Shelby A. Dahlen,Tyler F. Bernadyn,Alethia J. Dixon,Bo Sun,Jingsheng Xia,Elizabeth A. Owens,Patrick Osei-Owusu
Dual loss of regulator of G protein signaling 2 and 5 exacerbates ventricular myocyte arrhythmias and disrupts the fine-tuning of Gi/o signaling
RGS proteins,G protein signaling,Cardiac excitation-contraction coupling,Arrhythmia,Cardiomyopathy,cAMP,Catecholamine
Aims: Cardiac contractility, essential to maintaining proper cardiac output and circulation, is regulated by G protein-coupled receptor (GPCR) signaling. Previously, the absence of regulator of G protein signaling (RGS) 2 and 5, separately, was shown to cause G protein dysregulation, contributing to modest blood pressure elevation and exaggerated cardiac hypertrophic response to pressure-overload. Whether RGS2 and 5 redundantly control G protein signaling to maintain cardiovascular homeostasis is unknown. Here we examined how the dual absence of RGS2 and 5 (Rgs2/5 dbKO) affects blood pressure and cardiac structure and function. Methods and results: We found that Rgs2/5 dbKO mice showed left ventricular dilatation at baseline by echocardiography. Cardiac contractile response to dobutamine stress test was sex-dependently reduced in male Rgs2/5 dbKO relative to WT mice. When subjected to surgery-induced stress, male Rgs2/5 dbKO mice had 75% mortality within 72–96 h after surgery, accompanied by elevated baseline blood pressure and decreased cardiac contractile function. At the cellular level, cardiomyocytes (CM) from Rgs2/5 dbKO mice showed augmented Ca2+ transients and increased incidence of arrhythmia without augmented contractile response to electrical field stimulation (EFS) and activation of β-adrenergic receptors (βAR) with isoproterenol. Dual loss of Rgs2 and 5 suppressed forskolin-induced cAMP production, which was restored by Gi/o inactivation with pertussis toxin that also reduced arrhythmogenesis during EFS or βAR stimulation. Cardiomyocyte NCX and PMCA mRNA expression was unaffected in Rgs2/5 dbKO male mice. However, there was an exaggerated elevation of EFS-induced cytoplasmic Ca2+ in the presence of SERCA blockade with thapsigargin. Conclusions: We conclude that RGS2 and 5 promote normal ventricular rhythm by coordinating their regulatory activity towards Gi/o signaling and facilitating cardiomyocyte calcium handling.
Dual loss of regulator of G protein signaling 2 and 5 exacerbates ventricular myocyte arrhythmias and disrupts the fine-tuning of Gi/o signaling

Cardiac contractility, essential to maintaining proper cardiac output and circulation, is regulated by G protein-coupled receptor (GPCR) signaling. Previously, the absence of regulator of G protein signaling (RGS) 2 and 5, separately, was shown to cause G protein dysregulation, contributing to modest blood pressure elevation and exaggerated cardiac hypertrophic response to pressure-overload. Whether RGS2 and 5 redundantly control G protein signaling to maintain cardiovascular homeostasis is unknown. Here we examined how the dual absence of RGS2 and 5 (Rgs2/5 dbKO) affects blood pressure and cardiac structure and function.

We found that Rgs2/5 dbKO mice showed left ventricular dilatation at baseline by echocardiography. Cardiac contractile response to dobutamine stress test was sex-dependently reduced in male Rgs2/5 dbKO relative to WT mice. When subjected to surgery-induced stress, male Rgs2/5 dbKO mice had 75% mortality within 72–96 h after surgery, accompanied by elevated baseline blood pressure and decreased cardiac contractile function. At the cellular level, cardiomyocytes (CM) from Rgs2/5 dbKO mice showed augmented Ca2+ transients and increased incidence of arrhythmia without augmented contractile response to electrical field stimulation (EFS) and activation of β-adrenergic receptors (βAR) with isoproterenol. Dual loss of Rgs2 and 5 suppressed forskolin-induced cAMP production, which was restored by Gi/o inactivation with pertussis toxin that also reduced arrhythmogenesis during EFS or βAR stimulation. Cardiomyocyte NCX and PMCA mRNA expression was unaffected in Rgs2/5 dbKO male mice. However, there was an exaggerated elevation of EFS-induced cytoplasmic Ca2+ in the presence of SERCA blockade with thapsigargin.

We conclude that RGS2 and 5 promote normal ventricular rhythm by coordinating their regulatory activity towards Gi/o signaling and facilitating cardiomyocyte calcium handling.

Signaling downstream of heterotrimeric G proteins mediates the effects of several endogenous stimuli and G protein-coupled receptor (GPCR) agonists, including catecholamines, on cardiac structure and function. Aberrant G protein signaling is implicated in the pathogenesis of cardiac disorders including maladaptive hypertrophy leading to heart failure, and arrhythmias. Accordingly, pharmacological targeting of cardiac GPCR and their effectors is an established therapeutic approach to managing various cardiac disorders including ventricular arrhythmias and heart failure [1]. G protein signaling is fine-tuned by regulator of G protein signaling (RGS) proteins [2]. RGS proteins are GTPase activating proteins (GAPs) that accelerate the hydrolysis of GTP to GDP by the intrinsic GTPase activity of the Gα subunit. In so doing, RGS proteins facilitate the termination of signaling by regulating the magnitude and duration of G protein activity [3]. RGS proteins are widely expressed in vertebrates. In the heart, several members of the B/R4 family, including RGS1–5, 8, 13, 16, and 18 are expressed, at least at the mRNA level [4,5]. RGS proteins of the B/R4 family act as GAPs towards Gq/11 and Gi/o class G proteins. Among this family of RGS proteins, RGS2 and RGS5 are most noted for regulating β2-adrenergic receptor (β2AR)-mediated Gαi/o signaling within the myocardium [6-8]. In cardiomyocytes, the overexpression of RGS2 suppresses agonist-induced, β2AR-mediated Gαi signaling, a pathway implicated in ventricular arrhythmia, heart failure, and hypertension [7]. Similar to RGS2, RGS5 has also been shown to display relevance in proper cardiac function, specifically in the repolarization phase of cardiac contraction [9,10], hypertrophy and fibrosis [11,12], and maintenance of blood pressure homeostasis [13]. Thus, RGS2 and 5 seem to play overlapping roles in the cardiovascular system. However, it is unclear whether RGS2 and 5 redundantly control G protein signaling to maintain homeostasis in the cardiovascular system. To fill this knowledge gap, we generated mice dually lacking RGS2 and 5 (Rgs2/5 dbKO). In this study, we determined whether RGS2 and RGS5 show any compensatory or synergistic relationship in their regulation of cardiac structure and function. We report that Rgs2/5 dbKO mice develop spontaneous left ventricular dilatation in the absence of an experimental stressor. In addition, Rgs2/5 dbKO male mice are less responsive to βAR stimulation but are highly sensitive and succumb to surgery-related stress. Furthermore, the dual absence of RGS2 and 5 increases the susceptibility of ventricular myocytes to arrhythmias following βAR stimulation or pacing by electrical field stimulation, by mechanisms involving increased Gαi/o activity and impaired cytoplasmic calcium handling.

The generation of Rgs2 and Rgs5 null mice (Rgs2−/− or Rgs2 KO and Rgs5−/− orRgs5 KO) has been described previously [14,15]. The Rgs2 KO mouse model was obtained from Dr. Kendall Blumer at Washington University, with permission from Dr. Josef Penninger, while Rgs5 KO mice were obtained from Dr. John Kehrl at the NIH-NIAID. Both mouse lines have been backcrossed extensively into the C57BL/6 genetic background [15,16]. We performed Rgs2−/− x Rgs5−/− crosses to generate Rgs2−/−,Rgs5+/−; Rgs2+/− ,Rgs5−/−; and Rgs2+/−,Rgs5+/− mouse lines. We then crossed Rgs2+/−,Rgs5+/− mice to generate mice dually null for Rgs2 and 5 (Rgs2/5 dbKO). The Institutional Animal Care and Use Committees at Drexel University and Case Western Reserve University approved the protocols for all animal experiments performed in this study, in accordance with the U.S. animal welfare act. We used 2- to 5-month-old male and female mice in all experiments involving adult animals. The mice were provided access to food and water ad libitum in our institution’s animal facility at 22 °C and a 12-h light/dark cycle.

Blood pressure, heart rate, and cardiac electrocardiogram (ECG) were measured in conscious wild type (WT) and Rgs2/5 dbKO male and female mice using radiotelemetry, as we have previously described [17]. For conscious blood pressure measurement, HD-X10 radiotelemetry catheters and transmitters were implanted, as we have previously described [18]. Using Ponemah data acquisition system from Data Science International, recording of diurnal blood pressure and heart rate of conscious mice was initiated on day zero, i.e., immediately after the mice recovered from isoflurane anesthesia for the implantation of the radiotelemetry catheter. The acquired data were reduced to averages of continuous systolic blood pressure (SBP), diastolic pressure (DBP), mean blood pressure (MBP), and heart rate recordings in one-hour intervals over 24 h for 10 days, or until the mice died. For ECG monitoring, we used HD-X11 radiotelemeter implants. The mice were allowed to recover for at least one week from the transmitter implantation surgery prior to initiating recordings.

All imaging studies were performed using Vevo 2100 cardiac echocardiography system equipped with a 400-MHz linear transducer (VisualSonics Inc., Toronto, Canada). Mice were lightly anesthetized with isoflurane (1.5%) and placed supine on a heated pad equipped with ECG electrodes for continuous monitoring throughout the imaging procedure. After the chest hair was removed with hair removal cream, two-dimensional parasternal long- and short-axis views at the midventricular level, as well as 1-dimensional targeted M-mode tracings throughout the anterior and posterior LV walls were recorded, as previously described [19,20]. The reported data are mean values obtained from at least 6 mice of each group and analyzed for statistical significance by ANOVA or Student’s t-test, where appropriate.

Adult male and female mice were secured on a heated pad for echocardiography, as described above. A working solution of dobutamine hydrochloride (DOB, 10 μg/μl) was prepared from DOB stock (1 g/ml in DMSO) in 1:100 dilution in normal saline. After baseline recordings, the mice received intraperitoneal infusion of DOB (5 μg/kg/ min) for 1 min, using an infusion pump, as previously described [21]. Changes in left ventricular M-mode fractional shortening, in response to the infusion, were calculated offline.

We examined cardiac hemodynamics in isoflurane-anesthetized mice using closed-chest pressure-volume loop approach, as previously described [22]. Briefly, a 1.4F pressure-volume (PV) catheter (SPR 839; Millar Instruments, Inc.) was inserted into the right carotid artery and advanced past the aortic valve to lie longitudinally in the left ventricle. The mice were allowed to stabilize for 5 min while the isoflurane anesthetic was lowered from 3 to 1.5%. Data were acquired at baseline and during momentary occlusion of the inferior vena cava by compression of the abdomen with a dry cotton-tipped applicator. Shortly after the occlusion, a PE-10 catheter was placed in the right jugular vein for saline injection. Using a 1/3 cc insulin syringe, 10 μl of 20% saline was injected via the jugular vein for catheter calibration. At the end of the hemodynamics recording, blood was drawn for absolute volume calibration, according to the manufacturer’s protocol. Throughout the experiment, data were acquired at 1KHz and analyzed using Labchart 8.0 software (ADInstruments). We used 10–15 PV loops to calculate stroke work, cardiac output, stroke volume, arterial elastance, and relaxation time constant. End-systolic PV relation (a.ka. end-systolic elastance) was calculated from the slope of PV loops acquired during momentary vena cava occlusion.

This test employed only male mice. After the acquisition of baseline blood pressure (BP), heart rate, and ECG for 30 min, the mice received a bolus injection of ISO (0.75 μg/g, i.p.) and returned to their home cages, with continuous ECG and BP monitoring for 60 min. A day after the acute ISO injection, and following 30 min of baseline recordings, the mice were placed in 4-l beaker containing 1.2 l of pre-warmed water to initiate a 90-min forced swimming, with continuous recording of hemodynamics and ECG. An extra 30 min of data acquisition was obtained when the mice were returned to their home cages at the end of the forced-swim test.

Total RNA was extracted from heart apexes of male and female WT and Rgs2/5 dbKO mice via a Trizol extraction method (ThermoFisher Scientific) and utilizing an Omni Bead Ruptor 24 homogenizer (Omni International) with tissue homogenizer tubes. RNA was purified using the Purelink RNA Mini Kit (ThermoFisher) and then reverse-transcribed using a Maxima First Strand cDNA Synthesis Kit (ThermoFisher Scientific), all according to the manufacturer’s instructions. The primer probes listed in Table S4 were used in the real-time PCR assays with TaqMan gene expression master mix (ThermoFisher Scientific): The ΔCt method (where Ct is threshold cycle) was used to calculate mRNA expression after normalization to Gapdh expression.

Single cardiomyocytes were isolated using a simplified, Langendorff-free method adopted from Matthew Ackers-Johnson et al. [23]. EDTA buffer containing (in mM) 130 NaCl, 5 KCl, 0.5 NaH2PO4, 10 HEPES, 10 glucose, 10 BDM (Sigma-Aldrich), 10 Taurine, and 5 EDTA (pH 7.8 with NaOH) was prepared and stored at 4 °C up to two weeks. Perfusion buffer containing (in mM) 130 NaCl, 5 KCl, 0.5 NaH2PO4, 10 HEPES, 10 glucose, 10 BDM, 10 taurine, and 1 MgCl2 (pH 7.8 with NaOH) was prepared and stored at 4 °C fridge up to two weeks. Collagenase buffer containing 0.5 mg/ml collagenase type 2, 0.5 mg/ml collagenase type 4 (Worthington Biochemical Corporation), and 0.05 mg/ml protease XIV (Sigma-Aldrich) was prepared the day of the isolation. Stop buffer was made with 5% FBS in perfusion buffer on the day of isolation. Mice were anesthetized using isoflurane, and the chest was opened to expose the heart. Seven ml EDTA buffer was perfused into the right ventricle. The ascending aorta was clamped using a Ductus clamp, and the heart was removed by cutting behind the clamp. The heart was transferred into a 30-mm dish containing EDTA buffer and then perfused with another 10 ml EDTA through the left ventricle. The heart was transferred into another 30-mm dish containing perfusion buffer and perfused with 3 ml perfusion buffer through the left ventricle. The heart was then transferred into another 30-mm dish containing collagenase buffer and perfused with 30 ml collagenase buffer for at least 15 min. The excess tissue including the right ventricle, atria, etc. were excised. The remaining left ventricle was transferred to another 30 mm dish containing about 5 ml collagenase buffer and was gently separated into 1 mm pieces using forceps, before being drawn up into a 1 ml pipette tip with the tip cut to ensure a larger opening. The 1 mm pieces of tissue were drawn up and down the pipette tip for about 2 min. Five ml of stop buffer was added to the 30 mm dish, and the pieces of tissue were drawn up and down the pipette again for about a minute. The cell suspension was passed through a 100-μm filter into a 50 mL conical tube, and the filtered suspension was transferred to a 15-ml conical tube. Cells underwent 3 sequential rounds of Ca2+ reintroduction (0.2, 0.5, and 1 mM) in perfusion buffer by gravity settling for 15–20 min/round and were stored in perfusion buffer with 1 mM Ca2+ at room temperature until use.

Isolated cardiomyocytes were loaded with 10 μM Fluo-4 AM (Fisher Scientific) and 0.02% Pluronic F-127 (Life Technologies) for 20 min at room temperature in 1 mM Ca2+ perfusion buffer. Cells were centrifuged (rcf 50 xg for 2 min) at room temperature and resuspended in Tyrode’s solution containing (in mM) 140 NaCl, 5 KCl, 2 CaCl2, MgCl2, 10 HEPES, and 5.6 glucose (adjusted to pH 7.36 with NaOH). Cells were then transferred into a glass-bottom chamber mounted on the stage of an inverted microscope (Nikon Eclipse Ti). Calcium transients and contractility in single cardiomyocytes were recorded with IonOptix excitation-contraction coupling data acquisition system (IonOptix LLC, MA). After settling on the glass coverslip, cells were continuously perfused with Tyrode’s solution at a flow rate of 1.8 ml/min and paced with electric field stimulation (EFS) at 10 V voltage, 0.5 Hz throughout the experiment. After baseline recording, cardiomyocytes were perfused with the non-selective βAR agonist, isoproterenol (ISO, Sigma-Aldrich) at increasing concentrations (10−8 to 105 M) in Tyrode’s solution via a pump. Cardiomyocyte Ca2+ transients and contractile properties were analyzed using IonOptix data processing software. ISO-induced arrhythmic activity including premature ventricular contractions, tachycardia, and temporary pause were judged by both Ca2+ transient and contractility traces, and the incidence of arrhythmic activity in response to ISO was counted before and after the application of each concentration of ISO.

Following 20 min of incubation in Tyrode buffer containing 500 μM 3-isobutyl-1-methylxannthine (IBMX), freshly isolated left ventricular cardiomyocytes were stimulated at 37 °C for 5 min with vehicle or forskolin (1, 10, or 50 μM) in the absence or presence of the (α2-adrenergic receptor agonist, UK-14,304, or the Gi/o inhibitor, pertussis toxin (PTX, 200 or 750 ng/ml). Cells treated with PTX were pre-incubated with the toxin for 2 h prior to incubation with IBMX and subsequent stimulation with forskolin, as previously described [7,16]. Following forskolin stimulation, the cell suspension was quickly centrifuged at 4 °C, 3000 rpm for 10 min. Both the supernatant and pellet were quickly stored at −20 °C. cAMP levels were measured using a cAMP enzyme-linked immunosorbent assay by following the manufacturer’s protocol (R&D Systems, Inc., Minneapolis, MN).

All values are presented as mean ± s.e.m. All statistical analyses were performed using GraphPad Prism 8.1.1 software for Mac OS X (Graph-Pad Software, San Diego, CA). Where appropriate, we used unpaired Student’s t-test, repeated measures ANOVA (Analysis of variance), or mixed-effect model to determine significant interaction within and between groups. If the ANOVA or mixed-effect model yielded a significant effect of a factor or a significant interaction between multiple factors, Sidak post hoc analysis was used to determine significance of differences between groups, and results were considered significant if the P value was <0.05.

RGS2 and 5 are prominently expressed in cells of the cardiovascular and renal systems [24,25], and genetic ablation of either RGS protein is reported to cause hypertension in mice [13,26]. However, mice null for Rgs2 or Rgs5 have normal life span, and neither mouse model develops maladaptive cardiac hypertrophy or heart failure at baseline, despite exhibiting chronically elevated resting blood pressure [13,22]. These observations led us to hypothesize that the loss of RGS2 or 5 either triggers the activation of some yet unknown cardioprotective mechanism or increases the expression and/or function of other proteins that maintain the fine-tuning of G protein activity. To test these hypotheses, we sought to generate mice dually lacking RGS2 and 5. We crossed F1 double-heterozygous offspring resulting from single knockout (Rgs2−/− x Rgs5−/−) crosses (Supplementary Fig. S1A). The Rgs5−/− breeders were obtained from Dr. John Kehrl at the NIH-NIAID [15]. The Rgs2−/− mice, originally generated by Dr. Josef Penninger’s group [14], were generously provided by Dr. Kendall Blumer at Washington University. All F2 offspring from the Rgs2−/− x Rgs5−/− crosses, including those that were null for Rgs2 with one copy of Rgs5, those null for Rgs5 with one copy of Rgs2, and those null for Rgs2 and 5 (Rgs2/5 dbKO) were viable without showing any developmental or gross abnormalities. All genotypes were confirmed by PCR using genomic DNA extracted from tail clips (Supplementary Fig. S1B). Contrary to a previous report regarding the gravimetric characteristics of Rgs5−/− mice [15], we did not find any difference in body weight. Similarly, the dual loss of RGS2 and 5 did not result in any difference in body weight (Supplementary Table S1).

We examined the effects of the dual absence of RGS2 and 5 on baseline cardiac structure and function in male and female mice. We found, by gravimetric and echocardiographic assessments, that adult Rgs2/5 dbKO mice developed unprovoked left ventricular dilatation, which was prominent in adult male mice (Fig. 1A-E). Although heart weight-to-tibia length ratio was higher in Rgs2/5 dbKO mice, we found no difference in wet or dry cardiac tissue weight normalized to tibia length between the two genotypes (Supplementary Table S2). However, isolated cardiomyocytes from male Rgs2/5 dbKO mice had a higher length-to-width ratio compared to cells from WT mice (Supplementary Table S3). The dilated left ventricle phenotype was age-dependent, since 30-day old pups had normal hearts, except for apparent ventricular hypertrophy in young female Rgs2/5 dbKO mice (Fig. 1F & G). In adult male mice, heart rate measured from the anesthetized echocardiography data analysis was higher in WT relative to Rgs2/5 dbKO mice (WT: 442 ± 19 bpm vs. Rgs2/5 dbKO: 397 ± 12 bpm; P = 0.03). A similar trend was found in female mice; however, the difference did not reach statistical significance (WT: 432 ± 18 bpm vs. Rgs2/5 dbKO: 390 ± 22 bpm; P = 0.090). Despite left ventricular dilatation, lung weight-to-body weight ratio of adult Rgs2/5 dbKO mice was similar to that of WT control mice of the same sex (Fig. 1C), and no evidence of cardiac fibrosis was found (data not shown). In addition to echocardiography, we used closed-chest pressure-volume (PV) loop approach to further assess cardiac structure and function in WT and Rgs2/5 dbKO mice under isoflurane anesthesia. Heart rate was similar between WT and Rgs2/5 dbKO mice of both sexes (Fig. 2D). In male Rgs2/5 dbKO mice, percent ejection fraction (%EF) at baseline was low compared to WT cohort (Fig. 2 J), while stroke volume, cardiac output and slope of the end-systolic pressure-volume relationship (Ees) showed a decreasing trend (Fig. 2H, I, & K). Conversely, left ventricular systolic pressure, end-systolic volume (ESV) and arterial elastance (Ea) showed increasing trend in male Rgs2/5 dbKO mice (Fig. 2C, G & L). Interestingly, female Rgs2/5 dbKO mice showed a marked elevation of Ees relative to WT mice of the same sex (Fig. 2K). From a real-time PCR analysis, we did not find any marked changes in the expression of hallmark genes associated with maladaptive cardiac hypertrophy and fibrosis, including Nppa, Myh7b, Tgfb1, Col3a1, and Ctgf, except that female Rgs2/5 dbKO hearts showed decreased expression of Nppa and Ctgf (Supplementary Fig. S2). In contrast, we found significant changes in markers of cardiomyopathy. In female Rgs2/5 dbKO hearts, there was a significant decrease in Ttn, Tnnt2 and Tnni3, whereas male hearts showed a significant decrease in Tnnt2 and a trend towards lower expression level of Ttn (p = 0.1058) and increasing trend in the expression of Mybpc3 (p = 0.0671) (Supplementary Fig. S2, F-I). These findings are consistent with the hypothesis that the dual loss of RGS2 and 5 leads to a derangement in the homeostatic control of cardiac structure and function.

Because Rgs2/5 dbKO mice showed reduced %EF and decreasing trend in %FS at baseline, we Tested whether the dual absence of RGS2 and 5 affected cardiac contractile reserve. To this end, we performed acute intraperitoneal infusion of the selective β1AR agonist, dobutamine, while monitoring LV function by echocardiography under isoflurane anesthesia. As shown in Fig. 3, baseline percent fractional shortening (%FS) was low in male but not female Rgs2/5 dbKO mice compared to WT cohorts. Fig. 3 also shows that in male mice, dobutamine infusion caused a sharper increase in %FS, accompanied by a slightly steeper drop in heart rate relative to the response in Rgs2/5 dbKO cohort. In female mice, %FS prior to dobutamine infusion was similar between genotypes. As seen in male mice, dobutamine infusion caused a sharper increase in %FS in female WT mice, however heart rate appeared to decline slightly faster in Rgs2/5 dbKO relative to WT mice. These data indicated that myocardial responsiveness to βAR stimulation is impaired by the dual absence of RGS2 and 5, at least male mice.

Although gene expression analysis suggested the absence of maladaptive hypertrophy, we sought to examine baseline blood pressure to rule out ventricular hypertrophy resulting from potential pressure-overload. We assessed blood pressure by two approaches: using fluid-filled catheter under isoflurane anesthesia and in conscious, unrestrained condition by radiotelemetry approach involving a prior invasive surgery of telemeter implantation. Baseline blood pressure under anesthesia was slightly elevated in male but not female Rgs2/5 dbKO mice relative to WT cohorts (Fig. 4A). However, heart rate was similar between WT and Rgs2/5 dbKO mice (Fig. 4B). As shown in Fig. 4C, we observed ~70% mortality only in male Rgs2/5 dbKO mice during the recovery period following telemetry implantation surgery. To determine whether the high mortality rate in male Rgs2/5 dbKO mice was preceded by extreme hypertension resulting from surgery-induced stress, we initiated blood pressure and heart rate monitoring immediately after radiotelemeter implantation surgery. As shown in Fig. 4D, male mice generally exhibited greater elevation of SBP within the first 24–48 h after surgery. Both diastolic blood pressure and heart rate were similar between the two genotypes. The initial stress-induced increase in SBP was more robust in Rgs2/5 dbKO mice relative to wild type of the same sex. In male mice, diurnal blood pressure rhythm was re-established by day 4, but only SBP remained elevated in Rgs2/5 dbKO mice by day 9 post surgery. In contrast, diurnal blood pressure rhythm in female mice was re-established within 72 h post-surgery, with the hypertensive phenotype becoming more evident after day 6 (Fig. 4E). These results indicated that acute blood pressure elevation following surgery-induced stress was not sufficient to cause sex-dependent mortality of male Rgs2/5 dbKO mice.

To determine the cause of sudden death in male Rgs2/5 dbKO mice a few days after telemetry surgery, we examined whether the dual absence of RGS2 and 5 increased the incidence of fatal cardiac events in response to sudden spikes in circulating catecholamines triggered by surgery-induced stress. We simulated sudden catecholamine surge by acute systemic administration of isoproterenol (ISO) to conscious mice instrumented with ECG telemetry implants for continuous monitoring of blood pressure and cardiac electrical activity. Prior to ISO administration, SBP was elevated in Rgs2/5 dbKO male mice, whereas all baseline ECG parameters were similar between the two genotypes (Table 1). Administration of ISO normalized blood pressure, reducing SBP by ~30 and ~ 50 mmHg in WT and Rgs2/5 dbKO mice, respectively (Table 1). In addition to the effects on blood pressure, ISO administration reduced the height of T wave and increased ST interval in Rgs2/5 dbKO mice. In both WT and Rgs2/5 dbKO mice, the most prominent arrhythmic events observed at baseline included missed beats, ventricular ectopic (V Ectopic), single premature ventricular contractions (PVC Single), and sinus pause (Fig. 5A & 5B, blue tracings). Administration of ISO equally augmented the frequency of all the aforementioned arrhythmic events, except missed beats, which was reduced in Rgs2/5 dbKO mice (Fig. 5A & 5B, orange tracings). To replicate whole-body stress as occurs after surgery, the mice were subjected to 90 min of involuntary swimming after which ECG was monitored for 30 min. Only P-R interval among the ECG parameters was markedly increased. However, as shown in Fig. 5C and D, stress from involuntary swimming increased the incidence of V Ectopic, PVC Single, and sinus pause relative to baseline, in WT mice. In contrast, sinus pause was the most prominent arrhythmic event in Rgs2/5 dbKO mice following the swimming exercise (Fig. 5C & 5D, orange tracings). As happened after ISO administration, missed beats was markedly reduced after swimming. Together these results suggested that dual loss of RGS2 and 5 does not alter baseline arrhythmia burden but augments the occurrence of sinus pause due to stress.

To determine the underlying characteristics of the decreased myocardial contractility and arrhythmias resulting from the dual loss of RGS2 and 5 at the cellular level, we examined excitation-contraction coupling in vitro in freshly isolated adult left ventricular myocytes from male WT and Rgs2/5 dbKO mice. Application of electrical field stimulation (EFS) caused frequency-dependent increases in the speed of Ca2+ transients and contractility in cells from both genotypes (Fig. 6A). At the lowest EFS frequency (0.5 Hz), the maximal cardiomyocyte percent shortening (PS) was reduced in Rgs2/5 dbKO cells, despite a higher Ca2+ transient amplitude (Fig. 6B-E). In WT cells, the application of EFS caused frequency-dependent decreases in PS and Ca2+ transients. In contrast, PS in Rgs2/5 dbKO cardiomyocytes remained relatively unchanged, while Ca2+ transients continued to decrease relative to baseline level (Fig. 6D & E). Rgs2/5 dbKO cardiomyocytes showed prolonged contraction and cytoplasmic Ca2+ rise at 1–4 Hz, as determined by calculating the area under the curve of the respective signal traces from baseline to the peak for Ca2+ rise and contraction (Fig. 6F & G), and from the peak to baseline for relaxation and Ca2+ recycling (Fig. 6H & I). In addition, we noted that the rate of shortening of Rgs2/5 dbKO cardiomyocytes decreased relative to WT cells at higher EFS frequencies, particularly at 4 and 5 Hz (Fig. 6A-B, video #1 in supplementary data). The slowed contractile rate was interspersed with intermittent absence of Ca2+ transients and contractile response to the applied EFS (Fig 6A-B, arrows). Moreover, there were more Rgs2/5 dbKO relative to WT cardiomyocytes (WT: 13% vs. Rgs2/5 dbKO: 63%) that developed sustained abnormal Ca2+ and contractile rhythms at 3–5 Hz (video #2 in supplementary data). Some cells, however, resumed normal contractile rhythm upon the return of EFS to baseline frequency of 0.5 Hz.

RGS2 has been shown to negatively regulate β2AR-Gi/o signaling in the myocardium; accordingly, downregulation or the absence of RGS2 leads to decreased cardiomyocyte contractile response to βAR stimulation [7]. Because Gi/o is also regulated by RGS5 [3], and that the loss of RGS5 also predisposes to arrhythmias [9,10], we reasoned that decreased contractility and increased rate of ventricular myocyte arrhythmias following the dual absence of RGS2 and 5 could be due, at least partly, to augmented inhibitory Gi/o activity. To test this hypothesis, we compared Gi/o activity in freshly isolated cardiomyocytes from WT, Rgs2 KO, Rgs5 KO and Rgs2/5 dbKO male mice. In cellulo Gi/o activity was assessed by measuring forskolin-stimulated cAMP generation in freshly isolated adult ventricular myocytes. As shown in Fig. 7, direct adenylyl cyclase activation with forskolin induced concentration-dependent increases in cAMP levels in WT cells, and these were attenuated by activating Gi/o via the stimulation of the Gi/o-coupled α2-adrenergic receptor with UK-14,304 (1 μM). Conversely, inhibiting Gi/o by pre-incubation of the cells with pertussis toxin (PTX, 200 or 750 ng/ml) augmented forskolin-induced cAMP production in WT cells (Fig. 7). In Rgs2 KO, Rgs5 KO and Rgs2/5 dbKO cardiomyocytes, forskolin-induced cAMP production was reduced. Of note, the suppression of cAMP generation in response to 50 μM forskolin appeared to be less pronounced in Rgs5 KO cells relative to Rgs2 KO and Rgs2/5 dbKO cells. In addition, treatment with UK-14,304 had no effect on cAMP production in all three KO cell types. However, incubation of Rgs2/5 dbKO cardiomyocytes with PTX augmented forskolin-induced cAMP production. As was shown in Fig. 6, the application of EFS train triggered arrhythmias between 1 and 5 Hz in a fraction of cells from all genotypes, including cells from WT, Rgs2 KO, Rgs5 KO and Rgs2/5 dbKO mice. Fig. 8 shows the quantification of arrhythmia incidence during EFS application, with most arrhythmias occurring at 5 Hz (Fig. 8A). The proportion of cells showing no arrhythmias during EFS application was similar between Rgs2 KO, Rgs5 KO, and WT cells (Rgs2 KO, ~80 vs. Rgs5 KO, ~80 vs. WT, ~89%) but relatively lower in Rgs2 dbKO cells (Rgs2/5 dbKO, ~62% vs. WT, ~89%). Fig. 8A also shows that inactivation of Gi/o with 200 ng/ml PTX had little effect in WT cells but completely abolished arrhythmia incidence at all EFS frequencies in Rgs2/5 dbKO cells. However, when a higher concentration (750 ng/ml) of PTX was applied, arrhythmias were detected at 5 Hz in WT cells and at 1, 3, and 5 Hz in Rgs2/5 dbKO cells. By contrast, Fig. 8B shows that stimulation of cells with ISO induced arrhythmias at almost every concentration in all cells. Approximately 50% of WT cells developed no arrhythmias at all ISO concentrations. Notably, the percentage of Rgs2/5 dbKO cells that never developed arrhythmia at any ISO concentration was about half the percentage in either Rgs2 KO or Rgs5 KO cells. Pretreatment with 200 or 750 ng/ml PTX increased ISO-induced arrhythmia development by ~10% in WT cells, whereas the same pretreatment decreased arrhythmia development by ~15% in Rgs2/5 dbKO cells. These data together indicated that increased Gi/o activity due to the dual absence of RGS2 and 5 is involved in receptor and non-receptor-dependent arrhythmia development in ventricular cardiomyocytes.

Abnormal cytoplasmic Ca2+ handling has been implicated in ventricular myocyte arrhythmogenesis [27-31]. Because EFS-induced arrhythmias in cardiomyocytes from Rgs2/5 dbKO male mice was accompanied by elevated cytoplasmic Ca2+, we determined whether the dual absence of RGS2 and 5 affected the pathways involved in Ca2+ handling during excitation-contraction coupling. To determine whether altered Ca2+ extrusion pathways were involved in sustained elevation of cytoplasmic Ca2+, we treated isolated ventricular myocytes with thapsigargin to block SERCA, followed by the application of EFS at 10 Hz (10 V) for 25 s, while monitoring cytoplasmic Ca2+ rise and decay. Fig. 9 shows that the application of EFS train induced an almost instantaneous rise in Ca2+ transients and myocyte shortening, both of which plateaued for the duration of the stimulation in both genotypes (Fig. 9A). The overall cytoplasmic Ca2+rise, calculated as the area under the curve (AUC) of the fluorescence ratio-time curve for the duration of the EFS train, was elevated in Rgs2/5 dbKO relative WT to cells. However, the AUC of the myocyte length-time curve for the same period was similar between the two groups, indicating similar levels of cardiomyocyte contraction (Fig. 9A & B). We then assessed the rate of cytoplasmic Ca2+ clearance by performing first order exponential decay curve fitting. The parameters in the equations shown in Fig. 9C indicate that although cytoplasmic Ca2+ at the end of the EFS train was significantly higher in Rgs2/5 dbKO cells, the rate of Ca2+ removal from the cytoplasm, indicated by the time constant, was similar between the two groups. Using real-time PCR, we assessed myocardial mRNA levels of Atp2a2 (SERCA2a), and Slc8a1 (NCX1) and Atpb1 (PMCA1), genes encoding proteins for Ca2+ reuptake into the SR and extrusion from the cardiomyocyte cytoplasm, respectively, to determine whether the augmented EFS-induced cytoplasmic Ca2+ rise was due at least partly to changes in gene expression. Supplementary Figs. S2 and S3 show that the dual absence of Rgs2 and 5 did not affect the mRNA expression of SERCA2a, NCX1, and PMCA1 in freshly isolated cardiomyocytes. These results suggest that the exaggerated elevation of cytoplasmic Ca2+ in Rgs2/5 dbKO cells is not due to decreased expression or altered activity of proteins involved in Ca2+ removal during E-C coupling events.

The findings in this study reveal a novel understanding regarding the physiological relevance of the dual regulation of Gi/o signaling by RGS2 and 5 in the heart. Besides having mildly elevated or reduced resting blood pressure, mice lacking RGS2 or RGS5 alone do not show any baseline cardiac phenotype except when subjected to experimental stressors including pressure overload and cardiac pacing [12,26,32,33]. Previous studies have also reported that the absence of RGS2 or 5 increases the incidence of atrial tachyarrhythmia induced with muscarinic receptor stimulation [34,35], and ventricular tachyarrhythmia in response to programmed electrical stimulation of isolated hearts [9,10]. Our findings now show that the dual absence of RGS2 and 5 causes unprovoked left ventricular dilatation without cardiomyocyte hypertrophy and independently of the associated baseline blood pressure elevation. In addition, the dual absence of RGS2 and 5 increases susceptibility to stress-induced cardiac arrhythmia and death that are mediated at least partly by aberrant signaling via β-adrenergic receptors and Gi/o class G proteins. Moreover, our study suggests that RGS2 and 5 coordinate their regulatory activity towards Gi/o signaling to facilitate normal ventricular myocyte rhythm. Regulation of cardiac structure and function by the sympathetic nervous system is mediated by adrenergic receptors, including α- and β-adrenergic receptors (βAR) [36-38]. While α-adrenergic receptors couple mostly to Gq/11 and Gi/o class G proteins for mobilization of Ca2+ from the SR, βAR on the other hand couple to both stimulatory Gs and inhibitory Gi/o classes of heterotrimeric G proteins to regulate cAMP production [36,39]. Gq/11 signaling in many cell types, including cardiomyocytes, is fine-tuned by R4/B RGS proteins, most notably RGS2 and 5 [5,40,41]. Although specific RGS proteins that regulate Gs signaling are yet to be identified, there are emerging lines of evidence suggesting that RGS2 can regulate signaling downstream of this G protein class by interacting with adenylyl cyclase isoforms 5 and 6 in cells to negatively control cAMP production and affect cardiomyocyte contractile function [42-45]. Other lines of evidence also indicate that the inhibitory Gi/o class G protein is regulated by many RGS proteins from other subfamilies beside R4/B RGS proteins in the heart [46,47]. However, evidence to date indicate that the regulation of Gi/o by RGS2 and 5, individually, is critical to maintaining normal cardiac structure and function, since mice lacking RGS2 or 5 have been shown to be more sensitive to ISO-induced cardiac dysfunction and atrial fibrillation induced by cardiac pacing [6,7,9,10,35]. Despite these studies demonstrating the key roles of RGS2 and 5 in the myocardium, the fundamental questions that remain unexplored are how multiple RGS proteins coordinate their activity and, whether there is a functional redundancy in vivo in the fine-tuning of G protein signaling involved in the maintenance of normal physiology. Addressing these questions has been necessitated partly by previous studies showing that mice singly lacking RGS2 or 5 develop mild hypertension without any cardiac phenotype unless subjected to some experimental perturbation such as cardiac pacing or pressure-overload by transverse aortic constriction [12,22,35]. Because RGS2 and 5 both potently regulate Gq/11 and Gi/o, the dual absence of such key regulatory proteins was expected to be developmentally detrimental. However, Rgs2/5 dbKO mice are viable and appear normal and live to and beyond adulthood, suggesting that there is sufficient level of regulation by other proteins in the absence of potent regulators like RGS2 and 5 to ensure normal embryonic and postnatal development involving Gq/11 and Gi/o, signaling. Here, we found that unlike the hearts of adult single Rgs2 or 5 male KO mice [12,22], Rgs2/5 dbKO mouse hearts are dilated at baseline. This cardiac phenotype is not accompanied by the hallmark maladaptive hypertrophy or increases in the expression of maladaptive hypertrophy markers such as Nppa, Myh7b and Tgfb1, nor is there increased lung weight-to-body weight ratio as seen in male Rgs2−/− or Rgs5−/− mice subjected to left ventricular pressure-overload [12,22]. Interestingly, we found cardiomyocytes from adult male Rgs2/5 dbKO mice to be smaller in width but elongated relative to WT cells; however, there was no difference in cardiac weight between young and old mice (supplemental Table S1-3). These observations suggest that Rgs2/5 dbKO cardiomyocytes increase in length to compensate for failure to undergo physiologic hypertrophy associated with myocardial maturation process to meet increasing circulatory demands in adulthood. Such developmental defects could trigger ventricular dilatation in an attempt to compensate for low cardiac mass for the maintenance of basal contractile function. However, such compensatory structural changes resulting from the dual absence of RGS2 and 5 appear insufficient for maintaining normal basal function, as indicated by lower basal ejection fraction and decreased contractile response to dobutamine-induced cardiac stress. Furthermore, adult Rgs2/5 dbKO mice display elevated resting blood pressure and potential vascular stiffening indicated by elevated arterial elastance. These factors together increase left ventricular pressure-overload that usually triggers maladaptive cardiac hypertrophy, yet Rgs2/5 dbKO hearts do not show any hypertrophy at baseline. We find this outcome surprising because pressure-overload induces maladaptive cardiac hypertrophy partly via augmented Gq/11 signaling, and both RGS2 and 5 have potent GAP activity towards this G protein class, and their overexpression in the heart has been shown to be cardioprotective, blocking pressure-overload hypertrophy [12,48-50]. Thus, the presence of LV dilatation-only cardiac phenotype at baseline suggests that pro-hypertrophic Gq/11 signaling is somehow still tightly regulated by other GAPs in the dual absence of RGS2 and 5. Instead, the dilated left ventricle in Rgs2/5 dbKO mice, accompanied by changes in the expression of genes involved in Ca2+ sensing and cardiomyocyte contractility, and evident only in adult mice, is consistent with hypertrophic cardiomyopathy as opposed to pressure-overload hypertrophy and heart failure due to sustained hypertension [51]. Among the various forms of common cardiomyopathies known to date, the cardiac phenotype in Rgs2/5 dbKO mice is most consistent with dilated cardiomyopathy [51] that is more noticeable in male mice. In addition, the expression of cardiomyopathy markers, particularly titin and troponin T, is reduced in Rgs2/5 dbKO hearts of both sexes, and individual cardiomyocyte contractions are markedly decreased despite increased mobilization of intracellular Ca2+. The augmented Ca2+ mobilization, however, appears to contribute to arrhythmogenesis as Ca2+ recycling is impaired, leading to prolonged elevation of cytoplasmic Ca2+ concentration, which is known to be arrhythmogenic [30,52-54]. Taken together with the decrease in the expression of contractile genes, our data suggest that the cardiomyopathy in Rgs2/5 dbKO is accompanied by unraveling of the mechanisms that couple cardiac electrical excitation and contractility, thereby contributing to the marked decrease in baseline ejection fraction and fractional shortening. These deficiencies may reduce cardiac reserve and increase susceptibility to an adverse cardiac event upon exertion from increased physical activity such as exercise or acute increases in cardiac catecholamine levels. Indeed, this study shows that the dual inactivation of Rgs2 and 5 greatly elevates the susceptibility to factors known to trigger cardiac events. As reported above, surgery-induced stress on the heart is sufficient to cause death in male Rgs2/5 dBKO mice. Mechanistically, this is likely due to aberrant or unregulated βAR signaling resulting from sudden surges in cardiac sympathetic activation leading to exaggerated Ca2+ transients and prolonged elevation cytoplasmic Ca2+ level in ventricular myocytes. Consistent with this hypothesis, simulation of cardiac sympathetic overactivation and stress with acute administration of isoproterenol, or cardiomyocyte pacing, induced arrhythmia both in vivo and at single-cell levels, accompanied by substantial increases in cytoplasmic Ca2+, while blockade of Gi/o with pertussis toxin reduced arrhythmia incidents in Rgs2/5 dbKO cardiomyocytes. Moreover, forskolin-induced cAMP generation was suppressed in Rgs2/5 dbKO cardiomyocytes and was restored by inhibiting Gi/o with pertussis toxin. These findings indicate that abnormal activity and the loss of regulation of βAR-Gi/o signaling due to the dual absence of RGS2 and 5, at least partly, mediate cardiac arrhythmias in Rgs2/5 dbKO ventricular cardiomyocytes. Although RGS2 is known to be a more potent GAP for Gq/11 and Gi/o, in vitro [55], expression at the mRNA level is substantially less than RGS5 in the cardiovascular system [56-58]. This suggests that the regulatory roles of RGS2 and 5 do not overlap. Here, we discovered that arrhythmia incidence resulting from EFS or βAR stimulation in Rgs2/5 dbKO ventricular myocytes was roughly two times the level in either Rgs2 KO or Rgs5 KO myocytes. However, RGS2 appears to be more effective than RGS5 in finetuning Gi/o-dependent inhibition of adenylyl cyclase and thus cAMP generation in ventricular myocytes. Together, these findings suggest that RGS2 and 5 target different signaling pathways or different aspects of the same pathway that impinge on ventricular rhythm. In addition, the findings show that the relative strength of RGS2 and 5 as GAP towards Gi/o, in vivo may be different from previously established in vitro rank-order of GAP potencies B/R4 RGS proteins towards this inhibitory G protein class [55], as demonstrated in this study by the differences in the inhibition of forskolin-stimulated cAMP production upon Gi/o activation with (α2-adrenergic receptor stimulation. Whereas the current study focused on the role of the dual loss of RGS2 and 5 in ventricular arrhythmias and βAR–Gi/o signaling, previous reports by others have shown that mice null for Rgs2 are more susceptible to tachypacing-induced atrial arrhythmia due, at least partly, to unregulated muscarinic M3 receptor (M3R) signaling [34,35]. Since M3R couples mainly to Gq/11 class G proteins, such reports suggest that the dysregulation of Gq/11 by RGS2 and/or 5 in the myocardium could contribute to ventricular arrhythmia. However, this possibility is likely not the case in Rgs2/5 dbKO ventricular myocytes, since βAR, the primary mediator of cardiac sympathetic activity, do not couple to Gq/11 class G proteins. Thus, the findings here give novel insights into the mechanism by which Gi/o signaling is dually regulated by RGS2 and 5 to facilitate normal cardiac rhythm. The discovery that the loss of RGS5 has minimal effects on forskolin-induced cAMP generation yet capable of compensating for RGS2 deficiency in mitigating ventricular myocyte arrhythmia should spur additional studies to fully delineate how RGS2 and 5 regulate cellular responses dependent on Gi/o-adenylyl cyclase-cAMP signaling. Other notable observations in this study are the sex-related differences in the cardiac structural and functional changes resulting from the dual loss of RGS2 and 5. Although female Rgs2/5 dbKO mice displayed apparent cardiac hypertrophy, this was not accompanied by LV dilatation as in male mice or death following surgery-related stress. In addition, there was no effect of the dual absence of RGS2 and 5 on the response to cardiac stress test with acute dobutamine infusion. The lack of difference in the dobutamine stress test in female mice could be due to compensation by other RGS proteins. However, this possibility was ruled out as gene expression analysis showed no compensatory increase in the mRNA expression of other members of the B/R4 RGS family expressed in the cardiovascular system (Supplementary Fig. S4). Interestingly, we noted that in both sexes of Rgs2/5 dbKO mice, there was a substantial decrease in the mRNA expression of RGS4, which is prominently expressed in the myocardium and also acts a potent GAP towards Gi/o class G proteins [24,58,59]. We also noted that changes in gene markers of cardiomyopathy was more prominent in the hearts of female Rgs2/5 dbKO mice, suggesting potential sex differences in the pathogenesis of cardiomyopathy resulting from the dual absence of RGS2 and 5. The mechanisms mediating sex-related differences in the cardiac phenotype resulting from the dual absence of RGS2 and 5 was not explored in this study. However, it is noteworthy that numerous studies have established the cardioprotective role of ovarian hormones [60-62]. For instance, ovariectomized female rats subjected to chronic volume overload displayed eccentric cardiac hypertrophy accompanied by LV dilatation, which were reversed by exogenous estrogen treatment [62]. These previous observations warrant further studies to assess the potential interplay between RGS function and the protective role of sex hormones in the heart. In summary, this study demonstrates the critical role of the coordinated relationship between RGS2 and 5 in regulating signaling via Gi/o class G proteins to facilitate normal cardiac structure and function. A deeper probing of the coordination between the expression and function of RGS2 and 5 in other cells and organ systems is necessary to enhance the understanding of regulatory balance of RGS proteins for promoting homeostasis.