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Isoflurane Protects the Myocardium Against Ischemic Injury via the Preservation of Mitochondrial Respiration and Its Supramolecular Organization

Lotz, Christopher MD; Zhang, Jun PhD; Fang, Caiyun PhD; Liem, David MD, PhD; Ping, Peipei PhD, FAHA, FISHR

doi: 10.1213/ANE.0000000000000494
Cardiovascular Anesthesiology: Research Report

BACKGROUND: Isoflurane has been demonstrated to limit myocardial ischemic injury. This effect is hypothesized to be mediated in part via effects on mitochondria. We investigated the hypothesis that isoflurane maintains mitochondrial respiratory chain functionality, in turn limiting mitochondrial damage and mitochondrial membrane disintegration during myocardial ischemic injury.

METHODS: Mice (9–12 weeks of age) received isoflurane (1.0 minimum alveolar concentration) 36 hours before a 30-minute coronary artery occlusion that was followed by 24 hours of reperfusion. Cardiac mitochondria were isolated at a time point corresponding to 4 hours of reperfusion. 2,3,5-Triphenyltetrazoliumchloride staining was used to determine myocardial infarct size. Mitochondrial respiratory chain functionality was investigated using blue native polyacrylamide gel electrophoresis, as well as specific biochemical assays. Mitochondrial lipid peroxidation was quantified via the formation of malondialdehyde; mitochondrial membrane integrity was assessed by Ca2+-induced swelling. Protein identification was achieved via liquid chromatography mass spectrometry/mass spectrometry.

RESULTS: Thirty-one mice were studied. Mice receiving isoflurane displayed a reduced myocardial infarct size (P = 0.0011 versus ischemia/reperfusion [I/R]), accompanied by a preserved activity of respiratory complex III (P = 0.0008 versus I/R). Isoflurane stabilized mitochondrial supercomplexes consisting of oligomers from complex III/IV (P = 0.0086 versus I/R). Alleviation of mitochondrial damage after isoflurane treatment was further demonstrated as decreased malondialdehyde formation (P = 0.0019 versus I/R) as well as a diminished susceptibility to Ca2+-induced swelling (P = 0.0010 versus I/R).

CONCLUSIONS: Our findings support the hypothesis that isoflurane protects the heart from ischemic injury by maintaining the in vivo functionality of the mitochondrial respiratory chain. These effects may result in part from the preservation of mitochondrial supramolecular organization and minimized oxidative damage, circumventing the loss of mitochondrial membrane integrity.

Published ahead of print November 7, 2014.

From the Department of Physiology, Division of Cardiology, University of California, Los Angeles, Los Angeles, California.

Accepted for publication August 31, 2014.

Published ahead of print November 7, 2014.

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Christopher Lotz, MD, Departments of Physiology and Medicine, Division of Cardiology, Cardiovascular Research Laboratory, UCLA David Geffen School of Medicine, Suite 1–609 MRL Building, 675 Charles E. Young Dr. South, Los Angeles, CA 90095. Address e-mail to

The administration of volatile anesthetics elicits a protective cardiac phenotype effectively limiting the extent of myocardial ischemic injury.1–3 These beneficial effects can be observed immediately as well as 24 and 48 hours after myocardial ischemia.4 The former is referred to as the first window of cardioprotection and the latter the second window. Both windows are initiated via the preischemic administration of volatile anesthetics and follow a highly specific time course. Myocardial damage is contained subsequent to the initial administration of the volatile anesthetic in a first window of protection for approximately 2 hours. This is followed by a free interval before advancing into a second window of protection after 24 hours, which offers protection for up to another 48 hours.4 Multiple molecular mechanisms interact to confer this anesthetic-induced cardioprotection5 and several lines of data implicate that mitochondria serve as the final stage of survival.6

The normal stable interactions between the mitochondrial oxidative phosphorylation system, which is composed of 5 multisubunit protein complexes (I–V), and its associated respiratory supercomplexes are threatened by myocardial ischemia.7 Complex I is the most susceptible to ischemic damage,8 whereas the functionality of all respiratory complexes becomes compromised as ischemia progresses.9 In particular, complex III acts as a primary source for the production of large amounts of reactive oxygen species (ROS).10 Subsequent oxidative damage facilitates a sequence of deleterious events ultimately leading to an increased mitochondrial permeability and cell death.11 Volatile anesthetics elicit distinct actions on the mitochondrial membranes,12 as well as respiratory chain functionality within the first window of protection13–20 initiating a prosurvival process that attenuates these detrimental events. However, whether volatile anesthetics impact a salutary effect on the mitochondrial respiratory chain within the second window of myocardial protection is unclear.

The present study investigated the hypothesis that isoflurane alleviates the consequences of myocardial ischemia on mitochondrial respiratory complex and supercomplex functionality, that in turn prevents irreversible damage to mitochondrial membranes during a second window of protection.

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All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University of California, Los Angeles. All procedures conformed to the Guiding Principles in the Care and Use of Animals of the American Physiological Society. Figure 1 depicts the experimental workflow of the study.

Figure 1

Figure 1

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Volatile Anesthetic Protocol

Mice were randomly assigned to 4 different experimental groups. The animals were subjected to sham surgery (sham) or a 30-minute coronary artery occlusion (CAO) followed by 4 hours of ischemia/reperfusion (I/R). In additional groups, 1.0 minimum alveolar concentration (MAC) isoflurane was administered for 30 minutes during spontaneous breathing and discontinued 36 hours before sham surgery (isoflurane) or CAO (isoflurane + I/R), respectively. The animals were placed in an induction chamber saturated with an air/oxygen mixture. A calibrated infrared analyzer continuously measured the concentration of isoflurane. Anesthesia was discontinued after 30 minutes and the animals were allowed to emerge. Each animal was then housed overnight with food and water ad libitum. On day 2, the mice were euthanized at the end of each experimental protocol via cervical dislocation and the hearts quickly excised for further analyses.

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Murine Model of Myocardial Ischemic Injury and Infarct Size Analysis

Male mice (9–12 weeks of age) were subjected to myocardial I/R injury as previously described.3 In brief, mice were anesthetized with sodium pentobarbital (50 mg/kg body weight intraperitoneal) and the depth of anesthesia verified by recurrent testing of the palpebral reflexes and hindpaw withdrawal throughout the experiment. After performance of a left thoracotomy and pericardiotomy, the heart was exposed and a suture (8-0) was looped around the left anterior descending coronary artery approximately 1 to 3 mm from the tip of the left atrium.

Subsequently, 30 minutes of myocardial ischemia was induced by ligation of the suture (a 1–2-mm section of PE-10 tubing (Thermo Fisher Scientific, Waltham, MA) was placed between the suture and the artery to prevent damage to the vessel). Myocardial reperfusion was then induced by removal of CAO. After 4 hours of reperfusion, the anesthetized animals were euthanized via cervical dislocation. The heart was excised3 and the area at risk (AAR) for myocardial ischemic injury was delineated by perfusion of the left coronary artery with a 1% solution of Evans blue dye. Myocardial infarct size was determined by perfusion of the left coronary artery with a 1% solution of 2,3,5-triphenyltetrazoliumchloride in phosphate buffer (pH 4.4, 37°C). Infarct size was measured by planimetry (Adobe Photoshop; Adobe Systems Incorporated, San Jose, CA) and expressed as a percentage of the AAR.

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Isolation of Cardiac Mitochondria

Murine cardiac mitochondria were isolated as previously described.21 In all experimental groups, the anesthetized mice were euthanized by cervical dislocation at the end of the experimental protocol and the hearts quickly excised. Cardiac tissue was homogenized in a buffer containing 250 mM sucrose, 1 mM EGTA, 20 mM HEPES, pH 7.5, protease inhibitor (1 mL ready-to-use solution per 100 mL lysate, P8340; Sigma-Aldrich, St. Louis, MO) as well as phosphatase inhibitor (1 mL ready-to-use solution per 100 mL of extraction buffer, P5726; Sigma-Aldrich). After removal of the nuclear fraction and tissue debris by double-passage centrifugation at 800g for 7 minutes, the crude mitochondrial fraction was collected by centrifugation at 4000g for 20 minutes. All procedures were performed at 4°C. The structural/functional integrity of each preparation was validated as previously described.22

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Mitochondrial Permeability Assessment

Mitochondrial permeability transition was assessed by inducing mitochondrial swelling as previously described.3 In brief, freshly isolated cardiac mitochondria were resuspended in a buffer containing 120 mM KCl, 10 mM Tris-HCl, 20 mM 3-(N-morpholino) propansulfonic acid, and 5 mM KH2PO4 to a final mitochondrial protein concentration of 0.25 mg/mL. Protein concentrations were determined using a Bradford assay (Bio-Rad Laboratories, Hercules, CA). Opening of the mitochondrial permeability transition pore was initiated by the addition of 2 different concentrations of CaCl2 to give a free Ca2+ concentration of 50 or 200 μM, respectively, and determined after the decrease in light scattering at A520 that accompanies mitochondrial swelling at 25°C.23

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Spectrophotometric Assay of Respiratory Chain Complex Activity

The enzymatic function of each mitochondrial respiratory complex was measured using specific single-wavelength spectrophotometric assays. Each assay provides quantitative information concerning the maximal catalytic activities of the respective respiratory complex.

Mitochondrial respiratory complex I activity was measured as the reduction of nitrotetrazolium blue chloride (NTB) to formazan in the presence of reduced nicotinamide adenine dinucleotide (NADH) at 595 nm (NADH:NTB reductase assay).24,25 The assay mixture consisted of 10 mM Tris-HCl, pH 7.4; 2.44 μM NTB; 2 mM potassium cyanide (KCN); and 2 μM antimycin A. Samples were incubated in the reaction buffer for 5 minutes and the reaction was started with the addition of 90 μM NADH. Rates were monitored for 10 minutes with and without the addition of 10 μM of the specific complex I inhibitor diphenyleneiodonium (DIPH) to obtain the DIPH-insensitive rate.

Mitochondrial respiratory complex II activity was assayed as the reduction of NTB in the presence of phenazine methasulfate at 595 nm (succinate:NTB reductase assay).25 Samples were incubated for 5 minutes in an assay buffer containing 10 mM Tris-HCl, pH 7.4; 40 mM succinic acid; 2.44 μM NTB; 2 μM antimycin A; 10 μM DIPH; and 2 mM KCN. The reaction was started by the addition of 0.4 mM phenazine methasulfate in the presence or absence of 3 μM 3-nitropropionic acid to determine the 3-nitropropionic acid–insensitive background activity and the activity rates were monitored for 10 minutes.

Mitochondrial respiratory complex III activity was determined as the oxidation of decylubiquinol (DBH2) using cytochrome c (III) as an electron acceptor at 550 nm.26 DBH2 was synthesized according to the method used by Krahenbuhl et al.27 by reduction of decylubiquinone (10 mmol) with NaBH4 in 2 mL of a 1:1 ethanol:H2O mixture (v/v, pH 2). The assay was performed in a buffer containing 500 mM KH2PO4, pH 7.4; 2.5 mg/mL bovine serum albumin; 100 μg/mL rotenone; 2 mM KCN; 1 mM cytochrome c; and 2 mM DBH2. The reaction was started with the addition of 15 μg mitochondrial protein and the activity rates were followed for 2 minutes.

Mitochondrial respiratory complex IV activity was monitored as the rate of cytochrome c oxidation at 550 nm28 for 10 minutes, using a reaction buffer containing 100 mM NaPO4, pH 7.2, and 1 mg/mL 3,3’-diaminobenzidine. The reaction was started with the addition of 0.1 mM cytochrome c with or without 2 mM KCN to determine the KCN-insensitive background rate.

Mitochondrial respiratory complex V activity was determined as the decrease in NADH absorbance at 340 nm monitored for 2 minutes.29 Samples were incubated in a reaction buffer containing 100 mM KH2PO4/KOH, pH 8.0; 10 mM MgCL2; 350 μM NADH; 5 mM phosphoenolpyruvate; 4 μM rotenone; 4 mM KCN; 7 U/mL pyruvate kinase; 15 U/mL lactate dehydrogenase; and 5 mM adenosine triphosphate (ATP). The complex V inhibitor oligomycin (15 μM) was used to determine the oligomycin-insensitive background rate.

All assays were performed using a SpectraMax 190 spectrometer (Molecular Devices, Sunnyvale, CA) at 37°C.

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Mitochondrial Blue Native Gel Electrophoresis and In-Gel Activity Assays

Blue native polyacrylamide gel electrophoresis (BN-PAGE) was performed as previously described.30 Mitochondrial proteins (200 μg) were suspended in BN-PAGE buffer (20 mM imidazole, 100 mM 6-aminocaproic acid, 40% glycerol, and 0.1% Coomassie Blue G250, pH 7.5) and solubilized using the mild detergent digitonin with a detergent to protein ratio of 6 g/g for optimal visualization of mitochondrial supercomplexes. Coomassie Blue G250 was added to set a detergent/Coomassie ratio of 8 g/g in the final mixture. Electrophoresis was subsequently conducted for 12 hours in the cold (4°C) and the gels subjected to Coomassie staining before mass spectrometry or used for the analysis of in-gel catalytic activities, respectively.

The in-gel catalytic activity of mitochondrial complex I was assayed as the NADH:NTB-reductase activity using an assay buffer containing 1 mg/mL of NTB and 0.1 mg/mL of NADH added to 5 mM Tris-HCl, pH 7.4. After 3 to 5 minutes, the reaction was stopped using the fixing solution and the gels were scanned for densitometric quantitation.

Individual complex III/IV, as well as the supercomplexes containing complex III/IV catalytic activities, was investigated in-gel by a 30-minute incubation in an assay buffer containing 0.5 mg/mL diaminobenzidine and 5 mM cytochrome c dissolved in 10 mL of 50 mM sodium phosphate, pH 7.2. The reaction was stopped using the fixing solution and the gels were scanned for densitometric quantitation.

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Mitochondrial Lipid Peroxidation

Mitochondrial lipid peroxidation was assessed via the formation of malondialdehyde–thiobarbituric acid adducts as previously described.31 In brief, isolated mitochondria were suspended in a buffer (pH 3.4) containing 15% trichloroacetic acid, 0.375% thiobarbituric acid, and 0.25 N HCl. The sample was heated to 95°C for 15 minutes and allowed to cool before centrifugation (1000g, 10 minutes) and monitoring of the A535 at 25°C, respectively.

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Liquid Chromatography Tandem Mass Spectrometry

Mass spectrometry was used to identify mitochondrial respiratory complexes and supercomplexes subsequent to purification by BN-PAGE, respectively.32 The liquid chromatography–tandem mass spectrometry (LC-MS/MS) experiments were performed on a Proteomex LTQ tandem mass spectrometry instrument (Thermo Electron, San Jose, CA) equipped with a Surveyor pump system using a reversed-phase column (75 μm in diameter, 10 cm, BioBasic C18 5 μm particle size; New Objective, Woburn, MA). The flow rate was 5 μL/min for sample loading and 250 nL/min for separation. Buffer A consisted of 0.1% formic acid and 2% acetonitrile in water, whereas buffer B contained 0.1% formic acid and 20% water in acetonitrile. A shallow gradient was used for the analyses: at first a linear gradient from 5% B to 40% B over 70 minutes, increased to 100% B over 20 minutes, followed by 100% B for 9 minutes. The ion transfer tube of the linear ion trap was held at 200°C, the normalized collision energy was set at 35% for MS/MS, and the spray voltage was set at 1.9 kV, respectively. The mass spectrometer was operated in the data-dependent mode to switch automatically between MS and MS/MS acquisition. Survey full-scan MS spectra with 1 microscans (400–2000 m/z) were acquired, followed by 5 sequential scan events of MS/MS. Each subsequent MS/MS collision-induced dissociation fragmentation (at a target value of 10,000 ions) was performed on a precursor ion which was isolated using the data-dependent acquisition mode to automatically select ions sequentially with the top 5 highest intensities from the survey scan, using a 3.0-m/z isolation width. In acquisition mode, dynamic exclusion was used with 2 repeated counts within 10 seconds and an exclusion duration of 40 seconds. The spectra were searched with SEQUEST against the murine International Protein Index database and the following criteria were used for peptide identification: Xcorr, >2.0 (+1), >2.2 (+2), >3.8 (+3); DeltaCN >0.1; and Rsp = 1. All proteins were identified on the basis of ≥2 peptides.

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Statistical Analysis

Statistical analysis of data within and between groups was performed with 1-way and 2-way analysis of variance followed by post hoc least significant difference test with a Bonferroni correction of the significance levels (SPSS 16.0 Software; The Apache Software Foundation, Forest Hill, MD). Sample sizes were a priori calculated using the power analysis software G*Power for MAC (Heinrich-Heine-University, Düsseldorf, Germany).33 An effect size d of 2, an α error of 0.05, and a β error of 0.2 (power of 0.8) calculated minimum sample sizes of 4 per group. Changes were considered statistically significant when the P value was <0.01. Every reported P value is that after Bonferroni correction. A denominator of 3 was used in the Bonferroni correction with respect to the infarct size data, as well as a denominator of 6 regarding the analyses of mitochondrial respiratory complexes and supercomplexes, MDA formation, and Ca2+-induced swelling, respectively. All data are expressed as mean ± SD.

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Forty-nine mice were instrumented to obtain 45 successful experiments. Two mice had to be excluded due to cardiac arrest during reperfusion; 1 mouse was excluded from analysis due to incomplete Evans blue staining; and 1 mouse was excluded from analysis due to an AAR under 15% of left ventricular mass.

The results showing the extent of myocardial ischemic injury versus the AAR for the control and animals exposed to isoflurane is shown in Figure 2. Corroborating the results of our previous studies, the administration of isoflurane reduced the extent of myocardial ischemic injury induced 36 hours after its administration (P = 0.0011 versus I/R).

Figure 2

Figure 2

The mitochondrial respiratory complex activities for each experimental group are shown in Figure 3. Myocardial I/R injury reduced the activities of complex I (P = 0.0245 versus sham) and III (P = 0.0025 versus sham). Isoflurane treatment without I/R injury increased the activities of mitochondrial respiratory complexes II and IV compared with the sham group although these changes were not statistically significant (P = 0.0227 for complex II; P = 0.0140 for complex IV). Isoflurane treatment 36 hours before induction of myocardial ischemia resulted in a similar level of mitochondrial respiratory complex activities with respect to complexes II–IV compared with the sham group. Complex III activity was significantly higher in animals treated with isoflurane before I/R injury compared with those suffering from I/R injury without pharmacological preconditioning (P = 0.0008 sham versus isoflurane + I/R). A beneficial effect of isoflurane on complex I activity after I/R injury was not found. The activity of the complex was significantly reduced compared with the sham group (P = 0.0085 versus sham).

Figure 3

Figure 3

The gel electrophoresis of the respiratory chain complex proteins for the respective study groups is shown in Figure 4. Multiple high-molecular-weight bands indicating mitochondrial respiratory complexes associating with each other to form supercomplexes were observed on the BN-PAGE gels. These were identified as oligomers of complex I, III, and IV via LC-MS/MS. No significant difference in protein quantity of these supercomplexes was observed among experimental groups (Fig. 4, dashed box). In-gel respiratory complex activity assays showed decreased complex III/IV activities subsequent to ischemic injury (P = 0.0025 versus sham) (Fig. 4, upper right). This decrease was prevented by isoflurane, indicating preserved activities of respiratory complex III/IV assemblies (P = 0.0086 versus I/R).

Figure 4

Figure 4

Table 1

Table 1

The effects of each experimental condition on lipid peroxidation are shown in Figure 5. Mitochondrial lipid peroxidation was more than doubled after myocardial I/R injury (P = 0.0013 versus sham), indicating oxidative damage to the organelle. Isoflurane treatment prevented the increase in mitochondrial lipid peroxidation after I/R injury, hence preventing unfavorable free radical chain reactions (P = 0.0019 versus I/R).

Figure 5

Figure 5

Furthermore, isoflurane protected mitochondria against permeability transition during myocardial reperfusion. Myocardial I/R injury caused a significant increase in mitochondrial swelling (P = 0.00015 versus sham [200 μM Ca2+]), suggesting mitochondrial permeability transition. This phenomenon was abolished via the use of isoflurane (P = 0.0010 versus I/R [200 μM Ca2+]), indicating preserved mitochondrial integrity (Fig. 6).

Figure 6

Figure 6

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Myocardial ischemic injury is a self-perpetuating process because the mismatch of oxygen supply versus demand results in secondary cellular processes including damage to the mitochondrial ATP-producing machinery that further accentuates tissue injury after the reintroduction of oxygen.34 Volatile anesthetics protect the heart via multiple pharmacologic preconditioning mechanisms35,36 resulting in a reduction in the size of myocardial infarction, corroborated by the findings of this study.36–38 Our results further reveal that mitochondria have a pivotal role in mediating isoflurane-associated myocardial preconditioning during the second window of cardioprotection 36 hours after anesthetic exposure. We found that isoflurane protects the heart from ischemic injury by maintaining the in vivo function of mitochondrial respiratory complex III. The volatile anesthetic elicited beneficial effects onto mitochondrial respiratory complex III, as well as mitochondrial respiratory supercomplex III/IV activities. This resulted in minimized mitochondrial oxidative damage, circumventing the loss of mitochondrial membrane integrity.

Mitochondria have a well-recognized role in the final stage of anesthetic preconditioning. Hanley et al.,13 for example, showed that halothane, isoflurane, and sevoflurane inhibit mitochondrial respiratory complex I enzyme activity at a concentration of 2.0 MAC. Riess et al.14 found that the sevoflurane-induced inhibition of complex I is mediated by ROS. Furthermore, isoflurane altered mitochondrial electron transfer17 and prolonged the duration of NADH oxidation in state 3 respiration.18,19 Altered complex V activity has been linked to modified mitochondrial bioenergetics as well.20 However, all the above studies were conducted without prior episodes of myocardial ischemia. Investigations of the first window of anesthetic myocardial preconditioning demonstrated that the mitochondrial respiratory complex III mediated the generation of oxygen-derived free radicals16 and that both free radical scavengers and nitric oxide synthase inhibition prevented the sevoflurane-induced increase in NADH.15

Our study adds to these findings by showing that distinct changes within the function of the respiratory chain are in play during the second window of protection. We observed severe impairment in the activities of mitochondrial complex I and III after I/R injury (Fig. 3). This finding is of particular interest because complex III dysfunction results in a complete obstruction of the respiratory chain, limiting ATP production as well as effectuating a massive increase in oxidative stress.39 We found that isoflurane prevented these unfavorable effects of I/R injury, preserving the functionality of complex III, both in its free form and in association with complexes I, III, and IV. Although the extent to which the assembly of respiratory complexes to supramolecular structures contributes to the overall respiratory function is not completely understood, it is known that a loss of supercomplex formation causes mitochondrial dysfunction.40 In particular, the “oxidative string model” suggests that supercomplexes are only building blocks of higher-ordered electron transport chain strings. String dysfunction diminishes the oxidative capacity and is likely to account for an increased ROS generation.41,42 Furthermore, only a minority of complex I has been found in free form and it can be assumed that it is bound to complex III under physiological conditions.43 Supercomplex assembly organizes electron flux and facilitates the optimal use of available substrates.44

We identified supramolecular assemblies consisting of mitochondrial respiratory complexes I, III, and IV using BN-PAGE followed by LC-MS/MS. The presence of multiple high-molecular-weight protein bands on the BN-PAGE gels suggests different stochiometries of these supercomplexes. In this regard, the association of monomeric complex I (I1) with dimeric complex III (III2) and complex IV in different copy numbers (IVx) has been described as respirasomes.45 The related mitochondrial III-IV supercomplex activities showed a functional decline after I/R injury, indicating impaired thermodynamic and kinetic properties of the mitochondrial respiratory chain. Isoflurane attenuated these effects of I/R on the supramolecular assemblies suggesting preserved bioenergetic stability. However, the impact of these events within the cardioprotective response of isoflurane is ultimately defined by the fact that the maintenance of mitochondrial respiration also circumvents mitochondrial permeability transition.46

Preventing mitochondrial permeability transition due to I/R injury is an essential target of volatile anesthetic cardioprotection47 because cell death is inevitable after the disintegration of the mitochondrial membranes. Both anesthetic-induced preconditioning48,49 and postconditioning50 depend on the inhibition of mitochondrial permeability transition. In this regard, Ca2+-activated potassium channels,51 ROS and extracellular signal-related kinases 1/2,52 activation of protein kinase Cε,48 and endothelial nitric oxide synthase53 stabilize the mitochondrial membranes. Moreover, protective effects can be achieved via direct pharmacological inhibition of the mitochondrial permeability transition pore.54 Monitoring Ca2+-induced swelling provides a marker of mitochondrial permeability transition.55 Hence, the increased susceptibility to Ca2+-induced swelling after I/R injury, in conjunction with the observed increase in mitochondrial lipid peroxidation, reflects a vicious cycle of mitochondrial respiratory dysfunction, oxidative damage, and mitochondrial permeability transition.56 This deadly cascade was not initiated in animals pretreated with isoflurane, most likely due to the disruption of this vicious cycle at its onset via the preservation of mitochondrial ATP production.

The current results must be interpreted within the constraints of several potential limitations. Myocardial infarct size can be influenced by the size of the AAR and coronary collateral perfusion. In the present investigation, the AAR expressed as a percentage of total left ventricular mass was similar among groups. Because small rodents are reported to have little, if any, coronary collateral blood flow,57 it is highly improbable that vasodilatation during CAO influenced myocardial infarct size. Mitochondrial oxidative stress is difficult to study because free radicals are unstable. Furthermore, isolated mitochondrial preparations are performed under substrate and respiratory conditions that are defined experimentally and cannot perfectly replicate conditions in vivo. Blue native gel electrophoresis may lead to a shift in protein charge when using Coomassie-dye. Therefore, supramolecular assemblies of membrane protein complexes are dissociated to some extent. However, the use of digitonin for mitochondrial membrane permeabilization is a mild solubilization method helping to retain labile membrane protein complexes.

In summary, we used a stepwise approach combining in vivo experiments, BN-PAGE, in-gel complex activity measurements, as well as LC-MS/MS, to characterize the changes in mitochondrial respiratory physiology during myocardial I/R injury and isoflurane-induced protection. Our findings demonstrate that isoflurane confers cardioprotection 36 hours after exposure preserving the functionality of the mitochondrial respiratory chain, circumventing oxidative damage, and subsequent mitochondrial membrane disintegration.

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Name: Christopher Lotz, MD.

Contribution: This author designed the study, conducted the study, helped collect data, and prepared the manuscript.

Attestation: Christopher Lotz approved the final manuscript. The author attests to the integrity of the original data and the analysis reported in this manuscript. Christopher Lotz is the archival author.

Name: Jun Zhang, PhD.

Contribution: This author helped collect data.

Attestation: Jun Zhang approved the final manuscript.

Name: Caiyun Fang, PhD.

Contribution: This author helped collect data.

Attestation: Caiyun Fang approved the final manuscript.

Name: David Liem, MD PhD.

Contribution: This author helped collect data.

Attestation: David Liem approved the final manuscript.

Name: Peipei Ping, PhD, FAHA, FISHR.

Contribution: This author helped design and conduct the study.

Attestation: Peipei Ping approved the final manuscript. The author attests to the integrity of the original data and the analysis reported in this manuscript.

This manuscript was handled by: Charles W. Hogue, Jr, MD.

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