Supplementary Figure 3: Mitochondrial calcium uptake using isolated mitochondria and intact cells.

(a) Cardiac mitochondria were loaded with the calcium indicator Fluo-4FF. Calcium addition over the physiological (micromolar) range results in increasing calcium levels in mitochondria isolated from WT hearts. This uptake in WT mitochondria is blocked by Ru360 addition. MCU^−/− mitochondria lack any demonstrable uptake at low calcium concentrations. At higher Ca²⁺ concentrations, there is a small, non Ru360-inhibitable, increase in Fluo-4FF fluorescence in MCU^−/− mitochondria observed. Addition of EGTA (20 mM) returns fluorescence back to baseline, consistent with dye leakage as the basis of this non Ru360-inhibitable increase in Fluo-4FF fluorescence. (b) A similar experiment where the MCU^−/− mitochondria were briefly pelleted at the end of the experiment. All fluorescence seen with the MCU^−/− mitochondria appears to reside in the supernatant, suggesting again that the fluorescence observed following the addition of higher concentrations of calcium is most consistent with dye leakage out of the mitochondria. (c) Cardiac myocytes were isolated from WT or in MCU^−/− hearts and adult myocytes were loaded with Rhod-2 to measure mitochondrial calcium levels. Shown is a representative experiment following addition of KCl (50 mM) with average fluorescence calculated from WT myocytes (n = 9 cells) or MCU^−/− myocytes (n = 11 cells). (d) Similar analysis using caffeine (20 mM) as a stimulus to increase cytosolic calcium with subsequent imaging of WT myocytes (n = 16 cells) and MCU^−/− myocytes (n = 8 cells). All pooled data represents mean +/− S.E.M.