Atrial Tachyarrhythmia in Rgs5-Null Mice

Aims The aim of this study was to elucidate the effects of regulator of G-protein signaling 5 (Rgs5), a negative regulator of G protein-mediated signaling, on atrial repolarization and tachyarrhythmia (ATA) in mice. Methods and Results In present study, the incidence of ATA were increased in Rgs5−/− Langendorff-perfused mouse hearts during program electrical stimulation (PES) (46.7%, 7 of 15) and burst pacing (26.7%, 4 of 15) compared with wild-type (WT) mice (PES: 7.1%,1 of 14; burst:7.1%,1 of 14) (P<0.05). And the duration of ATA also shown longer in Rgs5−/− heart than that in WT, 2 out of 15 hearts exhibited sustained ATA (>30 s) but none of them observed in WT mice. Atrial prolonged repolarization was observed in Rgs5−/− hearts including widened P wave in surface ECG recording, increased action potential duration (APD) and atrial effective refractory periods (AERP), all of them showed significant difference with WT mice (P<0.05). At the cellular level, whole-cell patch clamp recorded markedly decreased densities of repolarizing K+ currents including IKur (at +60 mV: 14.0±2.2 pF/pA) and Ito (at +60 mV: 16.7±1.3 pA/pF) in Rgs5−/− atrial cardiomyocytes, compared to those of WT mice (at +60 mV Ito: 20.4±2.0 pA/pF; Ikur: 17.9±2.0 pF/pA) (P<0.05). Conclusion These results suggest that Rgs5 is an important regulator of arrhythmogenesis in the mouse atrium and that the enhanced susceptibility to atrial tachyarrhythmias in Rgs5−/− mice may contribute to abnormalities of atrial repolarization.


Introduction
Atrial tachyarrhythmia (ATA), characterized by abnormal, disorganized and rapid atrial electrical activity, is a major public health problem. In particular, atrial fibrillation, which affects 0.5% of people over the age of 50 and 10% of people over 80 [2], is a significant burden on health care resources and increases the risk of serious complications including embolic disease, heart failure and sudden cardiac death. Despite the development of diverse animal models and the application of a multitude of pharmacological and non-pharmacological therapies, understanding of the mechanism underlying ATA remained incomplete. The recent discovery of DNA variants associated with ATA provides new insights into the mechanisms underlying arrhythmias. Changes in the expression of multiple genes including KCNQ1, KCNE1 and KCNE2 have been shown predisposed to atrial fibrillation [1,4]. Thus, the upstream molecular-regulated targets of pro-arrhythmic substrate need to be better understood.
Rgs5 is a GTPase-activating protein (GAP) for Ga subunits and has an important role in the negative regulation of G-proteincoupled receptor (GPCR)-mediated signaling. Several studies have demonstrated that Rgs5 interacts with Ga(q) and Ga(i) in cardiovascular tissue and inhibits those acting via special GPCRs including AngII type 1 receptor (AT1R) and endothilin-1 receptors [5]. These GPCRs have been implicated in the development of cardiac remodeling during AF, and induced left atrial remodeling and fibrosis are dependent on the extracellular signal-regulated kinase (ERK) pathway [7]. Our previous work showed that Rgs5 2/2 mice promoted cardiac hypertrophy and fibrosis due to the activation of the MEK-ERK1/2 signaling pathway [15]. As an important regulator of atrial GPCRdependent signaling, RGS5 protein may potentially exhibit negative way in pro-arrhythmic substrate.
We report here that mice with a knockout of Rgs5 displayed susceptibility of ATA by electrical stimuli and had associated repolarization abnormalities. These observations may provide a new insight into understanding the molecular pathways underlying atrial arrhythmogenesis and for developing strategies for the treatment of atrial tachyarrhythmia.

Mouse experiments
All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and were approved by the Institutional Animal Care and Use Committee at Renmin Hospital of Wuhan University, China.
The generation and genotyping of Rgs5 2/2 mice (C57BL/6 background) have been described previously. Mice were provided with food and water and held on standard 12 hours light and dark cycles in temperature and humidity controlled house. Male Wildtype and Rgs5 2/2 mice aged 8 to 10 weeks were used in the studies.

ECG recordings
Telemetry ECG (Lead II) was continuously recorded in consciously moving mice by Telemetry ECG (DSI, US). The Pwave duration and amplitude (P dur and P amp ), PR interval and QRS duration was measured.

Echocardiography and histological analysis
Echocardiography was performed to assess left atrial diameter (LAD), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), ejection fraction (EF) and fractional shortening (FS). For histological analysis, hearts were excised and cut transversely close to the apex to visualize the left and right ventricles and atrial appendages. Sections of heart (4-5 mm thick) were prepared and stained with Picro Sirius Red (PSR) for collagen deposition [15].

Preparation of Langendorff-perfused hearts
The isolated heart was perfused with HEPES-buffered Tyrode's solution. Perfusion was commenced in a retrograde manner through the aorta at 2-2.5 ml/min. The hearts that did not recover to the regular spontaneous rhythm or had inreversible myocardial ischemia were discarded.

MAP and BEG recording
To examine the atrial electrical activity, the bipolar electrogram (BEG) was recorded from the epicardial surface of atrial appendage using a silver chloride (2 mm tip diameter) recording electrode. The paired platinum stimulating electrodes paced the epicardial surface of right atrial appendage and the stimulation used a 1 ms square-wave stimuli at three times excitation threshold.

Electrical stimulated protocol
The Programmed electrical stimulation (PES) protocol was used for atrial effective refractory period (AERP) and atrial-ventricular effective refractory period (AVERP) examinations. Sinus node recovery time (SNRT) was measured after a 2 s pacing train at a cycle length of 100 ms [16]. Inducibility of atrial tachyarrhythmia was tested by using both PES and burst pacing (2 ms pulses at 50 Hz, 2 s burst duration) [24], burst pacing used up to 3 minutes of pacing in both atrial locations.

Transmembrane action potential (TAP) recording
The samples of atrial myocardium were removed from heart and superfused with HEPES-buffered Tyrode's solution. TAP were recorded with borosilicate glass microelectrodes during regular pacing frequency of 1 Hz, 2 Hz, 3.3 Hz, 5 Hz and 6.7 Hz.

Isolation of atrial cardiac myocytes
Atrial myocytes were isolated by four steps perfused digestion. At the end of the perfusion, the whole ventricle was dissected from the heart, and the atria were placed in ice-cold KB solution. The temperature of the perfusion was maintained at 37uC. Isolated cardiac myocytes were stored in KB solution at 4uC until needed [28].

Cellular electrophysiology recording
All experiments were carried out at room temperature (20-22uC). The whole cell clamp recording protocols, data acquisition and analysis were described in Methods S1.

Real-time PCR analysis
RNA levels of the Tgfb1, Col1a1 and Col3a1 were determined by real-time PCR in Wild-type and Rgs5 2/2 atria. The mRNA expression of Rgs5 2/2 hearts was quantified relative to wild-type.

Statistical analysis
All data are expressed as mean 6 SEM. Statistical analysis performed with a Fisher exact test and Student's t test were completed by SPSS 16.0. A value of P,0.05 was considered significant. Patch-clamp data were analyzed using Origin 6.0 (Microcal Co. USA) for nonlinear curve fitting.

ECG recording and echocardiography determination
To analyze whether Rgs5 deficiency results in atrial electrical abnormalities, telemetry ECG were performed. The spontaneous atrial arrhythmias were not observed during 24 h continuous recording, and there was no difference between Rgs5 2/2 and wild-type mice in heart rate (HR) (P.0.05). However, the P wave usually showed widened in lead II of Rgs5 2/2 mice, and its duration (P dur ) was significantly different to that in wild-type mice (11.362.3 ms vs 9.462.4 ms, P,0.05) ( Table 1 and Fig. 1). Echocardiography was applied to confirm whether this prolonged P wave is associated with atrial chamber dilation. The left atrial diameter was similar between WT and Rgs5 2/2 mice (1.4360.03 mm vs 1.4060.08 mm, P.0.05). In addition, Rgs5 2/2 mice did not exhibit ventricular dysfunction by determination of left ventricular ejection fraction (LVEF) and fractional shortening (FS) ( Table 2).

Rgs5 2/2 mice have increased the duration of atrial repolarization
Monophasic action potentials (MAP) were obtained from isolated hearts with amplitudes ranging from 4.8 to 15.1 mV; the mean values did not significantly differ between the two groups. MAP duration (APD) from 30% to 90% was longer in Rgs5 2/2 than WT mice during regular atrial pacing (CL = 125 ms) (Fig. 1). Similarly, PES revealed longer AERP in  1 and Table 3). Transmembrane action potential (TAP) from Rgs5 2/2 and WT isolated sample of atrium was recorded at different paced CL due to very little spontaneous automaticity. Consistent with MAP findings, TAP recordings at Rgs5 2/2 samples showed increased APD 90 compared to WT group over all paced frequency from 1 Hz to 6.7 Hz. After-depolarization such as EAD was occurred frequently at 1 Hz and 2 Hz in Rgs5 2/2 group. Moreover, EAD developed in 6 of 11 atrial preparations (54.5%), but none of 10 WT preparations (P,0.05) (Fig. 2).     (Table 3 and Fig. 3). In addition, AVERP was recorded in each S1 paced CL, but there were no significant differences between Rgs5 2/2 and wild-type mice. However, the wenckebach cycle length (WCL) was significantly longer in Rgs5 2/2 mice than in wild-type mice (87.1620.6 ms vs 66.8611.9 ms, P,0.01) by decremental atrial pacing (Table 3).

Rgs5 2/2 heart increased the incidence and duration of atrial tachyarrhythmia
Atrial PES and burst pacing were applied to assess the arrhythmic tendency in Rgs5 2/2 (n = 15) and wild-type (n = 14) hearts. Recordings were obtained during PES at a stimulation frequency from 5 Hz to 10 Hz. The S 2 stimulus was continued until atrial refractoriness or arrhythmia occurred. A burst pacing procedure then applied high-frequency trains of S 1 stimuli to the right atrium. Each heart reverted to sinus rhythm after arrhythmia. The Rgs5 2/2 group showed a higher overall incidence of atrial tachyarrhythmia than the WT group through the procedures (P,0.05) (Fig. 4C).
The individual episodes of atrial arrhythmia were sorted by duration within the ranges ,10 s, 10-30 s, and .30 s. Most ATA lasted ,10 s in both groups, but the incidence of ,10 s ATA was significantly higher in the Rgs5 2/2 group than in the WT (P,0.05). Sustained ATA (.30 s) was induced in one Rgs5 2/2 heart in both stimulation procedures. In contrast, arrhythmia lasting .30 s was not recorded in WT hearts during either PES or burst pacing (Fig. 4D,E and Table 4).  The results revealed that the density of outward K + current was markedly changed in Rgs5 2/2 cardiomyocytes at room temperature (22uC). The I-V curves of total outward current (I Peak ) showed the current density at +60 mV was decreased in Rgs5 2/2 atrial cells compared to wild-type cells (53.565.4 pA/pF vs 40.164.0 pA/pF, P,0.05) (Fig. 5A). The  (Fig. 5C,E). In addition, the I-V curves of inwardly rectified K + current (I K1 ) showed similar densities at 2120 mV in Rgs5 2/2 and wild-type cardiomyocytes (216.662.5 pA/pF vs 218.662.1 pA/pF, P.0.05) (Fig. 5G). To determine whether this results are also convinced when data are obtained at more physiological temperatures, we performed the measurement at 37uC. As expected, the mean maximal density of I peak , I to and I Kur Figure 7. Channel kinetics of I to and I Kur were analyzed in Rgs5 2/2 and WT cardiomyocytes, respectively. The steady-state inactivation (A) and reactivation (B) of curves in I to plotted similar tendency between Rgs5 2/2 and wild-type atrial myocytes. However, the property of I Kur in Rgs5 2/2 cardiomyocytes showed markedly modification with significant difference of half-inactivated voltage (V 1/2 ) (C) and reactivated time constant (t) (D). Histogram summarized the statistical significance of V 1/2 and t in WT and Rgs5 2/2 groups (E, F). doi:10.1371/journal.pone.0046856.g007 Rgs5 2/2 Predispose to Atrial Arrhythmia PLOS ONE | www.plosone.org was significantly larger at 37uC than at room temperature (P,0.05) (Fig. 6). The difference of density between Rgs5 2/2 and WT also revealed significantly at 37uC in I peak , (at +60 mV: 44.364.2 pA/pF vs 59.264.7 pA/pF, P,0.05), I to (at +60 mV: 20.462.0 pA/pF vs 23.262.5 pA/pF, P,0.05) and I Kur (at +60 mV: 17.161.5 pF/pA vs 19.261.9 pF/pA, P,0.05).
To analyze the kinetics of I to and I kur , the properties of inactivation and recovery were assayed in each group (Fig. 7). For the voltage dependence of steady-state inactivation of I to , the halfinactivated voltage (V 1/2 ) and slope factor (k) showed no significant difference between Rgs5 2/2 and wild-type cells (V 1/2 : 263.161.0 mV vs 262.261.0 mV, P = 0.13; k: 13.060.8 mV vs 13.260.7 mV, P P.0.05). However, the recovery from inactivation of I to was significantly slower in Rgs5 2/2 myocytes than in the wild type (time constant: 107.5613.6 ms vs 95.1613.2 ms, P.0.05). For I kur , the V 1/2 (251.860.9 mV vs 255.860.9 mV, P,0.01) and the recovery time (356.9620.6 ms vs 577.0638.3 ms, P,0.01) were significantly different between WT and Rgs5 2/2 cardiomyocytes.

Main findings
The present study provided an important original insight into the atrial electrical remodeling of Rgs5 2/2 mice, As the consequence, we found that (i) Rgs5 2/2 significantly prolonged atrial repolarization associated with attenuated K + currents; (ii) the increased incidence and duration of atrial arrhythmia in Rgs5 2/2 mice may related to the prolonged repolarization.

Rgs5 and atrial repolarization
G proteins are key mediators of cardiac signal transduction, particularly following activation of G protein coupled receptor (GPCR). G proteins include a (Ga) and bc (Gbc) subunits. Different types of Ga can be activated by special receptors [18]. For example, Gai can be activated by muscarinic cholinergic receptors, Gas by b-adrenergic receptors, and Gaq by angiotensin II type 1 receptor (AT1R) and endothelin-1 receptor A (ET A ). RGS5, a member of the regulator of G-protein signaling protein super-family, acts as a GTPase-activating factor for a number of Gai-and Gaq-mediated pathways and as a negative regulator of GPCR signaling [5]. Our previous study demonstrated that Rgs5 transgenic mice were resistant to cardiac hypertrophy and fibrosis through inhibition of MEK-ERK1/2 signaling, whereas Rgs5 2/2 mice displayed the opposite phenotype in response to pressure overload [15]. Knockdown of RGS5 results in the selective upregulation of AT1R-mediated activation of ERK1/2 [17]. Previous studies have demonstrated a relationship between AT1R and K + ion in several tissues. AngII can inhibit several K + currents, including K v , K ATP , and B Kca , and its effects on K + currents can be reversed by blocking AT1R [9,11,23,[25][26]. Importantly, AT1R over-expressing mouse hearts had delayed repolarization and reduced density of several repolarizing K + currents, together with an increased incidence of spontaneous arrhythmia [14]. Thus, as a negative mediator of GPCR signaling pathways, Rgs5 may directly regulate atrial repolarization through K + currents.
As the primary repolarizing component in mouse heart, the voltage dependent K + current exhibits more powerful than other mammals [8]. Decreased K + currents may seriously prolong repolarizing phase. In present study, we observed the atrial action potential duration was prolonged in Rgs5 2/2 mice, which associated with greatly decreased K + currents including I Peak , I to and I Kur , but the inwardly rectified K + current (I K1 ) did not showed significant difference between Rgs5 2/2 and WT group. Rivard et al. demonstrated that prolongation of APD in AT1Roverexpressing mouse hearts was due to decreased I to and I Kur densities and alteration of their reactivation kinetics by chronic stimulation of the AT1R signaling pathway. Notably, these alterations preceded anatomic disturbances and were not a consequence of cardiac remodeling [20]. However, the reduced density of the I K1 current is more common in mouse heart with cardiac dysfunction and increased myocytes size [3]. In present study, Rgs5 2/2 heart did not show increased atrial size and cellular membrane capacitance compared to WT mice, and the function of Rgs5 2/2 heart was normal. Based on these observations, it has been concluded that reductions of I K1 are closely related to cardiomyocyte hypertrophy and may not be directly due to the increased stimulation of AT1R. Consistent with these notions, the markedly decreased densities of I Kur and I to in Rgs5 2/ 2 atria possibly contributed to the effect on the AT1R/Gq signaling pathway.

Rgs5 and atrial tachyarrhythmia
Toumi et al reported that Rgs2, another member of the RGS protein super-family, is expressed at high levels in mouse atrium and plays a critical role in atrial arrhythmias. Rgs2 2/2 mice were more susceptible to PES-induced atrial tachycardia and fibrillation (AT/AF), and a higher percentage of them were accompanied by heterogeneities in refractory periods and regional conduction block. This phenotype was likely due to the reentry mechanism [27]. Atrial tachyarrhythmia can be initialized by rapidly firing ectopic foci (e.g., from the pulmonary veins) and maintained by functional reentrant circuit (mother rotor) [12,19]. As a major risk factor for atrial arrhythmogenesis, prolonged repolarization forms the substrate for ectopic stimulation, which is generated from pulmonary and caval vein, cardiac ganglion or other atrial special tissues. Several studies have examined predisposition to atrial arrhythmia in mouse models of congenital long QT syndrome (LQT). Dautova et al. showed that in mice with a DKPQ knock-in LQT3 mutation, Langendorff-perfused hearts are more likely to develop atrial arrhythmia [13]. Lemoine et al. demonstrated that atrial arrhythmias induced by rapid rate alteration occurred significantly more often in the LQT3 mouse model, suggestive of after-depolarization-dependent arrhythmogenic mechanisms [21]. In present study, although we did not observe the spontaneous atrial arrhythmia during 24 h-ECG recording, the trigger activity including EAD was observed in Rgs5 2/2 atrial tissue, and the electrical stimulation (PES and burst pacing), which simulate a physiological ectopic excitation and is thought to more readily induce reentry than triggered activity [27], induced atrial arrhythmias were facilitated in Rgs5 2/2 heart. It suggested that Rgs5 2/2 induced prolonged repolarizaiton provided a potential substrate and the ectopic activity facilitated this effect for arrhythmogenesis in atria.
As the substrate for atrial arrhythmogenesis, interstitial fibrosis and atrial enlargement have been implicated in theoretical models. In present study, we did not observed significant sign of atrial enlarged or fibrosis. However, the prolonged P waves were recorded in Rgs5 2/2 conscious mice. As a sign of atrial enlargement, prolonged P wave was also seemed to be related to regional atrial conduction disturbances, which have been suggested to favour the development of AF [29]. Thus, the result suggested that the Rgs5 2/2 induced ATA was mainly dependent on the atrial electrophysiological modifications.
In addition, SNRT was measured to assess the function of the sinus node, which maintains the normal sinus rate. Several RGS mRNAs are expressed at high levels in the sinus node and atrioventricular nodal region and regulate parasympathetic tone of pacemaker myocytes. Rgs4 and Rgs6 are highly expressed in the sinus node, and hearts from mice deficient in these genes showed marked bradycardia with a sustained lower HR and prolonged RR intervals [6,10,22]. Alterations in RGS protein expression or activity could potentially contribute to diseases such as sick sinus syndrome. The Rgs5 2/2 hearts, which have significantly longer SNRT and cSNRT, had abnormal sinus function and thus promoted the maintenance of atrial ectopic rhythm.

Conclusion
In conclusion, the present study confirmed the relationship between the Rgs5 and atrial arrhythmogenesis, the Rgs5 2/2 facilitated ATA was due to the prolonged repolarization, and this electrophysiological alteration could be accounted for attenuated repolarizing K + currents.

Supporting Information
Methods S1 Supplemental Methods.