The cell biology of acute myocardial ischemia.

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Annu. Rev. Med. 1991. 42:225-46 Cupyright © 1991 by Annual Reviews Inc. All rights reserved

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THE CELL BIOLOGY OF ACUTE MYOCARDIAL ISCHEMIA!

Annu. Rev. Med. 1991.42:225-246. Downloaded from www.annualreviews.org by University of Sussex on 01/04/13. For personal use only.

Robert B. Jennings, M.D., and Keith A. Reimer, M.D., Ph.D. Department of Pathology, Duke University Medical Center, Box 3712, Durham, North Carolina 27710 KEY WORDS:

stunning, preconditioning, anaerobic glycolysis, adenine nucleo tide pool, lethal reperfusion injury, ion gradients, reversible injury,

irreversible injury AnSTRACT

The metabolic changes associated with the sudden onset of ischemia caused by occlusion of a major coronary artery include (a) cessation of aerobic metabolism, (b) depletion of creatine phosphate (CP), (c) onset of anaer obic glycolysis, and (d) accumulation of glycolytic products, such as lactate and alpha glycerol phosphate (IXGP), and catabolites of the nucleotide pools in the tissue. These changes are associated with contractile failure and electrocardiographic alterations. Since the demand of the myocardium for high-energy phosphate ( �P) exceeds the available supply, the net amount of ATP in tissue decreases. Eighty percent of the supply of �P utilized by severely ischemic tissue comes from anaerobic glycolysis using glycogen as the principal substrate. Early in ischemia, contractile activity utilizes ATP, but much of the continuing utilization of ATP by the ischemic tissue is energy wasted via the mitochondrial ATPase. A lesser quantity of ATP is used by ion transport ATPases. Metabolic changes slow as the duration of ischemia increases. Irreversibly injured myocytes exhibit (a) very low levels of ATP « 10% of control); (b) cessation of anaerobic glycolysis; (c) high levels ofH+ , AMP, INO, lactate, and IXGP; (d) a greatly increased osmolar load; (e) mitochondrial swelling and formation of amorphous matrix densities; and (f) disruption of the sarcolemma. The I Much of the work described herein was supported by grants HL23138 and HL274 1 6 from the National Heart, Lung, and Blood Institute of the National Institutes of Health.

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latter event is generally recognized as lethal, but its pathogenesis remains to be established. Most severely ischemic myocytes are dead in regional ischemia in the anesthetized open-chest dog heart after only 60 minutes of ischemia. Less severely ischemic myocytes in the mid- and subepicardial myocardium survive for as long as six hours. Virtually all myocytes destined to die in a zone of ischemia are irreversibly injured after six hours of ischemia have passed. Certain changes exhibited by myocytes injured by severe ischemia and reperfused late in the reversible phase of injury do not return to the control conditions for a period of days, while others rebound in only seconds to minutes. The adenine nucleotide pool still is not fully restored after four days of reperfusion. Stunning disappears after one to two days of reflow. The preconditioning effect is partially lost after two hours of reperfusion. The timing of its disappearance has not been fully established. Aerobic metabolism is restored after only a few minutes of reperfusion. Thus, reperfusion salvages injured myocardium and restores its structure and function to the control state at a variable rate. INTRODUCTION

Myocardial ischemia is present whenever the arterial flow cannot provide enough oxygen to meet the energy demands of the tissue, i.e. to maintain tissue function (1). The manifestations of altered function include cessation of contraction, alterations in membrane potential, and a variety of meta bolic changes most of which develop at or about the time that the oxygen supply becomes limiting. At this point, the myocardium shifts from aerobic to anaerobic metabolism. Irrespective of the functional changes, anaerobic glycolysis, with its accompanying accumulation of glycolytic inter mediates, especially lactate, is an invariable metabolic marker of the onset of acute ischemia. Thus, ischemia implies tissue hypoxia due to insufficient arterial flow and the presence of anaerobic glycolysis as the dominant energy-generating pathway.

General Features of Ischemia A complex series of metabolic changes is set into motion in myocardium suddenly made ischemic by occlusion of a major branch of a coronary artery. Most of these reactions are related to two general effects of the reduced arterial flow. The first is a reduction in the supply of O2 (hypoxia) and substrate available to the tissue, and the second is the accumulation in the tissue of the products of ischemic metabolism. Some of the metabolic changes of ischemia occur as a direct result of


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hypoxia. Thus, hypoxia in vivo or high-flow anoxia in vitro (2, 3) leads to many of the same metabolic changes seen in ischemia. However, some of the metabolic effects of high-flow hypoxia or anoxia differ from those of ischemia because hypoxia or anoxia with continued perfusion permits washout of ischemic metabolites. Also, exogenous substrates may be pro vided to hypoxic tissue, depending on the experimental conditions. Both washout and substrate delivery are absent or are greatly diminished during ischemia. These differences have an important impact on the metabolic and functional consequences of ischemia. Ischemia may occur in all degrees of severity, from only slight reduction in flow to complete absence of perfusion (total ischemia), and the degree of ischemia may vary within the vascular territory of an occluded artery. The degree of ischemia varies because of the heterogeneity in local delivery of blood flow that can be provided through collateral interarterial con nections after sudden occlusion of a major coronary artery. There are differences among species in regard to the adequacy of such preformed collateral anastomoses. Moreover, within those species that charac teristically have preformed coIIaterals, the extent of development varies among individuals. For example, virtually no collateral anastomoses are present in humans without significant coronary disease nor in some exper imental animals such as pigs, rabbits, and rats (4). On the other hand, collaterals are developed to a variable extent in normal dogs and cats, and in humans with ischemic heart disease. When there is collateral flow, it is distributed in a gradient such that the inner layer receives the least and the sub epicardium the most flow (5, 6). There is a clear relationship between the severity of the ischemia and the response of the tissue to injury. In the experimental animal, cell death begins to appear about 20 minutes after the onset of severe low-flow ischemia (flow < 10% of control). Most severely ischemic myocytes have died by 60 minutes (6, 7). Because the subendocardial zone is usually the most severely ischemic zone and because this zone also has higher energy requirements, this zone dies first. Tissue subjected to moderate-flow ischemia (10-35% of control flow) survives longer but dies by three to six hours in experimental studies. Mildly ischemic tissue (flow> 35%) may ultimately survive. The minimal degree of reduction in arterial flow required to induce ischemia in vivo is not precisely known because partial reductions in coronary blood flow create a heterogeneous pattern of myocardial perfusion, which cannot be resolved with currently available techniques (8). Much of our knowledge of the response of the myocyte to ischemic injury has been obtained in studies oftissue known to be severely or totally ischemic (9). Such tissue is readily identifiable and has the advantage of


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being uniformly injured. It has been difficult to identify zones of uniformly moderate-flow ischemia for direct study. Direct studies of the metabolic changes of acute ischemia in humans have been limited mostly to analyses of the changes occurring in totally ischemic myocardium after cardioplegia (10). On the other hand, interesting data have emerged from indirect studies using a variety of approaches such as arteriovenous differences in metabolite levels during coronary sinus catheterization (11, 12), PET imaging (13), nuclear magnetic resonance imaging (14, 15), and the effects of ischemia induced at the time of angioplasty (16, 17). The observations in such studies generally reflect changes in an overall ischemic region that is heterogeneous in terms of degrees of ischemia. METABOLIC CHANGES IN LOW-FLOW OR TOTAL ISCHEMIA

With the onset of severe ischemia, the O 2 present as oxyhemoglobin in the capillaries and as oxymyoglobin of the myocytes and tissue is exhausted in about 8-lO seconds, at which time oxidative phosphorylation essentially ceases (18, 19). Simultaneously, the terminal electron transport system becomes reduced and the ratio of NADHz to NAD increases markedly (8). At about the same time, effective contractions cease in the involved myocytes, their membrane potential begins to decrease, and electrocar diographic changes appear (20, 2 1).

Anaerobic Glycolysis Within 15-20 seconds of the occlusion, anaerobic glycolysis supervenes as the only significant source of new high-energy phosphate (�P) (22, 23). The initial high rate of anaerobic glycolysis still is only about one fourth of the myocardial aerobic glycolytic rate. Moreover, within 60 seconds, the glycolytic rate slows markedly, primarily because glyceraldehyde phos phate dehydrogenase (GPD) is inhibited by the high sarcoplasmic NADH 2/NAD ratio (24, 25) and low pH (26). Glycolysis proceeds at this slower rate for about 40-60 minutes and then ceases (27). When there is little or no arterial flow to provide exogenous glucose, glycogen is the chief substrate for anaerobic glycolysis. Glycogen is broken down to glucose-I-phosphate (GIP) through the action of phosphorylase and, when converted to lactate, yields 3 pmol of ATP per pmol of GIP entering glycolysis. During the first few seconds of ischemia, phosphorylase is converted to its more active form, phosphorylase a, by phosphorylase kinase. This activation is achieved quickly (28) through a complex series of reactions (29) initiated by an unknown stimulus. Cyclic AMP generated



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secondarily to catecholamine release appears to be involved (23, 28), although anoxia alone also contributes (23). The lactate produced by anaerobic glycolysis accumulates in the severely ischemic tissue because it is not further metabolized in the absence of O 2 and because it diffuses very slowly to the systemic circulation (9). Three other glycolytic intermediates, alpha glycerol phosphate (ocGP), glucose6-phosphate (G6P), and glucose-I-phosphate (G I P), also accumulate. Each is the product of a reaction proximal to glyceraldehyde phosphate dehydrogenase, the chief site of glycolytic inhibition (23). Glucose also accumulates in the ischemic tissue from the usual concentration of 1.7 to 2.0 to as high as 14 Ilmoles/g dry weight after 40 minutes of low-flow ischemia (9). The reason for this glucose accumulation is unknown. It may arise from dephosphorylation of GIP or G6P via the action of a phosphatase. However, the activity of G6P phosphatase (the phosphatase for the glucose intermediate that accumulates to the greatest extent) is very low in myocardium (23). Glucose also could be formed by the breakdown

of glycolipids or glycoproteins. Regardless of its source, it is clear that it is a product of intracellular metabolism. The later cessation of glycolysis in ischemia is attributed to the very low sarcoplasmic ATP concentration that occurs by the same time (27). In the absence of ATP, glycolysis is inhibited at the level of fructose-6-phosphate (F6P) because ATP is required to phosphorylate F6P to fructose-l ,6diphosphate (Fl,6P) via phosphofructokinase. Exhaustion of the glycogen supply most likely is not the reason why glycolysis ceases, because much glycogen often remains in tissue in which glycolysis has ceased. In zones of moderate-flow or high-flow ischemia, the pattern of metab olism is different from that described for severely ischemic myocardium. First, oxygen provided by higher amounts of collateral flow supports some aerobic glycolysis. Much more energy is produced per unit of completely oxidized glucose than is produced by anaerobic glycolysis. In addition, less lactate, H+, and other glycolytic intermediates are produced and more of what is produced is flushed out of the tissue. Thus, inhibition of anaerobic glycolysis is delayed and is perhaps less marked. Secondly, collateral flow provides the ischemic myocytes with additional glucose and other substrates for energy metabolism. These substrates supplement the finite supply of endogenous glycogen.

Energy Metabolism in Ischemia Reserves of P are scant in myocardium (30). There is only enough CP and A TP to support three or four efficient contractions. Although the rapid cessation of contraction markedly reduces the demand of the tissue �


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for'" P, most of the '" P of CP is utilized within the first 8 to 10 seconds to rephosphorylate ADP via the action of creatine kinase (31). This reaction, plus the provision of new ATP by anaerobic glycolysis, maintains myocyte ATP concentration during the first few minutes. Nevertheless, after only 5, 15, and 40 minutes, respectively, of severe subendocardial ischemia in the open-chest anesthetized dog heart, myocardial A TP decreases to 67%, 35%, and less than lO% of control (1, 32, 33). Quali tatively similar but less rapid depletion of'" P occurs in the less severely ischemic midmyocardium (34). The critical role of anaerobic glycolysis in providing ATP in severe ischemia is dramatically illustrated in experiments in which anaerobic glycolysis is inhibited by iodoacetate (lAA) (35). IAA covalently binds to GPD and inhibits glycolysis at this step. As a consequence, when the IAA poisoned heart is made ischemic, no glycolytic ATP is formed. In less than five minutes, the reserve supplies of '" P are depleted totally and the heart undergoes contracture-rigor. Noninhibited ischemic tissue does not suffer complete depletion of ATP nor develop contracture-rigor until 60 to 90 minutes of ischemia have passed. Tissue ADP concentration rises because the utilization of ATP exceeds its rate of synthesis (1). The consequences of this situation are shown diagrammatically in Figure 1. The '" P of ADP is salvaged via the action of adenylate kinase, which converts two ADP molecules into an ATP (which in turn is reutilized) and AMP. The latter molecule is broken down by 5'-nucleotidase to adenosine (ADO) and inorganic phosphate (Pi). ADO is deaminated to inosine (lNO) via adenosine deaminase (36). The nucleosides INO and ADO, in contrast to nucleotides, both can diffuse from the myocyte to the extracellular space where INO is degraded to hypoxanthine (HX) and xanthine (X). Because of the depressed flow, all of these catabolites of the adenine nucleotide pool are retained within the ischemic tissue (36). The proportion of the various catabolites of the adenine nucleotide pool found in myocardium varies with the duration of ischemia (27). Initially, ADP and AMP are increased markedly (1). ADO is always in relatively low concentration, presumably because sufficient ADO deaminase is present to break most of it down to INO as soon as it is formed. On the other hand, INO accumulates. After 40 minutes of severe ischemia in the dog heart, AMP is the predominant nucleotide, INO is the dominant nucleoside, and HX is beginning to appear in significant quantities. After several hours of ischemia, HX becomes the principal catabolite, together with a small quantity of X. During the first 60 minutes of low-flow regional ischemia, the entire adenine nucleotide pool can be recovered in the tissue in the form of the metabolites discussed above (27). However, in zones of moderate- or

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ADENOSINE + Pi • ADENOSINE .... DEAMINASE INOSINE + NH3 • NUCLEOSIDE • PHOSPHORYLASE HYPOXANTHINE • XANTHINE .... OXIDASE XANTHINE Figure 1 The large arrows on this diagram show the route of destruction of the adenine nucleotide pool in the ischemic dog heart. The demand of the tissue for ATP exceeds the supply from anaerobic glycolysis and reserves of P such as creatine phosphate (CP). As a consequence, ADP is produced in excess and accumulates in the tissue. Its P is captured by the action of adenylate kinase (MK), which converts ADP into ATP and AMP. The ATP formed from ADP by MK is reutilized and the AMP is converted to adenosine by 5' nucleotidase (S'ND) with the release of inorganic phosphate (Pi). Some AMP also is generated by reactions such as adenyl cyclase or fatty acid CoA synthetase. Adenosine is deaminated via adenosine deaminase to inosine plus ammonia. Inosine is further degraded via nucleoside phosphorylase and xanthine oxidase. When ATP has fallen to 1-2% of control, more than 80% of the adenine nucleotide pool can be recovered as inosine and hypoxanthine. See text for a brief description of species differences in adenine nucleotide breakdown. Reproduced from Reference 33 with permission. �

-

high-flow ischemia, some of the metabolites are washed to the systemic circulation. The distribution of catabolites of the adenine nucleotides is a function of the enzyme and substrate distribution in the heart. There is significant variation in enzyme levels between newborn and adult hearts, as well as variation among species (37-39). For example, rat heart as well as skeletal muscle of several mammalian species, including humans, contains sig nificant quantities of AMP deaminase. This enzyme converts AMP to inosine monophosphate (IMP). In these models, ischemia results in accumulation of IMP in the tissue, accompanied by production of less adenosine. Another example is that human and rabbit heart contain little or no xanthine oxidase (40-43). Hence, very little xanthine and virtually

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no uric acid are formed during ischemia in these species. In contrast, rat myocardium contains significant quantities of this enzyme, which converts hypoxanthine to xanthine and xanthine to uric acid (44).


The Supply versus Demandfor Energy in Severe Ischemia Studies of total ischemia in canine left ventricle revealed that, once tissue oxygen is depleted and during the ensuing period of progressive depletion of �P, 80% of the �P released and utilized originates from anaerobic glycolysis vs about 20% contributed by reserve �P (27). Several phos phorylation reactions of amino acid or Krebs cycle substrates can generate "-' P in an anoxic environment, but only when there is a continuous supply of such substrates available, a situation not found in low-flow ischemia. Howevcr, substrate phosphorylation reactions may be a more important source of energy in areas of moderate or mild ischemia (45). Ischemic myocardium has a greater potential supply of �P than it actually releases (1). For example, canine left ventricle contains 1.7-2.7 pmoles/g dry weight of extracellular glucose and anywhere from 1 50 to 350 pmole of glucose equivalents stored as glycogen. Thus, in the extreme example, if all this glucose were converted by anaerobic glycolysis to lactate, the tissue would contain 704 pmoles of lactate and would have released 1054 pmoles of P (1.5 pmol P per ,umol lactate produced). Nevertheless, this would be enough energy to support active contractile function for only about four minutes. Moreover, as noted earlier, glycolysis always becomes inhibited well before all myocardial glycogen has been exhausted. The highest tissue lactate concentrations actually observed in severe ischemia in canine hearts are in the range of 280-325 ,umoles/g dry weight, which is equivalent to the release of roughly 420 to 488 pmoles of �P. The reactions that generate demand for �P in ischemia include the myosin ATPase of the myofibrils, Na/K ATPase, the Ca2+ ATPases of the sarcolemma and sarcoplasmic reticulum, adenyl cyclase, fatty acid CoA synthetase, and other ATPases of the myocytes (46). The initial loss of P undoubtedly involves chiefly the energy expenditure of contractile activity through the myosin ATPase. However, effective contractions soon cease. Continued electrical activation results in ineffective attempts of the myocytes to contract, and the resulting ion fluxes necessitate continued activity of Ca 2+ and Na/K ATPases. Other A TP-requiring enzymes may also bc active while sufficient ATP is present in ischemic tissue. In addition, Rouslin et al (47) showed and Jennings et al (48) confirmed that much of the ATP utilization during ischemia is due to the action of the mitochondrial ATPase. In fact, inhibition of this ATPase with oligomycin greatly slows ATP depletion in totally ischemic myocardium (47, 48). �

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The mitochondrial ATPase is the same macromolecular complex as ATP synthetase (FI-FO particle) of the electron transport system, which when a proton gradient and O2 are present releases ATP during the process of oxidative phosphorylation (49). When the proton gradient is lost, the synthetase works in the reverse direction and breaks down ATP to ADP and Pi. Interestingly, in large animal hearts such as those of the dog and human, a natural protein inhibitor of the ATPase is present in cardiac mitochondria and binds to the ATPase when the mitochondrial pH decreases following the onset of ischemia (50). The presence of this inhibi tor has the fortuitous effect of slowing the rate of ATP depletion (51). Rodent hearts (mouse and rat) contain much less natural inhibitor in their mitochondria, and myocardial ATP depletion is correspondingly much faster during total ischemia in these species than it is in dog or human (51). The metabolic rate of ischemic myocardium is reflected by both lactate accumulation (rate of ATP release, or supply) and declining tissue ATP

concentration (rate of '" P utilization, or demand). Changes in the metabolic rate lead to parallel changes in both the rate of lactate accumulation and the rate of tissue ATP depletion (and to an inverse relationship between tissue lactate concentration and ATP concentration). Both aspects of isch emic energy metabolism (production and utilization) can be accelerated, e.g. by pacing the ischemic heart (52), or slowed, e.g. by instituting hypo thermia (53). Under no conditions, however, is ATP utilization selectively slowed or production selectively accelerated so that energy supply could match the demand. Theoretically, if a reversible inhibitor of the mito chondrial ATPase could be developed, its use might reduce ATP utilization sufficiently to maintain sarcoplasmic ATP for a much longer period of ischemia.

The Osmolar Load Thc intracellular metabolism of the ischemic myocytes generates a net increase in a variety of catabolites, some of which (e.g. H+ or the NH3 formed by the activity of adenosine deaminase) may be directly toxic. In addition, continued catabolism markedly increases the net number of molecules, i.e. the osmotic load of the myocyte (1). For example, the breakdown of 13.6 mosmoles of CP per liter of cell water to creatine and phosphate doubles the mosmoles. In addition, the progressive catabolism of cach molecule of ATP results in several smaller molecules. Catabolism of glycogen, which is essentially osmotically inactive, generates 17 mos moles per liter of cell water of lactate after only 5 minutes and as much as 102 mosmoles after 40 minutes of ischemia (1, 54).


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The osmolar load has been measured directly in total ischemia (55). It rises from 324 to 423 mosmoles/kg wet tissue, a value quite consistent with that estimated by adding the major known changes in energy metabolites (54). This intracellular osmotic load could cause substantial cell swelling if sufficient extracellular water was available. Indeed, transmission electron microscopy reveals that cell swelling occurs during ischemia, and ion specific electrodes implanted in the extracellular fluid show an increase in the concentration of extracellular ions (56). Nevertheless, in severe ischemia, total tissue water remains constant because of the absence of collateral flow, and cellular swelling must be limited to relatively small shifts of extracellular water into the cell (57). As a consequence of the increase in the osmolar load, there is a volume mediated adjustment of intracellular ion concentration. K + is exported to the extracellular fluid (58). Extracellular K + in the center of a zone of regional ischemia in the pig heart, where it has been measured with ion specific electrodes (59-61), begins to rise after 15 seconds of ischemia and reaches 11.5 mmol/liter after 7 minutes. This increase in extracellular fluid K + concentration undoubtedly contributes to the early electrocardio graphic changes of acute ischemia and may be related to arrhythmogenesis (60, 62). Potassium loss is a common response to osmotic swelling of myocytes and other cells (63, 64).

Ion Gradients Ion transport by the sarcolemma and sarcoplasmic reticulum requires ATP. These reactions depend on ATPases, most of which enzymes have a very low Km. Thus, a deficiency of sarcoplasmic ATP is unlikely to bc the explanation for the myocyte edema that develops early in ischemia. The fact that intracellular Na activity decreases slightly during the initial 10-15 minutes of ischemia (61) indicates that the Na/K ATPase is active and functioning. On the other hand, when severe depletion of ATP has occurred after longer periods of oxygen deficiency, intracellular Na+ does increase sub stantially (65), despite the presence of an intact sarcolemma. This obser vation suggests that very low ATP concentration can depress Na/K ATPase activity. Changes in intracellular Ca 2+ are difficult to evaluate because there are so many different systems involved in maintaining the more than 1000fold gradient between the extracellular fluid and the sarcoplasm. Within the myocyte, the sarcoplasmic reticulum and the mitochondria can both sequester and release Ca2+ . At the sarcolemma, Ca2+ transport via the Ca2+ ATPase, as well as Ca2+ exchange with Na+ or H+, takes place (66, 67). (Extrusion of Ca2+ via exchange with Na+ is a passive reaction but



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depends indirectly on ATP to maintain low intracellular concentration of Na+.) Sarcoplasmic Ca2+ could increase if Ca 2+ stores in the sarcoplasmic reticulum or mitochondria are released. In addition, Neely & Grotyohann (68) observed that Na+ jH+ exchange in the acidotic myocyte increases intracellular Na+, which causes an exchange of intracellular Na+ for extracellular Ca 2+ and an increase in intracellular Ca 2 + . Eowever, as long as Na+ can be removed from the sarcoplasm, the Ca2+ gradient is maintained as well. The fact the Ca 2+ concentration in the sarcoplasma increases under these conditions is supported by experiments of Steen bergen et al (69) using NMR and an intracellular marker of Ca2+ con centration, fluoro BAPTA. Similar observations are reported by Marban's group (70). Interestingly, there is no rise in intracellular Ca2+ until late in the reversible phase of ischemia, at which time Ca 2+ reaches a con centration of at least 10 11M. This late increase in Ca 2+ is thus due both to the indirect effects of tissue acidosis and to the late decline in activity of the NajK ATPase, which occurs when the supply of sarcoplasmic ATP nears exhaustion. Much less intracellular Ca2+ loading is found late in ischemia if NajH exchange is blocked by amiloride, an observation sug gesting that much of the increase in intracellular Ca 2+ is due to exogenous rather than endogenous Ca2+ (71). In severe or total ischemia, there is a finite amount of Ca2+ available to enter the myocyte from the extracellular space. If all of the extracellular calcium was ionized and entered the cell, the sarcoplasmic Ca2+ theo retically could rise to 8 x 10-4 M or 800-900 11M (54). However, because of the abundance of inorganic phosphate in the intracellular H 0 to bind 2 the Ca 2+ , it seems likely that this theoretical value would never be reached. Under conditions where significant collateral flow is present, much more Ca2+ is available to enter the myocyte. The increasing intracellular Ca2+ concentration has important conse quences in that certain phospholipases are activated by as little as 10 11 M Ca 2+ and others b y 100 1 1 M Ca 2+ (72, 73). I t i s likely, although not proven, that products of these reactions, such as lysophospholipids, cause serious intracellular problems (74). In addition, the protease calpain is activated by as little as 10 11M Ca2+ (75). STRUCTURAL CHANGES IN SEVERE ISCHEMIA

Myocyte Changes The metabolic changes of ischemia are accompanied by very few changes in ultrastructure during the early phase of the injury (76). The myofibrils of the myocytes generally show I-bands. These appear because the myofibrils


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cannot adequately resist the stretch imposed by systolic ventricular pres sure. The presence of anaerobic glycolysis is reflected by glycogen depletion. Also, the chromatin of the nucleus is aggregated peripherally and some mitochondria become swollen. After 30--40 minutes of severe ischemia, the injury becomes biologically irreversible. This transition to irreversibility is associated with new ultra structural changes (76). These include diffuse mitochondrial swelling, the appearance of tiny amorphous densities in the matrix space of virtually all mitochondrial profiles, the virtual absence of glycogen, marked peripheral aggregation of nuclear chromatin, and the appearance of discontinuities in the plasmalemma of the sarcolemma. The sarcolemmal disruption is considered to be the lethal event (I, 9).

Microvascular Injury The capillary endothelium is damaged by ischemia, but remains struc turally intact for many more minutes than the myocytes (77). Even early in the irreversible phase of myocyte injury, the capillary endothelium exhibits abundant pinocytotic vesicles and is indistinguishable from that found in well-perfused myocardium. Moreover, capillaries in reversibly

injured tissue withstand reperfusion with arterial blood and reactive hyper emic flows up to 10 times control flow, without developing signs of damage such as interstitial hemorrhage or thrombosis. Overt microvascular dam age does not appear until myocyte irreversibility is well advanced, i.e. after 60 or more minutes of severe in vivo ischemia. Severe microvascular damage precludes any reflow. REVERSIBLE AND IRREVERSIBLE INJURY

Definitions The early phase of ischemic injury to myocytes is reversible in the sense that eliminating the cause of the injury by restoring arterial flow prevents the death of myocytes destined to die had the ischemia been prolonged (78). Scattered myocyte death is detectable in low-flow ischemia 20- 25 minutes after the onset; on the other hand, in zones of moderate-flow ischemia, myocytes may not die for several hours. Cell death is detected by the failure of successful arterial reperfusion to prevent the death of the damaged myocytes. Thus, this injury is irreversible (78, 79). The term irreversible is useful during that period when death is difficult to detect by objective morphologic criteria. Over the past few years, a popular but unproved postulate has been that some injured myocytes are still viable immediately before reperfusion, but are killed by some deleterious aspect of reperfusion. This hypothetical



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concept is termed lethal reperfusion injury. Proposed mechanisms for reperfusion injury include direct myocyte injury or indirect injury caused by persistent or recurrent areas of ischemia because of microvascular injury (1). Proposed pathogenetic mechanisms include physical obstruction of capillaries by leukocytes (80) and endothelial injury caused by free radical generation emanating from the ischemic-reperfused myocytes (81, 82) and/or accumulating leukocytes (83, 84). Direct injury of myocytes by internally generated free radicals has also been proposed (81). Also, markedly accentuated influx orCa 2+ via the various exchange mechanisms discussed earlier (71) or rapid cellular swelling because of the afore mentioned osmotic load (osmolar load) may directly injure myocytes. In any event, lethal reperfusion injury (if it occurs) would be very important because then drug therapy might conceivably prevent or ameliorate it. Such therapy could be employed as an adjunct to reperfusion therapy.

Pathogenesis of the Irreversihle Injury The cause of the transition to irreversibility, though investigated exten sively, has not been established with certainty (9). No sudden metabolic change occurs to indicate that a myocyte is dead and cannot be salvaged by reperfusion. However, tissue early in the phase of irreversibility does have several characteristics, including (a) ATP < 10% of control; (b) high concentrations of H+, AMP, INO, and HX; (c) cessation of anaerobic glycolysis; (d) high lactate, aGP, G, and G6P and low glycogen; (e) mito chondrial swelling with amorphous matrix densities; and (f) a focally disrupted sarcolemma. All of the metabolic changes listed develop gradu ally during the reversible phase of injury but become marked in the irre versible phase. Howevcr, the structural changes in the mitochondria and sarcolemma are seen only during the phase of irreversible injury. The available evidence indicates that the final event from which the myocyte cannot recover is focal disruption of the sarcolemma (1, 9, 75, 85-89). The breaks in the plasmalemma allow critical intracellular enzymes and cofactors to leak from the myocyte into the extracellular fluid and allow intracellular and extracellular ions to come into equilibrium in the sarcoplasmic space. The cause of sarcolemmal disruption appears to be disaggregation or perhaps proteolytic digestion of the cytoskeletal attach ment complexes that hold the sarcolemma to the Z-bands of the myofibrils (9). This permits the formation of subsarcolemmal blebs of edema fluid over which the plasma membrane breaks. The weakening of the cyto skeletal attachment complex is associated with the disappearance ofvincu lin, one of the chief protein constituents of this complex (85) as well as other cytoskeletal proteins (87). However, injury of the sarcolemma and its cytoskeletal framework does not become obvious unless the membrane


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is mechanically stressed. During ischemia, one cause of mechanical stress is cell swelling, secondary to the osmotic load (85, 88, 89). It is interesting in this regard that the-plasma membrane of control myocytes can tolerate very marked cell swelling without developing bleb formation and mem brane disruption. Thus, the disruption of the sarcolemma is a two-stage process; the cytoskeletal and perhaps sarcolemmal changes must occur and a force such as cell swelling (1, 85, 88, 89) or contraction (90) is required to disrupt the membrane. Potential causes of membrane disruption, in addition to disaggregation or proteolytic digestion of the cytoskeletal attachment complexes, include (a) injury of the sarcolemma mediated by oxygen-derived free radicals (81, 91); (b) activation of phospholipases (92-94); and (c) detergent effects of metabolites such as acyl carnitine and CoA, which accumulate in the myocyte (95). While ischemia is present, O2 derived free radical attack seems unlikely because the ambient O2 is very low and the mitochondria contain abundant quantities of reduced cytochrome oxidase to bind any available ° 2- Endogenous phospholipases are present in the myocyte and Ca 2+ ion levels eventually rise to levels adequate to activate them (73) (see the section on ion gradients). Moreover, in vitr:o studies, in which phospholipids were labeled with radioactive arachidonic acid, showed that small quantities of this labeled fatty acid are released during ischemia (92). However, direct studies of phospholipid breakdown in ischemia (93, 94) showed little evidence of phospholipase activity until well after the mem brane was disrupted. Also, detergent effects seem unlikely because acyl CoA and acyl carnitine do not accumulate to the extent required in the absence of a continuous source of substrate (acyl groups). Thus, the two stage hypothesis is the most attractive at present. The role of ATP and �P depletion in the genesis of irreversibility remains an enigma. ATP is invariably very low in irreversibly injured myocytes. However, a causal relationship can only be established by ident ifying the reaction or reactions requiring ATP, the absence of which causes the sarcolemma or its supports to weaken (9, 36).

EFFECT OF REPERFUSION OF ISCHEMIC MYOCARDIUM

Reversible Injury Successful reperfusion of reversibly injured myocytes is associated with partial or complete restoration to the control state of many of the metabolic changes present in the ischemic myocytes (96, 97). The glycolytic inter mediates are washed out to the systemic circulation or are metabolized to



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CO and H20. Restoration of the adenylate charge and phosphorylation 2 potential, loss of mitochondrial swelling, resynthesis of about 6--8 Ilmoles of ATP/g dry weight from the ADP and AMP that accumulated in the tissue, and resumption of contraction all establish that oxidative phos phorylation has been restored (98). In addition, NMR studies using 31p spectra confirmed the results of direct analysis of metabolites in zones of low-flow ischemia (15, 99). However, the NMR results are diluted by the presence of less severely injured tissue in the mid- and subepicardial myocardium. After only 10--15 minutes of ischemia, the adenine nucleotide pool may not be fully repIeted for four days or more because the de novo synthesis of adenine nUcleotides is slow in myocardium (100-102). Edema persists in the myocytes for one or more hours and is associated with a net increase in the dry weight K + content of the tissue (98). Reversibly injured reperfused myocardium exhibits a prolonged con tractile dysfunction that has been termed myocardial stunning (103-106). Contractile function may require several days of reperfusion before it fully recovers. The cause of stunning has not been established. However, the effect can be reduced in severity by administration of free radical scav engers immediately before reperfusion (107, 108). Myocardium that has been subjected to one or several brief episodes (5, 10, or 15 minutes) of ischemia will tolerate a subsequent, more prolonged episode of ischemia much better than control myocardium. Thus, the amount of necrosis observed after 40 minutes of ischemia followed by reperfusion is much less when this ischemic insult has been preceded by one or more brief episodes of ischemia. This phenomenon is called ischemic preconditioning (109, 110). The mechanism of the preconditioning effect is unknown. However, it is clear that the demand for P is reduced in preconditioned ischemic tissue (110). Thus, ATP content of preconditioned myocardium is not depleted as quickly during the later episode of sustained ischemia compared with an equivalent ischemic episode in "virgin" myo cardium. The reduced metabolic demand of preconditioned ischemic myocardium may occur in part because the tissue also is stunned. However, pre conditioning begins to disappear by two hours of reperfusion after the initial brief ischemic episodes, whereas detectable recovery of contractile function has not begun in the same time period. Thus, while stunning may prove to be necessary for preconditioning, stunning alone is insufficient to explain the protective effect of preconditioning (96). �

Irreversible Injury Successful reperfusion of myocytes early in the phase of irreversible injury is associated with the sudden appearance of contraction-band necrosis


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(111) with its characteristic massive cell swelling, accumulation of Ca 2+ in the form of hydroxyapatite in the mitochondria, and massive contraction of the myofibrils (which aggregate into large contraction bands) (7). The contraction bands further disrupt the sarcolemma. We postulate that the entry of Ca 2+, from the blood reperfusing the tissue (112) through sar colemmal defects that are sufficiently large to be detected by electron microscopy, accentuates the Ca2+ overload as a result of Na-Ca and Na H exchange and increases the sarcoplasmic Ca 2+ to the point that massive contraction of the myofibrils occurs. At high concentrations of Ca 2+ , reoxygenated cardiac mitochondria actively accumulate some of the excess Ca2+ (7, 54). There is incontrovertible evidence that reactive oxygen metabolites induding superoxide and hydroxyl free radicals are formed in reperfused tissue (113-118). However, there is no agreement with respect to whether free radicals cause additional myocyte death or whether scavengers protect injured myocytes from such consequences (119, 120). More work is required to establish the role of 0 -derived free radicals in this form of 2 tissue injury.

The No-Reflow Phenomenon Reperfusion often is not totally successful after prolonged episodes of ischemia. The unperfused areas are termed areas of no reflow (77). In myocardium, no reflow is due to a combination of capillary damage (see the section on microvascular injury), the compression of capillaries by contracture-rigor occurring in the irreversibly injured myocytes (121, 122), and myocyte swelling (77). Hemorrhage is common, either adjacent to or sometimes within these areas. However, areas of no reflow are generally found in the center of zones of low-flow ischemia after episodes of injury exceeding the minimum time required to produce irreversible mycoyte injury (7). Thus, there is no evidence that the no-reflow effect causes additional myocyte injury or that prevention of the severe microvascular damage would salvage any additional myocytes.

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ment of cell injury in sustained isch emia. Circulation 82(Suppl. II): 2-12 10. Braimbridge, M. V., Chayen, J., Biten sky, L., Hearse, D. J., Jynge, P., Can kovic-Darracot, S. 1977. Cold car dioplegia or continuous coronary perfusion? Report on preliminary clini cal experience as assessed cyto chemically. 1. Thorac. Card. Surg. 74:

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Comparison of higher energy phos phate production, utilization and deple tion and of adenine nucleotide cata bolism in total ischemia in vitro vs. severe ischemia in vivo. Cire. Res. 49: 8 92-900 28. Wollenberger, A., Krause, E. 1 968. Metabolic control characteristics of the acutely ischemic myocardium. Am. J. Cardiol. 22: 349-59 29. Lamer, J. 1976. Mechanisms of regu lation of glycogen synthesis and break down. Cire. Res. 38(Suppl. I): 2-7 30. Gordon, E. E., Morgan, H . E. 1 986. Principles of metabolic regulation. In The Heart and Cardiovascular System: Scientific Foundations, ed. H. A. Foz

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T., Altschuld, R. A., Trewyn, R. W., Ansel, A. L., Lampka, K . , Brierly, G. P. 1984. Adenine nucleotide metabolism and com partmentization in isolated rat heart cells. Cire. Res. 54: 536-46 Swain, J. L., Holmes, E. W. 1 986. Nucleotide metabolism in cardiac mus cle. See Ref. 30, pp. 9 1 1-29 Downey, J. M . , Hearse, D. J., Yellon, D. M. 1 988. The role of xanthine oxidase during myocardial ischemia in several species including man. Mole cular and clinical events during post ischaemic reperfusion. J. Mol. Cel/. Cardiol. 2 1 0(Suppl. II): 55--63 Eddy, L. J., Stewart, J. R., Jones, H . P., Engerson, T. D., McCord, J. M., Downey, J. M. 1 987. Free radical-pro ducing enzyme, xanthine oxidase, is undetectable in human hearts. Am. J. Physiol. 253: H709-1 1 Grum, C. M., Ragsdale, R. A., Ketai, L. R., Shlafer, M. 1986. Absence of xanthine oxidase or xanthine dehydro genase in the rabbit myocardium. Bio

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injury: Pharmacologic implications. Circulation 74: 1-5 82. Ceeoni, C., Curello, S., Cargnoni, A.,

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in vitro. Effects of osmotic cell swelling on plasma membrane integrity. Circ. Res. 57: 864-75 86. Jennings, R. B., Reimer, K. A., Kinney, R. B., Steenbergen, C, Sharov, V. G., et al. 1987. Sarcolemmal damage in ischemia. In Myocardial Metabolism, ed. V. N. Smirnov, A. M. Katz , pp. 1 20-54. Zurich: Harwood 87. Ganote, C. E., VanderHeide, R. S. 1987. Cytoskeletal lesions in anoxic myocardial injury. A conventional and high-voltage electron-microscopic and immunofluorescence study. Am. J. Pathol. 1 29: 327-44 88. VanderHeide, R. S., Ganote, C. E. 1987. Increased myocyte fragility fol lowing anoxic injury. 1. Mol. Cell. Car dinl. 19: 1 085- 1 1 03 89. Ganote, C. E., VanderHeide, R. S. 1988. Irreversible injury of isolated adult rat myocytes. Osmotic fragility during metabolic inhibition. Am. J. . Pathol. 1 32: 2 1 2-22 90. Ganote, C. E., Lui, S. Y., Safavi, S., Kaltenbach, J. P. 198 1 . Anoxia, calcium, and contracture as mediators of myocardial enzyme release. J. Mol. Cell. Cardiol. 1 3 : 93-106 9 1 . Freeman, B. A., Crapo, J. D. 1982. Biology of disease. Free radicals and tissue injury. Lab. Invest. 47: 4 1 2-26 92. Chien, K. R . , Han, A., Buja, M . L., Willerson, J. To 1983. Accumulation of unesterified arachidonic acid in isch emic canine myocardium. Relationship to a phosphatidyicholine deacylation reacylation cycle and the depletion of membrane phospholipids. Cire. Res. 54: 3 1 3-22 93. Shaikh, N. A., Downar, E. 1 98 1 . Time course of changes in porcine myo cardial phospholipid levels during ischemia. A reassessment of the Iyso lipid hypothesis. Cire. Res. 49: 3 1 6-25 94. Steenbergen, C , Jennings, R. B. 1984. Relationship between Iysophospholipid accumulation and plasma membrane injury during total in vitro ischemia in dog heart. J. Mol. Cell. Cardiol. 16: 605-21 95. Neely, J. T., Garber, D., McDouugh, K., Idell-Wenger, J. 1979. Relationship between ventricular function and inter mediates of fatty acid metabolism dur ing myocardial ischemia. Effects of car nitine. In Ischemic Myocardium and Antianginal Drugs, ed. M. M. Winbury, Y. Abiko, pp. 225-34. New York: Raven 96. Jennings, R. B., Murry, c., Reimer, K. A. 1990. Myocardial effects of brief per-


iods of ischemia followed by reper fusion. Adv. Cardia!. 37: 7-3 1 97. Ellis, S. G., Henschke, C. I., Sandor, T., Wynne, J., Braunwald, E., Kloner, R. A. 1 983. Time course of functional and biochemical recovery of myo cardium salvaged by reperfusion. J. Am. Call. Cardio/. 1 : 1 047-55 98. Jennings, R. B., Schaper, J., Hill, M. L., Steenbergen, c., Reimer, K. A. 1 985. Effect of reperfusion late in the phase of reversible ischemic injury. Changes in cell volume, electrolytes, metab olites, and ultrastructure. Circ. Res. 56: 262-78


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Murry, C. E., Richard, V. J., Reimer, K. A., Jennings, R. B. 1 990. Ischemic preconditioning slows energy metab olism and delays ultrastructural dam age during sustained ischemia. Cire. Res. 66: 9 1 3-3 1 1 1 1 . Ganote, C. E. 1983. Cont raction band necrosis and irreversible myocardial injury. J. Mol. Cell. Cardial. 1 5: 67-73 1 1 2 . Shen, A. c., Jennings, R. B. 1 972. Kin etics of calcium accumulation in acute myocardial ischemic injury. Am. J.

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