Functional roles of a Ca2+-activated K+ channel in atrioventricular nodes

Abstract

Since the first description of the anatomical atrioventricular nodes (AVNs), a large number of studies have provided insights into the heterogeneity of the structure as well as a repertoire of ion channel proteins that govern this complex conduction pathway between the atria and ventricles. These studies have revealed the intricate organization of multiple nodal and nodal-like myocytes contributing to the unique electrophysiology of the AVN in health and diseases. On the other hand, information regarding the contribution of specific ion channels to the function of the AVN remains incomplete. We reason that the identification of AVN-specific ion channels may provide a more direct and rational design of therapeutic target in the control of AVN conduction in atrial flutter/fibrillation, one of the most common arrhythmias seen clinically. In this study, we took advantage of 2 genetically altered mouse models with overexpression or null mutation of 1 of a small conductance Ca2+-activated K+ channel isoform, SK2 channel, and demonstrated robust phenotypes of AVN dysfunction in these experimental models. Overexpression of SK2 channels results in the shortening of the spontaneous action potentials of the AVN cells and an increase in the firing frequency. On the other hand, ablation of the SK2 channel results in the opposite effects on the spontaneous action potentials of the AVN. Furthermore, we directly documented the expression of SK2 channel in mouse AVN using multiple techniques. The new insights may have important implications in providing novel drug targets for the modification of AVN conduction in the treatment of atrial arrhythmias.

Figure 1

Surface ECG recordings from WT, SK2+/T and SK2+/Δ mice. (A) Examples of ECG recordings in WT, SK2+/T and SK2+/Δ mice. (B) Examples of ECG recordings in SK2 Δ/Δ mice showing complete AV block with AV dissociation. (C) Summary data for PR intervals in WT, SK2+/T and SK2+/Δ mice in control conditions. There were also significant differences in the RR interval among the three groups of animals (135.0±7.8, 109.7±6.1 and 153.8±6.1 ms for WT, SK2 +/T and SK2 +/Δ, respectively. (D) Summary data for PR intervals after intraperitoneal injection of atropine and propranolol to abolish autonomic control of the heart (n=6, *p<0.05). There were no significant differences in the percent increase in PR intervals after autonomic blockade in WT, SK2+/T and SK2+/Δ mice (Lower Panel in D).

Figure 2

Spontaneous APs recorded from intact AVNs from WT, SK2+/T and SK2+/Δ mice. (A) Photomicrograph of endocardial view of typical AVN preparation from mouse hearts. RA, right atrium; IVC, inferior vena cava; RV, right ventricle; CS, coronary sinus. (B) Representative examples of spontaneous APs from intact AVNs showing an increase in the AVN firing frequency in SK2 +/T mice and a decrease in the firing frequency in SK2 +/Δ mice. (C) Summary data from the three groups of animals for DDR (mV/s), CL (ms), MDP (mV), APA (mV), V_max (V/s), APD₅₀ (ms), and APD₈₀ (ms). *P<0.05.

Figure 3

Whole-cell I_K,Ca density elicited using a holding potential of -55 mV in single AVN cells. I_K,Ca current density was obtained using the difference current before and after application of apamin (500 pmol/L) normalized to the cell capacitance. (A) Representative examples of whole-cell I_K,Ca recorded from single isolated AVN cells from WT, SK2 +/T and SK2 +/Δ mice. (B) Summary data for the current density-voltage relations of the apamin-sensitive current in AVN cells from WT (11.7±1.3 pF), SK2 +/T (10.6±2.0 pF) and SK2 +/Δ mice (13.9±1.6 pF). *p<0.05, n=6-9 cells for each group from 3 set of animals.

Figure 4

Subcellular distribution of SK2 channel in mouse AVN cells. (A) Photomicrographs of single isolated AVN cells (a&b). Atrial and ventricular myocytes are shown for comparison in the right panels (c&d, respectively). (B) Photomicrographs of confocal laser scanning microscopy of AVN cells (a) showing positive immunostaining with anti-Ca_v3.1 antibody followed by anti-rabbit IgG-FITC conjugated secondary antibody (green), (b) treatment with secondary antibody only as negative control and (c&d) absence of Ca_v3.1 staining in atrial and ventricular myocytes, respectively. The corresponding differential interference contrast (DIC) images are shown in the right panels. (C) Photomicrographs of confocal laser scanning microscopy of immunostaining of AVN cells, atrial and ventricular myocytes with anti-SK2 and anti-α-actinin2 antibodies: (a) double staining with anti-SK2 (green) and anti-α-actinin2 antibodies (red) in an AVN cell, (b) preincubation of the anti-SK2 antibody with antigenic peptide, (c) treatment with secondary antibodies only (anti-rabbit IgG-FITC conjugated and anti-mouse IgG-Texas Red conjugated antibodies) as additional negative control, (d&e) double staining with anti-SK2 (green) and anti-α-actinin2 antibodies (red) in single atrial and ventricular myocytes, respectively. (f) double staining with anti-SK2 and anti-α-actinin2 antibodies in atrial myocytes from SK2 Δ/Δ mice were used as a negative control showing lack of positive staining in SK2 Δ/Δ for SK2 channel. Merged images are shown in the right panels. (D) Photomicrographs of confocal laser scanning microscopy as in C except that anti-neurofilament 160 (NF 160) antibody (red) was used instead of anti-α-actinin2 antibody in AVN cells, atrial and ventricular myocytes. NF 160 was used as an AVN marker. Scale bars =10 μm.

Figure 5

Expression of SK2 channel protein in AVN. Photomicrographs of histologic sections of AVN and working myocardium from WT and SK2 Δ/Δ mice at low (A,B,C,D,E) and high magnification (A′,B′,C′,D′,E′). (A,A′) Sections stained with hematoxylin and eosin. (B,B′) Sections stained with Masson's trichrome. (C,C′) Expression of SK2 protein (peroxidase positive) in the AVN and working myocardiumin in WT animals. (D,D′) Absence of SK2 protein expression in the AVN and working myocardium in SK2 Δ/Δ mice. (E,E′) Treatment with secondary antibody only as negative control. AVN, atrioventricular node; Arrows indicate AVN/HB region; RA, right atrium; VS, ventricular septum. Scale Bars=50 μm.