ElResearch Advances Series

Transcription

1 ElResearch Advances Series 723 Mechanism of Myocardial "Stunning" Roberto Bolli, MD Downloaded from by guest on July 13, 2018 P ostischemic ventricular dysfunction, or myocardial "stunning,"' has recently become the focus of considerable attention among researchers and clinicians. There are two fundamental reasons for the explosive growth of interest in this phenomenon. First, it is now recognized that spontaneous reperfusion after coronary spasm or thrombosis is common in patients with coronary artery disease. This implies that postischemic myocardial stunning is likely to be an important component of the natural history of this disorder. Second, with the advent of interventional recanalization, an increasingly large number of patients are subjected to coronary reperfusion in an effort to preserve left ventricular function. The occurrence of myocardial stunning in these patients could significantly delay the benefits of reperfusion therapy. During the past decade, the efforts of experimental investigators have produced considerable progress in our understanding of the mechanism responsible for postischemic contractile dysfunction. This is clearly the central problem because it will not be possible to effectively prevent myocardial stunning unless its pathogenesis is elucidated. The purpose of this article is to critically review the various mechanisms proposed for postischemic dysfunction and their pathophysiological and clinical implications. The major unresolved issues and areas for future research will be identified, and an attempt will be made to integrate different theories into a unifying pathogenetic paradigm. Definition of Myocardial Stunning One cannot overemphasize the importance of a clear definition of myocardial stunning because this term is sometimes loosely applied to situations in which the persistence of contractile abnormalities in postischemic tissue is due to other causes (e.g., myocellular death). Postischemic dysfunction, or myocardial stunning, is the mechanical dysfunction that persists after reperfusion despite the absence of From the Section of Cardiology, Department of Medicine, Baylor College of Medicine, Houston, Tex. Supported in part by National Institutes of Health (NIH) grants HL and HL and NIH SCOR grant HL R.B. was a recipient of the Physician-Scientist Award, American College of Chest Physicians. Address for correspondence: Roberto Bolli, MD, Section of Cardiology, Baylor College of Medicine, 6535 Fannin, MS F-905, Houston, TX Received February 7, 1990; revision accepted May 8, irreversible damage. The essential point of this definition is that postischemic dysfunction, no matter how severe or prolonged, is a fully reversible abnormality. Diagnosis of myocardial stunning should not be made unless reasonable assurance can be provided that the tissue in question is still entirely viable. In accordance with this definition, myocardial stunning is a relatively mild, sublethal injury that must be kept quite distinct from myocardial infarction. It is unknown whether these two conditions share a common mechanism. Consequently, this review will discuss only results obtained in experimental preparations in which mechanical dysfunction is likely to be due to stunning rather than to cell death. The mechanism of lethal ischemia-reperfusion injury has been reviewed previously2-7 and is beyond the scope of this article. Classification of Myocardial Stunning Experimentally, the concept of myocardial stunning encompasses a wide variety of settings with major pathophysiological differences. A classification is in order because different mechanisms have been described in different experimental conditions. The available observations can be grouped into the following categories (Table 1). Myocardial Stunning After One Completely Reversible Ischemic Episode In the dog, a coronary occlusion lasting less than 20 minutes does not result in any myocardial necrosis2,8 but nevertheless produces prolonged abnormalities of contractile performance.8-14 This is the classic model of myocardial stunning, the one in which the phenomenon was originally described,9 and the one most commonly used in experimental investigations. Myocardial Stunning After Multiple Completely Reversible Ischemic Episodes Repeated brief (5 or 10 minutes) coronary occlusions have a cumulative effect on systolic function and result in prolonged contractile impairment despite absence of irreversible damage The largest decrease in contractility occurs after the first occlusion; with subsequent occlusions, the additional decrements in mechanical performance become progressively smaller.20 (Whether repetitive ischemia also has a cumulative effect on the time to complete recovery, however, remains unclear.) This model of

2 724 Circulation Vol 82, No 3, September 1990 Factors That Determine the Severity of Myocardial Stunning In conscious dogs undergoing a 15-minute coronary occlusion, there is a sensitive coupling between the degree of myocardial dysfunction after reperfu- Downloaded from by guest on July 13, 2018 TABLE 1. Classification of Myocardial Stunning and Evidence for Various Mechanisms Proposed in Experimental Animals Evidence for pathogenetic role of: Sarcoplasmic Reduced Damage of Oxygen reticulum Calcium calcium collagen Experimental setting radicals dysfunction overload sensitivity matrix Stunning due to decreased blood flow Regional ischemia One completely reversible ischemic episode + +? - - Multiple completely reversible ischemic episodes + +? - + One partly irreversible ischemic episode (subendocardial infarction)??? Global ischemia Isolated heart in vitro +? + + Cardioplegic arrest in vivo +???? Stunning due to increased 02 demands Exercise-induced ischemia -? +, Published studies support this mechanism; + +, published studies from multiple laboratories consistently support this mechanism, and evidence is also available in conscious animal preparations; -, published studies do not support this mechanism; ±, published studies are conflicting;?, no data are available. myocardial stunning differs from the single 10- or 15-minute occlusion model in that the mechanical dysfunction develops gradually and is associated with a considerably greater total ischemic burden (50-60 versus minutes). Myocardial Stunning After One Partly Irreversible Ischemic Episode (Subendocardial Infarction) In the dog, when reperfusion is instituted after a period of coronary occlusion of more than 20 minutes but less than 3 hours, the subendocardial portion of the region at risk is generally found to be infarcted, whereas variable quantities of subepicardial tissue remain viable.2 The recovery of function in this subepicardial region salvaged by reperfusion is, however, delayed for days or weeks.8,21-25 It is particularly difficult to evaluate the effect of therapy on this form of postischemic dysfunction because the reperfused region contains a complex admixture of necrotic subendocardium and stunned subepicardium, and the relative proportions of these two components are highly variable. Other confounding factors include the tethering of surviving myocytes by dead, nonfunctional tissue; the expansion of the infarcted region; and the progressive replacement of necrotic myocardium by scar. Myocardial Stunning After Global Ischemia in Isolated Hearts The occurrence of cell death in these preparations depends on species, temperature, duration of ischemia, and perfusate composition. Under selected conditions, isolated hearts reperfused after transient ischemia exhibit complete normalization of phosphocreatine content and intracellular ph,26-34 indicating that viability is generally preserved. Although in these models the reversibility of the contractile abnormalities cannot be verified, the metabolic recovery suggests that the injury is mostly nonlethal. Accordingly, despite the numerous obvious differences from ischemia in vivo, under selected circumstances isolated heart preparations can mimic myocardial stunning. In other cases, however, these preparations may be associated with significant cell death,35-44 and their relevance to myocardial stunning is questionable. Myocardial Stunning After Global Ischemia During Cardioplegic Arrest In Vivo Despite the use of hypothermic cardioplegia, global ischemia in intact animals is usually followed by prolonged contractile abnormalities The reversibility of these derangements has not been documented, but under carefully controlled conditions they are likely to be due mostly to stunning. Myocardial Stunning After Exercise-Induced Ischemia Stresses such as exercise provoke myocardial ischemia and dysfunction in animals with a flow-limiting coronary stenosis. With cessation of exercise, these contractile abnormalities persist.49 Thus, myocardial stunning can also occur after high-flow ischemia, in which the primary problem is an increase in oxygen requirements rather than a decrease in supply. Because of the many significant pathophysiological differences among these situations, one cannot assume that observations made in one setting necessarily apply to the others. An important, unresolved issue is whether all forms of stunning share a common pathogenesis.

3 Impairment of Sympathetic Neural Responsiveness After 25 minutes of coronary occlusion, the reperfused myocardium loses its inotropic responsiveness to sympathetic nervous stimulation, suggesting injury to the sympathetic-neural axis.69 However, no such defect is observed after 15 minutes of coronary occlusion.70 Furthermore, as noted above, myocardial stunning occurs in isolated (and thereby denervated) hearts. Although functional sympathetic denervation may contribute to postischemic dysfunction in certain situations in which contractility is strongly dependent on sympathetic drive, it seems unlikely that this abnormality plays a major role in most experimental preparations of myocardial stunning. Impairment of Myocardial Perfusion When myocardial stunning is produced by a 15- minute coronary occlusion, postreperfusion subepi- Downloaded from by guest on July 13, 2018 TABLE 2. Mechanisms Proposed for Myocardial Stunning Most likely mechanisms Generation of oxygen-derived free radicals Excitation-contraction uncoupling due to sarcoplasmic reticulum dysfunction Calcium overload Other proposed mechanisms Insufficient energy production by mitochondria Impaired energy use by myofibrils Impairment of sympathetic neural responsiveness Impairment of myocardial perfusion Damage of the extracellular collagen matrix Decreased sensitivity of myofilaments to calcium sion and the magnitude of blood flow reduction during the preceding period of ischemia, whereby even small differences in ischemic perfusion are associated with large differences in postischemic recovery.12 (In open-chest dogs, this correlation is weaker,50 probably because of the confounding effects of anesthesia and trauma.) Furthermore, the severity of stunning is greater in the inner layers of the left ventricular wall, which are the most severely ischemic, than in the outer layers.'4'51 Another important factor is the duration of flow deprivation: The longer the ischemic period, the greater the ensuing mechanical abnormalities.9'52 In conclusion, the severity of postischemic dysfunction is determined primarily by the severity and duration of the antecedent ischemia. This concept has two important implications. First, whatever may be the precise mechanism responsible for stunning, such mechanism must be initiated and modulated by perturbations associated with ischemia. Although stunning appears to be a form of "reperfusion injury" (see below), it is ischemia that "primes" the myocardium for the development of such injury. Second, any intervention that improves perfusion during ischemia would be expected to attenuate stunning after reflow. Reducing the severity of ischemia is probably the most effective way to reduce postischemic dysfunction. Mechanism of Myocardial Stunning Thus, in very general terms, the abnormalities of the postischemic myocardium are governed by the abnormalities occurring during ischemia. But what is the specific sequence of events whereby transient ischemia leads to prolonged depression of contractility? Several different hypotheses, which are not necessarily exclusive, have been proposed (Table 2) and are reviewed below. Bolli Mechanism of Stunned Myocardium 725 Insufficient Energy Production by Mitochondria ATP levels in the stunned myocardium are depressed and recover slowly,8, with a time course similar to that of contractile function.8'24 Thus, in the early 1980s, the hypothesis was advanced",54'55 that postischemic dysfunction may result from an inability of the myocardium to resynthesize enough high-energy phosphates to sustain contractile function, possibly because of loss of adenine nucleotide precursors. Numerous subsequent observations, however, have refuted this theory. First, no correlation is observed between myocardial ATP levels and recovery of contractility in several models of postischemic dysfunction.26'27,56-59 Second, the content of phosphocreatine in the stunned myocardium is normal or supranormal ("phosphocreatine overshoot"),8'26'27'3053'55'57'60 implying that the phosphorylation ability of the mitochondria is intact. Third, the stunned myocardium responds to inotropic stimuli with a marked and sustained increase in contractility, such that function exceeds the levels measured at baseline.17,30,61-66 This striking inotropic response occurs without a decrease in ATP or phosphocreatine stores,30,63 indicating that energy production is not inherently impaired and can keep pace with high metabolic demands. Fourth, manipulations that increase ATP levels in the stunned myocardium fail to increase mechanical function.33,67 In summary, the available evidence suggests that a defect in the rate of resynthesis of ATP by the mitochondria is not the primary cause of myocardial stunning. Impaired Energy Use by Myofibrils Myocardial stunning is associated with a decrease in myofibrillar creatine kinase activity68 and a reduction of the substrate (free ADP) used by the myofibrillar creatine kinase to produce ATP at the contraction site.30,68 Both of these abnormalities might disrupt normal energy use and thus have been postulated to contribute to postischemic dysfunction.68 This hypothesis, however, appears implausible because it does not explain the considerable contractile and metabolic reserve of the stunned myocardium.17'30'61-66 Indeed, the reversal of postischemic dysfunction by inotropic stimuli implies that the residual activity of myofibrillar creatine kinase is still sufficient to run the myofibrillar ATPase reaction at normal or supranormal rates.

4 726 Circulation Vol 82, No 3, September 1990 cardial blood flow is normal, whereas subendocardial flow is slightly decreased (-20%)."-14,71-73 The mechanism and significance of this phenomenon are unclear.72,73 Although it is possible, as speculated by Heyndrickx et al,1' that the reduced subendocardial perfusion may contribute to the impaired mechanical performance, two considerations suggest that it is not a major causative factor. First, there is no correlation between subendocardial blood flow and wall thickening in the postischemic myocardium.72,73 Second, the loss of systolic function (dyskinesis or akinesis) is out of proportion to the slight (-20%) decrease in subendocardial perfusion.72'73 When myocardial stunning is produced by repeated 5-minute coronary occlusions, blood flow to the postischemic myocardium, measured by radioactive microspheres, is generally normal.15-17,19 However, microspectrophotometry reveals increased veinto-vein heterogeneity of oxygen saturations, with an excess of vessels demonstrating very low oxygen saturations, suggesting that the stunned myocardium contains discrete microregions of either increased oxygen extraction or reduced flow.19 A nonuniform reduction of flow (microregions of ischemia intermixed with microregions of hyperemia) has also been invoked to explain the enhancement of function that is observed in stunned myocardium when perfusion is augmented.16 However, the findings that myocardium stunned by repeated 5-minute coronary occlusions has normal flow reserve'6 and intact microvascular patency'9 exclude nonuniform ischemia secondary to anatomical obstruction. Furthermore, the hypothesis of persistent ischemia as a cause of dysfunction does not explain the normal or near-normal contractile reserve of the postischemic myocardium.17'65,66 In summary, impaired perfusion (either uniform or nonuniform) is unlikely to be the primary pathogenetic mechanism in postischemic contractile failure. Damage of Extracellular Collagen Matrix Using a model of myocardial stunning produced by 12 5-minute coronary occlusions separated by 10 minutes of reperfusion, Eng's group has demonstrated that postischemic dysfunction is associated with severe ultrastructural damage of the extracellular collagen matrix74 and a modest (-10%) loss of collagen.75 These findings have led to the hypothesis that myocardial stunning may be due to a structural defect, namely, disruption of the mechanical coupling function provided by the extracellular collagen network.74 To fully evaluate this hypothesis, it will be important to explore the time course of the collagen changes and its relation to the time course of postischemic dysfunction. Because in conscious dogs undergoing one brief (c15 minutes) coronary occlusion most of the contractile recovery takes place in the first 3-4 hours of reperfusion9'12,13,52 (an interval too short for significant resynthesis or remodeling of the collagenous components), it seems improbable that structural damage to collagen plays a pathogenetic role in this model of myocardial stunning. Indeed, a recent study76 demonstrated that collagen is disrupted by 12 5-minute coronary occlusions but not by one 15-minute occlusion followed by reperfusion. In summary, collagen damage may contribute to myocardial stunning after multiple ischemic episodes but is unlikely to be a causative factor after a single completely reversible ischemic episode (Table 1). One possible explanation for this difference is that in the former setting, repetitive ischemia results in repetitive systolic bulging for a total duration of more than 15 minutes; this may cause greater mechanical stress and, consequently, greater damage of the extracellular collagen network. Decreased Sensitivity of Myofilaments to Calcium Using isolated ferret hearts subjected to 15 minutes of global ischemia at 370 C, Kusuoka et a127 observed that the stunned myocardium exhibits decreased responsiveness to calcium, as manifested by a decrease in the maximal calcium-activated force and a decrease in the myocardial sensitivity to extracellular calcium. They speculated that the reduced sensitivity to extracellular calcium could in turn be due to either a decrease in the intracellular free Ca21 concentration ([Ca]i) transient or a decrease in the sensitivity of myofilaments to calcium.27 The former possibility seems improbable because two recent studies with different techniques32'77 indicate that the calcium transient is (paradoxically) increased in the stunned myocardium after 1532 or 20 minutes77 of global ischemia at 370 C in isolated ferret hearts. Accordingly, it has been proposed32'77 that the fundamental mechanism for postischemic dysfunction is not insufficient availability of free cytosolic calcium during systole but instead is reduced sensitivity of the contractile apparatus to calcium. One problem with this hypothesis derived from in vitro studies is that it does not explain two observations made in vivo. First, when the stunned myocardium is challenged with inotropic stimuli, it exhibits a normal or near-normal contractile reserve.1765,66 Second, the apparent sensitivity of the stunned myocardium to intracoronary calcium is not decreased.65 If the primary problem was a reduced sensitivity of myofilaments to calcium, then the contractile response to exogenous calcium or to other inotropic agents (which act by raising [Ca]i) should be reduced. In summary, studies in vitro suggest that myocardial stunning is a result of reduced calcium sensitivity rather than reduced calcium availability, but studies in vivo are not readily reconcilable with this interpretation (Table 1). Calcium Overload Several lines of evidence point to a pathogenetic role of calcium overload in myocardial stunning. First, when isolated ferret hearts subjected to 15 minutes of global normothermic ischemia are reperfused with solutions containing low concentrations of calcium, the postischemic contractile abnormalities

5 are significantly attenuated.27 The results of this experiment, in which no intervention is applied during ischemia, indicate that calcium entry upon reperfusion is an important mechanism of myocardial stunning. A decrease in the severity of stunning is also observed in hearts pretreated with ryanodine, an inhibitor of cellular calcium overload.29 Second, exposure of isolated ferret hearts to a transient calcium overload in the absence of ischemia produces mechanical and metabolic abnormalities similar to myocardial stunning.78 Third, transient intracellular acidosis during early reperfusion can prevent myocardial stunning in isolated ferret hearts subjected to 15 minutes of global ischemia31 and open-chest dogs undergoing 15 minutes of coronary occlusion.79 Because acidosis antagonizes not only the influx of calcium into cells (by inhibiting the Na4-Ca21 exchange and the slow calcium channels) but also the intracellular binding of calcium,31 these results provide indirect evidence that a transient calcium overload contributes to postischemic dysfunction. Fourth, recent studies28'80'81 have shown that [Ca]i increases between 10 and 20 minutes of global ischemia in isolated hearts. This is the same duration of ischemia that results in myocardial stunning. In these models, [Ca]i returns to normal values within a few minutes after reperfusion,28,7780 but appears to remain transiently elevated during very early reflow.77'82 The mechanism for this rise in [Ca]i during ischemia remains uncertain. It may be secondary to decreased calcium uptake by the sarcoplasmic reticulum.83 It may also be mediated through the Na+-Ca2' exchange, secondary to a rise in intracellular Na+ during ischemia due to 1) metabolic inhibition of the Na+,K+-ATPase, and 2) acidosis and consequent Na+-H+ exchange Because the Na+- Ca2' exchange is inhibited by acidosis and intracellular ph in the stunned myocardium normalizes quickly after reflow 27,30'31'33'60 entry of calcium via the Na4-Ca2' exchange may be greatly increased upon reperfusion.84,86-88 Further research will be necessary to identify the precise circumstances leading to calcium overload. The mechanisms by which a transient calcium overload induces prolonged contractile dysfunction are also unclear. Increased cytosolic calcium can activate phospholipases and other degradative enzymes.6,34 In addition, elevation of cytosolic calcium could trigger production of oxygen radicals via xanthine oxidase89 (see below). The calcium overload theory is not incompatible with the fact that exogenous calcium ameliorates function in the stunned myocardium.27,65 The increase in [Ca]i is postulated to be a brief phenomenon immediately after reflow, following which there may be either a normalization of [Ca]i transients or a relative calcium deficiency (see "Excitation- Contraction Uncoupling Due to Sarcoplasmic Reticulum Dysfunction"). The early excessive [Ca]i levels could damage intracellular organelles concerned with contraction and thereby produce prolonged mechanical dysfunction. Bolli Mechanism of Stunned Myocardium 727 In summary, considerable evidence suggests that a transient calcium overload during early reperfusion contributes to the pathogenesis of myocardial stunning after global ischemia in vitro. The role of calcium overload in in vivo models of myocardial stunning remains to be defined (Table 1) and represents an important area for future investigations. Effect of calcium channel blockers. Calcium channel blockers have been used to gain insights into the role of altered calcium homeostasis in vivo, but unfortunately the results are difficult to interpret. Verapamil,59 diltiazem,90 nifedipine,91 nitrendipine,92 and amlodipine93 have been shown to improve recovery of function in regionally stunned myocardium in intact animals. However, it is unclear whether these beneficial effects reflected a direct protective action of the drugs or were mediated by favorable modifications of afterload, preload, heart rate, and regional myocardial blood flow, all of which could change the systolic properties of the stunned myocardium.16,59 A direct protective action was demonstrated by a recent study94 in which nifedipine enhanced the functional recovery of the stunned myocardium independently of any effect on systemic hemodynamics or regional myocardial blood flow. However, interpretation of these results is problematic because the beneficial effects of nifedipine were noted even when treatment was started 30 minutes after reperfusion,94 whereas myocardial stunning has been suggested to result from a transient calcium overload immediately after reflow.27 It is unlikely that nifedipine acted in a specific way to decrease [Ca]i because administration of calcium or inotropic agents (which raise [Ca]i) improves contractile function in the stunned myocardium.17,30,61-66 The mechanism of action of nifedipine in this intriguing study94 remains to be elucidated. Excitation-Contraction Uncoupling Due to Sarcoplasmic Reticulum Dysfunction Krause et a195 investigated this mechanism in a canine model of postischemic dysfunction produced by eight to 12 5-minute occlusions separated by 10-minute reflow periods. Sarcoplasmic reticulum isolated from the stunned myocardium demonstrated a decrease in the ability to transport calcium, concomitant with a reduction in the activity of the Ca2+,Mg2+-ATPase.95 These researchers proposed that a decrease in the amount of calcium stored in the sarcoplasmic reticulum as a result of a reduction in the calcium pump activity could diminish contractile protein activation through attenuated calcium release during systole.95 The concept of inadequate delivery of calcium to the contractile proteins, secondary to sarcoplasmic reticulum dysfunction, is an attractive hypothesis. The postulated relative calcium deficiency would be consistent with the observation that exogenous administration of calcium can return contractile function of stunned myocardium to preischemic levels.65 It would

6 728 Circulation Vol 82, No 3, September 1990 also be consistent with the notion that inotropic agents (which increase [Ca]i) can reverse myocardial stunning.17'30'61-66 However, since this hypothesis implies that the amplitude of the [Ca]i transient is decreased, it is not easily reconcilable with in vitro data32'77 (see above). Considerably more research is necessary to determine whether similar dysfunction of the sarcoplasmic reticulum occurs in other models of myocardial stunning (Table 1) and whether it is associated with decreased [Ca]i transients in vivo. In addition, the factors that cause injury to the sarcoplasmic reticulum remain to be elucidated. Generation of Oxygen-Derived Free Radicals Effect of antioxidants on myocardial stunning after a brief coronary occlusion. Approximately 7 years ago, a number of investigators, including ourselves, postulated that myocardial stunning is caused in part by the generation of reactive oxygen metabolites, such as superoxide anion (02 ), hydrogen peroxide (H202), and hydroxyl radical (.OH), and began a series of experiments designed to test this hypothesis. We used an open-chest canine preparation in which the left anterior descending coronary artery is occluded for 15 minutes and then reperfused.96 In the first study,97 we found that administration of superoxide dismutase (SOD) (an enzyme that catalyzes the dismutation of *02 to 02 and H202) and catalase (an enzyme that reduces H202 to 02 and H20) significantly enhanced recovery of function after reperfusion. Attenuation of myocardial stunning by SOD and catalase was also observed by other investigators50'57'98 using similar experimental preparations. In a more recent study99 we found that neither SOD nor catalase alone significantly improved recovery of function in the stunned myocardium; however, when the two enzymes were combined, contractile recovery was significantly greater than that observed in controls or in dogs receiving either enzyme alone. These results suggest that both *02- and H202 contribute to the cellular damage responsible for myocardial stunning and that combined administration of SOD and catalase is more likely to be effective against stunning than separate administration. The inability of SOD alone to mitigate postischemic dysfunction has also been observed in a pig model.100 Although both * 2- and H202 appear to contribute to stunning, it remains uncertain whether they do so by direct cytotoxicity or via formation of other species. * 2 and H202 can interact through the metal-catalyzed Haber-Weiss reaction to generate the highly reactive *OH radical.10' Consequently, accumulation of * 2 and H202 could produce postischemic dysfunction, at least in part, indirectly through generation of OH. To elucidate this problem, we evaluated dimethylthiourea, an -OH scavenger that is more effective than traditional *OH scavengers102 and does not react with * 2 or H202 in vitro.'03 We observed that dimethylthiourea produced a significant and sustained improvement in the function of the stunned myocardium.103 These results were further corroborated by studies with N-2-mercaptopropionyl glycine (MPG), a free radical scavenger that readily enters the intracellular space and is active orally. MPG was found to be a powerful scavenger of OH with no effect on *2 or H202 in vitro104 and to effectively attenuate myocardial stunning in vivo Taken together, these results suggest that the OH radical (or one of its reactive products) is a mediator of postischemic dysfunction and that the beneficial effects of SOD and catalase previously demonstrated5057'97-99 are due in part to prevention of OH generation. This conclusion is also consistent with the finding that the iron chelator desferrioxamine attenuates postischemic dysfunction.106"07 Because iron catalyzes the formation of OH (through the Haber-Weiss or Fenton mechanism) as well as the propagation of.oh-initiated lipid peroxidation,10' the protective effects of desferrioxamine are compatible with the view that OH is a mediator of myocardial stunning. The effectiveness of antioxidants that scavenge.oh or prevent its generation should not be construed as evidence that all of the damage initiated by oxygen is mediated via OH. It is true that H202 is a relatively nonreactive species and its toxicity may be due in large part to its reduction to OH.101 However, there is evidence that * 2 can directly cause cellular toxicity in vitro (Reference 108; reviewed in Reference 109). Furthermore, because catalase alone prevents formation of OH by removing H202, the fact that SOD has to be added to catalase to produce significant attenuation of stunning in vivo99 implies the existence of a component of damage specifically due to *02- It appears, therefore, that all three species ( 2-, H202, and *OH) are important in postischemic dysfunction (O2- and *OH as mediators of injury and H202 as a precursor of OH).99 Although the studies discussed above50,57,97-99, consistently support the oxyradical hypothesis, their significance is limited by the fact that they were all performed in open-chest animals. Thus, artifacts due to the combined effects of anesthesia, hypothermia, surgical trauma, volume and ionic imbalances, nonphysiological conditions, and associated neurohumoral perturbations, as well as other potentially confounding variables, cannot be excluded. It is therefore essential that the oxyradical hypothesis be tested in conscious animal preparations. In a recent experiment,"10 we observed that the combined administration of SOD and catalase significantly enhanced recovery of function after a 15-minute coronary occlusion in conscious, unsedated dogs and that this effect was sustained for 24 hours after reflow. The accelerated recovery was not followed by any subsequent deterioration, indicating that postischemic depression of contractility is not a useful "protective" response to injury. In summary, numerous investigations from several independent laboratories5057,97-99, , 10 uniformly suggest that oxygen metabolites play a significant role

7 in the genesis of myocardial stunning after a 15- minute period of ischemia, in both open-chest and conscious animals. The relative contributions of the various metabolites have not been definitively established. It is clear that a portion of the damage is mediated by OH; however, the available evidence suggests that myocardial stunning cannot be simplistically ascribed to a single oxygen species and that all of the three initial metabolites of oxygen (02, H202 and *OH) contribute to the cellular injury responsible for postischemic dysfunction. Iron also appears to play a role in stunning, presumably by catalyzing the formation of OH. Direct evidence for the oxyradical hypothesis. A major limitation of the studies reviewed heretofore50,57,97-99, ,110 is that all of the evidence provided by these investigations is indirect and therefore inconclusive. Clearly, to definitively confirm a causative role of oxygen metabolites in postischemic dysfunction, it is necessary to directly demonstrate and quantitate free radical generation in the stunned myocardium in the presence and absence of antioxidant interventions. Production of free radicals has been directly demonstrated in isolated rabbit or rat hearts undergoing global ischemia and reperfusion,42, but because of the numerous important differences in experimental conditions, results obtained in vitro cannot necessarily be extrapolated to the intact animal. We used the spin trap a-phenyl N-tert-butyl nitrone (PBN) and electron paramagnetic resonance (EPR) spectroscopy to detect and measure production of free radicals in our in vivo model of postischemic dysfunction (15-minute coronary occlusion in open-chest dogs).14 After infusion of PBN, EPR signals characteristic of PBN radical adducts were detected in the venous blood draining from the ischemic-reperfused vascular bed.15 The EPR signals were consistent with a mixture of different secondary lipid radicals, such as alkyl and alkoxy radicals. The myocardial production of radicals began during coronary occlusion but increased dramatically after reperfusion, peaking at 2-4 minutes. After this initial burst, production of radicals abated but did not cease, persisting for as long as 3 hours after reflow.1" There was a linear, positive relation between the magnitude of adduct production and the magnitude of ischemic flow reduction, indicating that the intensity of free radical generation after reflow is determined by the severity of the antecedent ischemia"15-the greater the degree of hypoperfusion, the greater the subsequent production of free radicals and, by inference, the severity of reperfusion injury. These findings imply that interventions that improve perfusion during ischemia will attenuate free radical reactions after reflow. Generation of free radicals in in vivo models of regional myocardial stunning has also been reported by Leiboff et al.1"6 In a subsequent study,17 we found that SOD plus catalase suppressed the production of free radicals in the stunned myocardium, indicating that these radi- Bolli Mechanism of Stunned Myocardium 729 cals are derived from univalent reduction of oxygen. Furthermore, the inhibition of free radical production was associated with inhibition of myocardial stunning.17 More recently, we observed that MPG or desferrioxamine administered just before reperfusion markedly attenuated myocardial stunning and the associated production of PBN adducts04,114; however, the same agents given 1 minute after reperfusion did not attenuate myocardial stunning or initial PBN adduct production. Thus, three different antioxidant interventions (SOD plus catalase, MPG, and desferrioxamine) reduced postischemic dysfunction at the same doses and under the same experimental conditions in which they reduced formation of PBN adducts. The correlation between the two effects suggests that the production of free radicals in the stunned myocardium plays a causal role in the depression of contractility. In summary, the results of the studies that have measured free radicals in the stunned myocardium provide direct evidence supporting a pathogenetic role of oxygen metabolites. Specifically, these studies indicate that 1) free radicals are produced in the stunned myocardium in the intact dog after reversible regional ischemia, 2) the univalent pathway of reduction of oxygen is the source of the radicals, and 3) inhibition of free radical reactions results in enhanced recovery of contractility (i.e., the radical reactions are necessary for postischemic dysfunction to occur). Effect of oxygen radicals on cardiac function. The ability of oxygen metabolites to depress myocardial function has been directly demonstrated in vitro and in vivo. Exposure of isolated rabbit interventricular septa,18 isolated rat"19 or rabbit'08 papillary muscles, and isolated rat or rabbit'23"124 hearts to free radical-generating solutions or pure H202 has uniformly resulted in decreased mechanical function and ATP levels (i.e., in changes similar to those observed in the stunned myocardium). In most of the studies in which an attempt was made to discern the relative roles of different oxygen species,118,119,122'123 it was found that the deleterious effects could be prevented by catalase or by *OH scavengers but not by SOD, suggesting that H202 or its byproduct, *OH, was the oxygen metabolite responsible for the observed depression of contractile function. In one study,108 however, SOD was protective, whereas catalase had no effect, implying that the major negative inotropic agent was *02. These in vitro observations have been recently expanded by Przyklenk et al,'25 who reported that infusion of xanthine oxidase plus purine plus iron-loaded transferrin, administered through a coronary vein in open-chest dogs, resulted in the development of significant wall motion abnormalities. In summary, it is clear that reactive oxygen species depress myocardial contractility both in vitro and in vivo. The precise role of each species remains to be defined. There is considerable evidence that the detrimental effects of * 2- and H202 on cardiac

8 730 Circulation Vol 82, No 3, September 1990 function are mediated in part by generation of *OH. This is in accordance with the results of in vivo studies,103-'07 which suggest an important role of *OH as a mediator of stunning. However, there is also evidence for a direct negative inotropic action of *02, which is also congruent with observations in vivo.99 Mechanism of oxyradical-mediated contractile dysfunction. The precise mechanism whereby oxygen metabolites depress contractile function remains speculative and represents one of the major unresolved issues pertaining to the pathogenesis of myocardial stunning. Free radicals are reactive species that have no specific target and can attack virtually all cellular components. In theory, every abnormality thus far described in the stunned myocardium (see above) could be caused by oxyradicals. At least two key cellular components, proteins and lipids, are likely to be involved in free radical-initiated reactions in the postischemic myocardium. Activated oxygen species can denaturate proteins and inactivate enzymes.126 Furthermore, they can produce peroxidation of the polyunsaturated fatty acids contained in cellular membranes,127 which would impair selective membrane permeability and interfere with the function of various cellular organelles. Evidence for the occurrence of lipid peroxidation in the stunned myocardium is provided by two recent studies. Romaschin et al128 observed increased myocardial concentration of hydroxy conjugated dienes (which are products of free fatty acid oxidation) during and after a 45-minute period of global normothermic ischemia in open-chest dogs. The maximal concentration of conjugated dienes was measured at 5 minutes of reflow. Importantly, the tissue examined after reperfusion was dysfunctional but not necrotic, thus representing stunned myocardium. Weisel et al129 subsequently reported that in patients undergoing cardioplegic arrest during coronary artery bypass surgery, there was myocardial release of conjugated dienes in the coronary sinus blood at 3 and 60 minutes of reperfusion, which was associated with a decrease in the myocardial concentration of the antioxidant a-tocopherol. Dysfunction of the sarcoplasmic reticulum may be an important mechanism whereby oxyradicals mediate the contractile abnormalities observed after reversible ischemia. As mentioned above, both calcium uptake and Ca2+,Mg2+-ATPase activity have been found to be significantly depressed in the stunned myocardium.95 A similar decrease in calcium uptake and Ca2',Mg2+-ATPase activity is observed in isolated sarcoplasmic reticulum after exposure to oxygen radicals,127,130 and oxyradical scavengers preserve sarcoplasmic reticulum function.'27,130 Other studies suggest that the sarcolemma may be a critical target of free radical-mediated damage. Oxyradicals have been shown to interfere with calcium transport and calcium-stimulated ATPase activity in the sarcolemma.'31132 Oxygen radicals have also been shown to interfere with the Na+- Ca2+ exchange133 and to inhibit the Na+,K+-ATPase activity.134 Furthermore, the sarcolemmal Na+,K+- ATPase activity is impaired in reperfused myocardium, and this impairment is prevented by antioxidants.135 Impairment of the Na+,K+-ATPase activity would result in Na+ overload, with consequent activation of the Na+-Ca2+ exchange activity.87'88 All of these observations imply that excessive production of oxyradicals could result in increased transarcolemmal calcium influx and cellular calcium overload. It is important to point out that the foregoing postulated mechanisms involve alterations in calcium homeostasis and thus would reconcile the oxyradical and calcium-overload hypotheses of stunning into one pathogenetic mechanism. Sources of oxygen radicals in the stunned myocardium. Among the various potential mechanisms of oxyradical generation in the stunned myocardium, only two have been explored thus far, namely, the enzyme xanthine oxidase and the activated neutrophil. We found that the xanthine oxidase inhibitor, allopurinol, produced a marked improvement in the functional recovery of the stunned myocardium.136 Furthermore, allopurinol inhibited the increased cardiac production of urate observed in control dogs during ischemia and early reperfusion, indicating effective inhibition of xanthine oxidoreductase.136 Attenuation of stunning has also been demonstrated with oxypurinol after and 90 minutes138 of ischemia and reperfusion. These data suggest that xanthine oxidase is one of the sources of the oxygen radicals that contribute to postischemic dysfunction in the dog. Whether this concept applies to humans remains uncertain because the myocardial content of xanthine oxidase is species dependent. Reports regarding the myocardial content of the enzyme in the human heart are conflicting In contrast to these earlier reports, in a recent study,'44 oxypurinol and amflutizole, two xanthine oxidase inhibitors, failed to mitigate stunning after 15 minutes of ischemia in dogs. Whether the discrepancy is due to different degrees of inhibition of xanthine oxidoreductase or to other reasons is unclear. Leukocytes are another potential source of oxygen metabolites.145,146 However, several investigators have found that myocardial stunning is not attenuated by depletion of neutrophils with antiserum71'147 or filtration,148 by administration of nafazatrom, which inhibits production of leukotrienes,149 by administration of antibodies to the adhesionpromoting Mol glycoprotein,150 and by administration of dextran, which inhibits leukocyte adherence to the endothelium.151 Recent studies have shown that the neutrophil content of the stunned myocardium after a 12-minute period of ischemia is decreased152 and that myeloperoxidase activity (a marker of neutrophils) in myocardium stunned by a 15-minute occlusion is not different from nonischemic myocardium.150 Furthermore, myocardial stun-

9 Bolli Mechanism of Stunned Myocardium 731 ning occurs in isolated heart preparations that are devoid of circulating neutrophils. Taken together, these results71'147-'52 suggest that granulocytes do not play a major role in the genesis of postischemic dysfunction after brief, reversible ischemia, although they appear to be major mediators of irreversible tissue injury and vascular obstruction after prolonged periods of ischemia.145" In contrast to the aforementioned findings, however, others have reported that depletion of granulocytes by Leukopak filtration prevents154 or alleviates155 postischemic dysfunction after a 15-minute coronary occlusion. The reasons for the differences between these studies'54"155 and the aforementioned investigations71" are unclear and warrant additional research.156 One possibility to be explored, raised by Engler and Covell,154 is that the relation between circulating neutrophil counts and myocardial damage may be nonlinear. In summary, in the canine model of myocardial stunning produced by one reversible ischemic episode, xanthine oxidase may be a source of free radicals (although the data are not entirely consistent), whereas the role of neutrophils remains controversial. Of course, many other processes could lead to formation of 2- and H202 during myocardial ischemia and reperfusion, including activation of the arachidonate cascade, autoxidation of catecholamines and other compounds, ischemia-induced damage of the electron transport chain in the mitochondria, and accumulation of reducing equivalents.127 It will be difficult to selectively assess the role of these potentially important mechanisms in a complex system such as the intact animal. Identification of the sources of oxyradicals in the stunned myocardium represents another major unresolved issue. Time course of free radical-induced damage in the stunned myocardium. When exactly in the ischemiareperfusion sequence that leads to stunning does the crucial free radical-induced damage take place? In a recent study,104 we observed that infusion of the antioxidant MPG attenuated postischemic dysfunction to a similar extent regardless of whether the infusion was started before ischemia or 1 minute before reperfusion; however, infusion started 1 minute after reflow was ineffective, suggesting that the critical radical-mediated injury occurs in the first few moments of reperfusion. We have subsequently obtained similar results with desferrioxamine.14 Furthermore, the spin trap PBN enhances contractile recovery even when the infusion is commenced 20 seconds before reflow; the magnitude of the protective effect is similar to that observed when the infusion is commenced before ischemia.1" Captopril, an angiotensin converting enzyme inhibitor with alleged * 2 scavenging properties, produces similar attenuation of postischemic dysfunction when the administration is started before ischemia or 2 minutes before reperfusion.157 The conclusion that a substantial portion of the cellular damage responsible for stunning occurs immediately after reflow is further corroborated by direct measurements of free radicals104"'15"17 and lipid peroxidation products128 in the stunned myocardium, both of which have shown a burst in the initial moments after restoration of flow. If free radical production is inhibited during this initial burst, postischemic dysfunction is mitigated; however, if free radical production is inhibited after the first 5 minutes of reperfusion (i.e., after the initial burst), no functional improvement is observed.04 These observations suggest that the free radicals important in causing myocardial stunning are those produced immediately after reflow. In summary, myocardial stunning appears to be, at least in part, a form of oxyradical-mediated "reperfusion injury." This concept may have significant therapeutic implications because it suggests that antioxidant therapies begun after the onset of ischemia could still be effective in preventing postischemic dysfunction; however, a delay in the implementation of such therapies until after reperfusion would result in loss of efficacy. Role of oxygen radicals in other forms of myocardial stunning (Table 1). Studies reviewed thus far used one brief (.15 minutes) coronary occlusion. In recent unpublished studies in our laboratory, we found that the *OH scavenger MPG markedly improves contractile recovery after 10 5-minute coronary occlusions separated by 10-minute reflow periods in open-chest dogs. Thus, oxyradicals also appear to contribute to the genesis of myocardial stunning after multiple brief ischemic episodes. Furthermore, numerous studies from the surgical literature suggest that oxygen radicals also contribute to postischemic dysfunction after global ischemia in in vivo models of cardioplegic arrest,45-48 and there is evidence that oxidative stress contributes to postoperative dysfunction in patients undergoing cardiac surgery.158 Finally, antioxidants consistently alleviate mechanical dysfunction after global ischemia in isolated hearts, but the relevance of these in vitro preparations to myocardial stunning is uncertain, as discussed above. Data regarding the role of oxygen radicals in myocardial stunning after a prolonged coronary occlusion are conflicting. Three studies failed to detect improvement in functional recovery with SOD and catalase after coronary occlusions lasting 1 hour,'59 90 minutes,160 or 2 hours16' in open-chest'6' or conscious dogs.'59 160Wel62 also observed that SOD fails to enhance recovery of contractility after a 2-hour coronary occlusion in anesthetized dogs. These results suggest that short-term administration of antioxidant enzymes is not effective in mitigating myocardial stunning associated with subendocardial infarction. It may be that the postischemic dysfunction that follows a prolonged, partly irreversible ischemic insult differs from the dysfunction that follows a short, completely reversible insult. However, other studies138"63 have shown that the cell-permeant antioxidants oxypurinol and N-

10 732 Circulation Vol 82, No 3, September 1990 acetylcysteine attenuate myocardial stunning independently of infarct size limitation in closed-chest dogs subjected to 90 minutes of coronary occlusion and 24 hours of reflow. Initial observations164 indicate that SOD and catalase do not alleviate exercise-induced stunning. In summary, there is strong evidence that oxyradicals contribute to postischemic dysfunction after global ischemia (in vitro as well as in vivo) and after multiple episodes of regional ischemia. They do not appear to contribute to exercise-induced postischemic dysfunction. The role of oxygen radicals in myocardial stunning after a prolonged, partly irreversible ischemic insult remains uncertain and represents a major unresolved problem. Elucidation of this issue will be difficult because the dysfunction is due in part to the presence of infarction and in part to the presence of stunning - a situation that complicates the evaluation of therapy, as discussed above. Integration of Different Hypotheses Myocardial stunning is probably a multifactorial process that involves complex sequences of cellular perturbations and the interaction of multiple pathogenetic mechanisms. Our understanding of this phenomenon is still fragmentary, and none of the theories discussed herein (Table 2) explains the entire cascade of events that culminates in postischemic contractile failure. For example, the oxyradical hypothesis does not explain how reactive oxygen species cause mechanical dysfunction, and the sarcoplasmic reticulum theory does not explain why this organelle is damaged. Integration of the various hypotheses is complicated by the fact that for the most part, each hypothesis has been developed in a different experimental preparation (Table 1). Nevertheless, it is important to emphasize that these hypotheses are not mutually exclusive and in fact may represent different parts of the same pathophysiological sequence. There is considerable evidence to suggest a link between generation of oxygen radicals and impaired calcium homeostasis in the setting of reperfusion injury. For example, the damage associated with the "calcium paradox" resembles that associated with the "oxygen paradox" and probably has a similar pathogenetic mechanism.165 Furthermore, as discussed above, oxyradicals generated upon reperfusion can cause dysfunction of the sarcoplasmic reticulum127,130 and alter calcium flux across the sarcolemma The result of these actions would be excitation-contraction uncoupling and cellular calcium overload In this regard, it has recently been demonstrated that reoxygenation of cultured myocytes results in Ca'+ overload, which can be greatly attenuated by antioxidant enzymes.85 Oxygen radicals could also damage the proteins of the contractile machinery in a manner that reduces their responsiveness to calcium. On the other hand, calcium overload may exaggerate oxyradical production by promoting the conversion of xanthine dehydrogenase to xanthine oxidase, which appears to be mediated by a calcium-dependent protease.89 A unifying hypothesis for the pathogenesis of myocardial stunning is proposed in Figure 1 (detailed description of the postulated mechanisms is provided in the figure legend). This paradigm is largely speculative but nevertheless based on the evidence available at this time and discussed in this review. According to this conceptual scheme (Figure 1), oxyradical generation, calcium overload, and sarcoplasmic reticulum dysfunction can be viewed as different aspects of the same pathogenetic mechanism, thereby reconciling the three major current hypotheses of myocardial stunning. In summary, the pathogenesis of myocardial stunning has not been definitively determined. Thus far, researchers have focused on the identification of individual mechanisms of injury. Three abnormalities (oxyradical generation, calcium overload, and excitation-contraction uncoupling) have emerged as likely contributing factors (although in different experimental preparations), and in vitro evidence suggests that they are interrelated. It is now essential to clarify the precise interactions among these three factors in vivo and the role that each of them plays in the various experimental settings of myocardial stunning. Clinical Implications There are numerous clinical settings in which the myocardium is subjected to transient ischemia, including unstable or variant angina, acute myocardial infarction with early reperfusion (either spontaneous or induced by thrombolytic therapy), openheart surgery with cardioplegic arrest, and cardiac transplantation. Recent observations suggest that these situations may be associated with myocardial stunning, which may be an important factor precipitating left ventricular failure with its attendant morbidity and mortality.1,166 Furthermore, in patients in whom silent episodes of ischemia recur at short intervals in the same territory, the myocardium might remain reversibly depressed for prolonged periods of time or even chronically.1 Elucidation of the mechanism responsible for postischemic dysfunction is indispensable for developing effective clinical therapies. The evidence discussed herein implies that myocardial stunning could be effectively attenuated by pharmacological manipulations targeted at specific pathogenetic mechanisms, for example, by antioxidant agents given before reperfusion. Compared with standard treatments for cardiac failure such as inotropic stimulation, antioxidants represent a novel approach; by interfering with the mechanism of cell injury, they might prevent rather than simply temporarily correct postischemic dysfunction. This approach also appears desirable because it is uncertain whether prolonged inotropic stimulation of stunned myocardium has deleterious effects.

11 REVERSIBLE ISCHEMIA / REPERFUSION Bolli Mechanism of Stunned Myocardium 733 IN over Xanthine oxidase?,? t Reducing equivalents eutrophil activation? * *-? tmitochondrial "univalent leak" Arachidonate cascade? -- "? Autoxidative processes _ SOD Ja+ *0 2 H2 2 rload I FeFI1 *OH. Downloaded from by guest on July 13, 2018 i Enzyme inactivation KhhO.u Lipid peroxidation Damage of: <eofjj t? OTHER S TRUCTURES /"? SARCOPLASMIC *****K (e.g., CONTRACTIIILE or COLLAGEN? SARCOLEMMA RETICULUM PROTEINSS MATRIX) + f+ + f 'Ca+" overload Altered Cat+ homeostasis I Sensitivity to Ca++ Excitation-contraction uncoupling- L.IMECHANICAL DYSFUNCTION..~ I Loss of mechanical coupling FIGURE 1. Proposed pathogenesis of postischemic myocardial dysfunction. This proposal integrates and reconciles different mechanisms into a unifyingpathogenetic hypothesis. Transient reversible ischemia followed by reperfusion could result in increased production of superoxide radicals ( 02 ) through several mechanisms, including 1) increased activity of xanthine oxidase, 2) activation ofneutrophils, 3) activation ofarachidonate cascade, 4) accumulation ofreducing equivalents during oxygen deprivation, 5) derangements of intramitochondrial electron transport system resulting in increased univalent reduction of oxygen, and 6) autoxidation of catecholamines and other substances. Superoxide dismutase (SOD) dismutates '02- to hydrogen peroxide (H202); in the presence of catalytic iron, '02- and interreact in a Haber- Weiss reaction to generate the hydroxyl radical (.OH). H202 can also generate *OH in the absence of '02 through a Fenton reaction provided that other substances (such as ascorbate) reduce Fe (III) to Fe (II). '02- and OH attack proteins and polyunsaturated fatty acids, causing enzyme inactivation and lipid peroxidation, respectively. In reversible ischemia, the intensity of this damage is not sufficient to cause cell death but is sufficient to produce dysfunction ofkey cellular organelles. Postulated targets offree radical damage include 1) the sarcolemma, with consequent loss of selective permeability, impairment of calcium-stimulated ATPase activity and calcium transport out of the cell, and impairnent of Na+K+-ATPase activity (the net result of these perturbations would be increased transarcolemmal calcium influx and cellular calcium overload); 2) the sarcoplasmic reticulum, with consequent impairment of calcium-stimulated A TPase activity and calcium transport (this would result in impaired calcium homeostasis- specifically, decreased calcium sequestration, which would contribute to increase free cytosolic calcium, and decreased calcium release during systole, which would cause excitation-contraction uncoupling); and 3) possibly other structures, such as the extracellular collagen matrix (with consequent loss of mechanical coupling) or the contractile proteins (with consequent decreased sensitivity to calcium). At the same time, reversible ischemia-reperfusion could cause cellular Na+ overload due to 1) inhibition of sarcolemmal Na+,K+-ATPase and 2) acidosis and Na+-H+ exchange. This could further exaggerate calcium overload by increased Na +-Ca2' exchange. An increase in free cytosolic calcium would activate phospholipases and other degradative enzymes and further exacerbate the injury to the aforementioned key subcellular structures (sarcolemma, sarcoplasmic reticulum, and contractile proteins). Thus, calcium overload could serve to amplify the damage initiated by oxygen radicals. In addition, calcium overload could in itself impair contractile performance and contribute to mechanical dysfunction. It is also possible that the increase in free cytosolic calcium could increase oxyradical production bypromoting conversion ofxanthine dehydrogenase to xanthine oxidase. The ultimate consequence ofthis complex series ofperturbations is a reversible depression of contractility. coronary occlusion, mitigation of stunning by antioxidants has been reproducibly observed by several independent laboratories. Similar protection has been recently demonstrated in the conscious animal, that is, in the most physiological experimental preparation available. Furthermore, generation of free radicals in the stunned myocardium has been directly demonstrated by spin trapping techniques, and attenm Summary Among the numerous mechanisms proposed for myocardial stunning, three appear to be more plausible: 1) generation of oxygen radicals, 2) calcium overload, and 3) excitation-contraction uncoupling. First, the evidence for a pathogenetic role of oxygen-derived free radicals in myocardial stunning is overwhelming. In the setting of a single 15-minute

12 734 Circulation Vol 82, No 3, September 1990 uation of free radical generation has been repeatedly shown to result in attenuation of contractile dysfunction. Numerous observations suggest that oxyradicals also contribute to stunning in other settings: after global ischemia in vitro, after global ischemia during cardioplegic arrest in vivo, and after multiple brief episodes of regional ischemia in vivo. Compelling evidence indicates that the critical free radical damage occurs in the initial moments of reflow, so that myocardial stunning can be viewed as a sublethal form of oxyradical-mediated "reperfusion injury." Second, there is also considerable evidence that a transient calcium overload during early reperfusion contributes to postischemic dysfunction in vitro; however, the importance of this mechanism in vivo remains to be defined. Third, inadequate release of calcium by the sarcoplasmic reticulum, with consequent excitationcontraction uncoupling, may occur after multiple brief episodes of regional ischemia, but its role in other forms of postischemic dysfunction has not been explored. It is probable that multiple mechanisms contribute to the pathogenesis of myocardial stunning. The three hypotheses outlined above are not mutually exclusive and in fact may represent different steps of the same pathophysiological cascade. Thus, generation of oxyradicals may cause sarcoplasmic reticulum dysfunction, and both of these processes may lead to calcium overload, which in turn could exacerbate the damage initiated by oxygen species. The concepts discussed in this review should provide not only a conceptual framework for further investigation of the pathophysiology of reversible ischemia-reperfusion injury but also a rationale for developing clinically applicable interventions designed to prevent postischemic ventricular dysfunction. Acknowledgment The excellent secretarial assistance of William LaCour is gratefully acknowledged. References 1. Braunwald E, Kloner RA: The stunned myocardium: Prolonged, postischemic ventricular dysfunction. Circulation 1982;66: Jennings RB, Reimer KA: Factors involved in salvaging ischemic myocardium: Effects of reperfusion of arterial blood. Circulation 1983;68(suppl I):I-25-I Simpson PJ, Lucchesi BR: Free radicals and myocardial ischemia and reperfusion injury. J Lab Clin Med 1987; 110: Becker LC, Ambrosio G: Myocardial consequences of reperfusion. Prog Cardiovasc Dis 1987;30: Kloner RA, Przyklenk K, Whittaker P: Deleterious effects of oxygen radicals in ischemia/reperfusion: Resolved and unresolved issues. Circulation 1989;80: Opie LH: Reperfusion injury and its pharmacologic modification. Circulation 1989;80: Engler R, Gilpin E: Can superoxide dismutase alter myocardial infarct size? Circulation 1989;79: Kloner RA, Ellis SG, Lange R, Braunwald E: Studies of experimental coronary artery reperfusion: Effects on infarct size, myocardial function, biochemistry, ultrastructure and microvascular damage. Circulation 1983;68(suppl I):I-8-I Heyndrickx GR, Millard RW, McRitchie RJ, Maroko PR, Vatner SF: Regional myocardial functional and electrophysiological alterations after brief coronary artery occlusion in conscious dogs. J Clin Invest 1975;56: Weiner JM, Apstein CS, Arthur JH, Pirzada FA, Hood WB Jr: Persistence of myocardial injury following brief periods of coronary occlusion. Cardiovasc Res 1976;10: Heyndrickx GR, Baig H, Nellens P, Leusen I, Fishbein MC, Vatner SF: Depression of regional blood flow and wall thickening after brief coronary occlusions. Am J Physiol 1978;234:H653-H Bolli R, Zhu WX, Thornby JI, O'Neill PG, Roberts R: Time-course and determinants of recovery of function after reversible ischemia in conscious dogs. Am J Physiol 1988; 254:H102-H Charlat ML, O'Neill PG, Hartley CJ, Roberts R, Bolli R: Prolonged abnormalities of left ventricular diastolic wall thinning in the "stunned" myocardium in conscious dogs: Time-course and relation to systolic function. J Am Coll Cardiol 1989;13: Bolli R, Patel BS, Hartley CJ, Thornby JI, Jeroudi MO, Roberts R: Nonuniform transmural recovery of contractile function in the "stunned" myocardium. Am J Physiol 1989; 257:H375-H Nicklas JM, Becker LC, Bulkley BH: Effects of repeated brief coronary occlusion on regional left ventricular function and dimension in dogs. Am J Cardiol 1985;56: Stahl LD, Aversano TR, Becker LC: Selective enhancement of function of stunned myocardium by increased flow. Circulation 1986;74: Becker LC, Levine JH, DiPaula AF, Guarnieri T, Aversano TR: Reversal of dysfunction in postischemic stunned myocardium by epinephrine and postextrasystolic potentiation. J Am Coll Cardiol 1986;7: Schroder E, Kieso RA, Laughlin D, Schroder M, Meng R, Kerber RE: Altered response of reperfused myocardium to repeated coronary occlusion in dogs. J Am Coll Cardiol 1987;10: Stahl LD, Weiss HR, Becker LC: Myocardial oxygen consumption, oxygen supply/demand heterogeneity, and microvascular patency in regionally stunned myocardium. Circulation 1988;77: Cohen MV, Downey JM: Myocardial stunning in dogs: Preconditioning effect and influence of coronary collateral flow. Am Heart J (in press) 21. Theroux P, Ross J Jr, Franklin D, Kemper WS, Sasayama S: Coronary arterial reperfusion: III. Early and late effects on regional myocardial function and dimensions in conscious dogs. Am J Cardiol 1976;38: Lavallee M, Cox D, Patrick TA, Vatner SF: Salvage of myocardial function by coronary artery reperfusion 1, 2, and 3 hours after occlusion in conscious dogs. Circ Res 1983; 53: Bush LR, Buja LM, Samowitz W, Rude RE, Wathen M, Tilton GD, Willerson JT: Recovery of left ventricular segmental function after long-term reperfusion following temporary coronary occlusion in conscious dogs: Comparison of 2- and 4-hour occlusions. Circ Res 1983;53: Ellis SG, Henschke CI, Sandor T, Wynne J, Braunwald E, Kioner RA: Time course of functional and biochemical recovery of myocardium salvaged by reperfusion. JAm Coll Cardiol 1983;1: Matsuzaki M, Gallagher KP, Kemper WS, White F, Ross J Jr: Sustained regional dysfunction produced by prolonged coronary stenosis: Gradual recovery after reperfusion. Circulation 1983;68: Taegtmeyer H, Roberts AFC, Raine AEG: Energy metabolism in reperfused heart muscle: Metabolic correlates to return of function. JAm Coll Cardiol 1985;6: Kusuoka H, Porterfield JK, Weisman HF, Weisfeldt ML, Marban E: Pathophysiology and pathogenesis of stunned myocardium: Depressed Ca2' activation of contraction as a

13 reperfusion injury by oxygen free radical scavengers. J Thorac Cardiovasc Surg 1983;86: Johnson DL, Horneffer PJ, Dinatale JM Jr, Gott VL, Gardner TJ: Free radical scavengers improve functional recovery of stunned myocardium in a model of surgical coronary revascularization. Surgery 1987;102: Gardner TJ: Oxygen radicals in cardiac surgery. Free Radic Biol Med 1988;4: Illes RW, Silverman NA, Krukenkamp IB, del Nido PJ, Levitsky S: Amelioration of postischemic stunning by deferoxamine-blood cardioplegia. Circulation 1989;80(suppl II1):III-30-III Homans DC, Sublett E, Dai XZ, Bache RJ: Persistence of regional left ventricular dysfunction after exercise-induced myocardial ischemia. J Clin Invest 1986;77: Gross GJ, Farber NE, Hardman HF, Warltier DC: Beneficial actions of superoxide dismutase and catalase in stunned myocardium of dogs. Am J Physiol 1986;250:H372-H O'Neill PG, Charlat ML, Hartley CJ, Roberts R, Bolli R: Nonuniform transmural response of the "stunned" myocardium to inotropic stimulation (abstract). J Am Coll Cardiol 1987;9:145A 52. Preuss KC, Gross GJ, Brooks HL, Warltier DC: Time course of recovery of "stunned" myocardium following variable periods of ischemia in conscious and anesthetized dogs. Am Heart J 1987;114: DeBoer FWV, Ingwall JS, Kloner RA, Braunwald E: Prolonged derangements of canine myocardial purine metabolism after a brief coronary artery occlusion not associated with anatomic evidence of necrosis. Proc Natl Acad Sci USA 1980;77: Reimer KA, Hill ML, Jennings RB: Prolonged depletion of ATP and of the adenine nucleotide pool due to delayed resynthesis of adenine nucleotides following reversible myocardial ischemic injury in dogs. J Mol Cell Cardiol 1981; 13: Swain JL, Sabina RL, McHale PA, Greenfield JC Jr, Holmes EW: Prolonged myocardial nucleotide depletion after brief ischemia in the open-chest dog. Am J Physiol 1982; 242:H818-H Neely JR, Grotyohann LW: Role of glycolytic products in damage to ischemic myocardium: Dissociation of adenosine triphosphate levels and recovery of function of reperfused ischemic hearts. Circ Res 1984;55: Przyklenk K, Kloner RA: Superoxide dismutase plus catalase improve contractile function in the canine model of the "stunned" myocardium. Circ Res 1986;58: Glower DD, Spratt JA, Newton JR, Wolfe JA, Rankin JS, Swain JL: Dissociation between early recovery of regional function and purine nucleotide content in postischaemic myocardium in the conscious dog. Cardiovasc Res 1987; 21: Przyklenk K, Kloner RA: Effect of verapamil on postischemic "stunned" myocardium: Importance of the timing of treatment. JAm Coll Cardiol 1988;11: Guth BD, Martin JF, Heusch G, Ross J Jr: Regional myocardial blood flow, function and metabolism using phosphorus-31 nuclear magnetic resonance spectroscopy during ischemia and reperfusion in dogs. J Am Coll Cardiol 1987;10: Mercier JC, Lando U, Kanmatsuse K, Ninomiya K, Meerbaum S, Fishbein MC, Swan HJC, Ganz W: Divergent effects of inotropic stimulation on the ischemic and severely depressed reperfused myocardium. Circulation 1982;66: Ellis SE, Wynne J, Braunwald E, Henschke CI, Sandor T, Kloner RA: Response of reperfusion-salvaged, stunned myocardium to inotropic stimulation. Am Heart J 1984;107: Arnold JMO, Braunwald E, Sandor T, Kloner RA: Inotropic stimulation of reperfused myocardium with dopamine: Effects on infarct size and myocardial function. J Am Coll Cardiol 1985;6: Bolli R, Zhu WX, Myers ML, Hartley CJ, Roberts R: Beta-adrenergic stimulation reverses postischemic myocar- Downloaded from by guest on July 13, 2018 consequence of reperfusion-induced cellular calcium overload in ferret hearts. J Clin Invest 1987;79: Steenbergen C, Murphy E, Levy L, London RE: Elevation in cytosolic free calcium concentration early in myocardial ischemia in perfused rat heart. Circ Res 1987;60: Porterfield JK, Kusuoka H, Weisman HF, Weisfeldt ML, Marban E: Ryanodine prevents the changes in myocardial function and morphology induced by reperfusion after brief periods of ischemia (abstract). Circ Res 1987;35:315A 30. Ambrosio G, Jacobus WE, Bergman CA, Weisman HF, Becker LC: Preserved high energy phosphate metabolic reserve in globally "stunned" hearts despite reduction of basal ATP content and contractility. J Mol Cell Cardiol 1987;19: Kitakaze M, Weisfeldt ML, Marban E: Acidosis during early reperfusion prevents myocardial stunning in perfused ferret hearts. J Clin Invest 1988;82: Kusuoka H, Koretsune Y, Chacko VP, Weisfeldt ML, Marban E: Excitation-contraction coupling in postischemic myocardium: Does failure of activator Ca21 transients underlie stunning? Circ Res 1990;66: Ambrosio G, Jacobus WE, Mitchell MC, Litt MR, Becker LC: Effects of ATP precursors on ATP and free ADP content and functional recovery of postischemic hearts. Am J Physiol 1989;256:H560-H Marban E, Koretsune Y, Corretti M, Chacko VP, Kusoka H: Calcium and its role in myocardial cell injury during ischemia and reperfusion. Circulation 1989;80(suppl IV):IV-17-IV Schlafer M, Kane PF, Kirsh MM: Superoxide dismutase plus catalase enhances the efficacy of hypothermic cardioplegia to protect the globally ischemic, reperfused heart. J Thorac Cardiovasc Surg 1982;83: Shlafer M, Kane PF, Wiggins VY, Kirsh MM: Possible role for cytotoxic oxygen metabolites in the pathogenesis of cardiac ischemic injury. Circulation 1982;66(suppl I):I-85- I Casale AS, Bulkley GB, Bulkley BH, Flaherty JT, Gott VL, Gardner TJ: Oxygen free radical scavengers protect the arrested globally ischemic heart upon reperfusion. Surg Forum 1983;34: Menasche P, Grousset C, Gauduel Y, Piwnica A: A comparative study of free radical scavengers in cardioplegic solutions: Improved protection with peroxidase. J Thorac Cardiovasc Surg 1986;92: Myers CL, Weiss SJ, Kirsh MM, Shepard BM, Shlafer M: Effects of supplementing hypothermic crystalloid cardioplegic solution with catalase, superoxide dismutase, allopurinol, or deferoxamine on functional recovery of globally ischemic and reperfused isolated hearts. J Thorac Cardiovasc Surg 1986;91: Ambrosio G, Weisfeldt ML, Jacobus WE, et al: Evidence for a reversible oxygen radical-mediated component of reperfusion injury: Reduction by recombinant human superoxide dismutase administered at the time of reflow. Circulation 1987;75: Ytrehus K, Gunnes S, Myklebust R, Mjos OD: Protection by superoxide dismutase and catalase in the isolated rat heart reperfused after prolonged cardioplegia: A combined study of metabolic, functional and morphometric ultrastructural variables. Cardiovasc Res 1987;21: Garlick PB, Davies MJ, Hearse DJ, Slater TF: Direct detection of free radicals in the reperfused rat heart using electron spin resonance spectroscopy. Circ Res 1987;61: Kramer JH, Arroyo CM, Dickens BF, Weglicki WB: Spintrapping evidence that graded myocardial ischemia alters postischemic superoxide production. Free Radic Biol Med 1987;3: Ambrosio G, Zweier JL, Jacobus WE, Weisfeldt ML, Flaherty JT: Improvement of postischemic myocardial function and metabolism induced by administration of desferrioxamine at the time of reflow: The role of iron in the pathogenesis of reperfusion injury. Circulation 1987;76: Stewart JR, Blackwell WH, Crute SL, Loughlin V, Greenfield LJ, Hess ML: Inhibition of surgically induced ischemia/ Bolli Mechanism of Stunned Myocardium 735

14 736 Circulation Vol 82, No 3, September 1990 dial dysfunction without producing subsequent functional deterioration. Am J Cardiol 1985;56: Ito BR, Tate H, Kobayashi M, Schaper W: Reversibly injured, postischemic canine myocardium retains normal contractile reserve. Cire Res 1987;61: Heusch G, Schafer S, Kroger K: Recruitment of inotropic reserve in "stunned" myocardium by the cardiotonic agent AR-L 57. Basic Res Cardiol 1988;83: Hoffmeister HM, Mauser M, Schaper W: Effect of adenosine and AICAriboside on ATP content and regional contractile function in reperfused canine myocardium. Basic Res Cardiol 1985;80: Greenfield RA, Swain JL: Disruption of myofibrillar energy use: Dual mechanisms that may contribute to postischemic dysfunction in stunned myocardium. Circ Res 1987;60: Ciuffo AA, Ouyang P, Becker LC, Levin L, Weisfeldt ML: Reduction of sympathetic inotropic response after ischemia in dogs: Contributor to stunned myocardium. J Clin Invest 1985;75: Heusch G, Frehen D, Kroger K, Schulz R, Thamer V: Integrity of sympathetic neurotransmission in stunned myocardium. JAppl Cardiol 1988;3: O'Neill PG, Charlat ML, Michael LH, Roberts R, Bolli R: Influence of neutrophil depletion on myocardial function and flow after reversible ischemia. Am J Physiol 1989;256: H341-H Bolli R, Triana JF, Jeroudi MO: Postischemic mechanical and vascular dysfunction (myocardial "stunning" and microvascular "stunning") and the effects of calcium channel blockers on ischemia/reperfusion injury. Clin Cardiol 1989; 12:III-16-III Bolli R, Triana JF, Jeroudi MO: Prolonged impairment of coronary vasodilation after reversible ischemia: Evidence for microvascular "stunning." Circ Res 1990;67: Zhao M, Zhang H, Robinson TF, Factor SM, Sonnenblick EH, Eng C: Profound structural alterations of the extracellular collagen matrix in postischemic dysfunctional ("stunned") but viable myocardium. JAm Coll Cardiol 1987;10: Charney RH, Takahashi S, Zhao M, Sonnenblick EH, Factor SM, Eng C: Collagen loss in the stunned myocardium (abstract). Circulation 1989;80(suppl II):II Whittaker P, Przyklenk K, Boughner DR, Kloner RA: Collagen damage in two different models of stunned myocardium (abstract). J Mol Cell Cardiol 1989;21(suppl II):S Guarnieri T: Direct measurement of [Ca2"Ji in early and late reperfused myocardium (abstract). Circulation 1989; 80(suppl II):II Kitakaze M, Weisman HF, Marban E: Contractile dysfunction and ATP depletion after transient calcium overload in perfused ferret hearts. Circulation 1988;77: Hori M, Kitakaze M, Sato H, Iwakura K, Gotoh K, Kusoka H, Kitabatake A: Transient acidosis by staged reperfusion prevents myocardial stunning (abstract). Circulation 1989; 80(suppl II):II Marban E, Kitakaze M, Kusoka H, Porterfield JK, Yue DT, Chacko VP: Intracellular free calcium concentration measured with `9F NMR spectroscopy in intact ferret hearts. Proc NatlAcad Sci USA 1987;84: Lee H-C, Smith N, Mohabir R, Chisin WT: Cytosolic calcium transients from the beating mammalian heart. Proc NatlAcad Sci USA 1987;84: Marban E, Kitakaze M, Koretsune Y, Yue DT, Chacko VP, Pike MM: Quantification of [Ca2+]i in perfused hearts: Critical evaluation of the SF-BAPTA and nuclear magnetic resonance method as applied to the study of ischemia and reperfusion. Circ Res 1990;66: Krause SM, Hess ML: Characterization of cardiac sarcoplasmic reticulum dysfunction during short-term normothermic global ischemia. Circ Res 1985;55: Lazdunski M, Frelin C, Vigue P: The sodium/hydrogen exchange system in cardiac cells: Its biochemical and pharmacological properties and its role in regulating internal concentrations of sodium and internal ph. J Mol Cell Cardiol 1985;17: Murphy JG, Smith TW, Marsh JD: Mechanisms of reoxygenation-induced calcium overload in cultured chick embryo heart cells. Am J Physiol 1988;254:H1133-H Tani M, Neely JR: Role of intracellular Na+ in Ca2' overload and depressed recovery of ventricular function of reperfused ischemic rat hearts: Possible involvement of H+-Na+ and Na+-Ca2' exchange. Circ Res 1989;65: Grinwald PM: Calcium uptake during postischemic reperfusion in the isolated rat heart: Influence of extracellular sodium. J Mol Cell Cardiol 1982;14: Renlund DG, Gerstenblith G, Lakatta EG, Jacobus WE, Kallman CH, Weisfeldt ML: Perfusate sodium during ischemia modifies postischemic functional and metabolic recovery in the rabbit heart. J Mol Cell Cardiol 1984;16: McCord JM: Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 1985;312: Taylor AL, Golino P, Eckels R, Pastor P, Buja LM, Willerson JT: Differential enhancement of postischemic segmental systolic thickening by diltiazem. J Am Coll Cardiol 1990; 15: Lamping KA, Gross GJ: Improved recovery of myocardial segment function following a short coronary occlusion in dogs by nicorandil, a potential new antianginal agent, and nifedipine. J Cardiovasc Pharmacol 1985;7: Warltier DC, Gross GJ, Brooks HL, Preuss KC: Improvement of postischemic, contractile function by the calcium channel blocking agent nitrendipine in conscious dogs. J Cardiovasc Pharmacol 1988;12(suppl 4):S120-S Dunlap E, Millard RW: Amlodipine, a new long-acting calcium channel blocking agent, improves recovery of "stunned" myocardium (abstract). Pharmacologist 1989; 31: Przyklenk K, Ghafari GB, Eitzman DT, Kloner RA: Nifedipine administered after reperfusion ablates systolic contractile dysfunction of postischemic "stunned" myocardium. J Am Coll Cardiol 1989;13: Krause SM, Jacobus WE, Becker LC: Alterations in cardiac sarcoplasmic reticulum calcium transport in the postischemic "stunned" myocardium. Circ Res 1989;65: Zhu WX, Myers ML, Hartley CJ, Roberts R, Bolli R: Validation of a single crystal for measurement of transmural and epicardial thickening. Am J Physiol 1986;251: H1045-H Myers ML, Bolli R, Lekich RF, Hartley CJ, Roberts R: Enhancement of recovery of myocardial function by oxygen free-radical scavengers after reversible regional ischemia. Circulation 1985;72: Murry CE, Richard VJ, Jennings RB, Reimer KA: Free radicals do not cause myocardial stunning after four 5 minute coronary occlusions (abstract). Circulation 1989;80(suppl II):II Jeroudi MO, Triana FJ, Patel BS, Bolli R: Effect of superoxide dismutase and catalase, given separately, on myocardial "stunning." Am J Physiol (in press) 100. Buchwald A, Klein HH, Lindert S, Pich S, Nebendahl K, Wiegand V, Kreuzer H: Effect of intracoronary superoxide dismutase on regional function in stunned myocardium. J Cardiovasc Pharmacol 1989;13: Halliwell PB, Gutteridge JMC: Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J 1984;219: Fox RB: Prevention of granulocyte-mediated oxidant lung injury in rats by a hydroxyl radical scavenger, dimethylthiourea. J Clin Invest 1984;74: Bolli R, Zhu WX, Hartley CJ, Michael LH, Repine J, Hess ML, Kukreja RC, Roberts R: Attenuation of dysfunction in the postischemic "stunned" myocardium by dimethylthiorea. Circulation 1987;76: Bolli R, Jeroudi MO, Patel BS, Aruoma 01, Halliwell B, Lai EK, McCay PB: Marked reduction of free radical generation and contractile dysfunction by antioxidant therapy begun at the time of reperfusion: Evidence that myocardial "stunning"

15 is a manifestation of reperfusion injury. Circ Res 1989; 65: Myers ML, Bolli R, Lekich RF, Hartley CJ, Michael LH, Roberts R: N-2-Mercaptopropionylglycine improves recovery of myocardial function following reversible regional ischemia. J Am Coll Cardiol 1986;8: Bolli R, Patel BS, Zhu WX, O'Neill PG, Charlat ML, Roberts R: The iron chelator desferrioxamine attenuates postischemic ventricular dysfunction. Am J Physiol 1987; 253:H1372-H Farber NE, Vercellotti GM, Jacob HS, Pieper GM, Gross GJ: Evidence for a role of iron-catalyzed oxidants in functional and metabolic stunning in the canine heart. Circ Res 1988;63: Schrier GM, Hess ML: Quantitative identification of superoxide anion as a negative inotropic species. Am J Physiol 1988;24:H138-H DiGuiseppi J, Fridovich I: Oxygen toxicity in Streptococcus sanguis: The relative importance of superoxide and hydroxyl radicals. J Biol Chem 1982;257: Triana JF, Unisa A, Bolli R: Antioxidant enzymes attenuate myocardial "stunning" in the conscious dog. FASEB J 1990; 4:A Zweier JL, Flaherty JT, Weisfeldt ML: Direct measurement of free radical generation following reperfusion of ischemic myocardium. Proc NatlAcad Sci USA 1987;84: Baker JE, Felix CC, Olinger GN, Kalyanaraman B: Myocardial ischemia and reperfusion: Direct evidence for free radical generation by electron spin resonance spectroscopy. Proc NatlAcad Sci USA 1988;85: Zweier JL: Measurement of superoxide-derived free radicals in the reperfused heart. J Biol Chem 1988;263: Bolli R, McCay PB: Use of spin traps in intact animals undergoing myocardial ischemia/reperfusion: A new approach to assessing the role of oxygen radicals in myocardial "stunning." Free Rad Res Comms 1990;9: Bolli R, Patel BS, Jeroudi MO, Lai EK, McCay PB: Demonstration of free radical generation in "stunned" myocardium of intact dogs with the use of the spin trap a-phenyl N-tertbutyl nitrone. J Clin Invest 1988;82: Leiboff RL, Arroyo CM, Schaer GL, Mergner GW, Kramer JH, Miller DL, Visner MS, Weglicki WB: Free radical generation in an in vivo model of regional myocardial stunning (abstract). FASEB J 1988;2:A Bolli R, Jeroudi MO, Patel BS, DuBose CM, Lai EK, Roberts R, McCay PB: Direct evidence that oxygen-derived free radicals contribute to postischemic myocardial dysfunction in the intact dog. Proc Natl Acad Sci USA 1989; 86: Burton KP, McCord JM, Ghai G: Myocardial alterations due to free-radical generation. Am JPhysiol 1984;246:H776-H Blaustein AS, Schine L, Brooks WW, Fanburg BL, Bing OHL: Influence of exogenously generated oxidant species on myocardial function. Am J Physiol 1986;250:H595-H Shattock MJ, Manning AS, Hearse DJ: Effects of hydrogen peroxide on cardiac function and postischaemic functional recovery in the isolated "working" rat heart. Pharmacology 1982;24: Ytrehus K, Myklebust R, Mjos OD: Influence of oxygen radicals generated by xanthine oxidase in the isolated perfused rat heart. Cardiovasc Res 1986;20: Miki S, Ashraf M, Salka S, Sperelakis N: Myocardial dysfunction and ultrastructural alterations mediated by oxygen metabolites. J Mol Cell Cardiol 1988;20: Jackson CV, Mickelson JK, Pope TK, Rao PS, Lucchesi BR: 02 free radical-mediated myocardial and vascular dysfunction. Am J Physiol 1986;251:H1225-H Goldhaber JI, Ji S, Lamp ST, Weiss JN: Effects of exogenous free radicals on electromechanical function and metabolism in isolated rabbit and guinea pig ventricle: Implications for ischemia and reperfusion injury. J Clin Invest 1988;83: Bolli Mechanism of Stunned Myocardium Przyklenk K, Whittaker P, Kloner RA: In vivo infusion of oxygen free radical substrates causes myocardial systolic, but not diastolic dysfunction. Am Heart J 1990;119: Davies KJA: Protein damage and degradation by oxygen radicals: I. General aspects. JBiol Chem 1987;262: Thompson JA, Hess ML: The oxygen free radical system: A fundamental mechanism in the production of myocardial necrosis. Prog Cardiovasc Dis 1986;28: Romaschin AD, Rebeyka I, Wilson GJ, Mickle DAG: Conjugated dienes in ischemic and reperfused myocardium: An in vivo chemical signature of oxygen free radical mediated injury. J Mol Cell Cardiol 1987;19: Weisel RD, Mickle DAG, Finkle CD, Tumiati LC, Madonik MM, Ivanov J, Burton GW, Ingold KU: Myocardial freeradical injury after cardioplegia. Circulation 1989;80(suppl III):III-14-III Rowe GT, Manson NH, Caplan M, Hess ML: Hydrogen peroxide and hydroxyl radical mediation of activated leukocyte depression of cardiac sarcoplasmic reticulum: Participation of the cyclooxygenase pathway. Circ Res 1983;53: Kaneko M, Beamish RE, Dhalla NS: Depression of heart sarcolemmal Ca2' pump activity by oxygen free radicals. Am J Physiol 1989;256:H368-H Kaneko M, Elimban V, Dhalla NS: Mechanism for depression of heart sarcolemmal Ca2' pump by oxygen free radicals. Am J Physiol 1989;257:H804-H Reeves JP, Bailey CA, Hale CC: Redox modification of sodium-calcium exchange activity in cardiac sarcolemmal vesicles. J Biol Chem 1986;261: Kramer JH, Mak IT, Weglicki WB: Differential sensitivity of canine cardiac sarcolemmal and microsomal enzymes to inhibition by free radical-induced lipid peroxidation. Circ Res 1984;55: Kim M-S, Akera T: 02 free radicals: Cause of ischemiareperfusion injury to cardiac Na+-K+-ATPase. Am J Physiol 1987;252:H252-H Charlat ML, O'Neill PG, Egan JM, Abernethy DR, Michael LH, Myers ML, Roberts R, Bolli R: Evidence for a pathogenetic role of xanthine oxidase in the "stunned" myocardium. Am J Physiol 1987;252:H566-H Holzgrefe HH, Gibson JK: Enhanced function recovery in the stunned canine myocardium by pretreatment with oxypurinol (abstract). JAm Coll Cardiol 1988;11:208A 138. Puett DW, Forman MB, Cates CU, Wilson BH, Hande KR, Friesinger GC, Virmani R: Oxypurinol limits myocardial stunning but does not reduce infarct size after reperfusion. Circulation 1987;76: Eddy LJ, Stewart JR, Jones HP, Engerson TD, McCord JM, Downey JM: Free radical-producing enzyme, xanthine oxidase, is undetectable in human hearts. Am J Physiol 1987; 253:H709-H Jarasch ED, Bruder G, Heid HW: Significance of xanthine oxidase in capillary endothelial cells. Acta Physiol Scand 1986;548(suppl): Muxfeldt M, Schaper W: The activity of xanthine oxidase in hearts of pigs, guinea pigs, rats, and humans. Basic Res Cardiol 1987;82: Huizer T, de Jong JW, Nelson JA, Czarnecki W, Serruys PW, Bonnier JJRM, Troquay R: Urate production by human heart. J Mol Cell Cardiol 1989;21: Grum CM, Gallagher KP, Kirsh MM, Shlafer M: Absence of detectable xanthine oxidase in human myocardium. J Mol Cell Cardiol 1989;21: Werns S, Ventura A, Li GC, Lucchesi BR: Amflutizole, a xanthine oxidase inhibitor, does not attenuate myocardial stunning in the canine heart (abstract). Circulation 1989; 80(suppl II):II Lucchesi BR, Mullane KM: Leukocytes and ischemiainduced myocardial injury. Ann Rev Pharmacol Toxicol 1986; 26: Engler R: Granulocytes and oxidative injury in myocardial ischemia and reperfusion. Fed Proc 1987;46:

16 738 Circulation Vol 82 No 3, September Shea MJ, Simpson PJ, Werns SW, Buda AJ, Alborzy- Khaghany AS, Hoff PT, Lucchesi BR: Effect of neutrophil depletion on recovery of "stunned" myocardium (abstract). Clin Res 1987;35:327A 148. Jeremy RW, Becker LC: Neutrophil depletion does not prevent myocardial dysfunction after brief coronary occlusion. JAm Coll Cardiol 1989;13: O'Neill PG, Charlat ML, Kim H-S, Pocius J, Michael LH, Hartley CJ, Roberts R, Bolli R: Lipoxygenase inhibitor nafazatrom fails to attenuate postischemic ventricular dysfunction. Cardiovasc Res 1987;21: Schott RJ, Nao BS, McClanahan TB, Simpson PJ, Stirling MC, Todd RF III, Gallagher KP: F(ab')2 fragments of anti-mol (904) monoclonal antibodies do not prevent myocardial stunning. Circ Res 1989;65: Kerber RE, Shasby DM, Seabold J, Kieso R, Fox-Eastham K: Does reduction of leukocyte accumulation in reperfused myocardium affect stunning (abstract)? Circulation 1989; 80(suppl II):II Go LO, Murry CE, Richard VJ, Jennings RB, Weischedel GR, Reimer KA: Myocardial neutrophil accumulation during reperfusion after reversible or irreversible ischemic injury. Am J Physiol 1988;255:H1188-H Engler RL, Dahlgren MD, Morris DD, Peterson MA, Schmid-Schonbein GW: Role of leukocytes in response to acute myocardial ischemia and reflow in dogs. Am J Physiol 1986;251:H314-H Engler R, Covell JW: Granulocytes cause reperfusion ventricular dysfunction after 15-minute ischemia in the dog. Circ Res 1987;61: Westlin W, Mullane KM: Alleviation of myocardial stunning by leukocyte and platelet depletion. Circulation 1989; 80: Kloner RA: Do neutrophils mediate the phenomenon of stunned myocardium? JAm Coll Cardiol 1989;13: Westlin W, Mullane KM: Does captopril attenuate reperfusion-induced myocardial dysfunction by scavenging free radicals? Circulation 1988;77(suppl I):I-30-I Ferrari R, Alfieri 0, Curello S, Ceconi A, Cargnoni A, Marzollo P, Pardini A, Caradonna E, Visioli 0: Occurrence of oxidative stress during reperfusion of the human heart. Circulation 1990;81: Asinger RW, Peterson DA, Elsperger KJ, Homans D: Longterm recovery of LV wall thickening after 1 hour of ischemia is not affected when superoxide dismutase and catalase are administered during the first 45 minutes of reperfusion (abstract). JAm Coll Cardiol 1988;2:163A 160. Nejima J, Knight DR, Fallon JT, Uemura N, Manders WT, Canfield DR, Cohen MV, Vatner SF: Superoxide dismutase reduces reperfusion arrhythmias but fails to salvage regional myocardial function or myocardium at risk in conscious dogs. Circulation 1989;79: Przyklenk K, Kloner RA: "Reperfusion injury" by oxygenderived free radicals? Effect of superoxide dismutase plus catalase, given at the time of reperfusion, on myocardial infarct size, contractile function, coronary microvasculature, and regional myocardial blood flow. Circ Res 1989;64: Patel BS, Jeroudi MO, O'Neill PG, Roberts R, Bolli R: Effect of human recombinant superoxide dismutase on canine myocardial infarction. Am J Physiol 1990;258: H369-H Forman MB, Puett DW, Cates CU, McCroskey DE, Beckman JK, Greene HL, Virmani R: Glutathione redox pathway and reperfusion injury: Effect of N-acetylcysteine on infarct size and ventricular function. Circulation 1988;78: Homans DC, Sublett E, Asinger R, Peterson D, Bache RJ: SOD+catalase does not attenuate regional left ventricular dysfunction following exercise induced ischemia (abstract). J Am Coll Cardiol 1988;78:II Hearse DJ, Humphrey SM, Bullock GR: The oxygen paradox and the calcium paradox: Two facets of the same problem? J Mol Cell Cardiol 1978;10: Patel B, Kloner RA, Przyklenk K, Braunwald E: Postischemic myocardial "stunning": A clinically relevant phenomenon. Ann Intem Med 1988;108:

17 Mechanism of myocardial "stunning". R Bolli Downloaded from by guest on July 13, 2018 Circulation. 1990;82: doi: /01.CIR Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX Copyright 1990 American Heart Association, Inc. All rights reserved. Print ISSN: Online ISSN: The online version of this article, along with updated information and services, is located on the World Wide Web at: published Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally in Circulation can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: Subscriptions: Information about subscribing to Circulation is online at: