Early phase of myocardial ischemic injury and infarction

The American Volume

24

Journal DECEMBER

Symposium

of Cardiology 6

on the Pre-Hospital

Phase of Acute Myocardial Part

Number

1969

Infarction.

II. Early Phase of Myocardial

Ischemic

Injury and Infarction* ROBERT

B. JENNINGS,

Chicago,

M.D.

Illinois

The acute effects of &hernia on myocardial tissue are reviewed in this paper with emphasis on how the events observed in experimental myocardial ischemic injury in dogs relate to the sequential changes occurring in the myocardium of man during the first few hours after the onset of acute infarction. The general effects of ischemia on myocardium include those which are secondary to a diminished local supply of substances such as oxygen and metabolites as well as changes resulting from the impaired diffusion of substances such as lactic acid and electrolytes from the poorly perfused ischemic tissue to the general circulation. Cell death (irreversible injury) first develops after 20 minutes of ischemia in areas of maximum injury in the dog heart and infarcts are not fully developed for 60 to 120 minutes after occlusion. Prior to the development of cell death, the severely ischemic cells are reversibly injured and show a variety of alterations from normal. These changes include depletion of supplies of glycogen and high energy phosphate, increased content of lactic acid and hydrogen, relaxation of myofibrils and failure of contraction. Cells which have just died show the same changes as well as mitochondrial, electrolyte, and nuclear defects. Which, if any, of the preceding changes causes the development of irreversibility in ischemit injury remains to be established. Some data is presented in support of the hypothesis that mitochondrial defects may be critical in the genesis of the irreversible state.

T

HE

CLINICAL

AND

PATHOLOGIC

EVENTS

OC-

curring during the early phase of acute myocardial infarction in man remain largely unknown. Knowledge of these events is important since the principal signs and symptoms of infarction arise from the ischemic myocardium and because therapy should be based on a

knowledge of the sequential changes occurring in the affected tissue. Since man in the early phase of infarction is not available for study, our information about the probable sequence of events occurring in the affected myocardium comes from the results of animal experiments on acute myocardial ischemic injury.

*From the Pathology Department, Northwestern University Medical School, Chicago, Ill. This study was supported in part by grants from the National Heart Institute (HE 80729) and the Chicago Heart Association. Northwestern Uni\-ersity Medical Address for reprints: Robert B. Jennings, M.D.. Pathology Department, school, 303 East Chicago Ave., Chicago, 111. 60611. 753

Jennings During the past decade we have done a series of studies on various aspects of acute myocardial ischemic injury in dogs. Most of our experiments have involved direct analysis of the injured tissue for changes occurring as a consequence of the sudden onset of ischemia. With few exceptions, we have employed the posterior papillary muscle infarct produced by occlusion of the circumflex branch of the left coronary artery near its origin.’ This preparation was used because the posterior papillary muscle is an easily sampled area of uniform maximal ischemia (injury) in the large posterolateral area of cyanosis produced by ligation of this vessel. (The upper two thirds of the posterior papillary muscle is uniformly and severely injured in all dogs that show cyanosis extending to within 1 cm. of the apex of the heart posterior1y.l) Sampling elsewhere in the ischemic tissue usually provides a mixture of injured and noninjured cells which is unsatisfactory for most types of study. Our principal aim has been to discover the events leading to the development of cell death in an area of acute myocardial ischemia. Certain aspects of these studies have been reviewed elsewhere.a-6 In this paper some of the difficulties inherent in transferring data from studies of infarction in animals to man are discussed, and the concept of reversible and irreversible injury in myocardial cells is presented. Next, the sequential changes occurring in severely ischemic reversibly and irreversibly injured cells are reviewed and, lastly, some possible causes of the development of irreversibility in ischemia are considered. The data presented illustrate an obvious but important concept about myocardial infarction: an acute myocardial infarct is a dynamic focus of changing metabolism, function and composition which does not become static until the infarct heals. APPLICABILITY OF ANIMAL DATA ON INFARCTIONTO HUMAN DISEASE Many of the changes occurring in an acute myocardial infarct produced by sudden occlusion of a coronary artery in an anesthetized dog with a thoracotomy are similar to the events occurring in man with the naturally occurring disease. However, there are some striking differences between the hearts of dogs and men with acute myocardial infarcts. Most of these differences are related to the fact that the unoccluded portion of the coronary arter-

ial tree is normal in the dog and almost always is diseased in man. Consequently, the extent of collateral arterial flow and indirectly the size of the infarct is largely dependent upon the magnitude of arteriosclerotic disease in the unoccluded vessels. If collateral how is reduced or insignificant, occlusion of a small artery may result in a large area of infarction. On the other hand, if collateral flow is abundant, occlusion of a large artery may not produce as large an infarct as one might expect. Myocardial cells die when the total flow of oxygenated arterial blood provided to them is reduced below that required to maintain viability. Thus, cell death occurs in both man and experimental animals if flow suddenly is reduced by the complete occlusion of a major vessel. In man, however, there is evidence from autopsy studies 7 that incomplete occlusion or extensive narrowing of the coronary arterial tree may give rise to episodes of acute myocardial ischemia marked enough to cause cell death. This may be especially true in the presence of conditions that may cause a transient decrease in coronary blood flow, such as arrhythmias or hemorrhagic shock. However, up to the present time, infarction without occlusion is a phenomenon that has not been reproduced in animals. With the exception of hemorrhage into an atherosclerotic plaque,8 sudden occlusion of a coronary artery in man in a fashion equivalent to that produced by a silk ligature in a dog probably occurs rarely, if at all. It seems probable that most natural occlusions are the result of the gradual accretion of thrombus over a patch of arteriosclerosis in the vessel wall, a process that almost certainly requires minutes and perhaps hours or days for completion. The thrombosis is the second stage of an acute coronary occlusion, the first stage being the development of the arteriosclerotic plaque on which the thrombus develops. Discovery of the pathogenesis of the thrombotic phenomenon will be a significant advance. However, the discovery of the cause or the means of prevention of arteriosclerosis is the most important unsolved problem relating to the prevention of myocardial infarction in man. Technics designed to mimic the develop ment of acute myocardial infarction in man or to alter the rate of development of collateral coronary arterial flow by slowly occluding a major coronary artery in an awake experimental animal with a closed thoracotomy have THE

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Myocardial Ischemic Injury and Infarction been reported by several investigatorsg-l1 Since the emphasis of our work has been on the changes developing in the injured tissue after carefully timed periods of injury, and because timing is difficult to establish in experiments employing slow occlusions, these latter studies will not be reviewed here. ISCHE~W.~ AND INFARCTION

Sudden occlusion of a major branch of a coronary artery in the dog is followed by an

immediate reduction in arterial blood flow through the myocardium supplied by the oceluded vessel. This reduction in blood flow is termed ischemia.” I believe that the arterial flow never ceases completely in the area of infarction; however, there is little direct evidence to support this concept aside from the observation that significant numbers of autogenous erythrocytes, labelled with ssP or slCr and injected 24 hours after occlusion, can be recovered from the tissue of the posterior papillary muscle infarct.’ Also, direct observation shows well oxygenated blood to be flowing through the circumflex artery distal to the point of occlusion. This arterial blood originates from small anastomoses between the occluded and unoccluded arteries of the myocardium.ls-14 However, it is impossible to measure the precise flow provided by collaterals to any particular area in the affected myocardium, except to note that, by definition, the flow is deficient. This intramyocardial collateral flow does not appear to be under great pressure, since restoration of arterial flow after a prolonged temporary occlusion of a major coronary artery in the dog is followed by the development of multiple hemorrhages in the infarct. Since these hemorrhages are not present before reflow, it seems likely that the arterial pressure in the injured vessels in the infarct was low during the ischemic episode.14 Tlzr results of experimental temporary occZu.rion,6J5s16 in which myocardium is subjected to episodes of ischemia of 5 to 60 minutes’ duration, show that all myocardial cells survive periods of severe ischemia lasting as long as 18 minutes. Restoration of the arterial *Ischemia has been defined because the word has been used loosely. Some authors have used it to describe in vitro experiments in which unperfused excised tissue is allowed to undergo autolysis. Ischemia literally means “to hold back blood” (Dorland’s Illustrated Medical Dictionary, ed. 20) _ This phenomenon is possible only if an intact circulatory system is present and operational. VOLUME

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blood flow to the ischemic tissue at any time during the first 18 minutes is followed by prompt restoration of metabolism and function of the injured cells as well as by marked reactive hyperemia. l6 Within a few seconds or, certainly, minutes after restoration of now, the affected tissue becomes indistinguishable by biochemical, morphologic, or functional technics from control myocardium. Thus, this tissue is reversibly injured. However, if the occlusion is maintained past 20 minutes, some of the severely ischemic cells die, even though the arterial flow has been restored to them. The injury to these cells is irreversible. Very few cells die after exposure to only 20 minutes of severe &hernia, but more and more cells die as the period of ischemia is extended; at 40 minutes an average of half of the cells and at 60 minutes almost all of the cells in the area of severe ischemia are dead. Irreversible injury is detectable only by special technics during the first hour after its development.lrJ* However, between 6 and 12 hours after the onset, gross and light microscopic evidence of necrosis becomes apparent. The area of irreversible injury now is an acute Later, the dead cells are myocardial infarct. phagocytosed, and the infarct is replaced by scar tissue. These concepts are summarized in Table I. Although the entire thickness of the posterolateral wall of the left ventricle of the dog becomes ischemic after occlusion of the circumflex artery, only a portion of the ischemic myocardium dies. This conclusion is based on both short- and long-term observations (Fig. I) which show that occlusion of this artery is followed by death of almost all cells in the subendocardial myocardium and a varying number of cells in the middle third of the myocardium; cells in the subepicardial myocardium usually survive. Examination of the subepicardial myocardium in the living heart days or weeks later reveals viable contracting pink myocardium with a normal structure in this region. The cells in the epicardial layer, even though ischemic during the acute phase, now are receiving an adequate collateral flow of arterial blood to support function, and had been reversibly injured during the acute phase. This conclusion is based on the assumption that cells in all layers of the myocardium are similar and that the increased frequency of severe injury in the subendocardial myocardium is due to factors other than innate differences in

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Figure 1. Cross section through the left zlentricle of a dog 106 days after ligation of the circumflex bwnch of the left coronary artery new its origin. The section was made at the level of the anterior (AP) and posterior papillary (PP) muscles. The posterior papillary muscle is uniformlv scarred and greatly reduced in size. Prior to occlusion it was approximately the same size as the anterior papillary muscle. Note that the subendocardial myocardium is scarred but that the subepicardial myocardium essentially is intact.4 Periodic acid Schiff stain after amylasc digestion. Magnification approximately X 2, increased by 11 per cent.

Table

Reversible injury

Irreversible injury

Myocardial infarct

I.

Events

Occurring

Period

of Ischemia

in Severely

Ischemic

Dog

Myocardium

Fate of Cells

Up to 19 minutes

Cells alive

20 to 30 minutes 31 to 60 minutes 61 min. to 12 hours

Few cells die More cells die All maximally ischemic to two hours

12 to 24 hours

Histologic evidence of necrosis inflammatory cell infiltration Phagocytosis and repair

1 day to 6 weeks

left ventricular myocardial cells. (Up to the present time, there is no evidence to indicate that different parts of the left ventricular myocardium differ significantly in composition, function, or structure.l? We have found the enzyme, electrolyte, and nitrogen content of

cells dead

by one

apparent; begins

subendocardial and epicardial myocardium be identica1.6~1Q~20) The

probabie

condition

to

of the myocardium

60 minutes after acute occlusion of the circumflex artery in the dog heart is diagrammed in Figure 2. The subendocardial myocardium is THE

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Figure 2. Diagram of the posterior wall of the dog left ventricle sectioned parallel to the long axis of the posterior papillary muscle (PP) and including a part of the apex and the posterior leaflet of the mitral valve. The shaded portion of the posterior wall is supplied by the circumflex branch of the left coronary artery. Immediately after occlusion of this vessel within 1.0 cm. of its origin, ischemia is most marked in the subendocardial and midmyocardial region, as evidenced by the fact that necrosis occurs in these inner zones. The endocardium, however, always is spared. The lightly cross-hatched subepicardial myocardium is believed to be less ischemic than the other zones. Cells in this region are injured reversibly, as are cells in the lightly crosshatched area at the juncture of the region supplied by the anterior descending and circumflex branches of the left coronary artery. Since no cells die in this lightly cross-hatched zone, and it is similar structurally, chemically, and metabolicallv , to the inner zonesli.l9,20. cell death is related best to differences in blood flow.

severely ischemic and contains many irreversibly injured cells. The middle third of the myocardium is less ischemic and contains a variable mixture of irreversibly and reversibly injured cells; the subepicardial myocardium is less ischemic and contains chiefly reversibly injured cells. Accordingly, it has been difficult to produce a so-called “through and through infarct” in the dog heart by occlusion of a single coronary artery. Disregarded in this consideration is the fact that dogs with especially large areas of ischemia tend to die of ventricular fibrillation. Ventricular arrhythmias, especially ventricular fibrillation, are common in both man and experimental animals with infarcts. In dogs, fibrillation is especially frequent during the first hour after the onset of myocardial ischemia.3 We have been puzzled by the unusually high incidence of ventricular fibrillation in dogs with temporary occlusions of 20 minutes’ duration; in 50 to 60 per cent of dogs treated in this fashion ventricular fibrillation will develop during the first 60 seconds after restoration of the arterial circulation. The significance of the unusual frequency of this arrhythmia at this time interval is unknown, but this period does correlate with the onset of cell death in the area of severe ischemia. Catecholamines may be involved in the development VOLUME

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of fibrillation in this model, since depletion of endogenous catecholamines by reserpine greatly reduces the incidence of ventricular fibrillation in this system.21 Clinical Zmplications: Therapeutic measures designed to increase the number of cells surviving in an area of ischemic injury in man should be taken immediately after the episode of infarction is diagnosed. It seems likely that most of the severely ischemic cells will have died by the time the average patient with an acute myocardial infarct is admitted to the hospital. Even though the severely damaged cells are dead, increased myocardial oxygenation is desirable to help prevent the death of less severely ischemic reversibly injured cells. Prevention of hypoxia in general, and of arrhythmias that cause a decrease in coronary blood flow and thus increase the degree of ischemia, is desirable. Hyperbaric oxygen therapy theoretically should decrease the number of cells dying in an infarct. I should emphasize, however, that changes in size of the infarct as a result of theoretical therapeutic considerations of this type are extremely difficult to demonstrate objectively in well controlled experiments. It seems likely that reversibly injured cells on the periphery of the infarct give rise to the arrhythmias that are common in acu’te

,

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758

myocardial infarction. Dead cells should be inactive; partially repolarized reversibly injured cells, on the other hand, should be unstable. Sodi-Pallares and his co-workers2avaa have suggested that therapy with glucose-potassium chloride-insulin may stabilize reversibly injured cells on the edge of the infarct, an attractive hypothesis that also is difficult to prove.24 There seems to be little doubt that it is possible to alter the intracellular milieu of living injured cells by therapy with glucose solutions.2j~26 EFFECTS

OF ISCHEMIA

REVERsIBLEINJURY

The broad outline of many of the events occurring in an area of acute ischemia now is fairly well known. 4,6,ar Myocardium is almost completely dependent on aerobic metabolism to produce enough energy to maintain the almost continuous contraction of the heart.28929 The large quantities of oxygen required are provided by the rich coronary arterial blood flow of the ventricular myocardium. Reduction in this blood supply induces local hypoxia; the myocardium also becomes deficient in exogenous substrates such as glucose, amino acids and fatty acids ordinarily supplied with the arterial blood. Moreover, end prodsucts of metabolism accumulate because the reduced blood flow slows egress of metabolites. The metabolism of the myocardial cells becomes anaerobic, and mitochondrial metabolism essentially ceases. Since oxygen no longer is available to combine with the hydrogen from substrates to form water, hydrogen and electrons from substrate metabolism accumulate in the terminal electron system of the mitochondria. Levels of nicotine adenine dinucleotide and the cytochromes become reduced;4ra” adenosine triphosphate (ATP) synthesis through oxidative phosphorylation ceases. Supplies of ATP are partially depleted as the muscle continues to contract for a few seconds after occlusion and supplies of creatine phosphate are quickly consumed.a19aa Adenosine diphosphate (ADP) , adenosine monophosphate (AMP), creatine and inorganic phosphate increase in concentration as ATP is hydrolyzed. Tissue oxygen tension decreases and myoglobin becomes reduced. Within 8 to 10 seconds the affected myocardium becomes cyanotic, and enough hydrogen accumulates to reduce intracellular artificial hydrogen acceptors such as methylene blue.4

Simultaneously, electrocardiographic changes appear. ;\naerobic glycolysis begins to be the principal source of energy; glycogen is utilized, and lactic acid and other glycolytic intermediates accumulate.25,26,29,34-37 The myocardium ceases contracting within the first 30 to 60 seconds after the onset of Tennant and Wiggers38 were the ischemia. first to investigate contractile changes in ischemia in detail. Many investigators39,40 subsequently have noted failure of contraction. The contractile failure is so marked with a large infarct that it can be seen with the naked eye. The affected myocardium bulges with each systole. The reason usually given for cessation of contraction is depletion of the intracellular high energy phosphate, but this may not be the case since ATP still is present in myocardium subjected to anoxia.29*a2 Katz41 has suggested, based on observations of Shfdler,da that the failure of contraction may be due to the effect of increased intracellular acidity on the ability of Ca++ to activate contraction. The temperature of the ischemic myocardium increases for the first few seconds after the onset of ischemia and then decreases to a level close to that of arterial blood. The change in temperature apparently reflects the decreased heat production associated with the cessation of aerobic metabolism.43 The myocardium continues to metabolize glycogen until supplies of this substrate become exhausted or anaerobic glycolysis is inhibited.20.44 Myocardial lactic acid accumulates quickly because it is the principal end product of glycogen degradation through anaerobic glycolysis. The increase in lactic acid is marked because Kreb’s cycle metabolism is not available to oxidize lactate to carbon dioxide and water and because clearance of metabolites from the area of ischemia is slowed by the reduced arterial flow. While the cells are reversibly injured, the mitochondria of the affected cells are in a situation in which exogenous substrate supplies from the blood are low. Intracellular endogenous substrate is available in the form of pyruvate, lactate and, presumably, fatty acids and amino acids. ADP supplies are adequate as long as some ATP is present to be hydrolyzed, and the oxygen supply, of course, is deficient. It seems likely that these mitochondria slowly use endogenous substrates in the area of ischemia whether or not much oxygen is present. However, true aerobic metabolism probTHE AMERICAN

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3. The upper micrograph shows a portion of a normal myocardial cell from the anterior superior septum of the left ventricle The myolibrils (arrows) are contracted and the sarcolemma (s) is scalloped. The chromatin of the nucleus (n) is evenly distributed. Mitochondria (m) are abundant and have a moderately dense matrix. Glycogen (g) is present in the sarcoplasm and between some myofilaments. Transverse tubules (t) are present at the Z lines of the myofibrils. nu=nucleolus. (X 9000, reduced by 25 per cent.) The lower micrograph is from the posterior papillary muscle of a dog subjected to 15 minutes of &hernia (reversible injury). The nucleus (n) shows dispersion of chromatin with probable early margination. The myofibrils are relaxed. Glycogen is diminished in amount. The mitochondria (m) are similar to control. (X 12,000, reduced by 25 per cent.) Both micrographs reproduced from JENNINGS et al.17 with permission of the publisher. Figure

ably continues to some extent in the area of ischemia, utilizing the variable quantities of oxyg en provided to the affected cells by the colla teral arterial flow. SirIce energy is required to maintain intracellu lar electrolyte distribution, it seems likely that some potassium and magnesium are lost VOLUME 24, DECEMBER 1969

from the cells to the interstitium and that :jome sodium and water enter the ischemic cells shortly after the onset of ischemia. Severa11 investigator+-47 have shown an acute increa se in coronary sinus potassium in blood drai ning areas of acute ischemia. This suggests that trolyte changes occur immediately and 13rob-

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760

Figure 4. Micrograph of a portion of an irreversibly injured cell in the posterior papillary (u) shows marked margination of muscle of a dog after 60 minutes of ischemia. The nucleus nuclear chromatin. Glycogen is virtually absent. The mitochondrial matrix is less dense than control. Prominent intramitochondrial granules are at the arrows. (X 12,000, reduced by 24 of the publisher. per cent.) Reproduced from JENNLNCS et al.17 with permission

ably coincidentally with the shift to anaerobic metabolism. However, such changes are not demonstrable by direct tissue analysis until ischemic injury has become irreversible20 Even with irreversible injury, electrolyte changes require time to become detectable. All of the effects of ischemia described so far appear during the phase of reversible injury and persist as long as cells remain in this condition. The structure of the affected cells is not significantly altered from normal (Fig. 3) except for depletion of glycogen, relaxation of myofibrils and possible changes in distribution of nuclear chromatin. However, further and more extensive changes develop in those reversibly injured cells which die in the infarct. IRREVERSIBLE

IN JURY

The early events occurring in cells that have just died in an acute infarct have been investigated in detail in the last 10 to 12 years. I plan to restrict my remarks to the acute phase of cell death and will not discuss the final events associated with necrosis, that is, phagocytosis and repair.4s Metabolic Changes: All of the changes occurring during the acute reversible phase of &hernia take place in the cells that have just died. After death, the cells lose their remaining K+, Mg+-. phosphate and, later, low molecular weight cofactors and certain enzymes to the interstitial space and eventually to the

circulation.20 Certain other substances, such as nicotine adenine dinucleotide, adenosine monophosphate and glycogen, are degraded, and changes in tissue concentrations of these substances often can be detected quickly.s1T3s Changes in the tissue concentration of those substances that are not altered through utilization or degradation in the ischemic cells can be demonstrated by direct analysis of the tissue after variable periods of time. Potassium and glutamic oxaloacetic acid transaminase are decreased in concentration or activity after one hour of injury, while stable membrane-bound enzyme systems manifest persistent activity for four or more hours after occlusion.rz Microscopic Changes: Although the structure of the irreversibly injured cells remains normal to routine technics of light microscopy for hours after cell death,49 histochemical technics reveal changes reflecting altered staining reactionsQ5~36 or increased utilization, destruction, or loss of tissue components. Decrease in stainable glycogen was one of the first such changes observed.35s36 Zugibe et al.60 have utilized some of these concepts about the development of early changes in dead myocardial cells to detect the presence of acute infarcts that could not be detected by standard technics at autopsy in man. On the other hand, alterations in the fine structure of cells that have just died are detectable quickly. Obvious changes appear in THEAMERICAN

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Myocardial Ischcmic Injury and Infarction 1

5%b-67

NO. IO

NO.20

PYRUVATE 120 UM

PYRUVATE 120uM

17

LV 60

PP MINUTES

OF

ISCHEMIA

Figure 5. Typical oxygraph tracings of respiration of mitochondria isolated52 from left ventricle (LV) and posterior papillary muscle (PP) of a dog with a 60 minute infarct. The posterior papillary muscle sample shows no respiratory control and little respiration. The numbers beside the tracing are microatoms of oxygen per mg. mitochondrial nitrogen. The medium contained 112 mM KCI, 10 mM potassium phosphate buffer (pH 7.4), 120 mM pyruvate and 3.2 mM ADP. Respiratory control ratio (RCR) is calculated by dividing the rate of respiration with substrate and phosphate acceptor present by the rate of respiration with substrate present and phosphate acceptor absent. Reproduced from JENNINGS et al.51 with permission from the publisher.

the severely ischemic irreversibly injured cells of the posterior papillary muscle infarct after 40 minutes of ischemia and are prominent after 60 minutes (Fig. 4). Characteristic features include peripheral aggregation of nuclear chromatin, marked relaxation of myofibrils, virtual absence of glycogen, and changes in mitochondrial morphologic features. The mitochondrial changes are particularly striking and consist of loss of matrix density, enlargement, occasional disruption, and accumulation of dense intramitochondrial granules. Changes in Mitochondria: The functional capability of the mitochondria of severely ischemic cells such as those illustrated in Figure 4, is greatly depressed.‘Jvsl Mitochondria isolated from such cells do not have the capacity

with pyruvate-malate are greatly depressed and are unassociated with respiratory control. These mitochondria also exhibit a depressed capacity to metabolize succinate, but the depression is not as great as that noted with pyruvate or alpha ketoglutarate. This is not unexpected since succinic dehydrogenase is a part of the structure of the cristae of the mitochondria and will function even in partially disrupted mitochondria.sl The mitochondria of the irreversibly injured cell also exhibit much greater fragility than mitochondria of control tissue. Special technics, such as predigestion of the muscle mince with a proteolytic enzyme,52 have to be used to isolate these fragile structures. Standard technics of isolation result in fragmentation* of the fragrespiration

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MINUTES Figure 6. Changes in lactic acid and glycogen concentrations of myocardium subjected to autcslysis in a moist chamber at 37°C. for varying time periods. The black squares are the average lactic acid and glycogen concentrations, expressed as percent of control, which were found in the posterior papillary muscle of four dogs subjected to 60 minutes of ischemia. Note that the tissue content of these substances is similar after one hour of ischemia or autolysis. This graph was modified from data published by HERDSON et a1.34 with permission of the publisher.

about 50 per cent as much mitochondrial nitrois isolated from the irreversibly injured cells (60 min.) as is isolated from control myocardium.sl All evidence so far obtained indicates that the mitochondrial changes observed in cells that have just entered a state of irreversible injury are irreparable. 61 The nature of the intramitochondrial granules in these cells is not established. Indirect evidences3 suggests that they might contain excess calcium.

gen

AUTOLYSIS

Many of the changes that occur in ischemic injury also develop in excised myocardium incubated at 37°C. in a moist chamber. This is not unexpected, since anaerobic glycolysis continues in excised tissue as it does in ischemic tissue.29v34 However, autolyzed tissue differs from ischemic tissue since it is not exposed to mechanical effects of the continued contrac-

tion of the viable heart and is not provided with oxygen from collateral arterial flow. Herdson et al.34 have compared the changes in autolysis with those of ischemia and have shown that the early phases are similar. Progress in studies of the mechanism of development of irreversibility in ischemic injury will be greatly accelerated if the lesions prove to be equivalent because of the abundance of tissue available for study in autolysis compared to that available in ischemic injury. The rate of accumulation of lactic acid and depletion of glycogen in tissue incubated at 37°C. is shown m Figure 6. Most of the lactic acid accumulates in the first hour, but some accumulation occurs during the subsequent two hours of incubation. Glycogen supplies, on the other hand, are almost exhausted at one hour.3+ Of interest relative to the comparison of ischemia and autolysis is the fact that the average value for glycogen and lactate in the posterior papillary muscle infarct, indicated by the black squares on the diagram, falls on the same curves as noted in autolysis. Changes in fine structure are very similar in vitro autolysis and in vivo ischemic injury of equivalent duration. An increase in sarcoplasmic space, depletion of glycogen, peripheral aggregation of nuclear chromatin, and mitochondrial changes including development of granules all are apparent. Moreover, similar changes in mitochondrial fragility and functional capacity are noted. These latter changes, however, appear to develop 15 to 30 minutes later than they do in ischemic injury.34 The electrolyte content of mitochondria isolated in sucrose ethylenediaminetetra-acetic acid (EDTA) from dog myocardium subjected to one, two and three hours of autolysis at 37°C. shows decreased Mg++ and K+ content and a 150 percent increase in Ca++ content after two hours of autolysis.52 Some evidence that the intramitochondrial granules of autolysis contain excess Ca+ + has been provided recently by Shen et al.53 DEVELOPMENT

OF

IRREVERSIBLE

INJURY

seems certain that oxygen deficiency is the primary cause of cell death in ischemic injury, although lack of nutrients ordinarily supplied to the myocardium by the blood also may be important causal factors. However, since all ischemic cells live in a state of severe oxygen deficiency for 19 minutes or more before dying, irreversible injury and cell death unIt

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Myocardial Ischemic Injury and Infarction doubteclly follow failure of a critical process tha( maintains the viability of these cells under conditions of prolonged severe ischemia. The elucidation of this critical event or series of events has been the subject of a long search in our laboratory. There are an infinite number of processes to consider in planning experiments designed to discover what factor or factors might be the first to indicate that a myocardial cell has died, even though most of its metabolic and structural equipment appears to be intact. As a general working principle, we assume that the injured cells die when the energy level within them decreases to a point no longer compatible with the maintenance of their structural and functional integrity. Therefore, one might expect cell death to occur when supplies of glycogen for anaerobic metabolism become exhausted, when a step of glycolysis is blocked, or when the intracellular pH decreases to a point below that compatible with viability of the cells because of accumulation of hydrogen and lactic acid in the area of ischemia.596 We54 have suggested in the past that insufficient metabolism to maintain viability of the injured cells may develop during the phase of reversible injury because of the loss of vital cofactors or enzymes through a leaky cell membrane. The structural in mitochondria

and functional changes noted of cells that have just died,

taken together with the importance of mitochondrial metabolism to maintenance of function of myocardial cells, suggest that mitochondrial damage may lead to the development of irreversibility. Our findings generally support the theory that a defect in mitochondrial function may be the event that ultimately causes myocardial cells to die. The pathogenesis of the mitochondrial defect observed in ischemic injury61 and in auto-

lysis34155 r is unknown. The development of the dense granules that are so prominent in the mitochondria of the dying heart suggest that after about 20 minutes of ischemia, a material, perhaps including a divalent cation like Ca++ or Mg++, begins either to redistribute within the mitochondria or actually is concentrated within them from the sarcoplasm. If active concentration of an inhibitor of oxidative phosphorylation like Ca++ is involved, then Ca++ either was not available earlier or its accumulation to toxic level was not reached for 20 minutes or more after the onset. Crystallization or binding of an essential cofactor VOLUME

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like inorganic phosphate or lMg++ in the granules also could produce irreversible mitochondrial failure, as could disruption of mitochondrial enzyme systems by swelling or the increasing intracellular acidity. The

e?Jent or events

causing

irreuersibility

remain unknown and will be difficult to discover in a system as complex as a myocardial cell. It is clear that phenomena not discussed here could be related causally to the mitochondrial failure. Deficient production of ATP, depletion of supplies of glycogen and other endogenous substrates, loss of enzymes, cofactors, and electrolytes to the extracellular space, accumulation of lactic acid and probable altered nuclear function, as well as other changes, all are occurring in the injured cells during the time they are teetering between life and death. Greater knowledge of these changes may allow improvements in therapy and provide basic information about processes involved in cell injury. ACKNOWLEDGMENTS Some of the recent work summarized in this papel was performed in collaboration with Drs. Peter B. Herdson, John P. Kaltenbach and Herbert M. Sommers. I am grateful for excellent technical assistance provided by Miss M. L. Hill and Mrs. C. Moore in biochemistry and Miss B. A. Moulton and Mrs. S. Safavi in electron microscopy.

REFERENCES 1. JENNINGS, R. B., WARTMAN, W. B. and ZIJDYK, Z. E. Production of an area of homogenous myocardial infarction in the dog. A.M.A. Arch. Path., 63:580, 1957. 2. JENNINGS, R. B. and WARTMAN, W. B. Reactions of myocardium to obstruction of the coronary arteries. M. Clin. North America, 41:3, 1957. 3. JENNINGS, R. B. Acute myocardial ischemic injury. Chicago Med., 63:9, 1961. 4. JENNINGS, R. B., KALTENBACH,J. P., SOMMERS, H. M., BAHR, G. F. and WARTMAN, W. B. Studies of the dying myocardial cell. In: Etiology of Myocardial Infarction, pp. 189-204. Edited by JAMES, T. N. and KEYES, J. W. Boston, 1963. Little, Brown. 5. JENNINGS, R. B., HERDSON, P. B. and KALTENBACH, J. P. Ischemic injury of myocardium: Structural and functional correlations during the acute phase. Proceedings of the Second Congress of the Academy of Medicine of Mexico, Mexico City, 1969, in press. 6. JENNINGS, R. B., SOMMERS, H. M., HERDSON, P. B. and KALTENBACH,J. P. Ischemic injury of myocardium. Ann. New York Acad. SC., 156:61, 1969. 7. EHRLICH, J. C. and SHINOHARA, Y. Low incidence of coronary thrombosis in myocardial infarction. Arch. Path., 78:432, 1964. 8. WARTMAN, W. B. Occlusion of the coronary ar-

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modif\ the consequences of acute coronary artery ligation. J. Thoracic 6 Cardiovas. Surg., 49:762, 1965. 25. RERAR. B., OAIACHI, A. and REBAR, J. Effects of glucose infusion on dog myocardial metabolism. Circulation Hrs., 7:977, 1959. 26. WEISSLER, A. M., ~~RLGER, F. .A.,BARA, N., SCARPELI.I, D. G.. LEIGHTON, R. F. and GALLIMORE, J. K. Role of anaerobic metabolism in the preservation of the functional capacity and structure of anoxic myocardium. ]. Clin. Invest., 47:403, 1968. 27. SHEUER, J. Myocardial metabolism in cardiac hypoxia. A?jr. ./. CarrlioE., 19:385, 1967. 28. BING, R. J. Metabolism of the heart. Harvey Lect., 1954-55, pp 27-70. 29. BING, R. J. Cardiac metabolism. P!qjsiol. Rev., 45: lil, 1965. 30. CHANCE,B., SCHOENER,B., KREJCI, K., R~~SSMAN,W.. WESEMANN, W., SCHNITGER, H. and B~CHER, T. Kinetics of fluorescence and metabolite changes in rat liver during a cycle of ischemia. Biochem. Ztschr., 341:325, 1965. 31. BRAASCH,W., GUDBJARNASON,S., PARI, P. S., RAVENS, K. G. and BINT., R. J. Early changes in energy metabolism in the myocardium following acute coronary artery occlusion in anesthetized dogs. Circulation Res., 23:429, 1968. 32. MANSFORD, K. R. L. and OPIE, L. H. Comparison of the effects of lack of substrate or of oxygen on isolated working rat heart. Proc. First International Conference on Influence of Exogenous ATP on Heart. Edited by SISKA, K. In press, 1968. 33. IX\IAI,S., RILEY, A. L. and BERNE, R. M. Effect of ischemia on adenine nucleotides in cardiac and skeletal muscle. Circulation Res., 15:443, 1964. 34. HERLISON,P. B., KALTENBACH,J. P. and JENNINGS, R. B. Fine structural and biochemical changes in dog myocardium during autolysis. Am. J. Path., in press. 35. YOKOYAMA, H. O., JENNINGS, R. B., CLABAUCH,G. F. and WARTMAN, W. B. Histochemical studies of early experimental myocardial infarction. A.M.A. Arch. Path., 59:347, 1955. 36. KENT, S. P. and DISEKER, M. Early myocardial ischemia: Study of histochemical changes in dogs. Lab. Invest., 4:398, 1955. 37. KLIONSBY, B. Myocardial ischemia and early infarction: A histochemical study. Am. ]. Path., 36:575, 1960. 38. TENNANT, R. and WIGGERS, C. J. The effect of coronary occlusion on myocardial contraction. Am. J. Physiol., 112:351, 1935. 39. PRINZMETAL, M., SCHWARTZ, L. L., CORDAY, E., SPRITZLER, R., BERGMAN, H. C. and KRUGER, H. E. Studies on the coronary circulation. VI. Loss of myocardial contractility after coronary artery occlusion. Ann. Int. Med., 31:429, 1949. 40. SAYEN, J. J., SHELDON, W. F., PIERCE, G. and Kuo, P. T. Polarographic oxygen; the epicardial electrocardiogram, and muscle contraction in experimental acute regional ischemia of the left ventricle. Circulation Rex, 6:779, 1958. 41. KATZ, A. Personal communication, 1969. 42. SCHKDLER, M. Proportionals Aktivierung von ATPase-Aktivitgt und Kontraktiansspannung THE AMERICAN JOURNAL OF CARDIOLOGY

Myocardial

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Ischemic

(lurch Calciumionen in isoliertcn contractile Strukturen vrrschiedeners Muskelarten. PfliigerA ,41&t. ges. Physiol., 296:70, 1967. SOUUERS. H. M.. WESSEL, H. U. and JENNINGS, R. 13. Myocardial temperature changes in acute esperimental infarction. Lab. Invest., 15:1982, 1966. COXN, L. J., WOOD. J. C. and MORALES, G. G. Rate of change in mrocardial glycogen and lactic acid following arrest of coronary circulation. Gil-ctcIn. tion Rec., i:721, 1959. HARRIS, .%. S., BISTENI, .a., RUSSEI.L, R. A., BRIGHAM, ,I. C. and FIRESTONE, J. E. Excitatory factors in ventricular tachycardia resulting from myocardial ischemia: Potassium a major excitant. Science, 119:200,1954. CHERBAKOFF, A.. T~Y.%x~, S. and HAMILTON, W. F. Relation between coronary sinus potassium and cardiac arrhythmia. Circulation Res., 5:517, 1957. REC.AN, T. J., HAR~IAN, M. A., LEHAN, P. H., BURKE, W. M. and OLDEWURTEL, H. A. Ventricular arrhythmias and K+ transfer during myocardial ischemia and intervention with procaine amide, insulin, or glucose solution. J. Clin. Znvest., 46: 1957. 1967. WARTMAN, W. B. Cardiomyopathy and myocardial degeneration: Problems of terminology in experimental and clinical pathology. Ann. New York Acad. SC., 156:7. IQFQ.

VOLUME

24, DECEMBER 1969

Injury

and Infarction

765

4Q. SOXII~RS, H. XI. and JE\xIU.\. R. 1%. Expel-imental acute rn)ocardial infrll( tio11. Ilistologic and histochemical studies of earl! m\ocardial infarcts produced b! tcmporar\ or per&ur~nt occlusion of a coronal-t arteq. f.nb. Ittwt., 13: 1491, 1964. 50. %UIRE. F. T., BELI., P., CONI.E\ , T. an(I STANDISH. 11. 1.. LIetermination of myocardial alterations at autopsy in the absence of gross and microscopic changes. rl~c/f. Pntil., 81:409, 1966. 51. JF~NIN(;S, R. B.. Hrtmsos, P. B. and SoUnrERs, H. 11. Structural and functional abnormalities in mitochondria isolated from ischemic dog myocardium. Lab. Invest., 20:548, 1969. 52. JE.\NIN(:s, R. B., HERDSON, P. B. and HILL, M. L. P\-ru~ate metabolism in rnitochondria isolated from dog myocardium. Lab. Invest., 20:537, 1969. 53. SHEN, A. C., JENNINGS, R. B., MOORE, C. B. and of mitochondria from OGATA, E. S. Electrolytes autolyring myocardium. Fed. Proc., 28:747, lQ69. 54. K~LTENBACH, J. P. and JENNINGS, R. B. Metabolism of ischernic cardiac muscle. Circu/nfiorl Xe.r., 8: 207. 1960. 55. WEDEI.L, J.. MERKER, H. and R‘ELBERT, I). Mitochondrienstruktur und Atmumgskettenphosphorlierung im Herzmuskel nach \ollstsndiger Kreislaufunterbrechang. ~‘irrho~~s Arc!/. Pnth. Amt., 338:355, 1965.