Figure 1.

Ca²⁺-independent pyruvate/malate-driven respiration in brain and heart mitochondria isolated from WT and MCU KO mice. A and B, respiratory traces of WT and MCU KO brain mitochondria in EGTA media supplemented with pyruvate, malate, ADP, and RR as indicated; arrows specify Ca²⁺ additions for a stepwise increase from ∼12 nm to 2 μm measured fluorimetrically using Fura-2. C, respiratory rates of WT brain mitochondria in the presence and absence of RR as indicated shown as mean ± S.E. (error bars) of n = 5 experiments plotted against free Ca²⁺ concentrations. The red arrow indicates significantly increased Ca²⁺-induced respiration in WT mitochondria (but not WT + RR) determined using two-way ANOVA with Dunnett's multiple-comparison post hoc test. *, p < 0.05. Gray area, significant difference between WT and WT + RR mitochondria determined using two-way ANOVA analysis with Sidak's multiple-comparison post hoc test and a p value of <0.05. White circles visualize the percentage of WT + RR respiration compared with WT calculated from the mean values (yellow area denotes 85–100% of WT respiration). D, respiratory rates of MCU KO brain mitochondria in the presence and absence of RR, as indicated, shown as mean ± S.E. of n = 5 experiments plotted against free Ca²⁺ concentrations. E and F, respiratory traces of WT and MCU KO heart mitochondria in EGTA media supplemented with pyruvate, malate, ADP, and RR as indicated; arrows specify Ca²⁺ additions for a stepwise increase from ∼12 nm to 2 μm. G, respiratory rates of WT heart mitochondria in the presence and absence of RR as indicated shown as mean ± S.E. of n = 5 experiments plotted against free Ca²⁺ concentrations. The red arrow indicates significantly increased Ca²⁺-induced respiration in WT mitochondria (but not WT + RR) determined using two-way ANOVA with Dunnett's multiple-comparison post hoc test. *, p < 0.05. Gray area, significant difference between WT and WT + RR mitochondria determined using two-way ANOVA with Sidak's multiple-comparison post hoc test and a p value of <0.05. White circles visualize the percentage of WT + RR respiration compared with WT calculated from the mean values (yellow area denotes 85–100% of WT respiration). H, respiratory rates of MCU KO heart mitochondria in the presence and absence of RR as indicated shown as mean ± S.E. of n = 5 experiments plotted against free Ca²⁺ concentrations.