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Oxidative stress and cardiac disease
PHYSIOLOGY IN MEDICINE In collaboration with The American Physiological Society, Thomas E. Andreoli, MD, Editor
Oxidative Stress and Cardiac Disease David J. Lefer, PhD, D. Neil Granger, PhD Reactive oxygen species (ROS) are formed at an accelerated rate in postischemic myocardium. Cardiac myocytes, endothelial cells, and infiltrating neutrophils contribute to this ROS production. Exposure of these cellular components of the myocardium to exogenous ROS can lead to cellular dysfunction and necrosis. While it remains uncertain whether ROS contribute to the pathogenesis of
myocardial infarction, there is strong support for ROS as mediators of the reversible ventricular dysfunction (stunning) that often accompanies reperfusion of the ischemic myocardium. The therapeutic potential of free radical-directed drugs in cardiac disease has not been fully realized. Am J Med. 2000;109:315–323. 䉷2000 by Excerpta Medica, Inc.
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readmission of oxygenated blood into previously ischemic myocardium can initiate a cascade of events that will paradoxically produce additional myocardial cell dysfunction and cell necrosis (7–10). This phenomenon, termed “reperfusion injury,” can be manifested either as reversible cardiac dysfunction (eg, myocardial stunning) or irreversible damage (eg, myocardial infarction). The cellular mechanisms involved in the pathogenesis of myocardial ischemia/reperfusion (I/R) injury are complex and involve the interaction of a number of cell types, including coronary endothelial cells, circulating blood cells (eg, leukocytes, platelets), and cardiac myocytes (11– 14), most of which are capable of generating reactive oxygen species (ROS). These ROS have the potential to injure vascular cells and cardiac myocytes directly, and can initiate a series of local chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated cardiomyocyte dysfunction and/or cytotoxicity (Figure 1). A key component of the amplification cascade that leads to irreversible tissue damage is the production of factors that promote the recruitment and activation of circulating inflammatory cells. Reactive oxygen species are molecules with unpaired electrons in their outer orbit. As a consequence, these molecules are very unstable and highly reactive, and they tend to initiate chain reactions that result in irreversible chemical changes in lipids or proteins. These potentially deleterious reactions can result in profound cellular dysfunction and even cytotoxicity. It is estimated that approximately 5% of the oxygen consumed by normal tissues are transformed into ROS. These basally generated ROS are efficiently detoxified by endogenous enzymatic free radical scavengers, such as superoxide dismutase, glutathione peroxidase, and catalase (15,16). However, under conditions associated with excess production of
t is estimated that approximately 60 million US citizens have cardiovascular disease, which accounts for almost half of all deaths in the United States. Nearly 14 million US citizens have coronary artery disease, and treatment-related costs for these diseases, including emergency room visits, cardiac catheterizations, coronary angioplasties, and bypass surgery, exceed $95 billion dollars per year (1,2). Ischemic heart disease resulting from coronary artery disease is devastating, with 1.5 million US citizens developing myocardial infarctions that account for nearly 200,000 deaths per year (1,2). Because there is a strong causal relationship between elevated serum cholesterol and coronary artery disease (3,4), and nearly 50% of US citizens are hypercholesterolemic according to present American Heart Association guidelines, it appears likely that acute myocardial infarctions will remain a major biomedical problem of national consequence for the foreseeable future. Coronary artery occlusion resulting from atherosclerotic plaques or vasospasm can result in a reduction in myocardial blood flow that is sufficiently prolonged or severe as to produce myocardial cell injury and necrosis, ultimately leading to diminished (and consequently fatal) cardiac function. The treatment of acute myocardial ischemia involves the use of thrombolytic agents (ie, tissue plasminogen activator, streptokinase) or percutaneous transluminal coronary balloon angioplasty (PTCA), which effectively restores blood flow to the ischemic myocardium. Although reperfusion of an occluded human coronary artery is known to reduce infarct size, preserve left ventricular function, and reduce overall mortality (5,6), it is now recognized that the From the Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, Louisiana. Requests for reprints should be addressed to David J. Lefer, PhD, Department of Molecular and Cellular Physiology, Louisiana Health Sciences Center, 1501 Kings Highway, Shreveport, Louisiana 71130. 䉷2000 by Excerpta Medica, Inc. All rights reserved.
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mutase) or genetic overexpression of these enzymes in experimental animals affords protection against reperfusion injury. A number of different experimental approaches have been used to detect ROS production in the postischemic myocardium. Electron paramagnetic resonance (EPR) spectroscopy is one of the most widely used methods for monitoring ROS generation in the heart. Studies based on this technique have clearly demonstrated a rapid and profound increase in ROS production after reperfusion of ischemic myocardium (17–20). Zweier and colleagues (19) were among the first to measure oxygen free–radical formation directly in isolated, Krebs buffer-perfused rabbit hearts under baseline conditions, during ischemia, and after reperfusion. They noted a marked and distinct EPR spectra in the ischemicreperfused heart that was consistent with maximum oxidant production at 10 to 30 seconds after reperfusion (Figure 2). These early studies suggested that superoxide anion is the predominant ROS produced after reperfusion and that endothelial cells represent an important source of the ROS. The latter contention was supported by two observations: 1) superoxide dismutase treatment reduces the reperfusion-induced EPR signal in isolated
Figure 1. Potential mechanisms of myocardial ischemia/reperfusion injury. Coronary artery occlusion followed by reperfusion results in the production of toxic reactive oxygen species. Reactive oxygen species can directly induce injury to cardiac myocytes, resulting in myocardial “stunning” and myocardial cell necrosis. In addition, oxygen radicals can also trigger a series of events that can ultimately lead to myocardial cell injury and necrosis by means of amplification of the inflammatory cascade involving the nuclear transcription factor kappa beta (NF-B), cytokine production, leukocyte– endothelial cell adhesion molecules, and infiltration of neutrophils.
ROS, such as inflammation or I/R, the flux of ROS generated by tissues can exceed the capacity of endogenous oxidant defense mechanisms to detoxify ROS and prevent deleterious radical-mediated reactions.
DETECTION OF ROS IN REPERFUSED MYOCARDIUM There are three major lines of evidence that implicate ROS in the pathogenesis of myocardial reperfusion injury: 1) ROS can be detected in postischemic myocardium, 2) exposure of myocardium to exogenous ROS results in myocyte and myocardial tissue dysfunction that is comparable to that elicited by I/R, and 3) pretreatment of animals with anti-oxidant enzymes (eg, superoxide dis316
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Figure 2. Electron paramagnetic resonance (EPR) spectra of ischemic-reperfused rat hearts with normal blood flow, after 10 minutes of ischemia, and after 10 minutes of ischemia and 10 seconds of reflow. Figure is redrawn from (reference 18). Reactive oxygen species (ROS) production significantly increases after ischemia and reperfusion.
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buffer-perfused hearts, and 2) monolayers of cultured human umbilical vein endothelial cells exposed to anoxia and reoxygenation also exhibit a burst of ROS production upon reoxygenation, as detected by EPR spectroscopy in vitro (21). EPR spectroscopy has also been used to demonstrate enhanced ROS production in experimental models of myocardial stunning (22). The EPR signals detected in venous blood draining the ischemic-reperfused heart suggest that secondary lipid radicals, such as alkyl and alkoxy radicals, are produced for as long as 3 hours after reflow, with maximal levels detected at 2 to 4 minutes after reperfusion. The existence of a positive, linear correlation between the magnitude of EPR radical signal detected after reperfusion and the severity of the ischemic insult suggests that the intensity of ROS production is dependent on the magnitude of the ischemic flow reduction. Because treatment with a combination of catalase and superoxide dismutase (SOD) reduces the EPR adduct signal detected after myocardial stunning, it appears likely that the ROS are derived from the univalent reduction of oxygen (22). An enhanced production of ROS after myocardial I/R in humans is also supported by different indirect measures of oxidant stress. For example, it has been shown that serum levels of vitamin E are reduced, whereas serum conjugated dienes and thiobarbituric acid reactive substances (TBARS) are elevated in patients undergoing coronary artery bypass graft (CABG) surgery (23). Similarly, electron spin resonance (ESR) spin trapping has been used in conjunction with CABG surgery to show that ROS are produced, with peak levels detected at 5 minutes and 25 minutes after reperfusion.
DIRECT EFFECTS OF ROS ON CARDIAC FUNCTION ROS have been shown to exert a direct inhibitory effect on myocardial function in vivo and in vitro. Indeed, exposure of the normal myocardium to ROS-generating systems or hydrogen peroxide alters myocardial function in a fashion that mimics reperfusion injury, including persistent cellular loss of K⫹, depletion of high-energy phosphates, elevated intracellular calcium concentration, loss of systolic force development, a progressive increase in diastolic tension, depressed metabolic function, and arrhythmias (22,24,25). The mechanism underlying the depressed myocardial contractility remains poorly understood. However, membranous components of mitochondria, sarcoplasmic reticulum, and sarcolemma may represent the most critical targets of ROS-mediated myocardial dysfunction. ROS have been shown to impair the function of isolated mitochondria and subsequently result in adenosine (ATP) depletion. Like I/R, exposure of
isolated sarcoplasmic reticulum to ROS results in a diminished calcium uptake and depresses Ca2⫹, Mg2⫹-ATPase activity. Similarly, ROS have been shown to reduce calcium-stimulated ATPase activity and depress calcium transport in the sarcolemma. Hence, the disturbances in calcium homeostasis that result from ROS interactions with cellular membranes could, at least hypothetically, explain some of the contractile abnormalities associated with I/R. The effects on ROS-generating systems on myocyte membranes likely reflect the propensity of ROS to interact with the protein and lipid components of these membranes (7,15). ROS-mediated reactions with proteins can result in the inactivation of key enzymes and ion transporters. Furthermore, the peroxidation of polyunsaturated fatty acid components of cell membranes can alter the permeability and selectivity of these membranes to specific ions and alter receptor function. Therefore, the combined actions of lipid peroxidation and protein oxidation could well explain the cellular alterations that lead to the depressed cardiac function in conditions associated with excessive production of ROS.
ANTIOXIDANT INTERVENTIONS AND REPERFUSION INJURY Myocardial Infarction (Irreversible Injury) in Animal Models The bulk of the evidence implicating ROS in the pathogenesis of myocardial I/R injury is based on experiments that examine the ability of free radical scavengers to alter the injury response. The results of a number of studies investigating antioxidant therapies in myocardial reperfusion injury are summarized in Table 1. Superoxide dismutase and catalase have received the most attention in this regard. The first assessment of antioxidant enzyme therapy in myocardial reperfusion injury was performed by Jolly et al (26), who studied a combination of SOD and catalase. This study (26) revealed that the combination of antioxidant enzymes significantly reduced myocardial infarct size in dogs after 90 minutes of coronary artery ischemia and 24 hours of reflow. Since this seminal report, there have been a large number of studies from different laboratories (26 –33) that have similarly demonstrated a beneficial effect of SOD and/or catalase in experimental models of myocardial I/R injury. There is a large and nearly equal number of reports that either describe a failure of SOD treatment to exert cardioprotection or demonstrate an early protective effect that waned with increasing duration of reperfusion (34 – 40). It has been suggested that the great disparity in results that were observed with SOD therapy is related to either the dose of the enzyme tested or the experimental conditions of the study (41). The variable results observed with
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Table 1. Antioxidant Therapy Agent/Enzyme
Mechanism of Action
References
Superoxide Dismutase (SOD) Catalase Polyethylene glycol (PEG)-SOD SMA-SOD SC-52608 N-2-mercaptopropionylglycine (MPG) N-acetylcysteine Dimethylthiourea (DMTU) Deferoxamine Desferrioxamine
Superoxide Scavenger Hydrogen Peroxide Scavenger Long Acting Form of SOD Long Acting Form of SOD SOD Mimetic General Free Radical Scavenger
26–41, 68–71, 73, 74 26, 68, 69, 71 42, 43 44 45 54–61
General Free Radical Scavenger General Free Radical Scavenger Iron Chelators
62 63 64–66
SOD and/or catalase in the postischemic myocardium have resulted in considerable skepticism about the therapeutic potential of antioxidant enzymes for acute myocardial infarction in humans. Conjugation of SOD with polyethylene glycol (PEG) increases both the plasma half-life and cellular uptake of the enzyme. PEG-SOD has been shown to be cardioprotective (42) against myocardial I/R injury in some studies, but there are also reports that do not support this conclusion (43). Because it has been suggested that PEG-SOD may in fact be too large to gain access into endothelial cells, where it can scavenge intracellular sources of superoxide (43), attention was redirected to developing other long-acting forms of SOD (44) as well synthetic low-molecular-weight SOD-mimetics (45). A novel long half-life preparation of SOD (SMA-SOD), which was injected every 12 hours during a 4-day reperfusion period in dogs subjected to myocardial I/R, was shown to provide dramatic and sustained reduction in myocardial necrosis (44). Low-molecular-weight SOD mimetics (45) have similarly yielded positive results in animal models of myocardial I/R. However, it remains unclear whether these agents act merely to delay, rather than to prevent, the myocardial necrosis. Another experimental strategy that has been used to assess the role of ROS in myocardial I/R injury is to assess the injury response in mutant mice in which a gene encoding a specific antioxidant enzyme has been either deleted or overexpressed. Transgenic mice that overexpress various isoforms of SOD have also been developed (46 – 49), as well as mice that either overexpress or lack the peroxide detoxifying enzymes, catalase, or glutathione peroxidase (50 –53). Recent advances in technology and physiological techniques for assessing cardiac function in small animals has enabled investigators to use these mutant mice to address the importance of endogenous antioxidant enzymes in the pathogenesis of I/R injury. For example, it has been shown that myocardial contractility is preserved in isolated, perfused hearts derived from mice that genetically overexpress endothelial cell superoxide dismutase (EC-SOD) (54). Similarly, mice that 318
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overexpress mitochondrial manganese-superoxide dismutase (MnSOD) are also protected against myocardial I/R (55). Glutathione peroxidase (GSHPx) is an important antioxidant enzyme that performs several vital functions, including the detoxification of lipid and nonlipid hydroperoxides, as well as hydrogen peroxide (50 –53). Transgenic mice that overexpress GSHPx appear to be resistant to myocardial I/R injury (53), whereas GSHPx knockout mice are more susceptible to myocardial reperfusion injury compared with their wild-type counterparts (56). Overall, the studies of myocardial reperfusion injury performed to date in mice with genetically altered levels of antioxidant enzymes more consistently yield results that support a role of ROS in myocardial I/R than animal studies that involve exogenous administration of antioxidant enzymes. This difference may reflect a need for substantial elevations in intracellular enzyme levels for cardioprotection against I/R that cannot be achieved by intravascular administration of exogenous enzyme. Alternative explanations for the consistently supportive results for ROS involvement obtained from mutant mice that either overexpress or lack a specific antioxidant enzyme include physiological compensations resulting from the gene insertion or deletion, and experimental designs that focus on myocardial injury responses only a few minutes or hours after reperfusion. Although antioxidant enzymes have been the preferred intervention for testing a role for ROS in myocardial I/R, difficulties related to the inability of these enzymes to achieve significant levels within cells have resulted in several studies that examine the cardioprotective effects of less specific antioxidant interventions. One frequently studied antioxidant agent is the sulfhydryl-containing amino acid derivative, N-2-mercaptoproprionyl glycine, (MPG), a safe and well-tolerated drug that is used clinically to reduce radiation-induced tissue injury (57,58). MPG is thought to work by directly reacting with freeradical species, promoting the resynthesis of glutathione, or acting as an alternative substrate for glutathione peroxidase, thereby limiting the cytotoxic effects of H2O2
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Figure 3. Mechanisms by which reactive oxygen species can induce myocardial contractile stunning after reversible coronary artery ischemia and reperfusion. Toxic oxygen species cause lipid and protein peroxidation in the myocardium, which can result in myocardial sarcolemmal damage and contractile protein damage. As a result of these events, Ca2⫹ overload and calcium desensitization can occur in cardiac myocytes in the ischemic-reperfused regions. These events ultimately reduce myocardial contractility and produce myocardia contractile stunning.
and lipid peroxides. This compound has been shown to reduce significantly myocardial infarct size both in the early moments (59,60) and for as long as 48 hours (61) after reperfusion. Other antioxidant agents that have been shown to exert some cardioprotective action in animal models of myocardial infarction include N-acetylcysteine (62), dimethylthiourea (63), and desferrioxamine (64). The cardioprotective effects of these agents have been used to support a role for catalytically active iron (chelated by desferrioxamine) and the hydroxyl radical (scavenged by dimethylthiourea and N-acetylcysteine) in myocardial I/R injury.
Myocardial Stunning (Reversible Injury) in Animal Models Myocardial stunning represents the reversible ventricular dysfunction that often accompanies reperfusion of the ischemic myocardium. In contrast to the controversial results that have been reported for the involvement of ROS in myocardial infarction, there is more uniform agreement that ROS contribute to the process of myocardial stunning (22,65– 67). Indeed, there is evidence implicating the superoxide anion, hydrogen peroxide, and the hydroxyl radical in postischemic myocardial stunning (65,66). Figure 3 summarizes some of the ROS-depen-
dent processes that may account for the mechanical dysfunction associated with myocardial stunning. In experimental models of myocardial stunning, hearts are subjected to very short periods of coronary artery ischemia (ie, 15 to 20 minutes) followed by hours or days of reperfusion. Cardiac contractile function is impaired in the absence of myocardial cell necrosis or infarction. However, when animals are pretreated with a combination of SOD and catalase (68 –71), the postischemic myocardial dysfunction is significantly attenuated. The effects of SOD and catalase given alone or in combination have been studied (69), and the results suggest that both agents are required to protect the myocardium fully. Furthermore, many of the nonspecific antioxidant interventions [eg, N-2-mercaptopropionylglycine (MPG), dimethylthiourea, and desferrioxamine] that have shown some efficacy in some models of myocardial infarction also afford protection against myocardial stunning (22,72).
Clinical Studies Using Antioxidants in Myocardial I/R Recombinant human superoxide dismutase (h-SOD) has been tested in two clinical trials of patients undergoing thrombolysis (73) or coronary angioplasty (74) for acute myocardial infarction. In a pilot clinical trial of 34 pa-
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Figure 4. Species of toxic oxygen radicals that are produced in the ischemic-reperfused myocardium. Reactive oxygen species can be generated within the ischemic-reperfused myocardium by means of uncoupling of the mitochondrial electron transport system. In addition, circulating polymorphonuclear leukocytes (PMNs) can generate superoxide anion by means of the reaction of NADPH with the enzyme NADPH oxidase. Finally, coronary endothelial cells contain the enzyme xanthine oxidase, which catalyzes the conversion of hypoxanthine to superoxide radical plus hydrogen peroxide after ischemia and reperfusion.
tients with acute anterior myocardial infarction receiving thrombolytic agents, h-SOD was randomly allocated to patients just before reperfusion (73). Arrhythmias and left ventricular function were monitored for a period of 3 to 4 weeks after thrombolytic therapy. Although a significant reduction in ventricular premature complexes was observed during early reperfusion, SOD treatment failed to demonstrate any significant improvement in left ventricular regional ejection fraction. A subsequent multicenter, randomized, placebo-controlled clinical trial was designed to test the hypothesis that oxidant-mediated myocardial reperfusion injury is attenuated by treatment with h-SOD before balloon angioplasty (74). Left ventricular function was analyzed using paired contrast ventriculograms and paired radionuclide ventriculograms at 6 and 10 days after angioplasty. Both h-SOD and placebotreated patients showed significant improvements in ventricular function after reperfusion, with no additional protection provided by treatment (74). Hence, the limited efforts to test the oxygen radical hypothesis of myocardial reperfusion injury in the clinical setting do not support the use of antioxidant enzymes in the treatment of patients who have a myocardial infarction. These negative findings, coupled with data derived from animal studies, suggest that if an antioxidant strategy is to be 320
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considered for emergency reperfusion therapy in the future, it is likely to involve low molecular compounds that are capable of both scavenging ROS and gaining access to the intracellular compartment (75).
ENZYMATIC AND CELLULAR SOURCES OF ROS IN MYOCARDIAL I/R Although it is now generally accepted that postischemic tissues, such as heart, skeletal muscle, liver, and intestine, are exposed to a significant oxidant stress upon reperfusion, the major source(s) of the ROS remain unknown. Nonetheless, several enzymatic sources of ROS have been proposed to explain I/R-induced production, including xanthine oxidase, prostaglandin biosynthetic enzymes, mitochondrial electron transport enzymes, and neutrophilic NADPH oxidase (15,16). Potential sources fo reactive oxygen species in the ischemic-reperfused myocardium are summarized in Figure 4. Among these potential sources, two have received considerable attention, xanthine oxidase and NADPH oxidase. Allopurinol, an inhibitor of xanthine oxidase, has been shown to improve functional recovery of the stunned myocardium (22) and to reduce infarct size (76) in dogs. There is also clinical
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evidence that allopurinol pretreatment improves postoperative cardiac performance in patients undergoing CABG (23). In contrast to some human tissues (eg, intestine, liver), which exhibit substantial xanthine oxidase activity, there is little or no detectable activity of this enzyme in human heart (76). Consequently, there is considerable skepticism about the importance of xanthine oxidase as a source of ROS in human myocardium. Recent attention has focused on activated neutrophils as a source of ROS in postischemic myocardium. There are numerous reports that describe a reduction in I/Rinduced myocardial necrosis in experimental animals that are either rendered neutropenic or that receive antibodies that immunoneutralize adhesion molecules that are critical for neutrophil recruitment (77). Similarly, mutant mice that are genetically deficient in certain leukocyte or endothelial cell adhesion molecules also exhibit smaller infarcts after myocardial I/R (78,79). Reports addressing the potential contribution of neutrophils to the phenomenon of myocardial stunning are less encouraging. Myocardial stunning is not accompanied by neutrophil accumulation in the heart, and interventions that prevent leukocyte-endothelial cell adhesion do not improve cardiac performance after ischemic insults that elicit reversible myocardial dysfunction (22). Endothelial cells, leukocytes, myocytes, mast cells, and macrophages are all capable of producing the ROS detected after reperfusion of the ischemic myocardium. In vitro models of I/R injury using monolayers of cultured endothelial cells suggest that vascular endothelium not only can generate the significant fluxes of ROS detected in postischemic tissues but also can adopt an inflammatory phenotype that promotes the recruitment and activation of leukocytes into postischemic tissue (80). Consequently, a mechanism that has been described in postischemic tissues invokes an initial burst of ROS production by endothelial cells, which subsequently promotes, by means of the formation of inflammatory mediators and transcription factor activation, the recruitment of activated neutrophils, which then account for a second, more prolonged phase of ROS production. Although there is substantial evidence implicating this mechanism in some tissues, it remains unclear whether endothelial cells assume such an important modulating role in the pathogenesis of myocardial I/R.
CONCLUSIONS Reactive oxygen species are generated at an accelerated level in the postischemic myocardium. Multiple cell types and different enzymes contribute to the enhanced ROS production and oxidant stress associated with ischemia and reperfusion. Although exogenously generated ROS are clearly able to damage the contractile machinery of
cardiac myocytes and can produce necrosis, it remains unclear whether the ROS generated at the time of reperfusion directly mediate the myocardial necrosis that characterizes a myocardial infarct. There appears to be less uncertainty, however, concerning the critical role of ROS in the genesis of myocardial stunning, a phenomenon of immense clinical significance. A better understanding of the actions of cell-permeant antioxidants in the setting of myocardial I/R is needed before the therapeutic potential of free radical– directed drugs can be fully realized. In view of the growing body of evidence that implicates inflammatory cells in the pathogenesis of myocardial reperfusion injury, further attention should be devoted to defining the role of ROS in the recruitment and activation of leukocytes in postischemic tissues.
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myocardium at risk in conscious dogs. Circulation. 1989;79:143– 153. Shirato C, Miura T, Downey JM. Superoxide dismutase (single dose) delays rather than prevents necrosis in reperfused rabbit heart. FASEB J. 1988;2:A918. Klein HH, Pich S, Lindert S, Buchwald A, Kreuzer H. Intracoronary superoxide dismutase for the treatment of “reperfusion injury”: a blind randomized placebo-controlled trial in ischemic-reperfused procine hearts. Bas Res Cardiol. 1998;83:141–148. Patel B, Jeroudi MO, O’Neill PG, Roberts R, Bolli R. Human superoxide dismutase fails to limit infarct size after 2 hours ischemia and reperfusion. Circulation. 1988;2:A918. Richard VJ, Murry CE, Jennings RB, Reimer KA. Therapy to reduce free radicals during early reperfusion does not limit the size of myocardial infarcts caused by 90 minutes of ischemia in dogs. Circulation. 1988;78:473– 480. Engler R, Gilpin E. Can superoxide dismutase alter infarct size? Circulation. 1989;79:1137–1142. Tamura Y, Chi L, Driscoll E, et al. Superoxide dismutase conjugated to polyethylene glycol provides sustained protection against myocardial ischemia/reperfusion injury in canine heart. Circ Res. 1988; 63:944 –959. Ooiwa BA, Jordan MC, Downey JM, McCord JM. PEG-SOD fails to limit infarct size in reperfused rabbit heart. Circulation. 1989;80:II294. Hori M, Goto K, Iwa K, et al. Effects of long-acting superoxide dismutase (SMA-SOD) on myocardial necrosis in coronary embolization in dogs. Circulation. 1988;78:II-372. Kilgore KS, Friedrichs GS, Johnson CR, et al. Protective effects of the SOD-mimetic SC-52608 against ischemia/reperfusion damage in the rabbit isolated heart. J Mol Cell Cardiol. 1994;26:995–1006. White CW, Avraham KB, Shanley PF, Groner Y. Transgenic mice with expression of elevated levels of copper-zinc superoxide dismutase are resistant to pulomary oxygen toxicity. J Clin Invest. 1991;87:2162–2168. Wispe JR, Warner BB, Clark JC, et al. Human Mn-superoxide dismutase in pulmonary epithelial cells of transgenic mice confers protection from oxgyen toxicity. J Biol Chem. 1992;267–23937– 23941. Ho Y-S, Vincent R, Dey MS, Slot JW, Crapo JD. Transgenic models for the study of lung antioxidant defense: enhanced manganesecontaining superoxide dismutase activity gives partial protection to B6C3 hybrid mice exposed to hyperoxia. Am J Respir Cell Mol Biol. 1998;18:538 –547. Yen H-C, Oberley TD, Vichitbandha S, Ho Y-S, St. Clair DK. The protective role of mangansese superoxide dismutase against adriamycin-induced acute cardiac toxicity in transgenic mice. J Clin Invest. 1996;98:1253–1260. Ho Y-S, Magnenat J-L, Bronson RT, et al. Mice deficient in cellular glutathione peroxidase develop normally and show no increased sensitivity to hyperoxia. J Biol Chem. 1997;272:16644 –16651. Jaeschke H, Ho Y-S, Fisher MA, Lawson JA, AF. Glutathione peroxidase-deficient mice are more susceptible to neutrophil-mediated hepatic parenchymal cell injury during endotoxemia: importance of an intracellular oxidant stress. Hepatology. 1999;29:443– 450. Beck MA, Esworthy RS, Ho Y-S, Chu F-F. Glutathione peroxidase protects mice from viral-induced myocarditis. FASEB J. 1998;12: 1143–1149. Yoshida T, Watanabe M, Engleman DT, et al. Transgenic mice overexpressing glutathione peroxidase are resistant to myocardial ischemia reperfusion injury. J Mol Cell Cardiol. 1996;28:1759 –1767. Chen EP, Bittner HB, Davis RD, Folz RJ, Van Trigt P. Extracellular superoxide dismutase transgene overexpression preserves postisch-
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68. Przyklenk K, Kloner RA. Superoxide dismutase plus catalase improves contractile function in the canine model of the “stunned myocardium.” Circ Res. 1986;58:148 –156. 69. Jeroudi MO, Triana FJ, Patel BS, Bolli M. Effect of superoxide dismutase and catalase, given seperately, on myocardial “stunning.” Am J Physiol. 1990;259:H889 –H890. 70. Buchwald A, Klein HH, Lindert et al. Effect of intracoronary superoxide dismutase on regional function in stunned myocardium. J Card Pharmacol. 1989;13:258 –264. 71. Gross GJ, Farger NE, Hardman HF, Warltier DC. Beneficial actions of superoxide dismutase and catalase in stunned myocardium of dogs. Am J Physiol. 1986;250:H373–H377. 72. Bolli R, Patel B, Zhu W, et al. The iron chelator desferrioxamine attenuates postischemic ventricular dysfunction. Am J Physiol. 1987;252:H1372–H1380. 73. Murohara Y, Yoshiki Y, Hattori R, Kawai C. Effects of superoxide dismutase on reperfusion arrhythmias and left ventricular function in patients undergoing thrombolysis for anterior wall acute myocardial infarction. Am J Cardiol. 1991;67:765–767. 74. Flaherty JT, Pitt B, Gruber JW, et al. Recombinant human superoxide dismutase (h-SOD) fails to improve recovery of ventricular function in patients undergoing coronary angioplasty for acute myocardial infarction. Circulation. 1994;89:1982–1991. 75. Maxwell I SRJ, Lip GYH. Reperfusion injury: a review of the pathophysiology, clinical manifestations and therapeutic options. Int J Cardiol. 1997;58:95–117. 76. Downey JM, Yellon DM. Do free radicals contribute to myocardial cell death during ischemia-reperfusion? In: Yellon DM, Jennings RB, eds. Myocardial Protection: Pathophysiology of Reperfusion and Reperfusion Injury. New York: Raven Press, 1992:35–57. 77. Lefer AM, Lefer DJ. The role of nitric oxide and cell adhesion molecules on the microcirculation in ischemia-reperfusion. Card Res. 1996;32:743–751. 78. Palazzo AJ, Jones SP, Anderson DC, Granger DN, Lefer DJ. Coronary endothelial P-selectin in the pathogenesis of myocardial ischemia-reperfusion injury. Am J Physiol. 1998;275:H1865–H1872. 79. Palazzo AJ, Jones SP, Girod WG, Anderson DC, Granger DN, Lefer DJ. Myocardial ischemia-reperfusion injury in CD18- and ICAM1-deficient mice. Am J Physiol. 1998;275:H2300 –H2307. 80. Granger DN. Ischemia-reperfusion: mechanisms of microvascular dysfunction and the influence of risk factors for cardiovascular disease. Microcirculation. 1999;6:167–178.
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Oxidative Stress and Cardiac Disease David J. Lefer, PhD, D. Neil Granger, PhD Reactive oxygen species (ROS) are formed at an accelerated rate in postischemic myocardium. Cardiac myocytes, endothelial cells, and infiltrating neutrophils contribute to this ROS production. Exposure of these cellular components of the myocardium to exogenous ROS can lead to cellular dysfunction and necrosis. While it remains uncertain whether ROS contribute to the pathogenesis of
myocardial infarction, there is strong support for ROS as mediators of the reversible ventricular dysfunction (stunning) that often accompanies reperfusion of the ischemic myocardium. The therapeutic potential of free radical-directed drugs in cardiac disease has not been fully realized. Am J Med. 2000;109:315–323. 䉷2000 by Excerpta Medica, Inc.
I
readmission of oxygenated blood into previously ischemic myocardium can initiate a cascade of events that will paradoxically produce additional myocardial cell dysfunction and cell necrosis (7–10). This phenomenon, termed “reperfusion injury,” can be manifested either as reversible cardiac dysfunction (eg, myocardial stunning) or irreversible damage (eg, myocardial infarction). The cellular mechanisms involved in the pathogenesis of myocardial ischemia/reperfusion (I/R) injury are complex and involve the interaction of a number of cell types, including coronary endothelial cells, circulating blood cells (eg, leukocytes, platelets), and cardiac myocytes (11– 14), most of which are capable of generating reactive oxygen species (ROS). These ROS have the potential to injure vascular cells and cardiac myocytes directly, and can initiate a series of local chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated cardiomyocyte dysfunction and/or cytotoxicity (Figure 1). A key component of the amplification cascade that leads to irreversible tissue damage is the production of factors that promote the recruitment and activation of circulating inflammatory cells. Reactive oxygen species are molecules with unpaired electrons in their outer orbit. As a consequence, these molecules are very unstable and highly reactive, and they tend to initiate chain reactions that result in irreversible chemical changes in lipids or proteins. These potentially deleterious reactions can result in profound cellular dysfunction and even cytotoxicity. It is estimated that approximately 5% of the oxygen consumed by normal tissues are transformed into ROS. These basally generated ROS are efficiently detoxified by endogenous enzymatic free radical scavengers, such as superoxide dismutase, glutathione peroxidase, and catalase (15,16). However, under conditions associated with excess production of
t is estimated that approximately 60 million US citizens have cardiovascular disease, which accounts for almost half of all deaths in the United States. Nearly 14 million US citizens have coronary artery disease, and treatment-related costs for these diseases, including emergency room visits, cardiac catheterizations, coronary angioplasties, and bypass surgery, exceed $95 billion dollars per year (1,2). Ischemic heart disease resulting from coronary artery disease is devastating, with 1.5 million US citizens developing myocardial infarctions that account for nearly 200,000 deaths per year (1,2). Because there is a strong causal relationship between elevated serum cholesterol and coronary artery disease (3,4), and nearly 50% of US citizens are hypercholesterolemic according to present American Heart Association guidelines, it appears likely that acute myocardial infarctions will remain a major biomedical problem of national consequence for the foreseeable future. Coronary artery occlusion resulting from atherosclerotic plaques or vasospasm can result in a reduction in myocardial blood flow that is sufficiently prolonged or severe as to produce myocardial cell injury and necrosis, ultimately leading to diminished (and consequently fatal) cardiac function. The treatment of acute myocardial ischemia involves the use of thrombolytic agents (ie, tissue plasminogen activator, streptokinase) or percutaneous transluminal coronary balloon angioplasty (PTCA), which effectively restores blood flow to the ischemic myocardium. Although reperfusion of an occluded human coronary artery is known to reduce infarct size, preserve left ventricular function, and reduce overall mortality (5,6), it is now recognized that the From the Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, Louisiana. Requests for reprints should be addressed to David J. Lefer, PhD, Department of Molecular and Cellular Physiology, Louisiana Health Sciences Center, 1501 Kings Highway, Shreveport, Louisiana 71130. 䉷2000 by Excerpta Medica, Inc. All rights reserved.
0002-9343/00/$–see front matter 315 PII S0002-9343(00)00467-8
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mutase) or genetic overexpression of these enzymes in experimental animals affords protection against reperfusion injury. A number of different experimental approaches have been used to detect ROS production in the postischemic myocardium. Electron paramagnetic resonance (EPR) spectroscopy is one of the most widely used methods for monitoring ROS generation in the heart. Studies based on this technique have clearly demonstrated a rapid and profound increase in ROS production after reperfusion of ischemic myocardium (17–20). Zweier and colleagues (19) were among the first to measure oxygen free–radical formation directly in isolated, Krebs buffer-perfused rabbit hearts under baseline conditions, during ischemia, and after reperfusion. They noted a marked and distinct EPR spectra in the ischemicreperfused heart that was consistent with maximum oxidant production at 10 to 30 seconds after reperfusion (Figure 2). These early studies suggested that superoxide anion is the predominant ROS produced after reperfusion and that endothelial cells represent an important source of the ROS. The latter contention was supported by two observations: 1) superoxide dismutase treatment reduces the reperfusion-induced EPR signal in isolated
Figure 1. Potential mechanisms of myocardial ischemia/reperfusion injury. Coronary artery occlusion followed by reperfusion results in the production of toxic reactive oxygen species. Reactive oxygen species can directly induce injury to cardiac myocytes, resulting in myocardial “stunning” and myocardial cell necrosis. In addition, oxygen radicals can also trigger a series of events that can ultimately lead to myocardial cell injury and necrosis by means of amplification of the inflammatory cascade involving the nuclear transcription factor kappa beta (NF-B), cytokine production, leukocyte– endothelial cell adhesion molecules, and infiltration of neutrophils.
ROS, such as inflammation or I/R, the flux of ROS generated by tissues can exceed the capacity of endogenous oxidant defense mechanisms to detoxify ROS and prevent deleterious radical-mediated reactions.
DETECTION OF ROS IN REPERFUSED MYOCARDIUM There are three major lines of evidence that implicate ROS in the pathogenesis of myocardial reperfusion injury: 1) ROS can be detected in postischemic myocardium, 2) exposure of myocardium to exogenous ROS results in myocyte and myocardial tissue dysfunction that is comparable to that elicited by I/R, and 3) pretreatment of animals with anti-oxidant enzymes (eg, superoxide dis316
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Figure 2. Electron paramagnetic resonance (EPR) spectra of ischemic-reperfused rat hearts with normal blood flow, after 10 minutes of ischemia, and after 10 minutes of ischemia and 10 seconds of reflow. Figure is redrawn from (reference 18). Reactive oxygen species (ROS) production significantly increases after ischemia and reperfusion.
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buffer-perfused hearts, and 2) monolayers of cultured human umbilical vein endothelial cells exposed to anoxia and reoxygenation also exhibit a burst of ROS production upon reoxygenation, as detected by EPR spectroscopy in vitro (21). EPR spectroscopy has also been used to demonstrate enhanced ROS production in experimental models of myocardial stunning (22). The EPR signals detected in venous blood draining the ischemic-reperfused heart suggest that secondary lipid radicals, such as alkyl and alkoxy radicals, are produced for as long as 3 hours after reflow, with maximal levels detected at 2 to 4 minutes after reperfusion. The existence of a positive, linear correlation between the magnitude of EPR radical signal detected after reperfusion and the severity of the ischemic insult suggests that the intensity of ROS production is dependent on the magnitude of the ischemic flow reduction. Because treatment with a combination of catalase and superoxide dismutase (SOD) reduces the EPR adduct signal detected after myocardial stunning, it appears likely that the ROS are derived from the univalent reduction of oxygen (22). An enhanced production of ROS after myocardial I/R in humans is also supported by different indirect measures of oxidant stress. For example, it has been shown that serum levels of vitamin E are reduced, whereas serum conjugated dienes and thiobarbituric acid reactive substances (TBARS) are elevated in patients undergoing coronary artery bypass graft (CABG) surgery (23). Similarly, electron spin resonance (ESR) spin trapping has been used in conjunction with CABG surgery to show that ROS are produced, with peak levels detected at 5 minutes and 25 minutes after reperfusion.
DIRECT EFFECTS OF ROS ON CARDIAC FUNCTION ROS have been shown to exert a direct inhibitory effect on myocardial function in vivo and in vitro. Indeed, exposure of the normal myocardium to ROS-generating systems or hydrogen peroxide alters myocardial function in a fashion that mimics reperfusion injury, including persistent cellular loss of K⫹, depletion of high-energy phosphates, elevated intracellular calcium concentration, loss of systolic force development, a progressive increase in diastolic tension, depressed metabolic function, and arrhythmias (22,24,25). The mechanism underlying the depressed myocardial contractility remains poorly understood. However, membranous components of mitochondria, sarcoplasmic reticulum, and sarcolemma may represent the most critical targets of ROS-mediated myocardial dysfunction. ROS have been shown to impair the function of isolated mitochondria and subsequently result in adenosine (ATP) depletion. Like I/R, exposure of
isolated sarcoplasmic reticulum to ROS results in a diminished calcium uptake and depresses Ca2⫹, Mg2⫹-ATPase activity. Similarly, ROS have been shown to reduce calcium-stimulated ATPase activity and depress calcium transport in the sarcolemma. Hence, the disturbances in calcium homeostasis that result from ROS interactions with cellular membranes could, at least hypothetically, explain some of the contractile abnormalities associated with I/R. The effects on ROS-generating systems on myocyte membranes likely reflect the propensity of ROS to interact with the protein and lipid components of these membranes (7,15). ROS-mediated reactions with proteins can result in the inactivation of key enzymes and ion transporters. Furthermore, the peroxidation of polyunsaturated fatty acid components of cell membranes can alter the permeability and selectivity of these membranes to specific ions and alter receptor function. Therefore, the combined actions of lipid peroxidation and protein oxidation could well explain the cellular alterations that lead to the depressed cardiac function in conditions associated with excessive production of ROS.
ANTIOXIDANT INTERVENTIONS AND REPERFUSION INJURY Myocardial Infarction (Irreversible Injury) in Animal Models The bulk of the evidence implicating ROS in the pathogenesis of myocardial I/R injury is based on experiments that examine the ability of free radical scavengers to alter the injury response. The results of a number of studies investigating antioxidant therapies in myocardial reperfusion injury are summarized in Table 1. Superoxide dismutase and catalase have received the most attention in this regard. The first assessment of antioxidant enzyme therapy in myocardial reperfusion injury was performed by Jolly et al (26), who studied a combination of SOD and catalase. This study (26) revealed that the combination of antioxidant enzymes significantly reduced myocardial infarct size in dogs after 90 minutes of coronary artery ischemia and 24 hours of reflow. Since this seminal report, there have been a large number of studies from different laboratories (26 –33) that have similarly demonstrated a beneficial effect of SOD and/or catalase in experimental models of myocardial I/R injury. There is a large and nearly equal number of reports that either describe a failure of SOD treatment to exert cardioprotection or demonstrate an early protective effect that waned with increasing duration of reperfusion (34 – 40). It has been suggested that the great disparity in results that were observed with SOD therapy is related to either the dose of the enzyme tested or the experimental conditions of the study (41). The variable results observed with
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Table 1. Antioxidant Therapy Agent/Enzyme
Mechanism of Action
References
Superoxide Dismutase (SOD) Catalase Polyethylene glycol (PEG)-SOD SMA-SOD SC-52608 N-2-mercaptopropionylglycine (MPG) N-acetylcysteine Dimethylthiourea (DMTU) Deferoxamine Desferrioxamine
Superoxide Scavenger Hydrogen Peroxide Scavenger Long Acting Form of SOD Long Acting Form of SOD SOD Mimetic General Free Radical Scavenger
26–41, 68–71, 73, 74 26, 68, 69, 71 42, 43 44 45 54–61
General Free Radical Scavenger General Free Radical Scavenger Iron Chelators
62 63 64–66
SOD and/or catalase in the postischemic myocardium have resulted in considerable skepticism about the therapeutic potential of antioxidant enzymes for acute myocardial infarction in humans. Conjugation of SOD with polyethylene glycol (PEG) increases both the plasma half-life and cellular uptake of the enzyme. PEG-SOD has been shown to be cardioprotective (42) against myocardial I/R injury in some studies, but there are also reports that do not support this conclusion (43). Because it has been suggested that PEG-SOD may in fact be too large to gain access into endothelial cells, where it can scavenge intracellular sources of superoxide (43), attention was redirected to developing other long-acting forms of SOD (44) as well synthetic low-molecular-weight SOD-mimetics (45). A novel long half-life preparation of SOD (SMA-SOD), which was injected every 12 hours during a 4-day reperfusion period in dogs subjected to myocardial I/R, was shown to provide dramatic and sustained reduction in myocardial necrosis (44). Low-molecular-weight SOD mimetics (45) have similarly yielded positive results in animal models of myocardial I/R. However, it remains unclear whether these agents act merely to delay, rather than to prevent, the myocardial necrosis. Another experimental strategy that has been used to assess the role of ROS in myocardial I/R injury is to assess the injury response in mutant mice in which a gene encoding a specific antioxidant enzyme has been either deleted or overexpressed. Transgenic mice that overexpress various isoforms of SOD have also been developed (46 – 49), as well as mice that either overexpress or lack the peroxide detoxifying enzymes, catalase, or glutathione peroxidase (50 –53). Recent advances in technology and physiological techniques for assessing cardiac function in small animals has enabled investigators to use these mutant mice to address the importance of endogenous antioxidant enzymes in the pathogenesis of I/R injury. For example, it has been shown that myocardial contractility is preserved in isolated, perfused hearts derived from mice that genetically overexpress endothelial cell superoxide dismutase (EC-SOD) (54). Similarly, mice that 318
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overexpress mitochondrial manganese-superoxide dismutase (MnSOD) are also protected against myocardial I/R (55). Glutathione peroxidase (GSHPx) is an important antioxidant enzyme that performs several vital functions, including the detoxification of lipid and nonlipid hydroperoxides, as well as hydrogen peroxide (50 –53). Transgenic mice that overexpress GSHPx appear to be resistant to myocardial I/R injury (53), whereas GSHPx knockout mice are more susceptible to myocardial reperfusion injury compared with their wild-type counterparts (56). Overall, the studies of myocardial reperfusion injury performed to date in mice with genetically altered levels of antioxidant enzymes more consistently yield results that support a role of ROS in myocardial I/R than animal studies that involve exogenous administration of antioxidant enzymes. This difference may reflect a need for substantial elevations in intracellular enzyme levels for cardioprotection against I/R that cannot be achieved by intravascular administration of exogenous enzyme. Alternative explanations for the consistently supportive results for ROS involvement obtained from mutant mice that either overexpress or lack a specific antioxidant enzyme include physiological compensations resulting from the gene insertion or deletion, and experimental designs that focus on myocardial injury responses only a few minutes or hours after reperfusion. Although antioxidant enzymes have been the preferred intervention for testing a role for ROS in myocardial I/R, difficulties related to the inability of these enzymes to achieve significant levels within cells have resulted in several studies that examine the cardioprotective effects of less specific antioxidant interventions. One frequently studied antioxidant agent is the sulfhydryl-containing amino acid derivative, N-2-mercaptoproprionyl glycine, (MPG), a safe and well-tolerated drug that is used clinically to reduce radiation-induced tissue injury (57,58). MPG is thought to work by directly reacting with freeradical species, promoting the resynthesis of glutathione, or acting as an alternative substrate for glutathione peroxidase, thereby limiting the cytotoxic effects of H2O2
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Figure 3. Mechanisms by which reactive oxygen species can induce myocardial contractile stunning after reversible coronary artery ischemia and reperfusion. Toxic oxygen species cause lipid and protein peroxidation in the myocardium, which can result in myocardial sarcolemmal damage and contractile protein damage. As a result of these events, Ca2⫹ overload and calcium desensitization can occur in cardiac myocytes in the ischemic-reperfused regions. These events ultimately reduce myocardial contractility and produce myocardia contractile stunning.
and lipid peroxides. This compound has been shown to reduce significantly myocardial infarct size both in the early moments (59,60) and for as long as 48 hours (61) after reperfusion. Other antioxidant agents that have been shown to exert some cardioprotective action in animal models of myocardial infarction include N-acetylcysteine (62), dimethylthiourea (63), and desferrioxamine (64). The cardioprotective effects of these agents have been used to support a role for catalytically active iron (chelated by desferrioxamine) and the hydroxyl radical (scavenged by dimethylthiourea and N-acetylcysteine) in myocardial I/R injury.
Myocardial Stunning (Reversible Injury) in Animal Models Myocardial stunning represents the reversible ventricular dysfunction that often accompanies reperfusion of the ischemic myocardium. In contrast to the controversial results that have been reported for the involvement of ROS in myocardial infarction, there is more uniform agreement that ROS contribute to the process of myocardial stunning (22,65– 67). Indeed, there is evidence implicating the superoxide anion, hydrogen peroxide, and the hydroxyl radical in postischemic myocardial stunning (65,66). Figure 3 summarizes some of the ROS-depen-
dent processes that may account for the mechanical dysfunction associated with myocardial stunning. In experimental models of myocardial stunning, hearts are subjected to very short periods of coronary artery ischemia (ie, 15 to 20 minutes) followed by hours or days of reperfusion. Cardiac contractile function is impaired in the absence of myocardial cell necrosis or infarction. However, when animals are pretreated with a combination of SOD and catalase (68 –71), the postischemic myocardial dysfunction is significantly attenuated. The effects of SOD and catalase given alone or in combination have been studied (69), and the results suggest that both agents are required to protect the myocardium fully. Furthermore, many of the nonspecific antioxidant interventions [eg, N-2-mercaptopropionylglycine (MPG), dimethylthiourea, and desferrioxamine] that have shown some efficacy in some models of myocardial infarction also afford protection against myocardial stunning (22,72).
Clinical Studies Using Antioxidants in Myocardial I/R Recombinant human superoxide dismutase (h-SOD) has been tested in two clinical trials of patients undergoing thrombolysis (73) or coronary angioplasty (74) for acute myocardial infarction. In a pilot clinical trial of 34 pa-
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Figure 4. Species of toxic oxygen radicals that are produced in the ischemic-reperfused myocardium. Reactive oxygen species can be generated within the ischemic-reperfused myocardium by means of uncoupling of the mitochondrial electron transport system. In addition, circulating polymorphonuclear leukocytes (PMNs) can generate superoxide anion by means of the reaction of NADPH with the enzyme NADPH oxidase. Finally, coronary endothelial cells contain the enzyme xanthine oxidase, which catalyzes the conversion of hypoxanthine to superoxide radical plus hydrogen peroxide after ischemia and reperfusion.
tients with acute anterior myocardial infarction receiving thrombolytic agents, h-SOD was randomly allocated to patients just before reperfusion (73). Arrhythmias and left ventricular function were monitored for a period of 3 to 4 weeks after thrombolytic therapy. Although a significant reduction in ventricular premature complexes was observed during early reperfusion, SOD treatment failed to demonstrate any significant improvement in left ventricular regional ejection fraction. A subsequent multicenter, randomized, placebo-controlled clinical trial was designed to test the hypothesis that oxidant-mediated myocardial reperfusion injury is attenuated by treatment with h-SOD before balloon angioplasty (74). Left ventricular function was analyzed using paired contrast ventriculograms and paired radionuclide ventriculograms at 6 and 10 days after angioplasty. Both h-SOD and placebotreated patients showed significant improvements in ventricular function after reperfusion, with no additional protection provided by treatment (74). Hence, the limited efforts to test the oxygen radical hypothesis of myocardial reperfusion injury in the clinical setting do not support the use of antioxidant enzymes in the treatment of patients who have a myocardial infarction. These negative findings, coupled with data derived from animal studies, suggest that if an antioxidant strategy is to be 320
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considered for emergency reperfusion therapy in the future, it is likely to involve low molecular compounds that are capable of both scavenging ROS and gaining access to the intracellular compartment (75).
ENZYMATIC AND CELLULAR SOURCES OF ROS IN MYOCARDIAL I/R Although it is now generally accepted that postischemic tissues, such as heart, skeletal muscle, liver, and intestine, are exposed to a significant oxidant stress upon reperfusion, the major source(s) of the ROS remain unknown. Nonetheless, several enzymatic sources of ROS have been proposed to explain I/R-induced production, including xanthine oxidase, prostaglandin biosynthetic enzymes, mitochondrial electron transport enzymes, and neutrophilic NADPH oxidase (15,16). Potential sources fo reactive oxygen species in the ischemic-reperfused myocardium are summarized in Figure 4. Among these potential sources, two have received considerable attention, xanthine oxidase and NADPH oxidase. Allopurinol, an inhibitor of xanthine oxidase, has been shown to improve functional recovery of the stunned myocardium (22) and to reduce infarct size (76) in dogs. There is also clinical
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evidence that allopurinol pretreatment improves postoperative cardiac performance in patients undergoing CABG (23). In contrast to some human tissues (eg, intestine, liver), which exhibit substantial xanthine oxidase activity, there is little or no detectable activity of this enzyme in human heart (76). Consequently, there is considerable skepticism about the importance of xanthine oxidase as a source of ROS in human myocardium. Recent attention has focused on activated neutrophils as a source of ROS in postischemic myocardium. There are numerous reports that describe a reduction in I/Rinduced myocardial necrosis in experimental animals that are either rendered neutropenic or that receive antibodies that immunoneutralize adhesion molecules that are critical for neutrophil recruitment (77). Similarly, mutant mice that are genetically deficient in certain leukocyte or endothelial cell adhesion molecules also exhibit smaller infarcts after myocardial I/R (78,79). Reports addressing the potential contribution of neutrophils to the phenomenon of myocardial stunning are less encouraging. Myocardial stunning is not accompanied by neutrophil accumulation in the heart, and interventions that prevent leukocyte-endothelial cell adhesion do not improve cardiac performance after ischemic insults that elicit reversible myocardial dysfunction (22). Endothelial cells, leukocytes, myocytes, mast cells, and macrophages are all capable of producing the ROS detected after reperfusion of the ischemic myocardium. In vitro models of I/R injury using monolayers of cultured endothelial cells suggest that vascular endothelium not only can generate the significant fluxes of ROS detected in postischemic tissues but also can adopt an inflammatory phenotype that promotes the recruitment and activation of leukocytes into postischemic tissue (80). Consequently, a mechanism that has been described in postischemic tissues invokes an initial burst of ROS production by endothelial cells, which subsequently promotes, by means of the formation of inflammatory mediators and transcription factor activation, the recruitment of activated neutrophils, which then account for a second, more prolonged phase of ROS production. Although there is substantial evidence implicating this mechanism in some tissues, it remains unclear whether endothelial cells assume such an important modulating role in the pathogenesis of myocardial I/R.
CONCLUSIONS Reactive oxygen species are generated at an accelerated level in the postischemic myocardium. Multiple cell types and different enzymes contribute to the enhanced ROS production and oxidant stress associated with ischemia and reperfusion. Although exogenously generated ROS are clearly able to damage the contractile machinery of
cardiac myocytes and can produce necrosis, it remains unclear whether the ROS generated at the time of reperfusion directly mediate the myocardial necrosis that characterizes a myocardial infarct. There appears to be less uncertainty, however, concerning the critical role of ROS in the genesis of myocardial stunning, a phenomenon of immense clinical significance. A better understanding of the actions of cell-permeant antioxidants in the setting of myocardial I/R is needed before the therapeutic potential of free radical– directed drugs can be fully realized. In view of the growing body of evidence that implicates inflammatory cells in the pathogenesis of myocardial reperfusion injury, further attention should be devoted to defining the role of ROS in the recruitment and activation of leukocytes in postischemic tissues.
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