Mitochondrial Permeability Transition Pore Assay

CRC of skeletal muscle mitochondria under energized conditions allowed measuring the mitochondrial permeability transition pore (mPTP) opening. Briefly, permeabilized fibers (5–6 mg wet weight) were incubated at 4 °C for 30 min under stirring in buffer R+ containing KCl (800 mM) to extract myosin, block the calcium uptake by the sarcoplasmic reticulum, and, thus, allow calcium uptake only by mitochondria. Then, fibers were washed 3 times for 10 min in CRC buffer (Tris-Base 20 mM, saccharose 150 mM, KCl 50 mM, KH₂PO₄ 2 mM, and succinate 5 mM, pH 7.4 at 23 °C) containing bovine serum albumin (2 mg/mL) and ethylene glycol-bis (β-aminoethyl ether)-N, N,N′,N′-tetraacetic acid (EGTA) (5 μM).
Permeabilized ghost fibers were incubated in a quartz tank with continuous stirring at 24 °C in 1 mL of CRC buffer containing a calcium green-5N fluorescent probe (5 µM; excitation 500 nm; emission 530 nm). The reaction was started by the addition of a calcium pulse (20 mM), followed by calcium pulses every 5 min until it was necessary. After each pulse, a peak of extramitochondrial calcium was recorded and a rapid uptake by the mitochondria was observed, resulting in a decrease in extramitochondrial calcium concentration to a near-basal level. When mitochondria reached the maximal calcium loading threshold, the opening of mPTP happens and mitochondrial calcium is released, resulting in an abrupt increase in extramitochondrial calcium concentration. The amount of calcium necessary to trigger the mPTP opening was calculated from a standard curve relating calcium concentrations to the fluorescence of calcium green-5N. At the end of the experiment, muscle fibers were gathered, dehydrated at 150 °C for 15 min, and weighed. Results were expressed as µmol/mg dry weight.