RGS6, but not RGS4, is the dominant regulator of G protein signaling (RGS) modulator of the parasympathetic regulation of mouse heart rate

Abstract

Parasympathetic activity decreases heart rate (HR) by inhibiting pacemaker cells in the sinoatrial node (SAN). Dysregulation of parasympathetic influence has been linked to sinus node dysfunction and arrhythmia. RGS (regulator of G protein signaling) proteins are negative modulators of the parasympathetic regulation of HR and the prototypical M2 muscarinic receptor (M2R)-dependent signaling pathway in the SAN that involves the muscarinic-gated atrial K(+) channel IKACh. Both RGS4 and RGS6-Gβ5 have been implicated in these processes. Here, we used Rgs4(-/-), Rgs6(-/-), and Rgs4(-/-):Rgs6(-/-) mice to compare the relative influence of RGS4 and RGS6 on parasympathetic regulation of HR and M2R-IKACh-dependent signaling in the SAN. In retrogradely perfused hearts, ablation of RGS6, but not RGS4, correlated with decreased resting HR, increased heart rate variability, and enhanced sensitivity to the negative chronotropic effects of the muscarinic agonist carbachol. Similarly, loss of RGS6, but not RGS4, correlated with enhanced sensitivity of the M2R-IKACh signaling pathway in SAN cells to carbachol and a significant slowing of M2R-IKACh deactivation rate. Surprisingly, concurrent genetic ablation of RGS4 partially rescued some deficits observed in Rgs6(-/-) mice. These findings, together with those from an acute pharmacologic approach in SAN cells from Rgs6(-/-) and Gβ5(-/-) mice, suggest that the partial rescue of phenotypes in Rgs4(-/-):Rgs6(-/-) mice is attributable to another R7 RGS protein whose influence on M2R-IKACh signaling is masked by RGS4. Thus, RGS6-Gβ5, but not RGS4, is the primary RGS modulator of parasympathetic HR regulation and SAN M2R-IKACh signaling in mice.

Keywords: G Proteins; GIRK/Kir3; Gene Knockout; Heart; Muscarinic; Patch Clamp Electrophysiology; Potassium Channels; RGS Proteins; Sinoatrial Node.

FIGURE 1.

Characterization of Rgs4^−/− mice. A, schematic depiction of the Rgs4 gene, which consists of five exons (boxes 1–5). The translation initiation (ATG) and termination (STOP) codons are shown in green and red, respectively. The gray regions in exons 1 and 2 (along with intervening intronic sequence) were replaced with the lacZ-containing cassette (LacO-SA-IRES-lacZ-Neo555G/Kan) to generate B6;129P2-Rgs4^tm1Dgen/J mice. B, schematic depiction of RGS4 mRNA, with exon boundaries denoted by vertical lines and numbers. The gray domain corresponds to the 58-bp fragment of coding sequence missing in the RGS4 mutant mice. Yellow highlighting shows the location of coding sequence for the catalytic (RGS) domain of RGS4. Arrows denote the positions and identities of the primer sets (A+/− and B+/−) used for quantitative RT-PCR. C, quantitative RT-PCR analysis of RGS4 expression in cardiac tissues (SAN and left atria) and hippocampi from wild-type and B6;129P2-Rgs4^tm1Dgen/J mice using the primer sets depicted in C. Expression levels were normalized within samples to GAPDH (the levels of which were comparable in all tissues examined) and to wild-type samples for each primer set. ***, p < 0.001 versus wild-type mice (within tissue and primer set by t test). D, immunoblotting for RGS4 in primary hippocampal cultures (10 days in vitro) from wild-type and B6;129P2-Rgs4^tm1Dgen/J mice. Cultures were pretreated with MG132 (50 μm) for 6 h prior to protein isolation. E, quantification of RGS4 immunoblotting data (n = three separate experiments). A significant impact of group was observed (F_3,11 = 29.8; p < 0.001). ***, p < 0.001 versus wild-type mice (untreated).

FIGURE 2.

Impact of RGS ablation on HR and CCh-induced bradycardia. A, HR in isolated hearts from wild-type (wt) and Rgs^−/− mice. A significant effect of genotype on HR was observed (F_3,94 = 2.7, p < 0.05; n = 4–12/genotype). B, impact of CCh on HR in wild-type and Rgs^−/− hearts (n = 4–8/genotype). Data are normalized to baseline HR. A significant impact of genotype was observed for normalized HR (F_3,101 = 21.0, p < 0.001). Hill coefficients for each curve are listed. A significant impact of genotype was observed for Hill coefficients (F_3,135 = 3.7, p = 0.013). Specifically, Rgs4^−/− was significantly different from Rgs4^−/−:Rgs6^−/− (*, p > 0.05). C, IC₅₀ values calculated from dose-response curves in B (F_3,135 = 38.1, p < 0.001). * and ***, p < 0.05 and 0.001, respectively, versus wild-type mice; +++, p < 0.001.

FIGURE 3.

Impact of RGS ablation on M₂R-I_KACh signaling in SAN cells. A, I_KACh currents evoked by CCh (10 μm) in SAN cells from wild-type and Rgs^−/− mice. Peaks were normalized to allow for comparison of deactivation kinetics. Scale bars = 10 s/400 pA. B, no impact of genotype on peak (F_3,36 = 2.0, p = 0.13) or steady-state (F_3,36 = 2.2, p = 0.10) current densities was observed (n = 8–16/genotype). pF, picofarads. C and D, a significant impact of genotype was observed for activation (F_3,35 = 5.0, p < 0.01) and deactivation (F_3,36 = 29.1, p < 0.001) kinetics of the CCh-induced current in wild-type and Rgs^−/− SAN cells. E, there was no impact of genotype on the acute desensitization of the CCh-induced I_KACh current (F_3,36 = 1.3, p = 0.29). F, concentration-response curves for CCh-induced I_KACh activation (steady-state current amplitudes normalized to response measured with 10 μm CCh) in wild-type and Rgs^−/− mice (n = 6–8/group). Hill coefficients for each curve are listed. No significant impact of genotype was observed for Hill coefficients (F_3,151 = 0.4, p = 0.8). G, EC₅₀ values calculated from concentration-response curves shown in F (F_3,151 = 24.8, p < 0.0001). H and I, activation and deactivation kinetics of the CCh-induced currents in wild-type and Rgs^−/− SAN cells. An interaction of genotype and dose was observed for activation kinetics (F_6,95 = 7.4, p < 0.001) but not deactivation kinetics (F_6,95 = 1.5, p = 0.18). However, a significant impact of group on deactivation kinetics was observed for both genotype (F_3,95 = 66.2, p < 0.001) and concentration (F_2,95 = 15.3, p < 0.001), so within-concentration comparisons were performed by one-way ANOVA. * and ***, p < 0.05 and 0.001, respectively, versus wild-type mice; + and +++, p < 0.05 and 0.001 respectively.

FIGURE 4.

Influence of another R7 RGS protein revealed by RGS4 ablation. A, I_KACh currents evoked by CCh (10 μm) in Rgs6^−/− and Gβ5^−/− SAN cells treated with vehicle or CCG-63802 (50 μm, delivered via the pipette). Peak currents were normalized for comparison. B, activation kinetics of the CCh-induced current in Rgs6^−/− (t₁₄ = 0.1, p = 0.9) or Gβ5^−/− (t₈ = 0.2, p = 1.0) SAN cells. C, deactivation kinetics of the CCh-induced current in Rgs6^−/− (t₁₈ = 3.0, p < 0.01) or Gβ5^−/− (t₆ = 0.3, p = 0.8) SAN cells. D, peak I_KACh current densities in Rgs6^−/− (t₁₅ = 1.7, p = 0.1) or Gβ5^−/− (t₉ = 0.4, p = 0.7) SAN cells. pF, picofarads. E, steady-state I_KACh current densities in Rgs6^−/− (t₁₆ = 1.1, p = 0.3) or Gβ5^−/− (t₈ = 0.2, p = 0.8) SAN cells. Note that only within-genotype comparisons were made (n = 4–12/genotype). ***, p < 0.001 versus vehicle for each genotype.

FIGURE 5.

RGS4 ablation in isoproterenol-stimulated hearts. A, HR in isolated hearts from wild-type (wt) and Rgs4^−/− mice stimulated with isoproterenol. HRs in isoproterenol-treated wild-type and Rgs4^−/− hearts were not significantly different (t₄ = 0.14, p = 0.9). bpm, beats/min. B, impact of CCh on HR in wild-type and Rgs4^−/− hearts (n = 3/genotype). Data are normalized to baseline HR. There was no significant impact of genotype observed for normalized HR (F_2,37 = 2.4, p = 0.1). Hill coefficients for each curve are listed and were not significantly different (t₃₇ = 1.8, p = 0.09). C, IC₅₀ values calculated from dose-response curves in B were not significant (t₃₇ = 1.6, p = 0.12).

FIGURE 6.

M₂R-I_KACh signaling evaluated by slow perfusion of CCh. Shown are summary data of I_KACh currents evoked by CCh (10 μm) in wild-type and Rgs4^−/− SAN cells, taken from experiments in which CCh was allowed to slowly fill the recording chamber by gravity flow and was washed out of the chamber by gravity flow of CCh-free bath solution. Although responses in SAN cells from Rgs4^−/− mice showed slightly faster kinetics and larger current densities, there were no significant genotype-dependent differences in any of the following parameters: activation rate (t₈ = 0.4, p = 0.7) (A), deactivation rate (t₇ = 0.7, p = 0.5) (B), peak (t₈ = 0.9, p = 0.4) or steady-state (t₇ = 1.5, p = 0.2) CCh-induced current densities (C), or acute desensitization of the CCh-induced I_KACh current (t₈ = 0.2, p = 0.8; n = 4–6/genotype) (D). pF, picofarads.