Dysfunction in Ankyrin-B Disrupts Heart Rate Regulation by Altering the Membrane Abundance of GIRK Channels

Figures

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  Figure 1. Impact of AnkB Deficiency on Heart Rate Response to Sympathetic/Parasympathetic Agonists (A and B) Representative surface ECG recordings from WT and AnkB+/− mice at baseline and after ISO injection. AnkB+/− mice display reduced HR compared to WT controls (WT: n = 20 mice, AnkB+/−: n = 17 mice). (C and D) Injection of ISO normalizes the differences in HR given a great net increase in HR in AnkB+/− mice (WT: n = 20 mice, AnkB+/−: n = 17 mice). (E and F) Injection of atropine abolishes the HR differences between AnkB+/− mice and their WT controls (WT: n = 10 mice, AnkB+/−: n = 8 mice). (G and H) Injection of CCH further reduces HR in AnkB+/− mice (WT: n = 10 mice, AnkB+/−: n = 9 mice). (I and J) Injection of propranolol reduces resting HR equivalently in both genotypes (WT: n = 8 mice, AnkB+/−: n = 8 mice). (K and L) Injection of propranolol plus atropine results in equivalent reductions at early time points but not in later time points in AnkB+/− mice (WT: n = 9 mice, AnkB+/−: n = 9 mice). Drug doses: 0.4 mg/kg ISO, 1 mg/kg atropine, 0.1 mg/kg CCH, 1 mg/kg propranolol. ΔHR denotes magnitude of HR change. Data are presented as mean ± SEM. Data were analyzed using unpaired Student's…

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  Figure 2. AnkB Deficiency Enhances Heart Rate Response to Sympathetic Signaling (A and B) Representative recordings from isolated hearts from AnkB+/− mice showing a reduction in HR compared to WT (WT: n = 20 mice, AnkB+/−: n = 20 mice). (C) ISO treatment increases HR in AnkB+/− mice to a level equivalent to control mice (WT: n = 20 mice, AnkB+/−: n = 20 mice). (D and E) AnkB+/− SAN cells show an increase in SAN firing rates in response to ISO at 50 nM compared to WT cells (WT: n = 6 mice, n = 13 cells; AnkB+/−: n = 6 mice, n = 14 cells). (F) AnkB+/− SAN cells show a higher net increase in SAN firing rates in response to 50 nM ISO compared to WT cells (WT: n = 6 mice, n = 13 cells; AnkB+/−: n = 6 mice, n = 14 cells). (G and H) SAN diastolic depolarization rate (DDR) is reduced in AnkB+/− cells compared to WT cells. (H) DDR is increased after ISO stimulation at 50 nM in AnkB+/− SAN cells compared to WT (WT: n = 6 mice, n = 13 cells, AnkB+/−: n = 6 mice, n = 14 cells). (I) AnkB+/− SAN cells show a higher net increase in DDR in response to 50 nM ISO compared to WT cells (WT: n = 6 mice, n = 13 cells; AnkB+/−: n = 6 mice, n = 14 cells). (J) Maximum diastolic potential (MDP) is not different…

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  Figure 3. AnkB+/− SAN Displays Enhanced Response to Parasympathetic Stimulation (A) HRs from Langendorff-perfused hearts of WT and AnkB+/− are equivalently suppressed by CCH in concentrations ranging from 0.1 to 10 μM (WT: n = 13 mice, AnkB+/−: n = 11 mice). (B) ISO stimulation in AnkB+/− hearts show an increased response to CCH treatment at 0.5, 1, 5, and 10 μM compared to WT hearts (WT: n = 10 mice, AnkB+/−: n = 10 mice). (C) In the presence of ISO baseline, sinus arrest in AnkB+/− hearts occurs faster in response to CCH compared to WT controls (WT: n = 12 mice, AnkB+/−: n = 11 mice). (D) AnkB+/− SAN cells show a reduced AP firing rate at baseline compared with WT SAN cells. 50 nM ISO increases firing rates in AnkB+/− SAN cells. Coapplication of CCH significantly reduces the AP firing rate at 0.1 μM CCH (WT: n = 5 mice, n = 15 cells; AnkB+/−: n = 6 mice, n = 14 cells). (E) Diastolic depolarization rate (DDR) of AnkB+/− SAN cells is increased upon 50 nM ISO but decreased at 0.1 and 0.5 μM CCH plus 50 nM ISO compared to WT SAN cells (WT: n = 5 mice, n = 15 cells; AnkB+/−: n = 6 mice, n = 14 cells). (F) Maximum diastolic potential (MDP) between WT and AnkB+/− SAN cells is not different (WT:…

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  Figure 4. AnkB Deficiency Is Independent of If but Alters IK,ACh Desensitization (A and B) Representative recordings of ICa,L and current-voltage relationship showing AnkB+/− SAN cells with a significant reduction in ICa,L density compared to WT SAN cells (WT: n = 6 mice, n = 10 cells; AnkB+/−: n = 7 mice, n = 12 cells). (C to E) No change in If density is noted at baseline, as well as following sympathetic (WT: n = 6 mice, n = 11 cells; WT ISO: n = 6 mice, n = 11 cells; AnkB+/−: n = 4 mice, n = 10 cells; AnkB+/− ISO: n = 4 mice, n = 10 cells) and sympathetic/parasympathetic activation (ISO + CCH) (WT: n = 7 mice, n = 12 cells; WT [ISO + CCH]: n = 7 mice, n = 12 cells; AnkB+/−: n = 6 mice, n = 9 cells; AnkB+/− [ISO + CCH]: n = 6 mice, n = 9 cells) between WT and AnkB+/− SAN cells. Significance is noted between treated and untreated within the same group. (F) No change in Ik density is noted between WT and AnkB+/− SAN cells (WT: n = 8 mice, n = 15 cells, AnkB+/−: n = 8 mice, n = 14 cells). (G) No change in IK,ACh density between WT and AnkB+/− SAN cells (WT: n = 8 mice, n = 15 cells; AnkB+/−: n = 8 mice, n = 14 cells). (H) Quantitative analysis showing that time-dependent decline of IK,ACh…

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  Figure 5. AnkB Associates With KACh Channel Subunits in Cardiac Lysates Atrial lysates from AnkB+/− and wild type (WT) mice were evaluated for GIRK1 and GIRK4 expression. (A and B) Immunoblots and quantitative analysis of AnkB expression normalized to GAPDH in atrial lysates (n = 8 mice/group). (C and D) Immunoblots and quantitative analysis of GIRK1 expression normalized to GAPDH in atrial lysates (n = 8 mice/group). (E and F) Immunoblots and quantitative analysis of GIRK4 expression normalized to GAPDH in atrial lysates (n = 8 mice/group). (G to J) Anti-GIRK1 Ig and anti-GIRK4 Ig co-immunoprecipitate ankyrin-B from whole heart and atrial lysates. There is no interaction between anti-AnkB and control IgG immunoprecipitates from any lysate. Co-immunoprecipitation (Co-IP) experiments were repeated 3 times. Data are presented as mean ± SEM. Data were analyzed using Mann-Whitney U test (B and D) and unpaired Student's t-test (F); ∗P < 0.05, ∗∗P < 0.01.

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  Figure 6. Ank2-cKO Mice Display Changes in Atrial GIRK1 Localization and Expression Without Changes in Atrial GIRK4 Localization and Expression (A and B) Representative atrial cryosection images and quantification from floxed and Ank2-cKO mice stained with GIRK1 (green), Wheat Germ Agglutinin (WGA; red) and overlap including DAPI nuclear stain (blue). Scale bars equal 20 μm. Images are representative of n = 3 mice/group, n = 5 to 6 sections/group. (C and D) Representative atrial cryosection images and quantification from floxed and Ank2-cKO mice stained with GIRK4 (green), WGA (red) and overlap including DAPI nuclear stain (blue). Scale bars equal 20 μm. Images are representative of n = 3 mice/group and n = 5 to 6 sections/group. Data are presented as mean ± SEM. Data were analyzed using generalized linear mixed-effects model (B, D); ∗∗∗P < 0.001.