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

GIRK channels stabilize RMP of NPY neurons.

(A) Brightfield illumination (Brightfield), fluorescent (FITC) illumination (Npy-hrGFP), fluorescent (TRITC) illumination (Alexa Fluor 594), and merged (Merge) images of targeted NPY neuron. Arrows indicate the cell targeted for whole-cell patch clamp recording. (B) Image demonstrates a depolarizing effect of tertiapin-Q. Dotted line indicates RMP. (C) Voltage deflections in response to small hyperpolarizing current steps (from −25 pA to 0 pA by 5 pA increments) before (control, black) and after (tertiapin-Q, red) the perfusion with tertiapin-Q as indicated by arrows in (B). (D) The voltage–current (V-I) relationship demonstrates increased input resistance by tertiapin-Q. Erev = reversal potential. (E) Lines and dots summarize effects of tertiapin-Q on RMP (from −47.7 ± 3.0 mV to −44.9 ± 2.1 mV, n = 11, df = 10, t = 2.787, p = 0.019). Red and black lines indicate changes of membrane potential in depolarized and nonresponsive neurons, respectively. (F) Lines and dots summarize effect of tertiapin-Q on input resistance (from 2.75 ± 0.27 GΩ to 3.03 ± 0.30 GΩ, n = 11, df = 10, t = 4.370, p = 0.001). Red and black lines indicate changes of input resistance in depolarized and nonresponsive neurons, respectively. (G, H) Lines and dots summarize effects of 100 nM tertiapin-Q (G) (from −41.2 ± 0.8 mV to −40.0 ± 1.1 mV, n = 11, df = 10, t = 2.040, p = 0.069) and 500 nM tertiapin-Q (H) (from −42.9 ± 1.2 mV to −40.5 ± 1.1 mV, n = 13, df = 12, t = 3.292, p = 0.006) on RMP. Red and black lines indicate changes of membrane potential in depolarized and nonresponsive neurons, respectively. (I) Histogram summarizes responses (no effects or depolarization) of NPY neurons to different concentrations of tertiapin-Q. (J) Bar graphs and dots summarize effects of K+ channel blockers. Each neuron was tested with only 1 K+ channel blocker. Data are presented as mean ± SEM. Paired t test was used for statistical analyses. *p p S1 Data. GIRK, G protein-gated inwardly rectifying K+; NPY, neuropeptide Y; RMP, resting membrane potential.</p

Contribution of GIRK2-containing channels to RMP and GABA_B-induced inhibition of NPY neurons.

(A) Traces demonstrate spontaneous firing and RMP of NPYG2WT (black) and NPYG2KO (red) neurons. Dotted line indicates RMP. (B, C) Bar graphs and dots summarize RMP (−47.9 ± 0.9 mV, n = 64, for NPYG2WT and −44.5 ± 0.7 mV, n = 41, for NPYG2KO, df = 103, t = 2.556, p = 0.012) (B) and input resistance (2.35 ± 0.11 GΩ, n = 64, for NPYG2WT and 2.78 ± 0.11 GΩ, n = 41, for NPYG2KO, df = 103, t = 2.590, p = 0.011) (C) of NPYG2WT (n = 64, black) and NPYG2KO (n = 41, red) neurons. (D) Image demonstrates a hyperpolarization of NPYG2WT neuron membrane potential by baclofen (10 μm). Arrows indicate interruptions to apply current step pulses. (E) Small hyperpolarizing current steps (from −50 pA to 0 pA by 10 pA increments) were applied before (control) and after (baclofen) applications of baclofen. (F) Voltage–current relationship demonstrates decreased input resistance and Erev close to EK. (G) Image demonstrates a hyperpolarization of NPYG2KO neuron membrane potential by baclofen (10 μm). (H) Summary of GABAB-induced hyperpolarization of NPYG2WT (black) and NPYG2KO (red) neurons. Changes of membrane potential by 10 μm baclofen was −11.9 ± 2.2 mV for NPYG2WT (n = 14) and −20.9 ± 2.4 mV for NPYG2KO (n = 8) (df = 20, t = 2.655, p = 0.015). Solid lines indicate fitting of dose-response curve (Hill slope = 1.0, Y = Bottom + (Top-Bottom)/(1+10^(logEC50-X)). Both hyperpolarizing and no responses were included for analyses. See Table 1 for hyperpolarizing responses only. Data are presented as mean ± SEM. Unpaired t test was used for statistical analyses. *p S3 Data. GIRK, G protein-gated inwardly rectifying K+; NPY, neuropeptide Y; RMP, resting membrane potential.</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

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

Dominant expression of <i>Girk2</i> over <i>Girk1</i> by the arcuate AgRP neurons.

(A) Image demonstrates DAPI (blue) and mRNA of Agrp (white), Girk1 (green), and Girk2 (magenta) detected by FISH experiments within the arcuate nucleus. 3V = third ventricle. Scale bar = 50 μm. (B) Magnified images of red rectangular area in (A). Dotted circles indicate Agrp (+) neurons (white) with Girk1 (green), Girk2 (magenta), or both Girk1 and Girk2 (yellow) mRNA. Scale bar = 10 μm. (C) Bar graph demonstrates numbers of Agrp (+) neurons in the arcuate nuclei of 3 wild-type mice. (D) Venn diagram demonstrates the numbers of Girk1- and/or Girk2-expressing Agrp (+) neurons. Data were pooled from neurons of 3 mice shown in (C), and 12 hypothalamic slices from each mouse (from bregma −1.58 mm to −2.02 mm) were included for analyses. The numerical data for Fig 2C can be found in S2 Data. AgRP, agouti-related peptide; FISH, fluorescence in situ hybridization; GIRK, G protein-gated inwardly rectifying K+.</p

Original data for the graphs in Fig 5.

Each tab includes data for individual panels of Fig 5. (XLSX)</p

Original data for the graphs in Figs 6 and S9.

Each tab includes data for individual panels of Figs 6 and S9. (XLSX)</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