5.4. Mitochondrial isolation procedure

Hearts allocated for mitochondrial isolation were placed in ice-cold isolation buffer (KE buffer: 0.18M KCL, 0.01M ethylenediaminotetraacetic acid (EDTA), pH 7.4, adjusted with 2M Tris) and mitochondria were isolated by differential centrifugation as described by Sordahl et al [80]. The mitochondrial pellet was divided in two: one half was dispersed in KE medium for immediate measurement of mitochondrial function, while the other half was dissolved in lysis buffer (see below) and stored at -80 °C for subsequent western blot analysis. Protein determination for mitochondrial functional studies was done by the technique of Lowry and coworkers [81].

Mitochondrial oxidative phosphorylation (oxphos) was measured polarographically at 27 °C using an oxygraph (Hansatech Instruments, Bannan UK) and Clark electrode. The mitochondrial incubation medium contained (in mM): sucrose 0.25, Tris-HCl 10, pH 7.4, K₂HPO₄ 8.5, and glutamate 5/malate 2 (substrates for Complex I) or palmitoyl-L-carnitine 0.45/malate 2 (fatty acid beta-oxidation substrate) (pH 7.4). ADP (250–350 nmoles) was added to initiate State 3 respiration. Parameters investigated were the ADP/O ratio, State 3 respiration (mitochondrial respiration in the presence of ADP), State 4 respiration (mitochondrial respiration in absence of ADP). Mitochondrial respiratory rates (states 3 and 4) were expressed as nAtoms oxygen uptake/mg protein/min. The amount of ADP added to the incubation system was obtained spectrophotometrically (molar extinction coefficient of ADP: 15.4 at 259nm). The oxidative phosphorylation rates (nmoles ATP produced/mg prot/min) were calculated as follows: QO₂ (state 3) x ADP/O ratio.