Mitochondrial Adenosine Triphosphate–regulated Potassium Channel Opening Acts as a Trigger for Isoflurane-induced Preconditioning by Generating Reactive Oxygen Species

All experimental procedures and protocols used in this investigation were reviewed and approved by the Animal Care and Use Committee of the Medical College of Wisconsin (Milwaukee, Wisconsin). Furthermore, all conformed to the Guiding Principles in the Care and Use of Animals  of the American Physiologic Society and were in accordance with the Guide for the Care and Use of Laboratory Animals  (National Academy Press, Washington, D.C., 1996).

Male New Zealand white rabbits weighing between 2.5 and 3.0 kg were anesthetized with intravenous sodium pentobarbital (30 mg/kg) as previously described. 11Additional doses of pentobarbital were titrated as required to ensure that pedal and palpebral reflexes were absent throughout the experiment. A tracheostomy was performed through a ventral midline incision, and the trachea was cannulated. The rabbits were ventilated with positive pressure using an air–oxygen mixture (fraction of inspired oxygen, 0.33). Arterial blood gas tensions and acid–base status were maintained within a normal physiologic range (pH, 7.35–7.45; Paco², 25–40 mmHg; and Pao², 90–150 mmHg) by adjusting the respiratory rate or tidal volume throughout the experiment. Body temperature was maintained with a heating blanket. Heparin-filled catheters were inserted into the right carotid artery and the left jugular vein for measurement of arterial blood pressure and fluid or drug administration, respectively. Maintenance fluids consisting of 0.9% saline (15 ml · kg⁻¹· h⁻¹) were continued for the duration of the experiment. A left thoracotomy was performed at the fourth intercostal space, and the heart was suspended in a pericardial cradle. A prominent branch of the left anterior descending coronary artery (LAD) was selected, and a silk ligature was placed around this artery approximately halfway between the base and the apex for the production of coronary artery occlusion and reperfusion in the myocardial infarct size experiments. Each rabbit was anticoagulated with 500 U heparin immediately before LAD occlusion. Coronary artery occlusion was verified by the presence of epicardial cyanosis and regional dyskinesia in the ischemic zone, and reperfusion was confirmed by observing an epicardial hyperemic response. A heparin-filled catheter was inserted into the left atrium for the subsequent administration of dihydroethidium in the experiments designed to detect ROS production. 11Hemodynamic parameters were continuously recorded on a polygraph throughout the experiments.

The experimental design for the myocardial infarct size experiments is illustrated in figure 1. Baseline systemic hemodynamic parameters were recorded 30 min after instrumentation was completed. Rabbits were randomly assigned to one of five experimental groups using a partial Latin square design. All rabbits underwent a 30-min LAD occlusion followed by 3 h reperfusion. The rabbits received an intravenous vehicle (0.9% saline) or the selective mitochondrial K^ATPchannel antagonist 5-hydroxydecanoate (5-HD; 5 mg/kg) in the presence or absence of 1.0 minimum alveolar concentration (MAC) isoflurane. 5-HD was administered either 10 min before or immediately after discontinuation of 1.0 MAC isoflurane (early or late timing, respectively). End-tidal concentrations of isoflurane were measured at the tip of the tracheostomy tube with an infrared anesthetic analyzer that was calibrated with known standards before and during experimentation. The end-tidal MAC value used for rabbits in the present investigation was 2.1%. 17 

Myocardial infarct size was measured as previously described. 18Briefly, at the end of each experiment, the LAD was reoccluded, and 3 ml of patent blue dye was injected intravenously. The left ventricular (LV) area at risk for infarction (AAR) was separated from surrounding normal areas (stained blue), and the two regions were incubated at 37°C for 20–30 min in 1% 2,3,5-triphenyltetrazolium chloride in 0.1 m phosphate buffer adjusted to a pH of 7.4. After overnight storage in 10% formaldehyde, infarcted and noninfarcted myocardia within the AAR were carefully separated and weighed. Infarct size was expressed as a percentage of the AAR. Rabbits that developed intractable ventricular fibrillation and those with an AAR less than 15% of LV mass were excluded from subsequent analysis.

In nine additional experimental groups, the rabbits received an intravenous vehicle (0.9% saline), the free radical scavengers N -acetylcysteine (150 mg/kg over 30 min) 11or N -2-mercaptopropionyl glycine (2-MPG; 1 mg · kg⁻¹· min⁻¹over 75 min), 11or 5-HD (5 mg/kg; early or late timing) in the presence or absence of 1.0 MAC isoflurane (fig. 2). The ROS probe dihydroethidium (2.2 mg) was rapidly injected into the left atrium 5 min before the administration of isoflurane or at the corresponding time point in rabbits that were not exposed to the volatile anesthetic. Isoflurane was discontinued after 30 min, and the rabbits were euthanized after 1 h with an overdose of pentobarbital. The heart was rapidly excised, and the LV was isolated, divided into four sections of equal size, and frozen in liquid nitrogen for subsequent analysis.

Reactive oxygen species were detected using dihydroethidium fluorescence as previously described. 11Dihydroethidium is oxidized by intracellular ROS to produce fluorescent ethidium that subsequently binds to DNA (ethidium–DNA), further amplifying the fluorescence. Briefly, cryostat sections (20 μm) of the LV were mounted on standard microscope slides. Using a laser fluorescence imaging system mounted on a confocal microscope, images were recorded and stored for subsequent off-line analysis on a computer workstation equipped with image analysis software. Use of the 40× objective yielded a 400× end magnification on a 292 × 195 μm²digital image (768 × 512 pixels). The signal-to-noise ratio was enhanced using the Kalman method. Excitation was produced using a krypton–argon laser at a wavelength of 488 nm, and emitted fluorescence was measured at 550 nm after long pass filtering. The pixel intensity of each myocyte nucleus was determined. Background was identified as an area without cells or with minimal cytosol fluorescence. In each rabbit, 20 Kalman-averaged images were obtained, and approximately 6–8 dihydroethidium-stained myocardial cells were analyzed in each image by subtraction of background fluorescence from the pixel intensity of the myocardial nuclei.

Statistical analysis of data within and between groups was performed with analysis of variance for repeated measures followed by Student–Newman–Keuls test. Changes within and between groups were considered statistically significant when the P  value was less than 0.05. All data are expressed as mean ± SEM.

Forty-nine rabbits were instrumented to obtain 39 successful myocardial infarct size experiments. Four rabbits were excluded because the AAR was less than 15% (one control, two isoflurane alone, and one isoflurane + late 5-HD). Six rabbits were excluded because of intractable ventricular fibrillation (two control, one isoflurane alone, one isoflurane + early 5-HD, and two isoflurane + late 5-HD).

No differences in baseline hemodynamics were observed between experimental groups (table 1). Isoflurane significantly (P < 0.05) decreased mean arterial pressure and rate–pressure product in the presence or absence of 5-HD. Hemodynamics returned to baseline values 15 min after isoflurane had been discontinued. Coronary artery occlusion and reperfusion produced similar decreases in mean arterial pressure and rate–pressure product in each experimental group.

Body weight, LV weight, AAR weight, and AAR–LV were similar between groups (table 2). Isoflurane reduced myocardial infarct size (19 ± 3% of the LV AAR; n = 8;fig. 3) as compared to control (38 ± 4%; n = 8). 5-HD alone did not affect infarct size (42 ± 3%; n = 7). 5-HD blocked the protective effects of isoflurane when administered before (37 ± 4%; n = 8) but not after (24 ± 3%; n = 8) administration of the volatile anesthetic.

Fifty-six rabbits were instrumented for ROS detection. Isoflurane produced similar hemodynamic effects (table 3) in the presence or absence of 2-MPG, N -acetylcysteine, or 5-HD and in the presence of dihydroethidium. Ethidium–DNA fluorescence was detected in all of the images examined (fig. 4). Rabbits exposed to 1.0 MAC isoflurane demonstrated enhanced fluorescence in myocardial nuclei as compared to the cytosol. The fluorescence intensity was greater (35 ± 5 vs.  2 ± 3 fluorescence units) in the rabbits that received isoflurane (n = 8) as compared to those that did not (n = 9;fig. 5). N -acetylcysteine and 2-MPG alone did not affect the fluorescence (11 ± 4 and 10 ± 3 fluorescence units, respectively; n = 5 for both groups), but these ROS scavengers blocked isoflurane-induced increases in fluorescence (12 ± 5 and 13 ± 4 fluorescence units, respectively; n = 6 for both groups;fig. 5). 5-HD alone did not affect fluorescence (8 ± 3 fluorescence units; n = 5). When 5-HD was administered before exposure to isoflurane, this mitochondrial K^ATPchannel antagonist inhibited increases in fluorescence produced by isoflurane (9 ± 2 fluorescence units; n = 6;fig. 6). In contrast, isoflurane-induced increases in fluorescence were partially preserved (20 ± 3 fluorescence units; n = 6) when 5-HD was administered after exposure to the volatile anesthetic.

It is clear that K^ATPchannels play a central role in anesthetic-induced preconditioning, 6–9but the mechanisms by which volatile agents produce myocardial protection through the opening of these channels are unkown. 19Isoflurane activates mitochondrial K^ATPchannels as measured by flavoprotein oxidation, 16and ROS generated by this agent have been implicated in the mechanism of anesthetic-induced preconditioning. 10–12Controversy exists regarding the temporal relationship between mitochondrial K^ATPchannel opening and ROS production in ischemic or pharmacologic preconditioning. 20Similarly, it remains unclear whether isoflurane-induced opening of the mitochondrial K^ATPchannel precedes or follows ROS generation. The present results indicate that 5-HD administered 10 min before exposure to isoflurane blocked myocardial protection, but 5-HD administered immediately after this agent had been discontinued did not affect infarct size. These data suggest that mitochondrial K^ATPchannel opening acts as a trigger of isoflurane-induced preconditioning in rabbits.

The role of K^ATPchannels as triggers or distal effectors of ischemic or pharmacologic preconditioning is controversial. 20The present results are similar to previous findings indicating that mitochondrial K^ATPchannel opening acts as a trigger for protection in ischemic or diazoxide-induced preconditioning. 14,15,21Pain et al.  14demonstrated that 5-HD administered during but not after a brief ischemic stimulus or the selective mitochondrial K^ATPchannel opener diazoxide blocked reductions in the extent of myocardial infarction. Diazoxide increased oxidation of the ROS probe MitoTracker® orange (Molecular Probes, Inc., Eugene, OR) in vitro , an action that was attenuated by pretreatment with 5-HD or the ROS scavengers N -acetylcysteine and 2-MPG. 15Diazoxide also directly increased ROS production as measured by the hydrogen peroxide probe 2′,7′-dichlorofluorescein diacetate 21in rat ventricular myocytes and isolated hearts. Two other mitochondrial K^ATPchannel openers, cromakalin and nicorandil, increased ROS production in the isolated rat heart, and 5-HD pretreatment blocked the increase in ROS. 22In contrast to these data suggesting that the opening of the mitochondrial K^ATPchannel is a trigger and not an end effector of preconditioning, Fryer et al.  23demonstrated that ischemic preconditioning was blocked by 5-HD whether this drug was administered before or after preconditioning stimuli. Similarly, diazoxide-induced protection was completely abolished when 5-HD was administered either 5 min before or after the mitochondrial K^ATPchannel opener in the rat. 24These findings suggest that mitochondrial K^ATPchannels function both as triggers and end effectors of preconditioning. 25 

Isoflurane opens K^ATPchannels to exert cardioprotective actions. 6–9In the current investigation, 5-HD was administered immediately after exposure to isoflurane in one experimental group, but this method may have been insufficient to completely block the mitochondrial K^ATPchannel opening by isoflurane during the subsequent ischemic insult. In contrast, pharmacologic preconditioning with diazoxide was blocked by the same dose of 5-HD (5 mg/kg intravenously), administered at a similar time as in the late–5-HD group in the current study, in an identical rabbit model of myocardial infarction in vivo . 26These findings support the contention that the pharmacokinetics of 5-HD administration alone do not explain our results and further support our conclusion that mitochondrial K^ATPchannel opening is a trigger for isoflurane-induced preconditioning.

Previous studies have demonstrated that 5-HD inhibits diazoxide-induced, 15,21acetylcholine-induced, 27and opioid-induced 28production of ROS that are associated with myocardial protection. The current findings support these previous data and indicate that 5-HD administered 10 min before exposure to isoflurane abolishes increases in ethidium–DNA fluorescence produced by the volatile anesthetic. Taken together with the results of the infarct size experiments, these results suggest that isoflurane first activates mitochondrial K^ATPchannels and then elicits the release of ROS. Recent evidence indicates that mitochondrial K^ATPchannel opening in response to diazoxide activates the extracellular signal-regulated kinase by an oxidant-dependent mechanism in monocytes. 29Moreover, ROS activate p38 mitogen-activated protein kinase and protein kinase C in cardiac myocytes 30and in isolated guinea pig hearts, 31respectively. These findings suggest that ROS production secondary to mitochondrial K^ATPchannel opening may be linked to subsequent activation of kinases implicated in the signal transduction responsible for protection against ischemic injury. However, the current investigation did not examine the downstream activation of these protein kinases in response to ROS generation during isoflurane-induced preconditioning. These objectives represent important goals of future research.

We previously reported that isoflurane generates ROS in rabbit myocardium in vivo  using ethidium–DNA fluorescence detected by laser confocal microscopy. 11The current results confirm and extend these previous findings 11and indicate that the ROS scavengers N -acetylcysteine and 2-MPG inhibit isoflurane-induced increases in fluorescence intensity at doses that also block reductions in myocardial infarct size produced by the volatile agent. N -acetylcysteine is a sulfhydryl-containing glutathione precursor that exerts antioxidant effects by contributing to glutathione synthesis, by serving as a glutathione peroxidase substrate, and by directly scavenging several oxygen-derived free radical species, primarily by the actions of reduced glutathione. 322-MPG also acts as a sulfhydryl donor to glutathione peroxidase, and several studies indicate that 2-MPG may be more specific for mitochondrial activity than N -acetylcysteine. 33–35The present results verify that isoflurane produces ROS, as detected by ethidium–DNA fluorescence, because these ROS are effectively scavenged, and fluorescence is abolished by N -acetylcysteine and 2-MPG.

The present results should be interpreted within the constraints of several potential limitations. The LV AAR for infarction and coronary collateral blood flow are important determinants of infarct size. The AAR was similar between experimental groups, and minimal coronary collateral blood flow has been reported previously in rabbits. 36Thus, it is unlikely that the current results were affected by the size of the AAR or the magnitude of coronary collateral perfusion. Isoflurane caused similar systemic hemodynamic effects in the presence or absence of 5-HD, and there were no differences in hemodynamics between groups after the volatile agent had been discontinued. Thus, the present results were independent of many of the hemodynamic determinants of myocardial oxygen consumption during administration of isoflurane or 5-HD. The rate–pressure product, an indirect index of myocardial oxygen consumption, was also similar between experimental groups. Experiments using dihydroethidium as an indicator of superoxide anion production may underestimate the rate of superoxide anion production because this ROS probe may catalyze the dismutation of superoxide anion. 37Cytochrome c  may also oxidize dihydroethidium. 37Increases in mitochondrial membrane permeability cause the release of several proteins, including cytochrome c , caspase precursors, adenylate kinase 2, and apoptosis-inducing factor, into the cytosol of apoptotic cells. 38These factors activate caspases that are known factors implicated in apoptosis. 39Nevertheless, it appears unlikely that cytochrome c  was released into the cytosol because the rabbits were not subjected to ischemia and reperfusion in the dihydroethidium experiments. Recent evidence suggests that pharmacologic agonists and antagonists of the mitochondrial K^ATPchannel, such as diazoxide and 5-HD, respectively, may have K^ATPchannel-independent actions on the mitochondrial electron transport chain. 40These findings increase the intriguing possibility that volatile anesthetics may also interact with components of the electron transport chain to induce a preconditioning effect. Possible direct effects of volatile anesthetics on electron transport chain complexes will require further investigation.

In summary, the present results indicate that isoflurane-induced myocardial protection requires activation of mitochondrial K^ATPchannels and generation of ROS. The findings suggest that mitochondrial K^ATPchannel opening acts as a trigger in an intracellular signaling pathway responsible for isoflurane-induced preconditioning by generating ROS in vivo .

The authors thank David Schwabe, B.S.E.E. (Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin), for technical assistance and Mary Lorence-Hanke, A.A. (Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin), for assistance in the preparation of the manuscript.