Cardiovascular Drugs and Therapy 1991;5:853-876 © Kluwer Academic Publishers, Boston. Printed in U.S.A.
Stunning: A Radical Re-view David J. Hearse Cardiovascular Research, The Rayne Institute, St Thomas" Hospital, London, UK
Summary. The recovery from trauma, whether ischemia or some other form of tissue injury, is never instantaneous; time is always required for repair and the return of normal metabolism and function. To what extent the delay in recovery of contractile activity (stunning) after a brief period of isehemia represents convalescence from ischemia-induced injury, as opposed to the expression of reperfusion-induced injury, is perhaps not as clear as the proponents of stunning would hope. Definitive evidence for a distinct reperfusion-induced pathology, which compromises the recovery of contractile function from the depressed state induced by ischemia, is elusive. If reperfusion-induced injury accounts for a significant proportion of stunning, then the molecular mechanisms responsible for initiating the event and those responsible for orchestrating the event at the level of the contractile protein are far from clear. Perturbations of calcium homeostasis are frequently cited as responsible for the depressed contractile state, however, some metabolic derangement must precede any pathologically induced ionic disturbance. In this connection, evidence indicates that free-radical-induced oxidant stress, during the early moments of reperfusion, may modify the activity of a number of thiol-regulated proteins that are directly, or indirectly, responsible for controlling the movement of calcium. Sarcolemmal sodium-calcium exchange and the calcium release channel of the sarcoplasmic reticulum may be activated, whereas the sarcolemmal calcium pump and sodium-potassium ATPase, together with the calcium pump of the sarcoplasmic reticulum, may be inhibited. Under the conditions prevailing during ischemia and reperfusion, this would be expected to promote an early intracellular calcium overload. It is difficult to reconcile such a change with the decreased inotropic state that characterizes stunning; however, it seems likely that the calicum overload is transient and that the stunned myocardium rapidly reestablishes normal levels of intracellular calcium. It is still difficult to explain adequately the reduced inotropic state; clearly, the mechanism of stunning is not quite as simple as its definition.
any more than a conveniently descriptive term for the natural, time-dependent recovery of traumatized tissue. In o t h e r words, to inquire w h e t h e r this t r a n s i e n t postischemic contractile deficit is a reflection of some unfavorable component of reperfusion, some injury sustained d u r i n g ischemia, or some combination of the two. This question will be e x p l o r e d b y r e v i e w i n g a n u m b e r of studies of ischemia and reperfusion in which anti-free-radical i n t e r v e n t i o n s have been used to enhance the postischemic r e c o v e r y of contractile function. From this, the hypothesis will be advanced
that oxidant stress-induced changes in the activity of thiol-regulated proteins may be critical in the initiation qf the ionic disturbances that occur during reperfusion and that may contribute to stunning and reperfusion arrhythmias.
Stunning: The Lure of the Cognomen Myocardial stunning is just one of an impressive
T h e publication of a focused issue of Cardiovascular Drugs and Therapy d e d i c a t e d to m y o c a r d i a l stunning
a r r a y of s e d u c t i v e t e r m s t h a t , in addition to "stunning the cardiologist" [1,2,], m a y increase the risk of lending c r e d i b i l i t y to an i n a d e q u a t e l y defined phenomenon m e r e l y b y giving it a name. In its simplest sense, m y o c a r d i a l s t u n n i n g could be considered as the expression of a postischemic " h a n g o v e r , " a phase of organ malfunction and r e c u p e r a t i o n t h a t i n e v i t a b l y followes a period of t r a u m a . W h e t h e r caused b y an excess of alcohol, a bout of influenza, or t r a n s i e n t ischemia, t h e r e can be few pathological s t a t e s in which a complete and i n s t a n t a n e o u s r e c o v e r y is o b s e r v e d as soon as t h e source of i r r i t a t i o n is r e m o v e d . Conditions such as ischemia, inflammation, and viral infections u p s e t , at a molecular level, the delicate balance of cellular m e t a b o l i s m and function. E n z y m e p a t h w a y s become d i s t u r b e d , toxins accumulate, and, not s u r p r i s i n g l y , cellular r e c o v e r y t a k e s time, particularly if p r o t e i n s y n t h e s i s and s t r u c t u r a l r e p a i r a r e required. W h a t t h e n is special about m y o c a r d i a l isch-
m i g h t s u g g e s t t h a t this is a p r o v e n e x a m p l e of r e p e r f u s i o n - i n d u c e d injury, of p r o b a b l e clinical relevance, t h a t is likely to c r e a t e a valuable t a r g e t for novel t h e r a p e u t i c interventions. One objective of this article is to question w h e t h e r myocardial stunning is
Address for correspondence and reprint requests: David J. Hearse, PhD, DSc, Cardiovascular Reseach, The Rayne Institute, St. Thomas' Hospital, London SE1 7EH, UK.
Cardiovasc Drugs Ther 1991;5:853-876 Key Words. stunning, myocardium, free radicals, oxidant stress, calcium, reperfusion, arrhythmias, ischemia
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emia? Why should we not consider the transient period of postischemic contractile and metabolic dysfunction simply as a phase in a process of progressive recovery--a form of myocardial convalescence? Why should the heart alone be expected to recover almost instantaneously from a condition as severe and complex as ischemia? Proponents of the stunning hypothesis would claim that the antecedent ischemia is not solely responsible for the depressed function and slow recovery, but that there is also some unfavorable component of reperfusion that limits the process. In support of this, they would argue that since the administration of certain drugs at the time of reperfusion can improve the rate of recovery, the interventions must be counteracting some unfavorable aspect of the reperfusion process itself. However, is this a sufficiently convincing argument for the existence of a distinct reperfusioninduced pathology that is capable of negating some of the benefits of reflow? To take an extreme analogy, it is perhaps akin to arguing that a hangover headache arises not from consuming too much alcohol but from stopping the drinking and that the curative aspirin comes to the rescue by countering the consequences of terminating the insult, rather than accelerating the recovery from it. Could it really be argued that, with myocardial stunning, the heart is suffering from withdrawal of ischemia?
Is S t u n n i n g a T r u e Reperfusion-Induced
Pathology?
A positive response to the last question implies the existence of reperfusion-induced injury. This concept is, however, becoming increasingly controversial [3-7], since it requires that, while reperfusion is overall beneficial, one or more components of the reflow process must be detrimental and act to slow the rate, or reduce the extent, of recovery. Arguments in support of reperfusion-induced injury usually derive from experiments in which interventions are given at the time of reperfusion and are shown to increase the rate of postischemic recovery. However, unless it can be shown that these agents actually prevent some unfavorable aspect of reperfusion (rather than simply acting as an inotropic stimulus or accelerating the natural, but slow, recovery process), then a reperfusion-mediated component of myocardial stunning remains unproven. Before focusing on the origins of myocardial stunning, it is therefore necessary to consider the controversy [6,7] over reperfusion-induced injury. Interest in this subject has been greatly stimulated by the advent of thrombolytic and angioplastic procedures for
the reperfusion of regionally ischemic human myocardium, by the growth of cardiac surgical procedures that necessitate reperfusion of the whole heart after extended periods of global ischemia, and by the realization that transient coronary spasm and reperfusion may be a frequent occurrence in humans. As stressed by Bolli [8], the occurrence of myocardial stunning in patients could significantly delay the benefits of reperfusion.
Reperfusion-induced injury: fact or pharmacologic fantasy? Without question, early reperfusion is an absolute prerequisite for the survival of ischemic tissue. However, it is widely believed that reperfusion may not be without hazard and may of itself induce injury [3,4,9-12], which may even kill potentially viable cells. Although there is no doubt that reperfusion can increase the apparent severity of tissue injury, some investigators question whether reperfusion can actually cause lethal cell injury [13,14]. They argue that, rather than creating injury de novo, reperfusion merely accelerates the expression of the injury that had already occurred during the preceding period of severe ischemia. In an attempt to resolve some of the confusion over reperfusion-induced injury and its existence, the present author has proposed [4,6] that the potentially unfavorable sequelae of reperfusion should be divided into the following four categories.
Reperfusion-induced arrhythmias. These may range in severity from ventricular premature beats to ventricular fibrillation; they occur within seconds [15,16] of the onset of reflow and have been observed in all species studied, including humans. Many contributory mechanisms have been proposed [16], but most recently, attention has been focused on the possibility that free-radical-induced oxidant stress acting on cellular ion regulating processes may be involved (for reviews see [17,18]).
Myocardial stunning. It is widely believed that reperfusion may precipitate a number of unfavorable (but nonlethal) cellular changes that, if given sufficient time, will revert to normal. The most widely discussed of these is mechanical stunning [8,19,20]. However, in this article the possibility will be considered that stunning might merely represent the slow recovery from the trauma of ischemia, rather than the occurrence of a specific reperfusion-induced pathology. A variety of candidates for the mechanism underlying postischemic contractile dysfunction have been
Stunning and Oxidant Stress
proposed. These include transient intracellular calcium overload, decreased sensitivity of the myofilaments to calcium, reduced ability to resynthesize high-energy phosphates, impaired sympathetic neural responsiveness, heterogeneous impairment of regional perfusion, loss of creatine kinase activity and impaired utilization of energy by the myofibrils, damage to the extracellular collagen matrix, and leukocyte activation. Bolli [5,8] has put forward arguments against a number of the above proposals and has claimed that, as with reperfusion-induced arrhythmias, the two most plausible mechanisms for myocardial stunning relate to free radical-induced injury and disturbances of calcium homeostasis. Lethal reperfusion-induced injury. The killing of cells by suboptimal reperfusion represents the most widely held concept of reperfusion-induced injury. However, as recently argued [4,6,7], there is little conclusive evidence for the existence of this form of reperfusion injury. Accelerated expression of necrosis. In this fourth sequela of reperfusion, the number of cells that die will not be increased, but the manner in which the necrosis develops may be different. Reperfusion-induced injury in tissue components other than the myocyte In enumerating possible forms of reperfusion-induced injury, it is important to acknowledge that the myocyte is not the only structure susceptible to injury during ischemia and reperfusion [6]. The microvasculature and the endothelium, the conducting system, and the connective tissue should all be considered [21-24] as possible targets. Thus a number of studies [6,21,23] have addressed the issue of stunning and the microvasculature. It could well be that the microvasculature and its endothelial lining, rather than the myocyte, play a determining role in the expression of some of the sequelae of reperfusion. Such an example could be the "no-reflow phenomenon" [25,26] in which the removal of a coronary occlusion does not lead to the restoration of coronary flow, despite the removal of occlusion.
T h e A s s o c i a t i o n B e t w e e n Free R a d i c a l s a n d Postischemic Contractile F u n c t i o n It is difficult to attribute the original study of free radicals in relation to cardiac contractile function to any single group of investigators. However, special mention should be made of the work of Guarnieri and
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colleagues, who in 1978 reported [27] that alphatocopherol could reduce enzyme leakage and improve the recovery of contractile function and mitochondrial metabolism in rabbit hearts subjected to 30 minutes of hypoxia and reoxygenation. In that study, and in another in 1980 [28], they provided strong collateral evidence for the proposition, first advanced by the present author [3,29,30], that oxygen-derived free radicals may play some role in determining the extent of contractile and metabolic recovery after a period of myocardial oxygen deprivation. Early support for this proposition is also to be found in a study from the author's laboratory [31] in which exogenous hydrogen peroxide was shown to impair postischemic functional recovery. Influenced by the work of the Italian group, Shlafer and colleagues in the United States extended investigations from crystalloid-perfused hypoxic hearts to studies involving ischemia and reperfusion with blood as well as crystalloid media [32-34]. Thus, in 1981 and 1982 Shlafer et al. [32,34], using the rabbit heart subjected to 2 hours of hypothermic ischemia, reported that superoxide dismutase (SOD) plus catalase, when added for only 5 minutes to a preischemic infusion solution and a postischemic reperfusion solution, reduced enzyme leakage and improved postischemic recovery of contractile function and mitochondrial metabolism. Similar results were also observed by the same authors [33] in studies with bloodperfused cat hearts. Potential relevance to cardiac surgery The work of Guarnieri and Shlafer provided an important stimulus for the evaluation of anti-free-radical interventions in the surgical arena. In 1982 and 1983, Stewart and colleagues, using dog hearts on cardiopulmonary bypass, reported [35,36] that SOD and mannitol improved the recovery of contractile function after 60 minutes of hypothermic global ischemia. Later, Stewart et al. [37] demonstrated a similar benefit with allopurinol; they also extended the concept to include cardiac transplantation [38]. Since 1983 there have been many studies [39-49] in the surgical literature supporting the view that antioxidant interventions, given before or after ischemia, improve the immediate postischemic recovery of cardiac contractile function. In contrast to the studies of antioxidants and infarct size (see below), there has been relatively little disagreement over the value of such interventions in models of surgical ischemia. Potential relevance to myocardial infarction While cardiac surgeons were exploring the potential benefits of anti-free-radical interventions as a means
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of improving the short-term recovery of the globally ischemic myocardium, cardiologists were assessing whether these agents conferred any benefit on the regionally ischemic myocardium. In 1984 Jolly et al. [50] studied postischemic contractile function and infarct size in dog hearts subjected to 90 minutes of coronary artery occlusion and 24 hours of reperfusion; SOD plus catalase was shown to afford considerable protection. Their study signalled the start of a period of intense interest (and controversy) over the possibility that antioxidant interventions might provide significant protection during evolving myocardial infarction. At least 50 (often conflicting) studies were reported in major journals (for reviews see [6,10, 51,52]; however, in the majority of these myocardial infarct size rather than postischemic contractile function was the primary end point.
Free Radicals a n d Myocardial Stunning Interest in the temporal contractile effects of oxygenderived free radicals and the value of antioxidant interventions during the early hours of reperfusion has been greatly stimulated by an excellent and extensive series of studies by Bolli and coworkers [8,20,21,5362]. In 1985 Bolli's group reported [61] that SOD and catalase, when given to open-chest dogs subjected to 15 minutes of regional ischemia and 2 hours of reperfusion, enhanced the postischemic recovery of function from 31.6 _+ 9.8% to 74.2 _+ 8.4%. Since that study, there have been many investigations (for reviews see [8,10]), from several laboratories (mostly employing the dog heart), into the ability of a wide variety of anti-free-radical interventions to enhance postischemic functional recovery. In some instances there is a good degree of agreement over efficacy and mechanisms; in others controversy exists [10]. Studies of anti.free radical interventions and stunning The manipulation of free radical production in the heart, or any other organ, can be achieved either by promoting the elimination of radicals (by supplementing endogenous antioxidant defenses) or by preventing their formation. In the following paragraphs studies of myocardial stunning using a variety of such approaches, targeted at both intracellular and extracellular sites, are summarized. Antioxidant enzymes. In the context of myocardial stunning, only SOD and/or catalase have been studied; in other areas (e.g., cardioplegia and global isch-
emia) enzymes such as peroxidase have also been investigated [45,63]. While there is some disagreement in the literature (usually from studies with extended periods of ischemia [64,65]), a considerable number of investigations now confirm that SOD, when used in combination with catalase, can accelerate the postischemic recovery of contractile function. Thus, in addition to the studies of Bolli's group [61], Gross et al. [66], Przyklenk and Kloner [67], and Murry et al. [68] have all reported positive results in the dog heart. In contrast, efficacy is in question in those studies in which SOD has been administered alone; for example, Buchwald et al. [69] gave SOD to pigs and were unable to attenuate stunning. Whether this was due to a species difference, to some variation in dose or administration protocol, or to the absence of catalase was not resolved. However, Bolli's group [70], using the dog, have also observed that SOD alone fails to protect against stunning; they argued that this provided evidence that both superoxide and hydrogen peroxide contributed to the cellular damage responsible for stunning. Koerner et al. [71], using the rabbit, have also failed to achieve protection with SOD alone, whereas SOD plus catalase was very effective. Contrasting with these results are the in vitro findings of Ambrosio and colleagues [72], in which human recombinant SOD alone, given at the time of reperfusion, afforded protection against the metabolic and functional consequences of stunning. The authors also claimed that catatase afforded no additional protection. Low molecular weight organic antioxidants. To assess the importance of superoxide, hydrogen peroxide, and hydroxyl radicals in the genesis of stunning, Bolli et al. [58] have studied the hydroxyl radical scavenger dimethylthiourea (DMTU) in the dog heart with 15 minutes of coronary artery occlusion and 4 hours of reperfusion. When given over 45 minutes (starting 30 minutes before occlusion), DMTU improved the recovery of postischemic wall thickening after 4 hours of reperfusion from 36 +- 13% to 67 +- 5%. Similar results have been reported [60,71] using mercaptopropionyl glycine (MPG), which is another organic antioxidant that readily gains access to the intracellular space. Bolli [8,55] argued that these findings, taken with those of the studies with antioxidant enzymes, provided evidence that the hydroxyl radical played a central role in the induction of injury. A number of other organic antioxidants have been positively assessed in relation to their ability to improve the postischemic recovery of contractile function; these include N~acetylcysteine [73,74], probucol [75], and alpha-tocopherol [76].
Stunning and Oxidant Stress
Cofactors for radical production. Further evidence of a role for the hydroxyl radical, together with additional support for an association between radicals and stunning, can be derived from reports [20,54,62, 77-80] that desferrioxamine can also attenuate myocardial stunning. Transition metals, such as iron and copper, are essential cofactors for the hydroxyl radical-producing Haber-Weiss reaction; desferrioxamine (which avidly binds iron) and other chelators have also been shown to reduce other types of reperfusion-induced injury, such as arrhythmias [81-83]. Inhibition of radical-generating processes. Studies [24,84,85] with allopurinol or oxypurinol, the inhibitors of xanthine oxido-reductase, provide further support for an association between reactive oxygen intermediates and myocardial stunning. However, some caution should be exercised in the interpretation of studies with these two agents, since they have been found [46] to be protective in hearts from species that are known to be deficient in xanthine oxido-reductase. In this connection, it is interesting that in studies [86] with amflutizole (a highly specific inhibitor of xanthine oxidase activity), an attenuation of stunning could not be demonstrated in the dog heart. Removal of radical sources. Many studies have focused on the possible importance of the leukocyte as a mediator of myocardial stunning, and considerable controversy has been generated (for reviews see [8,13,87,88]). In such studies, leukocytes have either been physically removed or their activity inhibited. This has been achieved in a number of ingenious manners, including the use of leukocyte filters, neutrophil antisera, inhibitors of leukotriene production, or the use of agents that inhibit leukocyte adherence. While some studies [89-92] have shown a beneficial effect on postischemic function, others [93-96] have failed to achieve any protection. This has led to the opinion [8] that leukocytes, or the radicals that they produce, are not involved in myocardial stunning. In any event, as Bolli pointed out [6,8], there is now evidence that in the early minutes of reperfusion after a brief period of ischemia, myocardial leukocyte content is reduced [97]. He went on to argue that this, together with the fact that stunning can be induced in leukocyte-free isolated perfused heart preparations, made it unlikely that leukocytes played a central role in myocardial stunning. However, this is not to deny their importance in other aspects of the pathogenesis of injury during ischemia and reperfusion. Sites and sources of radical production A very diverse group of antioxidant interventions appears to be able to improve postischemic functional
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recovery; some of these (e.g., SOD and catalase) are likely to be restricted to the extracellular space, others (e.g., DMTU and MPG) probably to gain access to the cytoplasm, while lipophilic agents (e.g., alpha tocopherol) are localized in membrane lipids. Since radicals can be produced at various intracellular and extracellular sites, and since all of these interventions exert partial protection, it would seem that radicalmediated injury is likely to arise as a consequence of the combined effects of multiple, independent, radical-producing pathways. If this is the case, then any single intervention is likely to attenuate only part of the injury. Potential sources of radical production are numerous and have been reviewed in detail elsewhere [7,18]; they include the mitochondria, catecholamines, the arachidonic acid pathway, hemoglobin, myoglobin, aldehyde oxidases, and leukocytes.
Time course of radical production A very important conclusion to emerge from the study by Bolli et al. [55] is that there appears to be an extremely narrow time window (less than 60 seconds) during which free radicals make their contribution to the overall stunning process. In this study, the antioxidant MPG, given 1 minute before reperfusion, was highly effective in reducing the early burst of radical production and attenuating stunning; however, when given I minute after reperfusion, little protection was observed. Further evidence supporting the occurrence of extremely rapid radical-mediated events comes from the many studies of reperfusion-induced arrhythmias [81,98-101] in which induction and protection can take place within the first 10 seconds of the restoration of flow. Although it is the production of radicals during the first 60 seconds of reperfusion that appears to be critical to stunning, this does not mean that radical production is necessarily a short-lasting phenomenon or that radicals produced later in the reperfusion process are unimportant to other aspects of the recovery process. Bolli et al. [57] have shown that production of radicals can persist for at least the first 3 hours of reperfusion, and although the level of production does not achieve that seen during the very early burst, it is nonetheless possible that it plays a contributory role in determining the extent or rate of postischemic recovery. Limitations of interpretation for many studies As is clear from the preceding paragraphs, during the past 5 years a substantial number of studies have been reported that, at first sight, appear to provide strong evidence for the existence of stunning as a discrete
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reperfusion-induced patholog3~. These studies also provide convincing evidence for a link between oxygen-derived free-radical production and the induction of the phenomenon. However, it is important to stress that a number of critical limitations require consideration before such firm conclusions can be drawn. Evidence for an involvement of free radicals is usually indirect. In the majority of studies cited thus far,
free radicals or their manipulation have not actually been measured. The association between radicals and stunning has generally been assumed on the basis of the known ability of the agents under test to promote the elimination, or to reduce the formation, of radical species. However, many, if not all, of the interventions studied will have secondary pharmacological or biochemical properties, and the possibility that it is these properties that confer the protection cannot automatically be ruled out.
Experimental requirements. Convincing evidence in support of an involvement of free radicals can only be derived from studies in which free radicals are measured, a relationship between free radical activity and the extent of stunning is demonstrated, and any successful intervention is shown to reduce the production of radicals with a commensurate increase in contractile function. Experiments are required in which drug-induced decreases in the production of radicals can be measured and can be shown to lead to an attenuation of stunning, and similarly, increases in radical production must be shown to exacerbate stunning. Rarely are these criteria fully met; however, some studies, particularly those from Bolli's group [55,56, 62], do fulfil these requirements. Although a number of indirect methods for assessing free-radical activity exist, they are often prone to artifact and do not help greatly in satisfying the above requirements. Radicals can only be adequately measured by the technique of electron spin resonance coupled with spin-trapping procedures [53,102]. In recent years a number of investigators have demonstrated that it is possible to detect and quantify the production of radicals in either coronary effluent or tissue from the hearts of a number of species, including the rat and the dog [53,55-57,102-113]. Thus, there is now convincing evidence for a burst of radical production during the early minutes of reperfusion after relatively short periods (e.g., 15 minutes) of regional or global ischemia (Figure 1). It has also been shown that the extent of this radical production is related to the duration or severity of the ischemia [57,105,108], that it can be attenuated by SOD and
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Fig. 1. Burst of free-radical productim~ (vis~alized by electron spin-resonance and spb~-trapping procedures) in the isolated rat heart after 15 minutes of ische~da. Adapted from [102] with permission.
other antioxidants [55,56,62,114], and that it occurs at a time during which the heart would be expected to be vulnerable to various forms of reperfusion-induced injury, including arrhythmias and stunning. While the exact identity of the primary radical species is yet to be resolved, there is reasonable evidence that, since the process is oxygen-dependent [102] and can be attenuated by SOD, oxygen-derived radicals are almost certainly involved. However, while they may be formed early in a complex cascade of radical reactions, they cannot necessarily be assumed to be the ultimate injurious species.
Electron spin-resonance and spin-trap studies of antioxidants and stunning. Bolli et al. [53,56,57] have used electron spin resonance, together with the spintrap alpha-phenyl N-tert-butyl nitrone (PBN), to define the time course and extent of radical production in the open-chest anesthetized dog during reperfusion after 15 minutes of coronary artery occlusion. Radical adducts, detected in the venous blood flowing from the reperfused tissue, increased suddenly at the time of reperfusion, peaked after 2 to 4 minutes, and then slowly declined. Production could be detected for as long as 3 hours after the initiation of reperfusion. Bolli et al. [53,56] went on to demonstrate that the extent of radical production could be reduced if the dogs were treated with SOD plus catalase, and furthermore, that this was accompanied by a reduction in the extent of stunning. Correlation between radical production and stunning. In their study with PBN, Bolli et al. [57] demon-
st~tt~t~it~gand Oxidant Stress
strated that there was a positive linear correlation between the magnitude of radical adduct production and the extent to which flow was reduced during the preceding ischemic period, lending further support to the concept that there is a relationship between the extent of radical production and the severity of antecedent ischemia. These conclusions are supported by the findings of other groups [105,113]. In additional studies with MPG and desferrioxamine, Bolli et al. [53,55,62] further showed that, so long as the drugs were available during the first minute of reperfusion, changes in radical adduct production and postischemic contractile dysfunction could be achieved.
Prooxidants and cardiac function. As mentioned earlier, support for the association between stunning and the production of radicals should come not only from studies in which radical production has been reduced, but also from studies in which enhanced production is shown to result in a greater impairment of contractile function. While few, if any, such studies have been carried out with coincident electron spin-resonance measurements, a number of investigations have shown exogenous radical production to exert an effect on contractile function. Thus, the administration of reactive oxygen intermediates (such as hydrogen peroxide), the application of freeradical-generating systems (such as hypoxanthine plus xanthine oxidase), or the use of promoters of free-radical production (such as iron) in a variety of experimental preparations have been shown to bring about profound and rapid reductions in contractile performance and/or changes in a variety of electrophysiological parameters [31,115-124]. In a number of these studies, SOD, catalase, or other antioxidants were shown to attenuate the detrimental effects of the free-radical-generating systems. One major difficulty in assessing the biological relevance of studies with free-radical-generating systems relates to the inability of investigators to quantify the extent of radical generation and then titrate this to a level that is thought to arise naturally from endogenous radical sources. Until this can be achieved, the possibility exists that massive excesses of radicals are being used in most studies and that this might exert a non-specific toxic effect. Arguing against this possibility are reports that some of the radical-induced changes in electrophysiology and contractile function can be attenuated by the coincident administration of various antioxidants [81]. Inappropriate timing of administration of anti-freeradical interventions. Regretably, many of the stud-
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ies of myocardial stunning and anti-free radical interventions have been characterized by protocols in which the intervention has been administered during, or even before, ischemia. It is well established that the postischemic recovery of contractile function is critically determined by the duration or the severity of the preceding period of ischemia. Thus, any intervention that possesses antiischemic properties (and thus has an ability to slow the rate or to reduce the extent of ischemic-induced injury) would be expected to enhance postischemic recovery. Since it is possible that some of the interventions studied possess antiischemic properties, then it is difficult to determine whether any beneficial effects on stunning are achieved by a direct effect upon some reperfusionmediated event or arise as a consequence of some indirect antiischemic effect. In a similar way, the ability of some interventions to reduce ischemic injury by modifying cellular metabolism or function prior to ischemia makes studies involving pretreatment very difficult to interpret.
Experimental requirements. This crucial problem of experimental design and interpretation can only be satisfactorily resolved by administering an intervention at the time of reperfusion. While this suffers the drawback that intervention may be ineffective because there was insufficient time for the agent to gain access to the tissue, it does allow positive results to be unequivocally attributed to an effect on some mechanism that operates during the reperfusion process. It should be stressed, however, that a satisfactory experimental design also demands that the agent be removed at a relatively early stage and the conferred protection be shown to persist after the last traces of the drug have been removed from the tissue. Unless this is done, it is impossible to exclude the possibility that the agent, as opposed to overcoming myocardial stunning, is exerting a simple inotropic effect, such as would be observed with the administration of calcium or a catecholamine [125], i.e., overriding rather than preventing myocardial stunning. Interventions given at the time of reperfusion and subsequently withdrawn. Fortunately some studies have been reported in which antioxidant interventions have been administered at the time of reperfusion. In a landmark study of this type, Bolli and colleagues [55] used four groups of dogs, each subjected to 15 minutes of coronary occlusion and 4 hours of reperfusion. In group 1, infusion of MPG was started 15 minutes before occlusion and terminated after 2 hours of reperfusion. In group 2, infusion of MPG was started 1 minute before reperfusion and again terminated
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after 2 hours of reperfusion. In group 3, infusion of MPG was started 1 minute after reperfusion. The postischemic recovery of contractile function was compared with that of control dogs receiving placebo (group 4). The recovery of function (postischemic wall thickening expressed as a percent of preischemic control) was much improved in groups 1 and 2 (50% and 47%, respectively, after 4 hours of reperfusion). In contrast, groups 3 and 4 showed little or no recovery, with wall thickening remaining at -29% and -25%, respectively. The results also indicated that there was no loss of protection after stopping the infusion of MPG in groups 1 and 2, suggesting that a sustained antistunning effect, rather than an inotropic stimulus, had been achieved. The same paper reported electron spin-resonance studies in hearts in which MPG was given either 1 minute before or 1 minute after reperfusion. Using PBN as a spin-trap agent, radical adduct production was shown to be very small in those hearts receiving MPG 1 minute before reperfusion, whereas in hearts given MPG 1 minute after reperfusion, adduct production was much greater, and in the first few minutes of reperfusion was similar to that in the MPG-free controls. As discussed earlier, this observation provides powerful evidence that the major events underlying the induction of stunning occur during the first 60 seconds of the reperfusion process. In a further study, Bolli et al. [57] explored the possibility, first raised by the author and his colleagues in studies of reperfusion-induced arrhythmias [126,127], that spin-trap agents themselves, by virtue of their ability to eliminate radicals from biological systems, might represent novel therapeutic agents in the control of myocardial stunning. In these studies [57], PBN was administered to dog hearts for a 10minute period starting 20 seconds before reperfusion. In comparison with PBN-free hearts, in which wall thickening after 3 hours of reperfusion was -58%, in the PBN group, despite the removal of the antioxidant, thickening had recovered to 16%. Most recently Bolli et al. [62] have reported a study in which desferrioxamine, administered at the time of reperfusion, attenuated stunning and reduced radical production.
Stunning: Convalescence or Reperfusion Pathology? In an early section of this article, the possibility was raised that myocardial stunning might be no more than the expression of the slow recovery of tissue from a period of trauma. Relatively few of the studies thus far reviewed help very much in determining whether this is so or whether stunning is truly a mani-
festation of damaging events that occur during the period of reperfusion.
The evidence for the phenomenon The strongest evidence in favor of the latter viewpoint can be derived from the study by Bolli et al. [57] in which PBN was administered as an antioxidant intervention for only 10 minutes, commencing 20 seconds before reperfusion. The substantial improvement in the rate of recovery of contractile function, despite only a transient exposure to the drug, provides a powerful argument for the concept of stunning as a true reperfusion pathology; this is further reinforced by the fact that PBN has no known positive inotropic properties. Conclusive proof that PBN is not acting directly on some ionic perturbation, or is acting in a "tonic" manner, or is accelerating some aspect of the recovery from ischemia-induced injury, would require further studies including the demonstration that the drug is completely cleared from the tissue but that the protection persists. What proportion of stunning can be attributed to reperfusion-induced injury? Figure 2 shows postischemic recovery profiles that are typical of many of the results discussed in the preceding sections. It is clear that while the use of a single antioxidant intervention considerably attenuates the degree of postischemic dysfunction, a substantial contractile deficit remains. Does this represent ischemiainduced injury, which is nonresponsive to the antioxidant and from which the heart will slowly recuperate, or is it additional reperfusion injury that the intervention failed to prevent? As discussed earlier, if different radical species, produced at different cellular sites (both lipophilic and hydrophilic), all contribute to stunning, then it would be surprising if a single intervention could completely prevent stunning. Bolli et al. [55], in discussing the relative contributions of "ischemia-associated" versus "reperfusionassociated" damage to postischemic dysfunction, have stated their opinion that "extensive additional injury occurs upon the restoration of flow and insofar as contractile function is concerned, such injury appears to be a major component of the damage." If reperfusion injury does account for a greater proportion of contractile dysfunction than is revealed through the application of a single antioxidant intervention, it would be reasonable to expect that combinations of different interventions, or the use of different doses, might result in more of the residual contractile deficit being eliminated. In addition, if, as is thought [8], other nonradical mechanisms are also involved in the genesis of stunning, then a combina-
Stunning and Oxidant Stress
papers in this issue of Cardiovascular Drugs and Therapy. As with so many of the adverse consequences of ischemia and reperfusion, it seems probable that multiple mechanisms are contributory to the pathogenesis of myocardial stunning. However, the two most frequently discussed candidates are freeradical-induced injury and disturbances of calcium homeostasis [8,26,146-153]. Unfortunately, some investigators view these two as alternatives, which is not necessarily the case.
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Stunning: A Radical Re-view David J. Hearse Cardiovascular Research, The Rayne Institute, St Thomas" Hospital, London, UK
Summary. The recovery from trauma, whether ischemia or some other form of tissue injury, is never instantaneous; time is always required for repair and the return of normal metabolism and function. To what extent the delay in recovery of contractile activity (stunning) after a brief period of isehemia represents convalescence from ischemia-induced injury, as opposed to the expression of reperfusion-induced injury, is perhaps not as clear as the proponents of stunning would hope. Definitive evidence for a distinct reperfusion-induced pathology, which compromises the recovery of contractile function from the depressed state induced by ischemia, is elusive. If reperfusion-induced injury accounts for a significant proportion of stunning, then the molecular mechanisms responsible for initiating the event and those responsible for orchestrating the event at the level of the contractile protein are far from clear. Perturbations of calcium homeostasis are frequently cited as responsible for the depressed contractile state, however, some metabolic derangement must precede any pathologically induced ionic disturbance. In this connection, evidence indicates that free-radical-induced oxidant stress, during the early moments of reperfusion, may modify the activity of a number of thiol-regulated proteins that are directly, or indirectly, responsible for controlling the movement of calcium. Sarcolemmal sodium-calcium exchange and the calcium release channel of the sarcoplasmic reticulum may be activated, whereas the sarcolemmal calcium pump and sodium-potassium ATPase, together with the calcium pump of the sarcoplasmic reticulum, may be inhibited. Under the conditions prevailing during ischemia and reperfusion, this would be expected to promote an early intracellular calcium overload. It is difficult to reconcile such a change with the decreased inotropic state that characterizes stunning; however, it seems likely that the calicum overload is transient and that the stunned myocardium rapidly reestablishes normal levels of intracellular calcium. It is still difficult to explain adequately the reduced inotropic state; clearly, the mechanism of stunning is not quite as simple as its definition.
any more than a conveniently descriptive term for the natural, time-dependent recovery of traumatized tissue. In o t h e r words, to inquire w h e t h e r this t r a n s i e n t postischemic contractile deficit is a reflection of some unfavorable component of reperfusion, some injury sustained d u r i n g ischemia, or some combination of the two. This question will be e x p l o r e d b y r e v i e w i n g a n u m b e r of studies of ischemia and reperfusion in which anti-free-radical i n t e r v e n t i o n s have been used to enhance the postischemic r e c o v e r y of contractile function. From this, the hypothesis will be advanced
that oxidant stress-induced changes in the activity of thiol-regulated proteins may be critical in the initiation qf the ionic disturbances that occur during reperfusion and that may contribute to stunning and reperfusion arrhythmias.
Stunning: The Lure of the Cognomen Myocardial stunning is just one of an impressive
T h e publication of a focused issue of Cardiovascular Drugs and Therapy d e d i c a t e d to m y o c a r d i a l stunning
a r r a y of s e d u c t i v e t e r m s t h a t , in addition to "stunning the cardiologist" [1,2,], m a y increase the risk of lending c r e d i b i l i t y to an i n a d e q u a t e l y defined phenomenon m e r e l y b y giving it a name. In its simplest sense, m y o c a r d i a l s t u n n i n g could be considered as the expression of a postischemic " h a n g o v e r , " a phase of organ malfunction and r e c u p e r a t i o n t h a t i n e v i t a b l y followes a period of t r a u m a . W h e t h e r caused b y an excess of alcohol, a bout of influenza, or t r a n s i e n t ischemia, t h e r e can be few pathological s t a t e s in which a complete and i n s t a n t a n e o u s r e c o v e r y is o b s e r v e d as soon as t h e source of i r r i t a t i o n is r e m o v e d . Conditions such as ischemia, inflammation, and viral infections u p s e t , at a molecular level, the delicate balance of cellular m e t a b o l i s m and function. E n z y m e p a t h w a y s become d i s t u r b e d , toxins accumulate, and, not s u r p r i s i n g l y , cellular r e c o v e r y t a k e s time, particularly if p r o t e i n s y n t h e s i s and s t r u c t u r a l r e p a i r a r e required. W h a t t h e n is special about m y o c a r d i a l isch-
m i g h t s u g g e s t t h a t this is a p r o v e n e x a m p l e of r e p e r f u s i o n - i n d u c e d injury, of p r o b a b l e clinical relevance, t h a t is likely to c r e a t e a valuable t a r g e t for novel t h e r a p e u t i c interventions. One objective of this article is to question w h e t h e r myocardial stunning is
Address for correspondence and reprint requests: David J. Hearse, PhD, DSc, Cardiovascular Reseach, The Rayne Institute, St. Thomas' Hospital, London SE1 7EH, UK.
Cardiovasc Drugs Ther 1991;5:853-876 Key Words. stunning, myocardium, free radicals, oxidant stress, calcium, reperfusion, arrhythmias, ischemia
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emia? Why should we not consider the transient period of postischemic contractile and metabolic dysfunction simply as a phase in a process of progressive recovery--a form of myocardial convalescence? Why should the heart alone be expected to recover almost instantaneously from a condition as severe and complex as ischemia? Proponents of the stunning hypothesis would claim that the antecedent ischemia is not solely responsible for the depressed function and slow recovery, but that there is also some unfavorable component of reperfusion that limits the process. In support of this, they would argue that since the administration of certain drugs at the time of reperfusion can improve the rate of recovery, the interventions must be counteracting some unfavorable aspect of the reperfusion process itself. However, is this a sufficiently convincing argument for the existence of a distinct reperfusioninduced pathology that is capable of negating some of the benefits of reflow? To take an extreme analogy, it is perhaps akin to arguing that a hangover headache arises not from consuming too much alcohol but from stopping the drinking and that the curative aspirin comes to the rescue by countering the consequences of terminating the insult, rather than accelerating the recovery from it. Could it really be argued that, with myocardial stunning, the heart is suffering from withdrawal of ischemia?
Is S t u n n i n g a T r u e Reperfusion-Induced
Pathology?
A positive response to the last question implies the existence of reperfusion-induced injury. This concept is, however, becoming increasingly controversial [3-7], since it requires that, while reperfusion is overall beneficial, one or more components of the reflow process must be detrimental and act to slow the rate, or reduce the extent, of recovery. Arguments in support of reperfusion-induced injury usually derive from experiments in which interventions are given at the time of reperfusion and are shown to increase the rate of postischemic recovery. However, unless it can be shown that these agents actually prevent some unfavorable aspect of reperfusion (rather than simply acting as an inotropic stimulus or accelerating the natural, but slow, recovery process), then a reperfusion-mediated component of myocardial stunning remains unproven. Before focusing on the origins of myocardial stunning, it is therefore necessary to consider the controversy [6,7] over reperfusion-induced injury. Interest in this subject has been greatly stimulated by the advent of thrombolytic and angioplastic procedures for
the reperfusion of regionally ischemic human myocardium, by the growth of cardiac surgical procedures that necessitate reperfusion of the whole heart after extended periods of global ischemia, and by the realization that transient coronary spasm and reperfusion may be a frequent occurrence in humans. As stressed by Bolli [8], the occurrence of myocardial stunning in patients could significantly delay the benefits of reperfusion.
Reperfusion-induced injury: fact or pharmacologic fantasy? Without question, early reperfusion is an absolute prerequisite for the survival of ischemic tissue. However, it is widely believed that reperfusion may not be without hazard and may of itself induce injury [3,4,9-12], which may even kill potentially viable cells. Although there is no doubt that reperfusion can increase the apparent severity of tissue injury, some investigators question whether reperfusion can actually cause lethal cell injury [13,14]. They argue that, rather than creating injury de novo, reperfusion merely accelerates the expression of the injury that had already occurred during the preceding period of severe ischemia. In an attempt to resolve some of the confusion over reperfusion-induced injury and its existence, the present author has proposed [4,6] that the potentially unfavorable sequelae of reperfusion should be divided into the following four categories.
Reperfusion-induced arrhythmias. These may range in severity from ventricular premature beats to ventricular fibrillation; they occur within seconds [15,16] of the onset of reflow and have been observed in all species studied, including humans. Many contributory mechanisms have been proposed [16], but most recently, attention has been focused on the possibility that free-radical-induced oxidant stress acting on cellular ion regulating processes may be involved (for reviews see [17,18]).
Myocardial stunning. It is widely believed that reperfusion may precipitate a number of unfavorable (but nonlethal) cellular changes that, if given sufficient time, will revert to normal. The most widely discussed of these is mechanical stunning [8,19,20]. However, in this article the possibility will be considered that stunning might merely represent the slow recovery from the trauma of ischemia, rather than the occurrence of a specific reperfusion-induced pathology. A variety of candidates for the mechanism underlying postischemic contractile dysfunction have been
Stunning and Oxidant Stress
proposed. These include transient intracellular calcium overload, decreased sensitivity of the myofilaments to calcium, reduced ability to resynthesize high-energy phosphates, impaired sympathetic neural responsiveness, heterogeneous impairment of regional perfusion, loss of creatine kinase activity and impaired utilization of energy by the myofibrils, damage to the extracellular collagen matrix, and leukocyte activation. Bolli [5,8] has put forward arguments against a number of the above proposals and has claimed that, as with reperfusion-induced arrhythmias, the two most plausible mechanisms for myocardial stunning relate to free radical-induced injury and disturbances of calcium homeostasis. Lethal reperfusion-induced injury. The killing of cells by suboptimal reperfusion represents the most widely held concept of reperfusion-induced injury. However, as recently argued [4,6,7], there is little conclusive evidence for the existence of this form of reperfusion injury. Accelerated expression of necrosis. In this fourth sequela of reperfusion, the number of cells that die will not be increased, but the manner in which the necrosis develops may be different. Reperfusion-induced injury in tissue components other than the myocyte In enumerating possible forms of reperfusion-induced injury, it is important to acknowledge that the myocyte is not the only structure susceptible to injury during ischemia and reperfusion [6]. The microvasculature and the endothelium, the conducting system, and the connective tissue should all be considered [21-24] as possible targets. Thus a number of studies [6,21,23] have addressed the issue of stunning and the microvasculature. It could well be that the microvasculature and its endothelial lining, rather than the myocyte, play a determining role in the expression of some of the sequelae of reperfusion. Such an example could be the "no-reflow phenomenon" [25,26] in which the removal of a coronary occlusion does not lead to the restoration of coronary flow, despite the removal of occlusion.
T h e A s s o c i a t i o n B e t w e e n Free R a d i c a l s a n d Postischemic Contractile F u n c t i o n It is difficult to attribute the original study of free radicals in relation to cardiac contractile function to any single group of investigators. However, special mention should be made of the work of Guarnieri and
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colleagues, who in 1978 reported [27] that alphatocopherol could reduce enzyme leakage and improve the recovery of contractile function and mitochondrial metabolism in rabbit hearts subjected to 30 minutes of hypoxia and reoxygenation. In that study, and in another in 1980 [28], they provided strong collateral evidence for the proposition, first advanced by the present author [3,29,30], that oxygen-derived free radicals may play some role in determining the extent of contractile and metabolic recovery after a period of myocardial oxygen deprivation. Early support for this proposition is also to be found in a study from the author's laboratory [31] in which exogenous hydrogen peroxide was shown to impair postischemic functional recovery. Influenced by the work of the Italian group, Shlafer and colleagues in the United States extended investigations from crystalloid-perfused hypoxic hearts to studies involving ischemia and reperfusion with blood as well as crystalloid media [32-34]. Thus, in 1981 and 1982 Shlafer et al. [32,34], using the rabbit heart subjected to 2 hours of hypothermic ischemia, reported that superoxide dismutase (SOD) plus catalase, when added for only 5 minutes to a preischemic infusion solution and a postischemic reperfusion solution, reduced enzyme leakage and improved postischemic recovery of contractile function and mitochondrial metabolism. Similar results were also observed by the same authors [33] in studies with bloodperfused cat hearts. Potential relevance to cardiac surgery The work of Guarnieri and Shlafer provided an important stimulus for the evaluation of anti-free-radical interventions in the surgical arena. In 1982 and 1983, Stewart and colleagues, using dog hearts on cardiopulmonary bypass, reported [35,36] that SOD and mannitol improved the recovery of contractile function after 60 minutes of hypothermic global ischemia. Later, Stewart et al. [37] demonstrated a similar benefit with allopurinol; they also extended the concept to include cardiac transplantation [38]. Since 1983 there have been many studies [39-49] in the surgical literature supporting the view that antioxidant interventions, given before or after ischemia, improve the immediate postischemic recovery of cardiac contractile function. In contrast to the studies of antioxidants and infarct size (see below), there has been relatively little disagreement over the value of such interventions in models of surgical ischemia. Potential relevance to myocardial infarction While cardiac surgeons were exploring the potential benefits of anti-free-radical interventions as a means
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of improving the short-term recovery of the globally ischemic myocardium, cardiologists were assessing whether these agents conferred any benefit on the regionally ischemic myocardium. In 1984 Jolly et al. [50] studied postischemic contractile function and infarct size in dog hearts subjected to 90 minutes of coronary artery occlusion and 24 hours of reperfusion; SOD plus catalase was shown to afford considerable protection. Their study signalled the start of a period of intense interest (and controversy) over the possibility that antioxidant interventions might provide significant protection during evolving myocardial infarction. At least 50 (often conflicting) studies were reported in major journals (for reviews see [6,10, 51,52]; however, in the majority of these myocardial infarct size rather than postischemic contractile function was the primary end point.
Free Radicals a n d Myocardial Stunning Interest in the temporal contractile effects of oxygenderived free radicals and the value of antioxidant interventions during the early hours of reperfusion has been greatly stimulated by an excellent and extensive series of studies by Bolli and coworkers [8,20,21,5362]. In 1985 Bolli's group reported [61] that SOD and catalase, when given to open-chest dogs subjected to 15 minutes of regional ischemia and 2 hours of reperfusion, enhanced the postischemic recovery of function from 31.6 _+ 9.8% to 74.2 _+ 8.4%. Since that study, there have been many investigations (for reviews see [8,10]), from several laboratories (mostly employing the dog heart), into the ability of a wide variety of anti-free-radical interventions to enhance postischemic functional recovery. In some instances there is a good degree of agreement over efficacy and mechanisms; in others controversy exists [10]. Studies of anti.free radical interventions and stunning The manipulation of free radical production in the heart, or any other organ, can be achieved either by promoting the elimination of radicals (by supplementing endogenous antioxidant defenses) or by preventing their formation. In the following paragraphs studies of myocardial stunning using a variety of such approaches, targeted at both intracellular and extracellular sites, are summarized. Antioxidant enzymes. In the context of myocardial stunning, only SOD and/or catalase have been studied; in other areas (e.g., cardioplegia and global isch-
emia) enzymes such as peroxidase have also been investigated [45,63]. While there is some disagreement in the literature (usually from studies with extended periods of ischemia [64,65]), a considerable number of investigations now confirm that SOD, when used in combination with catalase, can accelerate the postischemic recovery of contractile function. Thus, in addition to the studies of Bolli's group [61], Gross et al. [66], Przyklenk and Kloner [67], and Murry et al. [68] have all reported positive results in the dog heart. In contrast, efficacy is in question in those studies in which SOD has been administered alone; for example, Buchwald et al. [69] gave SOD to pigs and were unable to attenuate stunning. Whether this was due to a species difference, to some variation in dose or administration protocol, or to the absence of catalase was not resolved. However, Bolli's group [70], using the dog, have also observed that SOD alone fails to protect against stunning; they argued that this provided evidence that both superoxide and hydrogen peroxide contributed to the cellular damage responsible for stunning. Koerner et al. [71], using the rabbit, have also failed to achieve protection with SOD alone, whereas SOD plus catalase was very effective. Contrasting with these results are the in vitro findings of Ambrosio and colleagues [72], in which human recombinant SOD alone, given at the time of reperfusion, afforded protection against the metabolic and functional consequences of stunning. The authors also claimed that catatase afforded no additional protection. Low molecular weight organic antioxidants. To assess the importance of superoxide, hydrogen peroxide, and hydroxyl radicals in the genesis of stunning, Bolli et al. [58] have studied the hydroxyl radical scavenger dimethylthiourea (DMTU) in the dog heart with 15 minutes of coronary artery occlusion and 4 hours of reperfusion. When given over 45 minutes (starting 30 minutes before occlusion), DMTU improved the recovery of postischemic wall thickening after 4 hours of reperfusion from 36 +- 13% to 67 +- 5%. Similar results have been reported [60,71] using mercaptopropionyl glycine (MPG), which is another organic antioxidant that readily gains access to the intracellular space. Bolli [8,55] argued that these findings, taken with those of the studies with antioxidant enzymes, provided evidence that the hydroxyl radical played a central role in the induction of injury. A number of other organic antioxidants have been positively assessed in relation to their ability to improve the postischemic recovery of contractile function; these include N~acetylcysteine [73,74], probucol [75], and alpha-tocopherol [76].
Stunning and Oxidant Stress
Cofactors for radical production. Further evidence of a role for the hydroxyl radical, together with additional support for an association between radicals and stunning, can be derived from reports [20,54,62, 77-80] that desferrioxamine can also attenuate myocardial stunning. Transition metals, such as iron and copper, are essential cofactors for the hydroxyl radical-producing Haber-Weiss reaction; desferrioxamine (which avidly binds iron) and other chelators have also been shown to reduce other types of reperfusion-induced injury, such as arrhythmias [81-83]. Inhibition of radical-generating processes. Studies [24,84,85] with allopurinol or oxypurinol, the inhibitors of xanthine oxido-reductase, provide further support for an association between reactive oxygen intermediates and myocardial stunning. However, some caution should be exercised in the interpretation of studies with these two agents, since they have been found [46] to be protective in hearts from species that are known to be deficient in xanthine oxido-reductase. In this connection, it is interesting that in studies [86] with amflutizole (a highly specific inhibitor of xanthine oxidase activity), an attenuation of stunning could not be demonstrated in the dog heart. Removal of radical sources. Many studies have focused on the possible importance of the leukocyte as a mediator of myocardial stunning, and considerable controversy has been generated (for reviews see [8,13,87,88]). In such studies, leukocytes have either been physically removed or their activity inhibited. This has been achieved in a number of ingenious manners, including the use of leukocyte filters, neutrophil antisera, inhibitors of leukotriene production, or the use of agents that inhibit leukocyte adherence. While some studies [89-92] have shown a beneficial effect on postischemic function, others [93-96] have failed to achieve any protection. This has led to the opinion [8] that leukocytes, or the radicals that they produce, are not involved in myocardial stunning. In any event, as Bolli pointed out [6,8], there is now evidence that in the early minutes of reperfusion after a brief period of ischemia, myocardial leukocyte content is reduced [97]. He went on to argue that this, together with the fact that stunning can be induced in leukocyte-free isolated perfused heart preparations, made it unlikely that leukocytes played a central role in myocardial stunning. However, this is not to deny their importance in other aspects of the pathogenesis of injury during ischemia and reperfusion. Sites and sources of radical production A very diverse group of antioxidant interventions appears to be able to improve postischemic functional
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recovery; some of these (e.g., SOD and catalase) are likely to be restricted to the extracellular space, others (e.g., DMTU and MPG) probably to gain access to the cytoplasm, while lipophilic agents (e.g., alpha tocopherol) are localized in membrane lipids. Since radicals can be produced at various intracellular and extracellular sites, and since all of these interventions exert partial protection, it would seem that radicalmediated injury is likely to arise as a consequence of the combined effects of multiple, independent, radical-producing pathways. If this is the case, then any single intervention is likely to attenuate only part of the injury. Potential sources of radical production are numerous and have been reviewed in detail elsewhere [7,18]; they include the mitochondria, catecholamines, the arachidonic acid pathway, hemoglobin, myoglobin, aldehyde oxidases, and leukocytes.
Time course of radical production A very important conclusion to emerge from the study by Bolli et al. [55] is that there appears to be an extremely narrow time window (less than 60 seconds) during which free radicals make their contribution to the overall stunning process. In this study, the antioxidant MPG, given 1 minute before reperfusion, was highly effective in reducing the early burst of radical production and attenuating stunning; however, when given I minute after reperfusion, little protection was observed. Further evidence supporting the occurrence of extremely rapid radical-mediated events comes from the many studies of reperfusion-induced arrhythmias [81,98-101] in which induction and protection can take place within the first 10 seconds of the restoration of flow. Although it is the production of radicals during the first 60 seconds of reperfusion that appears to be critical to stunning, this does not mean that radical production is necessarily a short-lasting phenomenon or that radicals produced later in the reperfusion process are unimportant to other aspects of the recovery process. Bolli et al. [57] have shown that production of radicals can persist for at least the first 3 hours of reperfusion, and although the level of production does not achieve that seen during the very early burst, it is nonetheless possible that it plays a contributory role in determining the extent or rate of postischemic recovery. Limitations of interpretation for many studies As is clear from the preceding paragraphs, during the past 5 years a substantial number of studies have been reported that, at first sight, appear to provide strong evidence for the existence of stunning as a discrete
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reperfusion-induced patholog3~. These studies also provide convincing evidence for a link between oxygen-derived free-radical production and the induction of the phenomenon. However, it is important to stress that a number of critical limitations require consideration before such firm conclusions can be drawn. Evidence for an involvement of free radicals is usually indirect. In the majority of studies cited thus far,
free radicals or their manipulation have not actually been measured. The association between radicals and stunning has generally been assumed on the basis of the known ability of the agents under test to promote the elimination, or to reduce the formation, of radical species. However, many, if not all, of the interventions studied will have secondary pharmacological or biochemical properties, and the possibility that it is these properties that confer the protection cannot automatically be ruled out.
Experimental requirements. Convincing evidence in support of an involvement of free radicals can only be derived from studies in which free radicals are measured, a relationship between free radical activity and the extent of stunning is demonstrated, and any successful intervention is shown to reduce the production of radicals with a commensurate increase in contractile function. Experiments are required in which drug-induced decreases in the production of radicals can be measured and can be shown to lead to an attenuation of stunning, and similarly, increases in radical production must be shown to exacerbate stunning. Rarely are these criteria fully met; however, some studies, particularly those from Bolli's group [55,56, 62], do fulfil these requirements. Although a number of indirect methods for assessing free-radical activity exist, they are often prone to artifact and do not help greatly in satisfying the above requirements. Radicals can only be adequately measured by the technique of electron spin resonance coupled with spin-trapping procedures [53,102]. In recent years a number of investigators have demonstrated that it is possible to detect and quantify the production of radicals in either coronary effluent or tissue from the hearts of a number of species, including the rat and the dog [53,55-57,102-113]. Thus, there is now convincing evidence for a burst of radical production during the early minutes of reperfusion after relatively short periods (e.g., 15 minutes) of regional or global ischemia (Figure 1). It has also been shown that the extent of this radical production is related to the duration or severity of the ischemia [57,105,108], that it can be attenuated by SOD and
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other antioxidants [55,56,62,114], and that it occurs at a time during which the heart would be expected to be vulnerable to various forms of reperfusion-induced injury, including arrhythmias and stunning. While the exact identity of the primary radical species is yet to be resolved, there is reasonable evidence that, since the process is oxygen-dependent [102] and can be attenuated by SOD, oxygen-derived radicals are almost certainly involved. However, while they may be formed early in a complex cascade of radical reactions, they cannot necessarily be assumed to be the ultimate injurious species.
Electron spin-resonance and spin-trap studies of antioxidants and stunning. Bolli et al. [53,56,57] have used electron spin resonance, together with the spintrap alpha-phenyl N-tert-butyl nitrone (PBN), to define the time course and extent of radical production in the open-chest anesthetized dog during reperfusion after 15 minutes of coronary artery occlusion. Radical adducts, detected in the venous blood flowing from the reperfused tissue, increased suddenly at the time of reperfusion, peaked after 2 to 4 minutes, and then slowly declined. Production could be detected for as long as 3 hours after the initiation of reperfusion. Bolli et al. [53,56] went on to demonstrate that the extent of radical production could be reduced if the dogs were treated with SOD plus catalase, and furthermore, that this was accompanied by a reduction in the extent of stunning. Correlation between radical production and stunning. In their study with PBN, Bolli et al. [57] demon-
st~tt~t~it~gand Oxidant Stress
strated that there was a positive linear correlation between the magnitude of radical adduct production and the extent to which flow was reduced during the preceding ischemic period, lending further support to the concept that there is a relationship between the extent of radical production and the severity of antecedent ischemia. These conclusions are supported by the findings of other groups [105,113]. In additional studies with MPG and desferrioxamine, Bolli et al. [53,55,62] further showed that, so long as the drugs were available during the first minute of reperfusion, changes in radical adduct production and postischemic contractile dysfunction could be achieved.
Prooxidants and cardiac function. As mentioned earlier, support for the association between stunning and the production of radicals should come not only from studies in which radical production has been reduced, but also from studies in which enhanced production is shown to result in a greater impairment of contractile function. While few, if any, such studies have been carried out with coincident electron spin-resonance measurements, a number of investigations have shown exogenous radical production to exert an effect on contractile function. Thus, the administration of reactive oxygen intermediates (such as hydrogen peroxide), the application of freeradical-generating systems (such as hypoxanthine plus xanthine oxidase), or the use of promoters of free-radical production (such as iron) in a variety of experimental preparations have been shown to bring about profound and rapid reductions in contractile performance and/or changes in a variety of electrophysiological parameters [31,115-124]. In a number of these studies, SOD, catalase, or other antioxidants were shown to attenuate the detrimental effects of the free-radical-generating systems. One major difficulty in assessing the biological relevance of studies with free-radical-generating systems relates to the inability of investigators to quantify the extent of radical generation and then titrate this to a level that is thought to arise naturally from endogenous radical sources. Until this can be achieved, the possibility exists that massive excesses of radicals are being used in most studies and that this might exert a non-specific toxic effect. Arguing against this possibility are reports that some of the radical-induced changes in electrophysiology and contractile function can be attenuated by the coincident administration of various antioxidants [81]. Inappropriate timing of administration of anti-freeradical interventions. Regretably, many of the stud-
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ies of myocardial stunning and anti-free radical interventions have been characterized by protocols in which the intervention has been administered during, or even before, ischemia. It is well established that the postischemic recovery of contractile function is critically determined by the duration or the severity of the preceding period of ischemia. Thus, any intervention that possesses antiischemic properties (and thus has an ability to slow the rate or to reduce the extent of ischemic-induced injury) would be expected to enhance postischemic recovery. Since it is possible that some of the interventions studied possess antiischemic properties, then it is difficult to determine whether any beneficial effects on stunning are achieved by a direct effect upon some reperfusionmediated event or arise as a consequence of some indirect antiischemic effect. In a similar way, the ability of some interventions to reduce ischemic injury by modifying cellular metabolism or function prior to ischemia makes studies involving pretreatment very difficult to interpret.
Experimental requirements. This crucial problem of experimental design and interpretation can only be satisfactorily resolved by administering an intervention at the time of reperfusion. While this suffers the drawback that intervention may be ineffective because there was insufficient time for the agent to gain access to the tissue, it does allow positive results to be unequivocally attributed to an effect on some mechanism that operates during the reperfusion process. It should be stressed, however, that a satisfactory experimental design also demands that the agent be removed at a relatively early stage and the conferred protection be shown to persist after the last traces of the drug have been removed from the tissue. Unless this is done, it is impossible to exclude the possibility that the agent, as opposed to overcoming myocardial stunning, is exerting a simple inotropic effect, such as would be observed with the administration of calcium or a catecholamine [125], i.e., overriding rather than preventing myocardial stunning. Interventions given at the time of reperfusion and subsequently withdrawn. Fortunately some studies have been reported in which antioxidant interventions have been administered at the time of reperfusion. In a landmark study of this type, Bolli and colleagues [55] used four groups of dogs, each subjected to 15 minutes of coronary occlusion and 4 hours of reperfusion. In group 1, infusion of MPG was started 15 minutes before occlusion and terminated after 2 hours of reperfusion. In group 2, infusion of MPG was started 1 minute before reperfusion and again terminated
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after 2 hours of reperfusion. In group 3, infusion of MPG was started 1 minute after reperfusion. The postischemic recovery of contractile function was compared with that of control dogs receiving placebo (group 4). The recovery of function (postischemic wall thickening expressed as a percent of preischemic control) was much improved in groups 1 and 2 (50% and 47%, respectively, after 4 hours of reperfusion). In contrast, groups 3 and 4 showed little or no recovery, with wall thickening remaining at -29% and -25%, respectively. The results also indicated that there was no loss of protection after stopping the infusion of MPG in groups 1 and 2, suggesting that a sustained antistunning effect, rather than an inotropic stimulus, had been achieved. The same paper reported electron spin-resonance studies in hearts in which MPG was given either 1 minute before or 1 minute after reperfusion. Using PBN as a spin-trap agent, radical adduct production was shown to be very small in those hearts receiving MPG 1 minute before reperfusion, whereas in hearts given MPG 1 minute after reperfusion, adduct production was much greater, and in the first few minutes of reperfusion was similar to that in the MPG-free controls. As discussed earlier, this observation provides powerful evidence that the major events underlying the induction of stunning occur during the first 60 seconds of the reperfusion process. In a further study, Bolli et al. [57] explored the possibility, first raised by the author and his colleagues in studies of reperfusion-induced arrhythmias [126,127], that spin-trap agents themselves, by virtue of their ability to eliminate radicals from biological systems, might represent novel therapeutic agents in the control of myocardial stunning. In these studies [57], PBN was administered to dog hearts for a 10minute period starting 20 seconds before reperfusion. In comparison with PBN-free hearts, in which wall thickening after 3 hours of reperfusion was -58%, in the PBN group, despite the removal of the antioxidant, thickening had recovered to 16%. Most recently Bolli et al. [62] have reported a study in which desferrioxamine, administered at the time of reperfusion, attenuated stunning and reduced radical production.
Stunning: Convalescence or Reperfusion Pathology? In an early section of this article, the possibility was raised that myocardial stunning might be no more than the expression of the slow recovery of tissue from a period of trauma. Relatively few of the studies thus far reviewed help very much in determining whether this is so or whether stunning is truly a mani-
festation of damaging events that occur during the period of reperfusion.
The evidence for the phenomenon The strongest evidence in favor of the latter viewpoint can be derived from the study by Bolli et al. [57] in which PBN was administered as an antioxidant intervention for only 10 minutes, commencing 20 seconds before reperfusion. The substantial improvement in the rate of recovery of contractile function, despite only a transient exposure to the drug, provides a powerful argument for the concept of stunning as a true reperfusion pathology; this is further reinforced by the fact that PBN has no known positive inotropic properties. Conclusive proof that PBN is not acting directly on some ionic perturbation, or is acting in a "tonic" manner, or is accelerating some aspect of the recovery from ischemia-induced injury, would require further studies including the demonstration that the drug is completely cleared from the tissue but that the protection persists. What proportion of stunning can be attributed to reperfusion-induced injury? Figure 2 shows postischemic recovery profiles that are typical of many of the results discussed in the preceding sections. It is clear that while the use of a single antioxidant intervention considerably attenuates the degree of postischemic dysfunction, a substantial contractile deficit remains. Does this represent ischemiainduced injury, which is nonresponsive to the antioxidant and from which the heart will slowly recuperate, or is it additional reperfusion injury that the intervention failed to prevent? As discussed earlier, if different radical species, produced at different cellular sites (both lipophilic and hydrophilic), all contribute to stunning, then it would be surprising if a single intervention could completely prevent stunning. Bolli et al. [55], in discussing the relative contributions of "ischemia-associated" versus "reperfusionassociated" damage to postischemic dysfunction, have stated their opinion that "extensive additional injury occurs upon the restoration of flow and insofar as contractile function is concerned, such injury appears to be a major component of the damage." If reperfusion injury does account for a greater proportion of contractile dysfunction than is revealed through the application of a single antioxidant intervention, it would be reasonable to expect that combinations of different interventions, or the use of different doses, might result in more of the residual contractile deficit being eliminated. In addition, if, as is thought [8], other nonradical mechanisms are also involved in the genesis of stunning, then a combina-
Stunning and Oxidant Stress
papers in this issue of Cardiovascular Drugs and Therapy. As with so many of the adverse consequences of ischemia and reperfusion, it seems probable that multiple mechanisms are contributory to the pathogenesis of myocardial stunning. However, the two most frequently discussed candidates are freeradical-induced injury and disturbances of calcium homeostasis [8,26,146-153]. Unfortunately, some investigators view these two as alternatives, which is not necessarily the case.
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