Fig. 5

Suppression of arrhythmogenesis in freshly isolated murine RyR2^C4496R/WT cardiomyocytes by ezetimibe and disulfiram. (a) Representative confocal linescan recordings of intracellular Ca²⁺ in freshly isolated ventricular cardiomyocytes from RyR2^C4496R/WT mice. Cells were continuously pulsed at 0.5 Hz and spontaneous Ca²⁺ waves were analysed during 1.5 min after pulsing was stopped. Addition of isoprenaline (ISO) induced spontaneous Ca²⁺ waves which could be blocked by the addition of ezetimibe or disulfiram. (b) Quantitative analysis of the experiments in (a). Addition of ISO raised the propensity for spontaneous Ca²⁺ waves from 0.10 ± 0.04 waves per minute (n = 143 cells from 18 mice) to 0.73 ± 0.13 (n = 222 cells from 24 mice) in RyR2^C4496R/WT mice. Addition of MiCups lowered waves to 0.27 ± 0.06 under 0.1 μM ezetimibe (n = 64 cells from seven mice), 0.032 ± 0.02 under 1 μM ezetimibe (n = 35 cells from five mice) and, finally, 0.05 ± 0.03 under 10 μM ezetimibe (n = 80 cells from eight mice) and to 0.44 ± 0.14 under 0.1 (n = 49 cells from seven mice) and 0.15 ± 0.08 under 1 (n = 35 cells from six mice) for disulfiram (Kruskal–Wallis test). (c) Disruption of mitochondrial Ca²⁺ uptake by genetic ablation of mitochondrial Ca²⁺ uniporter (MCU) in MCU^−/− /RyR2^C4496R/WT mice abolished the effect of ezetimibe (Ez) and disulfiram (Dis) indicated by comparable values of 0.61 ± 0.15 (n = 42 cells from five mice, P > 0.05 Kruskal–Wallis-test) for ISO + 10 μM ezetimibe and 0.39 ± 0.20 (n = 47 cells from six mice, P > 0.05, Kruskal–Wallis-test) for ISO + 1 μM disulfiram compared to 0.59 ± 0.12 (n = 107 from 14 mice) under ISO alone. (d) Representative recording from electrically induced systolic Ca²⁺ transients showing normal transients (upper trace) and transients with secondary systolic Ca²⁺ elevations (SSCEs). (e) SSCEs were observed in 12.4 ± 4.7% (n = 121 cells from 14 mice) of all transients in the vehicle control and rose to 23.3 ± 4.4% after addition of ISO (n = 134 cells from 18 mice). Addition of ezetimibe dose-dependently suppressed SSCEs to a minimum of 0.0% under 10 μM ezetimibe (n = 53 cells from six mice). Disulfiram decreased SSCEs to 3.4 ± 2.2% at 0.1 μM (n = 42 cells from six mice), while cells treated with 1 μM showed SSCEs in 17.5 ± 6.5% of all transients (n = 32 cells from five mice, Kruskal–Wallis test). n.a. = not analysable