Contribution of small conductance K+ channels to sinoatrial node pacemaker activity: insights from atrial-specific Na+ /Ca2+ exchange knockout mice

Abstract

Key points: Repolarizing currents through K⁺ channels are essential for proper sinoatrial node (SAN) pacemaking, but the influence of intracellular Ca²⁺ on repolarization in the SAN is uncertain. We identified all three isoforms of Ca²⁺ -activated small conductance K⁺ (SK) channels in the murine SAN. SK channel blockade slows repolarization and subsequent depolarization of SAN cells. In the atrial-specific Na⁺ /Ca²⁺ exchanger (NCX) knockout mouse, cellular Ca²⁺ accumulation during spontaneous SAN pacemaker activity produces intermittent hyperactivation of SK channels, leading to arrhythmic pauses alternating with bursts of pacing. These findings suggest that Ca²⁺ -sensitive SK channels can translate changes in cellular Ca²⁺ into a repolarizing current capable of modulating pacemaking. SK channels are a potential pharmacological target for modulating SAN rate or treating SAN dysfunction, particularly under conditions characterized by abnormal increases in diastolic Ca²⁺ .

Abstract: Small conductance K⁺ (SK) channels have been implicated as modulators of spontaneous depolarization and electrical conduction that may be involved in cardiac arrhythmia. However, neither their presence nor their contribution to sinoatrial node (SAN) pacemaker activity has been investigated. Using quantitative PCR (q-PCR), immunostaining and patch clamp recordings of membrane current and voltage, we identified all three SK isoforms (SK1, SK2 and SK3) in mouse SAN. Inhibition of SK channels with the specific blocker apamin prolonged action potentials (APs) in isolated SAN cells. Apamin also slowed diastolic depolarization and reduced pacemaker rate in isolated SAN cells and intact tissue. We investigated whether the Ca²⁺ -sensitive nature of SK channels could explain arrhythmic SAN pacemaker activity in the atrial-specific Na⁺ /Ca²⁺ exchange (NCX) knockout (KO) mouse, a model of cellular Ca²⁺ overload. SAN cells isolated from the NCX KO exhibited higher SK current than wildtype (WT) and apamin prolonged their APs. SK blockade partially suppressed the arrhythmic burst pacing pattern of intact NCX KO SAN tissue. We conclude that SK channels have demonstrable effects on SAN pacemaking in the mouse. Their Ca²⁺ -dependent activation translates changes in cellular Ca²⁺ into a repolarizing current capable of modulating regular pacemaking. This Ca²⁺ dependence also promotes abnormal automaticity when these channels are hyperactivated by elevated Ca²⁺ . We propose SK channels as a potential target for modulating SAN rate, and for treating patients affected by SAN dysfunction, particularly in the setting of Ca²⁺ overload.

Keywords: Na+/Ca2+ exchange; sinoatrial node; small conductance K+ channels.

Figure 1. Expression and localization of SK channels in the SAN

A, qPCR measurements of SK1 (n = 6), SK2 (n = 6) and SK3 (n = 4) mRNA expression in tissue preparations of SAN dissected from WT and NCX KO mice. The results have been normalized to the expression of the housekeeper gene GAPDH in the same preparations. ^* P < 0.05, ^** P < 0.01 and ^*** P < 0.001, and ^# P < 0.05, ^## P < 0.01 and ^### P < 0.001 by two‐way ANOVA, with Sidak's and Tukey's post‐test. B, immunostaining of single SAN cells from WT and NCX KO tissues with antibodies against SK1, SK2, SK3 and α‐actinin2. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2. Expression and localization of SK channels in the atria

A, qPCR measuring the expression of SK1 (n = 6), SK2 (n = 6) and SK3 (n = 4) in atrial tissue preparations dissected from WT and NCX KO mice. The results have been normalized to the expression of the housekeeper gene GAPDH in the same preparation pools. ^** P < 0.01 and ^*** P < 0.001, and ^### P < 0.001 by two‐way ANOVA, with Sidak's and Tukey's post‐test. B, immunostaining SK1, SK2, SK3 and α‐actinin2 in single atrial cells dissociated from WT tissue. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3. Control staining of SAN cells, made using only the secondary antibody, but not the primary

[Color figure can be viewed at wileyonlinelibrary.com]

Figure 4. Localization of SK proteins

A, magnification of WT SAN cells shown in Fig. 1B and stained for the three SK channel isoforms (green) and α‐actinin2 (red). By measuring the spatial distribution of fluorescence intensity within the yellow rectangle drawn on the cell images, we obtained the spatial profile of the immunostaining expressed in arbitrary units (a.u.). This allowed us to compare the distribution of the three SK isoforms versus α‐actinin2 along the cells. The peaks in the histogram below the immunostaining correspond to the respective striped distribution of SK and α‐actinin2 proteins. B, as in A but for NCX KO cells shown in Fig. 1 B. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5. Example of SAN/atrial preparation for immunostaining

A, example of an SAN/atrial preparation showing the section plane (clear blue) used to obtain cryo‐slides to immunostain the whole preparation. B, immunostaining of a slide of the whole SAN/atrial preparation. Note reduced connexin 43 signal in the SAN region (indicated by the dotted white area in A) compared with the atria. CT, crista terminalis; IAS, interatrial septum; IVC, inferior vena cava; LA, left atrium; RA, right atrium; SVC, superior vena cava. C, control staining of SAN tissues, made using only the secondary antibody, but not the primary. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6. Expression and localization of SK channels in the whole SAN/atrial tissue preparation

A–C, immunostaining of SK1, SK2 and SK3, respectively, and α‐actinin2 and DAPI in sections cut through the WT and NCX KO SAN/atrial preparation so as to include both atria as well as the SAN (see Fig. 5). The positions of the SAN, RA and LA are indicated in the upper left corner (see Fig. 5 for more precise anatomy of the section). Insets 1, 2 and 3, magnification of SAN and RA regions shown in A, B and C. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 7. Whole cell measurement of SK current

Examples of K⁺ current recordings by patch clamp before and after apamin (Apa) in WT (A) and NCX KO (B) SAN cells. Currents were elicited using 500 ms voltage steps from a holding potential of −55 mV to a range of test potentials from −100 to +50 mV. Lower panels show the respective Apa‐sensitive currents after subtraction in WT and NCX KO. C, I–V relationship of the Apa‐sensitive currents recorded in WT (n = 10) and NCX KO (n = 7) SAN cells. ^** P < 0.01 and ^*** P < 0.001 by two‐way ANOVA, with Sidak's post‐test.

Figure 8. Effect of SK inhibition on spontaneous action potentials

A, examples of spontaneous action potentials (APs) recorded in WT SAN cells before and after exposure to apamin (Apa; 10 nmol l⁻¹). B and C, AP duration at 70% (APD70) and 90% (APD90) of repolarization, recorded in WT SAN cells, before and after exposure to Apa (n = 8). D, maximal diastolic potential (MDP) recorded in the same cells (n = 7). E, slope of diastolic depolarization phase (DDP) recorded in the same cells (n = 8).

Figure 9. AP shape in SAN cells treated with apamin

Example of APs recorded from a WT single SAN cell, before and after inhibition of SK current with Apa. Note the presence of aborted diastolic depolarization (DDP; arrows) after Apa exposure.

Figure 10. Effect of SK inhibition on induced action potentials

A, examples of electrically stimulated APs in WT SAN cells, before (black) and after (gray) exposure to apamin (Apa), respectively (n = 10). B, plots of summary data for APD70 and APD90 shown in A. C, examples of electrically stimulated APs in NCX KO SAN cells, before (black) and after (gray) exposure to apamin (Apa), respectively (n = 7). D, plots of summary data for APD70 and APD90 shown in C. ^* P < 0.05 and ^** P < 0.01 by paired t test.

Figure 11. Effect of SK inhibition on SAN tissue pacemaker activity

A, examples of spontaneous Ca²⁺ transients in SAN tissue from WT. B, average rates of Ca²⁺ transients in WT (n = 5) before and after exposure to apamin (Apa). C, examples of spontaneous Ca²⁺ transients in NCX KO whole SAN tissues. Note upper inset with enlarged scale to highlight the diastolic Ca²⁺ increase during the bursts (bottom) and spike adaptation (top). D, average rates of Ca²⁺ transients in NCX KO (n = 7) before and after exposure to Apa. E, F and G, adaptation degree (% of cycle length increase between the first and last spike interval of each burst; n = 7), burst firing rate (average of the rate during every burst of each sample recording; n = 7) and quiescent time (s min^–1 of quiescence during the recording; n = 6), in NCX KO SAN tissue before and after exposure to Apa. (See Methods for precise definition of the parameters.) ^* P < 0.05, ^** P < 0.01 and ^*** P < 0.001 by one‐way ANOVA with Sidak's post‐test and paired t test.

Figure 12. Recovery of NCX KO pacemaker activity under SK inhibition

Example of spontaneous Ca²⁺ transients recorded in NCX KO whole SAN tissue before and after exposure to apamin (Apa). Note the regular rate after Apa perfusion in this example.

Figure 13. Effect of BK inhibition on SAN tissue pacemaker activity

A, examples of spontaneous Ca²⁺ transients in SAN tissues from WT. B, average rates of Ca²⁺ transients in WT (n = 5), before and after exposure to paxilline (Pax). C, examples of spontaneous Ca²⁺ transients in NCX KO whole SAN tissues. D, average rate of Ca²⁺ transients in NCX KO (n = 6), before and after exposure to Pax. E, F and G, adaptation degree (% of cycle length increase between the first and last spike interval of each burst), burst firing rate (average of the rate during every burst of each sample recording) and quiescent time (s min^–1 of quiescence during the recording) in NCX KO SAN tissue (n = 6), before and after exposure to Pax. (See Methods for precise definition of these parameters.) ^* P < 0.05 by one‐way ANOVA with Sidak's post‐test.