Inhibition of Small Conductance Calcium-Activated Potassium (SK) Channels Prevents Arrhythmias in Rat Atria During β-Adrenergic and Muscarinic Receptor Activation

Figures

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  FIGURE 1. Flowchart. Graphic overview of the experimental design. After mounting of the right atrium in an organ bath and initiation of stimulated pacing, all experiments were allowed to stabilize for 30 min. Experiments were divided into three randomized sub-studies. (Top panel) Basal characterization of AP parameters without any premedication or antiarrhythmic treatment followed by induction of consecutive runs of AF by programmed atrial burst pacing. (Middle panel) Baseline AP recordings followed by pre-treatment with [ISO], AP characterization, and AF induction followed by antiarrhythmic treatment with [ICA]. (Lower panel) Baseline AP recordings followed by pre-treatment with [CCh], AP characterization, and AF induction before antiarrhythmic treatment with either [ICA], [Fleca], or [vehicle] with the purpose to benchmark the antiarrhythmic efficacy between the two drugs in the muscarinic activated atria.

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  FIGURE 2. Isoprenaline and carbachol (CCh) effects on frequency and AF duration. Recordings in the presence of no activators (basal, A), [ISO] (β-adrenergic, C), and [CCh] (muscarinic, E) activation. Burst pacing of the isolated right atrium leads to AF episodes of various lengths, all reverting spontaneously back to SR. Short (1–2 s) runs of AF are displayed to exemplify both initiation and termination of AF. (B,D,F) Diary plots of beating rate (1000 beats) from representative experiments, depicting the frequency during the induction and the free run of an arrhythmia before reverting back to SR. (G) AP frequencies in the entire group (mean ± SEM). (H) AF duration in the absence of [ISO] or [CCh] (basal) or in the presence either [CCh] or [ISO]. Statistical analysis was done using one-way ANOVA, comparing basal, CCh, and ISO. ∗p < 0.05.

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  FIGURE 3. Electrophysiological effects of [ISO]. (A) Diary plot depicting the effect of β-adrenergic activation on the pacemaker frequency (after application of [ISO] in an isolated rat right atrium). An immediate increase in intrinsic beating frequency was observed shortly after application of [ISO] to the perfusion buffer. After a transient peak, the frequency was maintained over time. (B) Bar graph shows the mean values ± SEM (n = 5) during basal intrinsic frequency and after [ISO] application. The effect of β-adrenergic activation on AP parameters (n = 9): (C) the resting membrane potential (RMP), (D) action potential duration (APD90), (E) amplitude (APA), (F) upstroke velocity (dV/dtmax), and (G) atrial effective refractory period (aERP), all measured before and after application of 1 μM [ISO] at a stimulated rate of 8 Hz. The calculated AP parameters were averaged from 10 consecutive APs before aERP measurements for each experiment. Statistical analysis was done using a Student’s paired t-test. ∗p < 0.05.

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  FIGURE 4. Electrophysiological effects of [CCh]. Diary plots from an experiment, 0–5 min after 1 μM [CCh] was applied, depicting the time dependence of [CCh] effects on (A) RMP and (B) APD90 as a function of action potentials (APs). The initial to full effect of CCh is abrupt and happens within 100–200 APs. Effect of muscarinic activation on AP parameters (n = 11): (C) Average of the resting membrane potential (RMP), (D) action potential duration at 90% repolarization (APD90), (E) action amplitude (APA), (F) maximum upstroke velocity (dV/dtmax), and (G) atrial effective refractory period (aERP), all measured before and after adding 1 μM CCh. Statistical analysis was done using a Student’s paired t-test. ∗p < 0.05, ∗∗p < 0.005, and ∗∗∗p < 0.001.

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  FIGURE 5. Electrophysiological effects of SK channel inhibition with [ICA] on [ISO] pre-stimulated atria (1 μM [ISO], 10 μM [ICA], n = 5). (A) Representative traces of action potentials. (B) Resting membrane potential (RMP), (C) atrial effective refractory period (aERP), (D) action potential duration at 90% repolarization (APD90), (E) action potential duration at 50% repolarization (APD50), (F) upstroke velocity (dV/dtmax), and (G) AF duration (s), at pacing rate 8 Hz. The significance level was found using a two-way ANOVA by comparing the average of 10 consecutive APs before aERP measurements at baseline and after application of drug. ∗p < 0.005 and ∗∗p < 0.01.

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  FIGURE 6. Electrophysiological effects of SK channel inhibition with [ICA] on [CCh] pre-stimulated atria (1 μM [CCh], 10 μM [ICA], n = 12). (A) Representative traces of action potentials. (B) Resting membrane potential (RMP), (C) atrial effective refractory period (aERP), (D) action potential duration at 90% repolarization (APD90), (E) action potential duration at 50% repolarization (APD50), (F) upstroke velocity (dV/dtmax), and (G) data on AF duration normalized to baseline (%), at pacing rates of 5 Hz. The significance level was found using a two-way ANOVA by comparing the average of 10 consecutive APs before each aERP measurement at baseline and after application of drug or vehicle. ∗p < 0.05, ∗∗p < 0.005, and ∗∗∗p < 0.001.

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  FIGURE 7. The effect of flecainide on CCh-pre-stimulated atria (1 μM [CCh], 1 μM [flecainide], n = 12). (A) Representative traces of action potentials. (B) Resting membrane potential (RMP), (C) atrial effective refractory period (aERP), (D) action potential duration at 90% repolarization (APD90), (E) action potential duration at 50% repolarization (APD50), (F) upstroke velocity (dV/dtmax), with the time-matched control group being unchanged (from 136 ± 12 to 137 ± 22 mV/s) and the flecainide group being reduced (from 152 ± 9 to 103 ± 6 mV/s), and (G) data on AF duration normalized to baseline (%), at pacing rates of 5 Hz. The significance is calculated using a two-way ANOVA by comparing the average of 10 consecutive APs before each aERP measurement at baseline and after application of drug or vehicle. ∗p < 0.05, ∗∗p < 0.005, and ∗∗∗p < 0.001.