Background Cardioprotection by volatile anesthetic-induced preconditioning (APC) involves activation of protein kinase C (PKC). The current study investigated the importance of APC-activated PKC in delaying mitochondrial permeability transition pore (mPTP) opening. Methods Rat ventricular myocytes were exposed to isoflurane in the presence or absence of nonselective PKC inhibitor chelerythrine or isoform-specific inhibitors of PKC-δ (rottlerin) and PKC-ε (myristoylated PKC-ε V1-2 peptide), and the mPTP opening time was measured using confocal microscopy. Ca2+-induced mPTP opening was measured in mitochondria isolated from rats exposed to isoflurane in the presence and absence of chelerythrine, or in mitochondria directly treated with isoflurane after isolation. Translocation of PKC-ε was assessed in APC and control cardiomyocytes by Western blotting. Results In cardiomyocytes, APC prolonged time necessary to induce mPTP opening (261±26 s APC vs. 216±27 s control; P<0.05), while chelerythrine abolished this delay to 213±22 s. The effect of isoflurane was also abolished when PKC-ε inhibitor was applied (210±22 s), but not in the presence of PKC-δ inhibitor (269±31 s). Western blotting revealed translocation of PKC-ε toward mitochondria in APC cells. The Ca2+ concentration required for mPTP opening was significantly higher in mitochondria from APC rats (45±8 μM mg-1 control vs. 64±8 μM mg-1 APC), and APC effect was reversed with chelerythrine. In contrast, isoflurane did not protect directly treated mitochondria. Conclusion APC induces delay of mPTP opening through PKC-ε-mediated inhibition of mPTP opening, but not through PKC-δ. These results point to the connection between cytosolic and mitochondrial components of cardioprotection by isoflurane.

-During reperfusion, the interplay between excess reactive oxygen species (ROS) production, mitochondrial Ca 2ϩ overload, and mitochondrial permeability transition pore (mPTP) opening, as the crucial mechanism of cardiomyocyte injury, remains intriguing. Here, we investigated whether an induction of a partial decrease in mitochondrial membrane potential (⌬⌿ m) is an underlying mechanism of protection by anesthetic-induced preconditioning (APC) with isoflurane, specifically addressing the interplay between ROS, Ca 2ϩ , and mPTP opening. The magnitude of APCinduced decrease in ⌬⌿m was mimicked with the protonophore 2,4-dinitrophenol (DNP), and the addition of pyruvate was used to reverse APC-and DNP-induced decrease in ⌬⌿ m. In cardiomyocytes, ⌬⌿ m, ROS, mPTP opening, and cytosolic and mitochondrial Ca 2ϩ were measured using confocal microscope, and cardiomyocyte survival was assessed by Trypan blue exclusion. In isolated cardiac mitochondria, antimycin A-induced ROS production and Ca 2ϩ uptake were determined spectrofluorometrically. In cells exposed to oxidative stress, APC and DNP increased cell survival, delayed mPTP opening, and attenuated ROS production, which was reversed by mitochondrial repolarization with pyruvate. In isolated mitochondria, depolarization by APC and DNP attenuated ROS production, but not Ca 2ϩ uptake. However, in stressed cardiomyocytes, a similar decrease in ⌬⌿m attenuated both cytosolic and mitochondrial Ca 2ϩ accumulation. In conclusion, a partial decrease in ⌬⌿m underlies cardioprotective effects of APC by attenuating excess ROS production, resulting in a delay in mPTP opening and an increase in cell survival. Such decrease in ⌬⌿m primarily attenuates mitochondrial ROS production, with consequential decrease in mitochondrial Ca 2ϩ uptake.cardioprotection; oxidative stress; mitochondria; reactive oxygen species DAMAGE DURING ISCHEMIA and reperfusion (I/R) of the heart involves complex processes where the cellular machinery itself becomes a source of deleterious mediators of injury. Such processes include excessive production of mitochondrial reactive oxygen species (ROS) and cellular Ca 2ϩ overload (6, 37), which trigger opening of the mitochondrial permeability transition pore (mPTP) (16, 19), a critical event in the transition towards cell death (18). The mPTP opening instantly dissipates mitochondrial membrane potential (⌬⌿ m ), ceases mitochondrial ATP production, and initiates cell death pathways (6).Studies suggest that most of the cell death occurs during reperfusion (51, 53), which can be attenuated with antioxidants (52), indicating an important role of ROS. In cardiomyocytes, ROS are primarily generated at complexes I and III of the mitochondrial electron transport chain (50). During I/R, accumulation of cytosolic Ca 2ϩ , which drives accumulation of mitochondrial Ca 2ϩ (45), is primarily attributed to insufficient ATP production and derangement of intracellular ion homeostasis (37). It is suggested that excess ROS production and mitochondrial Ca 2ϩ overload are mutually de...

Background Signal transduction cascade of anesthetic-induced preconditioning has been extensively studied, yet many aspects of it remain unsolved. Here we investigated the roles of reactive oxygen species (ROS) and mitochondrial uncoupling in cardiomyocyte preconditioning by 2 modern volatile anesthetics: desflurane and sevoflurane. Methods Adult rat ventricular cardiomyocytes were isolated enzymatically. The preconditioning potency of desflurane and sevoflurane was assessed in cell survival experiments by evaluating myocyte protection from the oxidative stress-induced cell death. ROS production and flavoprotein fluorescence, an indicator of flavoprotein oxidation and mitochondrial uncoupling, were monitored in real-time by confocal microscopy. The functional aspect of enhanced ROS generation by the anesthetics was assessed in cell survival and confocal experiments using the ROS scavenger Trolox. Results Preconditioning of cardiomyocytes with desflurane or sevoflurane significantly decreased oxidative stress-induced cell death. That effect coincided with increased ROS production and increased flavoprotein oxidation detected during acute myocyte exposure to the anesthetics. Desflurane induced significantly greater ROS production and flavoprotein oxidation than sevoflurane. ROS scavenging with Trolox abrogated preconditioning potency of anesthetics and attenuated flavoprotein oxidation. Conclusion Preconditioning with desflurane or sevoflurane protects isolated rat cardiomyocytes from oxidative stress-induced cell death. Scavenging of ROS abolishes the preconditioning effect of both anesthetics and attenuates anesthetic-induced mitochondrial uncoupling, suggesting a crucial role for ROS in anesthetic-induced preconditioning and implying that ROS act upstream of mitochondrial uncoupling. Desflurane exhibits greater effect on stimulation of ROS production and mitochondrial uncoupling than sevoflurane.

Backround 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, we investigated if mitochondria-targeted antioxidant (mitotempol) abolishes the cardioprotective effects of anesthetic preconditioning. Further, we 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 2h 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 to control experiments (60±4 %). Mitotempol abolished the cardioprotective effects of anesthetic preconditioning (60±9%). Isoflurane enhanced ROS generation in submitochondrial particles with NADH, 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.

Exercise reduces LV contractile deterioration in post-infarction heart failure and alleviates the extent of mitochondrial dysfunction, which is paralleled with preserved complex I activity.