency oscillations in heart rate that are attributed to parasympathetic actions occur on time frames faster than one every 6 seconds). Thus, the effect of RGS4 might not have much influence on these normal bursts of vagal activity given that there was no effect on the initial peak GIRK current. On the other hand, prolonged, pathological vagal signals might be enhanced dramatically in an RGS4–/– mouse (or a human with impaired RGS activity). The normal desensitization that occurs over a minute or two at low physiological acetylcholine concentrations would not occur to ameliorate the profound bradycardia induced by prolonged vagal output. This could lead to increased incidences of sinus bradycardia or arrest and potentially AF. It would be of interest to know if RGS4 is expressed at high levels in the AV node as well because both the RGS4–/– mouse and the mouse with an RGS-insensitive mutant Gi2 protein10 showed significant AV conduction delays and/or AV block.
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Given the substantial effects of the RGS4 knockout on muscarinic signaling in the mouse heart, it is clearly of interest whether the same will be true in humans. The results of Cifelli et al1 suggest that RGS4 should be considered as a high priority candidate gene for disorders that may depend on vagal tone. Most animal models of AF depend absolutely on a cholinergic stimulus to produce sustained AF.13,14 Similarly, human AF has been attributed to imbalances of autonomic activity with a significant number of AF patients identified as "vagotonic" AF.15,16 Similarly, a canine model of torsades de point also exhibited a cholinergic dependence.17 Certainly, it will be interesting to see whether the RGS4–/– mice are more susceptible to the induction of AF, which would be a counterpoint to the reduced AF seen in the Kir3.4 (GIRK4) knockout mouse.18
Is RGS4 the only RGS that modulates atrial function? That cannot be stated with certainty at this point. The effect of the RGS4 knockout on carbachol-induced bradycardia observed by Cifelli et al1 is quite similar in magnitude to the enhancement seen in mice with a Gi2 RGS-insensitive mutation.9 This suggests that RGS4 is the predominant RGS in the SA node for control of Gi2 effects on GIRK currents. The evaluation of other mouse RGS knockouts will be of interest. A recent study on RGS10 in rat atrial myocytes19 suggests that there may be a role for RGS10 as well. RGS10 is suggested to mediate a cross-talk between β-adrenergic and muscarinic effects. This is striking in light of the proposed role for combined sympathetic and parasympathetic effects in initiation of AF. In that study, however, short hairpin RNA knockdown of RGS10 did not slow the IK,Ach deactivation as seen with the RGS4–/– mice. So for now, it appears that RGS4 plays the starring role.
As with RGS2 and the follow-up mouse studies suggesting its role as a candidate gene for hypertension,20–23 there are likely to be numerous follow-up studies looking for mutations in RGS4 as a candidate gene in syndromes with a link to vagal or parasympathetic activity, such as vasovagal syncope, AF, and perhaps torsades de point. Furthermore, the novel differences seen here related to the role of RGS4 in immediate responses versus IK,Ach desensitization deserves further exploration in vivo. Does the RGS4–/– mouse show altered heart rate variability? Does it have differential effects on brief versus sustained vagal activation? As with most good studies, this work raises nearly as many questions as it answers.
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Acknowledgments |
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Sources of Funding
Supported by NIH grants R01-GM39561 and R01-DA23252.
Disclosures
None.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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Related Article:
Circ. Res. 2008 103: 527-535.
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