Ischemic preconditioning (IPC), originally described by Murry et al. (1), enhances myocardial tolerance against the consequences of I/R injury. Volatile anesthetics, including isoflurane (ISO), also exert IPC-like protection by reducing myocardial stunning, dysrhythmia, calcium loading, and infarct size (IS) in experimental animals and human subjects (2-4). Whereas the underlying mechanisms of anesthetic-induced preconditioning (APC) are still debatable, accumulating evidence indicates that production of reactive oxygen species (ROS), in particular superoxide anion (O2−), via opening of the mitochondrial adenosine triphosphate-sensitive potassium (KATP) channels initiate acute cardiac protection (2, 5, 6). The beneficial effect of APC is also associated with attenuated ROS production, improved mitochondrial respiration, and augmented ATP content at the time of reperfusion (6, 7). These observations imply that preservation of mitochondrial bioenergetics and oxidative homeostasis during reperfusion may be a key component of APC-mediated protection. Exactly how APC-induced O2− production attenuates ROS production, improves mitochondrial respiration, and elevates ATP content, leading to cardiac protection against I/R injury, nonetheless, has not been fully addressed.
Mitochondria play a central role in cell energetics via oxidative phosphorylation and orchestrate cellular viability under stress (8). In the heart during ischemia, significant generation of ROS, most likely from a mitochondrial source, compromises electron transport, which results in electron leak and further oxidative damage to lipids and proteins (9), leading to loss of mitochondrial integrity and function and cell death. Thus, an important element in myocardial protection is attenuation of mitochondrial oxidative stress during I/R injury (8, 10). Cytotoxic ROS can also be contained either by attenuating their generation or by reducing their effects (11). It is therefore reasonable to stipulate that preservation of electron transport system or activation of endogenous antioxidant defense mechanisms in the mitochondria may underlie APC-mediated cardiac protection by amelioration of I/R-induced mitochondrial damage. Moreover, ROS generated from opening of mitochondrial KATP may be part of this cellular scheme.
Using ISO as our representative anesthetic agent, the present study evaluated the hypothesis that APC induces myocardial protection against I/R by attenuation of excessive ROS and restoration of mitochondrial bioenergetics through up-regulation of manganese superoxide dismutase (MnSOD) expression and preservation of respiratory enzyme activity. Our results validated this hypothesis. We demonstrated that the cardioprotective effects of ISO preconditioning are attributed to postischemic up-regulation of mitochondrial MnSOD and increase in respiratory enzyme activity that reduces cardiac O2− surge during reperfusion, leading to preserved respiratory chain enzyme function and energy production that reduces IS. We also showed that it is likely that these beneficial effects are mediated by generation of O2− from ISO-induced mitochondrial KATP channel activation.
MATERIALS AND METHODS
Adult male Sprague-Dawley rats (10-12 weeks, 330-430 g) used in this study were purchased from the National Animal Experimental Center of the National Science Council, Taiwan. All experimental procedures were in accordance with the guidelines of our institutional animal care committee.
Rats were anesthetized initially via a single intraperitoneal administration of sodium pentobarbital (50 mg kg−1) for surgical preparation. After tracheotomy, rats were mechanically ventilated by a rodent respirator (ADS 1000; Angler Engineering Co., Hialeah, Fla) at 65 to 70 breaths per minute with room air supplemented with oxygen (Fio2, 40%-45%). Arterial pH, Pco2, and Po2 were monitored by a blood gas analyzer (CIBA CORNING 288; Diagnostics GmbH, Neuss, Germany) and were maintained within normal physiological range (pH 7.35-7.45; Pco2, 35-45 mmHg; Po2, 100-150 mmHg). The right femoral artery was cannulated for monitoring blood pressure and heart rate on a Gould polygraph (ES3400; Gould, Valley View, Ohio). Bilateral femoral veins were cannulated for saline and drug infusion. Body temperature was maintained at 37.5 ± 0.5°C by a heating pad. To maintain an anesthetic plane for the duration of the experiment, animals were supplemented after the completion of surgical preparation with intravenous infusion of midazolam at 1.5 mg kg−1 h−1. The only exception was the period when ISO was administered, during which the dose of midazolam was reduced to 0.15 mg kg−1 h−1. This benzodiazepine was chosen because it does not affect mitochondrial KATP channel activity (12), a key target of this study.
The heart was exposed via a left thoracotomy at the fifth intercostal space. The pericardium was removed, and the left appendage was adjusted to reveal the location of the left coronary artery. A ligature (5-0 PROLENE) was passed around this coronary artery at the proximity of its base, and both ends of the suture were threaded through a small propylene tube to make a snare for the performance of coronary occlusion and reperfusion (13). Epicardial cyanosis, akinesia or regional bulging, and progressive exhibition of marked arrhythmia verified the effectiveness of coronary artery occlusion (CAO). Reperfusion of the heart was visually confirmed by the appearance of hyperemia.
Study groups and experimental protocol
Figure 1 illustrates the treatment groups used in experiments that determined the myocardial IS. As a routine, systemic hemodynamics were recorded throughout the observation period, and all rats received 30 min of CAO followed by 120 min of reperfusion. In addition, rats were randomly assigned to receive 0.9% saline (I/R group), two cycles of 3 min of CAO followed by 5 or 10 min of reperfusion (IPC + I/R group) or 30-min inhalation of ISO (ISO-induced preconditioning [ISO-PC] + I/R group) at an end-tidal concentration of 1.4%, which corresponds to 1.0 minimum alveolar concentration (MAC) (13). The dose of ISO was adopted from previous studies (6, 14) that used this anesthetic for the purpose of preconditioning. A specific mitochondrial KATP channel blocker, 5-hydroxydecanoate (5-HD; 10 mg kg−1), the O2− scavenger 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (TEMPOL; 30 mg kg−1), or the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 1.3mg kg−1 min−1) was given alone (5-HD + I/R, TEMPOL + I/R, and L-NAME + I/R, respectively) or in association with ISO (5-HD + ISO-PC + I/R, TEMPOL + ISO-PC + I/R, and L-NAME + ISO-PC + I/R, respectively). 5-Hydroxydecanoate, TEMPOL, or L-NAME was given intravenously 10 min before ISO. Isoflurane was administered for 30 min and discontinued 10 min before CAO. End-tidal concentrations of ISO were measured at the tip of the tracheotomy tube with an anesthetic gas analyzer (Capnomac Ultima; Datex, Helsinki, Finland) that was calibrated with known standards before and during experimentation.
Determination of myocardial IS
At the end of the reperfusion period, the coronary artery was reoccluded, and 1% patent blue dye was injected via the femoral vein to reveal the in vivo area at risk (AAR). The rat was then killed with 15% KCl solution, and the heart was removed for 2,3,5-triphenyltetrazolium chloride staining to differentiate viable (deep red) and nonviable (pink or pale) tissue (15). For this purpose, the left ventricle (LV) was cut into six 2-mm cross-sectional slices and incubated at 37°C for 20 min in 1% 2,3,5-triphenyltetrazolium chloride in 0.1 M phosphate buffer (pH 7.4). The slices were then stored overnight in a 10% formaldehyde solution and subsequently photographed to determine the infract size (IS). Infarct size, AAR, and LV area were quantified by computerized planimetry (Image-Pro Plus software, version 4.5.1; Media Cybernetics, Inc., Silver Spring, Mass) and corrected for the weight of tissue slices. Area at risk was expressed as a percentage of the LV (AAR/LV), and IS was expressed as a percentage of the AAR (IS/AAR) (16).
Detection of superoxide anion
The lucigenin-enhanced chemiluminescence assay was used to determine O2− production in the AAR according to previously described and validated methods (17). Left-ventricle samples were obtained from separate groups of rats that were pretreated with 5-HD, TEMPOL, or L-NAME in the presence or absence of 1.0 MAC ISO. Hearts excised immediately after ISO exposure or 30 s after I/R were promptly processed for O2− measurements. Tissue was homogenized in a 20-mM sodium phosphate buffer (pH 7.4) containing 0.01 mM EDTA by a glass-to-glass homogenizer. The homogenate was subjected to low-speed centrifugation at 1,000 g at 4°C for 10 min to remove nuclei and unbroken cell debris. The pellet was discarded, and the supernatant was obtained immediately for O2− measurement. Background chemiluminescence in buffer (2 mL) containing lucigenin (5μM) was measured for 5 min. An aliquot of 100 μL of supernatant was then added, and the chemiluminescence was measured for 30 min at room temperature (Sirius Luminometer, Berthold, Germany). O2− levels were expressed as relative light units per second after subtraction of background activity and all assays were performed in triplicate. Specificity for O2− was determined by adding SOD (350U mL−1) into the incubation medium.
Measurement of nitrite and nitrate
Further experiments were performed to determine the changes of total NO content during ISO-PC. Rats were killed 0, 5, 10, 30, 60, or 120 min after 30 min of ISO (1.0 MAC) treatment, and LVs were rapidly dissected, frozen in liquid nitrogen, and homogenized in lysis buffer. After centrifugation (19,000g at 4°C) for 15 min, the supernatant was deproteinized using a Centricon-30 filtrator (Microcon YM-30; Bedford, Mass) and stored at −80°C until further processing. The level of total nitrite and nitrate (NOx) was determined with the purge system of a Sievers NO analyzer (NOA 280; Boulder, Colo) using modifications of the procedure described by Braman and Hendrix (18) based on chemiluminescence reaction. All assays were performed in triplicate and expressed as nanomoles per milligram of protein.
Isolation of mitochondria from cardiac tissues
To isolate the mitochondrial fraction, rats were killed after reperfusion, and the heart tissues in AAR were immediately minced in 5 volumes of cold buffer containing 2 mM HEPES (pH 7.4) 220 mM mannitol, 0.1 mM EDTA, 70 mM sucrose, and 0.5% (wt/vol) bovine serum albumin (BSA; buffer A) (19). The tissue samples were homogenized in a glass grinder and then centrifuged at 800 g for 10 min. The supernatant was filtered and centrifuged at 9,500 g for 20 min to generate the mitochondrial pellet. The pellet was washed with buffer A and then resuspended in buffer A without BSA to constitute the mitochondrial fraction. The entire procedure was performed at 4°C. The purity of the mitochondrial-rich fraction was verified by the expression of the mitochondrial inner membrane-specific protein cytochrome c oxidase (COX) (20). Mitochondrial protein concentration was determined using the Bradford method (21) (Bio-Rad, Hercules, Calif).
Mitochondrial MnSOD protein expression
Manganese superoxide dismutase protein was determined in cardiac mitochondria isolated at 30 s after reperfusion using Western blot analysis. Four micrograms of protein samples was separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane at 90 V for 2 h. The membrane was blocked with 5% skim milk and immunoblotted with a rabbit anti-MnSOD polyclonal antibody (StressGen Biotechnologies, Victoria, Canada) or COX (Oncogene, Boston, Mass), followed by incubation with horseradish peroxidase-conjugated goat antirabbit immunoglobulin G (Jackson Immunoresearch Laboratories, West Grove, Pa). Specific antigen-antibody complex was detected using an enhanced chemiluminescence detection system (GE Healthcare, Buckinghamshire, UK). The amount of detected protein was quantified by BIO-PROFIL Bio-1D Light V2000.00 software (Vilber Lourmat, Paris, France) and was expressed as the ratio to mitochondrial COX protein.
Measurement of mitochondrial MnSOD activity
The activity of MnSOD from the mitochondrial fraction was measured using a MnSOD assay kit (Calbiochem, San Diego, Calif), which uses 5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenso[c]fluorine as the substrate. This reagent undergoes alkaline autoxidation, which is accelerated by MnSOD, and yields a chromophore that absorbs maximally at 525 nm. A 50% inhibition is defined as 1 U MnSOD, and the specific activity was expressed as unit per milligram of mitochondrial protein (22).
Assays for activity of mitochondrial respiratory enzymes
All enzyme activity assays were performed immediately after mitochondrial isolation using a thermostatistically regulated spectrophotometer (ThermoSpectronic, Cambridge, UK) as described previously (23). At least triplicate determination was carried out for each tissue sample in all enzyme activity assays.
Nicotinamide adenine dinucleotide (reduced form; NADH) cytochrome c reductase (NCCR; marker enzyme for complex I and III) activity was determined by the reduction of oxidized cytochrome c measured at 550 nm, and was calculated as the difference in the presence or absence of rotenone. The activity was assayed in 50 mM K2HPO4 buffer (pH 7.4) containing 1.5 mM KCN, 1 mM β-NADH, and 100 μg of mitochondrial suspension in the presence or absence of 20 μM rotenone. The reaction was initiated after 2 min of stabilization by adding 0.1 mM cytochrome c, and absorbance at 550 nm was measured at 5-s intervals during the first 3 min at 37°C. The molar extinction coefficient of cytochrome c at 550 nm is 18,500 M cm−1.
Determination of succinate cytochrome c reductase (SCCR; marker enzyme for complex II and III) activity was performed in 40 mM K2HPO4 buffer (pH 7.4) containing 1.5 mM KCN, 20 mM succinate and 200 μg of mitochondrial suspension. After 5 min ofincubation at 37°C, the reaction was initiated by adding 50 μM cytochrome c, and absorbance at 550 nm was measured at 5-s intervals over the first 3 min.
Cytochrome c oxidase (COX, marker enzyme for complex IV) activity was measured by recording the oxidation of reduced cytochrome c at 550 nm. The activity of COX is defined as the first-order rate constant and is calculated from the known concentration of ferrocytochrome c and the amount of enzyme in the assay mixture. The activity was assayed in 10 mM K2HPO4 buffer (pH 7.4) containing 1.5 mM KCN, 20 mM succinate, and 200 μg of mitochondrial suspension. After 5 min of incubation at 30°C, the reaction was initiated by adding 45 μM ferrocytochrome c, and absorbance at 550 nm was measured at 5-s intervals during the first 3 min.
Assessment of ATP content
To determine myocardial ATP content, tissue samples in the AAR were rapidly dissected 5 min after reperfusion, frozen in liquid nitrogen, and then homogenized in lysis buffer. After centrifugation (19,000 g at 4°C) for 15 min, the protein concentration in the supernatant was measured by the Bradford method (21) (Bio-Rad), with BSA as the standard. Cellular ATP concentration was measured using an ATP bioluminescence assay (Roche Diagnostics GmbH, Penzberg, Germany) that detects light emission from a luciferase-mediated reaction in a tube luminometer. All assays were performed in triplicate, and tissue ATP levels were expressed as relative light units per second.
Rats were excluded from the study because of unacceptable blood gas values, malignant ventricular arrhythmia, or severe hypotension (<30 mmHg of systolic blood pressure). Hearts with a risk area less than 10% of the LV weight were also excluded from data analysis.
Data are presented as mean ± SEM. Statistical analysis was performed with SPSS statistical software package, version 10.0 (SPSS, Inc., Chicago, Ill). Differences were evaluated using one-way ANOVA followed by post hoc comparison with the Bonferroni method. Significant differences were determined at P < 0.05.
Two hundred three rats were used, of which 53 were used in successful IS experiments and 134 in biochemical studies. Three rats were excluded as a result of technical difficulties during surgical preparation. Eleven rats died of malignant ventricular arrhythmias during the 30-min occlusion. Two rats were not used because of a small region at risk (<10% of the LV).
Systemic hemodynamics changes
In our myocardial IS experiment, there were no significant differences among the nine treatment groups in baseline heart rate (HR), MAP, or rate-pressure product (RPP) (Table 1). These hemodynamic parameters were significantly decreased in rats that received ISO with or without 5-HD or TEMPOL but returned to baseline values 10 min after discontinuation of ISO and before coronary occlusion. Coronary artery occlusion and reperfusion also produced decreases in MAP and RPP in each experimental group. No significant differences in HR were found among groups at the preocclusion point during CAO or at reperfusion.
AAR and IS
The AAR mass (range, 0.15 ± 0.02-0.19 ± 0.03 g) and AAR as a percent of LV mass (range, 30.3 ± 2.6-35.7 ± 2.0%) did not differ significantly among the experimental groups (Table 2). Isoflurane (ISO-PC), similar to the protection in the IPC group, significantly reduced IS as compared with the I/R group (Fig. 2). Pretreatment with 5-HD or TEMPOL, but not L-NAME, abolished the protective effects of ISO. Infusion of 5-HD, TEMPOL, or L-NAME alone had no effect on IS.
Effects of ISO on O2− generation and NOx level
The myocardial O2− content, measured immediately after ISO exposure, was significantly greater as compared with those that did not receive the volatile agent (Fig. 3A). The increase in O2− level was inhibited when 5-HD or TEMPOL was administered before ISO exposure. In contrast, the total myocardial content of NOx in rats exposed to 30 min of ISO was not significantly changed throughout the 120-min observation period (Fig. 3B).
ATP content and O2− production on reperfusion
Compared with the untreated sham-control hearts, myocardial ATP content at the AAR in the I/R group was significantly reduced (Fig. 4A). Preconditioning with ISO significantly reversed this ATP reduction. This beneficial effect of ISO to restore ATP content was completely abrogated by 5-HD or TEMPOL, but not by L-NAME. Individual treatment alone had no direct effect on I/R-induced reduction in ATP content. Moreover, myocardial ATP content in nonischemic area of LV was not significantly different among experimental groups (data not shown).
At 30 s after reperfusion, the production of O2− was significantly increased in the I/R, but not in the ISO-PC group, when compared with the sham controls (Fig. 4B). 5-Hydroxydecanoate or TEMPOL, but not L-NAME, reversed the ISO-induced reduction in O2− production. 5-Hydroxydecanoate, TEMPOL, or L-NAME alone did not affect the I/R-induced O2−increase.
MnSOD expression and activity in isolated mitochondria after reperfusion
Mitochondrial MnSOD content, detected 30 s after reperfusion, was significantly increased to 55 ± 10% more than the sham-control hearts in the ISO-PC group, but only 13 ± 4% in the I/R group (Fig. 5). Enzyme activity of the mitochondrial SOD was also discernibly augmented in the heart that received ISO-PC (4.87 ± 0.43 U mg−1 protein; n = 5) compared with control (3.24 ± 0.36 U mg−1 protein; n = 5). 5-Hydroxydecanoate or TEMPOL completely abrogated the ISO-PC-promoted MnSOD up-regulation (Fig. 5) or the increase in enzyme activity.
Respiratory enzyme activity in isolated mitochondria after reperfusion
Compared with sham-control group, the activity of NCCR (Fig. 6A), SCCR (Fig. 6), or COX (Fig. 6C), detected 5 min after reperfusion, was significant decreased after I/R. Preconditioning with ISO reversed the depression of these respiratory enzyme activities from I/R injury. 5-Hydroxydecanoate or TEMPOL completely abrogated the ISO-PC-promoted preservation of mitochondrial respiratory enzyme activity.
The present study demonstrated that a brief exposure to ISO before ischemia significantly reduced myocardial IS after I/R that is associated with decreased O2− surge and improved cardiac ATP content. These beneficial effects of APC are attributed to the postischemic modulation of mitochondrial functions as evidenced by enhancement of MnSOD protein expression and enzyme activity, and preservation of respiratory enzyme activity in the heart. Our observations that these cellular and molecular adaptations were abrogated by 5-HD or TEMPOL indicate that they depend on opening of the mitochondrial KATP channels and generation of O2−. Because the early burst in myocardial O2− formation after ISO exposure was eliminated by 5-HD, O2− may constitute a key signal downstream to KATP channel activation in these protective processes induced by this volatile anesthetic. The observation that ISO-induced myocardial protection during I/R injury was not prevented by L-NAME, and myocardial NOx level was not altered by ISO exposure, suggest that NO signal plays a minor role in ISO-PC.
Cardiac mitochondria are highly vulnerable to oxidative stress after I/R, which orchestrates myocardial dysfunction by exhibiting reduced membrane potential, depressed electron transport chain (ETC) function, and impaired ATP synthesis (24). We found in this study that exposure to ISO effectively reversed the depressed cardiac ATP content and reduced IS during early reperfusion. This observation suggests that preservation of mitochondrial oxidative phosphorylation by APC plays an important role in protecting myocardial function after ischemia. In addition, a key finding in the present study was that the depressed respiratory enzyme activities after I/R were prevented by ISO-PC, indicating that APC acts to improve ETC function through the mitochondrial respiratory complexes, and thus restores ATP content. Together with its reported involvement in IPC (25), preservation of mitochondrial bioenergetics may represent an important common feature of APC and IPC, and is likely a critical cellular process of cardioprotection against I/R injury. The mechanisms by which APC enhances mitochondrial energetic capacity during reperfusion remain undefined. Recent experiments in isolated rat hearts demonstrated that APC with ISO induces up-regulation of the genes for NADH-ubiquinone dehydrogenase and COX subunit VIIa 3 (26). In addition, by limiting the decline in ATP content after ischemia, down-regulation of mitochondrial ATPase after exposure to ISO (26) may provide another possibility for better maintenance of ATP level after APC.
It is well known that oxidative stress introduced by increased O2− production during reperfusion after global ischemia induces irreversible cell damage by peroxidation of membrane lipids, denaturation of proteins, and strand breaks in DNA (11). Mitochondria are not only susceptible to oxidative damage but are also a major source of O2− during reperfusion, primarily from complexes I and III (27). Overproduction of ROS from attenuated ETC function after I/R may induce oxidative modification of mitochondrial proteins that include electron carriers, resulting in positive feedback that further depresses ETC activity and impair bioenergetic functions (27). It is therefore noteworthy that pharmacological treatments in the present study that significantly reversed the ISO-induced reduction in O2− production during reperfusion also abrogated the restoration of ATP content by APC. These findings are interpreted to indicate that the reduction in O2− formation after reperfusion elicited by APC constitutes an important cellular process that mediates cardioprotection against I/R injury through preservation of mitochondrial function.
Various endogenous free radical scavenging enzymes have been proposed to prevent O2− surge and mediate acute cardioprotection by IPC (28). The present study provided novel observations that suggest that postischemic up-regulation of mitochondrial MnSOD may underlie the ISO-induced cardioprotection against reperfusion injury. Along with reduced O2− production during early reperfusion, we found significantly increased mitochondrial MnSOD protein expression and enzyme activity in ISO-PC. Superoxide dismutase is a ubiquitous enzyme with a pivotal role in protecting cells against oxidative stress by catalyzing the conversion of O2− to hydrogen peroxide and molecular oxygen via the dismutation reaction (29). Three different SOD isoforms, including copper-zinc SOD in the cytosol, MnSOD primarily in the mitochondria, and extracellular SOD, might play distinct roles in I/R (29). Using various genetic approaches, mitochondrial MnSOD has a greater capacity than other isoforms in protecting hearts against ROS-induced injury (30, 31). It is therefore conceivable that the postischemic augmentation of MnSOD expression and enzyme activity in the heart after ISO exposure may reduce the surge of O2− during reperfusion, leading to preservation of mitochondrial bioenergetic function and cardioprotection. In support of this notion, overexpression of MnSOD, either in transgenic models or by adenoviral gene transfer, has provided direct evidence that MnSOD is a key component of cardioprotection during I/R injury (30, 31).
In contrast to the detrimental effects of excessive ROS on reperfusion, sublethal ROS generation is recognized as important triggers to initiate preconditioning protection (6). It is thus of interest that our results suggest that generation of O2− after ISO exposure plays an essential role in the development of acute preconditioning. The infarct-sparing effect by ISO during reperfusion is notably reversed by the O2− scavenger TEMPOL. That 5-HD administration before exposure to ISO-attenuated O2− formation during preconditioning further indicates that O2− formation is downstream to opening of mitochondrial KATP after APC. Moreover, at the molecular level, the present study demonstrated that the mitochondrial KATP channel-dependent O2− signaling may mediate APC by inducing up-regulation of MnSOD and preservation of mitochondrial functions in the heart during reperfusion injury. We found that 5-HD or TEMPOL, at a dose that effectively attenuated O2− burst immediately after ISO exposure, also suppressed the augmented MnSOD expression or enzyme activity and the restored ETC function and cardiac ATP content by the volatile anesthetic during reperfusion injury.
The cellular mechanisms that underlie the O2− burst after opening of mitochondrial KATP channels by APC are not clear. In this regard, opening of mitochondrial KATP channels has been proposed to cause partial dissipation of mitochondrial membrane potential via potassium influx, leading to uncoupling of ETC and generation of free radicals (5). The linkage between mitochondrial KATP channel-dependent O2− burst and MnSOD up-regulation after ISO exposure is yet to be identified. It was recently reported (32) that ROS activates a signal relay pathway in which the serine/threonine protein kinase D activates the transcription factor nuclear factor κB, leading to transcriptional up-regulation of MnSOD. Alternatively, other intracellular mediators activated by ROS may account for cardioprotection by APC. For example, hydrogen peroxide activates protein kinase C to restore contractility and limit myocardial infarction in rabbit hearts (33). In addition, mitochondrial-derived ROS activates ERK1/2 or p38 mitogen-activated protein kinase in monocytes (34) or cardiac myocytes (35). Reactive oxygen species also modulates mitochondrial KATP channel activity in guinea pig-isolated hearts that further amplifies activation of this channel (36).
We also found in the present study that NO is not as robust as O2− signaling in response to APC. Administration of L-NAME before exposure to ISO failed to affect the reduction in myocardial IS, enhanced ATP production, or reduced O2− production induced by ISO. Together with the lack of significant changes in myocardial NOx content after ISO exposure, it is deemed unlikely that NO signaling participates actively in ISO-PC. These results of our experiment are at variance with those of previous studies (37, 38), possibly because of differences in experimental protocols, animal species, or doses of NOS inhibitors used.
Because the design of the present study calls for the administration of only 30 min of ISO to induce preconditioning, it is imperative that our animals were properly maintained under an anesthetic plane. That this may not be a major confounding factor against the primary attribution of ISO-PC to cardioprotection is supported by at least three observations. First, control animals that were maintained by the same anesthetic management exhibited significant myocardial infarction on I/R injury. Second, midazolam does not affect mitochondrial KATP channel activity (12), a key step responsible for our observed cardioprotection, and the dose of this benzodiazepine was reduced to one-tenth during ISO-PC. Third, the dose of ISO used in this study, which corresponds to 1 MAC in human, has been reported to be protective in previous investigations (6, 14).
In summary, the present results demonstrate that brief exposure to ISO protects hearts from I/R injury by initiating a sequence of cellular adaptation that involves attenuation of ROS generation and restoration of energetic function during reperfusion. Specifically, we found that the cellular repertoires engaged in APC entailed postischemic augmentation of MnSOD expression and enzyme activity, preservation of mitochondrial ETC function, and maintenance of ATP content. At the molecular level, O2− generated from opening of mitochondrial KATP channel after ISO exposure seems to exert an inhibitory effect on postischemic ROS surge via up-regulation of MnSOD, leading to improved mitochondrial respiratory enzyme activities and maintained ATP content after reperfusion. On the other hand, NO does not seem to be involved in these acute processes. Our findings may have important implications for understanding the cellular and molecular mechanisms that underlie APC. The possibility that regulation of mitochondrial functions may be amenable to manipulation as therapeutic targets opens a new vista for thedevelopment of protective strategies against cardiac I/Rinjury.
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Isoflurane; superoxide anion; mitochondrial KATP channel; ATP production; I/R injury