Recent evidence indicates that volatile anesthetics exert protective effects during myocardial ischemia and reperfusion. The authors tested the hypothesis that sevoflurane decreases myocardial infarct size by activating adenosine triphosphate-sensitive potassium (K(ATP)) channels and reduces the time threshold of ischemic preconditioning necessary to protect against infarction.
Barbiturate-anesthetized dogs (n = 75) were instrumented for measurement of aortic and left ventricular pressures and maximum rate of increase of left ventricular pressure and were subjected to a 60-min left anterior descending (LAD) coronary artery occlusion followed by 3-h reperfusion. In four separate groups, dogs received vehicle or the K(ATP) channel antagonist glyburide (0.1 mg/kg intravenously), and 1 minimum alveolar concentration sevoflurane (administered until immediately before coronary artery occlusion) in the presence or absence of glyburide. In three additional experimental groups, sevoflurane was discontinued 30 min (memory) before the 60-min LAD occlusion or a 2-min LAD occlusion as an ischemic preconditioning stimulus was used with or without subsequent sevoflurane (with memory) pretreatment. Regional myocardial perfusion and infarct size were measured with radioactive microspheres and triphenyltetrazolium staining, respectively.
Vehicle (23 +/- 1% of the area at risk; mean +/- SEM) and glyburide (23 +/- 2%) alone produced equivalent effects on myocardial infarct size. Sevoflurane significantly (P < 0.05) decreased infarct size (13 +/- 2%). This beneficial effect was abolished by glyburide (21 +/- 3%). Neither the 2-min LAD occlusion nor sevoflurane followed by 30 min of memory were protective alone, but together, sevoflurane enhanced the effects of the brief ischemic stimulus and profoundly reduced infarct size (9 +/- 2%).
Sevoflurane reduces myocardial infarct size by activating K(ATP) channels and reduces the time threshold for ischemic preconditioning independent of hemodynamic effects in vivo.
PRECONDITIONING with single or multiple episodes of brief ischemia and reperfusion renders myocardium temporarily resistant to subsequent prolonged ischemia. 1The precise underlying mechanism(s) of ischemic preconditioning (IPC) has been the focus of intense investigation. Accumulating evidence indicates that adenosine triphosphate–sensitive potassium (KATP) channels, 2,3adenosine A1receptors, 4and protein kinase C 5–7are involved in IPC signal transduction. Notably, a threshold 8level of the ischemic stimulus must be present for IPC to occur, and combination of two independent subthreshold stimuli may exert protective effects. 9Recent evidence also suggests that KATPchannels and A1receptors modulate an acute memory phase characterized by increased tolerance to a subsequent prolonged coronary artery occlusion after a brief ischemic episode. 10
Previous investigations from this 11–14and other laboratories 15,16have demonstrated that volatile anesthetics, including isoflurane and halothane, exert cardioprotective effects during ischemia and reperfusion. The mechanisms for anesthetic-induced myocardial protection are similar to those observed during IPC and are mediated by KATPchannels, 13A1receptors, 14and protein kinase C. 15Like IPC, isoflurane has also been shown to produce an acute memory phase with a duration of at least 30 min. 13Whether the new volatile anesthetic sevoflurane exerts similar protective effects during myocardial ischemia and reperfusion is unknown. The present study tested the hypothesis that sevoflurane reduces experimental myocardial infarct size by activation of KATPchannels. Additionally, it is unknown whether volatile anesthetics affect the time threshold for IPC, allowing a stimulus of shorter duration to provide cardioprotection. KATPchannel activation has been shown to decrease this time threshold. 9Thus, the present investigation also tested the hypothesis that sevoflurane reduces the time threshold of IPC in vivo .
Methods and Materials
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. All procedures were in conformity with the Guiding Principles in the Care and Use of Animals 17of the American Physiologic Society and were performed in accordance with the Guide for the Care and Use of Laboratory Animals . 18
The experimental methods have been previously described in detail. 12Briefly, mongrel dogs of either sex (26 ± 1 kg; mean ± SEM) were fasted overnight, anesthetized with sodium barbital (200 mg/kg) and sodium pentobarbital (15 mg/kg), and ventilated with an air and oxygen mixture (fraction of inspired oxygen = 0.25) after tracheal intubation. Tidal volume and respiratory rate were adjusted to maintain arterial blood gas tensions within a physiologic range (p H, 7.39–7.44; carbon dioxide partial pressure, 28–35 mmHg; oxygen partial pressure, 140–210 mmHg). After calibration, a dual pressure transducer–tipped catheter was inserted into the aorta and left ventricle (LV)via the left carotid artery to measure aortic and LV pressures, respectively. The maximum rate of increase of LV pressure (+dP/dtmax) was obtained by electronic differentiation of the LV pressure waveform. The femoral vein and artery were cannulated for fluid administration and for the withdrawal of reference blood flow samples used in the calculation of regional myocardial blood flow, respectively. A thoracotomy was performed at the left fifth intercostal space. A heparin-filled catheter was inserted into the left atrial appendage for administration of radioactive microspheres. A 1.0-cm segment of the left anterior descending (LAD) coronary artery was dissected immediately distal to the first diagonal branch, and a silk ligature was positioned around the vessel to permit episodic coronary artery occlusion. Regional myocardial perfusion was measured in the ischemic (LAD) and normally (left circumflex coronary artery) perfused regions using the radioactive microsphere technique. 12Myocardial infarct size was determined with triphenyltetrazolium chloride staining at the completion of each experiment. 19End-tidal concentrations of sevoflurane were measured at the tip of the endotracheal tube by an infrared anesthetic gas analyzer that was calibrated with known standards before and during experimentation. The canine minimum alveolar concentration (MAC) value of sevoflurane used in the present investigation was 2.36%. 20Hemodynamic data were continuously monitored throughout the experiment, recorded with a polygraph, and digitized using a computer interfaced with an analog-to-digital converter.
The experimental design used in the present investigation is illustrated in figure 1. Dogs were randomly assigned to one of seven experimental groups. All dogs underwent a 60-min LAD occlusion followed by 3 h of reperfusion. Ninety minutes after completion of the surgical preparation and in four groups of experiments, dogs received either intravenous drug vehicle (50% polyethylene glycol and 50% ethyl alcohol) or the KATPchannel antagonist glyburide (0.1 mg/kg infusion over 10 min) in the presence and absence of a 30-min administration of 1 MAC end-tidal concentration sevoflurane. These experiments were designed to test the hypothesis that sevoflurane reduces myocardial infarct size by KATPchannel activation. To determine whether the use of sevoflurane is associated with an acute memory phase, another group of dogs was pretreated with 1 MAC sevoflurane for 30 min, which was then discontinued 30 min before the prolonged LAD occlusion. Finally, to test the hypothesis that sevoflurane reduces the time threshold of IPC, two additional groups of dogs were subjected to a brief (2-min) LAD occlusion 1 h before the prolonged LAD occlusion with or without a subsequent 30-min exposure to 1 MAC sevoflurane and 30 min of memory.
Statistical analysis of data within and between groups was performed with multiple analysis of variance for repeated measures followed by application of the Student t test with Duncan's correction for multiplicity. Linear regression analysis was performed to determine the relationship between transmural collateral blood flow and infarct size expressed as a percentage of the area at risk for infarction. Changes within and between groups were considered statistically significant at P < 0.05. All data are expressed as mean ± SEM.
Seventy-five dogs were instrumented to provide 58 successful experiments. Sixteen dogs were excluded from analysis because transmural collateral blood flow exceeded 0.2 ml · min−1· g−1in the ischemic zone during LAD occlusion (two control, five sevoflurane alone, four sevoflurane and glyburide, two sevoflurane with memory, two IPC alone, and one IPC and sevoflurane). One dog in the group that received sevoflurane with memory was excluded because intractable ventricular fibrillation occurred during LAD occlusion.
There were no differences in systemic hemodynamics during baseline conditions between experimental groups (tables 1–7). Sevoflurane alone significantly (P < 0.05) decreased heart rate, mean arterial and LV systolic pressures, rate–pressure product, and LV +dP/dtmax(table 2). Glyburide produced no hemodynamic effects (table 3), and there were no hemodynamic differences in glyburide- compared with vehicle-pretreated control dogs. Sevoflurane produced similar hemodynamic effects in the presence (table 4) or absence (table 2) of glyburide. When sevoflurane was discontinued 30 min before prolonged LAD occlusion in acute memory phase experiments, heart rate and rate–pressure product remained decreased during the following 30-min memory period compared with baseline values (table 5). A 2-min IPC briefly reduced LV systolic pressure and increased end-diastolic pressure but did not alter heart rate, mean arterial pressure, and rate–pressure product (table 6). Sevoflurane produced similar effects in the presence (table 7) or absence (table 5) of a preceding IPC stimulus. Prolonged LAD occlusion caused immediate and sustained increases in LV end-diastolic pressure and decreases in +dP/dtmaxin all groups. There were no differences in hemodynamics during coronary artery occlusion between groups. No differences in heart rate, mean arterial pressure, and rate–pressure product were observed between groups during reperfusion after the 1-h LAD occlusion.
Regional Myocardial Perfusion
Transmural myocardial blood flow in the ischemic (LAD) and normal (left circumflex coronary artery) regions is summarized in table 8. LAD occlusion caused equivalent decreases in transmural perfusion to the ischemic (LAD) zone in each group, and there were no differences in collateral blood flow between groups. Blood flow to the LAD region increased 1 h after final reperfusion in all groups.
Myocardial Infarct Size
Sevoflurane administered until immediately before coronary artery occlusion significantly reduced myocardial infarct size to 13 ± 2% of the area at risk compared with control experiments (23 ± 1%;fig. 2). Although glyburide alone had no effect on infarct size (23 ± 2%), the protective effects of sevoflurane were abolished by pretreatment with glyburide (21 ± 3%). Discontinuation of sevoflurane 30 min before the LAD occlusion (sevoflurane with memory) or a 2-min preconditioning stimulus did not produce cardioprotection (21 ± 4% and 24 ± 2%, respectively;fig. 3). In contrast, combined sevoflurane pretreatment and a 2-min period of IPC together after 30 min of memory caused significant reductions in myocardial infarct size (9 ± 2%;figs. 3 and 4). The relationship between infarct size and transmural collateral blood flow during threshold of IPC experiments is shown in figure 4. IPC and sevoflurane followed by 30 min of memory reduced the extent of myocardial infarction at any given level of collateral blood flow and shifted the regression relation downward as compared with unprotected dogs (control, sevoflurane with memory, and IPC groups). The area at risk for myocardial infarction was similar between groups (control, 41 ± 2; sevoflurane, 40 ± 2; glyburide, 40 ± 2; sevoflurane plus glyburide, 46 ± 2; sevoflurane plus memory, 44 ± 2; IPC, 44 ± 2; IPC plus sevoflurane plus memory, 45 ± 1% of the LV).
Substantial experimental evidence suggests that KATPchannels play an important role in mediating the protective effects of IPC. 21–23Activation of these channels by pharmacologic agents mimics IPC 24and reduces the time threshold necessary to produce IPC in dogs. 9Consistent with this hypothesis, the KATPchannel antagonist glyburide 21,22abolishes IPC. Volatile anesthetics 13,15,16have been shown to reduce ischemia–reperfusion injury by activating KATPchannels and to produce an acute memory phase, 13characteristics that are also distinctive of IPC. The present results indicate that sevoflurane causes anesthetic-induced preconditioning, 16reducing myocardial infarct size by a KATPchannel–mediated mechanism. The present findings confirm and extend our previous results with isoflurane 13,25and support the contention that KATPchannel activation may represent an end-effector of a signal transduction pathway responsible for anesthetic-induced preconditioning. Further in vitro investigation is required to clarify whether sevoflurane or other volatile anesthetics directly stimulate KATPchannels, 26alter the ATP sensitivity of such channels, 27or trigger a signaling cascade that indirectly leads to KATPchannel activation.
Ischemic preconditioning with a 2-min LAD occlusion was not sufficient to reduce myocardial infarct size in the present investigation. These data confirm the majority of previous findings indicating that a threshold stimulus > 3 min is generally required for IPC to occur. Miura et al. 28demonstrated no protective effects of 2-min IPC in anesthetized rabbits. A similar lack of protection was shown for preconditioning periods of 2.5 and 3 min but not 5 min in dogs. 9,24,29In contrast, Ovize et al. 30demonstrated that a 2.5-min coronary artery occlusion reduced infarct size in dogs subjected to 60-min coronary artery occlusion and reperfusion; however, a substantially smaller area at risk for infarction (20% of the LV) was present in this study 30compared with the present study and other investigations. 9,24,29
The present investigation is the first to examine whether a volatile anesthetic is capable of lowering the time threshold for IPC. Neither the 2-min LAD occlusion alone nor administration of sevoflurane followed by a 30-min memory period afforded protection against infarction, but the combination of brief LAD occlusion and remote sevoflurane exposure caused a dramatic reduction of infarct size (9 ± 2% of the area at risk for infarction). Previous studies have demonstrated that although two individual subthreshold stimuli are insufficient to produce preconditioning when administered separately, their combination often provides a full protective effect. The KATPchannel opener bimakalim, 9dipyridamole, 28allosteric enhancement of A1receptor activity, 29and activation of bradykinin B2receptors 31have been shown to lower the time threshold required for IPC. Because the protective effects of volatile anesthetics have also been attributed to KATPchannels 12,27and A1receptors, 14,15these agents may have the capability to enhance a subthreshold stimulus and produce IPC as well.
Despite convincing evidence for synergistic actions of various stimuli of IPC, the precise underlying mechanisms remain unclear. Yao and Gross 9demonstrated the capability of enhanced activation of KATPchannels to sensitize the myocardium and lower the threshold for ischemia necessary to produce IPC. Miura et al. 28and Auchampach and Gross 32suggested that elevation of interstitial adenosine concentrations may enhance the effect of IPC. It is unknown if sevoflurane increased interstitial concentrations of adenosine in the present investigation. We have previously shown that isoflurane decreases interstitial adenosine concentration and blunts the increase in adenosine that occurs during a brief period of coronary artery occlusion. 14Despite decreases in interstitial adenosine concentrations during isoflurane, the cardioprotective effects of this anesthetic were attenuated by an A1receptor antagonist. 14Similarly to isoflurane, sevoflurane may have activated KATPchannels or A1receptors and thereby enhanced the insufficient stimulus of a 2-min LAD occlusion to produce IPC in the present investigation.
Discontinuation of sevoflurane 30 min before the prolonged LAD occlusion produced no subsequent protective effects, indicating that this volatile anesthetic does not cause an acute memory period. This finding contrasts with our previous results with isoflurane, where persistent cardioprotective actions were observed when this volatile agent was discontinued 30 min before the prolonged LAD occlusion. 13The cellular mechanisms responsible for the induction and maintenance of the acute memory period of IPC and isoflurane anesthesia are not completely understood; however, evidence suggests that pharmacologic activation of both KATPchannels and A1receptors is required for the acute memory phase of IPC to occur. 10,33We have shown that isoflurane-induced cardioprotection is mediated through both KATPchannels 12,13,25and A1receptors. 14Thus, isoflurane may contribute to a memory period by mechanisms similar to those observed during memory and IPC. Memory associated with isoflurane anesthesia may have also required the persistent presence of residual amounts of this agent in the myocardium that continued to open KATPchannels for at least 30 min after discontinuation. However, systemic hemodynamics returned to baseline values, and end-tidal isoflurane concentrations were undetectable 30 min after discontinuation of this agent. 13Nevertheless, it remains possible that the substantially lower blood-gas solubility of sevoflurane may have resulted in less residual drug in the myocardium than isoflurane after a 30 min washout period and may partially account for the absence of memory after exposure to sevoflurane.
The mechanism or extent of KATPchannel opening may be different between volatile agents. As discussed previously, volatile anesthetics may directly activate KATPchannels or trigger a series of intracellular events leading to KATPchannel opening as the final common pathway. However, the direct effects of volatile anesthetics on KATPchannel opening using patch clamp methodology are as yet unclear, 26,27and whether isoflurane and sevoflurane produce differential actions on intracellular signal transduction pathways, leading to KATPactivation, has yet to be examined. Sustained activity of protein kinase C may contribute to maintenance of the memory of IPC 29and may be observed during ischemia and reperfusion injury. Both isoflurane-34and halothane-induced 15myocardial protection may also be causally linked to enhanced protein kinase C activity to varying degrees. However, whether sevoflurane activates protein kinase C is unknown.
The present findings must be interpreted within the constraints of several possible limitations. Sevoflurane-induced decreases in heart rate and myocardial contractility during ischemia may have produced favorable alterations in myocardial oxygen supply–demand relations, thereby contributing to a reduction in infarct size. However, halothane has been shown to exert protective effects even during mechanical arrest produced by cardioplegia, 35indicating that preferential alterations in myocardial metabolism are not solely responsible for the antiischemic actions of this anesthetic. Glyburide also blocked the cardioprotection afforded by sevoflurane without affecting the hemodynamic actions of this anesthetic. Nevertheless, coronary venous oxygen tension was not measured and myocardial oxygen consumption was not determined in the present investigation, and favorable changes in myocardial metabolism during administration of sevoflurane cannot be completely excluded. We have recently shown that sevoflurane selectively dilates coronary collateral vessels independent of KATPchannels in a canine model of enhanced collateral development. 36Such a preferential increase in coronary collateral blood flow may have contributed to sevoflurane-mediated protection in the present investigation. However, transmural collateral perfusion during LAD occlusion was similar between experimental groups conducted in the present investigation in dogs with relatively poor coronary collateral vasculature, indicating that sevoflurane-induced coronary collateral dilation was not responsible for the beneficial effects of this agent on infarct size. Furthermore, sevoflurane reduced the threshold of IPC at any given level of collateral blood flow. Interpretation of the present findings should also be qualified because only a single end-tidal concentration of sevoflurane was used, and the possibility that higher sevoflurane concentrations may have produced more pronounced reductions in infarct size or a memory response also cannot be excluded. Finally, we were unable to specifically evaluate the role of sarcolemmal versus mitochondrial KATPchannels to mediate sevoflurane-induced cardioprotection because glyburide is a nonselective antagonist of these channels. 37,38It has been suggested that mitochondrial KATPchannels may be primarily responsible for the protective effects of IPC 39; however, the relative contribution of mitochondrial versus sarcolemmal KATPchannels during anesthetic-induced cardioprotection is currently unknown.
In summary, the present results indicate that sevoflurane reduces experimental myocardial infarct size by activating KATPchannels. Discontinuation of sevoflurane 30 min before a prolonged coronary artery occlusion affords no inherent myocardial protection but reduces the time required for a brief ischemic stimulus to exert cardioprotective effects.
The authors thank Todd Schmeling and David Schwabe for technical assistance, and Drs. Werner F. List and Helfried Metzler (Department of Anesthesiology and Intensive Care Medicine, University of Graz, Austria) for their gracious support.