Background:The role of endothelial nitric oxide synthase (eNOS) in isoflurane postconditioning (IsoPC)-elicited cardioprotection is poorly understood. The authors addressed this issue using eNOS−/− mice. Methods:In vivo or Langendorff-perfused mouse hearts underwent 30 min of ischemia followed by 2 h of reperfusion in the presence and absence of postconditioning produced with isoflurane 5 min before and 3 min after reperfusion. Ca2+-induced mitochondrial permeability transition (MPT) pore opening was assessed in isolated mitochondria. Echocardiography was used to evaluate ventricular function. Results:Postconditioning with 0.5, 1.0, and 1.5 minimum alveolar concentrations of isoflurane decreased infarct size from 56 ± 10% (n = 10) in control to 48 ± 10%, 41 ± 8% (n = 8, P < 0.05), and 38 ± 10% (n = 8, P < 0.05), respectively, and improved cardiac function in wild-type mice. Improvement in cardiac function by IsoPC was blocked by NG-nitro-l-arginine methyl ester (a nonselective nitric oxide synthase inhibitor) administered either before ischemia or at the onset of reperfusion. Mitochondria isolated from postconditioned hearts required significantly higher in vitro Ca2+ loading than did controls (78 ± 29 &mgr;m vs. 40 ± 25 &mgr;m CaCl2 per milligram of protein, n = 10, P < 0.05) to open the MPT pore. Hearts from eNOS−/− mice displayed no marked differences in infarct size, cardiac function, and sensitivity of MPT pore to Ca2+, compared with wild-type hearts. However, IsoPC failed to alter infarct size, cardiac function, or the amount of Ca2+ necessary to open the MPT pore in mitochondria isolated from the eNOS−/− hearts compared with control hearts. Conclusions:IsoPC protects mouse hearts from reperfusion injury by preventing MPT pore opening in an eNOS-dependent manner. Nitric oxide functions as both a trigger and a mediator of cardioprotection produced by IsoPC.

Background:Cardioprotection by volatile anesthetic-induced preconditioning (APC) involves activation of protein kinase C (PKC). This 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-&dgr; (rottlerin) and PKC-ϵ (myristoylated PKC-ϵ V1–2 peptide), and the mPTP opening time was measured by 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), and 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-&dgr; 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 &mgr;m · mg−1 control vs. 64 ± 8 &mgr;m · 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-&dgr;. These results point to the connection between cytosolic and mitochondrial components of cardioprotection by isoflurane.

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 two 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.