1 Mariannick Marcil, Karine Bourduas, Alexis Ascah, Yan Burelle ...

Ventricular tissue was minced with scissors in 5 ml of buffer A supplemented with 0.2 % fatty
acid free bovine serum albumin (BSA) and homogenized using a Polytron tissue tearer (~3 sec
at a setting of 3). The homogenate was then incubated with the protease Nagarse (1.5 mg/g) for
5 min and further homogenized at the same settings. The homogenate volume was completed to
30 ml with Buffer A + 0.2 % BSA and centrifuged at 800 x g for 10 min. The pellet was discarded
and the supernatant was decanted and centrifuged at 10 000 x g for 10 min. The pellet obtained
was re-suspended in buffer B (in mM: 300 sucrose, 0.5 EGTA, 10 Tris-HCl, pH 7.3) and
centrifuged at 10 000 x g for 10 min. After repeating this washing step twice, the final
mitochondrial pellet was re-suspended in 0.3 ml of buffer B to a protein concentration of ~20
mg/ml. All procedures were carried out at 4 o C. Protein determinations were performed using
the bicinchoninic acid method (Pierce, Rockford, IL, USA), with bovine serum albumin as a
standard.
Mitochondrial respiration
Mitochondrial oxygen consumption was measured polarographically at 22 °C, using Clark-type
electrodes (Oxygraph, Hansatech Instruments, Kings Lynn, England). Experiments were started
with the addition of 0.30 mg mitochondria in 1ml of buffer C (in mM: 125 KCl, 10 KH2PO4, 0.05
EGTA, 10 Tris (hydroxymethyl)-aminomethane - 3-(N-morpholino)propanesulfonic acid (Tris-
MOPS), 2.5 MgCl2). Respiratory substrates feeding complex I (glutamate-malate 5:2.5 mM),
complex II (succinate 5 mM) or complex IV (TMPD-Ascorbate 0.1:1 mM) were added in the
incubation medium. All substrates were free acids buffered to pH 7.3 with Tris. Experiments for
the complex II were made in presence or absence of the complex I inhibitor rotenone (1QM)
(Figure 1). The medium was then supplemented with 0.25 mM ADP to measure maximal rate of
oxidative phosphorylation (VADP). When respiration reached state 4 following complete
phosphorylation of ADP, 0.5 QM oligomycin was added in order to measure oligomycin-
insensitive respiration (Voligo), which eliminates the contribution of slow turnover of adenylates to
6