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Anesthesiology Research Laboratories, Departments of *Anesthesiology and
Physiology, and
Cardiovascular Research Center, The Medical College of Wisconsin, Milwaukee, Wisconsin;
Department of Biomedical Engineering, Marquette University, Milwaukee, Wisconsin; and
||Research Service, Veterans Affairs Medical Center, Milwaukee, Wisconsin
Address correspondence and reprint requests to David F. Stowe, MD, PhD, M4280, 8701 Watertown Plank Rd., Medical College of Wisconsin, Milwaukee, WI 53226. Address e-mail to dfstowe{at}mcw.edu
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IMPLICATIONS: Reactive oxygen species (ROS) are implicated in triggering anesthetic preconditioning (APC). The ROS superoxide (O2·-) was measured continuously in guinea pig isolated hearts. Sevoflurane directly increased O2·- but led to attenuated O2·- formation during ischemia. This demonstrates triggering of APC by ROS and clarifies the mechanism of cardioprotection during ischemia.
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Introduction |
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There is now evidence that significant ROS formation occurs during ischemia before reperfusion (6–8) . The mitochondrial electron transport chain is believed to be the principle source of ROS during ischemia in cardiac cells (7) and may also be the source of the postulated ROS formation during anesthetic exposure, because anesthetics are known to alter several indices of electron transport chain function (4,9,10) . We have recently shown in intact hearts that APC leads to more normalized nicotinamide-adenine dinucleotide (NADH) (4) and attenuated mitochondrial Ca2+ loading (5) during isch-emia and that APC leads to reduced ROS release from intact hearts (3) and to less ROS formation in isolated mitochondria during reperfusion (11). These findings suggest that improved mitochondrial bioenergetics during ischemia or reperfusion is a feature of APC and is likely to underlie the global functional and structural preservation. Suppressed electron transport chain activity (i.e., more normalized NADH) (4) during ischemia would lead to decreased superoxide (O2·-) formation, the principle progenitor ROS formed by mitochondrial enzyme complexes. Thus, a volatile anesthetic may paradoxically stimulate O2·- formation, leading to activation of protective mechanisms that ultimately lead to decreased O2·- formation by the mitochondrion during ischemia.
There is evidence to link ROS with several mediators implicated in preconditioning, particularly the mitochondrial adenosine triphosphate-sensitive potassium (mKATP) channel (12). There is conflicting evidence, however, on the relative sequence of ROS formation and mKATP opening (12,13) . We and others have shown that the cardioprotective effects of APC can be inhibited by either ROS scavengers (2,3) or mKATP channel blockers (5,14–16) . In this study, we hypothesized first that O2·- is generated during anesthetic exposure and that this is not inhibited by mKATP channel blockade, indicating an initiating role for ROS and a downstream role for the mKATP channel in APC. We hypothesized second that the O2·- generation then leads to preservation of electron transport chain function via mKATP opening during subsequent ischemia, i.e., manifested by reduced O2·- not only during reperfusion, but also during ischemia. To examine these hypotheses, we measured O2·- formation in real time in isolated guinea pig hearts by spectrofluorometry via a probe placed at the left ventricular free wall and the fluorescent probe dihydroethidium (DHE).
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Diastolic and systolic left ventricular pressure (LVP) were measured isovolumetrically by using a transducer connected to a saline-filled latex balloon placed in the left ventricle. Balloon volume was adjusted to a diastolic LVP of 0 mm Hg during the initial equilibration period. Coronary inflow (CF) was measured by an ultrasonic flowmeter (Transonic T106X; Transonic, Ithaca, NY). Atrial and ventricular bipolar leads were used to measure spontaneous heart rate. CF and coronary venous Na+, K+, Ca2+, PO2, pH, and PCO2 were measured off-line with an intermittently self-calibrating analyzer (Radiometer ABL 505; Radiometer, Copenhagen, Denmark). Coronary sinus PO2 tension (PvO2) was also measured continuously on-line with a Clark electrode (Model 203B; Instech, Plymouth Meeting, PA). Myocardial oxygen consumption was calculated as CF/heart weight (g) · (PaO2 - PvO2) · 24 mL/µL of oxygen at 760 mm Hg.
Sevoflurane was bubbled into the perfusate with an agent-specific vaporizer. Sevoflurane concentrations in KR were measured by gas chromatography from samples taken anaerobically from the inflow line just proximal to the flowprobe. Global ischemia was achieved by clamping the inflow line. If ventricular fibrillation occurred on reperfusion, a bolus of lidocaine (250 µg) was given via a side port of the aortic inflow cannula. After 120 min of reperfusion, hearts were removed, and the ventricles were cut into six transverse sections and stained with 1% 2,3,5-triphenyltetrazolium chloride in 0.1 M KH2PO4 buffer (pH 7.4; 38°C). Tissue was stored in 10% formaldehyde for 48 h before dissection in a blinded fashion. Infarct size was expressed as a percentage of total ventricular weight (3). The average total ventricular weight was 1.7 ± 0.1 g (mean ± SEM), with no differences among groups.
We have previously described in detail our application of fluorescence techniques in intact hearts for measurements of cytosolic Ca2+ (17), mitochondrial Ca2+ (5), Na+ (17), and NADH (4,16) . Briefly, the distal end of a trifurcated fiberoptic cable (optical surface area 3.85 mm2) was placed against the LV free wall through a hole in the tissue bath. The fiberoptic cable was connected to a modified spectrophotometer (SLM Aminco-Bowman II; Spectronic Instruments, Urbana, IL). The fluorescent dye DHE (Molecular Probes, Eugene, OR) was dissolved in dimethyl sulfoxide containing 16% (wt/vol) Pluronic I-127 (Sigma Chemical, St. Louis, MO) and made up in 300 mL of KR, for a final concentration of 10 µM DHE and 10-3 M dimethyl sulfoxide. DHE enters cells and, when oxidized by ROS with a relative selectivity for O2·- (7), is converted to ethidium (ETH); ETH intercalates with DNA, causing the nucleus to exhibit red fluorescence. The fluorescent intensity (FI) of ETH (ETH FI) was measured in a light-blocking Faraday cage at an emission wavelength of 590 nm (bandwidth, 4 nm), amplified by a photomultiplier tube (700 V), and recorded after excitation with a 150 W xenon arc lamp filtered at 540 nm (bandwidth, 4 nm). The excitation wavelength penetrates the whole 4 mm of the ventricular wall.
In preliminary experiments (n = 4), background changes in FI (DHE vehicle) were determined for each experimental protocol. 5-Hydroxydecanoate (5-HD) and manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP; OxisResearch, Portland, OR) had no effect on background fluorescence, and ischemia/reperfusion had a minimal (nonsignificant) effect (<5% of the value obtained with DHE). All subsequent recorded values of ETH FI were adjusted for the minimal change in background fluorescence obtained during ischemia and reperfusion. Hearts were loaded with DHE in KR for 25 min, followed by washout of residual DHE with standard KR for 15 min. Loading of DHE was found to increase diastolic LVP approximately 8% and to increase CF approximately 10%; the effect on diastolic LVP was partly (but incompletely) reversed by washout, and CF returned to baseline values. DHE loading increased FI from 0.04 ± 0.01 (mean ± SEM) before loading to 2.1 ± 0.3 arbitrary units after washout. Each washout value was adjusted to 0 arbitrary units for normalizing FI values for all experiments.
In preliminary experiments (n = 8), the specificity of ETH FI for O2·- and the effects of possible sources of artifact, including movement-induced changes in LVP, pH, and flow, were studied. A rate increase to 375 bpm by isoproterenol and a decrease to 125 bpm by labetalol did not affect ETH FI, nor did pH in the range 6.2–8.0 or mechanically altered flow between 5 and 20 mL · g-1 · min-1. Brief pharmacologic arrest induced with adenosine had no effect on FI. Endogenous generation of O2·- by using the mitochondrial electron transport chain inhibitors rotenone (10 µM) and antimycin A (10 µM) was found to increase ETH FI by 21% and 82% from baseline, respectively. The administration of H2O2 (10–100 µM) did not affect ETH FI, whereas H2O2 caused significant dose-dependent increases (3%–36%) in the FI of dichorohydro-fluoroscein, which has a higher specificity for H2O2 and hydroxyl radical (OH·-) than for O2·-.
There were 6 experimental groups (n = 8 per group) and a time-control group (n = 6). Each experiment lasted 190 min after a 30-min equilibration period, a 25-min DHE-loading period, and a 15-min washout period. The ischemia control group (ISC) underwent only 30 min of ischemia and 120 min of reperfusion. Cardiac preconditioning consisted of two 5-min pulses of sevoflurane with an intervening 5-min perfusion period with KR and a 20-min perfusion period before ischemia (APC). The inflow sevoflurane concentration was 0.52 ± 0.02 mM (mean ± SEM) (or 3.6%) at 37°C (there was no difference among groups exposed to sevoflurane). In the APC plus MnTBAP and APC plus 5-HD groups, 20 µM of the ROS scavenger MnTBAP or 200 µM of the putative mKATP channel blocker 5-HD was given from 5 min before the first preconditioning pulse, during the intervening washout period, and for 5 min after the conclusion of the second APC pulse before ischemia. In the MnTBAP and 5-HD groups, either drug was given continuously for 25 min without sevoflurane, followed by a 15-min washout before ischemia. ETH FI was sampled for 100 ms during each recording. This was repeated every 12 s throughout the entire experimental protocol, for a total fluorescence recording time of 95 s. In four additional hearts, the effect of 0.82 ± 0.03 mM (mean ± SEM) sevoflurane (5.7%) on ETH FI was evaluated in the absence and presence of 20 µM MnTBAP or 200 µM 5-HD.
All data are expressed as mean ± SE. Within-group data were compared with those of a time-control group at the following time points: baseline (0 min), during and after the preconditioning stimuli, during ischemia at 5-min intervals, during reperfusion at 5-min intervals to 15 min of reperfusion, and at 60 and 120 min of reperfusion. Among-group data were compared at the same time points. Comparisons were made by using analysis of variance with the Student-Newman-Keuls test as the post hoc test (Prism Version 3.0a; GraphPad Software Inc, San Diego, CA). Differences among means were considered statistically significant at P < 0.05 (two tailed).
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Discussion |
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Intracellular oxygen decreases during ischemia, but absolute anoxia does not occur in the intact heart (18). Decreases in substrate gradients and electron transport chain dysfunction during ischemia lead to O2·- production from residual oxygen by enzyme complexes of the electron transport chain (7,8) . Prior evidence of ROS formation during ischemia included the finding of oxidant injury in cardiac tissues subject to ischemia without reperfusion (19). More recently, ROS formation during ischemia has been demonstrated directly in cardiomyocytes (7,8) and in intact animals (6). Most evidence points to the mitochondrion as the prime source of ROS within myocytes (7,20) , although other sources are possible, including endothelial xanthine oxidase and macrophages. We have previously shown that APC decreases the reperfusion release of dityrosine, a marker of oxidization by ROS and reactive nitrogen species (3). This study extends these findings by identifying O2·- as the initial ROS modulated by APC and by showing that significant alterations to O2·- production occur during ischemia, as well as during reperfusion.
Abrogation of APC-induced infarct-size reduction by ROS scavengers has been demonstrated previously (2,3) . This implies an effect of anesthetics to generate ROS, as directly demonstrated here and as previously suggested on the basis of indirect measures in blood vessels (21,22) . Because of the limits of our detection system for ETH fluorescence, we had to use a larger concentration of sevoflurane to demonstrate an increase in the fluorescent signal produced by sevo- flurane. However, the smaller concentration was sufficient to induce preconditioning, and we have shown previously that a larger concentration similarly induces APC (5). The relative quantity of intracellular ROS formed in response to anesthetic exposure at the larger dose was <10% of that during initial ischemia; at the smaller concentration used to initiate APC, it was probably proportionately smaller and outside our detection limit. Although we cannot exclude the possibility, on the basis of our direct measurements, that O2·- generation did not occur at the smaller concentration of sevoflurane, at this smaller concentration the O2·- scavenger MnTBAP was found to inhibit preconditioning, strongly suggesting the presence of O2·- and confirming its triggering role.
It is not known how anesthetics generate ROS, although reported effects of anesthetics on indices of mitochondrial electron transport chain function (4,9,10) strongly implicate the electron transport chain as the site of action. It was suggested more than 25 years ago that volatile anesthetics might interfere with NADH oxidoreductase activity (10), and subsequently NADH was shown to increase in response to volatile anesthetics in rat hearts (23). Nonetheless, our measurements do not exclude the possibility of alternative mechanisms of ROS formation; for example, radical intermediates of halothane are produced by hepatocytes (24). A similar reaction in myocytes with sevoflurane appears unlikely to us, however.
Several reports demonstrate the effect of ROS to induce preconditioning (2,3,8) , but it is not known how ROS, generated by anesthetics or by other stimuli, lead to preconditioning. ROS are protean cellular messengers in the heart, and there is evidence to link ROS with several mediators implicated in preconditioning, including the mKATP channel (12), protein kinase C (12), and others. Our results with the putative mKATP channel blocker 5-HD suggest that this channel is not required for O2·- generation in response to anesthetic exposure, because ETH FI during sevoflurane exposure was unaffected by concomitant administration of 5-HD. Attenuation of O2·- formation during subsequent ischemia by APC may require opening of this channel, however, because ETH FI during ischemia and reperfusion in the APC plus 5-HD group was similar to that in the ISC group. This is consistent with previous work that suggested that the preconditioning pathway involves the opening of the mKATP channel in response to ROS (12). Alternatively, it has been suggested that the converse occurs, i.e., that mKATP channel opening leads to the formation of ROS (13). The opening of the mKATP channel has been proposed to optimize mitochondrial bioenergetics by causing partial dissipation of the mitochondrial membrane potential and subsequent swelling of the mitochondrial matrix (25). Nonetheless, it is important to note that non-mKATP channel effects of 5-HD have been reported recently (26), and further work will be required to delineate the role of mKATP channels in preconditioning.
Limitations of our measure of O2·- are that an exact calibration technique is unavailable and that we cannot determine the specific reactive species that triggers preconditioning. It is possible that downstream products of O2·-, such as H2O2 or OH·, are the specific trigger of the pathway that leads to APC. Indeed, there is evidence in cardiomyocytes that dismutation of O2·- is a necessary step for preconditioning (8). Moreover, OH· has been reported to decrease ETH FI (7) and could have contributed to the decreased ETH FI that we observed during early reperfusion, as similarly reported by Becker et al. (7) during reoxygenation of anoxic myocytes.
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Summary |
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Acknowledgments |
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The authors thank the following for their contributions to this study: James Heisner, Dr. Ming Tao Jiang, and Anita Tredeau. Portions of this work have appeared in abstract form (Kevin LG, Novalija E, Camara AK, et al. Formation of reactive oxygen species during ischemic and anesthetic preconditioning in isolated hearts. Anesthesiology 2002;96:A80).
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