Intracellular Signaling by Reactive Oxygen Species during Hypoxia in Cardiomyocytes*

Abstract

Cardiomyocytes suppress contraction and O₂ consumption during hypoxia. Cytochrome oxidase undergoes a decrease in V _max during hypoxia, which could alter mitochondrial redox and increase generation of reactive oxygen species (ROS). We therefore tested whether ROS generated by mitochondria act as second messengers in the signaling pathway linking the detection of O₂ with the functional response. Contracting cardiomyocytes were superfused under controlled O₂ conditions while fluorescence imaging of 2,7-dichlorofluorescein (DCF) was used to assess ROS generation. Compared with normoxia (PO₂ ∼ 107 torr, 15% O₂), graded increases in DCF fluorescence were seen during hypoxia, with responses at PO₂ = 7 torr > 20 torr > 35 torr. The antioxidants 2-mercaptopropionyl glycine and 1,10-phenanthroline attenuated these increases and abolished the inhibition of contraction. Superfusion of normoxic cells with H₂O₂ (25 μm) for >60 min mimicked the effects of hypoxia by eliciting decreases in contraction that were reversible after washout of H₂O₂. To test the role of cytochrome oxidase, sodium azide (0.75–2 μm) was added during normoxia to reduce theV _max of the enzyme. Azide produced graded increases in ROS signaling, accompanied by graded decreases in contraction that were reversible. These results demonstrate that mitochondria respond to graded hypoxia by increasing the generation of ROS and suggest that cytochrome oxidase may contribute to this O₂ sensing.