Figure 5.

The role of Ca²⁺ and of uncoupling in the effect of ETH129. (a and b) Isolated mouse adult cardiomyocytes were co-loaded with calcein, CoCl₂ and TMRM at 37 °C and then incubated in a Tyrode’s buffer. After 5 min incubation, 20 µM ETH129 (arrow) was added to the medium. Prior to experiments, cells were incubated for 40 min in Tyrode’s medium devoid of Ca²⁺ supplemented with 20 nM ryanodine to deplete sarcoplasmic reticulum Ca²⁺ stocks. (c) Isolated mouse adult cardiomyocytes were co-loaded with calcein, CoCl₂ and propidium iodide at 37 °C and then incubated in a Tyrode’s buffer. After 5 min incubation 1 µM FCCP (arrow) was added to the medium. Fluorescence values were obtained by averaging pixel intensities after background substraction and were normalised as a percentage of the initial value. Each trace represents the mean ± SEM of 3–5 experiments. (d) Mitochondrial membrane potential in isolated cardiac mitochondria treated with increasing concentrations of ETH129 was followed by measuring the uptake/release of the fluorescent dye rhodamine 123. Control: 1 µM FCCP was added to depolarise mitochondrial membrane potential. Curves are representative of at least 4 independent experiments.