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

Original data for the graphs in Figs 4 and S7 and S8.

Each tab includes data for individual panels of Figs 4 and S7 and S8. (XLSX)</p

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

Original data for the graphs in Figs 2 and S3.

Each tab includes data for individual panels of Figs 2 and S3. (XLSX)</p

Effects of K⁺ channel blockers on RMP of NPY neurons.

Related to Fig 1. (A) Trace demonstrates depolarizing effects of linopirdine and XE991, M channels blockers. (B) Trace demonstrates no effects of PK-THPP, a TASK-3 channel blocker. (C) Trace demonstrates no effects of spadin, a TREK-1 channel blocker. (D) Trace demonstrates no effects of tolbutamide, a KATP channel blocker. (E–H) Bar graphs and dots summarize effects on RMP change of linopirdine and XE991 (from −40.4 ± 0.7 mV to −39.5 ± 0.7 mV, n = 12, df = 11, t = 1.650, p = 0.127) (E), PK-THPP (from −42.5 ± 1.0 mV to −42.1 ± 0.8 mV, n = 12, df = 11, t = 0.890, p = 0.393) (F), spadin (from −41.9 ± 1.1 mV to −42.3 ± 1.0 mV, n = 13, df = 12, t = 1.866, p = 0.087) (G), and tolbutamide (from −42.2 ± 0.7 mV to −41.7 ± 0.8 mV, n = 13,df = 12, t = 1.879, and p = 0.085) (H). Red and black lines indicate changes of membrane potential in depolarized and nonresponsive neurons, respectively. Data are presented as mean ± SEM. Paired t test was used for statistical analyses. ns = not significant. The numerical data for S2E–S2H Fig can be found in S1 Data. (TIF)</p

Effects of CGP54626 on NPY^G2WT neurons.

Related to Fig 3. (A) Image demonstrates no effects of CGP54626 on NPYG2WT neurons. Dotted line indicates RMP. (B) Lines and dots summarize effects of CGP54626 on RMP (from −42.9 ± 0.8 mV to −43.2 ± 0.8 mV, n = 12, df = 11, t = 2.191, p = 0.051). (C) Lines and dots summarize effect of CGP54626 on input resistance (from 2.68 ± 0.20 GΩ to 2.71 ± 0.21 GΩ, n = 12, df = 11, t = 0.519, p = 0.614). Paired t test was used for statistical analyses. ns = not significant. The numerical data for S6B and S6C Fig can be found in S3 Data. (TIF)</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

Expression of <i>Girk</i> mRNA by arcuate AgRP neurons.

Related to Fig 2. (A) Graph demonstrates percentage of Agrp (+) neurons that express mRNA of Girk1 and/or Girk2. Girk1 (green): Girk1-containing Agrp (+) neurons; Girk2 (magenta): Girk2-containing Agrp (+) neurons; Girk1 and Girk2 (gray): Agrp (+) neurons containing both Girk1 and Girk2. n = 3. (B) Graph demonstrates percentage of Agrp (+) neurons that express mRNA of Girk1 and/or Girk3. Girk1 (green): Girk1-containing Agrp (+) neurons; Girk3 (cyan): Girk3-containing Agrp (+) neurons; Girk1 and Girk3 (gray): Agrp (+) neurons containing both Girk1 and Girk3. n = 3. (C) Graph demonstrates percentage of Agrp (+) neurons that express mRNA of Girk1 and/or Girk4. Girk1 (green): Girk1-containing Agrp (+) neurons; Girk4 (orange): Girk4-containing Agrp (+) neurons; Girk1 and Girk4 (gray): Agrp (+) neurons containing both Girk1 and Girk4. n = 3. (D) Graph demonstrates percentage of Agrp (+) neurons that express mRNA of Girk2 and/or Girk3. Girk2 (magenta): Girk2-containing Agrp (+) neurons; Girk3 (cyan): Girk3-containing Agrp (+) neurons; and Girk2 and Girk3 (gray): Agrp (+) neurons containing both Girk2 and Girk3. n = 3. Data are presented as mean ± SEM. Twelve hypothalamic slices from each mouse (from bregma −1.58 mm to −2.02 mm) were included for analyses. See text for specific values. The numerical data for S3A–S3D Fig can be found in S2 Data. (TIF)</p

Role of GIRK2-containing GIRK channels in GABA_B-activated K⁺ current recorded from NPY neurons.

Related to Fig 3. (A) Image demonstrates outward currents by local application of 100 μm baclofen. Voltage ramp pulses (from −120 mV to −10 mV, 100 mV/s) were applied as indicated by arrows, a and b, to obtain current responses, Ia and Ib. (B) Image demonstrates current–voltage (I-V) relationship of baclofen-activated currents (IBac); IBac was calculated by subtracting current responses (Ib- Ia) obtained in (A). (C) Rectification index was calculated by obtaining the ratio of amplitudes at −120 mV (I-120 mV) and −60 mV (I-60 mV) in 12 NPY neurons. (D, E) Images demonstrate IBac recorded from NPYG2WT (black) and NPYG2KO (red) neurons using 10 μm (D) or 100 μm (E) baclofen. (F, G) Image summarizes normalized amplitudes of IBac recorded from NPYG2WT (black) and NPYG2KO (red) neurons using 10 μm baclofen (1.4 ± 0.1 pA/pF, n = 32, for NPYG2WT and 1.4 ± 0.1 pA/pF, n = 23, for NPYG2KO, df = 53, t = 0.276, p = 0.783) (F) and 100 μm baclofen (1.8 ± 0.1 pA/pF, n = 53, for NPYG2WT and 1.8 ± 0.2 pA/pF, n = 26, for NPYG2KO, df = 77, t = 0.021, and p = 0.984) (G). Data are presented as mean ± SEM. Unpaired t test was used for statistical analyses. ns = not significant. The numerical data for S4C, S4F, and S4G Fig can be found in S3 Data. (TIF)</p

Deletion of GIRK2, but not GIRK1, leads to increased Fos expression by the arcuate AgRP neurons.

(A) Images demonstrate Fos IHC results from AgrptdTomato, AgrptdTomato/Girk1KO, and AgrptdTomato/Girk2KO mice, as indicated. 3V = third ventricle. Scale bar = 50 μm. (B) Bar graphs and dots summarize proportion of Fos-expressing AgRP neurons in AgrptdTomato (56.0 ± 3.2%, n = 6, black), AgrptdTomato/Girk1KO (64.6 ± 2.6%, n = 4, gray), and AgrptdTomato/Girk2KO (71.7 ± 4.8%, n = 4, red). Twelve hypothalamic slices from each mouse (from bregma −1.46 mm to −2.06 mm) were included for analyses. Data are presented as mean ± SEM. Ordinary one-way ANOVA with Bonferroni correction was used for statistical analyses (df = 2, F2, 11 = 4.961, p = 0.029). *p S4 Data. AgRP, agouti-related peptide; GIRK, G protein-gated inwardly rectifying K+; IHC, immunohistochemistry.</p