Hypothermia enhances reactive oxygen species generation from complex III with succinate and rotenone but not with pyruvate

Hypothermia is widely used to protect hearts against ischemia reperfusion injury. However, hypothermia has been shown to increase the production of reactive oxygen species (ROS) derived primarily from mitochondria. We examined the effect of temperature on ROS production from complex III in isolated mitochondria using complex I substrate pyruvate (10 mM) or complex II substrate succinate (10 mM) with the complex I blocker rotenone (10 μM) (SR). Mitochondria were isolated from guinea pig hearts (n=8) by differential centrifugation. Hydrogen peroxide (H 2 O 2 ) release rate was measured using Amplex Red + horseradish peroxidase at 37°C, 27°C, and 17°C. The uncoupler CCCP (4 μM) was given to increase electron flow followed by antimycin A (5 μM) to block complex III. Data are mean ± sem. Compared to its basal level in the presence of the substrate, H 2 O 2 release rate increased with antimycin A by 71±12, 15±2, and 13±1 times at 37°C, 27°C, 17°C, respectively with pyruvate, and by 4±0, 5±0, and 13±2 times at 37 v C, 27°C, 17°C respectively with SR. These results demonstrate that the lower the temperature, the more ROS is generated from complex III with SR and the less with pyruvate. This could suggest that ROS generation from complex III is both substrate and temperature dependent.