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

Additional file 1: Table S1. of GNB5 mutation causes a novel neuropsychiatric disorder featuring attention deficit hyperactivity disorder, severely impaired language development and normal cognition

Clinical summary of the study patients. Clinical characteristics of the five patients included in this study with homozygous GNB5 mutation. (DOCX 14 kb

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

Effect of Gβ5 on dopamine-mediated activation of D2R-coupled G protein signaling as measured by a fast kinetic BRET assay.

<p><b>A.</b> Average BRET dose-repsonse curve elicited by the application of 10 pM - 10 µM concentrations of dopamine to cells expressing only D2R (black trace) and cells transiently coexpressing low (dark grey) and high (light grey) levels of Gβ5. D2R stimulation by dopamine application leads to the dissociation of the G protein heterotrimer into Gβγ-Venus and GTP-bound Gαo subunits. Free Gβγ-Venus interacts with masGRK3ct-NanoLuc to produce a BRET signal. The black trace is from HEK293 cells that did not coexpress Gβ5 and the dark and light grey traces are from HEK293 cells transiently coexpressing two different levels (l, for low and h, for high) of Gβ5 (mean ± SEM; n = 4). <b>B.</b> Quantification of the maximal amplitude (E_max) of the BRET signal elicited by the application of dopamine in the cells described above. The E_max only significantly differed between D2R and D2R + Gβ5 (h) (*p<0.01, ANOVA followed by Tukey’s post-hoc test). <b>C.</b> Quantification of the average EC₅₀ derived for the dopamine mediated activation of D2R in the cells described in D. The response at each dose is expressed as a percent of the average maximal response (n = 4). The EC₅₀ (in nM) for D2R (black bar), D2R + Gβ5 (l, dark grey bar), and D2R + Gβ5 (h, light grey bar) was 19.8±0.825, 21.3±0.863, and 25.4±0.431 (mean ± SEM), respectively. The EC₅₀ only significantly differed between D2R and D2R + Gβ5 (h) (*p<0.01, ANOVA followed by Tukey’s post-hoc test). <b>D.</b> Averaged traces (± SEM) of changes in the BRET signal (ΔBRET or the BRET response) over time obtained from HEK293 cells transfected with cDNA for D2R, Gαo, Venus-Gβγ, masGRK3ct-NanoLuc and treated sequentially with dopamine (10 nM) and haloperidol (100 µM). <b>E.</b> Quantification of the deactivation kinetics of the dopamine-elicited BRET response after application of the D2R antagonist, haloperidol (100 µM).</p

Coexpression of D2R enhances the stability of Gβ5.

<p><b>A.</b> Representative image of a Western blot which depicts Gβ5 cellular expression levels levels (upper panel) or total cellular protein (lower panel), from HEK293 cells transiently expressing either Gβ5 alone or Gβ5 coexpressed with D2R, at times, t = 0, 3, or 6 hr after treatment with cycloheximide (100 µM). <b>B.</b> Quantification of the reduction in cellular Gβ5 levels after treatment of cells with cycloheximide. The Gβ5 levels at times 3 and 6 hr after cycloheximide treatment are expressed as a percentage of the levels of Gβ5 measured in cells that were not treated with cycloheximide (mean ± SEM; n = 4, *p<0.05, t-test, comparing to the cells that did not coexpress D2R).</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

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

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

Targeting of Gβ5 to the TX100-insoluble fraction upon coexpression of D2-like dopamine receptors, D2R and D4R.

<p><b>A.</b> Representative image of a Western blot depicting the segregation of Gβ5 (upper panels) and total protein (lower panels) into TX100-soluble (S) and insoluble (I) biochemical fractions prepared from HEK293 cells transfected with cDNAs for the indicated proteins. <b>B.</b> Quantification of the relative levels of Gβ5 segregating into TX100-soluble (white bars) and TX100-insoluble (black bars) biochemical fractions expressed as percentage of the total cellular Gβ5 signal from the respective cellular samples (mean ± SEM; n = 4, *p<0.01, t-test relative to cells expressing Gβ5 alone. <b>C.</b> Representative image of a Western blot depicting the segregation of the respective FLAG-tagged dopamine receptor proteins, D2R and D4R, into TX100-soluble (S) and insoluble (I) biochemical fractions prepared from HEK293 cells transfected with the indicated cDNAs. <b>D.</b> Quantification of the relative levels of D2R and D4R segregating into TX100-soluble (white bars) and TX100-insoluble (black bars) biochemical fractions prepared from cell samples indicated in C (mean ± SEM; n = 4). <b>E.</b> Representative image of a Western blot depicting the segregation into TX100-soluble (S) and insoluble (I) biochemical fractions of transiently expressed KRAS-BL (upper panels) and total protein (lower panels) in HEK293 cells and effect of transient coexpression of D2R on such segregation. <b>F.</b> Quantification of the relative levels of KRAS-BL segregating into TX100-soluble (white bars) and TX100-insoluble (black bars) biochemical fractions (mean ± SEM; n = 4).</p