Mechanisms of Cardioprotection by Volatile Anesthetics

Received from the Departments of Anesthesiology, Pharmacology and Toxicology, and Medicine (Division of Cardiovascular Diseases), Medical College of Wisconsin, Milwaukee, Wisconsin; the Clement J. Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin; and the Department of Biomedical Engineering, Marquette University, Milwaukee, Wisconsin.A RAPIDLY growing body of evidence indicates that volatile anesthetics protect myocardium against reversible and irreversible ischemic injury. Identifying the mechanisms by which volatile agents mediate these antiischemic actions is the subject of intense research. This objective has been difficult to accomplish because volatile anesthetics also profoundly affect cardiovascular function. Volatile agents reduce arterial and coronary perfusion pressure, cause dose-related depression of myocardial contractility, produce coronary vasodilation, affect electrophysiologic function, and modify autonomic nervous system activity to varying degrees. Therefore, the antiischemic effects of volatile anesthetics may be mediated, at least in part, by favorable alterations in myocardial oxygen supply-demand relations, preservation of energy-dependent cellular functions, and increased coronary blood flow. However, it seems unlikely that changes in myocardial metabolism and coronary perfusion caused by volatile anesthetics are solely responsible for protection against ischemic damage. Instead, several endogenous signal transduction pathways, acting through the adenosine triphosphate (ATP)-sensitive potassium (KATP) channel and involving the generation of reactive oxygen species (ROS), have been implicated in mediating the antiischemic actions of volatile anesthetics. The experimental and clinical findings documenting the phenomenon of volatile anesthetic preconditioning against ischemic injury of myocardium are evaluated. Recent findings in vitro and in vivo that seek to define the intracellular mechanisms responsible for these beneficial actions are also summarized.The antiischemic effects of volatile anesthetics were initially proposed more than 20 yr ago. Lowenstein et al. 1demonstrated that halothane reduced ST segment elevation in a canine model of brief coronary artery occlusion. These data were consistent with the hypothesis that exposure to halothane reduced acute ischemic injury. A subsequent study by this research group also demonstrated that halothane reduced myocardial infarct size when administered before prolonged coronary artery occlusion in dogs. 2Lactate production was decreased in the presence as compared with the absence of enflurane during demand-induced ischemia produced by a critical coronary artery stenosis and ventricular pacing when coronary perfusion pressure was maintained. 3These results suggested that myocardial metabolism may be improved by administration of a volatile agent during an ischemic episode independent of alterations in hemodynamics. The relative importance of these early findings was initially overshadowed 4by a series of reports published in the mid-1980s 5–8suggesting that isoflurane may be capable of producing an abnormal redistribution of coronary blood flow away from ischemic toward normal myocardium. 9–11This “coronary steal” phenomenon was attributed to the coronary vasodilating properties of isoflurane that are known to occur primarily in arterioles of less than 100 μm in diameter. 12Isoflurane was thought to be capable of directly producing myocardial ischemia in susceptible patients with “steal-prone” coronary artery anatomy under certain hemodynamic conditions 13in a fashion similar to that of potent coronary vasodilators (e.g. , adenosine, chromonar, dipyridamole).The implication that isoflurane might produce myocardial ischemia through such a steal mechanism was subsequently dispelled by several investigations conducted in animal models 14–17and humans with coronary artery disease. 18–20For example, isoflurane did not selectively redistribute blood flow away from the collateral-dependent region in a chronically instrumented canine model of multivessel coronary artery disease. 15In contrast, adenosine produced marked coronary steal by preferentially shunting blood flow away from collateral-dependent myocardium in this model. 15Other studies 14,16–20also suggested that isoflurane-induced hypotension may reduce myocardial perfusion, but true coronary steal did not occur when coronary perfusion pressure was maintained. Subsequent investigations with the newer volatile anesthetics sevoflurane 21,22and desflurane 23showed that these drugs also did not reduce or abnormally redistribute coronary collateral blood flow. Therefore, despite initial concerns, volatile anesthetics were subsequently shown to be relatively weak coronary vasodilators that are incapable of causing coronary steal under the vast majority of clinical conditions. 24Contrary to the hypothesis that the use of volatile anesthetics may be potentially deleterious in some patients with coronary artery disease, many laboratory and clinical investigations conducted since the resolution of the coronary steal controversy have convincingly shown that volatile anesthetics protect the heart against ischemia and reperfusion injury. In addition to previously cited studies suggesting that halothane 1,2and enflurane 3exerted antiischemic effects, halothane was also shown to preserve contractile function and ultrastructural integrity during cardioplegic arrest. 25This latter study was of considerable interest because these data indicated that halothane was capable of exerting a cardioprotective effect completely independent of improvements in myocardial oxygen supply-demand balance. In addition, halothane, 26enflurane, 26desflurane, 26–28and sevoflurane 26–31have been shown to reduce myocardial damage when administered during reperfusion after prolonged coronary occlusion or cardioplegic arrest. Another study showed that preservation of high-energy phosphate concentrations was coupled to the protective effects of enflurane. 32Isoflurane and desflurane did not depress but modestly enhanced left ventricular diastolic function during acute coronary occlusion in dogs. 33Halothane, 34–36enflurane, 34,37,38isoflurane, 34,35,37and sevoflurane 39were shown to improve the functional recovery of isolated hearts subjected to global ischemia and reperfusion. Halothane and isoflurane markedly augmented the recovery of regional contractile function of stunned myocardium in vivo . 40,41Halothane 2and isoflurane 42reduced myocardial infarct size in dogs, and this beneficial action was found to persist despite discontinuation of the volatile anesthetic before coronary artery occlusion. 42The myocardium acted as if it had “remembered” the previous exposure to the volatile agent. This phenomenon was termed anesthetic-induced preconditioning (APC) 43and was characterized by a short-term memory phase similar to that observed during ischemic preconditioning (IPC).Anesthetic-induced preconditioning has also been described in other animal species, including rats 44and rabbits. 43The efficacy of APC conferred by isoflurane to reduce infarct size has been shown to be dose dependent in rats, 44an animal model with minimal coronary collateral flow. 45High concentrations of isoflurane may also have greater efficacy to protect myocardium during conditions of low coronary collateral blood flow in dog myocardium. 46Similarly, isoflurane and sevoflurane dose-dependently preserved the viability of isolated cardiac myocytes during ischemia. 47Isoflurane has been shown to elicit cardioprotective effects after discontinuation for 15 min or 30 min before coronary artery occlusion in rats and rabbits 43,44or dogs, 42respectively. In contrast, sevoflurane did not exert antiischemic actions after a 30-min washout period. 48Taken together, these data suggest that the memory period associated with APC may differ between volatile anesthetics and among species. Interestingly, recent findings showed that isoflurane reduced myocardial damage when administered 24 h before coronary artery occlusion and reperfusion in rabbit hearts in vivo . 49Pretreatment with isoflurane also preserved endothelial and vascular smooth muscle cell viability 12–48 h after cytokine-induced injury. 50Therefore, volatile anesthetics also produce a late phase (i.e. , a second window) of myocardial protection similar to IPC. In addition, sevoflurane reduced the duration of a brief ischemic episode required to protect against infarction during IPC. 48Sevoflurane also enhanced cardioprotection when administered 24 h after an initial IPC stimulus. 51These important findings showed that administration of a volatile anesthetic combined with a brief ischemic event synergistically protects myocardium against subsequent damage as well.Additional data about the effects of volatile agents on the coronary circulation also stand in contrast with the conclusions implicated by the coronary steal hypothesis. These results indicated that volatile agents are certainly not deleterious to but may instead exert beneficial actions on coronary collateral perfusion to ischemic myocardium. Volatile anesthetics have been shown to produce coronary vasodilation by activating KATPchannels 39,52–55or by favorably affecting intracellular Ca2+homeostasis in vascular smooth muscle. 56Halothane attenuated reductions in coronary collateral perfusion associated with acute coronary occlusion and also improved the myocardial oxygen supply-demand relation in collateral-dependent myocardium. 57In addition, halothane reduced cyclical changes in coronary blood flow and prevented the development of platelet thrombi in the presence of a critical coronary artery stenosis. 58Sevoflurane increased collateral blood flow to ischemic myocardium when perfusion pressure was maintained. 21,59Sevoflurane also improved the functional recovery of coronary vascular reactivity and nitric oxide release in isolated hearts after global ischemia. 39Lastly, volatile anesthetics attenuated neutrophil and platelet aggregation 60and also inhibited cytokine-induced cell death 50,61after ischemia-reperfusion injury in vitro . The results of these studies collectively show the protection against ischemia and reperfusion injury may be at least partially based on favorable effects of volatile agents on coronary perfusion.The precise mechanisms responsible for volatile anesthetic-induced protection against ischemic injury remain unclear despite extensive study. Although it is clear that volatile anesthetics may indirectly improve myocardial oxygen supply-demand relations or enhance coronary collateral perfusion, it is equally clear that these actions are not entirely responsible for the antiischemic effects of these agents. This contention is emphasized by findings showing that volatile anesthetics conferred protection during cardioplegic arrest 25and during reperfusion, 26–30conditions in which myocardial oxygen supply-demand relations play little if any role. Similarly, isoflurane and sevoflurane increased the viability of isolated cardiac myocytes, 47and sevoflurane 62and desflurane 63improved contractility of isolated cardiac muscle exposed to simulated ischemia. These results were initially attributed to reductions in excessive intracellular Ca2+concentrations during ischemia and reperfusion 64produced by partial inhibition of Ca2+channel activity. 65–68However, this relatively generic Ca2+hypothesis did not address the precise mechanisms or provide deeper insight into the intracellular processes by which volatile anesthetics exert protective effects in the intact heart.The signal transduction pathways involved in APC bear striking similarity to those responsible for IPC. It is hypothesized that volatile anesthetics stimulate a trigger that initiates a cascade of events leading to activation of an end-effector that is responsible for resistance to injury. To date, adenosine type 1 (A1) receptors, 34,69,70protein kinase C (PKC), 34,71,72inhibitory guanine nucleotide binding (Gi) proteins, 73ROS, 74–76and mitochondrial and sarcolemmal KATP(mito KATPand sarc KATP, respectively) channels 42,77–79have been shown to mediate APC (fig. 1). KATPchannels are heteromultimeric complexes containing an inward-rectifying potassium (Kir) channel and a sulfonylurea receptor (SUR). 80Pharmacologic and recombinant techniques indicate that sarc KATPand mito KATPchannels 81,82are composed of the Kir6.2/SUR2A and Kir6.1/SUR1 isoforms, 83respectively. KATPchannel opening was initially implicated as the central end-effector during APC, 84similar to the findings during studies of the mechanisms responsible for IPC. 85,86Isoflurane and sevoflurane preserved myocardial viability in a cellular model of ischemia, and this protective effect was abolished by the selective mito KATPchannel antagonist 5-hydroxydecanoate (5-HD) but not the selective sarc KATPchannel antagonist HMR-1098. 47Isoflurane, 69sevoflurane, 62and desflurane 63but not halothane 69enhanced the recovery of contractile force of isolated human right atrial trabeculae after hypoxia and reoxygenation. The nonselective KATPchannel blocker glyburide (glibenclamide) or 5-HD inhibited this protective effect. HMR-1098 also attenuated the beneficial actions produced by sevoflurane in isolated human atria. 62Glyburide blocked the enhanced recovery of contractile function produced by isoflurane in stunned myocardium in vivo . 41,87Reductions in canine myocardial infarct size produced by isoflurane 42and the ATP-sparing effects of this agent 88have been shown to be blocked by glyburide as well. 5-HD also inhibited preconditioning by isoflurane in rats 44and rabbits. 77Both 5-HD and HMR-1098 abolished the protective effects of desflurane against ischemia and reperfusion injury in dogs, 78supporting a role for both mito KATPand sarc KATPchannels in APC. In contrast, another study showed that HMR-1098 did not modify desflurane-induced preconditioning in isolated human right atria in vitro . 63Therefore, some controversy continues to exist about the relative contribution of sarc KATPand mito KATPchannels in APC.Carefully conducted in vitro experiments suggest that volatile anesthetics are capable of modifying KATPchannel activity. Isoflurane stimulated outward K+current through sarc KATPchannels in isolated ventricular myocytes during patch clamping. 89,90Volatile anesthetics also reduced sarc KATPchannel sensitivity to inhibition by ATP, thereby increasing open state probability. 91In contrast, other patch clamp results suggested that volatile agents alone did not open KATPchannels. Isoflurane did not affect sarc KATPchannel current in human atrial cells 69and also inhibited sarc KATPchannel activity in rabbit ventricular myocytes. 91However, some volatile anesthetics were able to enhance sarc KATPchannel current by facilitating channel opening after initial activation. 89,90Isoflurane enhanced sarc KATPchannel opening in the presence of the mitochondrial uncoupler 2,4-dinitrophenol, the KATPchannel opener pinacidil, and the protein tyrosine kinase (PTK) inhibitor genistein in a whole cell patch clamp model. 89,92Activation of PKC, 90adenosine receptors, 93and phosphatidylinositol kinase 93seemed to be necessary for this process to occur. Isoflurane also directly opened sarc KATPchannels during intracellular acidosis, a condition that is known to occur during ischemia. 94These data suggest that volatile anesthetics may not directly interact with sarc KATPchannels but instead may affect other signaling elements that modulate sarc KATPchannel activity. In contrast with the findings with isoflurane, halothane had no effect on pinacidil-induced increases in sarc KATPchannel current and even inhibited KATPchannel current that had been maximally activated by 2,4-dinitrophenol. 89The anesthetic specificity for APC remains to be well characterized, although studies such as these do suggest important differences in efficacy may exist among individual agents.The ability of volatile anesthetics to directly open mito KATPchannels has also been examined. Isoflurane and sevoflurane increased mitochondrial flavoprotein oxidation, an index of mito KATPchannel activity, in guinea pig cardiac myocytes. 95This process was inhibited by 5-HD. 95Flavoprotein fluorescence may not be entirely specific for mito KATPchannel opening, 96but isoflurane has also been shown to directly activate mito KATPchannels reconstituted in lipid bilayers. 97In contrast with these intriguing findings, 97Zaugg et al. 47recently demonstrated that although isoflurane or sevoflurane did not directly enhance flavoprotein oxidation in rat ventricular myocytes, these volatile agents did potentiate increases in fluorescence produced by the selective mito KATPchannel agonist diazoxide. These results suggested that volatile anesthetics may not directly open but instead act to prime mito KATPchannels, thus enhancing their ability to open in response to an agonist. Sarc KATPchannels may also be linked to the function of the mitochondrial inner membrane. For example, ROS generated by mitochondria may act to open sarc KATPchannels. 982,4-Dinitrophenol-induced activation of sarc KATPchannel current was reversible and accompanied by nicotinamide adenine dinucleotide oxidation, suggesting the existence of cross-talk between mito KATPand sarc KATPchannels. 99Taken as a whole, the preponderance of evidence collected to date implies that volatile anesthetics do not necessarily directly open KATPchannels but instead prime the activation of these channels in both sarcolemmal and mitochondrial membranes.Adenosine triphosphate-sensitive potassium channels in vascular smooth muscle cells have been shown to be essential regulators of coronary vascular tone when ATP production is reduced. 100Volatile anesthetic-induced coronary vasodilation 39,52–55was attenuated by glyburide, indicating an important role for KATPchannels in this process. These data suggest that the beneficial actions of volatile agents during myocardial ischemia may be partially attributed to increased oxygen supply mediated via KATPchannel-dependent coronary vasodilation. However, sevoflurane increased coronary collateral blood flow in the presence of glyburide in vivo , indicating that volatile anesthetics enhance collateral perfusion independent of KATPchannel activation. 59In fact, sevoflurane-induced increases in collateral perfusion were recently shown to occur as a result of Ca2+-regulated potassium and not KATPchannel activation. 101Based on these findings and results obtained in isolated cardiac myocytes where blood flow is not a factor, 47it seems highly unlikely that myocardial protection produced by volatile anesthetics is solely related to favorable alterations in coronary vascular tone mediated by KATPchannels.Volatile anesthetics may activate parallel or redundant signaling pathways that involve KATPchannel opening to generate a physiologically meaningful cellular response. The sequential activation of several intracellular elements within a given transduction pathway may facilitate signal amplification and interaction between other redundant signaling systems. For example, administration of isoflurane in the presence of the KATPchannel openers nicorandil 102or diazoxide 103markedly enhances protection against ischemic injury beyond that observed with either drug alone. Several receptor-mediated events and intracellular signaling elements that converge on the KATPchannel have been implicated in APC. Pretreatment with pertussis toxin abolished any reduction in infarct size produced by isoflurane, indicating that Giproteins are linked to the signal transduction pathways that mediate APC. 73In contrast, pertussis toxin did not alter the beneficial effects of direct KATPchannel opening produced by nicorandil. These data strongly support the contention that volatile anesthetics modulate KATPchannel activity through second messenger signaling.Halothane-induced protection against infarction was completely abolished by blockade of the adenosine A1receptor. 34The nonselective adenosine receptor antagonist 8-(p-sulfophenyl)-theophylline abolished isoflurane-induced preconditioning in rabbits. 79The selective A1receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine partially attenuated the beneficial effects of isoflurane in canine stunned myocardium. 70A role for adenosine receptors in APC was also identified in isolated human right atrial myocardium in vitro . 69Isoflurane eliminated increases in interstitial adenosine during repetitive periods of coronary artery occlusion and reperfusion using a microdialysis technique. 70These findings suggest that ATP preservation and a subsequent reduction of adenosine released into the interstitium occur during isoflurane anesthesia. 70In addition, the data imply that volatile agents may either directly activate A1receptors or indirectly enhance A1receptor sensitivity to diminished endogenous adenosine concentrations. 70These results were also similar to those observed during IPC 104or bimakalim-induced pharmacologic preconditioning. 105The preservation of cardiac myocyte viability during ischemia produced by volatile anesthetics was also sensitive to adenosine receptor and Giprotein inhibition in rats. 47Stimulation of the δ1-opioid receptor has been shown to produce a cardioprotective effect that is abolished by selective opioid antagonists 106–110or KATPchannel blockers. 107,108,110–113The acute and delayed phases of IPC are also mediated by activation of the δ1-opioid receptor. 114Recent results indicated that the combined administration of isoflurane and selective δ1-opioid receptor agonists TAN-67 or BW373U86 potentiated KATPchannel opening and enhanced protection against myocardial ischemia and reperfusion injury. 103Combined administration of isoflurane and morphine, a μ receptor agonist with δ1receptor agonist properties, also reduced the extent of myocardial infarction to a greater degree than either drug alone. 44This beneficial effect was shown to be mediated by mito KATPchannels and opioid receptors. Interestingly, the nonselective opioid antagonist naloxone abolished isoflurane-induced preconditioning. 44These intriguing data suggest an important link between volatile anesthetics and the opioid family of G protein-coupled receptors. Another recent study also indicated that halothane competitively inhibited the ligand-binding site of G protein-coupled receptors. 115Adrenergic receptor blockade was shown to abolish desflurane-induced preconditioning in isolated human right atria 63but had no effect on the antiischemic actions of sevoflurane in isolated rat cardiac myocytes. 47Overall, APC seems to be associated with the activation of separate receptor-mediated pathways that are linked to Giproteins.Translocation and phosphorylation of multiple protein kinases are known to be involved in signal transduction pathways involved in protecting myocardium against cell death after ischemia and reperfusion. 116–118In particular, PKC is an essential component of the signaling pathways associated with preserving cellular viability. 119The diverse PKC isoform family is a large group of serine/threonine protein kinases that are distinguished by variable regulatory domains and cofactors and also display diverse tissue and species distributions. 120Activation of G protein-coupled receptors (e.g. , A1, 121,122bradykinin, 123,124δ1opioid 125,126) stimulate PKC during IPC. Volatile anesthetics have also been shown to stimulate PKC translocation and activity, 127possibly by interacting with the regulatory domain of the enzyme. 128Inhibition of PKC attenuated isoflurane-enhanced recovery of contractile function in canine stunned myocardium. 71The antiischemic actions of halothane were abolished by selective PKC antagonism in rabbits. 34The δ and ε isoforms of PKC translocated to mitochondria and sarcolemma, respectively, 10 min after discontinuation of isoflurane in isolated rat hearts. 129In contrast, isoflurane stimulated translocation of PKC-δ and -ε to sarcolemmal and mitochondrial membranes, respectively, in the in vivo rat heart. 72These discrepancies may be attributed to differences in experimental model or time of tissue sampling. The microtubule depolymerizing drug, colchicine, prevented isoflurane-induced reductions in myocardial infarct size in rabbits, 130suggesting that an intact cytoskeleton is essential for translocation of these protein kinases. 72,129Recent findings strongly suggest that volatile anesthetic-induced PKC activation is required to open KATPchannels and produce myocardial protection. For example, the nonselective PKC antagonist chelerythrine abolished sevoflurane-induced increases in mito KATPchannel activity in rat ventricular myocytes and prevented protection against simulated ischemia. 47Patch clamp experiments showed that isoflurane did not facilitate KATPchannel opening in excised membrane patches but enhanced KATPchannel current in a whole cell with PKC were by other studies showing that adenosine and PKC increased KATPchannel activity. PKC have been identified on KATPchannels, indicating a for phosphorylation and activation of the channel by the enzyme. KATPchannel opening also after PKC activation during IPC in isolated rabbit hearts. contrast, recent evidence indicates that 5-HD inhibited PKC that mito KATPchannel opening may be of PKC activation. Therefore, a system between PKC and KATPchannel activation may occur during kinase C has been shown to stimulate protein kinases volatile anesthetics may modulate several of these critical intracellular signaling independent of direct receptor activation as well. and pharmacologic preconditioning have been shown to be mediated by activation of PKC, recent showed that the inhibitor A and the inhibitor abolished isoflurane-induced preconditioning in rats. family an important role in signal transduction from the cell and the and has been strongly implicated in the and of cell death (i.e. , mediated IPC of myocardium in dogs, and activation of was also associated with phosphorylation and translocation of protein in vivo . contrast with the findings during recent data suggest that may not play a role in APC in isolated rat hearts. volatile anesthetics modulate activity of or more intracellular kinases to produce APC, and it seems that activation of PKC is critical to of ROS are released during reperfusion of ischemic myocardium that damage responsible for intracellular depress contractile function, and produce membrane damage. isoflurane, and enflurane have been shown to the effects of ROS on left ventricular pressure development in isolated hearts. decreased generation in the ischemic rat halothane had a similar effect in dogs. protective effects of sevoflurane were associated with reduced an of ROS and reactive species. results support the hypothesis that volatile anesthetics reduce the release of deleterious of ROS associated with coronary artery occlusion and reperfusion. Isoflurane also inhibited production by activated an action that independent of KATPchannel addition, isoflurane and sevoflurane have been shown to abolish activated myocardial effects were associated with reductions in production and neutrophil to coronary vascular Therefore, volatile anesthetics also to exert beneficial actions by injury during contrast with data a role of large of other findings strongly suggest that a of preconditioning including brief ischemia, direct mito KATPchannel and volatile stimulate a of ROS that signaling events and produce protection from subsequent ischemic injury. example, with low concentrations of ROS have been shown to the beneficial actions of IPC. administered before or during brief ischemia markedly attenuated the protective effect of the preconditioning on infarct findings indicate that IPC is mediated by of ROS released during the preconditioning stimulus. The beneficial actions of sevoflurane against ischemic damage were abolished by of and inhibition of nitric oxide results suggest that may act to trigger APC and indicated that nitric oxide may on reperfusion to reduce injury. ROS attenuated isoflurane-induced reductions in myocardial infarct size in rabbits also inhibited the beneficial effects of direct mito KATPchannel activation. has been shown to directly in vivo independent of an ischemic episode by use of the and data for the time that volatile anesthetics were capable of