Anesthetic-induced Preconditioning Research Articles

Repetitive Treatment with Volatile Anesthetics Does Not Affect the In Vivo Plasma Concentration and Composition of Extracellular Vesicles in Rats.

Background: Anesthetic-induced preconditioning (AIP) with volatile anesthetics is a well-known experimental technique to protect tissues from ischemic injury or oxidative stress. Additionally, plasmatic extracellular vesicle (EV) populations and their cargo are known to be affected by AIP in vitro, and to provide organ protective properties via their cargo. We investigated whether AIP would affect the generation of EVs in an in vivo rat model. Methods: Twenty male Sprague Dawley rats received a repetitive treatment with either isoflurane or with sevoflurane for a duration of 4 or 8 weeks. EVs from blood plasma were characterized by nanoparticle tracking analysis, transmission electron microscopy (TEM) and Western blot. A scratch assay (H9C2 cardiomyoblast cell line) was performed to investigate the protective capabilities of the isolated EVs. Results: TEM images as well as Western blot analysis indicated that EVs were successfully isolated. The AIP changed the flotillin and CD63 expression on the EV surface, but not the EV concentration. The scratch assay did not show increased cell migration and/or proliferation after EV treatment. Conclusion: AIP in rats changed the cargo of EVs but had no effect on EV concentration or cell migration/proliferation. Future studies are needed to investigate the cargo on a miRNA level and to investigate the properties of these EVs in additional functional experiments.

The Effect of Argon in Anesthetic-Induced Myocardial Preconditioning

The Effect of Argon in Anesthetic-Induced Myocardial Preconditioning

The Role of Macrophage Migration Inhibitory Factor in Anesthetic-Induced Myocardial Preconditioning

IntroductionAnesthetic-induced preconditioning (AIP) is known to elicit cardioprotective effects that are mediated at least in part by activation of the kinases AMPK and PKCε as well as by inhibition of JNK. Recent data demonstrated that the pleiotropic cytokine macrophage migration inhibitory factor (MIF) provides cardioprotection through activation and/or inhibition of kinases that are also known to mediate effects of AIP. Therefore, we hypothesized that MIF could play a key role in the AIP response.MethodsCardiomyocytes were isolated from rats and subjected to isoflurane preconditioning (4 h; 1.5 vol. %). Subsequently, MIF secretion and alterations in the activation levels of protective kinases were compared to a control group that was exposed to ambient air conditions. MIF secretion was quantified by ELISA and AIP-induced activation of protein kinases was assessed by Western blotting of cardiomyocyte lysates after isoflurane treatment.ResultsIn cardiomyocytes, preconditioning with isoflurane resulted in a significantly elevated secretion of MIF that followed a biphasic behavior (30 min vs. baseline: p = 0.020; 24 h vs. baseline p = 0.000). Moreover, quantitative polymerase chain reaction demonstrated a significant increase in MIF mRNA expression 8 h after AIP. Of note, activation of AMPK and PKCε coincided with the observed peaks in MIF secretion and differed significantly from baseline.ConclusionsThese results suggest that the pleiotropic mediator MIF is involved in anesthetic-induced preconditioning of cardiomyocytes through stimulation of the protective kinases AMPK and PKCε.

The Role of Cyclooxygenase-1 and -2 in Sevoflurane-Induced Postconditioning Against Myocardial Infarction

Cyclooxygenase (COX)-2 mediates ischemic pre- and postconditioning as well as anesthetic-induced preconditioning. However, the role of COX-1 and -2 in anesthetic-induced postconditioning has not been investigated. We evaluated the role of COX-1 and -2 in sevoflurane-induced postconditioning in vivo. Pentobarbital-anaesthetized male C57BL/6 mice were subjected to 45 minutes of coronary artery occlusion and 3 hours of reperfusion. Animals received either no intervention, the vehicle dimethyl sulfoxide (DMSO, 10 µL/g intraperitoneally), acetylsalicylic acid (ASA, 5 µg/g intraperitoneally), the selective COX-1 inhibitor SC-560 (10 µg/g intraperitoneally), or the selective COX-2 inhibitor NS-398 (5 µg/g intraperitoneally). 1.0 MAC (minimum alveolar concentration) sevoflurane was administered for 18 minutes during early reperfusion either alone or in combination with ASA, SC-560, and NS-398. Infarct size was determined with triphenyltetrazolium chloride. Statistical analysis was performed using 1-way and 2-way analyses of variance with post hoc Duncan testing. The infarct size in the control group was 44% ± 9%. DMSO (42% ± 7%), ASA (36% ± 6%), and NS-398 (44% ± 18%) had no effect on infarct size. Sevoflurane (17% ± 4%; P < .05) and SC-560 (26% ± 10%; P < .05) significantly reduced the infarct size compared with control condition. Sevoflurane-induced postconditioning was not abolished by ASA (16% ± 5%) and SC-560 (22% ± 4%). NS-398 abolished sevoflurane-induced postconditioning (33% ± 14%). It was concluded that sevoflurane induces postconditioning in mice. Inhibition of COX-1 elicits a myocardial infarct size reduction and does not abolish sevoflurane-induced postconditioning. Blockade of COX-2 abolishes sevoflurane-induced postconditioning. These results indicate that sevoflurane-induced postconditioning is mediated by COX-2.

Anesthetic-Induced Myocardial Preconditioning and Some Biochemical Markers for Cardiac and Coronary Failures after Aortocoronary Bypass Surgery

Objective: to provide a rationale for the efficiency of sevoflurane-induced cardiac preconditioning (CPC), by assessing the pattern of recovery of heart rate and by estimating troponin I levels and changes in NT-proBNP concentrations in patients undergoing aortocoronary bypass surgery (ACBS) under extracorporeal circulation (EC). Subjects and methods. Sixty patients aged 60.6±8 years (M±&) were examined after elective ACBS using EC and divided into two groups of 30 patients each: 1) inhalation induction and maintenance of anesthesia (IIMA) with sevoflurane and fentanyl, with CPC being simulated; 2) total intravenous anesthesia (TIA) with propofol and fentanyl. Inhalation induction of sevoflurane anesthesia was performed in the IIMA group. Ten minutes before aortic ligation, the dose of the anesthetic was increased up to 2 MAC for CPC. Inhaled anesthetics were not used in the TIA group. The authors assessed the pattern of cardiac performance recovery and estimated the level of NT-proBNP 24 and 48 hours after tracheal intubation and that of troponin I following 24 hours of the intubation. Results. Defibrillation was required in one patient from the TIA group who developed ventricular fibrillation. The baseline levels of NT-proBNP were comparable in both groups. Following 24 hours, its level was more than thrice higher in the TIA group than that in the IIMA one (p<0.05). By the end of 2 days, the concentration of NT-proBNP continued to rise (up to 480% of the baseline level) in the TIA group and returned to the preoperative values in the IIMA group (p=0.05). Twenty-four hours after tracheal intubation the level of troponin I was insignificantly higher in the TIA group than that in the IIMA group (p=0.1). Conclusion. Sevoflurane has cardioprotective properties in preventing and/or reducing the degree of heart failure after ACBS using EC. There is a need to continue the study in increased cohort to provide evidence that sevoflurane-induced CPC can lower cardiomyocyte damage due to ischemia/perfusion. Key words: anesthetic preconditioning, sevoflurane, NT-proBNP, troponin I, heart failure.

Commentary on: Volatile anesthetic-induced preconditioning

Commentary on: Volatile anesthetic-induced preconditioning

Volatile anesthetic-induced preconditioning

The myocardium has an innate ability to protect itself from ischemic events. This protection occurs when the myocardium is exposed to a brief ischemic period prior to a more extreme ischemic event. This is termed ischemic preconditioning. Ischemic preconditioning induces a series of molecular pathways that protect the cardiac myocyte; first, for a period of 1-6 hours (early preconditioning) and, also, for a second period from 24-72 hours (delayed phase). The early preconditioning is mediated by the release of adenosine which induces a protective signal that is related to the mitochondrial KATP channel activation and activation of the δ-opioid and bradykinin receptors. The delayed phase is related to the induction of inducible nitric oxide synthase, superoxide dismutase and heat-shock proteins. Indirect evidence indicates that O2-derived free radicals are involved in the delayed phase, as noted in the early preconditioning phase. Applying ischemic preconditioning to clinical practice can be dangerous and difficult to implement in a controlled fashion. However, recent studies have shown that the use of volatile anesthetics, such as sevoflurane, isoflurane and desflurane, can mimic the early phase of ischemic preconditioning through a multi-pathway signaling of mitochondrial KATP channels. This important finding can easily be applied to clinical practice for patients undergoing surgery. It can also be significantly important for patients undergoing off-pump cardiac bypass surgery or cardiac bypass surgery where there is no cross-clamp or cardioplegia used where the probability of myocardial ischemia is greatly increased. This report will, therefore, discuss the mechanism, safety and efficacy of volatile anesthetics as inducers of cardiac preconditioning.

Preconditioning by Isoflurane Elicits Mitochondrial Protective Mechanisms Independent of Sarcolemmal KATP Channel in Mouse Cardiomyocytes

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.

Quantitative characterization of changes in the cardiac mitochondrial proteome during anesthetic preconditioning and ischemia.

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 (18)O-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.

Endothelial Nitric Oxide Synthase Mediates the First and Inducible Nitric Oxide Synthase Mediates the Second Window of Desflurane-Induced Preconditioning

Nitric oxide synthases (NOSs) mediate the first window of anesthetic-induced preconditioning (APC). The authors tested the hypothesis that endothelial NOS (eNOS) mediates the first window and inducible NOS (iNOS) mediates the second window of APC. Randomized, prospective, blinded laboratory investigation. Experimental laboratory. Mice. Mice were subjected to a 45-minute coronary artery occlusion (CAO) and a 180-minute reperfusion. C57BL/6 mice received desflurane, 1.0 minimum alveolar concentration, for 30 minutes or 12, 24, 48, or 96 hours before CAO. In eNOS(-/-) and iNOS(-/-) mice, desflurane was given 30 minutes and 48 hours before CAO. In the control groups, no desflurane was administered. Myocardial infarct size (IS) was determined after staining with Evans blue and triphenyltetrazolium chloride. The second window of APC was detectable at 48 hours but not at 12, 24, and 96 hours after preconditioning. In the control groups, IS was not different among the wild-type (50 ± 10%), eNOS(-/-) (52 ± 14%), and iNOS(-/-) (46 ± 10%) mice. The IS decreased significantly (p < 0.05) when desflurane was administered 30 minutes (10 ± 6%) or 48 hours (16 ± 7%) before CAO in wild-type mice, 48 hours (21 ± 13%) before CAO in eNOS(-/-) mice, and 30 minutes (13 ± 6%) before CAO in iNOS(-/-) mice. Desflurane given 30 minutes before CAO in eNOS(-/-) mice (60 ± 10%) and 48 hours before CAO in iNOS(-/-) mice (48 ± 21%) did not decrease the IS significantly compared with controls. Endothelial NOS and iNOS work independently to mediate the first and second windows of APC, respectively. Endothelial NOS is not necessary to trigger the second window of APC.

Abstract P094: Anesthetic Preconditioning in Human Cardiomyocytes Derived from Type II Diabetic Patient-Induced Pluripotent Stem Cells in a Varying Glucose Environment

Volatile anesthetic-induced preconditioning (APC) has been shown to pharmacologically precondition the myocardium, providing protection from an ischemia-reperfusion injury. However, APC is attenuated or even eliminated in diabetic individuals and in the presence of acute hyperglycemia with the underlying mechanism(s) being unknown. In this study, we used ventricular cardiomyocytes (CMs) differentiated from non-diabetic and type II diabetic-induced pluripotent stem cells (N-iPSCs and DM-iPSCs, respectively) to investigate the influence of a high glucose environment and genetic background on APC. Differentiated CMs were identified using cardiac-specific immunostaining and expression of green fluorescent protein under the transcriptional control of cardiac promoter myosin light chain-2v, a genetic construct delivered by lentiviral vector. N-iPSC- and DM-iPSC-derived CMs were exposed to varying glucose environments (11 mM and 25 mM). Confocal microscopy was utilized to measure mitochondrial membrane potential and mitochondrial permeability transition pore opening (mPTP) in CMs. The volatile anesthetic isoflurane depolarized mitochondria in N-iPSC-derived CMs via opening of the mitochondrial adenosine triphosphate-sensitive potassium channel; however, isoflurane depolarized mitochondria to a significantly lower level in diabetic-derived cardiomyocytes and in the presence of 25 mM glucose. APC delayed mPTP opening in both N-iPSC- and DM-iPSC-derived CMs in 11 mM glucose environment only. We have established an in vitro model based on directed differentiation of ventricular CMs from N-iPSCs and DM-iPSCs that allows us the unique opportunity to conduct comparative studies to address the inability of diabetic individuals to be preconditioned with anesthetics. Our preliminary results indicate for the first time that both a high glucose environment and a diabetic background have detrimental effects on the efficiency of APC to protect CMs from an ischemia-reperfusion injury.

Role of the O-linked β-N -acetylglucosamine in the Cardioprotection Induced by Isoflurane

Cardiac protection by volatile anesthetic-induced preconditioning and ischemic preconditioning have similar signaling pathways. Recently, it was reported that augmentation of protein modified with O-linked β-N-acetylglucosamine (O-GlcNAc) contributes to cardiac protection. This study investigated the role of O-GlcNAc in cardiac protection induced by anesthetic-induced preconditioning. O-GlcNAc-modified proteins were visualized by immunoblotting. Tolerance against ischemia or reperfusion was tested in vivo (n = 8) and in vitro (n = 6). The opening of the mitochondrial permeability transition pore (mPTP) upon oxidative stress was examined in myocytes treated with calcein AM (n = 5). Coimmunoprecipitation and enzymatic labeling were performed to detect the mitochondrial protein responsible for the mPTP opening. Isoflurane treatment and the consequent augmentation of O-GlcNAc concentrations reduced the infarct size (26 ± 5% [mean ± SD], P < 0.001) compared with the control. The protective effect of O-GlcNAc was eliminated in the group pretreated with the O-GlcNAc transferase inhibitor alloxan (39 ± 5%, P < 0.001). Myocyte survival also showed the same result in vitro. Formation of the mPTP was abrogated in the isoflurane-treated cells (86 ± 4%, P < 0.001) compared with the control and alloxan-plus-isoflurane-treated cells (57 ± 7%, P < 0.001). Coimmunoprecipitation and enzymatic labeling studies revealed that the O-GlcNAc-modified, voltage-dependent anion channel restained the mPTP opening. Isoflurane induced O-GlcNAc modification of mitochondrial voltage-dependent anion channel. This modification inhibited the opening of the mPTP and conferred resistance to ischemia-reperfusion stress.

Suppression of Ischemic and Reperfusion Ventricular Arrhythmias by Inhalational Anesthetic-Induced Preconditioning in the Rat Heart

Inhalational anesthetic-induced preconditioning (APC) has been shown to reduce infarct size and attenuate contractile dysfunction caused by myocardial ischemia. Only a few studies have reported the effects of APC on arrhythmias during myocardial ischemia-reperfusion injury, focusing exclusively on reperfusion. Accordingly, the aim of the present study was to examine the influence of APC on ventricular arrhythmias evoked by regional no-flow ischemia. APC was induced in adult male Wistar rats by 12-min exposures to two different concentrations (0.5 and 1.0 MAC) of isoflurane followed by 30-min wash-out periods. Ventricular arrhythmias were assessed in the isolated perfused hearts during a 45-min regional ischemia and a subsequent 15-min reperfusion. Myocardial infarct size was determined after an additional 45 min of reperfusion. The incidence, severity and duration of ventricular arrhythmias during ischemia were markedly reduced by APC. The higher concentration of isoflurane had a larger effect on the incidence of ventricular fibrillation than the lower concentration. The incidence of ventricular tachycardia and reversible ventricular fibrillation during reperfusion was also significantly reduced by APC; the same was true for myocardial infarct size. In conclusion, we have shown that preconditioning with isoflurane confers profound protection against myocardial ischemia- and reperfusion-induced arrhythmias and lethal myocardial injury.

Isoflurane‐induced cardioprotection: role of sarcolemmal KATP channels and mitochondria

We investigated the role of sarcKATP channels and mitochondria in anesthetic-induced preconditioning (APC) using a knockout mouse model with deleted Kir6.2 subunit, the pore-forming part of sarcKATP channel (Kir6.2 C57BL/6 KO). Flavoprotein (FP) fluorescence, reactive oxygen species (ROS) production, and mitochondrial membrane potential, were monitored in isolated myocytes by laser scanning confocal microscopy during exposure to 1 MAC isoflurane. In the presence of isoflurane FP fluorescence increased to 180% and 190% of baseline in cardiomyocytes of WT and Kir6.2 KO mice, respectively. ROS increased to 118% and 124% of baseline in WT and Kir6.2 KO myocytes. Mitochondrial membrane potential decreased to 87% and 86% of baseline in WT and Kir6.2 KO. Oxidative stress induced cell death in 31% and 44% of WT and Kir6.2 KO. APC attenuated cell death to 21% and 35% in WT and Kir6.2 KO myocytes. Deletion of pore-forming subunit of sarcKATP channel did not alter characteristic mitochondrial responses to isoflurane in Kir6.2 KO cardiomyocytes. APC is preserved in the Kir6.2 KO cardiomyocytes, but their susceptibility to oxidative stress appears greater under basal conditions. Thus, sarcKATP channels are an important component of a cardioprotective pathway independent of mitochondria-mediated protection. Supported by P01GM066730 (ZJB) from the NIH, Bethesda, MD.

Diabetic rats are unable to elicit cardioprotection by isoflurane: role of mitochondrial dysfunction

We investigated whether differences in mitochondrial genome play a role in anesthetic-induced preconditioning (APC) in the diabetic heart model with “swapped” mitochondria. These rats have identical nuclear genome, but some bear Wistar (T2DNmtWistar) while others bear Fawn Hooded Hypertensive (T2DNmtFHH) mitochondrial genome. The production of reactive oxygen species (ROS) and changes in flavoproteins (FPs) fluorescence were monitored from ventricular myocytes in the presence of 1 MAC isoflurane by laser scanning confocal microscopy. Opening of mitochondrial permeability transition pore (mPTP) was detected by tetramethylrhodamine dye. During isoflurane, ROS were increased by 165% and 107% in T2DNmtFHH and T2DNmtWistar cardiomyocytes, respectively. In addition, isoflurane induced oxidation of mitochondrial FPs in T2DNmtFHH by 94% and by 35% in T2DNmtWistar myocytes. Compared to respective controls, APC increased the mPTP opening time in T2DNmtWistar but not in T2DNmtFHH myocytes. Our findings suggest that mitochondrial genome-related differences in the function of respiratory chain complexes have an impact on anesthetic sensitivity of the mitochondria. This in part could explain that APC protection against mPTP opening is compromised in T2DNmtFHH but not in T2DNmtWistar cardiomyocytes. Supported by P01GM066730 (ZJB) from the NIH, Bethesda, MD.

Propofol Inhibits Desflurane-Induced Preconditioning in Rabbits

The authors tested the hypothesis that ischemic and desflurane-induced preconditioning are blocked by propofol. A prospective, randomized, vehicle-controlled study. A university research laboratory. New Zealand white rabbits (n = 52). Pentobarbital-anesthetized rabbits were subjected to 30 minutes of coronary artery occlusion followed by 3 hours of reperfusion. Rabbits received 0.0 (control) or 1.0 minimum alveolar concentration of desflurane (30 minutes' duration and a 30-minute memory period) or ischemic preconditioning (5 minutes of ischemia and a 30-minute memory period) in the absence or presence of propofol (10 mg/kg/h intravenously) or its vehicle (10% Intralipid emulsion; B Braun, Melsungen, Germany). The myocardial infarct size was measured with triphenyltetrazolium staining. Statistical analysis was performed with 1-way and 2-way analysis of variance when appropriate, followed by a post hoc Duncan test. Data are mean ± standard deviation. Myocardial infarct size was 56% ± 8% in control animals (n = 7). Desflurane significantly (p < 0.05) reduced the infarct size to 37% ± 6% (n = 7). Desflurane-induced preconditioning was blocked by propofol (65% ± 10%, n = 7) but not by its vehicle (45% ± 11%, n = 5). Propofol and its vehicle alone had no effect on the infarct size (62% ± 8% [n = 6] and 58% ± 3% [n=5], respectively). Ischemic preconditioning reduced infarct size in the absence or presence of propofol to 24% ± 7% (n = 7) and 29% ± 12% (n = 6). Desflurane-induced preconditioning markedly reduced infarct size and was blocked by propofol, whereas ischemic preconditioning was not blocked by propofol. The results suggest an important interference between propofol and anesthetic-induced preconditioning and might explain some contradictory findings in studies in humans.

Isoflurane Preconditioning Elicits Competent Endogenous Mechanisms of Protection from Oxidative Stress in Cardiomyocytes Derived from Human Embryonic Stem Cells

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. 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. 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. 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.

Mitochondrial depolarization underlies delay in permeability transition by preconditioning with isoflurane: roles of ROS and Ca2+

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.

Response to Letter Regarding Article, “Reactive Oxygen Species–Induced Stimulation of 5′AMP-Activated Protein Kinase Mediates Sevoflurane-Induced Cardioprotection”

To the Editor: AMP kinase (AMPK) activation represents a unique molecular event in cardioprotection against ischemia/reperfusion injury, potentially linking myocardial metabolism to a number of cardioprotective signaling pathways such as nitric oxide and reactive oxygen species signaling. In a recently published article in Circulation , Lamberts et al1 demonstrated that AMPK activation contributed to the signaling cascade in the powerful cardioprotection conferred by sevoflurane-induced preconditioning. This finding is intriguing because AMPK activation has the potential to overcome the loss of anesthetic-induced preconditioning during diabetes mellitus.2 The published report provides important information and useful insights, particularly regarding the triangle of AMPK signaling, reactive oxygen species generation, …

Letter by Lotz et al Regarding Article, “Reactive Oxygen Species–Induced Stimulation of 5′ AMP-Activated Protein Kinase Mediates Sevoflurane-Induced Cardioprotection”

To the Editor: AMP kinase (AMPK) activation represents a unique molecular event in cardioprotection against ischemia/reperfusion injury, potentially linking myocardial metabolism to a number of cardioprotective signaling pathways such as nitric oxide and reactive oxygen species signaling. In a recently published article in Circulation , Lamberts et al1 demonstrated that AMPK activation contributed to the signaling cascade in the powerful cardioprotection conferred by sevoflurane-induced preconditioning. This finding is intriguing because AMPK activation has the potential to overcome the loss of anesthetic-induced preconditioning during diabetes mellitus.2 The published report provides important information and useful insights, particularly regarding the triangle of AMPK signaling, reactive oxygen species generation, …