4.2. Isolation of Mitochondria from Rat Liver, Heart, and Brain

All manipulations with animals, before the isolation of the organs, were performed in accordance with the Helsinki Declaration of 1975 (revised in 1983), national requirements for the care and use of laboratory animals, and protocol 9/2020 of 17.02.2020 approved by the Commission on Biological Safety and Bioethics at the ITEB RAS. Adult male Wistar rats were decapitated after anesthesia with CO₂. Rat liver mitochondria (RLM) were isolated by a standard differential centrifugation procedure [62]. The homogenization medium contained 220 mM mannitol, 70 mM sucrose, 10 mM HEPES (pH adjusted to 7.4 with Trizma Base), 1 mM EGTA, and 0.05% BSA. The mitochondrial pellet was washed three times with a medium devoid of EGTA and BSA. Final pellets were resuspended in this medium to yield ~70 mg protein/mL.

Rat heart mitochondria (RHM) were isolated in the same buffer in a similar way except the mitochondrial pellet was washed twice. The concentration of isolated RHM was approximately 20 mg protein/mL.

Rat brain mitochondria (RBM) were isolated as described in [63] with minor modifications. The homogenization medium contained 320 mM sucrose, 10 mM Tris (pH adjusted to 7.4 with Trizma Base), 0.5 mM EGTA and 0.5 mM EDTA. The brain was homogenized in a Potter homogenizer (800 rpm, 20 passages). The homogenate was centrifuged twice for 4 min at 2000× g and each time the precipitate was discarded. A “сrude” mitochondrial pellet containing synaptosomes, myelin, and non-synaptic RBM was obtained by sedimentation from supernatant at 12,500× g for 11 min. Then, RBM was purified by the sedimentation (17,500× g for 11 min) in a discontinuous Percoll gradient (3, 10, 15, and 24% Percoll in the isolation medium). The fraction of nonsynaptic mitochondria was collected and washed once in an isolation medium free of EGTA (11,500× g, 11 min). The concentration of isolated RBM was approximately 20 mg protein/mL.

Measurements were performed at 37 °C either in a KCl-based medium (KCl-BM) or in a sucrose-based medium (S-BM). KCl-BM contained 125 mM KCl, 20 mM sucrose, 10 mM HEPES (pH adjusted to 7.3 with Trizma Base), 2 mM KH₂PO₄, 2 mM MgCl₂, 5 mM succinate, and 2.5 µM rotenone. S-BM was composed of 280 mM sucrose, 10 mM HEPES (pH 7.3), 2 mM KH₂PO₄, 2 mM MgCl₂, 5 mM succinate, and 2.5 µM rotenone. Other experimental details are given in the figures and figure legends. Mitochondrial protein was assayed by the Biuret method using BSA as a standard [64].