The use of CalciumOrange-5N as a specific marker of mitochondrial Ca2+ in mouse skeletal muscle fibers

We report the use of the fluorescent dye CalciumOrange-5N (CaOr-5N) as a specific mitochondria Ca2+ marker in enzymatically dissociated mouse FBD muscle fibers. Using laser scanning confocal microscopy and the dyes Mitotracker Green (MTG), di-8-ANEPPS and endoplasmic reticulum tracker green (ERTG), we determined the relative position of mitochondria, transverse tubules and sarcoplasmic reticulum in the sarcomere. Comparison with electron micrographies showed that mitochondria are mostly present at both sides of Z lines and near the triads located at the A-I band border. CaOr-5N fluorescence was mainly distributed in mitochondria, highly co-localised with MTG and basically excluded from the A band space. ERTG localised mostly between the two t-tubules present in each sarcomere. We studied the effect of the protonophore FCCP using CaOr-5N to measure mitochondrial Ca2+ and JC-1 dye to measure mitochondria inner membrane potential (Delta Psi(m)). After FCCP treatment, the CaOr-5N fluorescence diminished by about 33% in 80 s, while JC-1 fluorescence diminished by 36% in 200 s. Our results show the loss of Ca2+ from mitochondria when Delta Psi(m) is depolarised and demonstrate the usefulness of CaOr-5N to mark mitochondrial [Ca2+](m).