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Aims: Previous studies indicate that nitric oxide derived from endothelial nitric oxide synthase (eNOS) serves as both trigger and mediator in anaesthetic cardiac preconditioning. The mechanisms underlying regulation of eNOS by volatile anaesthetics have not been fully understood. Therefore, this study examined the role of vascular endothelial growth factor (VEGF) in isoflurane cardiac preconditioning., Methods and Results: Wistar rats underwent 30 min of coronary artery occlusion followed by 2 h of reperfusion. Isoflurane given prior to ischaemia/reperfusion significantly decreased myocardial infarct size from 60 ± 1% in control to 40 ± 3% (n = 8 rats/group, P < 0.05). The beneficial effects of isoflurane were blocked by neutralizing antibody against VEGF (nVEGF). Coronary arterial endothelial cells (ECs) alone or together with cardiomyocytes (CMs) were subjected to hypoxia/reoxygenation injury. The expression of VEGF and eNOS was analysed by western blot, and nitric oxide was measured by ozone-based chemiluminescence. In co-cultured CMs and ECs, isoflurane administered before hypoxia/reoxygenation attenuated lactate dehydrogenase activity and increased the ratio of phosphorylated eNOS/eNOS and nitric oxide production. The protective effect of isoflurane on CMs was compromised by nVEGF and after VEGF in ECs was inhibited with hypoxia inducible factor-1α short hairpin RNA (shRNA). The negative effect of hypoxia inducible factor-1α shRNA was restored by recombinant VEGF., Conclusion: Isoflurane cardiac preconditioning is associated with VEGF regulation of phosphorylation of eNOS and nitric oxide production.

Background: Diabetes impairs the cardioprotective effect of volatile anesthetics, yet the mechanisms are still murky. We examined the regulatory effect of isoflurane on microRNA-21, endothelial nitric-oxide synthase, and mitochondrial respiratory complex I in type 2 diabetic mice., Methods: Myocardial ischemia/reperfusion injury was produced in obese type 2 diabetic (db/db) and C57BL/6 control mice ex vivo in the presence or absence of isoflurane administered before ischemia. Cardiac microRNA-21 was quantified by real-time quantitative reverse transcriptional-polymerase chain reaction. The dimers and monomers of endothelial nitric-oxide synthase were measured by Western blot analysis. Mitochondrial nicotinamide adenine dinucleotide fluorescence was determined in Langendorff-perfused hearts., Results: Body weight and fasting blood glucose were greater in db/db than C57BL/6 mice. Isoflurane decreased left ventricular end-diastolic pressure from 35 ± 8 mmHg in control to 23 ± 9 mmHg (P = 0.019, n = 8 mice/group, mean ± SD) and elevated ±dP/dt 2 h after post-ischemic reperfusion in C57BL/6 mice. These beneficial effects of isoflurane were lost in db/db mice. Isoflurane elevated microRNA-21 and the ratio of endothelial nitric-oxide synthase dimers/monomers and decreased mitochondrial nicotinamide adenine dinucleotide levels 5 min after ischemia in C57BL/6 but not db/db mice. MicroRNA-21 knockout blocked these favorable effects of isoflurane, whereas endothelial nitric-oxide synthase knockout had no effect on the expression of microRNA-21 but blocked the inhibitory effect of isoflurane preconditioning on nicotinamide adenine dinucleotide., Conclusions: Failure of isoflurane cardiac preconditioning in obese type 2 diabetic db/db mice is associated with aberrant regulation of microRNA-21, endothelial nitric-oxide synthase, and mitochondrial respiratory complex I.

Background: Hyperglycemia can blunt the cardioprotective effects of isoflurane in the setting of ischemia-reperfusion injury. Previous studies suggest that reactive oxygen species (ROS) and increased mitochondrial fission play a role in cardiomyocyte death during ischemia-reperfusion injury. To investigate the role of glucose concentration in ROS production and mitochondrial fission during ischemia-reperfusion (with and without anesthetic protection), we used the novel platform of human-induced pluripotent stem-cell (iPSC)-derived cardiomyocytes (CMs)., Methods: Cardiomyocyte differentiation from iPSC was characterized by the expression of CM-specific markers using immunohistochemistry and by measuring contractility. iPSC-CMs were exposed to varying glucose conditions (5, 11, and 25 mM) for 24 hours. Mitochondrial permeability transition pore opening, cell viability, and ROS generation endpoints were used to assess the effects of various treatment conditions. Mitochondrial fission was monitored by the visualization of fragmented mitochondria using confocal microscopy. Expression of activated dynamin-related protein 1, a key protein responsible for mitochondrial fission, was assessed by Western blot., Results: Cardiomyocytes were successfully differentiated from iPSC. Elevated glucose conditions (11 and 25 mM) significantly increased ROS generation, whereas only the 25-mM high glucose condition induced mitochondrial fission and increased the expression of activated dynamin-related protein 1 in iPSC-CMs. Isoflurane delayed mitochondrial permeability transition pore opening and protected iPSC-CMs from oxidative stress in 5- and 11-mM glucose conditions to a similar level as previously observed in various isolated animal cardiomyocytes. Scavenging ROS with Trolox or inhibiting mitochondrial fission with mdivi-1 restored the anesthetic cardioprotective effects in iPSC-CMs in 25-mM glucose conditions., Conclusions: Human iPSC-CM is a useful, relevant model for studying isoflurane cardioprotection and can be manipulated to recapitulate complex clinical perturbations. We demonstrate that the cardioprotective effects of isoflurane in elevated glucose conditions can be restored by scavenging ROS or inhibiting mitochondrial fission. These findings may contribute to further understanding and guidance for restoring pharmacological cardioprotection in hyperglycemic patients., Competing Interests: The authors declare no conflicts of interest.

Background: The role of microRNA-21 in isoflurane-induced cardioprotection is unknown. The authors addressed this issue by using microRNA-21 knockout mice and explored the underlying mechanisms., Methods: C57BL/6 and microRNA-21 knockout mice were echocardiographically examined. Mouse hearts underwent 30 min of ischemia followed by 2 h of reperfusion in vivo or ex vivo in the presence or absence of 1.0 minimum alveolar concentration of isoflurane administered before ischemia. Cardiac Akt, endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) proteins were determined by Western blot analysis. Opening of the mitochondrial permeability transition pore (mPTP) in cardiomyocytes was induced by photoexcitation-generated oxidative stress and detected by rapid dissipation of tetramethylrhodamine ethyl ester fluorescence using a confocal microscope., Results: Genetic disruption of miR-21 gene did not alter phenotype of the left ventricle, baseline cardiac function, area at risk, and the ratios of phosphorylated-Akt/Akt, phosphorylated-eNOS/eNOS, and phosphorylated-nNOS/nNOS. Isoflurane decreased infarct size from 54 ± 10% in control to 36 ± 10% (P < 0.05, n = 8 mice per group), improved cardiac function after reperfusion, and increased the ratios of phosphorylated-Akt/AKT, phosphorylated-eNOS/eNOS, and phosphorylated-nNOS/nNOS in C57BL/6 mice subjected to ischemia-reperfusion injury. These beneficial effects of isoflurane were lost in microRNA-21 knockout mice. There were no significant differences in time of the mPTP opening induced by photoexcitation-generated oxidative stress in cardiomyocytes isolated between C57BL/6 and microRNA-21 knockout mice. Isoflurane significantly delayed mPTP opening in cardiomyocytes from C57BL/6 but not from microRNA-21 knockout mice., Conclusions: Isoflurane protects mouse hearts from ischemia-reperfusion injury by a microRNA-21-dependent mechanism. The Akt/NOS/mPTP pathway is involved in the microRNA-21-mediated protective effect of isoflurane.

Background: Anesthetic cardioprotection reduces myocardial infarct size after ischemia-reperfusion injury. Currently, the role of microRNA in this process remains unknown. MicroRNAs are short, noncoding nucleotide sequences that negatively regulate gene expression through degradation or suppression of messenger RNA. In this study, the authors uncovered the functional role of microRNA-21 (miR-21) up-regulation after anesthetic exposure., Methods: MicroRNA and messenger RNA expression changes were analyzed by quantitative real-time polymerase chain reaction in cardiomyocytes after exposure to isoflurane. Lactate dehydrogenase release assay and propidium iodide staining were conducted after inhibition of miR-21. miR-21 target expression was analyzed by Western blot. The functional role of miR-21 was confirmed in vivo in both wild-type and miR-21 knockout mice., Results: Isoflurane induces an acute up-regulation of miR-21 in both in vivo and in vitro rat models (n = 6, 247.8 ± 27.5% and 258.5 ± 9.0%), which mediates protection to cardiomyocytes through down-regulation of programmed cell death protein 4 messenger RNA (n = 3, 82.0 ± 4.9% of control group). This protective effect was confirmed by knockdown of miR-21 and programmed cell death protein 4 in vitro. In addition, the protective effect of isoflurane was abolished in miR-21 knockout mice in vivo, with no significant decrease in infarct size compared with nonexposed controls (n = 8, 62.3 ± 4.6% and 56.2 ± 3.2%)., Conclusions: The authors demonstrate for the first time that isoflurane mediates protection of cardiomyocytes against oxidative stress via an miR-21/programmed cell death protein 4 pathway. These results reveal a novel mechanism by which the damage done by ischemia/reperfusion injury may be decreased.

Mitochondrial dysfunction contributes to cardiac ischemia-reperfusion (IR) injury but volatile anesthetics (VA) may alter mitochondrial function to trigger cardioprotection. We hypothesized that the VA isoflurane (ISO) mediates cardioprotection in part by altering the function of several respiratory and transport proteins involved in oxidative phosphorylation (OxPhos). To test this we used fluorescence spectrophotometry to measure the effects of ISO (0, 0.5, 1, 2mM) on the time-course of interlinked mitochondrial bioenergetic variables during states 2, 3 and 4 respiration in the presence of either complex I substrate K(+)-pyruvate/malate (PM) or complex II substrate K(+)-succinate (SUC) at physiological levels of extra-matrix free Ca(2+) (~200nM) and Na(+) (10mM). To mimic ISO effects on mitochondrial functions and to clearly delineate the possible ISO targets, the observed actions of ISO were interpreted by comparing effects of ISO to those elicited by low concentrations of inhibitors that act at each respiratory complex, e.g. rotenone (ROT) at complex I or antimycin A (AA) at complex III. Our conclusions are based primarily on the similar responses of ISO and titrated concentrations of ETC. inhibitors during state 3. We found that with the substrate PM, ISO and ROT similarly decreased the magnitude of state 3 NADH oxidation and increased the duration of state 3 NADH oxidation, ΔΨm depolarization, and respiration in a concentration-dependent manner, whereas with substrate SUC, ISO and ROT decreased the duration of state 3 NADH oxidation, ΔΨm depolarization and respiration. Unlike AA, ISO reduced the magnitude of state 3 NADH oxidation with PM or SUC as substrate. With substrate SUC, after complete block of complex I with ROT, ISO and AA similarly increased the duration of state 3 ΔΨm depolarization and respiration. This study provides a mechanistic understanding in how ISO alters mitochondrial function in a way that may lead to cardioprotection., (Copyright © 2013. Published by Elsevier B.V.)

Cardiac mitochondria and the sarcolemmal (sarc)KATP channels contribute to cardioprotective signaling of anesthetic-induced preconditioning. Changes in mitochondrial bioenergetics influence the sarcolemmal ATP-sensitive K (sarcKATP) channel function, but whether this channel has impacts on mitochondria is uncertain. We used the mouse model with deleted pore-forming Kir6.2 subunit of sarcKATP channel (Kir6.2 KO) to investigate whether the functional sarcKATP channels are necessary for isoflurane activation of mitochondrial protective mechanisms. Ventricular cardiomyocytes were isolated from C57Bl6 wild-type (WT) and Kir6.2 KO mouse hearts. Flavoprotein autofluorescence, mitochondrial reactive oxygen species production, and mitochondrial membrane potential were monitored by laser-scanning confocal microscopy in intact cardiomyocytes. Cell survival was assessed using H2O2-induced stress. Isoflurane (0.5 mM) increased flavoprotein fluorescence to 180% ± 14% and 190% ± 15% and reactive oxygen species production to 118% ± 2% and 124% ± 6% of baseline in WT and Kir6.2 KO myocytes, respectively. Tetramethylrhodamine ethyl ester fluorescence decreased to 84% ± 6% in WT and to 86% ± 4% in Kir6.2 KO myocytes. This effect was abolished by 5HD. Pretreatment with isoflurane decreased the stress-induced cell death from 31% ± 1% to 21% ± 1% in WT and from 44% ± 2% to 35% ± 2% in Kir6.2 KO myocytes. In conclusion, Kir6.2 deletion increases the sensitivity of intact cardiomyocytes to oxidative stress, but does not alter the isoflurane-elicited protective mitochondrial mechanisms, suggesting independent roles for cardiac mitochondria and sarcKATP channels in anesthetic-induced preconditioning by isoflurane.

Short application of the volatile anesthetic isoflurane at reperfusion after ischemia exerts strong protection of the heart against injury. Mild depolarization and acidification of the mitochondrial matrix are involved in the protective mechanisms of isoflurane, but the molecular basis for these changes is not clear. In this study, mitochondrial respiration, membrane potential, matrix pH, matrix swelling, ATP synthesis and -hydrolysis, and H(2)O(2) release were assessed in isolated mitochondria. We hypothesized that isoflurane induces mitochondrial depolarization and matrix acidification through direct action on both complex I and ATP synthase. With complex I-linked substrates, isoflurane (0.5mM) inhibited mitochondrial respiration by 28 ± 10%, and slightly, but significantly depolarized membrane potential and decreased matrix pH. With complex II- and complex IV-linked substrates, respiration was not changed, but isoflurane still decreased matrix pH and depolarized mitochondrial membrane potential. Depolarization and matrix acidification were attenuated by inhibition of ATP synthase with oligomycin, but not by inhibition of mitochondrial ATP- and Ca(2+)-sensitive K(+) channels or uncoupling proteins. Isoflurane did not induce matrix swelling and did not affect ATP synthesis and hydrolysis, but decreased H(2)O(2) release in the presence of succinate in an oligomycin- and matrix pH-sensitive manner. Isoflurane modulated H(+) flux through ATP synthase in an oligomycin-sensitive manner. Our results indicate that isoflurane-induced mitochondrial depolarization and acidification occur due to inhibition of the electron transport chain at the site of complex I and increased proton flux through ATP synthase. K(+) channels and uncoupling proteins appear not to be involved in the direct effects of isoflurane on mitochondria., (Copyright © 2012. Published by Elsevier B.V.)

Modulation of mitochondrial free Ca(2+) ([Ca(2+)](m)) is implicated as one of the possible upstream factors that initiates anesthetic-mediated cardioprotection against ischemia-reperfusion (IR) injury. To unravel possible mechanisms by which volatile anesthetics modulate [Ca(2+)](m) and mitochondrial bioenergetics, with implications for cardioprotection, experiments were conducted to spectrofluorometrically measure concentration-dependent effects of isoflurane (0.5, 1, 1.5, 2mM) on the magnitudes and time-courses of [Ca(2+)](m) and mitochondrial redox state (NADH), membrane potential (ΔΨ(m)), respiration, and matrix volume. Isolated mitochondria from rat hearts were energized with 10mM Na(+)- or K(+)-pyruvate/malate (NaPM or KPM) or Na(+)-succinate (NaSuc) followed by additions of isoflurane, 0.5mM CaCl(2) (≈200nM free Ca(2+) with 1mM EGTA buffer), and 250μM ADP. Isoflurane stepwise: (a) increased [Ca(2+)](m) in state 2 with NaPM, but not with KPM substrate, despite an isoflurane-induced slight fall in ΔΨ(m) and a mild matrix expansion, and (b) decreased NADH oxidation, respiration, ΔΨ(m), and matrix volume in state 3, while prolonging the duration of state 3 NADH oxidation, respiration, ΔΨ(m), and matrix contraction with PM substrates. These findings suggest that isoflurane's effects are mediated in part at the mitochondrial level: (1) to enhance the net rate of state 2 Ca(2+) uptake by inhibiting the Na(+)/Ca(2+) exchanger (NCE), independent of changes in ΔΨ(m) and matrix volume, and (2) to decrease the rates of state 3 electron transfer and ADP phosphorylation by inhibiting complex I. These direct effects of isoflurane to increase [Ca(2+)](m), while depressing NCE activity and oxidative phosphorylation, could underlie the mechanisms by which isoflurane provides cardioprotection against IR injury at the mitochondrial level., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

Mitochondrial bioenergetic studies mostly rely on isolated mitochondria thus excluding the regulatory role of other cellular compartments important for the overall mitochondrial function. In intact cardiomyocytes, we followed the dynamics of electron fluxes along specific sites of the electron transport chain (ETC) by simultaneous detection of NAD(P)H and flavoprotein (FP) fluorescence intensities using a laser-scanning confocal microscope. This method was used to delineate the effects of isoflurane, a volatile anesthetic and cardioprotective agent, on the ETC. Comparison to the effects of well-characterized ETC inhibitors and uncoupling agent revealed two distinct effects of isoflurane: uncoupling-induced mitochondrial depolarization and inhibition of ETC at the level of complex I. In correlation, oxygen consumption measurements in cardiomyocytes confirmed a dose-dependent, dual effect of isoflurane, and in isolated mitochondria an obstruction of the ETC primarily at the level of complex I. These effects are likely responsible for the reported mild stimulation of mitochondrial reactive oxygen species (ROS) production required for the cardioprotective effects of isoflurane. In conclusion, isoflurane exhibits complex effects on the ETC in intact cardiomyocytes, altering its electron fluxes, and thereby enhancing ROS production. The NAD(P)H-FP fluorometry is a useful method for exploring the effect of drugs on mitochondria and identifying their specific sites of action within the ETC of intact cardiomyocytes., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Human embryonic stem cell (hESC)-derived cardiomyocytes potentially represent a powerful experimental model complementary to myocardium obtained from patients that is relatively inaccessible for research purposes. We tested whether anesthetic-induced preconditioning (APC) with isoflurane elicits competent protective mechanisms in hESC-derived cardiomyocytes against oxidative stress to be used as a model of human cardiomyocytes for studying preconditioning., Methods: H1 hESC cell line was differentiated into cardiomyocytes using growth factors activin A and bone morphogenetic protein-4. Living ventricular hESC-derived cardiomyocytes were identified using a lentiviral vector expressing a reporter gene (enhanced green fluorescent protein) driven by a cardiac-specific human myosin light chain-2v promoter. Mitochondrial membrane potential, reactive oxygen species production, opening of mitochondrial permeability transition pore, and survival of hESC-derived cardiomyocytes were assessed using confocal microscopy. Oxygen consumption was measured in contracting cell clusters., Results: Differentiation yielded a high percentage (∼85%) of cardiomyocytes in beating clusters that were positive for cardiac-specific markers and exhibited action potentials resembling those of mature cardiomyocytes. Isoflurane depolarized mitochondria, attenuated oxygen consumption, and stimulated generation of reactive oxygen species. APC protected these cells from oxidative stress-induced death and delayed mitochondrial permeability transition pore opening., Conclusions: APC elicits competent protective mechanisms against oxidative stress in hESC-derived cardiomyocytes, suggesting the feasibility to use these cells as a model of human cardiomyocytes for studying APC and potentially other treatments/diseases. Our differentiation protocol is very efficient and yields a high percentage of cardiomyocytes. These results also suggest a promising ability of APC to protect and improve engraftment of hESC-derived cardiomyocytes into the ischemic heart.

During reperfusion, the interplay between excess reactive oxygen species (ROS) production, mitochondrial Ca(2+) 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 (DeltaPsi(m)) is an underlying mechanism of protection by anesthetic-induced preconditioning (APC) with isoflurane, specifically addressing the interplay between ROS, Ca(2+), and mPTP opening. The magnitude of APC-induced decrease in DeltaPsi(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 DeltaPsi(m). In cardiomyocytes, DeltaPsi(m), ROS, mPTP opening, and cytosolic and mitochondrial Ca(2+) 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(2+) 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(2+) uptake. However, in stressed cardiomyocytes, a similar decrease in DeltaPsi(m) attenuated both cytosolic and mitochondrial Ca(2+) accumulation. In conclusion, a partial decrease in DeltaPsi(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 DeltaPsi(m) primarily attenuates mitochondrial ROS production, with consequential decrease in mitochondrial Ca(2+) uptake.

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.

Background: We investigated whether reversal of intraoperative atelectasis with the lung recruitment maneuver (RM) affects desflurane arterial concentrations during bariatric surgery., Methods: After anesthetic induction and maintenance with propofol, patients were randomized to receive alveolar RM at intervals (RM group) or not (controls). Desflurane 6% was initiated, and rate of increase of alveolar desflurane concentration (ratio of end-expiratory to inspiratory concentrations, F(A)/F(I)) and desflurane blood concentrations were measured in both groups. Blood and end-tidal desflurane concentrations were also measured after the discontinuation of anesthesia., Results: The RM group had higher intraoperative Pao(2)/Fio(2) compared with the control group (both, P < 0.001). During induction, the rate of increase in blood desflurane concentrations was rapid in both groups. At comparable mechanical ventilation settings, median times to achieve 0.5 mM (approximately 3%) were 2.1 and 1.59 min (P = 0.09) in the control and RM group, respectively. The times to achieve 0.7 mM (approximately 4.2%) desflurane were 15.9 and 9.3 min in the control and RM group, respectively (P = 0.08). Desflurane blood concentrations tended to be higher during the first 30 min after induction in the RM group (P = 0.066). During maintenance or emergence, the blood desflurane concentrations were not different between control and RM groups. Consequently, the time to eye opening did not differ between groups., Conclusion: Although the RM during bariatric surgery represents an effective method for improving intraoperative oxygenation, it does not significantly affect the desflurane blood concentrations during anesthesia or its elimination during emergence.

Background: Clinical trials suggest that anesthetic-induced preconditioning (APC) produces cardioprotection in humans, but the mechanisms of APC and significance of aging for APC in humans are not well understood. Here, the impact of age on the role of two major effectors of APC, mitochondria and sarcolemmal adenosine triphosphate-sensitive potassium (sarcKATP) channels, in preconditioning of the human atrial myocardium were investigated., Methods: Right atrial appendages were obtained from adult patients undergoing cardiac surgery and assigned to mid-aged (MA) and old-aged (OA) groups. APC was induced by isoflurane in isolated myocardium and isolated cardiomyocytes. Mitochondrial oxygen consumption measurements, myocyte survival testing, and patch clamp techniques were used to investigate mitochondrial respiratory function and sarcKATP channel activity., Results: After in vitro APC with isoflurane, the respiratory function of isolated mitochondria was better preserved after hypoxia-reoxygenation stress in MA than in OA. In isolated intact myocytes, APC significantly decreased oxidative stress-induced cell death in MA but not in OA, and isoflurane protection from cell death was attenuated by the sarcKATP channel inhibitor HMR-1098. Further, the properties of single sarcKATP channels were similar in MA and OA, and isoflurane sensitivity of pinacidil-activated whole cell KATP current was no different between MA and OA myocytes., Conclusion: Anesthetic-induced preconditioning with isoflurane decreases stress-induced cell death and preserves mitochondrial respiratory function to a greater degree in MA than in OA myocytes; however, sarcKATP channel activity is not differentially affected by isoflurane. Therefore, effectiveness of APC in humans may decrease with advancing age partly because of altered mitochondrial function of myocardial cells.

Introduction: Anesthetic preconditioning (APC) with volatile anesthetics improves recovery of contractile function and reduces calcium overload after ischemia/reperfusion (I/R). Mitochondrial and sarcolemmal K(ATP) channel openings have been implicated in APC-induced cardioprotection. In this study, we investigated the effect of APC on major calcium cycling proteins and its relation to K(ATP) channels., Methods: Isolated perfused rat hearts were divided into seven groups: Time control (n = 10), ischemia control (n = 8), APC (n = 8), Mitochondrial K(ATP) inhibitor 5-hydroxydecanoate (5-HD, 200 microM, n = 8), Sarcolemmal K(ATP) inhibitor HMR1098 (HMR, 20 microM, n = 8), and APC plus 5-HD or APC plus HMR1098 (n = 8 each). APC was initiated by administering 1.5% isoflurane for 15 min, followed by a 15 min washout before 30 min of myocardial ischemia and 60 min of reperfusion. Ca2+-release channels (RyR2), Ca2+-adenosine triphosphatase (SERCA2a), phospholamban, plasma membrane Ca2+ ATPase, and sodium-calcium exchanger in the homogenate were determined by Western blot assay., Results: APC improved contractile recovery (left ventricular developed pressure, +dP/dt, -dP/dt) after I/R, which was blocked by 5-HD and HMR. I/R depressed the density of RyR2, SERCA2a, and phospholamban, with no changes in the density of plasma membrane Ca2+ ATPase and sodium-calcium exchanger. APC reversed I/R-induced degradation of RyR2 and SERCA2a in the presence or absence of 5HD and HMR., Conclusions: I/R-induced depression in cardiac performance is associated with a down-regulation of the major sarcoplasmic reticulum Ca2+-cycling proteins. Anesthesia preconditioning with isoflurane prevents I/R-related degradation of the RyR2 and SERCA2a in the sarcoplasmic reticulum. However, this effect was independent of its activation of K(ATP) channels.

Background: Activation of the mitochondrial adenosine triphosphate (ATP)-sensitive K+ channel (mitoK(ATP)) has been proposed as a critical step in myocardial protection by isoflurane-induced preconditioning in humans and animals. Recent evidence suggests that reactive oxygen species (ROS) may mediate isoflurane-mediated myocardial protection. In this study, we examined the direct effect of isoflurane and ROS on human cardiac mitoK(ATP) channels reconstituted into the lipid bilayers., Methods: Inner mitochondrial membranes were isolated from explanted human left ventricles not suitable for heart transplantation and fused into lipid bilayers in symmetrical potassium glutamate solution (150 mM). ATP-sensitive K+ currents were recorded before and after exposure to isoflurane and H2O2 under voltage clamp., Results: The human mitoK(ATP) was identified by its sensitivity to inhibition by ATP and 5-hydroxydecanoate. Addition of isoflurane (0.8 mM) increased the open probability of the mitoK(ATP) channels, either in the presence or absence of ATP inhibition (0.5 mM). The isoflurane-mediated increase in K+ currents was completely inhibited by 5-hydroxydecanoate. Similarly, H2O2 (200 microM) was able to activate the mitoK(ATP) previously inhibited by ATP., Conclusions: These data confirm that isoflurane, as well as ROS, directly activates reconstituted human cardiac mitoK(ATP) channel in vitro, without apparent involvement of cytosolic protein kinases, as commonly proposed. Activation of the mitoK(ATP) channel may contribute to the myocardial protective effect of isoflurane in the human heart.

Ischemic cardiac injury can be substantially alleviated by exposing the heart to pharmacological agents such as volatile anesthetics before occurrence of ischemia-reperfusion. A hallmark of this preconditioning phenomenon is its memory, when cardioprotective effects persist even after removal of preconditioning stimulus. Since numerous studies pinpoint mitochondria as crucial players in protective pathways of preconditioning, the aim of this study was to investigate the effects of preconditioning agent isoflurane on the mitochondrial bioenergetic phenotype. Endogenous flavoprotein fluorescence, an indicator of mitochondrial redox state, was elevated to 195 +/- 16% of baseline upon isoflurane application in intact cardiomyocytes, indicating more oxidized state of mitochondria. Isoflurane treatment also elicited partial dissipation of mitochondrial transmembrane potential, which remained depolarized even after anesthetic withdrawal (tetramethylrhodamine fluorescence intensity declined to 83 +/- 3 and 81 +/- 7% of baseline during isoflurane exposure and washout, respectively). Mild uncoupling, with preserved ATP synthesis, was also detected in mitochondria that were isolated from animals that had been previously preconditioned by isoflurane in vivo, revealing its memory nature. These mitochondria, after exposure to hypoxia and reoxygenation, exhibited better preserved respiration and ATP synthesis compared with mitochondria from nonpreconditioned animals. Partial mitochondrial depolarization was paralleled by a diminished Ca(2+) uptake into isoflurane-treated mitochondria, as indicated by the reduced increment in rhod-2 fluorescence when mitochondria were challenged with increased Ca(2+) (180 +/- 24 vs. 258 +/- 14% for the control). In conclusion, isoflurane preconditioning elicits partial mitochondrial uncoupling and reduces mitochondrial Ca(2+) uptake. These effects are likely to reduce the extent of the mitochondrial damage after the hypoxic stress.

Background: Activation of the cardiac sarcolemmal adenosine triphosphate-sensitive potassium (KATP) channel during metabolic stress initiates cellular events that preserve cardiac performance. Previous studies showed that halogenated anesthetics prime KATP channels under whole cell voltage clamp and act in intracellular pH (pHi)-dependent manner on KATP channels in excised membrane patches. However, it is not known how halogenated anesthetics interact with these channels., Methods: The authors evaluated the effect of pHi and isoflurane on the KATP channel subunits, the pore-forming inward rectifier Kir6.2, and the regulatory sulfonylurea receptor SUR2A, using HEK293 cells as a heterologous expression system. Single channel activity was recorded in the inside-out patch configuration., Results: At pHi 7.4, isoflurane had negligible effect on activity of wild-type Kir6.2/SUR2A, but at pHi 6.8, the channel open probability was increased by isoflurane (0.177 +/- 0.077 to 0.364 +/- 0.164). By contrast, the open probability of truncated Kir6.2DeltaC26, which forms a functional channel without SUR2A, was attenuated by isoflurane at both pHi 7.4 and pHi 6.8. Coexpression of Kir6.2DeltaC26 with SUR2A restored pHi sensitivity of channel activation by isoflurane. Site-directed mutagenesis within the Walker motifs of SUR2A abolished isoflurane activation of KATP channel at pHi 6.8. In addition, the pancreatic-type channels expressing sulfonylurea receptor SUR1 could not be activated by isoflurane., Conclusions: The nucleotide binding domains of SUR2A play a crucial role in isoflurane facilitation of the KATP channel activity at moderately acidic pHi as would occur during early ischemia. These findings support direct and differential interaction of isoflurane with the subunits of the cardiac sarcolemmal KATP channel.

Background: Cardiac preconditioning, including that induced by halogenated anesthetics, is an innate protective mechanism against ischemia-reperfusion injury. The adenosine triphosphate-sensitive potassium (K(ATP)) channels are considered essential in preconditioning mechanism. However, it is unclear whether K(ATP) channels are triggers initiating the preconditioning signaling, and/or effectors responsible for the cardioprotective memory and activated during ischemia-reperfusion., Methods: Adult rat cardiomyocytes were exposed to oxidative stress with 200 microM H(2)O(2) and 100 microM FeSO4. Myocyte survival was determined based on morphologic characteristics and trypan blue exclusion. To induce preconditioning, the myocytes were pretreated with isoflurane. The involvement of sarcolemmal and mitochondrial K(ATP) channels was investigated using specific inhibitors HMR-1098 and 5-hydroxydecanoic acid. Data are expressed as mean +/- SD., Results: Oxidative stress induced cell death in 47 +/- 14% of myocytes. Pretreatment with isoflurane attenuated this effect to 26 +/- 8%. Blockade of the sarcolemmal K(ATP) channels abolished the protection by isoflurane pretreatment when HMR-1098 was applied throughout the experiment (50 +/- 21%) or only during oxidative stress (50 +/- 12%), but not when applied during isoflurane pretreatment (29 +/- 13%). Inhibition of the mitochondrial K(ATP) channels abolished cardioprotection irrespective of the timing of 5-hydroxydecanoic acid application. Cell death was 42 +/- 23, 45 +/- 23, and 46 +/- 22% when 5-hydroxydecanoic acid was applied throughout the experiment, only during isoflurane pretreatment, or only during oxidative stress, respectively., Conclusion: The authors conclude that both sarcolemmal and mitochondrial K(ATP) channels play essential and distinct roles in protection afforded by isoflurane. Sarcolemmal K(ATP) channel seems to act as an effector of preconditioning, whereas mitochondrial K(ATP) channel plays a dual role as a trigger and an effector.

Background: The early memory of anesthetic-induced preconditioning (APC) is a period when myocardial protection continues even after removal of the anesthetic. Because adenosine triphosphate-sensitive potassium (KATP) channels are important mediators of APC, the authors investigated the hypothesis that the memory involves channel priming by isoflurane via a long-term modulation of the sensitivity to intracellular adenosine nucleotides., Methods: Ventricular cardiomyocytes were obtained from the rat hearts after 30 min in vivo APC with 1.4% isoflurane and from control non-APC rat hearts. Whole cell and excised inside-out patch clamp techniques were used to study the sarcolemmal KATP channel. Membrane expression of KATP channel proteins, the pore-forming inward rectifier Kir6.2, and the regulatory sulfonylurea receptor SUR2A were assessed in APC and non-APC hearts by Western blotting., Results: Activation of whole cell KATP current by isoflurane was enhanced after in vivo APC. At the single-channel level, this was paralleled by a 12-fold decrease in adenosine 5'-triphosphate sensitivity and a 3-fold decrease in adenosine 5'-diphosphate sensitivity, without changing the probability of channel opening or single-channel conductance. The membrane expression of Kir6.2 and SUR2A subunits was not altered by in vivo APC. A direct in vitro application of isoflurane to excised membrane patches increased the channel open probability and produced a 4-fold decrease in adenosine 5'-triphosphate sensitivity only of channels in non-APC myocytes., Conclusions: In vivo APC by isoflurane decreases sensitivity of the sarcolemmal KATP channel to inhibition by adenosine 5'-triphosphate and decreases adenosine 5'-diphosphate sensitivity. These effects persist even after discontinuation of the anesthetic, suggesting a possible novel factor that may contribute to the mechanism of early memory of APC.

Background: Volatile anesthetics attenuate apoptosis. The underlying mechanisms remain undefined. The authors tested whether isoflurane reduces apoptosis in cardiomyocytes subjected to oxidative or inflammatory stress by enhancing Akt and B-cell lymphoma-2 (Bcl-2)., Methods: Adult and neonatal rat ventricular myocytes and atrial HL-1 myocytes were exposed to hypoxia, hydrogen peroxide, or neutrophils with or without isoflurane pretreatment. The authors assessed cell damage and investigated apoptosis using mitochondrial cytochrome c release, caspase activity, and TUNEL assay. They determined expression of phospho-Akt and Bcl-2 and tested their involvement by blocking phospho-Akt with wortmannin and Bcl-2 with HA14-1., Results: Isoflurane significantly reduced the cell damage and apoptosis induced by hypoxia, H2O2, and neutrophils. Isoflurane reduced hypoxia-induced mitochondrial cytochrome c release in HL-1 cells by 45 +/- 12% and caspase activity by 28 +/- 4%; in neonatal cells, it reduced caspase activity by 43 +/- 5% and TUNEL-positive cells by 50 +/- 2%. Isoflurane attenuated H2O2-induced caspase activity in HL-1 cells by 48 +/- 16% and TUNEL-positive cells by 78 +/- 3%; in neonatal cells, it reduced caspase activity by 30 +/- 3% and TUNEL-positive cells by 32 +/- 7%. In adult cardiomyocytes exposed to neutrophils, isoflurane decreased both mitochondrial cytochrome c and caspase activity by 47 +/- 3% and TUNEL-positive cells by 25 +/- 4%. Isoflurane enhanced phospho-Akt and Bcl-2 expression. Wortmannin and HA14-1 prevented the action of isoflurane (53 +/- 8% and 54 +/- 7% apoptotic cells vs. 18 +/- 1% without blockers)., Conclusions: Isoflurane protects cardiomyocytes against apoptosis induced by hypoxia, H2O2, or activated neutrophils through Akt activation and increased Bcl-2 expression. This suggests that a reduction in apoptosis contributes to the cardioprotective effects of isoflurane.

Background: Cardioprotective effects of volatile anesthetics in anesthetic-induced preconditioning involve activation of the cardiac sarcolemmal adenosine triphosphate-sensitive potassium (sarcKATP) channels. This study addressed the memory phase of anesthetic preconditioning by investigating whether brief exposure to isoflurane produces lasting sensitization of the sarcKATP channel and whether protein kinase C mediates this effect., Methods: Whole cell sarcKATP channel current (IKATP) was monitored from single isolated rat ventricular cardiomyocytes. Pinacidil was used to open the channel, and the magnitude of activated IKATP was an indicator of channel's ability to open. Involvement of protein kinase C was investigated using chelerythrine and isoform-specific peptide inhibitors and activators of protein kinase C-delta and protein kinase C-epsilon., Results: The mean density of IKATP elicited by pinacidil (5 microm) in anesthetic-free conditions was 3.8 +/- 3.7 pA/pF (n = 11). After 10 min of exposure to isoflurane (0.56 mm) and 10 or 30 min of anesthetic washout, pinacidil-elicited IKATP was increased to 15.6 +/- 11.3 pA/pF (n = 12; P < 0.05) and 11.8 +/- 3.9 pA/pF (n = 6; P < 0.05), respectively. In the presence of chelerythrine (5 microm), isoflurane did not potentiate channel opening, and IKATP was 6.6 +/- 4.6 pA/pF (n = 11). Application of protein kinase C-delta peptide inhibitor also abolished isoflurane-induced sensitization of sarcKATP channel, and IKATP was 7.7 +/- 5.4 pA/pF (n = 12). In contrast, protein kinase C-epsilon peptide inhibitor did not affect channel sensitization, and pinacidil-elicited current was 14.8 +/- 9.6 pA/pF (n = 12). Interestingly, when both protein kinase C-delta and protein kinase C-epsilon activators were applied instead of isoflurane, they sensitized the channel to the same extent as isoflurane (18.9 +/- 7.2 pA/pF, n = 11, and 18.6 +/- 11.1 pA/pF, n = 10, respectively)., Conclusion: Isoflurane induces prolonged sensitization of the sarcKATP channel to opening that persists even after anesthetic withdrawal. Our results indicate that protein kinase C-delta, rather than protein kinase C-epsilon, is a likely mediator of isoflurane effects, although both protein kinase C-delta and protein kinase C-epsilon can modulate the channel function.

Protein kinase C (PKC)-dependent signaling pathways may be involved in the "memory" effect of anesthetic and ischemic preconditioning, which facilitates activation of cardioprotective adenosine triphosphate (ATP)-sensitive potassium channels during later ischemic challenge and ATP depletion. Using patch-clamp techniques, we found that exposure of isolated guinea pig cardiomyocytes to 1 mM of isoflurane after phorbol ester stimulation of PKC facilitates the induction of larger (P < or = 0.05) sarcolemmal K(ATP) channel currents (IKATP) during cell dialysis with 0.5, compared to 1.0, mM of ATP in the pipette (10 +/- 5 versus 2 +/- 1 pA/pF in five and six cells, respectively). A PKC inhibitor, bisindolylmaleimide, abolished the induction of IKATP by a second brief isoflurane exposure under these conditions. A diacylglycerol PKC activator applied via the pipette elicited concentration-related activation of IKATP. The diacylglycerol alone (0.5 microM) elicited I(KATP), averaging 5 +/- 3 pA/pF in nine cells. Briefly treating myocytes on the microscope stage with isoflurane, followed by washout and patching with the same diacylglycerol solution, elicited larger (P < or = 0.01) IKATP, averaging 40 +/- 9 pA/pF (10 cells), with an onset 48 +/- 2 min after anesthetic pretreatment. Facilitation of IKATP by isoflurane during the reduction of intracellular ATP is dependent on PKC, whereas "preconditioning" myocytes with isoflurane causes persistent changes in sarcolemmal KATP channel function, which enhance the induction of IKATP by a diacylglycerol.

Background: Cardioprotection by volatile anesthetic-induced preconditioning is known to involve intracellular signaling pathways. Recent studies have shown that protein kinase C (PKC) plays an important role in anesthetic-induced preconditioning. In this study, the effects of the activation of specific isozymes of PKC, specifically PKC-epsilon and -delta, on the modulation of the sarcolemmal adenosine triphosphate-sensitive potassium (sarcKATP) channel by isoflurane were investigated., Methods: The sarcKATP current was measured in ventricular myocytes isolated from guinea pig hearts using the whole cell configuration of the patch clamp technique. Peptides that induced the translocation of specific PKC isozymes were used to activate PKC-epsilon and PKC-delta., Results: Under whole cell conditions, isoflurane alone was unable to elicit the opening of the sarcKATP channel. Pretreatment with the specific PKC-epsilon activator, PP106, primed the sarcKATP channel to open in the presence of isoflurane. The resulting sarcKATP current densities in the presence of 0.88 mm isoflurane were 6.5 +/- 6.0 pA/pF (n = 7) and 40.4 +/- 18.2 pA/pF (n = 7) after pretreatment with 100 and 200 nm PP106, respectively. The PKC-epsilon antagonist PP93 abolished this effect. A scrambled peptide of the PKC-epsilon activator PP105 did not prime the sarcKATP channel. The PKC-delta activator PP114 was significantly less effective in priming the sarcKATP channel. 5-Hydroxydecanoate significantly attenuated the effect of the PKC-epdsilon activator on the sarcKATP channel. In addition, immunohistochemical analysis showed that the PKC-epsilon isoform translocated to both the mitochondria and sarcolemma after anesthetic-induced preconditioning, whereas the PKC-delta isoform translocated to the mitochondria., Conclusion: The PKC-epsilon isozyme primed the sarcKATP channel to open in the presence of isoflurane. The PKC-delta isozyme was significantly less effective in modulating the isoflurane effect on this channel.

Background: Myocardial protection by volatile anesthetics involves activation of cardiac adenosine triphosphate-sensitive potassium (K(ATP)) channels. The authors have previously shown that isoflurane enhances sensitivity of the sarcolemmal K(ATP) channel to the opener, pinacidil. Because reactive oxygen species seem to be mediators in anesthetic preconditioning, the authors investigated whether they contribute to the mechanism of the sensitization effect by isoflurane., Methods: Ventricular myocytes were isolated from guinea pig hearts for the whole cell patch clamp recordings of the sarcolemmal K(ATP) channel current (I(KAPT)). Free radical scavengers N-acetyl-L-cysteine, carnosine, superoxide dismutase, and catalase were used to investigate whether reactive oxygen species mediate isoflurane facilitation of the channel opening by pinacidil. A possible role of the mitochondrial K(ATP) channels was tested using a blocker of these channels, 5-hydroxydecanoate., Results: The mean density (+/- SEM) of I(KAPT) elicited by pinacidil (20 microM) was 18.9 +/- 1.8 pA/pF (n = 11). In the presence of isoflurane (0.55 mM), the density of pinacidil-activated I(KAPT) increased to 38.5 +/- 2.4 pA/pF (n = 9). Concurrent application of isoflurane and N-acetyl-L-cysteine decreased the sensitization effect by isoflurane in a concentration-dependent manner, whereby the densities of I(KAPT) were 32.6 +/- 1.4 (n = 6), 26.2 +/- 2.3 (n = 6), and 19.4 +/- 2.1 pA/pF (n = 8) at 100, 250, and 500 microM N-acetyl-L-cysteine, respectively. Concurrent application of isoflurane and carnosine (100 microM), superoxide dismutase (100 U/ml), or catalase (100 U/ml) attenuated the densities of I(KAPT) to 27.9 +/- 2.6, 27.2 +/- 2.9, and 25.9 +/- 2.2 pA/pF, respectively. None of the scavengers affected activation of I(KAPT) by pinacidil alone. 5-Hydroxydecanoate (100 microM) did not alter the sensitization effect by isoflurane, and the density of I(KAPT) in this group was 37.1 +/- 3.8 pA/pF (n= 6)., Conclusion: These results suggest that reactive oxygen species contribute to the mechanism by which isoflurane sensitizes the cardiac sarcolemmal K(ATP) channel to the opener, pinacidil.

Unlabelled: The precise mechanism of isoflurane and mitochondrial adenosine triphosphate-sensitive potassium channel (mitoK(ATP)) interaction is still unclear, although the mitoK(ATP) is involved in isoflurane-induced preconditioning. We examined the role of various intracellular signaling systems in mitoK(ATP) activation with isoflurane. Mitochondrial flavoprotein fluorescence (MFF) was measured to quantify mitoK(ATP) activity in guinea pig cardiomyocytes. To confirm isoflurane-induced MFF, cells were exposed to Tyrode's solution containing either isoflurane (1.0 +/- 0.1 mM) or diazoxide and then both drugs together (n = 10 each). In other studies, the following drugs were each added during isoflurane administration: adenosine or the adenosine receptor antagonist 8-(p-sulfophenyl)-theophylline (SPT); the protein kinase C (PKC) activators phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu); the PKC inhibitors polymyxin B and staurosporine; the tyrosine kinase inhibitor lavendustin A; or the mitogen-activated protein kinase inhibitor SB203580 (n = 10 each). Isoflurane potentiated MFF induced by diazoxide (100 micro M), and diazoxide also increased isoflurane-induced MFF. PMA (0.2 micro M), PDBu (1 micro M), and adenosine (100 micro M) induced MFF. However, SPT (100 micro M), polymyxin B (50 micro M), staurosporine (200 nM), lavendustin A (0.5 micro M), and SB203580 (10 micro M) all failed to inhibit the effect of isoflurane. Our results show that isoflurane, adenosine, and PKC activate mitoK(ATP). However, our data do not support an action of isoflurane through pathways involving adenosine, PKC, tyrosine kinase, or mitogen-activated protein kinase. These results suggest that isoflurane may directly activate mitoK(ATP)., Implications: Our results show that isoflurane activates mitochondrial adenosine triphosphate-sensitive potassium (mitoK(ATP)) channels, but not through pathways involving adenosine, protein kinase C, tyrosine kinase, or p38 mitogen-activated protein kinase. Isoflurane may directly activate mitoK(ATP) channels.

Unlabelled: The slowly activating delayed-rectifier potassium current, IKs, is a major outward current responsible for the repolarization of the cardiac action potential (AP). Dysfunction of this channel can lead to AP prolongation, resulting in the long QT syndrome. We hypothesized that anesthetic-induced AP prolongation is caused by inhibition of IKs, in addition to the inhibition of IKr (rapidly activating delayed-rectifier potassium channel current), a condition often found in drug-induced AP prolongation. The whole-cell patch clamp technique was used to study the effects of isoflurane on IKs and IKr recorded from guinea pig single ventricular myocytes. The effect of protein kinase C on IKs inhibition by isoflurane was also investigated. Isoflurane inhibited IKs in a concentration- and temperature-dependent manner. The inhibitory effects of isoflurane at clinically relevant concentrations of 0.3 and 0.6 mM were greater at 22 degrees C than at 36 degrees C. Voltage-dependent activation of IKs was not affected at these concentrations. IKs deactivation kinetics were accelerated by isoflurane at 22 degrees C but not at 36 degrees C. Isoflurane inhibition of IKs was significantly greater than that of IKr. Protein kinase C activation enhanced IKs but did not suppress the inhibitory effect of isoflurane. Our results suggest that IKs inhibition is one of the mechanisms underlying anesthetic-induced AP and QT prolongation. Because most of the ion channel studies on anesthetic effects are conducted at room temperature, the temperature-dependent effect on IKs confirms the importance of anesthetic experiments conducted at physiological temperature., Implications: The effects of a volatile anesthetic, isoflurane, were determined on a cardiac potassium channel current, IKs, a major ionic component underlying the cardiac action potential. The result shows that IKs is significantly inhibited by isoflurane. This may contribute to anesthetic-induced changes in the electrocardiogram, particularly the prolongation of the QT interval.

Activation of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels is critical in myocardial protection induced by preconditioning with volatile anesthetics or brief periods of ischemia. In this study, we characterized rat mitoK(ATP) channels reconstituted in lipid bilayers and examined their direct regulation by isoflurane. Mitochondria and the inner membrane fraction were isolated from rat ventricles and fused into lipid bilayers. On the basis of their inhibition by 5-hydroxydecanoate (5-HD)/ATP or activation by diazoxide, mitoK(ATP) channels of several conductance states were observed in symmetrical (150 mM) potassium glutamate (26, 47, 66, 83, and 105 pS). Isoflurane (0.8 mM) increased the cumulative open probability from 0.09 +/- 0.02 at baseline to 0.50 +/- 0.09 (P < 0.05, n = 5), which was inhibited by 5-HD. Isoflurane caused a dose-dependent rightward shift in ATP inhibition of mitoK(ATP) channels, which increased the IC(50) for ATP from 335 +/- 4 to 940 +/- 34 microM at 0.8 mM (P < 0.05, n = 5 approximately 8). We conclude that direct activation of the mitoK(ATP) channel by isoflurane is likely to contribute to volatile anesthetic-induced myocardial preconditioning.

Background: Volatile anesthetics can protect the myocardium against ischemic injury by opening the adenosine triphosphate (ATP)-sensitive potassium (K(atp)) channels. However, direct evidence for anesthetic-channel interaction is still limited, and little is known about the role K(atp) channel modulators play in this effect. Because pH is one of the regulators of K(atp) channels, the authors tested the hypothesis that intracellular pH (pHi) modulates the direct interaction of isoflurane with the cardiac K(atp) channel., Methods: The effects of isoflurane on sarcolemmal K(atp) channels were investigated at pHi 7.4 and pHi 6.8 in excised inside-out membrane patches from ventricular myocytes of guinea pig hearts., Results: At pHi 7.4, intracellular ATP (1-1,000 microm) inhibited K(atp) channels and decreased channel open probability (Po) in a concentration-dependent manner with an IC(50) of 8 +/- 1.5 microm, and isoflurane (0.5 mm) either had no effect or decreased channel activity. Lowering pHi from 7.4 to 6.8 enhanced channel opening by increasing Po and reduced channel sensitivity to ATP, with IC shifting from 8 +/- 1.2 to 45 +/- 5.6 microm. When applied to the channels activated at pHi 6.8, isoflurane (0.5 mm) increased Po and further reduced ATP sensitivity, shifting IC(50) to 110 +/- 10.0 microm., Conclusions: Changes in pHi appear to modulate isoflurane interaction with the cardiac K(atp) channel. At pHi 6.8, which itself facilitates channel opening, isoflurane enhances channel activity by increasing Po and reduces sensitivity to inhibition by ATP without changing the unitary amplitude of single channel current or the conductance. These results support the hypothesis of direct isoflurane-K(atp) channel interaction that may play a role in cardioprotection by volatile anesthetics.

Background: Cardioprotective effects of isoflurane are partially mediated by the sarcolemmal adenosine triphosphate-sensitive potassium (sarcK ATP ) channel. The authors tested the hypothesis that isoflurane sensitizes sarcK ATP channels to a potassium channel opener, pinacidil, adenosine- and phospholipid-mediated pathways., Methods: Activation by pinacidil of the K ATP current (I KATP ) was monitored in guinea pig ventricular myocytes at 0.5 and 5 mm intracellular ATP in the whole cell configuration of the patch clamp technique. The sensitization effect was evaluated by pretreating each myocyte with isoflurane (0.57 +/- 0.04 mm) before application of pinacidil (5 micro m) in the continued presence of the anesthetic. To investigate whether intracellular signaling pathways may be involved in isoflurane sensitization, the authors used the adenosine receptor antagonist theophylline (100 micro m) and the phosphatidylinositol kinase inhibitor wortmannin (100 micro m)., Results: The density of pinacidil-activated I KATP was higher at 0.5 mm ATP (20.7 +/- 3.2 pA/pF) than at 5 mm ATP (2.0 +/- 0.3 pA/pF). At 0.5 mm ATP, pretreatment with isoflurane caused an increase in density of pinacidil-activated I KATP (42.4 +/- 6.2 pA/pF) and accelerated the rate of current activation (from 5.4 +/- 1.2 to 39.0 +/- 7.9 pA. pF(-1). min(-1) ). Theophylline attenuated current activation by pinacidil (9.4 +/- 3.9 pA/pF) and abolished the sensitization effect of isoflurane on I KATP (10.0 +/- 2.5 pA/pF). Wortmannin did not alter pinacidil activation of I KATP (13.2 +/- 1.7 pA/pF) but prevented sensitization by isoflurane (15.8 +/- 4.5 pA/pF)., Conclusions: These results suggest that isoflurane increases sensitivity of cardiac sarcK ATP channels to the potassium channel opener pinacidil. Blockade of adenosine receptors or phosphatidylinositol kinases abolishes the sensitization effect, suggesting that the adenosine and phospholipid signaling pathways may be involved in the actions by isoflurane.

Background: Cardiac adenosine triphosphate-sensitive potassium (K(ATP)) channels and protein tyrosine kinases (PTKs) are mediators of ischemic preconditioning, but the interaction of both and a role in myocardial protection afforded by volatile anesthetics have not been defined., Methods: Whole cell and single channel patch clamp techniques were used to investigate the effects of isoflurane and the PTK inhibitor genistein on the cardiac sarcolemmal K(ATP) channel in acutely dissociated guinea pig ventricular myocytes., Results: At 0.5 mm internal ATP, genistein (50 microm) elicited whole cell K(ATP) current (22.5 +/- 7.9 pA/pF). Genistein effects were concentration-dependent, with an EC50 of 32.3 +/- 1.4 microm. Another PTK inhibitor, tyrphostin B42, had a similar effect. The inactive analog of genistein, daidzein (50 microm), did not elicit K(ATP) current. Isoflurane (0.5 mm) increased genistein (35 microm)-activated whole cell K(ATP) current from 14.5 +/- 3.1 to 32.5 +/- 6.6 pA/pF. Stimulation of receptor PTKs with epidermal growth factor, nerve growth factor, or insulin attenuated genistein and isoflurane effects, and the protein tyrosine phosphatase inhibitor orthovanadate (1 mm) prevented their actions on K(ATP) current. In excised inside-out membrane patches, and at fixed 0.2 mm internal ATP, genistein (50 microm) increased channel open probability from 0.053 +/- 0.016 to 0.183 +/- 0.039, but isoflurane failed to further increase open probability (0.162 +/- 0.051) of genistein-activated channels. However, applied in the presence of genistein and protein tyrosine phosphatase 1B (1 microg/ml), isoflurane significantly increased open probability to 0.473 +/- 0.114., Conclusions: These results suggest that the PTK-protein tyrosine phosphatase signaling pathway may be one of the regulators of cardiac sarcolemmal K(ATP) channel and may play a role in modulating its responsiveness to isoflurane. Relative importance of this modulation for cardioprotection by volatile anesthetics remains to be established.

Background: The mechanism underlying isoflurane modulation of cardiac electrophysiology is not well understood. In the present study, the authors investigated the effects of isoflurane on the cardiac action potential (AP) characteristics. The results were correlated to modulation of the L-type calcium (I(Ca,L)), the delayed-rectifier potassium (I(Kdr)), and the inward-rectifier potassium (I(Kir)) currents., Methods: Single ventricular myocytes were enzymatically isolated from guinea pig hearts. The current clamp and whole cell voltage clamp configurations of the patch clamp technique were used to monitor the cardiac AP and ionic currents, respectively. A dynamic AP voltage protocol that mimicked changes in membrane potential during an AP was used to monitor the I(Ca,L), I(Kdr) and I(Kir)., Results: Isoflurane produced a concentration-dependent, biphasic effect on the AP duration (APD). At 0.6 mm (1.26 vol%), isoflurane significantly increased APD50 and APD90 by 50.0 +/- 7.6% and 48.9 +/- 7.2%, respectively (P < 0.05; n = 6). At 1.0 mm (2.09 vol%), isoflurane had no significant effect on APD (n = 6). In contrast, at 1.8 mm (3.77 vol%), isoflurane decreased APD50 and APD90 by 38.3 +/- 5.4% and 32.2 +/- 5.5%, respectively (P < 0.05; n = 7). The inhibitory effects of isoflurane on I(Kdr) chord conductance were greater than those on I(Ca,L) (P < 0.05; n = 6/group). Both I(Ca,L) inactivation and I(Kdr) activation kinetics were accelerated by isoflurane. Isoflurane had no significant effects on I(Kir) chord conductance (n = 6)., Conclusion: At the lower anesthetic concentration, the prolongation of the APD may be the result of the dominant inhibitory effects of isoflurane on I(Kdr). At the higher concentration, the shortening of the APD may be caused by the inhibitory effects on I (Ca,L) combined with the isoflurane-induced acceleration of I(Ca,L) inactivation kinetics. Because I(Kdr) is significantly inhibited by isoflurane, I(Kir) appears to be the major repolarizing current, which is minimally affected by isoflurane.

Background: Volatile anesthetics have cardioprotective effects that mimic ischemic preconditioning, including the involvement of adenosine triphosphate-sensitive potassium (K(ATP)) channels. However, evidence for a direct effect of volatile anesthetic on the K(ATP) channel is limited. In this study, the effects of isoflurane on the cardiac sarcolemmal K(ATP) channel were investigated., Methods: Single ventricular myocytes were enzymatically isolated from guinea pig hearts. Whole cell and single-channel configurations, specifically the cell-attached and inside-out patch mode, of the patch clamp technique were used to monitor sarcolemmal K(ATP) channel current., Results: In the cell-attached patch configuration, 2,4-dinitrophenol (150 microm) opened the sarcolemmal K(ATP) channel. Isoflurane (0.5 mm) further increased channel open probability and the number of active channels in the patch. In contrast, in the inside-out patch experiments, isoflurane had no significant effect on the K(ATP) channel activated by low ATP (0.2-0.5 mm). In addition, isoflurane had no effect on the K(ATP) channel when activated by adenosine diphosphate, adenosine + guanosine triphosphate, bimakalim, and 2,4-dinitrophenol under inside-out patch configurations. When K(ATP) current was monitored in the whole cell mode, isoflurane alone was unable to elicit channel opening. However, during sustained protein kinase C activation by 12,13-dibutyrate, isoflurane activated the K(ATP) current that was sensitive to glibenclamide. In contrast, isoflurane had no effect on the K(ATP) channel activated by 12,13-dibutyrate in a cell-free environment., Conclusions: Isoflurane facilitated the opening of the sarcolemmal K(ATP) channel in the intact cell, but not in an excised, inside-out patch. The isoflurane effect was not due to a direct interaction with the K(ATP) channel protein, but required an intracellular component, likely including the translocation of specific protein kinase C isoforms. This suggests that the sarcolemmal K(ATP) channel may have a significant role in anesthetic-induced preconditioning.

Background: The cardiac adenosine triphosphate-sensitive potassium (K(ATP)) channel is activated during pathophysiological episodes such as ischemia and hypoxia and may lead to beneficial effects on cardiac function. Studies of volatile anesthetic interactions with the cardiac K(ATP) channel have been limited. The goal of this study was to investigate the ability of volatile anesthetics halothane and isoflurane to modulate the cardiac sarcolemmal K(ATP) channel., Methods: The K(ATP) channel current (I(KATP)) was monitored using the whole cell configuration of the patch clamp technique from single ventricular cardiac myocytes enzymatically isolated from guinea pig hearts. I(KATP) was elicited by extracellular application of the potassium channel openers 2,4-dinitrophenol or pinacidil., Results: Volatile anesthetics modulated I(KATP) in an anesthetic-dependent manner. Isoflurane facilitated the opening of the K(ATP) channel. Following initial activation of I(KATP) by 2,4-dinitrophenol, isoflurane at 0.5 and 1.3 mm further increased current amplitude by 40.4 +/- 11.1% and 58.4 +/- 20.6%, respectively. Similar results of isoflurane were obtained when pinacidil was used to activate I(KATP). However, isoflurane alone was unable to elicit K(ATP) channel opening. In contrast, halothane inhibited I(KATP) elicited by 2,4-dinitrophenol by 50.6 +/- 5.8% and 72.1 +/- 11.6% at 0.4 and 1.0 mm, respectively. When I(KATP) was activated by pinacidil, halothane had no significant effect on the current., Conclusions: The cardiac sarcolemmal K(ATP) channel is differentially modulated by volatile anesthetics. Isoflurane can facilitate the further opening of the K(ATP) channel following initial channel activation by 2,4-dinitrophenol or pinacidil. The effect of halothane was dependent on the method of channel activation, inhibiting I(KATP) activated by 2,4-dinitrophenol but not by pinacidil.

Isoflurane mimics the cardioprotective effect of acute ischemic preconditioning with an acute memory phase. We determined whether isoflurane can induce delayed cardioprotection, the involvement of ATP-sensitive potassium (K(ATP)) channels, and cellular location of the channels. Neonatal New Zealand White rabbits at 7-10 days of age (n = 5-16/group) were exposed to 1% isoflurane-100% oxygen for 2 h. Hearts exposed 2 h to 100% oxygen served as untreated controls. Twenty-four hours later resistance to myocardial ischemia was determined using an isolated perfused heart model. Isoflurane significantly reduced infarct size/area at risk (means +/- SD) by 50% (10 +/- 5%) versus untreated controls (20 +/- 6%). Isoflurane increased recovery of preischemic left ventricular developed pressure by 28% (69 +/- 4%) versus untreated controls (54 +/- 6%). The mitochondrial K(ATP) channel blocker 5-hydroxydecanoate (5-HD) completely (55 +/- 3%) and the sarcolemmal K(ATP) channel blocker HMR 1098 partially (62 +/- 3%) attenuated the cardioprotective effects of isoflurane. The combination of 5-HD and HMR-1098 completely abolished the cardioprotective effect of isoflurane (56 +/- 5%). We conclude that both mitochondrial and sarcolemmal K(ATP) channels contribute to isoflurane-induced delayed cardioprotection.

Mitochondria are critical modulators of cell function and are increasingly recognized as proximal sensors and effectors that ultimately determine the balance between cell survival and cell death. Volatile anesthetics (VA) are long known for their cardioprotective effects, as demonstrated by improved mitochondrial and cellular functions, and by reduced necrotic and apoptotic cell death during cardiac ischemia and reperfusion (IR) injury. The molecular mechanisms by which VA impart cardioprotection are still poorly understood. Because of the emerging role of mitochondria as therapeutic targets in diseases, including ischemic heart disease, it is important to know if VA-induced cytoprotective mechanisms are mediated at the mitochondrial level. In recent years, considerable evidence points to direct effects of VA on mitochondrial channel/transporter protein functions and electron transport chain (ETC) complexes as potential targets in mediating cardioprotection. This review furnishes an integrated overview of targets that VA impart on mitochondrial channels/transporters and ETC proteins that could provide a basis for cation regulation and homeostasis, mitochondrial bioenergetics, and reactive oxygen species (ROS) emission in redox signaling for cardiac cell protection during IR injury. [ABSTRACT FROM AUTHOR]

Changes in mitochondrial bioenergetics have been proposed to be critical for triggering and effecting anesthetic-induced preconditioning (APC) against cardiac ischemia and reperfusion injury. The objective of this study was to analyze changes in mitochondrial protein levels and link those changes to potential functional changes. A 18O-labeling method was applied for relative comparison of cardiac mitochondrial samples from control and isoflurane exposed rats before and after ischemia and reperfusion. Wistar rats were exposed to isoflurane for 30 min (APC) or did not receive the anesthetic (control). Rats were subjected to 30 min coronary occlusion and 15 min reperfusion without (ischemia) or after APC (ischemia + APC). The following comparisons were made: control vs. APC, control vs. ischemia, and APC vs. ischemia + APC. Proteins were analyzed by liquid chromatography-mass spectrometry. A total of 98 proteins currently annotated as mitochondrial proteins in the UniProt database were positively identified from three replicate experiments. Most of the changes during APC and ischemia occur in complexes of the electron transport chain. Overall, fewer changes in ETC complexes were found when comparing APC with APC + ischemia than when comparing control and ischemia. This corresponds to the preservation of bioenergetics due to APC after ischemia and reperfusion as indicated by preserved ATP level and generation. APC itself induced changes in complex I, but those changes were not correlated with activity changes in mitochondria after APC. Thus, a proteomic mass spectral approach does not only assess quantitative changes without prior knowledge of proteins, but also allows insight into the mechanisms of ischemia and reperfusion injury and APC. [ABSTRACT FROM AUTHOR]

During reperfusion, the interplay between excess reactive oxygen species (ROS) production, mitochondrial Ca2+ 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 (ΔΨm) is an underlying mechanism of protection by anesthetic-induced preconditioning (APC) with isoflurane, specifically addressing the interplay between ROS, Ca2+, and mPTP opening. The magnitude of APC-induced decrease in δΨm was mimicked with the protonophore 2.4-dinitrophenol (DNP), and the addition of pyruvate was used to reverse APC- and DNP-induced decrease in ΔΨm. In cardiomyocytes. ΔΨm, ROS, mPTP opening, and cytosolic and mitochondrial Ca2+ were measured using confocal microscope, and cardiomyocyte survival was assessed by Trypan blue exclusion. In isolated cardiac mitochondria, antimycin A-induced ROS production and Ca2+ 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 Ca2+ uptake. However, in stressed cardiomyocytes, a similar decrease in ΔΨm attenuated both cytosolic and mitochondrial Ca2+ accumulation. In conclusion, a partial decrease in ΔΨm 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 ΔΨm primarily attenuates mitochondrial ROS production, with consequential decrease in mitochondrial Ca2+ uptake. [ABSTRACT FROM AUTHOR]

Activation of mitochondrial ATP-sensitive K[sup +] (mitoK[sub ATP]) channels is critical in myocardial protection induced by preconditioning with volatile anesthetics or brief periods of ischemia. In this study, we characterized rat mitoK[sub ATP] channels reconstituted in lipid bilayers and examined their direct regulation by isoflurane.. Mitochondria and the inner membrane fraction were isolated from rat ventricles and fused into lipid bilayers. On the basis of their inhibition by 5-hydroxydecanoate (5-HD)/ATP or activation by diazoxide, mitoK[sub ATP] channels of several conductance states were observed in symmetrical (150 mM) potassium glutamate (26, 47, 66, 83, and 105 pS). Isoflurane (0.8 mM) increased the cumulative open probability from 0.09 ± 0.02 at baseline to 0.50 ± 0.09 (P < 0.05, n = 5), which was inhibited by 5-HD. Isoflurane caused a dose-dependent rightward shift in ATP inhibition of mitoK[sub ATP] channels, which increased the IC[sub 50] for ATP from 335 ± 4 to 940 ± 34 μM at 0.8 mM (P < 0.05, n = 5∼8). We conclude that direct activation of the mitoK[sub ATP] channel by isoflurane is likely to contribute to volatile anesthetic-induced myocerdial preconditioning. [ABSTRACT FROM AUTHOR]

Anesthetic-induced preconditioning (APC) is a phenomenon whereby a brief exposure to volatile anesthetics protects the heart from a prolonged ischemic episode. APC is cardioprotective reducing infarct size. In contrast, its effect on cardiac rhythm has not been established. Our previous studies suggested that APC can trigger arrhythmias. Functional changes in the voltage-gated ion channels may underlie this arrhythmogenic effect, but the consequence of APC on these channels is unknown. In the present study, we investigated the impact of APC on the cardiac sodium channel. Preconditioned (APC) Wistar rats were administered inhaled isoflurane (1 MAC) for 30 minutes followed by a 30-minute recovery period, prior to thoracotomy and excision of the heart. Control rats (NON) were not treated with isoflurane. Patch clamp studies on isolated myocytes indicated that APC triggered a 51% increase in the sodium channel current density, compared to the control group (-8.7±0.5 pA/pF and -13.2±1.2 pA/pF, respectively; n=12,13). Western blotting showed that APC treatment did not alter the protein expression of the pore-forming Nav1.5 α-subunit or the regulatory Nav2 β-subunit. Immunoprecipitation using a phospho-serine antibody and subsequent immunoblotting with an Nav1.5 α-subunit antibody indicated that APC treatment decreased the phosphorylation of the Nav1.5 α-subunit. This novel finding suggests that APC triggers changes in the functional state of the cardiac sodium channel. [ABSTRACT FROM AUTHOR]

The preservation of mitochondrial function after ischemia is involved in the mechanism of anesthetics-induced cardioprotection. Nitric oxide (NO) is recognized as a trigger or mediator of anesthetic-induced pre-and post-conditioning. In the present study, we investigated the modulatory effect of NO on the effect of isoflurane on complex I-linked mitochondrial respiration. Respiration rates were measured in isolated rat heart mitochondria polarographically with a computer-controlled Clark-type O2 electrode using complex I substrates glutamate and malate. To test the involvement of NO on the isoflurane-induced changes of mitochondrial respiration, NO donor SNAP and NO synthase inhibitor L-NIO were added with or without isoflurane (0.25 mM) before ADP initiated state 3 respiration. Isoflurane attenuated state 3 respiration (82±6% of control). L-NIO (10 µM) or SNAP (1 µM) alone had no effect on state 3 respiration (101±7%, 100±5% of control, respectively). In addition, L-NIO and SNAP did not alter the attenuation of isoflurane on state 3 respiration (84±7%, 85±5% of control, respectively). We conclude that isoflurane attenuates mitochondrial state 3 respiratory rate under complex I substrates glutamate and malate. In addition, this effect of isoflurane-induced attenuation of state 3 respiration is independent of mitochondrial NO. [ABSTRACT FROM AUTHOR]

Abstract: Modulation of mitochondrial free Ca2+ ([Ca2+]m) is implicated as one of the possible upstream factors that initiates anesthetic-mediated cardioprotection against ischemia–reperfusion (IR) injury. To unravel possible mechanisms by which volatile anesthetics modulate [Ca2+]m and mitochondrial bioenergetics, with implications for cardioprotection, experiments were conducted to spectrofluorometrically measure concentration-dependent effects of isoflurane (0.5, 1, 1.5, 2mM) on the magnitudes and time-courses of [Ca2+]m and mitochondrial redox state (NADH), membrane potential (ΔΨm), respiration, and matrix volume. Isolated mitochondria from rat hearts were energized with 10mM Na+- or K+-pyruvate/malate (NaPM or KPM) or Na+-succinate (NaSuc) followed by additions of isoflurane, 0.5mM CaCl2 (≈200nM free Ca2+ with 1mM EGTA buffer), and 250μM ADP. Isoflurane stepwise: (a) increased [Ca2+]m in state 2 with NaPM, but not with KPM substrate, despite an isoflurane-induced slight fall in ΔΨm and a mild matrix expansion, and (b) decreased NADH oxidation, respiration, ΔΨm, and matrix volume in state 3, while prolonging the duration of state 3 NADH oxidation, respiration, ΔΨm, and matrix contraction with PM substrates. These findings suggest that isoflurane''s effects are mediated in part at the mitochondrial level: (1) to enhance the net rate of state 2 Ca2+ uptake by inhibiting the Na+/Ca2+ exchanger (NCE), independent of changes in ΔΨm and matrix volume, and (2) to decrease the rates of state 3 electron transfer and ADP phosphorylation by inhibiting complex I. These direct effects of isoflurane to increase [Ca2+]m, while depressing NCE activity and oxidative phosphorylation, could underlie the mechanisms by which isoflurane provides cardioprotection against IR injury at the mitochondrial level. [Copyright &y& Elsevier]

During reperfusion, the interplay between excess reactive oxygen species (ROS) production, mitochondrial Ca2+ 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 (ΔΨm) is an underlying mechanism of protection by anesthetic-induced preconditioning (APC) with isoflurane, specifically addressing the interplay between ROS, Ca2+, and mPTP opening. The magnitude of APC-induced decrease in δΨm was mimicked with the protonophore 2.4-dinitrophenol (DNP), and the addition of pyruvate was used to reverse APC- and DNP-induced decrease in ΔΨm. In cardiomyocytes. ΔΨm, ROS, mPTP opening, and cytosolic and mitochondrial Ca2+ were measured using confocal microscope, and cardiomyocyte survival was assessed by Trypan blue exclusion. In isolated cardiac mitochondria, antimycin A-induced ROS production and Ca2+ 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 Ca2+ uptake. However, in stressed cardiomyocytes, a similar decrease in ΔΨm attenuated both cytosolic and mitochondrial Ca2+ accumulation. In conclusion, a partial decrease in ΔΨm 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 ΔΨm primarily attenuates mitochondrial ROS production, with consequential decrease in mitochondrial Ca2+ uptake. [ABSTRACT FROM AUTHOR]