Ischemic preconditioning preserves mitochondrial membrane potential and limits reactive oxygen species production

Background: Mitochondrial superoxide radical (O₂ ^•) production increases after cardiac ischemia/reperfusion (IR). Ischemic preconditioning (IPC) preserves mitochondrial function and attenuates O₂^• production, but the mechanism is unknown. Mitochondrial membrane potential (mΔΨ) is known to affect O ₂^• production; mitochondrial depolarization decreases O₂^• formation. We examined the relationship between O₂^• production and mΔΨ during IR and IPC. Materials/methods: Rat hearts were subjected to Control or IPC. Mitochondria were isolated at end equilibration (End EQ), end ischemia (End I), and end reperfusion (End RP). mΔΨ was measured using a tetraphenylphosphonium electrode. Mitochondrial O₂^• production was measured by electron paramagnetic resonance using DMPO spin trap. Cytochrome c levels were measured using high-pressure liquid chromatography. Results: IPC preserved mΔΨ at End I (-156 ± 5 versus -131 ± 6 mV, P <0.001) and End RP (-168 ± 2 versus -155 ± 2 mV, P <0.05). At End RP, IPC attenuated O₂^• production (2527 ± 221 versus 3523 ± 250 AU/mg protein, P <0.05). IPC preserved cytochrome c levels (351 ± 14 versus 269 ± 16 picomoles/mg protein, P <0.05) at End RP, and decreased mitochondrial cristae disruption (10% ± 4% versus 33% ± 7%, P <0.05) and amorphous density formation (18% ± 4% versus 28% ± 1%, P <0.05). Conclusion: We conclude that IPC preserves mΔΨ, possibly by limiting disruption of mitochondrial inner membrane. IPC also decreases mitochondrial O₂^• production and preserves mitochondrial ultrastructure after IR. While it was previously held that slight decreases in mΔΨ decrease O ₂^• production, our results indicate that preservation of mΔΨ is associated with decreased O₂^• and preservation of cardiac function in IPC. These findings indicate that the mechanism of IPC may not involve mΔΨ depolarization, but rather preservation of mitochondrial electrochemical potential.