Myocardial ischemia and infarction: Anatomic and biochemical substrates for ischemic cell death and ventricular arrhythmias

Myocardial Ischemia and Infarction: Anatomic a n d Biochemical Substrates for Ischemic Cell Death a n d Ventricular Arrhythmias KEITHA. REIMER,MD, PHD,AND RAYMOND E. IDEKER,MD, PHD Myocardial ischemia is a widespread cause of morbidity and the number one cause of death in the economically developed countries of the world. In the United States, approximately 25 per cent of all deaths are attributed to " h e a r t attacks"; of these deaths, about one half occur suddenly outside the hospital setting. Although there are many possible causes of sudden unexpected death, the majority undoubtedly are cardiogenic, owing to ventricular arrhythmias that occur either in the setting of transient myocardial ischemia or in some phase of an evolving myocardial infarct. Following hospitalization for an acute myocardial infarct, the majority of deaths have anatomically definable explanations (i.e., loss of sufficient myocardium to cause cardiac pump failure or specific anatomic complications of infarction such as cardiac rupture). In addition, despite the development of many potent antiarrhythmic drugs, some patients still die of ventricular fibrillation during the acute phase o f myocardial infarction; others, even after the infarct has undergone replacement by scarring, stiffer f r o m r e c u r r e n t chronic v e n t r i c u l a r tachycardia that can lead to hemodynamic collapse and/or ventricular fibrillation. The reduction of overall cardiac pump function in myocardial infarction may have several contributory causes; the most important cause surely is the loss of contractile mass. It has been shown, for example, that among patients with fatal myocardial infarcts, those who had died o f cardiogenic shock usually had lost at least 30 per cent and often 40 per cent or more of their left ventricular mass) Some of the gross anatomic determinants of myocardial infarct size are known, as are many subcellular biochemical and structural consequences of acute ischemic injury. However, the subcellular conditions dictating that an ischemic myocyte has become "irreversibly injured" and thus destined for necrosis have not been defined completely. T h e electrophysiologic explanations for most ventricular arrhythmias are reentrant conduction, enhanced automaticity o f an injured focus, or triggered activity of an injured focus. 2 However, the underlying anatomic and/or biochemical explanations Received from the Departmentof Patholog)',Duke University Medical Center, Durham. Supported in part by NIH grants HL 27416, 28138, 17670, and 28429. Address correspondence and reprint requests to Dr. Reimer: Department of Pathology, Duke University Medical Center, Durham, NC 27710. 0046-8177/87 $0.00 + .25 462

for these mechanisms of acute or chronic ventricular arrhythmias have not been completely elucidated. In general terms, the pathogenesis of ventricular arrhythmias in the acute phase of infarction may include: 1) altered properties of the sarcolemma or altered trans-sarcolemmal ionic gradients in individual myocytes; and 2) tile three-dimensional arrangement of myocytes with such altered properties that might permit reentrant conduction. In the case of chronic ventricular tachycardia, the explanation may lie either in the.persistence of altered membrane properties in surwwng myocytes within an infarcted region, or in the anatomic interrelationship of viable myocytes within a region o f scar, which could permit reentrant conduction. If the pathogeneses of ischemic cell death and of the ventricular arrhythmias associated with ischemia or reperfusion are to be learned, it is necessary to understand the general biology of myocardial infarction with respect to both the three-dimensional evolution of myocyte injury and the morphologic or biochemical changes in individual myocytes during the acute phase of myocardial ischemic injury. In the following sections, we provide an overview o f both aspects of myocardial ischemic injury. Much of what we infer to be true of human myocardial infarcts has been studied only directly in experimental animal models; our review therefore relies heavily on such experimental results. THREE-DIMENSIONAL ANALYSIS OF ISCHEMIA AND INFARCTION

Causes of Acute Myocardial Infarction Although patients who develop myocardial infarction often have diffuse coronary atherosclerosis, with narrowing o f more than one coronary artery, most infarcts involve m y o c a r d i u m supplied' by a single coronary artery. Moreover, ahhough dynamic (transient) causes of myocardial ischemia such as coronary spasm 3 or platelet aggregation 4 have been observed to cause myocardial infarction, it is now generally believed, on tile basis of coronary angiographic studies in patients with very acute myocardial infarcts, that most infarctions (80 to 90 per cent) are caused by coronary artery thrombi. 5 The lower incidence of coronary thrombi in many older autopsy studies 6 probably is erroneous, perhaps because of spontaneous thrombolysis between the onset of infarction and death of the patient, and/or the failure of pathologists in cases of sudden cardiac death to

MYOCARDIALISCHEMIAAND INFARCTION[Reimer & Ideke0

distinguish deaths associated with acute myocardial infarction from deaths resulting from primary ventricular arrhythmias. T h e most common reason that a thrombus suddenly develops at a particular site in a coronary artery, which may have been narrowed by atherosclerotic plaque for years, is that the plaque has ruptured. This exposes arterial blood to the underlying collagen and acellular debris contained within the lipid pool of tile plaque. Several studies have shown that when coronary thrombi are serially sectioned, most are found to be associated with sites of plaque rupture. 7-9 Additional contributory factors to coronary thrombosis may include stasis owing to coronary spasm and/or platelet aggregation. 4 Infarction Versus The "Area at Risk"

Occlusion of a major coronary artery results in ischemia throughout the anatonfic region supplied by that artery. In some animal species such as pigs or baboons, and in patients without pre-existing coronary a t h e r o s c l e r o t i c disease (in w h o m occlusion might be caused by coronary embolism), there may be virtually no collateral a n a s t o m o s e s b e t w e e n branches of the different coronary arteries. In such c i r c u m s t a n c e s , ischemia is severe, if not total, throughout the occluded vascular bed. In contrast, in some species such as the dog, and in human patients with long-standing c o r o n a r y atherosclerosis, pref o r m e d collateral a n a s t o m o s e s do exist b e t w e e n !arger branches o f the major coronary arteries either m the interventricular septum or on the epicardial surface of the free wall of the ventricles. 1~ The classical concept of myocardial ischemia was analogous to a target, with a bull's-eye center of severe ischemia, s u r r o u n d e d by three-dimensional b o r d e r zones o f progressively less severe ischemia with a gradual transition to nonischemic myocardium. Experimental and human autopsy studies have not supported this concept. It is true that when collateral blood flow is available to an ischemic vascular bed, it is distributed unevenly throughout the affected myocardium; collateral flow is preferentially shunted to the subepicardial zone so that a transmural gradient exists with ischemia most severe in the subendocardial zone. 11 This collateral flow gradient is caused by a gradient in intramyocardial pressure that impedes capillary flow when the limits o f metabolic vasoregulation are exceeded. However, despite whether or not surface collateral connections are present, the small penetrating arterial branches that course from epicardium toward the endocardium are essentially end arteries, without functionally important microvascular anastomoses between adjacent vascular beds. 12 Thus, there is a sharp interface, without a broad transitional border zone between capillaries that are unperfllsed 9versus those that are perfused via adjacent, unoccluded arteries. Transmural differences in metabolic activity, ind e p e n d e n t of coronary collateral blood flow, also 463

exist, so that for a given reduction in collateral blood flow, high-energy phosphates are d e p l e t e d m o r e quickly in the subendocardial zone./3 As a consequence o f either the transmural gradient o f collateral blood flow or o f metabolic activity, or both, irreversible injury o f ischemic myocytes occurs first in the subendocardial zone and progressively involves more o f the midepicardial and subepicardial zones (fig. 1). 14 This transmural wavefront of cell death begins in the subendocardial region between 15 and 30 minutes after the onset o f ischemia; the final transmural extent o f an infarct is established by three to six hours o f complete coronary occlusion in experimental studies using anesthetized dogs. However, in this experimetal model, ll as well as in h u m a n patients, ~4 infarcts seldom involve the entire vascular area at risk. A variable proportion of the subepicardial zone often survives in proportion to the amount o f collateral blood flow available. Border Zones and Interfaces

In either experimental or human myocardial infarcts, the b o u n d a r y between infarct and spared subepicardial myocytes often is highly irregular, with peninsulas o f surviving myocytes surrounding penetrating arteries, interspersed with peninsulas of infarct projecting toward t h e epicardium (fig. 2A). In addition, although no broad border zones of viable myocytes survive on either the endocardial or lateral borders o f an infarct, a thin rim of myocytes within the ischemic area at risk may survive just beneath the endocardium and at the lateral edges of the infarct (fig. 2A). 11.14A5 Most likely, this result occurs because o f diffusion o f sufficient oxygen and substrate from the ventricular cavity and the nonischemic microvasculature at the ischemic/nonischemic interface. T h e lateral boundaries between ischemic and nonischemic vascular regions can be irregular at the microscopic level. In the days and weeks that follow the acute onset of myocardial infarction, necrotic myocytes and capillaries attract an inflammatory response composed predominantly of neutrophils during tile first three to four days, followed by increasing numbers of macrophages. In larger infarcts, in which the microvasculature as well as myocytes have undergone necrosis, repair must occur from the periphery toward tile center and is achieved by tile ingrowth of granulation tissue with progressive conversion to a dense fibrous scar. T h e general features and time course of such infarct repair are well known. 16 It is also known that myocardial scars are smaller than the original mass of necrotic muscle that they replace, 17 contributing to myocardial wall thinning during infarct repair. However, the factors influencing the intensity o f the inflammatory response and the quantitative relationship o f scar size and geometry to the initial size and geometry of the infarct have not been evaluated thoroughly. The occlusion of a major coronary artery in dogs stimulates the development and/or growth of func-

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( Vi a ble ) FIGURE t. Progression of cell death versus time after circumflex coronary occlusion in dogs. Necrosis occurs first in the subendocardial myocardium. With longer occlusions, a wavefront of cell death moves from the subendocardial zone across the wall to involve progressively more of the transmural thickness of the ischemic zone. In contrast, the lateral margins in the subendocardial region of the infarct are established as early as 40 minutes after occlusion and are sharply defined by the anatomic boundaries of the ischemic bed. AP, anterior papillary muscle; PP, posterior papillary muscle. [Reproduced with permission from Reimer KA, Jennings RB: Lab Invest 40:633, 1979.)

tionally important collateral anastomoses between branches of adjacent coronary arteries. Substantial increases in collateral blood flow have been observed within four days after an acute coronary occlusion in dogs; ]~ it seems likely that collateral growth is similarly rapid in humans. Thus, perfusion of surviving myocytes in the subepicardial zone of the area at risk probably improves rapidly; on the other hand, the degree and rapidity of recovery of contractile function of surviving myocytes are concerns that have yet to be completely resolved. It is generally recognized that loss of a large mass of myocardium is followed by compensatory hypertrophy of remaining viable myocytes.~8 In the thin rim o f myocardium that survives just beneath the endocardial surface of an infarct region, and in a thin rim at the lateral interfaces between ischemic and nonischemic regions, arterial blood

flow probably is not restored easily; myocytes in these locations may show persistent subcellular changes thought to indicate a chronic ischemic state. One such change is the deposition of large quantities of neutral fat (fig. 2B)19; lipid droplets can be detected histologically using special stains for fat such as oil red O. Increased numbers of lipid droplets also can be seen by electron microscopy. Accumulation of lipid d e p e n d s on uptake o f exogenous free fatty acids and is most apparent in a thin rim o f viable myocytes at the lateral interface between necrotic and nonischemic myocytes) 9 This is evidence of limited mitochondrial oxidation of fatty acids, coupled with abundant availability of glycerol phosphate from the glycolytic pathway; the latter molecule is the backbone on which fatty acids are placed to form triglycerides. Thus, lipid accumulation is indirect evidence for relative impairment of oxidative metabolism and

FIGURE 2. A, Myocardial scar representing a remote myocardial infarct in humans. The section encompasses the full transmural thickness of the left ventricle from endocardium [top] to epicardium [bottom). Although the scar has replaced much of the myocardium in this section, the fibrotic scar tissue is interspersed with residual viable myocardium especially in the subepioardial zone and around larger blood vessels (BV). Also, there is a subendocordial rim eight to 10 myocyles thick [between arrows], which survived the earlier ischemic event. Myocytes in the subendocardium, subepicardium, and around penetrating arteries commonly survive in the region of a myocardial infarct. [Masson's connective-tissue stain, x t4.) [Reproduced with permission from Bolick DR et al: Circulation 74:1266, 1986.) B, Chronic sublethal injury of myocytes [lower left) immediately adjacent to necrotic myocytes [upper right) is evident by the presence of many small, darkly stained sarcoplasmic lipid droplets. This illustration is from the edge of a two-day-old myocardial infarct induced by coronary occlusion in a dog. The arrows indicate the interface between necrotic and viable myocytes. (oil red 0 stain, x 400.) [Reproduced with permission from Herfklns RJ et al: Radiology 157[P):147,1985 [abstract].) C, Typical features of moderately severe "myocytolysis" involving the subendocardial spared zone [between arrows) overlying an organizing myocardial infarct [MI). This illustration is from an autopsied human heart. The subendooardial myocytes, although apparently viable by virtue of retained nuclei, have undergone degeneration, with vacuolated sarcoplasm, apparently owing to loss of much of the myofibrillar apparatus. (Masson's connective-tissue stain. • 130.] [Reproduced with permission from Bolick DR et al: Circulation 74:1266, 1986.] D, Typical histologic features of contraction-band necrosis. A few viable myocytes are present in the lower-left corner for comparison. In contrast to coagulation necrosis, which may not become detectable for several hours after the onset of myocardial infarction, contraction-band necrosis develops almost immediately on reperfuston of irreversibly injured myocytes. This illustration is from a patient who died two hours after coronary bypass surgery. [Heidenhain's variant of Mallory's connective-tissue stain, x 350.] [Reproduced with permission from Reimer KA, Jennings RB: In Fozzard et al [eds]: The Heart and Cardiovascular System. Raven Press,1986.]

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suggests that the oxygen supply is somehow limited. Whether electrical properties of a cardiac myocyte are affected by increased intracellular lipid deposits or by the altered metabolic pathways of which such deposits are symptomatic is unknown. Another type of morphologic change that often c a n be seen in the subendocardial spared zone even months or years after an acute infarction is myocytolysis (fig. 2C). This term refers to d e g e n e r a t e d myocytes in which part or all of the sarcoplasmic contactile machinery has been lost, leaving a clear, vacuolated sarcoplasm. Whether such myocytes persist in such a degenerate state indefinitely, eventually undergo necrosis, or finally recover is uncertain. T h e electrical properties o f such cells also are tmknown. Whether the presence o f large numbers of myocytolyic cells in the subendocardial spared zone is a predisposing factor in the genesis of chronic postinfarction ventricular arrhythmias is an unresolved issue that merits further study. ACUTE CELLULAR CONSEQUENCES OF MYOCARDIAL ISCHEMIA

T h e early metabolic consequences of ischemia have been of considerable interest because one or more individual or interrelated metabolic pathways must cause 1) contractile dysfunction; 2) altered electrical properties that form the basis of cardiac arrhythmias; and/or 3) irreversible cellular injury event u a t i n g in m y o c a r d i a l necrosis. T h e m e t a b o l i c changes that develop in severe or total ischemia have been more readily characterized than the effects of moderate or mild degrees of ischemia, because it is easy to produce tissue with uniformly severe or total ischemia and quite difficuh to produce uniformly mild ischemia. This article emphasizes the changes observed in severe ischemia. Compared with severe ischemia, soine of the metabolic consequences o f mild ischemia may develop more slowly but be qualitatively similar; other important metabolic consequences of ischemia may be qualitatively different in zones of severe versus mild ischemia. Energy Metabolism Within a few seconds after occlusion of a major coronary artery, tissue oxygen content decreases and mitochondrial oxidative metabolism becomes inhibited. Flux through the respiratory chain slows, and as the net level of reduced nicotinamide adenine dinucleotide (NADH) increases, Krebs citric acid cycle and fatty acid oxidation also become inhibited. Concomitantly with the inhibition of oxidative reactions, anaerobic glycolysis becomes accelerated; in severe myocardial ischemia, the sarcoplasmic glycogen is the major substrate for this pathway. Thus, the early metabolic consequences o f ischemia 2~ include declining reserves of glycogen and progressive accumulation of lactate, the end product of anaerobic glycolysis. 466

By comparison with oxidative metabolism, anaerobic glycolysis is an inefficient source o f high-energy phosphate; 38 molecules of adenosine triphosphate (ATP) can be produced by the complete oxidation of glucose, whereas only three can be produced from conversion o f one glucosyl unit of glycogen to lactate. Even maximally stimulated rates o f anaerobic glycolysis can produce no more than about 7 per cent o f the h i g h - e n e r g y p h o s p h a t e needs o f n o r m a l working myocardium. Moreover, the rate of anaerobic glycolysis, initially accelerated by tissue hypoxia, soon slows markedly in severely ischemic myocytes because of end-product inhibition of glyceraldehyde3-phosphate dehydrogenase by NADH, hydrogen ion, and lactate. 22 Even though contractile function, the major energy-dependent process of working myocardium, is suppressed quickly during ischemia, the basal rate of A T P use continues to outstrip the capacity of myocardium to produce ATP. Thus, a major hallmark of ischemia is the progressive depletion of high-energy phosphate reserves. Creatine phosphate is depleted most quickly and is nearly gone within 1 to 3 minutes o f severe ischemia. Approximately 50 per cent of myocardial A T P stores are degraded within the first 10 minutes of severe ischemia, and the remainder is degraded more slowly, ls,23 In addition to the degradation of ATP to adenosine diphosphate (ADP), there is progressive loss of the adenine nucleotide pool because the ADP is converted to adenosine monophosphate (AMP) by the adenylate kinase reaction. AMP is progressively degraded to adenosine, inosine, hypoxanthine, and xanthine. T h e s e catabolites accumulate in severely ischemic tissue but are gradually lost if some collateral flow is available and are quickly washed away if the tissue is reperfused. ~4 Accumulation of Metabolic End Products Ahhough many of the deleterious effects of ischemia may be caused by the conseqnences of oxygen deprivation (hypoxia) per se, other consequences of ischemic injury may relate to the deleterious effects of catabolite accumulation. As already noted, lactate and the purine nucleosides and bases rapidly acc u m u l a t e in severely ischemic tissue. 24 Acidosis increases as a c o n s e q u e n c e o f several catabolic pathways, including anaerobic glycolysis, lip01ysis, and ATP hydrolysis. 2:)" Ammonia accumulates as a result of deamination of adenosine, amino acids, and so forth, and inorganic phosphate accumulates as highenergy phosphates are degraded. Acidosis may contribute to the inhibition of contractile functions, a potential advantage in delaying high-energy phosphate depletion. However, as noted earlier, acidosis also inhibits a variety of metabolic pathways including anaerobic glycolysis2~ and has been shown to have deleterious uhrastructural consequences including the aggregation of nuclear chromatin and the formation o f mitochondrial amorphous matrix densities. 27 Lactate accumulation also

MYOCARDIALISCHEMIAAND INFARCTION[Reimer & Ideker)

has been associated with tfltrastructural and functional evidence o f cellular injury2S; whether the observed deleterious effects are because of lactate per se or the accompanying intracellular acidosis is difficult to differentiate. Even in the absence of toxicity of individual catabolites, the combined effect of the aforementioned catabolic pathways is a substantial intracellular osmotic load that may contribute to cell swelling. Cell swelling has been proposed as a cause of the rupture of the sarcolemma, which occurs in irreversible ischemic damage after the sarcolemma or its cytoskeletal supports already have been weakened by other consequences o f ischemia. 29 Lipid and Protein Catabolism

Myocardial lipolysis is increased in ischemia, apparently through a catecholamine-dependent mechanism. Thus, increased but small quantities of fatty acids, acyl coenzyme A, and acyl carnitine accumulate even in severely ischemic tissue. 39 In areas of nfild ischemia, fatty acid uptake from the plasma also is facilitated and, coupled with the limited capacity for fatty acid oxidation and the abundant supplies of caglycerol phosphate from glycolysis, can result in accumulation o f intracellular lipid. 31 Increased concentrations of fatty acid esters can be detrimental to myocytes in a number of ways. s2,3s For example, acyl coenzyme A inhibits adenine nucleotide translocase, an enzyme that is responsible for export of A T P from mitochondria to sarcoplasm. Fatty acids also inhibit mitochondrial respiration and uncouple oxidative phosphorylation. It also has been postulated that fatty acid esters such as lysophospholipids may act as detergents and disrupt cellular membranes. 34 Arachidonic acid is one of the fatty acids that accumulate in ischemic myocardium; arachidonic acid is a major component of phospholipids, and its accumulation in the tissue is indirect evidence for catabolism o f cellular membranes during ischemia. 35 Free Radicals

In the last few years, much attention has focused on the idea that production of free radicals may be increased during myocardial ischemia and/or in the early period following reperfusion, and that free radicals such as superoxide or hydroxyl radicals could contribute to myocyte injury. ~6 Free radicals are highly reactive molecular species that can cause detrimental alterations o f proteins, nucleic acids, and lipids. For example, free radicals can cause lipid peroxidation and thereby alter the properties of sarcolemmal or other cellular m e m b r a n e s Y There are a number of potential sources for increased free-radical production in ischemia. The xanthine oxidase reaction (which converts h y p o x a n t h i n e to xanthine with formation of superoxide) has been of particular interest 3s because of the known rapid catabolism of adenine nucleotides to nucleosides and bases in ischemia, 3~ of which hypoxanthine and xanthine are sub467

strates for the xanthine oxidase reaction. Neutrophils are another potential source o f free-radical injury. Neutrophils participate in the i n f l a m m a t o r y response to injured myocytes and capillaries; their beneficial role in the initial phases of infarct repair may be counterbalanced by the injurious effects of the local release of superoxide anions and lysosomal enzymes in the vicinity of myocytes that are still viable. 4~ Ion Gradients and Altered Membrane Properties

One of the earliest events in ischemia is the net loss o f a small proportion of intracellular myocyte potassium. 41 This early potassium loss begins within seconds after the onset o f ischelnia, before substantial loss of high-energy phosphates has occurred, and is apparently caused by increased potassium efflux; influx o f potassium via the sodium-potassium adenosine triphosphatase (ATPase) pump is initially not altered. Although the mechanism for the increased potassium efflux is not completely understood, it has been shown to be depolarization dependent. 42 The early potassium loss is not accompanied by a gain of intracellular sodium, 43 and because electroneutrality must be maintained, potassium loss may occur in conjunction with cellular extrusion o f anions such as phosphate and lactate that are produced by ischemic metabolism.43, 44 Maintenance of electrolyte gradients is energy dependent, and although the Michaelis constant (Kin) of the sodium-potassium ATPase for A T P is low, marked A T P depletion in ischemia eventually results in its inhibition. Net sodium influx and a further potassium loss occur. The loss of ion transport activity also may contribute to net influx of water and consequent cell swelling. Ischemia also may lead to increased cytosolic calcium because of increased influx from the extracellular space or sarcoplasmic reticulum, or because of insufficient energy for extrusion. 45,4~ A cellular calcium overload could have serious consequences for the myocyte; calcium activates a variety of proteases, lipases, and phospholipases and may augment ATP depletion by activating a variety of ATPases. In addition, excess calcium inhibits mitochondrial respiration. A n u m b e r of electrophysiologic changes can be recorded through unipolar electrodes early in ischemia; these changes include both T Q depression and ST segment elevation, cellular changes that summate to produce the ST segment elevation seen on body surface electrocardiograms recorded with AC-coupled amplifiers. 44 These changes occur within 15 to 30 seconds after the onset of ischemia. In addition, ischemia causes shortening of the action-potential duration, slowed intramyocardial conduction, and increased dispersion of refractoriness. 47,4s T h e subcellular bases for these changes are not well understood but are thought by various investigators to be the c o n s e q u e n c e s o f m e t a b o l i c a n d e l e c t r o l y t e changes, 49,5~ including extracellular hyperkale-

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mia, 49,51 intracellular acidosis, local catecholamine release, 52 fatty acid, 53 or lysophospholipid accumulation, 54 and/or cellular calcium overload. 55

tion bands composed o f the agglutinated myofibrillar proteins of several adjacent sarcomeres. 6~ T h e formation of these contraction bands probably is related to the massive calcium overload of cells that have severe damage to the sarcolemma. Additional evidence for calcium overload in these myocytes include the deposition within mitochondria of granular and crystalline deposits o f calcium and phosphate (which are distinct from the amorphous densities described previously). 6~

Neurophysiologic Changes

Endogenous norepinephrine is released from adrenergic nerve terminals in ischemic myocardium within the first hour after the onset of occlusion. 56 Simultaneously, the numbers of exposed 13-receptors detected in membrane fractions of homogenized cardiac myocytes increase. 57 The cause of excessive norepinephrine release is uncertain; however, neuronal reuptake of norepinephrine may be decreased because it is an ATP-dependent process, s6

Ultrastructura[ Features o f I s c h e m i c Injury to Microvasculature

Ultrastructural evidence of myocyte injury generally preceeds ultrastructural evidence o f injury to vessels. 61,63 Nevertheless, ischemia does injure blood vessels. 63-66 Mild injury may be manifest by increased capillary permeability. ~t Severer injury causes endothelial cell swelling; cytoplasmic blebs protrude into capillary lumens and may cause capillary obstruction. 65 As noted earlier, in large infarcts, both myocytes and the microvasculature undergo necrosis, resulting in a central core of infarct that persists for weeks while infarct repair proceeds from the edge toward the center by growth of new granulation tissue. Reperfusion of ischemic myocardium having microvascular injury may result in extensive intramyocordial hemorrhage. Moreover, in zones where capillaries have become obstructed by endothelial blebs, 65 neutrophils, 67 microthrombi, 6s or external compression from rigorous myocytes, 69 and/or interstitial e d e m a , m i c r o v a s c u l a r r e p e r f u s i o n m a y n o t be achieved despite restoration of flow through a previously occluded coronary artery. This is referred to as the "no-reflow phenomenon. ''65

Ultrastructural Features o f I s c h e m i c Injury to Myocytes

Within the first few minutes after the onset of ischemia, ultrastructural changes develop and include cellular and mitochondrial swelling, progressive loss of sarcoplasmic glycogen particles, and mild margination of nuclear chromatin. 23,2~,5s These early changes have been shown to be reversible in experimental studies, in that restoration of coronary blood flow after coronary occlusion up to 15 minutes in duration results in rapid recovery of myocyte ultrastructure even in the most severely ischemic zone. 59 With a longer duration of ischemia, the aforementioned changes become progressively more pronounced. In addition, two uhrastructural features of injury develop that have been associated with the transition to irreversible injury. These are the development of amorphous densities within the matrix of mitochondria, and of breaks in the trilaminar unit membrane o f the sarcolemma (fig. 3). 20,21 In the absence of reperfusion, these ultrastructural features persist; eventually, coagulation necrosis becomes manifest at the light microscopic level. Reperfusion of myocytes containing mitochondrial amorphous matrix densities and sarcolemmal breaks by electron microscopy, even though they still appear viable at the light microscopic level, results in contraction band necrosis, which can be detected within m i n u t e s even by light m i c r o s c o p y (fig. 2D); 6~ the development of necrosis despite reperfusion was the initial basis for defining ischemic inj u r y as "irreversible". 59 Contraction band necrosis is characterized at the ultrastructural level by much worse disruption of the sarcolemma, by massive calcium influx to the sarcoplasm, and by destruction of the myofibrillar apparatus with formation of contrac-

PATHOGENESIS OF IRREVERSIBLEISCHEMIC CELL INJURY

Elucidation of a specifiC sequence of metabolic reactions that culminate in the transition from reversible to irreversible cellular injury has proved to be difficult despite studies in many different laboratories directed toward this problem. 2~ In general, two experimental approaches have been used. T h e first has b e e n to c o m p a r e s t r u c t u r a l a n d m e t a b o l i c changes in myocytes known to be in the late reversible phase o f injury with changes in mycoytes known to have already entered the irreversible phase of injury. A second general approach has been to inter-

FIGURE 3, Characteristic changes in severely ischemic left ventricular myocytes that were irreversibly injured by 40 minutes of in-viva ischemia. Top,The sarcolemma is distended by two swollen mitochondria (M), each of which contains amorphous matrix densities (amd). In regions where the sarcolemma is intact, a trilaminar plasmalemma [PL) is visible along the overlying basal lamina (BL). Between the thick arrows are prominent breaks in the plasmalemma. In other areas, no identifiable plasmalemma is present. (x 150,000.) (Reproduced with permission from Reimer KA, Jennings RB: In Fozzard HM et al (eds]: The Head and Cardiovascular System. New York, Raven Press, 1986, p 1133.) Bottom Relatively small gap in the plasmalemma [thin arrow) and another much larger gap [between two thick arrows). Note that the attachment between the plasmalemma and the underlying myofibril at the level of the Z line (Z) is broken. [Perfusion fixation with glutaraIdehyde and paraformaldehyde followed by postosmication, x 62,500.) (Reproduced with permission from Jennings RB, Hawkins HI<: In Wildenthal K (ec0: Degradative Processes in Heart and Skeletal Muscle. Amsterdam, ElsevierlNodh Holland, 1980, p 295.]

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Il

FIGURE 4 (tap), Some potential pathways leading to sarcolemmal damage, which form the basis for various hypotheses of events leading to irreversible [schemic cell injury. In general terms, two major facets of ischemia are the inadequate production of ATP and the accumulation of potentially noxious cafabolites. Declining ATP content could have many adverse consequences including loss of sodium and potassium gradients, calcium overload, and activation of endogenous phospholipases or proteases. Calcium overload also could cause activation of phospholipases or proteases; the latter could damage the sarcolemma and/or its cytoskelefal supports. Accumulation of catabolifes such as lactate, hydrogen ion and reduced nicotinamlde adenine dinucleotide inhibit anaerobic glycolysis and thereby inhibit ATP production in ischemia. Products of lipid degradation may act as detergents and damage cell membranes. Adenine nucteosides and bases accumulate and might be a major source of free radicals via the xanthine oxidase reaction. In addition, accumulating catabolifes are an intrace!lular osmotic load, which may accentuate cell swelling and facilitate the rupture of already weakened membranes. At present, the relative importance of these various pathways in the pathogenesis of ischemic cell death has not been established. Moreover, many reactions, which have not been studied as well and are not included on this diagram, occur in ischemic myocardium; it is not even certain that the most important pathways are illustrated. [Reproduced with permission from Reimer KA, Jennings RB: In Fozzard HM et al [eds]: The Heart and Cardiovascular System. New York, Raven Press, 1986, p 1133.] FIGURE 5 [bottom]. Components of myocardial ischemia, infarction, and repair. Myocardial infarction is o dynamic process in which cardiac myocytes undergo necrosis and eventual replacement by scar. Myocyte necrosis occurs as a direct consequence of ischemlc injury. In addition, microvascular damage occurs; it is uncertain whether or not and in what circumstances microvascutar damage exacerbates ischemia. The inflammatory response is essential for the eventual repair of an infarct. It is possible, but unproved, that the inflammatory response may exacerbate myocyte injury and contribute to overall infarct size. [Reproduced with permission from Reimer KA, Jennings RB: In Hearse DJ, YeIIon DM (eds]: Therapeutic Approaches to Myocardial Infarct Size Limitation. New York, Raven Press, 1984, p 163.) 470

MYOCARDIALISCHEMIAAND INFARCTION[Reimer & Ideker]

grouped into three distinct phases. 47 The first phase (phase I) o f e n h a n c e d v e n t r i c u l a r vulnerability begins with the first five minutes after the onset of ischemia and persists for about 30 minutes. After this phase, there is generally a quiescent period. Following this dysrhythmia-free interval, ventricular dysrhythmias begin again; phase II begins as early as two or three hours after the onset of ischemia and continues through the first day or so of infarction. A third phase (phase III) of late arrhythmias may develop days to weeks after the acute onset of infarction. Similar phases of dysrhythmias may occur clinically, with phase I being analogous to ttmt subset of sudden cardiac death that occurs early in the prehospital phase of acute myocardial infarction. Phase II dysrhythmias may be analogous to the early hospital phase of myocardial infarction, and phase III may be analogous to chronic ventricular dysrhythmias, which develop in patients with older myocardial infarcts. Ventricular arrhythmias also may be induced by reperfusion of ischemic myocardium. 72 Reperfusion arrhythmias characteristically occur within the first minute following reopening of an occluded artery. The likelihood that reperfusion arrhythmias, in particular ventricular fibrillation, will occur depends on the duration of preceding occlusion. For example, in anesthetized dogs with circumflex occlusion, reperfusion within the first five to 10 minutes after occlusion rarely produces ventricular fibrillation; however, ventricular vulnerability increases markedly by 15 minutes o f ischemia. R e p e r f u s i o n at this time (when all o f the ischemic myocytes still are viable) causes ventricular fibrillation in 50 per cent of such dogs. With longer periods of ischemia, the incidence of reperfusion ventricular fibrillation gradually decreases to about 25 per cent by 40 minutes of ischemia and to less than 5 per cent by three hours.

vene with a therapy designed to interrupt a metabolic reaction suspected to have critical importance in the pathogenesis of cell death, with the desired outcome being the prevention of cell death. Several hypotheses center on disruption of the sarcolemma as the proximate cause o f cell death. Figure 4 is a complicated diagram illustrating a simplified overview o f a few intracellular processes that may occur in ischemic myocytes and result in sarcolemmal rupture and cell death. In general terms, two major facets of ischemia are the inadequate production of high-energy phosphates and the accumulation o f potentially noxious catabolites. Declining ATP content has many consequences including loss of sodium and potassium gradients, calcium overload, and possible activation o f endogenous phospholipases or proteases. Calcium overload may also contribute to activation of phospholipases and/or proteases that could damage either the sarcolemma or its cytoskeletal supports. Accumulation of catabolites such as lactate and N A D H inhibit ATP production. Products o f lipid d e g r a d a t i o n may act as detergents to damage cell membranes. Adenine nucleosides and bases accumulate and may be a major source of free radicals via the xanthine oxidase reaction. In addition, accumulating catabolites are an intracellular osmotic load, which may accentuate cell swelling and facilitate rupture of already weakened membranes. In the setting of reperfusion, the prognosis of individual myocytes d e p e n d s not only on their internal state at the onset of reperfusion but also on the state o f the s u p p o r t i n g m i c r o v a s c u l a t u r e a n d perhaps on the manner of reperfusion, and/or the nature of the inflammatory response that may be elicited by d a m a g e d myocytes or capillaries (fig. 5). 21,7~ It is possible that some myocytes that are potentially salvageable at the onset of reperfusion are killed by some aspect of reperfusion (i.e., so-called reperfusion injury). 71 It also is hypothetically possible that microvascular damage results in perpetuation of ischemia in some areas where myocytes might have been salvageable were it not for the vascular injury. However, currently available data generally support the view that severe microvascular damage is a late phenomenon in areas where myocyte death already has occurred. Finally, whereas the inflammatory response is a necessary part of the process of infarct repair, the inflammatory response also could exacerbate myocyte or microvascular injury. U n d e r what conditions any of these reactions, or others not illustrated in figures 4 and 5, may be of seminal importance in the pathogenesis of ischemic cell death requires further elucidation.

Electrophysiologic Basis of Ventricular Arrhythmias

In general terms, ventricular arrhythmias are considered to arise from enhanced automaticity of localized regions of myocytes, triggered activity caused by after-depolarizations, or altered electrical conduction pathways that permit reentrant electrical circuits. 2'47'73 Enhanced automaticity and triggered activity must reflect altered membrane properties of injured but still viable myocytes. Reentrant pathways are thought to occur because of unidirectional conduction block followed by late activation, via alternative pathways, of myocytes distal to the initial block. Conduction then proceeds in the opposite direction across the region of unidirectional block to reenter the tissues proximal to the blocked region. For example, such a conduction circuit might occur because of altered electrical properties of injured myocytes that result in delayed repolarization of some myocytes that are therefore still refractory to an initial electrical wavefront but become excitable when conduction returns via a more circuitous or slower path.

PATHOGENESIS OF ACUTE OR CHRONIC VENTRICULAR ARRHYTHMIAS General Biology of Ventricular Arrhythmias in Ischemia or Reperfusion

Ventricular arrhythmias that occur following c o r o n a r y occlusion in animal models have been 471

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Reentry may occur in the presence or absence of a central anatomic obstacle of nonconducting tissue 2 and may be initiated because of differences in conduction caused by fiber orientation. 74 T h e detailed pathogenetic mechanism o f ventricular arrhythmia probably is different for each of the three different phases of arrhythmia and may be different for occlusion versus r e p e r f u s i o n arrhythmias. For example, in the phase I arrhythmias, the general mechanism has been postulated to be reentry within the still viable but acutely ischemic myocardium. 47 However, it is difficult to differentiate between enhanced automaticity versus reentry mechanisms by experimental techniques. Moreover, it has been reported that arrhythmias in phase I have a bimodal distribution (permitting subdivision into phase Ia and Ib) and that two distinct mechanisms apply to these subphases of arrhythmogenesis. 75,76 In phase II, when most or all of the ischemic myocytes at risk trove died and become nonelectrogenic, most ventricular arrhythmias are thought to arise because of enhanced automaticity of surviving border cells such as subendocardial Purkinje s cells. 47 However, recent evidence raises the possibility that these arrhythmias are caused by triggered activity. 77 For the late arrhythmias of phase III, the mechanism is thought to be reentry but may in some cases be enhanced automaticity of surviving Purkinje's fibers or of surviving myocytes at the lateral edges of the infarct. Reentry may occur via circuitous pathways involving the spared subendocardial zone and/or isolated surviving myocytes within a developing or completed scar. Reperfusion arrhythmias are now generally thought to result from a reentrant mechanism caused by the marked dispersion of refractoriness, which is characteristic of acutely reperfused myocardium. 7s Metabolic Basis for Altered Electrical Properties of Acutely Ischemic Myocardium

j u s t as the metabolic consequences of ischemia must initiate a sequence of events that results in the death of severely ischemic myocytes, so too, the metabolic consequences of ischemia must initiate changes in the electrical properties of the sarcolemma that give rise to ventricular arrhythmias. Much research has been devoted to identifying metabolic ctmnges that are associated either with an increased incidence of ventricular fibrillation in intact animal preparations or in reduced ventricular fibrillation thresholds in intact or isolated heart preparations. In dogs with coronary occlusions, the likelihood of ventricular fibrillation is greatest when collateral flow is low; v9 thus, there is a general relationship between the likelihood of arrhythmia and increasing severity of ischemia. However, the ionic and/or metabolic mechanisms for enhanced arrhythmogenicity have not been defined completely. A n u m b e r o f investigators have f o c u s e d on changes in trans-sarcolemmal ion gradients in isch-

472

emia. As noted earlier, there is a net efflux o f K + from the myocyte within seconds after the onset of ischemia. 41 M o r e o v e r , the m a i n t e n a n c e o f Na + and K + gradients depends on s o d i u m - p o t a s s i u m ATPase p u m p activity. Thus, the inadequate production of ATP eventually must result in loss of cellular potassium and gain of sodium. Calcium homeostasis also is dependent directly or indirectly on adequate supplies of ATP. 46 Calcium extrusion from the sarcoplasm is accomplished in part by calcium efflux coupled with sodium influx, an exchange that depends indirectly on ATP because ATP is required to maintain low intracellular sodium via the s o d i u m potassium ATPase. Several studies have shown that increased extracellular potassium concentration is arrhythmogenic. 4%51 Other studies have provided evid e n c e f a v o r i n g a l t e r a t i o n s in the slow calcium channel and/or a cellular calcium overload in the genesis of ventricular arrhythmias. 55,s~ The release of endogenous norepinephrine in ischemic myocardium, and specifically the [3-adrenergic-mediated production of cyclic AMP, may enhance arrhythmias through increased sarcoplasmic calcium concentrations or through other undefined mechanisms. 52 Alternatively, o~-adrenergic stimulation also has been implicated in the genesis o f arrhythmias. 81 Other studies have focused on the possible a r rhythmogenic role of alterations to the composition o f the lipid/bilayer per se. For example, increased concentrations o f fatty acids, 53 thought to have detergent action on cell membranes, have been shown to be arrhythmogenic. More recently, a number of studies have focused on phospholipid catabolism. Phospholipase activation at the sarcolemma might occur because of reduced levels of ATP and/or elevated sarcoplasmic calcium concentration. This could cause phospholipid loss and lysophospholipid accumulation in the plasmalemma. ~5,54 Ahhough recent studies have shown that the absolute quantities o f phospholipid degraded to lysophospholipids in the first h o u r a f t e r the onset Of ischemia are quite small, s2 it is possible that locally increased lysophospholipid concentration in the cell m e m b r a n e could provide a structural basis for the altered electrical properties that give rise to arrhythmias. In addition, arachidonic acid, 83 one of the fatty acids released by phospholipase action, is the precursor for prostaglandin synthesis; the many potent effects of various prostaglandins still are being defined. It has been reported that thromboxane may be arrhythmogenic, whereas prostacyclin may have antiarrhythmic properties, s4 Recently, the possible role of free radicals in ischemic injury has received considerable attention. Although many studies have focused on free-radical injury as a possible cause o f myocyte death (described previously), recent studies also have shown protection against ventricular fibrillation with a variety of free-radical scavengers administered to ischemic rat hearts, s5

MYOCARDIALISCHEMIAAND INFARC11ON[Reimer & Ideker] Structural Basis for Chronic Postinfarction Ventricular Arrhythmias V e n t r i c u l a r t a c h y c a r d i a is a r e l a t i v e l y f r e q u e n t c o m p l i c a t i o n in t h e late p h a s e o f m y o c a r d i a l i n f a r c t i o n . M o s t o f t h e s e a r r h y t h m i a s a r e t h o u g h t to h a v e r e e n t r a n t e l e c t r o p h y s i o l o g i c m e c h a n i s m s , 86-88 b u t structural features that distinguish infarcts inducing c h r o n i c a r r h y t h m i a s v e r s u s i n f a r c t s w i t h o u t this c o m p l i c a t i o n h a v e n o t b e e n d e f i n e d c l e a r l y . I t is r e c o g n i z e d t h a t s u c h a r r h y t h m i a s a r e m o r e l i k e l y to o c c u r w i t h i n c r e a s i n g i n f a r c t size. as I t a l s o h a s b e e n r e p o r t e d t h a t v e n t i c u l a r a n e u r y s m s w i t h e n d o c a r d i a i fib r o e l a s t o s i s a r e m o r e l i k e l y to b e a s s o c i a t e d w i t h a r rhythmias than are aneurysms lined by mural t h r o m b u s ; 89 t h e m e c h a n i s m f o r t h i s d i f f e r e n c e , i f r e a l , is u n c l e a r . S p e c i f i c s t r u c t u r a l f e a t u r e s o f i n f a r c t s that could facilitate reentrant pathways include presence of a patchy infarct with scattered, viable myocytes p e r s i s t i n g w i t h i n a r e a s o f scar, 86,9~ a n d a l a r g e extent of a subendocardial zone of spared myocytes. 9L99 S u b e n d o c a r d i a l s p a r i n g is p r e s e n t in m o s t i n f a r c t s i f t h e r e is n o m u r a l t h r o m b u s in t h e p e r i - i n f a r c t i o n p e r i o d . S p a r e d s u b e n d o c a r d i u m is a likely site f o r a t l e a s t p a r t o f t h e r e e n t r a n t p a t h w a y , b e c a u s e s u r g i c a l r e m o v a l o f this tissue f r e q u e n t l y p r e v e n t s r e c u r r e n c e s o f v e n t r i c u l a r t a c h y c a r d i a . 92 I n a d d i t i o n , s t r u c t u r a l c h a n g e s s u c h as m y o c y t o l y s i s , w h i c h o c c u r s w i t h v a r i a b l e d e g r e e s o f s e v e r i t y in m y o c y t e s in t h e s u b e n d o c a r d i a l s p a r e d z o n e , m i g h t c o n t r i b u t e to r e e n t r a n t p a t h w a y s ; it is n o t k n o w n w h e t h e r c o n d u c t i o n t h r o u g h m y o c y t o l y t i c cells is a b n o r m a l .

CONCLUSIONS M u c h h a s b e e n l e a r n e d a b o u t t h e g e n e r a l bio l o g y o f m y o c a r d i a l i n f a r c t i o n , in t e r m s o f t h e c a u s e s of infarction and determinants of ultimate infarct size. I n a d d i t i o n , m u c h h a s b e e n l e a r n e d a b o u t t h e acute and chronic cellular changes induced by severe or milder degrees of myocardial ischemia. Nevertheless, t h e p a t h o g e n e s i s o f t h e t r a n s i t i o n f r o m r e v e r s ible to i r r e v e r s i b l e cell i n j u r y a n d t h e c e l h d a r basis for either acute or chronic ventricular arrhythmias r e m a i n to b e e l u c i d a t e d fully.

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