Figure 5. Mitochondrial O₂^.− and ΔΨ_m in response to increased exogenous O₂^.− in saponin-permeabilized cardiomyocytes.

Myocytes were loaded with TMRM (100 nM) and CM-H₂DCFDA (2 µM) for at least 20min and imaged using two-photon laser scanning fluorescence microscopy (see Materials and Methods). After loading, the excess dye was washed out and the cells were briefly superfused with a permeabilizing solution (saponin) as previously described [10]. After permeabilization, the myocytes were continuously perfused with an intracellular solution containing GSH∶GSSG at a ratio of 300∶1. The TMRM was included in the medium to avoid depletion of the probe during depolarization-repolarization cycles. A) The TMRM and CM-DCF images of a permeabilized cardiomyocyte at time zero after loading and before (top row image) or after permeabilization and 5min imaging under control conditions (Control, second row) or the presence of KO₂ (10 µM, third row; 20 µM, fourth row; 30 µM, fifth row) after 3min equilibration in each case. RIRR-mediated ΔΨ_m depolarization without a permeability transition occurs at the two lower concentrations, while loss of the CM-DCF probe (∼500MW) from the mitochondrial matrix due to PTP opening occurs at 30 µM KO₂. B) The rates of O₂^.− accumulation as a function of KO₂ concentration. Slopes were calculated when the linear rate of change of the CM-DCF signal stabilized under each condition.