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.

Background The role of endothelial nitric oxide synthase (eNOS) in isoflurane postconditioning (IsoPC)-elicited cardioprotection is poorly understood. We 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 ischemia and 3 min after reperfusion. Ca2+-induced mitochondrial permeability transition 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 NOS inhibitor) administered either prior to ischemia or at the onset of reperfusion. Mitochondria isolated from postconditioned hearts required significantly higher in vitro Ca2+ loading than control (78 ± 29 vs. 40 ± 25 μM CaCl2 mg protein-1, n = 10, P < 0.05) to open the mitochondrial permeability transition pore. Hearts from eNOS-/- mice displayed no marked differences in infarct size, cardiac function, and sensitivity of mitochondrial permeability transition pore to Ca2+, compared to the wild-type hearts. However, IsoPC failed to alter infarct size, cardiac function or the amount of Ca2+ necessary to open the mitochondrial permeability transition pore in mitochondria isolated from the eNOS-/- hearts compared to 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.

Painful axotomy decreases KATP channel current (IKATP) in primary afferent neurons. Because cytosolic Ca 2؉ signaling is depressed in injured dorsal root ganglia (DRG) neurons, we investigated whether Ca 2؉ -calmodulin (CaM)-Ca 2؉ /CaM-dependent kinase II (CaMKII) regulates IK ATP in large DRG neurons. Immunohistochemistry identified the presence of K ATP channel subunits SUR1, SUR2, and Kir6.2 but not Kir6.1, and pCaMKII in neurofilament 200 -positive DRG somata. Single-channel recordings from cell-attached patches revealed that basal and evoked IK ATP by ionomycin, a Ca 2؉ ionophore, is activated by CaMKII. In axotomized neurons from rats made hyperalgesic by spinal nerve ligation (SNL), basal K ATP channel activity was decreased, and sensitivity to ionomycin was abolished. Basal and Ca 2؉ -evoked K ATP channel activity correlated inversely with the degree of hyperalgesia induced by SNL in the rats from which the neurons were isolated. Inhibition of IK ATP by glybenclamide, a selective KATP channel inhibitor, depolarized resting membrane potential (RMP) recorded in perforated whole-cell patches and enhanced neurotransmitter release measured by amperometry. The selective K ATP channel opener diazoxide hyperpolarized the RMP and attenuated neurotransmitter release. Axotomized neurons from rats made hyperalgesic by SNL lost sensitivity to the myristoylated form of autocamtide-2-related inhibitory peptide (AIPm), a pseudosubstrate blocker of CaMKII, whereas axotomized neurons from SNL animals that failed to develop hyperalgesia showed normal IK ATP inhibition by AIPm. AIPm also depolarized RMP in control neurons via K ATP channel inhibition. Unitary current conductance and sensitivity of K ATP channels to cytosolic ATP and ligands were preserved even after painful nerve injury, thus providing opportunities for selective therapeutic targeting against neuropathic pain.calcium ͉ calmodulin ͉ potassium channels ͉ dorsal root ganglia ͉ neuropathic pain A fter peripheral nerve injury, phenotypic changes in axons and the corresponding somata in the dorsal root ganglia (DRG) lead to membrane hyperexcitability, which results in neuropathic pain (1, 2). These alterations include decreased Ca 2ϩ influx via voltage-gated calcium channels (VGCC), reduced [Ca 2ϩ ] i , and diminished Ca 2ϩ -induced Ca 2ϩ release (3, 4). Additionally, ATPsensitive potassium (K ATP ) channels in large axotomized DRG neurons from rats with hyperalgesia exhibit decreased opening (5, 6). K ATP channels in cardiac myocytes and pancreatic ␤-cells are modulated by cytosolic Ca 2ϩ (7,8), but it remains unknown whether Ca 2ϩ affects neuronal K ATP channels.Cytosolic Ca 2ϩ regulates neuronal channels and other targets via multiple mechanisms (9), including the Ca 2ϩ -calmodulin (CaM)-Ca 2ϩ /CaM-dependent kinase II (CaMKII) pathway (10). CaMKII, which is abundant in neurons (11), assembles into a dodecameric holoenzymatic structure (12, 13). Ca 2ϩ /CaM activates CaMKII subunits, triggering autophosphorylation at T286 residues (14) that increase the affinit...

Background and purpose:The volatile anaesthetic isoflurane protects the heart from ischaemia and reperfusion (I/R) injury when applied at the onset of reperfusion [anaesthetic postconditioning (APoC)]. However, the mechanism of APoC-mediated protection is unknown. In this study, we examined the effect of APoC on mitochondrial bioenergetics, mitochondrial matrix pH (pHm) and cytosolic pH (pHi), and intracellular Ca 2+ . Experimental approach: Cardiac mitochondria from Wistar rats were isolated after in vivo I/R with or without APoC (1.4%-vol isoflurane, 1 minimum alveolar concentration), and mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential (DYm), and oxygen consumption were assessed. In isolated cardiomyocytes and isolated mitochondria I/R injury was produced in vitro, with or without APoC (0.5 mM isoflurane). Intracellular Ca 2+ , pHm, pHi and DYm were monitored with SNARF-1, TMRE and fluo-4, respectively. Myocyte survival was assessed when APoC was induced at pH 7.4 and 7.8. In isolated mitochondria oxygen consumption and ATP synthesis were measured. Key results: In vivo APoC protected against mPTP opening, slowed mitochondrial respiration and depolarized mitochondria. APoC decreased the number of hypercontracted cardiomyocytes at pH 7.4, but not at pH 7.8. APoC attenuated intracellular Ca 2+ accumulation, maintained lower pHm, and preserved DYm during reoxygenation. Isoflurane did not affect the regulation of cytosolic pH. In mitochondria, APoC preserved ATP production rate and respiration. Conclusions and implications: At reperfusion, APoC inhibited mitochondrial respiration, depolarized mitochondria and acidified pHm. These events may lead to inhibition of mPTP opening and, consequently, to preserved DYm and ATP synthesis. This reduces intracellular Ca 2+ overload and cell death.