Figure 4

Response of freeze-thawed mitochondria to Ca²⁺-induced mPT.

Rat liver mitochondria (2 mg protein ml⁻¹) were incubated with TMRM (5 μM) in the presence of succinate (10 mM) and rotenone (1 μM). (a) TMRM fluorescence pre-read (ex. 560/em. 590 nm) revealed no significant effect on ER-000444793 (50 μM) or CsA (10 μM) after 10 minutes incubation. Statistical significance was calculated using a one-way ANOVA corrected for multiple comparisons using Tukey method (NS P > 0.05; GraphPad Prism). Mitochondria were incubated with (b) ER-000444793 or (c) CsA in a 2-fold dilution series for 10 minutes prior to the addition of a CaCl₂ bolus (150 μM). TMRM fluorescence was measured after 20 minutes incubation. Results are expressed as % depolarisation, normalised to DMSO (0% inhibition) and CsA (5 μM; 100% inhibition). (d–g) Rat liver mitochondria (1 mg protein ml⁻¹) were incubated with Fluo-4FF (0.35 μM) in the presence of succinate (10 mM) and rotenone (1 μM). Pulses of CaCl₂ (10 μM) were added sequentially and extra-mitochondrial fluorescence measured. Representative raw traces of Ca²⁺ uptake in the presence of (d) ER-000444793 and (e) CsA demonstrate complete Ca²⁺ uptake prior to concentration-dependent, Ca²⁺-induced mPT. (f,g) Area under the curve was calculated between 240–840 seconds and results are expressed as % inhibition, normalised to DMSO (0% inhibition) and CsA (5 μM; 100% inhibition). Data are expressed as means (±s.d.) of at least three independent experiments. Curves fitting used a 4-parameter logistic equation (GraphPad Prism). Raw traces are representative of at least three independent experiments. Abbreviations: NS; not significant, CsA; cyclosporin A, TMRM; tetramethylrhodamine methylester. a.u; arbitrary unit.