Fluorescence assay for mitochondrial permeability transition in cardiomyocytes cultured in a microtiter plate

Mitochondrial permeability transition pore (MPTP) is a voltage-dependent, large-conductance channel of the inner mitochondrial membrane with an important role in a range of pathophysiological conditions. To facilitate studies of pharmacological pore modulation, we describe an assay in a model using neonatal cardiomyocytes in a 96-well microtiter plate format. In the presence of mitochondrial membrane potential Delta Psi m, accumulation of rhodamine-123 in mitochondria (40,000 cells/well, 2.6 microM rhodamine-123) caused fluorescence signal quenching. Following substitution of dye-free buffer, dequenching occurred on the distribution of rhodamine-123 into the extracellular volume. The addition of a small buffer volume containing digitonin (final concentration 10 microg/ml) and Ca(2+) (final concentrations up to 100 microM free Ca(2+)) caused dequenching (Delta F) due to Delta Psi m dissipation by MPTP, as evidenced by inhibition in the presence of cyclosporin A (0.2-2 microM) and facilitation by pH 6.2. Delta F due to Delta Psi m-dissipating agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or alamethicin (10 microM) was insensitive to either pH or cyclosporin A. Inhibition of Ca(2+)-induced (but not of FCCP- or alamethicin-induced) Delta F by glycogen synthase kinase 3beta (GSK3 beta) antagonist SB216763 and adenosine, acting at the level of intracellular signaling and plasma membrane receptors, respectively, is shown to illustrate potential applications of this assay. Limitation of the assay to cells with energized mitochondria is stressed.