Genetic Loss of IK1 Causes Adrenergic-Induced Phase 3 Early Afterdepolariz ations and Polymorphic and Bidirectional Ventricular Tachycardia

Abstract

Background: Arrhythmia syndromes associated with KCNJ2 mutations have been described clinically; however, little is known of the underlying arrhythmia mechanism. We create the first patient inspired KCNJ2 transgenic mouse and study effects of this mutation on cardiac function, I_K1, and Ca²⁺ handling, to determine the underlying cellular arrhythmic pathogenesis.

Methods: A cardiac-specific KCNJ2-R67Q mouse was generated and bred for heterozygosity (R67Q^+/-). Echocardiography was performed at rest, under anesthesia. In vivo ECG recording and whole heart optical mapping of intact hearts was performed before and after adrenergic stimulation in wild-type (WT) littermate controls and R67Q^+/- mice. I_K1 measurements, action potential characterization, and intracellular Ca²⁺ imaging from isolated ventricular myocytes at baseline and after adrenergic stimulation were performed in WT and R67Q^+/- mice.

Results: R67Q^+/- mice (n=17) showed normal cardiac function, structure, and baseline electrical activity compared with WT (n=10). Following epinephrine and caffeine, only the R67Q^+/- mice had bidirectional ventricular tachycardia, ventricular tachycardia, frequent ventricular ectopy, and/or bigeminy and optical mapping demonstrated high prevalence of spontaneous and sustained ventricular arrhythmia. Both R67Q^+/- (n=8) and WT myocytes (n=9) demonstrated typical n-shaped I_K1IV relationship; however, following isoproterenol, max outward I_K1 increased by ≈20% in WT but decreased by ≈24% in R67Q^+/- (P<0.01). R67Q^+/- myocytes (n=5) demonstrated prolonged action potential duration at 90% repolarization and after 10 nmol/L isoproterenol compared with WT (n=7; P<0.05). Ca²⁺ transient amplitude, 50% decay rate, and sarcoplasmic reticulum Ca²⁺ content were not different between WT (n=18) and R67Q^+/- (n=16) myocytes. R67Q^+/- myocytes (n=10) under adrenergic stimulation showed frequent spontaneous development of early afterdepolarizations that occurred at phase 3 of action potential repolarization.

Conclusions: KCNJ2 mutation R67Q^+/- causes adrenergic-dependent loss of I_K1 during terminal repolarization and vulnerability to phase 3 early afterdepolarizations. This model clarifies a heretofore unknown arrhythmia mechanism and extends our understanding of treatment implications for patients with KCNJ2 mutation.

Keywords: action potential; heart; potassium channels; sarcoplasmic reticulum; tachycardia, ventricular.

Figure 1:

R67Q^+/− mice develop paroxysmal bi-directional VT following administration of caffeine and epinephrine. A. Tracing from a Holter monitor of the patient with R67Q mutation showing BiVT and PMVT. B. Hematoxylin and Eosin staining of whole heart from WT and R67Q^+/−. Gross histology revealed no significant difference between WT and R67Q^+/− in heart structure and size. Scale bar is 500μm. Insert scale is 50μm C. Quantification of arrhythmic events observed in WT and R67Q^+/− during ECG analysis. D. ECG recorded from anesthetized WT (top panel) and R67Q^+/− (bottom panel) mice at baseline for 5 minutes. Normal sinus rhythm was observed in both groups. Epinephrine (2mg/kg) and caffeine (120mg/kg) were administered by IP injection and ECG recorded for up to 30 minutes. WT showed faster sinus rhythm (top right panel), whereas R67Q^+/− mice developed paroxysm of bi-directional ventricular tachycardia (bottom right panel taken at 10 minutes post-injection). E. Upper trace shows a representative PVC observed in WT mice following injection of caffeine and epinephrine. PVC is highlight by the arrow. Lower trace shows representative polymorphic ventricular tachycardia observed in R67Q^+/− following injection. N=8 animals per group. Bi-VT – bi-directional ventricular tachycardia; VT – ventricular tachycardia; pVCs – premature ventricular contractions; IP – intraperitoneal injection.

Figure 2:

Epinephrine and caffeine administration results in ventricular arrhythmia in Langendorff-perfused R67Q^+/− heart. A. Sinus rhythm pseudo ECG (pECG) from intact WT heart (black trace) and optical action potential (OAP, blue trace). B. Activation map for sinus rhythm from intact WT heart. C. Sinus rhythm pseudo ECG (pECG) from intact heart (black trace) and optical action potential (OAP, blue trace). D. Spontaneous ventricular tachycardia following adrenergic stimulation captured on pECG and OAP. E. Activation map for sinus rhythm from intact heart. F. Focal activity activation maps from OAP beats VT1–4. Initiation of focal activity is indicated with a black asterisk. Lower right panel shows progression to focal reentry in beats VT5 and VT6. Insert shows reentry activation pattern and OAP delay between a, b and c. N=3/group.

Figure 3:

I_K1 fails to increase following isoproterenol. A. Baseline current voltage relationship for WT (black squares, N=5, n=9), R67Q^+/− (red squares, N=5, n=8). Following isoproterenol (ISO) perfusion for 5 minutes, WT outward current (black open circles, N=5, n=9) increased, however R67Q^+/− outward current (red open circles, N=5, n=8) decreased in response to adrenergic stress. Currents shown are calculated following barium subtraction. Barium is perfused for 2 minutes following final ISO measurement. B. Absolute current values at −50mV following ISO treatment: R67Q^+/− outward I_K1 (0.71 ± 0.08 pA/pF) is significantly decreased compared to WT (1.62 ± 0.38 pA/pF) following isoproterenol treatment (p<0.05). C. Peak outward current was determined at −50 mV and the percentage change for each cell from baseline was calculated. The delta from baseline showed a 19.5 ± 2.7% increase in outward current at −50mV in WT cells (black, N=5, n=9), whereas R67Q^+/− cells (red, N=5, n=8) showed a 23.7 ± 8.3% decrease in outward current at −50 mV. *** p<0.001. Students t-test was used to determine significance difference between groups (3B), or Two-way repeated measures ANOVA with post-hoc Bonferroni correction was used (3C).

Figure 4:

R67Q^+/− myocytes have prolonged APD₉₀ at baseline and following adrenergic stress. A. Representative action potentials from WT (left, upper), WT + isoproterenol (ISO) (right, upper), R67Q^+/− (left, lower), and R67Q^+/− + ISO (right, lower) at 2, 4 and 6Hz. Scale bar represents 100 ms and 10 mV.

Figure 5:

R67Q^+/− does not impact Ca²⁺ handling. A. Representative calcium transient line-scans from WT and R67Q^+/− myocytes. Cells were paced for 30s at 1Hz, 15s at 2Hz and 15s at 3Hz, followed by a pulse of 10 mM caffeine to measure SR content. B. WT and R67Q^+/− myocytes had comparable SR calcium loads at baseline (8.48 ± 0.70 ΔF/F₀ vs. 8.23 ± 0.73 ΔF/F₀, respectively, p>0.1) and following treatment with ISO (14.17 ± 0.84 ΔF/F₀ vs. 14.75 ± 0.49 ΔF/F₀). (WT - N=4, n=18; R67Q^+/− - N=4, n=16). C. No significant difference was observed in calcium transient amplitude between WT (black circles) and R67Q^+/− (red circles) at baseline or following ISO (WT, black squares, R67Q^+/−, red squares) (WT - N=4, n=18; R67Q^+/− - N=4, n=16). D. 50% decay time was not significantly different between WT and R67Q^+/− at baseline or following ISO. (WT - N=4, n=18; R67Q^+/− - N=4, n=16). E. Velocity of the transient (dF/dt_max) is not significantly different between WT and R67Q^+/− at baseline or following ISO. (WT - N=4, n=18; R67Q^+/− - N=4, n=16). F. Ratiometric calcium transient measurements show no significant difference in diastolic calcium between WT and R67Q^+/− VMs (p>0.05). (WT – N=6, n=7; R67Q^+/− - N=4, n=10). Two-way repeated measures ANOVA with post-hoc Bonferroni correction was used (5B-F).

Figure 6:

Adrenergic-dependent phase 3 EADs and spontaneous Ca²⁺ oscillations in R67Q^+/− mice. A. Representative traces from action potential recordings (left) and calcium transient measurements (right) showing phase 3 EADs and spontaneous Ca²⁺ release events, respectively, from isolated R67Q^+/− ventricular myocytes. B. R67Q^+/− myocytes had more EADs at baseline (23 ± 14.2 events) and following ISO (42.8 ± 19.6 events) compared to WT (0 events at baseline, 1.5 ± 1.5 events following ISO) (left panel) during action potential measurements and increased spontaneous release events following ISO during calcium transient measurements (right panel). C. Analysis of take off potential revealed depolarized potential compared to classical phase 2 EADs, with delayed onset from stimulus. D. Linear regression of take off potential vs peak voltage reached by phase 3 EADs in R67Q^+/− myocytes. (Animals/cells for EAD analysis: WT – N=3, n=7; R67Q^+/− - N=2, n=5. Animals/cells for spontaneous event analysis - WT – N=6, n=7; R67Q^+/− - N=4, n=10). Two-way repeated measures ANOVA with post-hoc Bonferroni correction was used (6B), or non-linear regression (6C, 6D).

Figure 7:

Proposed arrhythmia mechanism for KCNJ2 mutation: Adrenergic loss of I_K1 causes a critical change in membrane voltage that decreases repolarization reserve and drives the development of phase 3 EAD.