Figure 1.

MCU^-/- mice lack MCU expression and evidence for rapid mitochondrial calcium uptake. a) Besides their size, MCU^-/- mice (on right) lack a discernible phenotype. b) MCU^-/- mice are smaller than WT mice (mean +/- S.E.M., p<0.01 by ANOVA; n=14 female WT and n=13 female MCU^-/- mice). c) MCU mRNA expression by RT-PCR analysis in various tissues of WT, heterozygous and MCU^-/- mice (n=3 animals per genotype, mean +/- S.E.M., *p<0.05, **p<0.01 by ANOVA compared to WT expression). d) MCU protein expression in various tissues using a rabbit polyclonal antibody generated against the C-terminus of MCU. Tubulin is used as a loading control. e) Assessment of mitochondrial calcium levels using the fluorescent mitochondrial calcium sensor Fluo-4FF. Calcium addition over the physiological (micromolar) range results in increasing calcium levels in mitochondria isolated from WT skeletal muscle. This uptake in WT mitochondria is inhibited by Ru360 addition. MCU^-/- mitochondria lack any demonstrable uptake. Shown is one experiment that is representative of three similar experiments. Inset-Western blot analysis of MCU expression in purified WT and MCU^-/- mitochondria with cytochrome C oxidase subunit IV (isoform 1) used as a loading control. f) Similar experiment performed using cardiac mitochondria. At higher Ca²⁺ concentrations, there is a small, non Ru360-inhibitable, increase in Fluo-4FF fluorescence in MCU^-/- mitochondria observed. g) Parallel assessment of extramitochondrial calcium measurements demonstrating that only WT cardiac mitochondria appear capable of calcium uptake.