Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

Abstract

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established(1), accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca(2+) homeostasis(2), bioenergetics and redox signaling( 3). mtPTP opening is triggered by matrix Ca(2+) but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids(4). In vitro, mtPTP opening can be achieved by increasing Ca(2+) concentration inside the mitochondrial matrix through exogenous additions of Ca(2+) (calcium retention capacity). When Ca(2+) levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca(2+) release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function. Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca(2+) handling (uptake and release, as measured by Ca(2+) concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca(2+) measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca(2+), and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.