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.</p

The effects of the Rgs6 on HRV are mediated by the I_KACh and are influenced by the m₂R activity.

<p>A, Schematic representation of the pathway targeted both genetically and pharmacologically. Abbreviations are: atropine (Atro), carbamylcholine (CCh). B, Effect of m₂R blockade by atropine on HRV in wild-type (black; n = 7) and <i>Rgs6^−/−</i> hearts (red; n = 10). No significant effect of drug was observed in wild-type hearts. C, Increased sensitivity of <i>Rgs6^−/−</i> hearts to m₂R stimulation and its rescue by I_KACh (<i>Girk4</i>) ablation. Increasing concentrations of CCh were applied to isolated perfused hearts (n = 4–6 per genotype). D, m₂R stimulation non-proportionately increased HRV in <i>Rgs6^−/−</i> hearts. Hearts (n = 3–6 per genotype) were perfused with CCh (∼IC₁₀ concentration) identified from dose-response studies, followed by measurement of changes in the RMSSD parameters. Symbols: * P<0.05 vs wild-type, #P<0.05 vs treatment.</p

Abnormal sinus arrhythmia in a human subject with dysfunctional RGS6.

<p>A, HRV measured in humans carrying variants in <i>RGS6</i> and 11 age-matched control subjects (wt, black). Lines represent upper (2σ) and lower (−2 σ) 95% confidence thresholds as determined by the 2σ rule. <i>Insert</i>: domain structure of RGS6 protein. Arrows show localization of corresponding variants. B, Representative tachograms of RR intervals from a control subject (black) and a subject heterozygous for the p.Val13LeufsX11 variant in the <i>RGS6</i> gene (red) determined from continuous Holter recordings. C, Schematics of the assay design to study effects of mutations on the RGS6 function. Stimulation of the m₂R by ACh results in the dissociation of Gμo from the heterotrimer. Released Gβγ subunits tagged with Venus become available for the interaction with Nluc8-tagged GRK reporter producing the BRET signal. D. Representative responses to sequential application of ACh (10 µM) and atropine (1 mM) recorded in the presence of the indicated constructs. The BRET signals averaged from 4 experiments were plotted as individual data points. <i>E</i>, Catalytic activity of RGS6 defined by the <i>k</i>_GAP parameter. To determine the <i>k</i>_GAP values, the deactivation rate constant measured in the absence of RGS6 was subtracted from values measured in the presence of RGS6. Symbols: ***, p<0.001 (n = 4).</p

The influence of M₂R-I_KACh signaling on in vivo HR and HRV.

<p>Summary of baseline and post-CCh (300nM CCh) in-vivo HR (A) and HRV (B) in WT, <i>Rgs6</i>^<i>-/-</i>, and <i>Girk4</i>^<i>-/-</i> 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 <i>Rgs6</i>^<i>-/-</i> mice post-CCh. ‘$’ denotes statistical significance of p < 0.05 between <i>Girk4</i>^<i>-/-</i> and <i>Rgs6</i>^<i>-/-</i> 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 <i>Girk4</i>^<i>-/-</i> and <i>Rgs6</i>^<i>-/-</i>. Statistics performed using Fisher’s exact test). (D) Representative examples of ECG data during control and demonstrating episodes of arrhythmia in WT and <i>Rgs6</i>^<i>-/-</i>, and no arrhythmia in <i>Girk4</i>^<i>-/-</i> mice post CCh. n = 8, 8, 6 for WT, <i>Rgs6</i>^<i>-/</i>-, and <i>Girk4</i>^<i>-/-</i>_, respectively.</p

The effect of GIRK4 and RGS6 ablation on APD₈₀.

<p>(A) Change in average APD₈₀ with decreasing BCL in WT, <i>Girk4</i>^<i>-/-</i>, and <i>Rgs6</i>^<i>-/-</i> hearts. (‘#’ denotes statistical significance of p < 0.05 between WT and <i>Girk4</i>^<i>-/-</i>. ‘$’ denotes statistical significance of p < 0.05 between WT and <i>Rgs6</i>^<i>-/-</i>). (B) Representative 2D APD₈₀ maps from WT, <i>Girk4</i>^<i>-/-</i>, and <i>Rgs6</i>^<i>-/-</i> 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, <i>Girk4</i>^<i>-/-</i>, and <i>Rgs6</i>^<i>-/-</i> 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, <i>Rgs6</i>^<i>-/-</i>, and <i>Girk4</i>^<i>-/-</i>_, respectively. All statistics performed using 1-way ANOVA.</p

Essential Role of the m₂R-RGS6-I_KACh Pathway in Controlling Intrinsic Heart Rate Variability

<div><p>Normal heart function requires generation of a regular rhythm by sinoatrial pacemaker cells and the alteration of this spontaneous heart rate by the autonomic input to match physiological demand. However, the molecular mechanisms that ensure consistent periodicity of cardiac contractions and fine tuning of this process by autonomic system are not completely understood.</p><p>Here we examined the contribution of the m₂R-I_KACh intracellular signaling pathway, which mediates the negative chronotropic effect of parasympathetic stimulation, to the regulation of the cardiac pacemaking rhythm. Using isolated heart preparations and single-cell recordings we show that the m₂R-I_KACh signaling pathway controls the excitability and firing pattern of the sinoatrial cardiomyocytes and determines variability of cardiac rhythm in a manner independent from the autonomic input. Ablation of the major regulator of this pathway, Rgs6, in mice results in irregular cardiac rhythmicity and increases susceptibility to atrial fibrillation. We further identify several human subjects with variants in the <i>RGS6</i> gene and show that the loss of function in RGS6 correlates with increased heart rate variability. These findings identify the essential role of the m₂R-I_KACh signaling pathway in the regulation of cardiac sinus rhythm and implicate RGS6 in arrhythmia pathogenesis.</p></div

Rgs6 and Girk4 have opposite effects on HRV in isolated hearts.

<p>A, Average HR in hearts isolated from wild-type (wt, n = 36), <i>Rgs6</i>^−/− (n = 52), and <i>Girk4</i>^−/− (n = 19) mice. B, ECG traces recorded in isolated wild-type (black), <i>Rgs6^−/−</i> (red), and <i>Girk4^−/−</i> (green) hearts. Note rhythm irregularity in <i>Rgs6</i>^−/− hearts. C, Quantification of sinoatrial dysfunction events. D–F, Representative tachograms of baseline ECG in wild-type (black), <i>Rgs6</i>^−/− (red), and <i>Girk4</i>^−/− (green) hearts. G–I, Key HRV parameters in the time and frequency domains from ECG recordings. J–L, Non-linear HRV analysis by Poincare plots for wild-type (J), <i>Rgs6^−/−</i> (K), and <i>Girk4^−/−</i> (L) hearts. Symbols: * P<0.05, ** P<0.01, ***P<0.001 vs. wild-type.</p

The effect of GIRK4 and RGS6 ablation on CV.

<p>(A) Average CV as a function of BCL in WT, <i>Girk4</i>^<i>-/-</i>, and <i>Rgs6</i>^<i>-/-</i> hearts. (B) Representative 2D activation maps from WT, <i>Girk4</i>^<i>-/-</i>, and <i>Rgs6</i>^<i>-/-</i> 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, <i>Girk4</i>^<i>-/-</i>, and <i>Rgs6</i>^<i>-/-</i>. n = 8, 5, 8 for WT, <i>Rgs6</i>^<i>-/-</i>, and <i>Girk4</i>^<i>-/-</i>_, respectively. Statistics performed using 1-way ANOVA.</p

Ablation of <i>Rgs6</i> reduces excitability of sinoatrial cells and disrupts their automaticity.

<p>A, Resting membrane potential measured immediately after obtaining whole-cell access in wild-type (wt), <i>Rgs6^−/−</i>, and <i>Girk4^−/−</i> SAN cells. B, Inward currents evoked by application of acetylcholine (ACh, 100 µM) in SAN cells from wild-type (black), <i>Rgs6^−/−</i> (red) and <i>Girk4^−/−</i> (green, no current) mice. C, Summary of steady-state ACh-induced deactivation kinetics of I_KACh in wild-type and <i>Rgs6^−/−</i> SAN cells (n = 11–15 cells/genotype). D, Representative traces of spontaneous calcium oscillations recorded from wild-type (black; n = 14) and <i>Rgs6^−/−</i> (red, n = 20) SAN cells. Arrows show skipped beats. E, Quantification of SAN arrhythmic events defined as more than 15% change in duration of peak-to-peak interval of spontaneous calcium oscillations in wild-type (n = 11) and <i>Rgs6^−/−</i> (n = 17) SAN cells. F, Reduced frequency of spontaneous calcium oscillations recorded in <i>Rgs6^−/−</i> SAN cardiomyocytes as compared to wild-type (n = 14–20 cells/genotype). G, Increased variability of spontaneous calcium oscillations in <i>Rgs6^−/−</i> SAN cells as determined by increase in RMSSD values (n = 14–20 cells per genotype). Symbols: *P<0.05; **P<0.01; ***P<0.001.</p

Inactivation of <i>Rgs6</i> disrupts cardiac rhythm in mice.

<p>A, Representative tachograms of RR intervals from wild-type (black) and <i>Rgs6^−/−</i> (red) mice recorded by ECG radiotelemetry. B and C, Summary of HRV analysis in conscious, freely-moving mice. D, Burst pacing induced AF in <i>Rgs6^−/−</i> but not in wild-type mice. Note an irregular rhythm with no discernible P waves in the <i>Rgs6^−/−</i> recording. E, Quantification of AF induction probability. Symbols: *, P<0.05.</p