Expression and relevance of the G protein-gated K+ channel in the mouse ventricle

Abstract

The atrial G protein-gated inwardly rectifying K⁺ (GIRK) channel is a critical mediator of parasympathetic influence on cardiac physiology. Here, we probed the details and relevance of the GIRK channel in mouse ventricle. mRNAs for the atrial GIRK channel subunits (GIRK1, GIRK4), M2 muscarinic receptor (M₂R), and RGS6, a negative regulator of atrial GIRK-dependent signaling, were detected in mouse ventricle at relatively low levels. The cholinergic agonist carbachol (CCh) activated small GIRK currents in adult wild-type ventricular myocytes that exhibited relatively slow kinetics and low CCh sensitivity; these currents were absent in ventricular myocytes from Girk1^-/- or Girk4^-/- mice. While loss of GIRK channels attenuated the CCh-induced shortening of action potential duration and suppression of ventricular myocyte excitability, selective ablation of GIRK channels in ventricle had no effect on heart rate, heart rate variability, or electrocardiogram parameters at baseline or after CCh injection. Additionally, loss of ventricular GIRK channels did not impact susceptibility to ventricular arrhythmias. These data suggest that the mouse ventricular GIRK channel is a GIRK1/GIRK4 heteromer, and show that while it contributes to the cholinergic suppression of ventricular myocyte excitability, this influence does not substantially impact cardiac physiology or ventricular arrhythmogenesis in the mouse.

Figure 1

Expression of I_KACh-dependent signaling elements in mouse atria and ventricle. mRNA levels of GIRK1 (t₁₄ = 9.4, ***P < 0.001), GIRK4 (t₁₄ = 21.2, ***P < 0.001), M₂R (t₁₃ = 18.6, ***P < 0.001), and RGS6 (t₁₄ = 6.7, ***P < 0.001), and in atria and ventricle from adult C57BL/6J mice, compared using an unpaired Student’s t-test. mRNA levels of each target were normalized to the level of GAPDH mRNA measured in each sample. Ventricular mRNA levels for each target was normalized to the level present in atrial samples (n = 7–8 samples per tissue, per target).

Figure 2

Carbachol-induced GIRK currents in adult mouse ventricular myocytes. (a) Whole-cell currents (V_hold = −70 mV) evoked by carbachol (CCh, 100 μM) in a high-K⁺ bath solution (containing 5 μM BaCl₂) in adult ventricular myocytes from wild-type, Girk1^−/−, Girk4^−/−, and Rgs6^−/− mice. Scale: 0.1 nA/10 s. (b) Summary of maximal CCh-induced current density in adult ventricular myocytes from wild-type (n = 28 cells/6 mice), Girk1^−/− (n = 23 cells/4 mice), Girk4^−/− (n = 21 cells/4 mice), and Rgs6^−/− mice (n = 24 cells/6 mice); open symbols overlapping the bars denote individual data points. One-way ANOVA analysis revealed an effect of genotype on CCh-induced current density (F_3,92 = 110.6, P < 0.001). Symbols: **^, ***P < 0.01 and 0.001, respectively, vs. wild-type. (c,d) Summary of activation (c; t₄₄ = 0.7, P = 0.51) and deactivation (d); t₄₄ = 1.1, P = 0.28) kinetics for the CCh-induced current in ventricular myocytes from wild-type (n = 25 cells/6 mice) and Rgs6^−/− mice (n = 21 cells/6 mice), compared across genotypes using an unpaired Student’s t-test. (e) Summary of EC₅₀ values derived from concentration-response experiments for the CCh-induced current in adult ventricular myocytes from wild-type vs. Rgs6^−/− mice. The EC₅₀ for current activation by CCh did not differ between wild-type (4.8 ± 0.6 μM, n = 11 cells/4 mice) and Rgs6^−/− (4.9 ± 0.4 μM, n = 19 cells/4 mice) ventricular myocytes, as determined using an unpaired Student’s t-test (t₂₈ = 0.20, P = 0.84).

Figure 3

GIRK channel contribution to the cholinergic regulation of mouse ventricular myocyte repolarization and excitability. (a) Change in rheobase evoked by CCh (100 μM) in adult wild-type (n = 19 cells/5 mice) and Girk4^−/− (n = 26 cells/6 mice) ventricular myocytes, compared using an unpaired Student’s t-test (t₄₃ = 5.1, ***P < 0.001). (b) Action potentials evoked by current injection in wild-type and Girk4^−/− ventricular myocytes, at baseline (solid line) and in the presence of CCh (100 μM, dashed line). Scale: 20 mV/25 ms. (c–f) Summary of the percentage change in APD₂₀ (t₂₉ = 0.37, P = 0.72), APD₅₀ (t₂₉ = 0.38, P = 0.71), APD₇₀ (t₂₉ = 0.97, P = 0.34), and APD₉₀ (t₂₉ = 3.5, **P < 0.01) evoked by CCh in wild-type (n = 17 cells/6 mice) and Girk4^−/− (n = 14 cells/5 mice) ventricular myocytes, compared using an unpaired Student’s t-test.

Figure 4

Characterization of mice lacking GIRK channels in the ventricle. (a,b) Sections of the heart from MLC2VCre(−):Ai14-tdTomato and MLC2VCre(+):Ai14-tdTomato mice, labeled with the nuclear stain DAPI (left panels), showing restricted Cre-dependent gene expression (tdTomato, right panels) in the ventricle of MLC2VCre(+) mice. (c) Whole-cell currents (V_hold = − 70 mV) evoked by CCh (100 μM) in a high-K⁺ bath solution (containing 5 μM BaCl₂) in adult ventricular myocytes from MLC2VCre(−):Girk1^fl/fl and MLC2VCre(+):Girk1^fl/fl mice. Scale: 0.2 nA/10 s. (d) Summary of CCh-induced current densities in MLC2VCre(−):Girk1^fl/fl (n =1 1 cells/2 mice) and MLC2VCre(+):Girk1^fl/fl (n = 15 cells/2 mice) ventricular myocytes (VM), compared with an unpaired Student’s t-test (t₂₄ = 7.5, ***P < 0.001). (e) Summary of CCh-induced current densities in adult SAN cells from MLC2VCre(−):Girk1^fl/fl (n = 9 cells/3 mice) and MLC2VCre(+):Girk1^fl/fl (n = 7 cells/2 mice) mice, compared with an unpaired Student’s t-test (t₁₄ = 1.1, P = 0.31).

Figure 5

HR analysis of constitutive and ventricle-specific Girk^−/− mice. (a,b) Segments of ECG recordings from anesthetized wild-type (a) and Girk4^−/− (b) mice, at baseline and after injection of CCh (1.0 mg/kg i.p.). Scale: 1 s. (c) Summary of HR data at baseline and after injection of CCh for wild-type (n = 12) and Girk4^−/− (n = 13) mice. Two-way ANOVA analysis revealed an interaction between genotype and treatment (F_1,23 = 54.8, P < 0.001). Symbols: **^,***P < 0.01 and 0.001, respectively, vs. baseline (within genotype); ^##P < 0.01 vs. wild-type (within treatment). (d) Summary of HR data at baseline and after injection of CCh for MLC2VCre(+):Girk1^fl/fl (n = 13) and MLC2VCre(−):Girk1^fl/fl (n = 12) littermates. There was a significant main effect of treatment (F_1,23 = 173.1, P < 0.001), but no main effect of genotype (F_1,23 = 3.6, P = 0.070), or interaction between genotype and treatment (F_1,23 = 0.09, P = 0.77). Symbols: ***P < 0.001 vs. baseline (within genotype).

Figure 6

Time-domain HRV analysis of constitutive and ventricle-specific Girk^−/− mice. (a,b) RR tachograms for anesthetized wild-type (a) and Girk4^−/− (b) mice, at baseline and following injection of CCh (1.0 mg/kg i.p.). Scale: 10 s. (c) Summary of RMSSD data at baseline and following injection of CCh for wild-type (n = 12) and Girk4^−/− (n=13) mice. Two-way ANOVA analysis revealed an interaction between genotype and treatment (F_1,23 = 158.7, P < 0.001). Symbols: ***P < 0.001 vs. baseline (within genotype); ^###P < 0.001 vs. wild-type (within treatment). (d) Summary of RMSSD data at baseline and following injection of CCh for MLC2VCre(−):Girk1^fl/fl (n = 13) and MLC2VCre(+):Girk1^fl/fl (n = 12) mice. There was a significant main effect of treatment (F_1,23 = 96.3, P < 0.001), but no main effect of genotype (F_1,23 = 3.2, P = 0.087) or interaction between genotype and treatment (F_1,23 = 0.66, P = 0.43). Symbols: ***P < 0.001 vs. baseline (within genotype).

Figure 7

Frequency domain HRV analysis in constitutive and ventricle-specific Girk^−/− mice. (a) Power in the VLF ( < 0.4 Hz) range at baseline and following injection of CCh (1.0 mg/kg i.p.). Two-way ANOVA analysis revealed an interaction between genotype and treatment (F_1,23 = 36.3, P < 0.001) for wild-type (n = 12) and Girk4^−/− (n = 13) mice (left panel). While main effects of treatment (F_1,23 = 51.0, P < 0.001) and genotype (F_1,23 = 13.77, P < 0.01) were observed for MLC2VCre(−):Girk1^fl/fl (n = 13) and MLC2VCre(+):Girk1^fl/fl (n = 12) mice (right panel), there was no interaction between genotype and treatment (F_1,23 = 0.01, P = 0.90). Symbols: ***P < 0.001 vs. baseline (within genotype); ^###P < 0.001 vs. wild-type (within treatment). (b) Power in the LF (0.4–1.5 Hz) range at baseline and following injection of CCh (1.0 mg/kg i.p.). Two-way ANOVA analysis revealed an interaction between genotype and treatment (F_1,23 = 41.6, P < 0.001) for wild-type and Girk4^−/− mice (left panel). While main effects of treatment (F_1,23 = 113.1, P < 0.001) and genotype (F_1,23 = 8.8, P < 0.01) were observed for MLC2VCre(−):Girk1^fl/fl and MLC2VCre(+):Girk1^fl/fl mice (right panel), there was no interaction between genotype and treatment (F_1,23 = 0.02, P = 0.88). Symbols: ***P < 0.001 vs. baseline (within genotype); ^##,###P < 0.01 and 0.001, respectively, vs. wild-type (within treatment). (c) Power in the HF (1.5–5.0 Hz) range at baseline and following injection of CCh (1.0 mg/kg i.p.). Two-way ANOVA analysis revealed an interaction between genotype and treatment (F_1,23 = 37.5, P < 0.001) for wild-type and Girk4^−/− mice (left panel). There were no main effects of treatment (F_1,23 = 2.7, P = 0.11) or genotype (F_1,23 = 1.8, P = 0.19) for MLC2VCre(−):Girk1^fl/fl and MLC2VCre(+):Girk1^fl/fl mice (right panel), nor was there an interaction between genotype and treatment (F_1,23 = 0.8, P = 0.38). Symbols: ***P < 0.001 vs. baseline (within genotype). ^##P < 0.01 vs. wild-type (within treatment).

Figure 8

Susceptibility to pacing-induced ventricular arrhythmias in constitutive and ventricle-specific Girk^−/− mice. (a) Recording of a VT episode after infusion of CCh (3 μM) and burst pacing of the left ventricle. Scale: 0.5 V/0.2 s. (b) Summary of the number of mice exhibiting VT/VF episodes after burst pacing of the left ventricle at baseline (left) and after CCh perfusion (right). Fisher’s exact test revealed no differences in the occurrence of VT/VF episodes between wild-type and Girk4^−/− mice, either at baseline (P = 1.0) or after CCh administration (P = 1.0). We did not observe any incidents of VT/VF in MLC2VCre(−):Girk1^fl/fl or MLC2VCre(+):Girk1^fl/fl mice, either at baseline or after CCh administration.