SK4 K+ channels are therapeutic targets for the treatment of cardiac arrhythmias

Figures

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  Figure 1. SK4 channels are expressed in hiPSC‐CMs derived from a healthy normal individual and a CPVT2 (CASQ2‐D307H) patient Representative traces of an hiPSC‐CM derived from a healthy normal individual following a voltage ramp under the indicated conditions. Solution 1 included 300 μM CdCl2, 25 μM ZD7288, and 10 μM E‐4031.Representative traces of an hiPSC‐CM derived from a CPVT2 (CASQ2 D307H) patient.Scatter plot of the TRAM‐sensitive current densities measured at +60 mV with 1 or 5 μM TRAM‐34. Current densities were 1.00 ± 0.25 pA/pF in normal (n = 19) and 1.39 ± 0.29 pA/pF in CPVT2 (n = 18). Not statistically different (two‐tailed unpaired t‐test).Average traces of the TRAM‐34‐sensitive currents using 1 μM TRAM‐34 of normal (n = 4) and CPVT2‐derived hiPSC‐CMs (n = 5). For clarity, the SEM bars are shown for every mV.Representative Western blots of beating EB lysates from a normal individual and a CPVT2 (CASQ2 D307H) patient showing immunoreactive SK4 protein (≈50 KDa).

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  Figure 2. Blockade of SK4 channels by 1 μM TRAM‐34 reduces arrhythmias recorded in hiPSC‐CMs derived from a CPVT2 (CASQ2‐D307H) patient Representative traces of spontaneous APs recorded in a hiPSC‐CM derived from a normal individual under the indicated conditions.Histograms of statistical data of the beating rate, DD slope, and MDP of hiPSC‐CMs from a normal individual. One‐way ANOVA followed by Tukey's multiple comparison test. For rate (normalized to Control), *P < 0.05, n = 24; for DD slope (normalized to Control), **P < 0.01 and ***P < 0.0001, n = 24; for MDP, ***P < 0.0001, n = 24. Bars and error bars are mean ± SEM.Representative traces of spontaneous APs recorded in a hiPSC‐CM derived from a CPVT2 patient under the indicated conditions.Histograms of statistical data of the beating rate, APD50, and DADs of hiPSC‐CMs from a CPVT2 patient. One‐way ANOVA followed by Tukey's multiple comparison test. For the rate, P = ns, n = 21; for the APD50, P = ns, n = 19; for the DADs, **P < 0.01, n = 19. Bars and error bars are mean ± SEM.

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  Figure 3. SK4 channels are expressed in SAN cells from WT and CASQ2‐D307H KI mice Representative traces of a SAN cell from WT mice following a voltage ramp under the indicated conditions.Representative traces of a SAN cell from CASQ2‐D307H KI mice.Scatter plot of the TRAM‐sensitive current densities measured at +60 mV with 1 or 5 μM TRAM‐34. Current densities were 2.82 ± 0.63 pA/pF in WT (n = 15) and 2.36 ± 0.89 pA/pF in CASQ2‐D307H KI mice (n = 16). Not statistically different (two‐tailed unpaired t‐test).Average traces of the TRAM‐34‐sensitive currents using 1 μM TRAM‐34 of WT (n = 7) and CASQ2‐D307H KI mice (n = 4). For clarity, the SEM bars are shown for every mV.Representative Western blots of heart lysates from WT and CASQ2‐D307H KI mice showing the immunoreactive bands of SK4, CASQ2, and β‐actin proteins in SAN, right and left atrial appendages, and right and left ventricles.Quantification of the SK4 channel immunoreactive protein (normalized to β‐actin) in different heart regions (n = 3). Not statistically different (two‐tailed unpaired t‐test). Error bars are SEM. Source data are available online for this figure.

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  Figure 4. Inhibition of SK4 channels by 2 μM TRAM‐34 lessens the arrhythmic phenotype of SAN cells from CASQ2‐D307H KI mice Representative traces of spontaneous APs recorded in a SAN cell from WT mice under the indicated conditions.Histograms of statistical data of the beating rate, DD slope, and MDP of SAN cells from WT mice. One‐way ANOVA followed by Tukey's multiple comparison test. For rate, *P < 0.05, n = 6; for DD slope, *P < 0.05, **P < 0.01, n = 7; for MDP, *P < 0.05, n = 5. Bars and error bards are mean ± SEM.Representative traces of spontaneous APs recorded in a SAN cell from CASQ2‐D307H KI mice under the indicated conditions.Histograms of statistical data of the beating rate, and MDP of SAN cells from CASQ2‐D307H KI mice. One‐way ANOVA followed by Tukey's multiple comparison test. For the rate, P is not significant, n = 5; for DADs, **P < 0.01, n = 5. Bars and error bards are mean ± SEM.

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  Figure 5. Isoproterenol leads to abnormal SAN calcium transients, which are improved with TRAM‐34 Left, representative traces of spontaneous calcium transients recorded ex vivo in intact SAN tissue preparations from WT mice under the indicated conditions. Right: data summary of calcium transient rate (one‐way ANOVA: **P < 0.01, ***P < 0.001, n = 12). Bars and error bars are mean ± SEM.Representative traces of spontaneous calcium transients recorded from intact SAN of CASQ2 D307H KI mice.Representative traces of different types of calcium transient abnormalities recorded in intact SAN from CASQ2 D307H KI mice, termed as “local Ca2+ release”, “double‐humped transients”, “large‐stored released Ca2+ waves”, and “calcium alternans”.Data summary of the arrhythmic calcium transients in SAN from CASQ2 D307H KI under the indicated conditions.

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  Figure 6. Blockade of SK4 channels by TRAM‐34 improves the ECG arrhythmic features of CASQ2‐D307H KI and CASQ2 KO mice under rest conditions Representative ECG recording following IP injection of vehicle (upper) and 20 mg/kg TRAM‐34 (lower) in WT mice at rest. Sequential vehicle and TRAM‐34 injections were performed on the same animal.Data summary of heart rate (paired t‐test; ***P = 0.0003, n = 10) and PR interval (paired t‐test; ***P = 0.0004, n = 10) in WT mice at rest. Error bars: ± SEM.Representative ECG recording following IP injection of vehicle (upper) and 20 mg/kg TRAM‐34 (lower) in CASQ2‐D307H KI mice at rest.Data summary of heart rate (paired t‐test; ***P < 0.0001, n = 12) and PR interval (paired t‐test; ***P < 0.0001, n = 12) in CASQ2‐D307H KI mice at rest. Error bars: ± SEM.Representative ECG recording following IP injection of vehicle (upper) and 20 mg/kg TRAM‐34 (lower) in CASQ2 KO mice at rest.Data summary of heart rate (paired t‐test; **P = 0.004, n = 7 mice) and PR interval (paired t‐test; **P = 0.004, n = 7) in CASQ2 KO mice at rest. Error bars: ± SEM.

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  Figure 7. Blockade of SK4 channels by TRAM‐34 improves the ECG arrhythmic features of CASQ2‐D307H KI and CASQ2 KO mice during treadmill exercise Representative ECG recording following intraperitoneal injection of vehicle (upper) and 20 mg/kg TRAM‐34 (lower) in WT mice during treadmill exercise.Data summary of heart rate (paired t‐test; ***P = 0.001, n = 10) and PR interval (paired t‐test; ***P = 0.0005, n = 10) in WT mice during exercise. Error bars: ± SEM.Representative ECG recording following IP injection of vehicle (upper) and 20 mg/kg TRAM‐34 (lower) in CASQ2‐D307H KI mice during treadmill exercise.Data summary of heart rate (paired t‐test; ***P = 0.0004, n = 11) and PR interval (paired t‐test; **P = 0.0099, n = 11) in CASQ2‐D307H KI mice during exercise. Error bars: ± SEM.Representative ECG recording following IP injection of vehicle (upper) and 20 mg/kg TRAM‐34 (lower) in CASQ2 KO mice during treadmill exercise.Data summary of heart rate (paired t‐test; *P = 0.0165, n = 7) and PR interval (paired t‐test; **P = 0.0042, n = 7) in CASQ2 KO mice during exercise. Error bars: ± SEM.