Calcium uptake, swelling, and calcium content.

As previously described (27), isolated mitochondria or MEFs were resuspended in a buffer containing 125 mM KCl, 2 mM K₂HPO₄, 10 μM EGTA, 1 mM MgCl₂, 20 mM HEPES at pH 7.2, 5 mM glutamate, and 5 mM malate. For MEFs, 0.004% digitonin was added for cell permeabilization. Calcium uptake was measured with the fluorescent calcium indicator Calcium Green-5N (Thermo Fisher Scientific) at a final concentration of 1 μM. For quantifying uptake rates, slopes were calculated from linear fits of Calcium Green-5N traces during the first 2 postpeak minutes using GraphPad Prism software. Calcium-induced mitochondrial swelling was measured in isolated mitochondria as a decrease in absorbance at 540 nm using a microplate reader (FLUOstar Omega, BMG Labtech).

Measurement of intramitochondrial calcium content was performed as previously described (12, 27). Briefly, liver and heart mitochondria were isolated continuously in the presence of 2 μM Ru360 (Calbiochem) and 10 μM CGP-37157 (Tocris) to inhibit mitochondrial exchange of calcium during the isolation. For total matrix calcium in liver mitochondria, mitochondria were washed in isolation buffer without EGTA, and then pelleted and diluted in 0.6N HCl, homogenized, and sonicated. After heating for 30 minutes at 95°C and centrifuging for 5 minutes at 10,000 g, supernatants were brought to neutral pH. Calcium concentration was determined spectrophotometrically using the O-Cresolphthalein Complexone calcium assay kit (Cayman Chemical). For free matrix calcium in heart mitochondria, mitochondria were loaded with 20 μM Fluo-4 AM (Thermo Fisher Scientific) for 30 minutes at room temperature and then washed twice with mitochondrial isolation buffer, and then twice more with buffer without EGTA. Finally, mitochondria were resuspended in buffer containing 137 mM KCl, 20 mM HEPES, and 2 mM K₂HPO₄ at pH 7.15, and baseline fluorescence was measured on a microplate reader (FLUOstar Omega, BMG Labtech). The F_max value was obtained by addition of 5 μM ionomycin (Tocris) and 10 μM calcium, followed by the F_min value by addition of 1 mM EGTA.