Proarrhythmic effect of blocking the small conductance calcium activated potassium channel in isolated canine left atrium

Abstract

Background: Small conductance calcium activated potassium (SKCa) channels are voltage insensitive and are activated by intracellular calcium. Genome-wide association studies revealed that a variant of SKCa is associated with lone atrial fibrillation in humans. Roles of SKCa in atrial arrhythmias remain unclear.

Objective: To determine roles of SKCa in atrial arrhythmias.

Methods: Optical mapping using the isolated canine left atrium was performed. The optical action potential duration (APD) and induction of arrhythmia were evaluated before and after the addition of specific SKCa blockers-apamin or UCL-1684.

Results: SKCa blockade significantly increased APD₈₀ (188 ± 19 ms vs 147 ± 11 ms; P<.001). The pacing cycle length thresholds to induce 2:2 alternans, and wave breaks were prolonged by SKCa blockade. Increased APD heterogeneity was observed after the SKCa blockade, as measured by the difference between the maximum and the minimum APD (39 ± 4 ms vs 26 ± 5 ms; P<.05), by standard deviation (12.43 ± 2.36 ms vs 7.49 ± 1.47 ms; P<.001), or by coefficient of variation (6.68% ± 0.97% vs 4.90% ± 0.84%; P<.05). No arrhythmia was induced at baseline by an S1-S2 protocol. After SKCa blockade, 4 of 6 atria developed arrhythmia.

Conclusions: SKCa blockade promotes arrhythmia and prolongs the pacing cycle length threshold of 2:2 alternans and wave breaks in the canine left atrium. The proarrhythmic effect could be attributed to increased APD heterogeneity in the canine left atrium. This study provides supportive evidence of genome-wide association studies showing association of KCNN3 and lone atrial fibrillation.

Figure 1. Effect on atrium by SK_Ca blockade

A. Configuration of isolated canine left atrium preparation and corresponding APD change. The black square identifies the optically imaged region. The corresponding APD is shown in the left panel. Note that the pattern of ADP prolongation is very heterogeneous. LOM, Ligament of Marshall. LAA, left atrium appendage. B. Relationship between PCL and APD₈₀. A biphasic relationship between APD and PCL was observed at baseline. APD increased as PCL increased from 250ms to 500ms. When PCL was longer than 500ms, APD shortened. SK_Ca blockade abolished this phenomenon (N=9, 5 with apamin treatment and 4 with UCL-1684 treatment). Comparison was performed between baseline and SK_Ca blockade (apamin and UCL-1684). Star (*) denotes the post test p-values following ANOVA test. C. Percentage of APD prolongation by SK_Ca blockade at different PCLs. SK_Ca blockade showed a tendency to prolong APD in all PCLs. The effect was most significant at long PCLs. Star (*) denotes the p-values from post test when compared with 1500ms *, p<0.05, **, p< 0.01. ***, P< 0.001

Figure 2. Increased APD heterogeneity by SK_Ca blockade

A. Examples of APD map at different PCLs. The orientation of LAA is illustrated by the diagram. Increased APD heterogeneity is demonstrated by increased color gradient in APD maps after blockade by UCL-1684. ΔAPD reveals heterogeneous prolongation of SKCa blockade. B, C, and D. Quantification of APD heterogeneity. All three measurements indicate that SK_Ca blockade increases APD heterogeneity (N=9, 5 with apamin treatment and 4 with UCL-1684 treatment). Comparison was performed between baseline and SKCa blockade (apamin and UCL-1684). Star (*) denotes the post test p-values following ANOVA test. *, p< 0.05. **, P< 0.01. ***, P< 0.001

Figure 3. Detection of SK2 and its relationship with APD prolongation

A. Top left is an illustration showing the 8 predefined regions which the LAA was dissected into. Top right shows percentage of APD prolongation by SK_Ca blockade in different regions (n=9, 5 with apamin treatment and 4 with UCL-1684 treatment). Insets are representative ΔAPD maps from two different LAAs showing heterogeneous APD prolongation by SK_Ca blockade. B. SK_Ca protein expression at different regions (n=5). A representative Western blotting image is shown in the inset. C. Relationship of SK2 proteins and APD prolongation. The abundance of SK2 protein is plotted against the percentage of corresponding APD prolongation (n=3). Among them, #1 and #2 were treated with apamin while #3 was treated with UCL-1684. The formula and value of R² are displayed. The plotting from all points yields a significant linear correlation with R² equaling to 0.2388 (p= 0.0154). *, p< 0.05., ***, P< 0.001

Figure 4. Development of 2:2 alternans and wave breaks

A. Representative traces of 2:2 alternans. Data were acquired from an atrium at baseline. Corresponding isochronal maps and APD maps from beat 1 and beat 2 are shown. Note that there is a region showing conduction delay (#4 and #5). The site that developed 2:2 alternans lies within the region showing conduction delay. B. Ratio of consecutive beats showing conduction efficiency. Beat 2/beat 1, beat 4/beat3, and beat 6/beat5 from Figure 3A are plotted. The conduction efficiency decreases gradually from #1 to #5. C. SK_Ca blockade prolonged the PCL threshold of 2:2 alternans and wave breaks. Wave breaks could only be induced in 5 atria at baseline while it was induced in all atria after SK_Ca blockade. *, p< 0.05. ***, P< 0.001

Figure 5. Induction of reentry arrhythmia by SK_Ca blockade

A. APD maps and the corresponding ΔAPD map from the same atrium showing the pattern of APD change after SK_Ca blockade. Maps were generated from PCL of 1500ms. B. Representative arrhythmia induced by S1–S2 protocol. Additional pacing (S2) after a stimuli was used to induce arrhythmia. S1, regular pacing at 400ms, S2, additional pacing 160ms after a S1 pacing. No arrhythmia could be induced at baseline while 4 out of 6 atria developed arrhythmia after SK_Ca blockade. a) Isochronal maps showing the conduction patterns. Star symbol (*) indicates the pacing site. Only parts could be activated, as revealed by S2 isochrone. b) Corresponding voltage maps and phase maps. Arrows in voltage map show the propagation of reentry. Time label represents time interval after S2 pacing.