All measurements were carried out using a Fiber Optic Spectrofluorometer (Ocean Optics) in a partially open continuously stirred cuvette at room temperature (22–24°C). Mitochondria were isolated from rabbit hearts as described previously 9 (link) and added (0.5–1.0 mg/ml) to incubation buffer containing 100 mM KCl, 10 mM HEPES, pH 7.4 with Tris, followed by addition of 2.5 mM Pi, and substrates as indicated. In some experiments ADP was added in combination with hexokinase, glucose and MgCl2 to ensure a continuous ADP load to mitochondria, similarly to that in active cardiac cells.
O2 delivery was regulated by adjusting stirring speed (settings 1 to 10, corresponding to 60 to 1100 RPM). The effect of stirring speed on the rate of O2 diffusion into buffer, and how this was balanced by O2 consumption by respiring mitochondria to regulate buffer [O2] levels, is shown in theonline supplement . Alternatively, O2 delivery could be increased rapidly and significantly by injecting a small amount of compressed air into the buffer.
Mitochondria O2 consumption was measured continuously by monitoring buffer O2 content via a fiber optic oxygen sensor FOXY-AL300 (Ocean Optics). The O2 sensor responded to O2 changes at the level > 1 μM (=0.1 kPa = 0.75 mm Hg) (seeonline supplement ).
Mitochondrial membrane potential (Δψm) was estimated using tetramethylrhodamine methyl ester (TMRM, 200 nM) in the buffer solution 9 (link).
ROS production was monitored from reduced dichlorofluorescin (H2DCF) oxidation (ex/em 490/525 nM) after incubating mitochondria with H2DCF diacetate (10 μM) and washing away extramitochondrial dye. Alternatively, O2•− production was measured using MitoSOX red (ex/em 510/580 nm) 10 (link) H2O2 release from mitochondria was measured using Amplex Red Hydrogen Peroxide Assay Kit (Invitrogen) (ex/em 560/590 nm) (seesupplement Fig. S4 ).
Mitochondrial NADH autofluorescence was recorded at excitation/emission (340/460 nm) wavelengths. The NADH signal was calibrated by making mitochondria anoxic with N2 to fully reduce NAD, and subsequently adding O2 and FCCP to fully oxidize NAD.
Mitochondrial iron uptake or release of bound iron in the matrix was determined by monitoring quenching of the Phen Green or calcein fluorescence by chelatable iron 11 (link). Mitochondria were incubated with 10 μM Phen Green FL of 5 μM Calcein AM for 15 min at room temperature, washed and fluorescence was measured at 490 nm excitation, 520 nm emission. “Dequenching” was accomplished with 200 μM 1,10,-phenanthroline.
Aconitase activity was determined by an increase in NADPH fluorescence after adding reaction mixture and permeabilizing inner membrane with alamethicin as described 12 (link).
Changes in buffer •NO level released from Diethylamine NONOate sodium salt (DETA NONO) or S-nitroso-N-acetylpenicillamine (SNAP) were monitored electrochemically using a •NO electrode (World Precision Instruments, Sarasota, FL).
O2 delivery was regulated by adjusting stirring speed (settings 1 to 10, corresponding to 60 to 1100 RPM). The effect of stirring speed on the rate of O2 diffusion into buffer, and how this was balanced by O2 consumption by respiring mitochondria to regulate buffer [O2] levels, is shown in the
Mitochondria O2 consumption was measured continuously by monitoring buffer O2 content via a fiber optic oxygen sensor FOXY-AL300 (Ocean Optics). The O2 sensor responded to O2 changes at the level > 1 μM (=0.1 kPa = 0.75 mm Hg) (see
Mitochondrial membrane potential (Δψm) was estimated using tetramethylrhodamine methyl ester (TMRM, 200 nM) in the buffer solution 9 (link).
ROS production was monitored from reduced dichlorofluorescin (H2DCF) oxidation (ex/em 490/525 nM) after incubating mitochondria with H2DCF diacetate (10 μM) and washing away extramitochondrial dye. Alternatively, O2•− production was measured using MitoSOX red (ex/em 510/580 nm) 10 (link) H2O2 release from mitochondria was measured using Amplex Red Hydrogen Peroxide Assay Kit (Invitrogen) (ex/em 560/590 nm) (see
Mitochondrial NADH autofluorescence was recorded at excitation/emission (340/460 nm) wavelengths. The NADH signal was calibrated by making mitochondria anoxic with N2 to fully reduce NAD, and subsequently adding O2 and FCCP to fully oxidize NAD.
Mitochondrial iron uptake or release of bound iron in the matrix was determined by monitoring quenching of the Phen Green or calcein fluorescence by chelatable iron 11 (link). Mitochondria were incubated with 10 μM Phen Green FL of 5 μM Calcein AM for 15 min at room temperature, washed and fluorescence was measured at 490 nm excitation, 520 nm emission. “Dequenching” was accomplished with 200 μM 1,10,-phenanthroline.
Aconitase activity was determined by an increase in NADPH fluorescence after adding reaction mixture and permeabilizing inner membrane with alamethicin as described 12 (link).
Changes in buffer •NO level released from Diethylamine NONOate sodium salt (DETA NONO) or S-nitroso-N-acetylpenicillamine (SNAP) were monitored electrochemically using a •NO electrode (World Precision Instruments, Sarasota, FL).
