Substrate-dependent modulation of oxidative phosphorylation in isolated mitochondria following in vitro hypoxia and reoxygenation injury.

Abstract

Background/objectives: Previous studies using isolated mitochondria have provided new insight into the mechanisms and interventions for ischemia and reperfusion (I/R) injury. In in vitro experiments involving isolated mitochondria, hypoxia and reoxygenation (H/R) has been widely used to mimic I/R injury. However, in in vitro H/R mitochondrial experiments, the effects of various substrates on mitochondrial oxidative phosphorylation are unclear. In the present study, the effects of in vitro I/R injury on mitochondrial oxidative phosphorylation under different substrate conditions were investigated.

Methods: Hypoxia was achieved following complete consumption of oxygen by mitochondria isolated from rat heart tissue in an experimental chamber. The H/R protocol involved 30 min hypoxia followed by 15 min reoxygenation in a chamber opened to the atmosphere. Mitochondrial respiration and respiratory control ratio (RCR) were measured.

Results: When pyruvate/malate were used as substrates, H/R significantly decreased state 3 respiration (28.2±12 nmol O2/min/mg protein) and RCR (2.7±0.8) compared with the control (121.4±32.5 nmol O2/mg protein/min and 7.8±1.2, respectively). In contrast, when succinate was used without rotenone, H/R significantly increased state 3 respiration (57.0±11.2 nmol O2/mg protein/min) and RCR (2.0±0.3) compared with the control (48.2±12.3 nmol O2/mg protein/min and 1.3±0.2, respectively).

Conclusions: The present study demonstrated that mitochondrial oxidative phosphorylation can be modulated by H/R in vitro depending on substrate conditions.