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Metabolism of the Ischemic Heart
Symposium on Coronary Heart Disease
Metabolism of the Ischemic Heart Radovan Zak, Ph.D.,* and Murray Rabinowitz, M.D.**
Therapy in acute myocardial infarction is directed toward overcoming lifethreatening situations until reparative processes become effective and the patient reaches a new stable state. Treatment and prevention of arrhythmias, improvement of hemodynamic function, and minimization of the metabolic needs of the heart are the principles underlying conservative management of an infarction. A fundamental objective of therapy, in addition to the treatment or prevention of catastrophic arrhythmias, shock, or congestive failure, is to maintain the viability of those regions of the myocardium which have sustained injury that is potentially reversible and minimize the extent of muscle death. In this article we will review some aspects of metabolism, synthesis, and reparative processes of cardiac muscle and their adjustment in ischemia and hypoxia, with the aim of providing a background for the rational treatment of myocardial infarction. The events which follow occlusion of a coronary artery are complex and variable, and depend on the extent of deprivation of the blood supply to cardiac tissues. Regions of the myocardium to which the supply of nutrients and oxygen is grossly inadequate become necrotic. In the injured tissue surrounding the infarcted regions there probably exist a continuous gradient of oxygen tension and degrees of ischemia. Reflecting these variations in oxygen tension, substrate utilization may also vary greatly. The tissue closest to the infarct may depend almost completely on glycolytic energy, whereas in distant, normally perfused tissue, oxidative metabolism may be unaltered, or may even continue at greater than normal rates 4 because of hypertrophy at these sites. A continuous spectrum of differences in metabolism probably exists, especially early From the Departments of Medicine (Cardiology) and Biochemistry, and the Argonne Cancer Research Hospital, University of Chicago, Pritzker School of Medicine, Chicago, Illinois "Associate Professor, Department of Medicine; Research Associate, Department of Biochemistry *"Professor of Medicine and Biochemistry This investigation was supported in part by the Myocardial Infarction Research Unit (Contract PH-43-NHLI-68-1334), Grants HL-9172 and HL-4442 from the National Heart and Lung Institute, United States Public Health Service, and a grant from. the Chicago and Illinois Heart Association. The Argonne Cancer Research Hospital is operated by the University of Chicago for the United States Atomic Energy Commission.
Medical Clinics of North America- Vo!. 57, No. 1, January 1973
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in myocardial ischemia. We will now examine selected areas of myocardial metabolism which have particular bearing on the changes that occur in ischemia and infarction. Several other reviews have been published.3. 42-45. 49
SOME CHARACTERISTICS OF CARDIAC TISSUE METABOLISM The normal heart derives its energy almost exclusively from oxidative metabolism. In contrast with skeletal muscle, in which bursts of activity are supported in part by glycolysis, with breakdown of glycogen and formation of lactic acid, no evidence of lactic acid formation in the normal heart has been obtained, even during periods of greatly enhanced myocardial activity. The adenosine triphosphate (ATP) required for muscle contraction is derived predominantly from mitochondrial oxidation of carbohydrate and lipid substrates. Glucose, free fatty acids, lactate, pyruvate, acetate, and acetoacetate can all be utilized by the intact heart. 3 Acetate, short-chain fatty acids, lactate, pyruvate, and ketones appear to be oxidized in preference to glucose. 43 .49 This depends on the relative concentrations of other available nutrients, and upon the levels of hormones such as insulin which may affect glucose uptake. Although complex relationships for selective substrate uptake by the heart have been postulated, the major principle that emerges from a study of arteriovenous differences is that heart tissue is non-fastidious as to the nutrients it will utilize, and can oxidize almost any substrate available to it. It is also clear that the metabolism of the heart is finely regulated so that uptake and utilization of substrate are closely adjusted to energy utilization. The mechanism of this regulation will be discussed below. Another characteristic of heart muscle metabolism which is especially relevant to the problem of myocardial infarction is that the adult myocardium has a substantial capacity for growth, i.e., for synthesis, accumulation, or repair of its constituents. When work load is acutely increased by such procedures as constriction of the ascending aorta, a 30 to 50 per cent increase in muscle mass may result within 24 to 48 hoursP' 29. 53 The capacity of the heart for the synthesis of protein, nucleic acids, and lipids is therefore substantial. Furthermore, components within the muscle cell such as mitochondria, muscle proteins, and membranes normally turn over rapidly.I6. 37. 51. 52 Cell components are constantly being destroyed and resynthesized, although the cell itself may live as long as the organism. This leads to a plasticity of structure within a cell and allows the cell to adapt to changing conditions and to chronically altered work loads. Although the cardiac muscle cell retains a capacity for substantial repair and growth, one fundamental limitation is present. The adult cardiac muscle cell has apparently lost the ability to divide and multiply.70 Dead cardiac muscle cells in an infarct cannot be replaced by multiplication of other muscle cells but instead are replaced by connective tissue cells. In contrast with skeletal muscle, 55.56 regeneration of the adult cardiac muscle cell does not occur.
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Adjustment of Myocardial Oxidative Metabolism to Energy Used The predominance of aerobic metabolism in the heart is reflected structurally by the large number of mitochondria which are packed between the muscle fibers. Over 35 per cent of the myocardial volume is occupied by mitochondria, as judged by electron microscopyY.48 The considerable potential of cardiac muscle for oxidative metabolism, the very effective mechanisms adjusting the rate of glycolysis to the rate of oxidation, and the regulation of oxidative metabolism by ATP utilization account for the absence of a significant degree of aerobic glycolysis. The ATP formed by oxidative processes is used predominantly to provide energy for cardiac muscle contraction. Other energy-requiring processes, such as ion transport, uptake of Ca++ by the sarcoplasmic reticulum leading to muscle relaxation, and synthesis and maintenance of cellular constituents, constitute only a small fraction (5 to 10 per cent) of cardiac energy needs. The rate of ATP utilization, as reflected by oxygen consumption, is dependent on wall tension and inotropic state of the heart muscle, and to a lesser extent on the degree of shortening of the muscle. 5 Oxygen consumption in the left ventricle increases considerably when aortic pressure is raised, or when intrinsic contractility is increased by catecholamines or by paired stimuli, but only small changes are attributed to changes in stroke output.5 It is apparent that therapeutic maneuvers in myocardial infarction, which seek to increase systemic blood pressure to improve perfusion of ischemic myocardium or which attempt to improve cardiac function by administration of inotropic drugs such as catecholamines, carry with them the danger of increasing myocardial energy requirements. The rate of mitochondrial oxidative metabolism is adapted to energy requirements largely through the mechanism of respiratory control, which has been elucidated by Chance and his colleagues.7 In mitochondria in the intact muscle cell or isolated by suitable methods, electron transport is almost completely dependent on the availability of a phosphate acceptor, in the form of adenosine diphosphate (ADP). Physiologically the ADP is formed from the hydrolysis of ATP during muscle contraction, ion transport, or other energy-requiring processes. Oxidation of substrate by the mitochondria is inhibited if ADP is not available. Thus if ATP utilization is decreased, less ADP is formed and the rate of oxidative metabolism is reduced. In the controlled state (State 4 of Chance/ i.e., inhibition because of nonavailability of ADP), analysis of the oxidation-reduction state of the cytochromes and the pyridine nucleotides indicates that electron transport is inhibited at three sites, presumably the three loci for coupled high energy phosphate synthesis. The addition of ADP, in the presence of adequate amounts of substrate, results in a release in the inhibition of electron transport, and in the synthesis of ATP (State 3 of Chance). By this process the rates of oxidative metabolism and of ATP synthesis are closely adjusted to the rate of ATP utilization. It is of interest that cardiac muscle's capacity of ATP synthesis is far in excess of resting requirements. The heart is capable of supplying ATP needs during the greatest possible physiologic increments of cardiac work. The level of the cardiac mitochondrial functional mass appears to
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be quite sensitive to the chronic level of cardiac work. An increment in cardiac work produced by prolonged exercise or by experimental aortic stenosis results in an increment in mitochondrial functional mass,33,48,50,51,53 so that oxidative capacity is maintained. The mechanism by which synthesis and degradation are controlled by the level of cardiac work is still unclear. Regulation of Glycolysis in Normal and Ischemic Heart Many factors contribute to survival and restoration of myocardial function following a coronary occlusion. Among the most important are: (1) the ability of the ischemic and hypoxic myocardium to alter its normal pattern of oxidative metabolism and utilize, to a limited extent, energy derived from glycolysis; and (2) the remarkable ability of the myocardium to repair organelles and cellular structures that are injured. Normal cardiac function cannot be maintained for more than a brief period of time, perhaps a minute, in the absence of oxygen. The rate of glycogen breakdown and formation of ATP by glycolysis is insufficient to satisfy the energy demands of the normal heart. The energy formed by glycogen breakdown and subsequent glycolysis, however, can help overcome brief periods of complete myocardial ischemia or hypoxia. 69 Glycolytic ATP formation contributes to cell survival when the oxygen supply is restricted but not completely cut off. Just as oxidative metabolism is sensitively adjusted to need by the respiratory control mechanisms, the normal rate of glycolysis is closely controlled by the rate of oxidation, Adjustment of the rate of glycolysis to oxidative rate and metabolic needs is maintained largely through allosteric control of key enzymes of the glycolytic pathway by the level of adenine nucleotides, inorganic phosphate, and perhaps by Krebs cycle intermediates. 4o ,65-67,69 ATP and its breakdown products ADP, AMP, and inorganic phosphate greatly affect the activity of rate-limiting enzymes in the glycolytic cycle, and serve either to stimulate or to inhibit glycolysis. The principal site of control appears to be the enzyme phosphofructokinase. Other secondary control sites may be pyruvate kinase and glyceraldehydephosphate dehydrogenase. Control sites are localized by study of concentrations of glycolytic intermediates under varying metabolic conditions. 40 ,65,66,69 When examined under conditions that inhibit the rate of glycolysis, tissue concentrations of substrates and products of these enzymes are substantially displaced from the equilibrium values determined for these enzymes in vitro.65 Thus the enzymes are either inhibited or present in limiting quantities. After exposure to hypoxia54,66,69 or other experimental devices that stimulate overall glycolytic rates, concentrations of the substrate decline while concentration of the product increases and equilibrium is more closely approached. This reciprocal change in concentration of reactants has been referred to as a cross-over point6.7 and indicates a control site. Details of the control mechanism have also been elucidated by studies of the enzymatic properties of the isolated controlling enzyme, phosphofructokinase.31.32 Enzyme activity is inhibited by ATP and greatly enhanced by ADP, AMP, inorganic phosphate, and the product of the re-
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action 1-6-fructose-diphosphate. Phosphofructokinase activity, therefore, is enhanced during breakdown of ATP and accumulation of ADP, AMP, or inorganic phosphate, paralleling the changes of mitochondrial oxidative activity. In this way the rate of glycolysis is adjusted to provide the proper amount of pyruvate to the oxidative pathways. In the isolated perfused heart,4o.66 or in the intact dog heart,54.69 hypoxia leads to a rapid activation of glycolysis. Hypoxia leads to a rapid fall in creatine-phosphate and a rise in inorganic phosphate levels. ATP levels fall somewhat later in hypoxia, because of the buffering of creatine-phosphate. The enzymes controlling glycolysis, such as phosphofructokinase, are activated by the increased levels of 5' AMP, ADP, inorganic phosphate, and 3' ,5' cyclic AMP and by the decreased concentrations of ATP. Because of the presence of creatine-phosphate, which stabilizes the adenine nucleotide system, changes in inorganic orthophosphate concentration rather than in 5' AMP play the important role in releasing restrictions on glycolysis early in hypoxia. 69 Later, however, the other factors come into play, as well. Phosphofructokinase is also affected by other factors, such as the level of citrate. Citrate inhibits activity of the purified enzyme30.32 as well as inhibiting in vivo. 14 •67 Citrate inhibition of phosphofructokinase may be the mechanism by which glycolysis is inhibited when the heart is subsisting predominantly on nonfermentable substrates such as nonesterified fatty acids, as is the case after fasting, or between meals. Cyclic AMP may also stimulate phosphofructokinase activity in the heart. 31 .32 In this way catecholamines may lead to increased rates of glycolysis independent of accumulation of other stimulating intermediates.
CONTROL OF GLYCOGENOLYSIS IN THE NORMAL AND ISCHEMIC HEART The amount of glycogen present in cardiac tissue is considerably less than that present in skeletal muscleY·42 Glycogen concentration is particularly high in conduction tissue but its function there is still quite unclear. Glycogen breakdown does not appear to provide the normal heart with significant nutrient, even at high levels of cardiac work.3.11.42 During severe hypoxia, however, glycogenolysis proceeds at significant rates. Myocardial activity begins to decline 15 to 30 seconds after complete anaerobiosis, and contraction disappears within 1 to 2 minutes. 6o The nonbeating, arrested heart may survive for considerably longer periods, however, using energy derived from glycogen breakdown and glycosis, since energy utilization is reduced by more than 70 per cent. Glycogenolysis is catalyzed by the enzyme phosphorylase which is present in two forms, a and b, in muscle. 26 . 35. 36 Phosphorylase b is almost inactive under normal conditions, since it requires a high concentration of AMP for activity. Phosphorylase a, however, acts independently of AMP concentration. In normal heart 99 per cent of phosphorylase is in the b form,25.68 which is inactive at normal AMP concentrations. Phosphorylase b is converted to phosphorylase a by the enzyme phosphorylase b kinase. The conversion is accompanied by phosphorylation of phos-
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phorylase b. In turn, phosphorylase b kinase is activated by another enzyme, phosphorylase b kinase kinase. 63 During the earliest stage of hypoxia (15 to 20 seconds) phosphorylase b is rapidly converted to phosphorylase a 25 ,68 probably through catecholamine stimulation of adenyl cyclase.69 This early increase is inhibited by beta-adrenergic blockers. The .cyclic AMP in turn activates phosphorylase b kinase kinase, phosphorylase kinase, and phosphorylase. Within a minute after hypoxia begins, however, the levels of AMP and inorganic phosphate increase significantly, so that phosphorylase b is also maximally active. 35 ,36 The rate of glycogen breakdown at this time is greater than can be accounted for by phosphorylase a concentration. Thus allosteric activation of phosphorylase.b is the more important factor in glycogen breakdown beyond the very earliest period of anoxia.
OTHER METABOLIC EFFECTS OF ISCHEMIA During ischemia or myocardial infarction, an increase of serum free fatty acids has been noted. 27 An association between the level of free fatty acids and increased frequency of arrhythmia has also been reported,41 and the administration of free fatty acids has been reported to increase arrhythmias in the experimental ischemic animal,28 The increase in free fatty acids is probably due to an increase in catecholamine activity during myocardial infarction. Increased free fatty acids may lead to increased oxygen requirement, perhaps by uncoupling oxidative phosphorylation at high free fatty acid levels. Ischemic areas of muscle cannot use free fatty acid because of the tissue hypoxia. Ischemic myocardium was found to extract relatively more glucose and less free fatty acid than controls. 46 As might be expected, hypoxic tissue is more dependent on glycolysis for its energy requirements. It has been suggested that glucose infusion may be useful to maintain the integrity of severely hypoxic areas. Free fatty acids decrease contractility in the isolated rat papillary muscle preparation. 22 These depressant effects were observed only in anaerobic muscle, and were not present in aerobic conditions. Glucose reversed the depressant effects of free fatty acid. The mechanisms underlying these observations are unclear. An increase in hexosemonophosphate-shunt activity of injured heart muscle has been observed both in necrotic areas and in adjacent non-infarcted regions of the left ventricle. 19 ATP and norepinephrine contents fell in non-infarcted as well as in infarcted heart muscle after experimental coronary artery occlusion in the dog. 21 Ischemic regions in which injury is reversible apparently extend considerably beyond the boundary of the infarct.
SYNTHETIC PROCESSES IN CARDIAC MUSCLE Although contractile activity ceases soon after coronary artery occlusion, presumably because of the development of acidosis and ATP depletion, the viability of cardiac muscle may be maintained for as long as an
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hour.23 If circulation is restored before this time, complete functional and structural recovery may ensue. Ischemic tissue that is partially perfused very likely may survive for long periods of time, supported in part by glycolysis, until collateral circulation is established. Hypoxia and acidosis undoubtedly lead to damage to cellular organelles and structures which must be repaired if the cell is to survive. The considerable capacity of heart muscle to repair injured subcellular structures plays a very important role in the maintenance of the viability of tissue surrounding the infarct. It is apparent from studies on amino acid incorporation into proteins following experimental myocardial infarction that repair processes are promptly and maximally stimulated. 20.29 It is only recently that the considerable capacity of the myocardium for synthesis of proteins, nucleic acid, and other macromolecules has been appreciated. Most of our information has come from studies of turnover of myocardial components in the normal heart, and from studies of cardiac growth in hypertrophy stimulated by experimental procedures producing increased pressure or volume work loads upon the heart. Although the cardiac muscle cell is extremely stable, with a lifetime of years or decades, its components turn over quite rapidly. In heart an exponential decay of myosin specific activity has been observed which, when corrected for accumulation of myosin during growth, yields a halflife of about 8 to 11 days.37.50.51 The decay of other muscle proteins, actin, tropomyosin, and troponin appears to be similar to that of myosin. 50 Unit assembly and destruction of the myofibril appears to be likely. Similarly, mitochondrial proteins turn over rapidly, with a half-life of 5 to 6 days in heart.! Several mitochondrial cytochromes, cytochromes b, c, and aa 3 , all turn over with the same half-life,l·lo.52 indicating a synchronous turnover in the inner mitochondrial membrane. The outer mitochondrial membrane and the matrix enzymes, however, turn over more rapidly.9.52 The turnover and synthesis of other myocardial components such as membranes, soluble enzymes or ribosomes have not been extensively studied as yet. The capacity of cardiac muscle for synthesis must be maintained at a relatively high level merely to maintain the integrity of the cell components that are rapidly turning over. The rapid turnover may be viewed as a mechanism that endows the cell with a functional and structural flexibility. An increase or decrease of any cell component can be produced by change in the rates of synthesis or degradation. In case of a severely increased work load, for example, remarkable degrees of synthesis of cardiac muscle components can be attained in a short time. Myocardial proteins are increased by 30 to 50 per cent within 36 or 48 hours after experimental constriction of the ascending aortaY' 39. 53 Protein synthesis is preceded and accompanied by enhanced RNA synthesisY' 24. 39 Nuclear RNA polymerase activity increases very early after increasing cardiac work load. 39 Mitochondria appear to be synthesized preferentially during the earliest periods of acute pressure-induced hypertrophy, and, proportional to other cell components later. 51 After prolonged hypertrophy, mitochondrial mass may decline. 34 Myofibrillar proteins also are rapidly synthesized. The accumulation of mitochondrial and perhaps myofibrillar
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proteins appears to be secondary to decreased rate of destruction as well as to increased synthesis during hypertrophy. Similarly, mitochondrial accumulation in hearts of hypothyroid rats treated with thyroid hormone is also due to decreased destruction as well as to increased synthesis. 15 Thus accumulation of myocardial elements can be the result of alteration in either the rate of synthesis or of degradation, or both processes. Myocardial ischemia stimulates myocardial repair processes. 20 Increased rates of incorporation of labeled amino acid into myocardial proteins in regions surrounding the infarction have been observed. Reparative processes apparently commence rapidly in the injured myocardium, and surrounding normal muscle may hypertrophy to compensate for the decreased efficiency produced by the necrotic and fibrotic tissue. Exposure of rats to a 6 hour period of 4 per cent oxygen results in selective destruction of mitochondria within the viable myocardial cell. Myosin and membrane proteins appear to be less susceptible than mitochondria to the destructive effect of low oxygen tensions. After return of the animals to normal oxygen tension a rapid resynthesis of the mitochondria destroyed by hypoxia ensues. ABSENCE OF REGENERATION IN ADULT CARDIAC MUSCLE One of the most important features of cardiac muscle relevant to myocardial infarction is that the destroyed muscle cells are not replaced. This contrasts with the situation in skeletal muscle, in which complete regeneration is observed after necrosis secondary to intense freezing by liquid nitrogen. 55, 56 In skeletal muscle, primitive mononuclear cells reappear, divide, fuse and differentiate into mature muscle cells. The myoblasts are thought to originate either from nuclei of injured muscle cells which discard their cytoplasm and de-differentiate in mononuclear cells,55,56 or from "satellite" cells which may be regarded as primitive myoblasts reJained in the adult tissue. Undifferentiated myoblasts have been described in cardiac muscle cells of young rats. 6I Weinstein and Hay, however, dispute their existence,64 suggesting that cardiac mitotic cell division normally occurs in differentiated cells. No substantiated example of true regeneration of adult ventricular myocardial cells has been reported in higher organisms, but regeneration has been described in hearts of very young animals. 57 In the adult myocardium, destroyed muscle cells are replaced by connective tissue cells that retain their capacity for cell multiplication. The altered metabolism of the infarcted areas may reflect the metabolism of replacing connective tissue rather than that of regenerating muscle. It has been reported that following coronary ligation in the rat, DNA synthesis and mitosis are stimulated in atrial but not in ventricular muscle cells. 58 Apparently the proliferative activity of ventricular muscle is more strongly inhibited than that of atrial muscle cells in the adult heart. The adult ventricular myocardium probably does not irreversibly lose its potential for DNA synthesis and mitosis, but conditions for stimulation have not as yet been found. It is also of note that the amphibian ventricular myocardium retains its ability to regenerate. 59
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The limitation of DNA synthesis in the adult heart muscle is also apparent in studies of cardiac hypertrophy. Large increases in cardiac DNA have been observed after experimental aortic constriction, but further analysis shows that this increase is primarily due to connective tissue hyperplasia,8, 17, 18,33,38 and to a very small extent to an increased frequency of polyploidy in the muscle cellsP There is no increase in the number of muscle cells, and mitosis in muscle cells is rarely observed. Cardiac enlargement produced by anemia or aortic banding in the newborn rat, however, results in a true hyperplasia, with increased numbers of muscle cells.62 The processes controlling and restricting muscle cell proliferation and DNA synthesis are now under study in many laboratories. It is not unlikely that when they become better understood, methods will be developed to reinitiate muscle cell division in the adult heart. It may then be possible to replace infarcted tissue with a functioning contractile myocardium.
REFERENCES 1. Aschenbrenner, v., Druyan, R., Albin, R., and Rabinowitz, M.: Heme a, cytochrome c and total protein turnover in mitochondria from rat heart and liver. Biochem. J., 119:157, 1970. 2. Aschenbrenner, V., Zak, R., Cutilletta, A. F., and Rabinowitz, M.: Effect of hypoxia on degradation of mitochondrial components in rat cardiac muscle. Amer. J. Physiol., 221 :1418, 1971. 3. Bing, R. J.: Cardiac metabolism. Physiol. Rev., 45:171, 1965. 4. Braasch, W., Gudbjarnason, S., Puri, P. S., et al.: Early changes in energy metabolism in the myocardium follOwing acute coronary artery occlusion in anesthetized dogs. Circ. Res., 23:429, 1968. 5. Braunwald, E.: The determinants of myocardial oxygen consumption. The Physiologist, 12:65,1969. 6. Chance, B., Holmes, W., and Higgins, J.: Localization of interaction sites in multi-component transfer systems: Theorem derived from analogues. Nature, 182:1190, 1958. 7. Chance, B., and Williams, G. R.: The respiratory chain and oxidative phosphorylation. In Nord, F. F., ed.: Advances in Enzymology. New York, Interscience Publishers, Inc., 17:65, 1955. 8. Crane, W. A. J., and Dutta, L. P.: Utilization of tritiated thymidine for deoxyribonucleic acid synthesis by the lesion of experimental hypertension in rat. J. Pathol. Bacteriol., 86:83, 1963. 9. DeBernard, B., Getz, G. S., and Rabinowitz, M.: The turnover of the protein of the inner and outer mitochondrial membrane of rat liver. Biochim. Biophys. Acta, 193:58, 1969. 10. Druyan, R., DeBernard, D., and Rabinowitz, M.: Turnover of cytochromes labeled with 1)aminolevulinic acid-3 H in rat liver. J. BioI. Chem., 244:5874,1969. 11. Evans, G.: The glycogen content of the rat heart. J. Physiol., 82:468, 1934. 12. Fanburg, B. L.: Experimental cardiac hypertrophy. New Eng. J. Med., 282:723,1970. 13. Fanburg, B. L., and Posner, B. I.: Ribonucleic acid synthesis in experimental cardiac hypertrophy in rats. I. Characterization and kinetics oflabeling. Circ. Res., 23: 123,1968. 14. Garland, P. B., Randle, P. J., and Newsholme, E. A.: Citrate as an intermediary in the inhibition of phosphofructokinase in rat heart muscle by fatty acids, ketone bodies, pyruvate, diabetes and starvation. Nature, 200:169, 1963. 15. Gross, N. J.: Control of mitochondrial turnover under the influence of thyroid hormone. J. Cell BioI., 48:29, 1971. 16. Gross, N. J., Getz, G. S., and Rabinowitz, M.: Apparent turnover of mitochondrial deoxyribonucleic acid and mitochondrial phospholipids in the tissues of the rat. J. BioI. Chem., 244:1552, 1969. 17. Grove, D., Nair, K. G., and Zak, R.: Biochemical correlates of cardiac hypertrophy. Ill. Changes in DNA content; the relative contributions of polyploidy and mitotic activity. Circ. Res., 25:463, 1969. 18. Grove, D., Zak, R., Nair, K. G., and Aschenbrenner, V.: Biochemical correlates of cardiac
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47. Page, E., Polimeni, P. I., Zak, R, et al.: Myofibrillar mass in rat and rabbit muscle. Correlation of microchemical with steriological measurements in normal and hypertrophied heart. Circ. Res., 30:430, 1972. 48. Poche, R, Moraes de Mello Mattos, C., Rebarz, H. W., and Stoepel, K.: Uber das Verhaltnis Mitochondrien: Myofibrillen in den Herzmuskelzellen der Ratte bei Druckhypertrophie des Herzens. Virchows Arch. Path. Anat., 344:100, 1968. 49. Rabinowitz, M.: Control of metabolism and synthesis of macromolecules in normal and ischemic heart. J. Mol. Cell. CardioI., 2:277, 1971. 50. Rabinowitz, M.: Protein synthesis and turnover in normal and hypertrophied heart. Amer. J. CardioI., 1972, in press. 51. Rabinowitz, M., Aschenbrenner, V., Albin, R, et al.: Synthesis and turnover of heart mitochondria in normal, hypertrophied and hypoxic rat. In Alpert, N. R, ed.: Cardiac Hypertrophy. New York, Academic Press, 1971, p. 283. 52. Rabinowitz, M., and Swift, H.: Mitochondrial nucleic acids and their relation to the biogenesis of mitochondria. PhysioI. Rev., 50:376, 1970. 53. Rabinowitz, M., and Zak, R: Biochemical and cellular changes in cardiac hypertrophy. Ann. Rev. Med., 23:245,1972. 54. Regan, D. M., Davis, W. W., Morgan, H. E., and Park, C. R: The regulation of hexokinase and phosphofructokinase activity in heart muscle. Effects of alloxan diabetes, growth hormone, cortisol, and anoxia. J. BioI. Chem., 239:43, 1964. 55. Reznik, M.: Origin of myoblasts during skeletal muscle regeneration. Electron microscopic observations. Lab. Invest., 20:353, 1969. 56. Reznik, M.: Thymidine-H' uptake by satellite cells of regenerating skeletal muscle. J. Cell BioI., 40:568, 1969. 57. Robledo, M.: Myocardial regeneration in young rats. Amer. J. Pathol., 32:1215,1956. 58. Rumyantsev, P. P., and Mirakjan, V. 0.: Reactive synthesis of DNA and mitotic division in atrial heart muscle cells following ventricle infarction. Experientia, 24:1234, 1968. 59. Rumyantsev, P. P., and Shmantsar, I. A.: SubmicroscopiC patterns of reactive changes in frog heart muscle cells during regeneration. Cytologia (Leningrad), 10: 1234, 1968. 60. Sayen, J. J., Sheldon, W. F., Peirce, G., and Kuo, P. T.: Polarographic oxygen, the epicardial electrocardiogram and muscle contraction in experimental acute regional ischemia of the left ventricle. Circ. Res., 6:779, 1958. 61. Shafiq, S. A., Gorycki, M. A., and Mauro, A.: Mitosis during postnatal growth in skeletal and cardiac muscle of the rat. J. Anat., 103:135, 1968. 62. Wachtlova, M., and Rakusan, K.: Different types of cardiomegaly and its blood supply. Folia Facultatis Medicae Universitatis Comenianae Bratislaviensis, 1972, in press. 63. Walsh, D. S., Perkins, J. P., and Krebs, E. G.: An adenosine 3',5'-monophosphate-dependent protein kinase from rabbit skeletal muscle. J. BioI. Chem., 243:3763, 1968. 64. Weinstein, R B., and Hay, E. D.: Deoxyribonucleic acid synthesis and mitosis in differentiated cardiac muscle cells of chick embryos. J. Cell BioI., 47:310, 1970. 65. Williamson, J. R.: Glycolytic control mechanisms. I. Inhibition of glycolysis by acetate and pyruvate in the isolated, perfused rat heart. J. BioI. Chem., 240:2308, 1965. 66. Williamson, J. R: Glycolytic control mechanisms. 11. Kinetics of intermediate changes during the ~erobic-anoxic transition in perfused rat heart. J. BioI. Chem., 241 :5026, 1966. 67. Williamson, J. R: Glycolytic control mechanisms. Ill. Effects of iodoacetamide and fluoracetate on glucose metabolism in the perfused rat heart. J. BioI. Chem., 242:4476, 1967. 68. Wollenberger, A., and Krause, E. G.: Activation of a-glucan phosphorylase and related metabolic changes in dog myocardium following arrest of blood flow. Biochim. Biophys. Acta, 67:337, 1963. 69. Wollenberger, A., and Krause, E. G.: Metabolic control characteristics of the acutely ischemic myocardium. Amer. J. CardioI., 22:349, 1968. 70. Zak, R: Cell proliferation during cardiac growth. Amer. J. CardioI., 1972, in press. Department of Medicine, Box 407 Pritzker School of Medicine University of Chicago 950 East 59th Street Chicago, Illinois 60637 (Dr. Rabinowitz)
Metabolism of the Ischemic Heart Radovan Zak, Ph.D.,* and Murray Rabinowitz, M.D.**
Therapy in acute myocardial infarction is directed toward overcoming lifethreatening situations until reparative processes become effective and the patient reaches a new stable state. Treatment and prevention of arrhythmias, improvement of hemodynamic function, and minimization of the metabolic needs of the heart are the principles underlying conservative management of an infarction. A fundamental objective of therapy, in addition to the treatment or prevention of catastrophic arrhythmias, shock, or congestive failure, is to maintain the viability of those regions of the myocardium which have sustained injury that is potentially reversible and minimize the extent of muscle death. In this article we will review some aspects of metabolism, synthesis, and reparative processes of cardiac muscle and their adjustment in ischemia and hypoxia, with the aim of providing a background for the rational treatment of myocardial infarction. The events which follow occlusion of a coronary artery are complex and variable, and depend on the extent of deprivation of the blood supply to cardiac tissues. Regions of the myocardium to which the supply of nutrients and oxygen is grossly inadequate become necrotic. In the injured tissue surrounding the infarcted regions there probably exist a continuous gradient of oxygen tension and degrees of ischemia. Reflecting these variations in oxygen tension, substrate utilization may also vary greatly. The tissue closest to the infarct may depend almost completely on glycolytic energy, whereas in distant, normally perfused tissue, oxidative metabolism may be unaltered, or may even continue at greater than normal rates 4 because of hypertrophy at these sites. A continuous spectrum of differences in metabolism probably exists, especially early From the Departments of Medicine (Cardiology) and Biochemistry, and the Argonne Cancer Research Hospital, University of Chicago, Pritzker School of Medicine, Chicago, Illinois "Associate Professor, Department of Medicine; Research Associate, Department of Biochemistry *"Professor of Medicine and Biochemistry This investigation was supported in part by the Myocardial Infarction Research Unit (Contract PH-43-NHLI-68-1334), Grants HL-9172 and HL-4442 from the National Heart and Lung Institute, United States Public Health Service, and a grant from. the Chicago and Illinois Heart Association. The Argonne Cancer Research Hospital is operated by the University of Chicago for the United States Atomic Energy Commission.
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in myocardial ischemia. We will now examine selected areas of myocardial metabolism which have particular bearing on the changes that occur in ischemia and infarction. Several other reviews have been published.3. 42-45. 49
SOME CHARACTERISTICS OF CARDIAC TISSUE METABOLISM The normal heart derives its energy almost exclusively from oxidative metabolism. In contrast with skeletal muscle, in which bursts of activity are supported in part by glycolysis, with breakdown of glycogen and formation of lactic acid, no evidence of lactic acid formation in the normal heart has been obtained, even during periods of greatly enhanced myocardial activity. The adenosine triphosphate (ATP) required for muscle contraction is derived predominantly from mitochondrial oxidation of carbohydrate and lipid substrates. Glucose, free fatty acids, lactate, pyruvate, acetate, and acetoacetate can all be utilized by the intact heart. 3 Acetate, short-chain fatty acids, lactate, pyruvate, and ketones appear to be oxidized in preference to glucose. 43 .49 This depends on the relative concentrations of other available nutrients, and upon the levels of hormones such as insulin which may affect glucose uptake. Although complex relationships for selective substrate uptake by the heart have been postulated, the major principle that emerges from a study of arteriovenous differences is that heart tissue is non-fastidious as to the nutrients it will utilize, and can oxidize almost any substrate available to it. It is also clear that the metabolism of the heart is finely regulated so that uptake and utilization of substrate are closely adjusted to energy utilization. The mechanism of this regulation will be discussed below. Another characteristic of heart muscle metabolism which is especially relevant to the problem of myocardial infarction is that the adult myocardium has a substantial capacity for growth, i.e., for synthesis, accumulation, or repair of its constituents. When work load is acutely increased by such procedures as constriction of the ascending aorta, a 30 to 50 per cent increase in muscle mass may result within 24 to 48 hoursP' 29. 53 The capacity of the heart for the synthesis of protein, nucleic acids, and lipids is therefore substantial. Furthermore, components within the muscle cell such as mitochondria, muscle proteins, and membranes normally turn over rapidly.I6. 37. 51. 52 Cell components are constantly being destroyed and resynthesized, although the cell itself may live as long as the organism. This leads to a plasticity of structure within a cell and allows the cell to adapt to changing conditions and to chronically altered work loads. Although the cardiac muscle cell retains a capacity for substantial repair and growth, one fundamental limitation is present. The adult cardiac muscle cell has apparently lost the ability to divide and multiply.70 Dead cardiac muscle cells in an infarct cannot be replaced by multiplication of other muscle cells but instead are replaced by connective tissue cells. In contrast with skeletal muscle, 55.56 regeneration of the adult cardiac muscle cell does not occur.
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Adjustment of Myocardial Oxidative Metabolism to Energy Used The predominance of aerobic metabolism in the heart is reflected structurally by the large number of mitochondria which are packed between the muscle fibers. Over 35 per cent of the myocardial volume is occupied by mitochondria, as judged by electron microscopyY.48 The considerable potential of cardiac muscle for oxidative metabolism, the very effective mechanisms adjusting the rate of glycolysis to the rate of oxidation, and the regulation of oxidative metabolism by ATP utilization account for the absence of a significant degree of aerobic glycolysis. The ATP formed by oxidative processes is used predominantly to provide energy for cardiac muscle contraction. Other energy-requiring processes, such as ion transport, uptake of Ca++ by the sarcoplasmic reticulum leading to muscle relaxation, and synthesis and maintenance of cellular constituents, constitute only a small fraction (5 to 10 per cent) of cardiac energy needs. The rate of ATP utilization, as reflected by oxygen consumption, is dependent on wall tension and inotropic state of the heart muscle, and to a lesser extent on the degree of shortening of the muscle. 5 Oxygen consumption in the left ventricle increases considerably when aortic pressure is raised, or when intrinsic contractility is increased by catecholamines or by paired stimuli, but only small changes are attributed to changes in stroke output.5 It is apparent that therapeutic maneuvers in myocardial infarction, which seek to increase systemic blood pressure to improve perfusion of ischemic myocardium or which attempt to improve cardiac function by administration of inotropic drugs such as catecholamines, carry with them the danger of increasing myocardial energy requirements. The rate of mitochondrial oxidative metabolism is adapted to energy requirements largely through the mechanism of respiratory control, which has been elucidated by Chance and his colleagues.7 In mitochondria in the intact muscle cell or isolated by suitable methods, electron transport is almost completely dependent on the availability of a phosphate acceptor, in the form of adenosine diphosphate (ADP). Physiologically the ADP is formed from the hydrolysis of ATP during muscle contraction, ion transport, or other energy-requiring processes. Oxidation of substrate by the mitochondria is inhibited if ADP is not available. Thus if ATP utilization is decreased, less ADP is formed and the rate of oxidative metabolism is reduced. In the controlled state (State 4 of Chance/ i.e., inhibition because of nonavailability of ADP), analysis of the oxidation-reduction state of the cytochromes and the pyridine nucleotides indicates that electron transport is inhibited at three sites, presumably the three loci for coupled high energy phosphate synthesis. The addition of ADP, in the presence of adequate amounts of substrate, results in a release in the inhibition of electron transport, and in the synthesis of ATP (State 3 of Chance). By this process the rates of oxidative metabolism and of ATP synthesis are closely adjusted to the rate of ATP utilization. It is of interest that cardiac muscle's capacity of ATP synthesis is far in excess of resting requirements. The heart is capable of supplying ATP needs during the greatest possible physiologic increments of cardiac work. The level of the cardiac mitochondrial functional mass appears to
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be quite sensitive to the chronic level of cardiac work. An increment in cardiac work produced by prolonged exercise or by experimental aortic stenosis results in an increment in mitochondrial functional mass,33,48,50,51,53 so that oxidative capacity is maintained. The mechanism by which synthesis and degradation are controlled by the level of cardiac work is still unclear. Regulation of Glycolysis in Normal and Ischemic Heart Many factors contribute to survival and restoration of myocardial function following a coronary occlusion. Among the most important are: (1) the ability of the ischemic and hypoxic myocardium to alter its normal pattern of oxidative metabolism and utilize, to a limited extent, energy derived from glycolysis; and (2) the remarkable ability of the myocardium to repair organelles and cellular structures that are injured. Normal cardiac function cannot be maintained for more than a brief period of time, perhaps a minute, in the absence of oxygen. The rate of glycogen breakdown and formation of ATP by glycolysis is insufficient to satisfy the energy demands of the normal heart. The energy formed by glycogen breakdown and subsequent glycolysis, however, can help overcome brief periods of complete myocardial ischemia or hypoxia. 69 Glycolytic ATP formation contributes to cell survival when the oxygen supply is restricted but not completely cut off. Just as oxidative metabolism is sensitively adjusted to need by the respiratory control mechanisms, the normal rate of glycolysis is closely controlled by the rate of oxidation, Adjustment of the rate of glycolysis to oxidative rate and metabolic needs is maintained largely through allosteric control of key enzymes of the glycolytic pathway by the level of adenine nucleotides, inorganic phosphate, and perhaps by Krebs cycle intermediates. 4o ,65-67,69 ATP and its breakdown products ADP, AMP, and inorganic phosphate greatly affect the activity of rate-limiting enzymes in the glycolytic cycle, and serve either to stimulate or to inhibit glycolysis. The principal site of control appears to be the enzyme phosphofructokinase. Other secondary control sites may be pyruvate kinase and glyceraldehydephosphate dehydrogenase. Control sites are localized by study of concentrations of glycolytic intermediates under varying metabolic conditions. 40 ,65,66,69 When examined under conditions that inhibit the rate of glycolysis, tissue concentrations of substrates and products of these enzymes are substantially displaced from the equilibrium values determined for these enzymes in vitro.65 Thus the enzymes are either inhibited or present in limiting quantities. After exposure to hypoxia54,66,69 or other experimental devices that stimulate overall glycolytic rates, concentrations of the substrate decline while concentration of the product increases and equilibrium is more closely approached. This reciprocal change in concentration of reactants has been referred to as a cross-over point6.7 and indicates a control site. Details of the control mechanism have also been elucidated by studies of the enzymatic properties of the isolated controlling enzyme, phosphofructokinase.31.32 Enzyme activity is inhibited by ATP and greatly enhanced by ADP, AMP, inorganic phosphate, and the product of the re-
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action 1-6-fructose-diphosphate. Phosphofructokinase activity, therefore, is enhanced during breakdown of ATP and accumulation of ADP, AMP, or inorganic phosphate, paralleling the changes of mitochondrial oxidative activity. In this way the rate of glycolysis is adjusted to provide the proper amount of pyruvate to the oxidative pathways. In the isolated perfused heart,4o.66 or in the intact dog heart,54.69 hypoxia leads to a rapid activation of glycolysis. Hypoxia leads to a rapid fall in creatine-phosphate and a rise in inorganic phosphate levels. ATP levels fall somewhat later in hypoxia, because of the buffering of creatine-phosphate. The enzymes controlling glycolysis, such as phosphofructokinase, are activated by the increased levels of 5' AMP, ADP, inorganic phosphate, and 3' ,5' cyclic AMP and by the decreased concentrations of ATP. Because of the presence of creatine-phosphate, which stabilizes the adenine nucleotide system, changes in inorganic orthophosphate concentration rather than in 5' AMP play the important role in releasing restrictions on glycolysis early in hypoxia. 69 Later, however, the other factors come into play, as well. Phosphofructokinase is also affected by other factors, such as the level of citrate. Citrate inhibits activity of the purified enzyme30.32 as well as inhibiting in vivo. 14 •67 Citrate inhibition of phosphofructokinase may be the mechanism by which glycolysis is inhibited when the heart is subsisting predominantly on nonfermentable substrates such as nonesterified fatty acids, as is the case after fasting, or between meals. Cyclic AMP may also stimulate phosphofructokinase activity in the heart. 31 .32 In this way catecholamines may lead to increased rates of glycolysis independent of accumulation of other stimulating intermediates.
CONTROL OF GLYCOGENOLYSIS IN THE NORMAL AND ISCHEMIC HEART The amount of glycogen present in cardiac tissue is considerably less than that present in skeletal muscleY·42 Glycogen concentration is particularly high in conduction tissue but its function there is still quite unclear. Glycogen breakdown does not appear to provide the normal heart with significant nutrient, even at high levels of cardiac work.3.11.42 During severe hypoxia, however, glycogenolysis proceeds at significant rates. Myocardial activity begins to decline 15 to 30 seconds after complete anaerobiosis, and contraction disappears within 1 to 2 minutes. 6o The nonbeating, arrested heart may survive for considerably longer periods, however, using energy derived from glycogen breakdown and glycosis, since energy utilization is reduced by more than 70 per cent. Glycogenolysis is catalyzed by the enzyme phosphorylase which is present in two forms, a and b, in muscle. 26 . 35. 36 Phosphorylase b is almost inactive under normal conditions, since it requires a high concentration of AMP for activity. Phosphorylase a, however, acts independently of AMP concentration. In normal heart 99 per cent of phosphorylase is in the b form,25.68 which is inactive at normal AMP concentrations. Phosphorylase b is converted to phosphorylase a by the enzyme phosphorylase b kinase. The conversion is accompanied by phosphorylation of phos-
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phorylase b. In turn, phosphorylase b kinase is activated by another enzyme, phosphorylase b kinase kinase. 63 During the earliest stage of hypoxia (15 to 20 seconds) phosphorylase b is rapidly converted to phosphorylase a 25 ,68 probably through catecholamine stimulation of adenyl cyclase.69 This early increase is inhibited by beta-adrenergic blockers. The .cyclic AMP in turn activates phosphorylase b kinase kinase, phosphorylase kinase, and phosphorylase. Within a minute after hypoxia begins, however, the levels of AMP and inorganic phosphate increase significantly, so that phosphorylase b is also maximally active. 35 ,36 The rate of glycogen breakdown at this time is greater than can be accounted for by phosphorylase a concentration. Thus allosteric activation of phosphorylase.b is the more important factor in glycogen breakdown beyond the very earliest period of anoxia.
OTHER METABOLIC EFFECTS OF ISCHEMIA During ischemia or myocardial infarction, an increase of serum free fatty acids has been noted. 27 An association between the level of free fatty acids and increased frequency of arrhythmia has also been reported,41 and the administration of free fatty acids has been reported to increase arrhythmias in the experimental ischemic animal,28 The increase in free fatty acids is probably due to an increase in catecholamine activity during myocardial infarction. Increased free fatty acids may lead to increased oxygen requirement, perhaps by uncoupling oxidative phosphorylation at high free fatty acid levels. Ischemic areas of muscle cannot use free fatty acid because of the tissue hypoxia. Ischemic myocardium was found to extract relatively more glucose and less free fatty acid than controls. 46 As might be expected, hypoxic tissue is more dependent on glycolysis for its energy requirements. It has been suggested that glucose infusion may be useful to maintain the integrity of severely hypoxic areas. Free fatty acids decrease contractility in the isolated rat papillary muscle preparation. 22 These depressant effects were observed only in anaerobic muscle, and were not present in aerobic conditions. Glucose reversed the depressant effects of free fatty acid. The mechanisms underlying these observations are unclear. An increase in hexosemonophosphate-shunt activity of injured heart muscle has been observed both in necrotic areas and in adjacent non-infarcted regions of the left ventricle. 19 ATP and norepinephrine contents fell in non-infarcted as well as in infarcted heart muscle after experimental coronary artery occlusion in the dog. 21 Ischemic regions in which injury is reversible apparently extend considerably beyond the boundary of the infarct.
SYNTHETIC PROCESSES IN CARDIAC MUSCLE Although contractile activity ceases soon after coronary artery occlusion, presumably because of the development of acidosis and ATP depletion, the viability of cardiac muscle may be maintained for as long as an
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hour.23 If circulation is restored before this time, complete functional and structural recovery may ensue. Ischemic tissue that is partially perfused very likely may survive for long periods of time, supported in part by glycolysis, until collateral circulation is established. Hypoxia and acidosis undoubtedly lead to damage to cellular organelles and structures which must be repaired if the cell is to survive. The considerable capacity of heart muscle to repair injured subcellular structures plays a very important role in the maintenance of the viability of tissue surrounding the infarct. It is apparent from studies on amino acid incorporation into proteins following experimental myocardial infarction that repair processes are promptly and maximally stimulated. 20.29 It is only recently that the considerable capacity of the myocardium for synthesis of proteins, nucleic acid, and other macromolecules has been appreciated. Most of our information has come from studies of turnover of myocardial components in the normal heart, and from studies of cardiac growth in hypertrophy stimulated by experimental procedures producing increased pressure or volume work loads upon the heart. Although the cardiac muscle cell is extremely stable, with a lifetime of years or decades, its components turn over quite rapidly. In heart an exponential decay of myosin specific activity has been observed which, when corrected for accumulation of myosin during growth, yields a halflife of about 8 to 11 days.37.50.51 The decay of other muscle proteins, actin, tropomyosin, and troponin appears to be similar to that of myosin. 50 Unit assembly and destruction of the myofibril appears to be likely. Similarly, mitochondrial proteins turn over rapidly, with a half-life of 5 to 6 days in heart.! Several mitochondrial cytochromes, cytochromes b, c, and aa 3 , all turn over with the same half-life,l·lo.52 indicating a synchronous turnover in the inner mitochondrial membrane. The outer mitochondrial membrane and the matrix enzymes, however, turn over more rapidly.9.52 The turnover and synthesis of other myocardial components such as membranes, soluble enzymes or ribosomes have not been extensively studied as yet. The capacity of cardiac muscle for synthesis must be maintained at a relatively high level merely to maintain the integrity of the cell components that are rapidly turning over. The rapid turnover may be viewed as a mechanism that endows the cell with a functional and structural flexibility. An increase or decrease of any cell component can be produced by change in the rates of synthesis or degradation. In case of a severely increased work load, for example, remarkable degrees of synthesis of cardiac muscle components can be attained in a short time. Myocardial proteins are increased by 30 to 50 per cent within 36 or 48 hours after experimental constriction of the ascending aortaY' 39. 53 Protein synthesis is preceded and accompanied by enhanced RNA synthesisY' 24. 39 Nuclear RNA polymerase activity increases very early after increasing cardiac work load. 39 Mitochondria appear to be synthesized preferentially during the earliest periods of acute pressure-induced hypertrophy, and, proportional to other cell components later. 51 After prolonged hypertrophy, mitochondrial mass may decline. 34 Myofibrillar proteins also are rapidly synthesized. The accumulation of mitochondrial and perhaps myofibrillar
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proteins appears to be secondary to decreased rate of destruction as well as to increased synthesis during hypertrophy. Similarly, mitochondrial accumulation in hearts of hypothyroid rats treated with thyroid hormone is also due to decreased destruction as well as to increased synthesis. 15 Thus accumulation of myocardial elements can be the result of alteration in either the rate of synthesis or of degradation, or both processes. Myocardial ischemia stimulates myocardial repair processes. 20 Increased rates of incorporation of labeled amino acid into myocardial proteins in regions surrounding the infarction have been observed. Reparative processes apparently commence rapidly in the injured myocardium, and surrounding normal muscle may hypertrophy to compensate for the decreased efficiency produced by the necrotic and fibrotic tissue. Exposure of rats to a 6 hour period of 4 per cent oxygen results in selective destruction of mitochondria within the viable myocardial cell. Myosin and membrane proteins appear to be less susceptible than mitochondria to the destructive effect of low oxygen tensions. After return of the animals to normal oxygen tension a rapid resynthesis of the mitochondria destroyed by hypoxia ensues. ABSENCE OF REGENERATION IN ADULT CARDIAC MUSCLE One of the most important features of cardiac muscle relevant to myocardial infarction is that the destroyed muscle cells are not replaced. This contrasts with the situation in skeletal muscle, in which complete regeneration is observed after necrosis secondary to intense freezing by liquid nitrogen. 55, 56 In skeletal muscle, primitive mononuclear cells reappear, divide, fuse and differentiate into mature muscle cells. The myoblasts are thought to originate either from nuclei of injured muscle cells which discard their cytoplasm and de-differentiate in mononuclear cells,55,56 or from "satellite" cells which may be regarded as primitive myoblasts reJained in the adult tissue. Undifferentiated myoblasts have been described in cardiac muscle cells of young rats. 6I Weinstein and Hay, however, dispute their existence,64 suggesting that cardiac mitotic cell division normally occurs in differentiated cells. No substantiated example of true regeneration of adult ventricular myocardial cells has been reported in higher organisms, but regeneration has been described in hearts of very young animals. 57 In the adult myocardium, destroyed muscle cells are replaced by connective tissue cells that retain their capacity for cell multiplication. The altered metabolism of the infarcted areas may reflect the metabolism of replacing connective tissue rather than that of regenerating muscle. It has been reported that following coronary ligation in the rat, DNA synthesis and mitosis are stimulated in atrial but not in ventricular muscle cells. 58 Apparently the proliferative activity of ventricular muscle is more strongly inhibited than that of atrial muscle cells in the adult heart. The adult ventricular myocardium probably does not irreversibly lose its potential for DNA synthesis and mitosis, but conditions for stimulation have not as yet been found. It is also of note that the amphibian ventricular myocardium retains its ability to regenerate. 59
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The limitation of DNA synthesis in the adult heart muscle is also apparent in studies of cardiac hypertrophy. Large increases in cardiac DNA have been observed after experimental aortic constriction, but further analysis shows that this increase is primarily due to connective tissue hyperplasia,8, 17, 18,33,38 and to a very small extent to an increased frequency of polyploidy in the muscle cellsP There is no increase in the number of muscle cells, and mitosis in muscle cells is rarely observed. Cardiac enlargement produced by anemia or aortic banding in the newborn rat, however, results in a true hyperplasia, with increased numbers of muscle cells.62 The processes controlling and restricting muscle cell proliferation and DNA synthesis are now under study in many laboratories. It is not unlikely that when they become better understood, methods will be developed to reinitiate muscle cell division in the adult heart. It may then be possible to replace infarcted tissue with a functioning contractile myocardium.
REFERENCES 1. Aschenbrenner, v., Druyan, R., Albin, R., and Rabinowitz, M.: Heme a, cytochrome c and total protein turnover in mitochondria from rat heart and liver. Biochem. J., 119:157, 1970. 2. Aschenbrenner, V., Zak, R., Cutilletta, A. F., and Rabinowitz, M.: Effect of hypoxia on degradation of mitochondrial components in rat cardiac muscle. Amer. J. Physiol., 221 :1418, 1971. 3. Bing, R. J.: Cardiac metabolism. Physiol. Rev., 45:171, 1965. 4. Braasch, W., Gudbjarnason, S., Puri, P. S., et al.: Early changes in energy metabolism in the myocardium follOwing acute coronary artery occlusion in anesthetized dogs. Circ. Res., 23:429, 1968. 5. Braunwald, E.: The determinants of myocardial oxygen consumption. The Physiologist, 12:65,1969. 6. Chance, B., Holmes, W., and Higgins, J.: Localization of interaction sites in multi-component transfer systems: Theorem derived from analogues. Nature, 182:1190, 1958. 7. Chance, B., and Williams, G. R.: The respiratory chain and oxidative phosphorylation. In Nord, F. F., ed.: Advances in Enzymology. New York, Interscience Publishers, Inc., 17:65, 1955. 8. Crane, W. A. J., and Dutta, L. P.: Utilization of tritiated thymidine for deoxyribonucleic acid synthesis by the lesion of experimental hypertension in rat. J. Pathol. Bacteriol., 86:83, 1963. 9. DeBernard, B., Getz, G. S., and Rabinowitz, M.: The turnover of the protein of the inner and outer mitochondrial membrane of rat liver. Biochim. Biophys. Acta, 193:58, 1969. 10. Druyan, R., DeBernard, D., and Rabinowitz, M.: Turnover of cytochromes labeled with 1)aminolevulinic acid-3 H in rat liver. J. BioI. Chem., 244:5874,1969. 11. Evans, G.: The glycogen content of the rat heart. J. Physiol., 82:468, 1934. 12. Fanburg, B. L.: Experimental cardiac hypertrophy. New Eng. J. Med., 282:723,1970. 13. Fanburg, B. L., and Posner, B. I.: Ribonucleic acid synthesis in experimental cardiac hypertrophy in rats. I. Characterization and kinetics oflabeling. Circ. Res., 23: 123,1968. 14. Garland, P. B., Randle, P. J., and Newsholme, E. A.: Citrate as an intermediary in the inhibition of phosphofructokinase in rat heart muscle by fatty acids, ketone bodies, pyruvate, diabetes and starvation. Nature, 200:169, 1963. 15. Gross, N. J.: Control of mitochondrial turnover under the influence of thyroid hormone. J. Cell BioI., 48:29, 1971. 16. Gross, N. J., Getz, G. S., and Rabinowitz, M.: Apparent turnover of mitochondrial deoxyribonucleic acid and mitochondrial phospholipids in the tissues of the rat. J. BioI. Chem., 244:1552, 1969. 17. Grove, D., Nair, K. G., and Zak, R.: Biochemical correlates of cardiac hypertrophy. Ill. Changes in DNA content; the relative contributions of polyploidy and mitotic activity. Circ. Res., 25:463, 1969. 18. Grove, D., Zak, R., Nair, K. G., and Aschenbrenner, V.: Biochemical correlates of cardiac
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