Fig. 3

Direct transfer of Ca²⁺ from the sarcoplasmic reticulum into mitochondria in cardiomyocytes. (a) Representative recordings of mitochondrial (mt)-Ca²⁺ uptake (black line) in permeabilized HL-1 cardiomyocytes. Superfusion with 10-mM caffeine induced uptake of Ca²⁺ released from the SR into mitochondria, which was enhanced by 1-μM ezetimibe and 1-μM disulfiram. Ruthenium red as a blocker of RyR, VDAC and mitochondrial Ca²⁺ uniporter was used to block SR-mitochondria Ca²⁺ transfer as a negative control. (b) Ezetimibe and disulfiram enhanced SR-mitochondria (Mito) Ca²⁺ transfer dose dependently from 0.17 ± 0.01 (n = 26 biological replicates from nine experiments) in vehicle-treated control cells to maximum values of ΔF/F₀ = 0.41 ± 0.04 at 50 nM for ezetimibe (n = 19 replicates from seven experiments) and from 0.15 ± 0.01 (n = 20 replicates from seven experiments) to 0.37 ± 0.04 at 100 nM for disulfiram (n = 9 replicates from nine individual experiments) and at significantly lower concentrations then the established MiCUps kaempferol and efsevin (ezetimibe: EC₅₀ = 25.5 nM, disulfiram: EC₅₀ = 36.8 nM, kaempferol: EC₅₀ = 3.7 μM, efsevin: EC₅₀ = 2.9 μM), while no significant effect of honokiol could be observed. (c) Ezetimibe (Ez) and disulfiram (Dis) did not alter SR Ca²⁺ release as assessed by fura-2 fluorescence in intact HL-1 cardiomyocytes after addition of 10-mM caffeine. The baseline fura-2 fluorescence ratio (R_340nm/380nm) was 0.75 ± 0.03 for vehicle-treated cells, 0.72 ± 0.03 for cells treated with 1-μM ezetimibe and 0.79 ± 0.03 for cells treated with 1-μM disulfiram. Release of Ca²⁺ from the SR induced a change in fluorescence (ΔR) of 1.07 ± 0.09 for vehicle-treated cells, 1.02 ± 0.08 for cells treated with 1-μM ezetimibe and 0.96 ± 0.06 for cells treated with 1-μM disulfiram (n = 34 replicates from six experiments, ANOVA)