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

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.