Isoflurane differentially modulates mitochondrial reactive oxygen species production via forward versus reverse electron transport flow: implications for preconditioning

Abstract

Background: Reactive oxygen species (ROS) mediate the effects of anesthetic precondition to protect against ischemia and reperfusion injury, but the mechanisms of ROS generation remain unclear. In this study, the authors investigated if mitochondria-targeted antioxidant (mitotempol) abolishes the cardioprotective effects of anesthetic preconditioning. Further, the authors investigated the mechanism by which isoflurane alters ROS generation in isolated mitochondria and submitochondrial particles.

Methods: Rats were pretreated with 0.9% saline, 3.0 mg/kg mitotempol in the absence or presence of 30 min exposure to isoflurane. Myocardial infarction was induced by left anterior descending artery occlusion for 30 min followed by reperfusion for 2 h and infarct size measurements. Mitochondrial ROS production was determined spectrofluorometrically. The effect of isoflurane on enzymatic activity of mitochondrial respiratory complexes was also determined.

Results: Isoflurane reduced myocardial infarct size (40 ± 9% = mean ± SD) compared with control experiments (60 ± 4%). Mitotempol abolished the cardioprotective effects of anesthetic preconditioning (60 ± 9%). Isoflurane enhanced ROS generation in submitochondrial particles with nicotinamide adenine dinucleotide (reduced form), but not with succinate, as substrate. In intact mitochondria, isoflurane enhanced ROS production in the presence of rotenone, antimycin A, or ubiquinone when pyruvate and malate were substrates, but isoflurane attenuated ROS production when succinate was substrate. Mitochondrial respiratory experiments and electron transport chain complex assays revealed that isoflurane inhibited only complex I activity.

Conclusions: The results demonstrated that isoflurane produces ROS at complex I and III of the respiratory chain via the attenuation of complex I activity. The action on complex I decreases unfavorable reverse electron flow and ROS release in myocardium during reperfusion.

Figure 1

Experimental protocols used for the production of coronary artery occlusion (OCC) and reperfusion in rats treated with saline vehicle (CON), tempol (TEM), mitotempol (MT), and with and without 1.0 minimum alveolar concentration (MAC) isoflurane (ISO). n=8/group.

Figure 2

Myocardial infarct size expressed as a percentage of the left ventricular area at risk (AAR) in rats pretreated with saline vehicle (Con), tempol (TEM) and mitotempol (MT), and in the absence or presence of isoflurane (ISO). Data are presented as mean±standard deviation . n=8/group. *P<0.05 versus Con.

Figure 3

Reactive oxygen species (ROS) production in isolated mitochondria with pyruvate and malate (P/M) as substrates. Representative traces show that rotenone (A), antimycin-A (B), and ubiquinone (C) induced ROS production to a larger extent in the presence compared with the absence (Con) of isoflurane (Iso). (D) Summary of the recordings. Summary data are mean±SD. P/M alone; n=10/group. Rotenone, antimycin-A and ubiquinone; n=8/group. *P<0.05 versus corresponding Con, #P<0.05 versus respective Con with P/M alone.

Figure 4

Reactive oxygen species (ROS) production in isolated mitochondria with succinate (Succ) as substrate. The effects of antimycin-A (A) and ubiquinone (B) on complex II-linked ROS production in the presence or absence (Con) of isoflurane (Iso) were analyzed after reverse electron flow induced-ROS was blocked by rotenone. Iso reduced reverse electron flow induced-ROS production in the absence of rotenone, however, Iso had no effect in the presence of rotenone, and antimycin A or ubiquinone. (C) Summary of recordings. Data are presented as mean±SD, n=8/group, *P<0.05 versus corresponding Con, #P<0.05 versus the respective Con with Succ alone, §P<0.05 versus respective Con with rotenone.

Figure 5

Reactive oxygen species (ROS) production in submitochondrial particles (SMP) with complex I (NADH) and complex II (succinate) substrates in the presence or absence (Con) of isoflurane (Iso). Representative traces (A) show the effects of Iso on ROS production using the different substrates (S). Iso induced ROS generation only when NADH was used as substrate, but had no effect when succinate (Succ) was used. (B) Summary data are mean±SD, n=8/group, *P<0.05 versus Con.

Figure 6

Effect of isoflurane (Iso) on mitochondrial respiration using substrates pyruvate/malate (P/M), succinate (Succ), and adenosine diphosphate (ADP) in the absence or presence of Iso. Representative traces demonstrate the effect of Iso on oxygen consumption using P/M (A), Succ (B) and Succ+rotenone (C). Iso decreased mitochondrial respiration compared to the control group (Con) when P/M were used (D). When Succ was used in the absence of rotenone, Iso significantly increased mitochondrial respiration (E). Iso had no significant effect on mitochondrial respiration when Succ was used in the presence of rotenone (F). Summary data are mean±SD. P/M; n=10/group. Succinate and Succinate+Rotenone; n=8/group. *P<0.05 versus Con.

Figure 7

The effects of (Iso) on the activity of the electron transport chain complexes. Enzymatic activity for each complex during Iso is expressed relative to the absence (Con) of Iso. Data are mean±SD, n=8/group, *P<0.05 versus (Con).