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Open Access

Peer-reviewed

Research Article

The influences of the M2R-GIRK4-RGS6 dependent parasympathetic pathway on electrophysiological properties of the mouse heart

  • Kanchan Kulkarni,

    Roles Formal analysis, Investigation, Writing – original draft, Writing – review & editing

    Affiliation Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota, United States of America

    ⨯
  • Xueyi Xie,

    Roles Investigation, Methodology, Writing – review & editing

    Affiliation Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota, United States of America

    ⨯
  • Ezequiel Marron Fernandez de Velasco,

    Roles Investigation, Methodology, Writing – review & editing

    Affiliation Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota, United States of America

    ⨯
  • Allison Anderson,

    Roles Investigation, Writing – review & editing

    Affiliation Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota, United States of America

    ⨯
  • Kirill A. Martemyanov,

    Roles Conceptualization, Writing – review & editing

    Affiliation Department of Neuroscience, The Scripps Research Institute, Jupiter, Florida, United States of America

    ⨯
  • Kevin Wickman,

    Roles Conceptualization, Resources, Supervision, Writing – review & editing

    Affiliation Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota, United States of America

    ⨯
  • Elena G. Tolkacheva

    Roles Conceptualization, Resources, Writing – review & editing

    talkacal@umn.edu

    Affiliation Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota, United States of America

    ⨯

Correction

6 Jul 2018: Kulkarni K, Xie X, Marron Fernandez de Velasco E, Anderson A, Martemyanov KA, et al. (2018) Correction: The influences of the M2R-GIRK4-RGS6 dependent parasympathetic pathway on electrophysiological properties of the mouse heart. PLOS ONE 13(7): e0200553. https://doi.org/10.1371/journal.pone.0200553 View correction

Abstract

A large body of work has established the prominent roles of the atrial M2R-IKACh signaling pathway, and the negative regulatory protein RGS6, in modulating critical aspects of parasympathetic influence on cardiac function, including pace-making, heart rate (HR) variability (HRV), and atrial arrhythmogenesis. Despite increasing evidence of its innervation of the ventricles, and the expression of M2R, IKACh channel subunits, and RGS6 in ventricle, the effects of parasympathetic modulation on ventricular electrophysiology are less clear. The main objective of our study was to investigate the contribution of M2R-IKACh signaling pathway elements in murine ventricular electrophysiology, using in-vivo ECG measurements, isolated whole-heart optical mapping and constitutive knockout mice lacking IKACh (Girk4–/–) or RGS6 (Rgs6-/-). Consistent with previous findings, mice lacking GIRK4 exhibited diminished HR and HRV responses to the cholinergic agonist carbachol (CCh), and resistance to CCh-induced arrhythmic episodes. In line with its role as a negative regulator of atrial M2R-IKACh signaling, loss of RGS6 correlated with a mild resting bradycardia, enhanced HR and HRV responses to CCh, and increased propensity for arrhythmic episodes. Interestingly, ventricles from mice lacking GIRK4 or RGS6 both exhibited increased action potential duration (APD) at baseline, and APD was prolonged by CCh across all genotypes. Similarly, CCh significantly increased the slope of APD restitution in all genotypes. There was no impact of genotype or CCh on either conduction velocity or heterogeneity. Our data suggests that altered parasympathetic signaling through the M2R-IKACh pathway can affect ventricular electrophysiological properties distinct from its influence on atrial physiology.

Citation: Kulkarni K, Xie X, Fernandez de Velasco EM, Anderson A, Martemyanov KA, Wickman K, et al. (2018) The influences of the M2R-GIRK4-RGS6 dependent parasympathetic pathway on electrophysiological properties of the mouse heart. PLoS ONE 13(4): e0193798. https://doi.org/10.1371/journal.pone.0193798

Editor: Alexander V. Panfilov, Universiteit Gent, BELGIUM

Received: September 27, 2017; Accepted: February 20, 2018; Published: April 18, 2018

Copyright: © 2018 Kulkarni et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper.

Funding: This work was supported by the National Science Foundation grant CAREER PHY-125541 (EGT); National Science Foundation grant DCSD-1662250 (EGT); National Institute of Health grant HL128790 (EGT); National Institute of Health grant HL105550 (KW and KAM) and the National Institute of Health grant HL139090 (AA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The autonomic nervous system (ANS) plays an important role in regulating cardiac electrical activity. Both branches of the ANS, namely the sympathetic and parasympathetic systems, directly innervate the heart and influence cardiac electrophysiology. While stimulation of the sympathetic system increases cardiac contractility, heart rate (HR) and conductivity, stimulation of the parasympathetic system has an opposite effect. In many disease conditions, including hypertension and chronic heart failure, there is an imbalance between these two branches of the ANS [1–4].

Parasympathetic modulation of cardiac output is mediated primarily by acetylcholine (ACh) release from the vagus nerve which activates M2 muscarinic receptors (M2R). In sino-atrial nodal cells and atrial myocytes, the activation of M2R triggers the release of inhibitory G proteins, which then proceed to modulate the activity of multiple downstream targets. Among these is the activation of the G protein-gated inwardly rectifying K+ channel IKACh, a heterotetrameric complex formed by GIRK1 and GIRK4 subunits which exerts a prominent effect on both HR and HR variability (HRV) [5, 6]. Girk4–/–mice, which exhibit a complete loss of IKACh activity, are less sensitive to carbachol (CCh)-induced bradycardia, show reduced HRV, and a resistance to pacing-induced atrial fibrillation [7]. Previous studies have also shown that M2R-IKACh signaling is negatively regulated by regulator of G protein signaling (RGS) proteins [8–10]. In particular, a dimer consisting of the atypical G protein ß subunit Gß5 and RGS6, a member of the R7 RGS family of RGS proteins, has been shown to prominently accelerate IKACh deactivation kinetics in sinoatrial nodal cells and atrial myocytes. Loss of RGS6 in mice results in an increase in IKACh activity which, in contrast to Girk4–/–mice, correlates with an increase in CCh induced bradycardia, increase in HRV, and increase in susceptibility to pacing induced AF [8–10].

While the M2R-IKACh signaling pathway is a critical mediator of parasympathetic influence on the atria, growing evidence suggests that a similar signaling pathway may exist in the ventricle [11]. For example, GIRK4 mRNA and protein are present, albeit at lower levels than in atria, in the ventricle of many species [8, 11, 12]. In addition, pharmacologic blockade or genetic ablation of GIRK4 correlates with diminished influence of ACh on action potential duration (APD) and resting membrane potential in rodent ventricular tissue. Similarly, RGS6 has been detected in the ventricle [8–10], however its impact on ventricular physiology is less well defined. Given the growing appreciation for parasympathetic innervation of the ventricles [13–16], there has been interest in understanding the molecular components which mediate parasympathetic input on ventricular physiology and arrhythmogenesis.

The main objective of our study was to gain deeper insight into the impact of parasympathetic signaling on ventricular electrophysiology. To this end, we used in-vivo ECG measurements and ex-vivo optical mapping to evaluate cardiac physiology in mice lacking GIRK4 or RGS6. Our findings show that RGS6 exerts a prominent influence on key supraventricular and ventricular physiological variables, and that its relationship with IKACh-dependent signaling likely differs between the atria and ventricle.

Materials and methods

All experiments were approved by and done in accordance with the guidelines of the Institutional Animal Care and Use Committee at the University of Minnesota. Girk4-/- and Rgs6-/- lines were created as previously described [10, 17]. Mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and housed at University of Minnesota's Research Animal Resources (RAR) facility. Mice were group-housed on a 12-h light/dark cycle, and given free access to food and water. Experiments were done on 2–3 month old mice of either gender. At the end of the experiments, animals were sacrificed by exsanguination while under anesthesia (ketamine/xylazine).

In-vivo ECG measurements

Mice were anesthetized with ketamine and xylazine (35 and 5 mg/kg i.p., respectively) and ECG recordings were performed using IX-ECG12 recorder interfaced with Labscribe software (v2, iWorx Systems Inc., Dover, New Hampshire). After 20 min of stabilization period, 10 min of baseline ECG was recorded. Subsequently, 300 nM of CCh was administered via tail vein injection (ex-vivo EC50 = 0.2 uM [18]). The final concentration of CCh was estimated using approximated blood volume based on body weight of each mouse measured at the beginning of the experiment. CCh has been reported to have a relatively long half-life, and a duration of action lasting 4–8 hours in humans, with topical administration [19]. Hence, after a 5 min waiting period for the effect of CCh to reach equilibrium, 10 min of ECG was recorded. The mice were then sacrificed by exsanguination. The 10 min baseline and post-CCh intervals of ECG recordings were used to calculate HR and HRV, and to identify the occurrence of arrhythmic episodes induced by administration of CCh. Any noise artifacts were removed through data filtering and only clean segments of ECG data were used. A maximum HR threshold was set to 1000 bpm for baseline and mean baseline HR + 25 bpm for evaluating effect of CCh, in order to eliminate artifacts and erroneous spikes in HR. HRV was calculated as a ratio of standard deviation of HR to mean HR, as described previously [20]. Number of mice that exhibited arrhythmic episodes after injection of CCh were also quantified. An arrhythmic episode was defined as an occurrence of either bradycardia (at least 25 bpm < mean post CCh HR) or tachycardia (at least 25 bpm > post CCh HR) that lasted for more than a 2-s duration.

Ex-vivo optical mapping experiments

Isolated ex-vivo whole heart optical mapping experiments were performed as described previously [21–23]. Briefly, mice were first heparinized (55 U/10g) to avoid blood clots during heart excision. Ketamine and xylazine (100 mg/kg and 10 mg/kg i.p., respectively) were administered to anesthetize the animal, after which hearts were quickly excised and immersed in cold cardioplegic solution (in mM): glucose 280, KCl 13.44, NaHCO3 12.6, and mannitol 34. The aorta was isolated under microscope for cannulation and the hearts were retrogradely perfused using Langendorff perfusion setup with warm (37±1°C) Tyrode’s solution (in mM): NaCl 130, CaCl2 1.8, KCl 4, MgCl2 1.0, NaH2PO4 1.2, NaHCO3 24, glucose 5.5, and pH 7.4. After 15 min of stabilization, a bolus of the voltage-sensitive dye (di-4-ANEPPS, 5 μg/mL) was injected into the hearts. Two green lasers (532 nm, Shanghai Dream Lasers Tech., Shanghai, China) were used to excite the hearts and the fluorescence signal emitted was recorded from the left ventricular (LV) surface by a fast high-resolution (14-bit, 80x80 pixels, 1000 frames/s) CCD camera (Little Joe, RedShirt Imaging, SciMeasure). Mechanical uncoupler Blebbistatin (10 μM) was added to the perfusate to reduce motion artifacts when required. Background fluorescence was eliminated during analysis by using spatial (3 x 3 pixels) and temporal convolution filters.

The hearts were paced at progressively decreasing basic cycle lengths (BCL), starting from 150 ms down to 90 ms in steps of 10 ms. At each BCL, 40 stimuli were applied to reach steady state, and optical movies corresponding to the last 10 stimuli were recorded and later analyzed. After capturing baseline recordings, hearts were injected with a bolus of CCh, either 300 nM or 3 μM. After 10 min of stabilization, similar pacing protocol was repeated and optical movies were captured.

Ex-vivo parameter measurements

Optical movies were analyzed to characterize the electrophysiological properties of the LV. The APD was calculated at 80% (APD80) repolarization. For each mouse, average of 5 action potentials was used to calculate mean APD80 at each pixel. Two dimensional (2D) APD maps were then constructed as described previously [21–23] to demonstrate the spatial distribution of APD. For each heart and at each BCL, average APD value for the entire LV surface was then calculated from individual pixel values. To investigate the dispersion of repolarization in LV, APD80 heterogeneity was calculated based on the heterogeneity index μ as, where X is APD80, X95 and X5 represent the 95th and 5th percentiles of the APD80 distribution, and X50 is the median of the APD80 distribution. Conduction velocity (CV) was calculated as described previously [23]. Specifically, the distributions of activation times measured at (dV/dt)max for the spatial regions of 3 x 3 pixels were fitted with the plane, and gradients of activation times were calculated for each plane. The magnitude of the local CV was calculated for each pixel. APD restitution curves were generated by plotting mean APD80 as a function of mean diastolic interval (DI = BCL–APD80). The maximum slope of APD restitution, Smax, was calculated by fitting with 2nd degree polynomial and evaluating its derivative at the lowest DI value for each run. Mean Smax was then calculated for each genotype both at baseline and post administration of CCh.

Statistics

All data are presented as mean ± standard error (SEM). Statistical comparisons were analyzed using 1-way ANOVA and Fisher’s exact test, as appropriate (Origin Software, Northampton, MA, USA). In addition, 2-way ANOVA was performed between genotype, CCh dosage and APD80 to investigate the role of GIRK4 or RGS6 in modulating the effects of CCh on APD prolongation. P<0.05 was the set level of significance.

Results

In-vivo effect of parasympathetic modulation on HR and HRV

To confirm the impact of M2R-IKACh signaling on atrial physiology, HR and HRV was assessed in WT, Girk4–/–, and Rgs6–/–mice at baseline and after CCh injection using in-vivo ECG recordings from anesthetized mice. Consistent with previous reports, Rgs6-/- mice exhibited significantly lower baseline HR compared to WT mice (Fig 1A). Conversely, Girk4-/- mice displayed slightly higher baseline HR, however, this effect was not significant (Fig 1A). Injection of CCh evoked a significant decrease in HR for both WT and Rgs6-/- mice, whereas no significant change in HR was seen for Girk4-/- mice (Fig 1A). Furthermore, CCh significantly increased HRV in both WT and Rgs6-/- mice, while this effect was absent in Girk4-/- mice (Fig 1B). In addition, Girk4-/- mice were resistant to the onset of arrhythmias post injection of CCh, as opposed to both Rgs6-/- and WT mice (Fig 1C). These results confirm previously reported phenotypes describing the prominent role the M2R-IKACh signaling pathway plays in mediating atrial physiology and how RGS6 is a critical regulator of its influence.

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Fig 1. The influence of M2R-IKACh signaling on in vivo HR and HRV.

Summary of baseline and post-CCh (300nM CCh) in-vivo HR (A) and HRV (B) in WT, Rgs6-/-, and Girk4-/- mice. (‘*’ denotes statistical significance of p < 0.05 between baseline and CCh within the same genotype. ‘&’ denotes a statistically significant (p < 0.05) difference for both baseline and CCh when comparing between two genotypes). ‘#’ denotes statistical significance of p < 0.05 between WT and Rgs6-/- mice post-CCh. ‘$’ denotes statistical significance of p < 0.05 between Girk4-/- and Rgs6-/- mice post-CCh. Statistics performed using 1-way ANOVA.) (C) Quantification of the total number of mice that exhibited arrhythmias post CCh. (‘*’ denotes statistical significance of p < 0.05 between Girk4-/- and Rgs6-/-. Statistics performed using Fisher’s exact test). (D) Representative examples of ECG data during control and demonstrating episodes of arrhythmia in WT and Rgs6-/-, and no arrhythmia in Girk4-/- mice post CCh. n = 8, 8, 6 for WT, Rgs6-/-, and Girk4-/-, respectively.

https://doi.org/10.1371/journal.pone.0193798.g001

Ex-vivo optical mapping assessment of M2R-IKACh signaling in the mouse ventricle

While the atrial influence of M2R-IKACh has been well described [8–10], only recently have there been suggestions of an IKACh-like channel in the ventricle [11, 12, 16]. To investigate the influence of M2R-IKACh on ventricular physiology, we utilized optical mapping of the LV, a technique which allows for the simultaneous examination of multiple characteristics of ventricular electrophysiology, such as APD, CV, APD heterogeneity μ and slope of APD restitution curve, Smax.

Action potential duration

Optical movies taken from the LV were used to assess the impact of GIRK4 or RGS6 ablation on ventricular APD80. Notably, and consistent with the expected impact of K+ channels on repolarization, hearts from Girk4–/–mice showed a significant prolongation in baseline APD80 relative to WT controls at almost all BCLs (Fig 2A). Surprisingly, Rgs6-/- mice also exhibited prolonged APD80, and this effect was significant only at higher BCLs (Fig 2A). Consistent with previous studies [16, 24, 25], CCh stimulation significantly increased APD80 across all genotypes and in a dose-dependent fashion (Fig 2C–2E). Since 3 μM CCh terminated beating in Rgs6-/- mice, only the lower concentration of CCh (300 nM) was shown in these hearts. Collectively, these data suggest that while IKACh impacts baseline ventricular APD in a predictable fashion, the influence of RGS6 is likely IKACh-independent. Furthermore, statistical analysis of the effect of CCh on APD across genotypes that was performed using 2-way ANOVA, did not reveal any significant differences. This suggests that although CCh significantly prolonged APD80 in all genotypes, the signaling pathway did not seem to meaningfully impact the effect of CCh on ventricular APD80.

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Fig 2. The effect of GIRK4 and RGS6 ablation on APD80.

(A) Change in average APD80 with decreasing BCL in WT, Girk4-/-, and Rgs6-/- hearts. (‘#’ denotes statistical significance of p < 0.05 between WT and Girk4-/-. ‘$’ denotes statistical significance of p < 0.05 between WT and Rgs6-/-). (B) Representative 2D APD80 maps from WT, Girk4-/-, and Rgs6-/- hearts, constructed at BCL = 120 ms both at baseline and post-CCh injection. Representative action potential traces are shown at baseline (top panel, pixels marked by *) and post-CCh (bottom panel, pixels marked by Δ). (C-E) The effect of CCh on APD80 at decreasing BCL in WT, Girk4-/-, and Rgs6-/- hearts. (‘*’ denotes statistical significance of p < 0.05 between baseline and 300nM CCh; ‘&’ denotes statistical significance of p < 0.05 between baseline and 3uM CCh). n = 8, 5, 8 for WT, Rgs6-/-, and Girk4-/-, respectively. All statistics performed using 1-way ANOVA.

https://doi.org/10.1371/journal.pone.0193798.g002

Slopes of APD restitution curve (Smax)

The slope of APD restitution curve describes the relationship between APD and preceding DI, and a steep maximum slope Smax > 1 is associated with an increase in arrhythmia susceptibility [26, 27]. Our results indicate that CCh administration yielded a Smax > 1 in all mice, indicating a pro-arrhythmic effect [26, 27], irrespective of genotype (Fig 3). In the case of WT and Girk4-/- hearts, 3 μM CCh yielded a significantly larger Smax, but there was no significant effect of 300 nM CCh (Fig 3). In contrast, hearts from Rgs6-/- mice showed significant steepening of Smax at 300 nM CCh (Fig 3).

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Fig 3. The effect of GIRK4 and RGS6 ablation on Smax.

Summary of the baseline and post-CCh maximum slopes of APD restitution, Smax, in WT (n = 8), Girk4-/- (n = 8) and Rgs6-/- (n = 5) mice. (‘*’ denotes statistical significance of p < 0.05 between post-CCh and baseline for the same genotype. Statistics performed using 1-way ANOVA).

https://doi.org/10.1371/journal.pone.0193798.g003

CV and APD80 heterogeneity, μ

Next, we measured the LV CV, a measurement of APD propagation across the ventricle, with decreasing BCL. We found that there was no impact of genotype on baseline CV values (Fig 4A). Similarly, no significant effect of CCh on LV CV was observed in any of the genotypes (Fig 4C–4E). Representative activation maps demonstrate smooth propagation of action potentials for all the three groups both at baseline and post CCh (Fig 4B). The change in LV APD80 heterogeneity, μ, was also measured, given the critical role heterogeneity plays in arrhythmogenesis. Again, there were no significant differences in APD heterogeneity observed between the three mice groups, both at baseline and post injection of CCh (Fig 5A–5D).

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Fig 4. The effect of GIRK4 and RGS6 ablation on CV.

(A) Average CV as a function of BCL in WT, Girk4-/-, and Rgs6-/- hearts. (B) Representative 2D activation maps from WT, Girk4-/-, and Rgs6-/- hearts, constructed at BCL = 150 ms both at baseline and post-CCh injection. (C-E) The effect of CCh on CV at different BCL in WT, Girk4-/-, and Rgs6-/-. n = 8, 5, 8 for WT, Rgs6-/-, and Girk4-/-, respectively. Statistics performed using 1-way ANOVA.

https://doi.org/10.1371/journal.pone.0193798.g004

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Fig 5. The effect of GIRK4 and RGS6 ablation on APD heterogeneity, μ.

(A) Average APD heterogeneity, μ, as a function of BCL in WT, Girk4-/-, and Rgs6-/- hearts. (B-D) The effect of CCh on μ at different BCL in WT, Girk4-/-, and Rgs6-/- hearts. n = 8, 5, 8 for WT, Rgs6-/-, and Girk4-/- respectively. Statistics performed using 1-way ANOVA.

https://doi.org/10.1371/journal.pone.0193798.g005

Discussion

In this study, we investigated the impact of the M2R-IKACh signaling pathway on ventricular electrophysiology using both Girk4–/–and Rgs6–/–mice. There is a body of published evidence supporting the contention that a GIRK4-containing GIRK channel is expressed in the mouse ventricle, and that it mediates the effects of cholinergic input on ventricular myocyte excitability [11, 12]. Our current work supports a critical role for this signaling pathway on the parasympathetic regulation of HR and HRV, while also suggesting intriguing differences in the roles of key pathway elements on the parasympathetic regulation of ventricular physiology. Indeed, our efforts support the emerging perspective that RGS6 impacts multiple electrophysiological parameters in the ventricle, likely apart from any influence it has on signaling involving a ventricular IKACh-like channel.

Our major findings are as follows: (1) Ablation of Girk4 and RGS6 can lead to electrophysiological remodeling and affect overall cardiac dynamics. (2) Parasympathetic stimulation of Girk4-/- and Rgs6-/- mice leads to a dose-dependent prolongation in APD80 and steepening of APD restitution. (3) While the M2R-Girk4-RGS6 pathway plays an important role in regulating cardiac dynamics, the effects of parasympathetic stimulation of the ventricles could be mediated via M2R-IKACh independent pathways.

In-vivo ECG

Our in-vivo ECG data confirmed many previously reported phenotypes of Girk4-/- and Rgs6-/- mice, highlighting the opposing influences that RGS6 and IKACh exert on atrial physiology. Rgs6-/- mice displayed significantly lower baseline HR as well as exaggerated responses to CCh-induced bradycardia and increases in HRV, all of which have been previously reported [5–11]. Conversely, Girk4-/- mice displayed blunted responses to the impact of CCh on HR and HRV. While the baseline HR in the Girk4-/- mice was higher than that of the WT mice, this effect was not significant. Lastly, we also show that Rgs6-/- mice were more susceptible to CCh-induced arrhythmic episodes, whereas Girk4-/- mice were resistant. These data further emphasize the contrasting roles IKACh and RGS6 play in mediating parasympathetic influence on atrial physiology.

Ex-vivo results

To characterize the potential influence of IKACh and RGS6 on ventricular electrophysiology, we employed, for the first time, a high resolution optical mapping of the ventricle to analyze 2D APD80, CV and APD heterogeneity. As expected, we observed a prolongation in APD80 at baseline in Girk4-/- mice, consistent with previous reports and the presence of a functional IKACh-like channel in ventricular myocytes [11]. Although previous studies have investigated the effect of RGS6 ablation on atrial APD [9, 10], its role and impact on ventricular electrophysiology is relatively unknown. This is the first study to investigate the effect of RGS6 ablation on APD restitution and characterize its effect on ventricular APD at different pacing rates. Surprisingly, hearts from Rgs6-/- mice also showed a prolongation in APD80, particularly at higher BCLs. Our results strongly indicate that RGS6 does play an important role in ventricular repolarization, while also arguing that this influence is not mediated via its influence on a ventricular IKACh-like channel. This is also supported by the fact that at 300 nM of CCh, there was a significant increase in the slope of APD restitution in Rgs6-/- mice, but no difference between WT and Girk4–/–mice. Given the lack of impact GIRK4 ablation had on this parameter, this further suggests an IKACh-independent mechanism for RGS6 influence in mediating ventricular physiology. Indeed, RGS6 has been shown to impact cardiac physiology independent of its GTPase activity via reactive oxygen species (ROS)-dependent mechanisms [28–30]. Specifically, RGS6 mediates doxorubicin-induced cardiotoxicity through a ROS mechanism. Furthermore, it has also been reported that loss of RGS6 predisposes the ventricle to pro-death signaling through a β2 Adrenergic and G protein-coupled receptor kinase 2-dependent signaling mechanism [29]. Hence, it is possible that the pronounced effect of CCh in Rgs6-/- mice could be the result of indirect effects of parasympathetic stimulation or accentuated antagonism.

CCh increased APD80 in hearts from WT, Girk4–/–, and Rgs6-/- mice, consistent with previously reported effects of vagal nerve stimulation (VNS) on ventricular APD. The APD80 after CCh injection, at both 300 nM and 3 μM, was not significantly different across all genotypes. This indicates that M2R-IKACh signaling does not significantly mediate the influence of CCh on APD80. This is also supported by the fact that there was no difference in the CCh increase in the slopes of APD restitution between WT and Girk4–/–mice. CCh did increase the slope of APD restitution in Rgs6-/- mice at a lower concentration than WT and Girk4–/–mice. This suggests an IKACh-independent mechanism for RGS6 influence in mediating ventricular physiology. Indeed, supporting our current finding, it has been shown previously that the anti-arrhythmic effects of vagal stimulation can occur via nitric oxide, independent of muscarinic receptor activation [24, 25]. Additionally, there was no significant effect of either GIRK4 or RGS6 ablation on CV or APD heterogeneity. Since CV is predominantly affected by sodium channel activity, a lack of significant effect of IKACh on ventricular conduction was not surprising.

Recently, significant interest has been placed on understanding the molecular mechanisms underlying vagal input to the ventricles given innovative investigations into the efficacy of VNS as a therapy for metabolic and cardiovascular diseases. Multiple studies have reported beneficial effects of VNS in restoring autonomic imbalance by parasympathetic activation of the healthy [31] and diseased [32–35] hearts. One previous study [31] suggests that chronic VNS leads to beneficial electrophysiological remodeling of the heart and therefore has a potential to treat cardiovascular diseases such as hypertension and myocardial ischemia. Therefore, a full understanding of the molecular mechanisms underlying vagal effects on cardiac physiology, which include M2R-IKACh signaling, is necessary. In that regard, this study contributes to understanding the effects of parasympathetic modulation on ventricular electrophysiology.

Limitations

Since the in-vivo ECGs in this study were acquired under anesthesia, baseline HRs were much lower than what is observed in conscious mice (Fig 1A) [36]. However, similar quantities of anesthesia were administered for all mice and effect of CCh was compared to baseline recordings in the same animal with no change in anesthesia administration during the experiment. In addition, a 20-minute stabilization window was used for each mouse prior to recording baseline HR, to enable the effect of anesthesia to stabilize. Hence, although the overall lower HRs could be attributed to the effect of anesthesia in each mouse [37] the differences in HRs observed can be attributed to the effect of genotype and CCh administration. Furthermore, the current study utilizes knock out mice with GIRK4 or RGS6 completely ablated in both the atria and ventricles. Future studies could be performed utilizing a pharmacological IKACh blocker to investigate the relative importance of this pathway under varying conditions. In addition, ventricle and atria specific GIRK4 or RGS6 knock out models could be utilized to isolate the electrophysiological effects observed, to atrial or ventricular signaling pathways.

Acknowledgments

The authors would also like to thank Nicholas Carlblom for his assistance with the experiments.

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