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

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.

Near infrared light protects cardiomyocytes from hypoxia and reoxygenation injury by a nitric oxide dependent mechanism.

Photobiomodulation with near infrared light (NIR) provides cellular protection in various disease models. Previously, infrared light emitted by a low-energy laser has been shown to significantly improve recovery from ischemic injury of the canine heart. The goal of this investigation was to test the hypothesis that NIR (670 nm) from light emitting diodes produces cellular protection against hypoxia and reoxygenation-induced cardiomyocyte injury. Additionally, nitric oxide (NO) was investigated as a potential cellular mediator of NIR. Our results demonstrate that exposure to NIR at the time of reoxygenation protects neonatal rat cardiomyocytes and HL-1 cells from injury, as assessed by lactate dehydrogenase release and MTT assay. Similarly, indices of apoptosis, including caspase 3 activity, annexin binding and the release of cytochrome c from mitochondria into the cytosol, were decreased after NIR treatment. NIR increased NO in cardiomyocytes, and the protective effect of NIR was completely reversed by the NO scavengers carboxy-PTIO and oxyhemoglobin, but only partially blocked by the NO synthase (NOS) inhibitor L-NMMA. Mitochondrial metabolism, measured by ATP synthase activity, was increased by NIR, and NO-induced inhibition of oxygen consumption with substrates for complex I or complex IV was reversed by exposure to NIR. Taken together these data provide evidence for protection against hypoxia and reoxygenation injury in cardiomyocytes by NIR in a manner that is dependent upon NO derived from NOS and non-NOS sources.

Role of heat shock protein 90 and endothelial nitric oxide synthase during early anesthetic and ischemic preconditioning.

BACKGROUND: Nitric oxide is known to be essential for early anesthetic preconditioning (APC) and ischemic preconditioning (IPC) of myocardium. Heat shock protein 90 (Hsp90) regulates endothelial nitric oxide synthase (eNOS) activity. In this study, the authors tested the hypothesis that Hsp90-eNOS interactions modulate APC and IPC. METHODS: Myocardial infarct size was measured in rabbits after coronary occlusion and reperfusion in the absence or presence of preconditioning within 30 min of isoflurane (APC) or 5 min of coronary artery occlusion (IPC), and with or without pretreatment with geldanamycin or radicicol, two chemically distinct Hsp90 inhibitors, or N-nitro-L-arginine methyl ester, a nonspecific nitric oxide synthase NOS inhibitor. Isoflurane-dependent nitric oxide production was measured (ozone chemiluminescence) in human coronary artery endothelial cells or mouse cardiomyocytes, in the absence or presence of Hsp90 inhibitors or N-nitro-L-arginine methyl ester. Interactions between Hsp90 and eNOS, and eNOS activation, were assessed with immunoprecipitation, immunoblotting, and confocal microscopy. RESULTS: APC and IPC decreased infarct size (by 50% and 59%, respectively), and this action was abolished by Hsp90 inhibitors. N-nitro-L-arginine methyl ester blocked APC but not IPC. Isoflurane increased nitric oxide production in human coronary artery endothelial cells concomitantly with an increase in Hsp90-eNOS interaction (immunoprecipitation, immunoblotting, and immunohistochemistry). Pretreatment with Hsp90 inhibitors abolished isoflurane-dependent nitric oxide production and decreased Hsp90-eNOS interactions. Isoflurane did not increase nitric oxide production in mouse cardiomyocytes, and eNOS was below the level of detection. CONCLUSION: The results indicate that Hsp90 plays a critical role in mediating APC and IPC through protein-protein interactions, and suggest that endothelial cells are important contributors to nitric oxide-mediated signaling during APC.