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A histochemical and electron microscopic study of of epinephrine-induced myocardial necrosis
Journal of Molecule
and
Cellu.!ar Cardiology (1970)
1, 1 l-22
A Histochemical and Electron of Epinephrine-induced 1’. J. FERRANS,*
R. G. HIBBS, J. J. WALSH
H. AND
Microscopic Myocardial
Study of Necrosis
S. WEILY, D. G. WEILBAECHER, G. E. BURCH
of Medicine and Anatomy of the Tulane University School of Medicine the Seamen’s Memorial Reaeurch Laboratories of the United States Public Health ServiceHospital, New Orleans,Louisiana, U.S.A.
Departments
and
V. J. FERRANS, R. G. HIBBS, H. S. WEILY, D. G. WEILBAECEER, J. J. WALEX AND G. E. Brmca. AHistochemicalend ElectronMicroscopic Studyof Epinephrine-induced Myocardiel Necrosis, Journal of Molecular and CeUulur Cardiology (1970) 1, 11-22. A histochemical rmd electron microscopic study wes made of the effects oflarge doses of epinephrine on the rat heart. Diffuse changes observed in the initial phases of epinephrine toxicity consisted of the deposition of lipid droplets in the myocardiel fibres, an increase in the activity of oxidative enzymes, and 8 rapid fall in myocardial glycogen. These alterations gradually subsided except in certtrin areas, mostly in the subendocardium, in which foci of myocardiml necrosis developed with marked fatty degeneration and total loss of oxidative enzyme activity. Electron microscopy revealed swelling of the sarcopleamio reticulum, the T-system, tmd the mitochondria. The mitochondrial cristae often developed angulations in their ordinr&ly straight, parallel surfties. These clnmges appeared to be largely reversible. Alter&ions of the contractile elements were found only in association with myocardisl necrosis and were characterized by hyalinizcttion and loss of the stristions.
1. Introduction Catecholaminesplay a crucial role in the physiologic regulation of cardiovascular functions. However, the excessive secretion of catecholamines or the potentiation of their inotropic and vasomotor actions can produce or aggravate various manifestations of cardiovascular disease.These effects have been documented clinically in pheochromocytomas [55], in hyperthyroidism [16] and in angina pectoris [42, 431. Overdosesof epinephrine and other cateoholamines produce focal necrosesin the heart and other organs and sensitize the myocardium to the necrotizing effect of other agents [6, 43, 46, 55, 571. Since little is known of the histochemical and ultrastructural changesinduced in the myocardium by epinephrine, the present studies were undertaken as part of an investigationof the responsesofthe heart muscleto various sympathomimetic agents.
2. Materials
and Methods
Adult male rats of the Holtzmann strain were given one subcutaneousinjection of 1 mg/kg of epinephrine in the form of a 1 : 1000 aqueous solution. The animals *Requests for reprints National Heert end Lung
should be addressed to Dr Victor J. Ferrets, Section Institute, Nation81 Institutes of Health, Bethesda, Md. 11
of Pathology, 20014, U.S.A.
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were sacrificed by decapitation at 30 min, 1, 2, 4, 8, 12, 16, 20, 24, 48 and 72 h after injection. Tissues samples were collected for electron microscopy from the free wall and the papillary muscles of the left ventricle. The remainder of the heart was used for the histochemical studies. The following histochemical techniques were performed on cryostat sections: benzpyrene-caffeine was used for the fluorescence microscopy of lipids; the PAS reaction, for the localization of carbohydrates, and a series of methods for the demonstration of the activity of the following enzymes: succinic dehydrogenase, DPN-diaphorase, isocitric dehydrogenase, malic dehydrogenase, lactic dehydrogenase, glutamic dehydrogenase, ethanol dehydrogenase and cytochrome oxidase. Details of these methods have been given elsewhere [la]. Phosphorylase was studied by the procedure of Takeuchi and Kuriaki [56]. For electron microscopy, the tissues were cut into l/2 mm cubes, fixed in phosphate-buffered osmium tetroxide, dehydrated in a graded series of alcohols and propylene oxide, and embedded in Maraglas [15]. Sections were cut with an LKB Ultrotome, stained with uranyl acetate and lead citrate, and examined with an RCA EMU 3C electron microscope. 3. Results Histochemical observations Clywgen As visualized by the PAS reaction, cardiac muscle glycogen was fairly evenly distributed throughout the cytoplasm of the cardiac muscle fibers. A pronounced, sustained fall in glycogen was noted following the administration of epinephrine. This change was evident, particularly in the subendocardial fibers, 30 mm after injection and persisted for 24 h, after which a gradual reappearance of glycogen was noted in all areas except in those that were necrotic.
Study of benzpyrene-stained sections of normal rat heart [Plate l(a)] revealed a pale blue fluorescence, due to the presence of phospholipids, in the muscle fibers, in the capillary endothelium, and in the fibroblasts and macrophages of the valvular and subendocardial connective tissues. In the muscle cells the fluorescence was localized to the intercalated discs, the sarcoplasm, and the mitochondria. Neutral lipid droplets, present only in an occasional cell, measured 0.1 to 1~ in diameter and exhibited a brighter fluorescence than the remaining cellular elements. In some cells these droplets formed parallel, longitudinally oriented rows in the interfibrillary spaces. As early as 30 min after the injection of epinephrine, large numbers of small lipid droplets were observed in some groups of fibers in the subendocardium and immediately adjacent areas. By 4 h the degree of fatty change [Plate l(b)] had
EPINEPHRINE-INDUCED
MYOCARDIAL
NECROSIS
13
increased and the intracellular distribution of the droplets had become more diffuse. The number and size of these droplets increased up to about 20 h [Plate l(c), (d), (e)], at which time some of the areas of lipid deposition were cotiuent and contained necrotic fibers laden with very large lipid droplets. After 24 h, the amount of neutral lipid gradually diminished [Plate l(f)], and by 72 h it was normal throughout most of the myocardium.
S,u.ccinic dehydrogenase
(SDH)
The staining reaction for this enzyme was localized in the mitochondria [Plate 2(a)], A particularly intense reaction was observed in the perinuclear areas, due to the large numbers of mitochondria present in these sites. The nuclei and intercalated discs remained unstained. A change in SDH activity was noted 30 min after the administration of epinephrine. At that time, and up to 4 h, an increase in staining was observed throughout the myocardium in association with very dark, granular formazan deposits [Plate 2(b)]. After 6 h the increased staining was no longer prominent; instead, there were spotty areas of diminished staining and disruption of the orderly mitochondrial pattern. At 24 h, foci of necrosis [Plate 2(c) and (d)] with grossmitochondrial swelling [Plate 3(b), (c) and (d)] and loss of the normal banding pattern were evident in the subendocardial fibres. Some of these cells showed hyaline necrosiswith little or no accumulation of fat [Plates 2(d) and 3(b)]. However, a great deal of fat was found in most of the necrotic cells. Inflammatory cells showed faint SDH activity in the form of a few perinuclear formazan deposits. After 48 h most fibers appeared normal [Plate 3(f)] except those in the areas of necrosis,which showed very little staining. Formazan depositswere frequently encountered at the lipid-water interface of the fat droplets [Plate 3(c)]. This artifactual pattern, which simulated closely that of mitochondrial swelling, was eliminated [Plate 3(d) and (e)] by extracting the sectionswith acetone at -20°C prior to incubation in the SDH medium.
DPN-diaphorme
and DPN-
and TPN-linked
dehydrogenases
In the normal rat heart, the intensity of the reactions for theseenzymes, on a scale from 0 to 4+, was estimated as follows : DPN-diaphorase, 4+ ; isocitric dehydrogenase,3+ ; lactic dehydrogenase,3+ ; malic dehydrogenase,2+ ; ethanol dehydrogenase,trace, and glutamic dehydrogenase, 1+. DPN-diaphorase, lactic dehydrogenase[Plate 3(a)], and isocitric dehydrogenasewere localized in the mitochondria of the cardiac muscle fibres and in the cytoplasm of endocardial and connective tissuecells. The reaction for malic dehydrogenasewas fainter, and its mitochondrial localization was not as sharp as that of the other enzymes; macrophages and connective tissue cells showed only a weak reaction for malic dehydrogenase. The reactions for glutamic and ethanol dehydrogenase showed faint staining in the
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cytoplasm of muscle and connective tissue cells, but no definite intracellular localization. The changes observed in the activity of these enzymes after the administration of epinephrine were very similar to those described for SDH, i.e. areas of increased staining with a precipitate of dark formazan granules, followed by the development of necrotic foci with little or no activity. Cytochrome
oxidme
The distribution of this enzyme in normalmyocardium was similar to that of SDH ; however, its histochemical reaction product was much more granular in character. In contrast to the pyridine nucleotide-linked enzymes, no initial rise in the activity of cytochrome oxidase was observed following treatment with epinephrine. A decreasein activity was observed as musclenecrosisdeveloped, but interpretation of results was rendered somewhat difficult by the affinity of the reaction product for the lipid droplets in the tissue.
The intensity of the staining reaction for phosphorylase showedpatchy, irregular variations in the normal rat heart. A definite but transient increasein this reaction was observed after the administration of epinephrine. This was followed by a gradual decreaseuntil 24 h, at which time a subnormal level of staining was evident throughout the heart. Areas of myocardial necrosisshowed very little or no staining. A progressive return of enzymatic activity occurred in non-necrotic areas at 48 and 72 h.
Comparison with control animals [Plate 4(a)] showed that the earliest alteration consistently observed in the myocardium was a reduction in the number of glycogen granules. This change was first noted 30 min after the administration of epinephrine and persisted for 24 h [Plate 4(b)]. Throughout that period the muscle fibres were almost completely devoid of stainable glycogen, and the small amount that remained was found between the myofibrils in the form of widely spaced clusters of dark granules. After 24 h, myocardial glyoogen gradually increased, and at 72 h it was roughly normal. Necrotic areasusually contained very little or no glyoogen. During the period of glycogen depletion, numerous small lipid droplets were found between the myofibrils and in the perinuclear sarcoplasm [Plate 5(a)]. These droplets reached an average size of 0.5 ,X in 24 h. A few of the droplets were enclosedby single layered membranesclosely resembling those of the sarooplasmic reticulum; most of them, however, appeared to be free in the sarooplasm,without discernible membranes. The largest number of lipid droplets was found at 24 h; somewhat fewer were encountered at 48 h, and at 72 h the number of lipid droplets was only slightly greater than in the controls.
PLATE 1. Fluorescence photomicrographs of the myooardium of normal and epinephrinetreated rats. Sections stained with benzpyrene for the fluorescence microscopy of lipids. (a) Normal heart. Longitudinal section showing the distribution of lipids in normal cardiac muscle fibres. The mitochondria form parallel rows in the interflbrillary spaces, and small clumps in the perinuclear area. Note the intercalated discs and the unstained nuclei. ( x 400.) (b) One hour after epinephrine. Note the patchy areas of fatty change in the cytoplasm of the cardiac muscle fibres (X 400.) (c) Sixteen hours after epinephrine. The lipid droplets are now much larger and numerous, and their distribution is now more diffuse. ( x 400.) (d) Twenty hours after epinephrine. Cross-se&ion of myocardial fibres showing edema and lipid deposits. Note the lipid droplets immediately under the cell membrane. ( x 1000.) (e) Twenty hours after epinephrine. Cross-section through subendocardial fibres laden with large numbers of lipid droplets. ( x 100.) (f) Forty-eight hours after epinephrine. Comparison with (e) shows a marked decrease in the amount of lipid present in the muscle fibres. ( x 100.) PLAT'E 2. Succinic dehydrogenase activity in the myocardium of normal and epinephrinetreat& rats. (a) Normal heart. The staining reaction is localized in the mitochondria. ( x 400.) (b) Four hours after epinephrine. There is a marked increase in the granularity of the reation. ( X 400.) (c) Twenty hours after epinephrine. Necrotic fibres with a marked decrease in succinic dehydrogenase activity are seen adjacent to fibres that show increased staining. ( x 35.) (d) Twenty hours after epinephrine. Area of hyaline necrosis showing varying degrees of cellular damage and partial loss of enzymic activity. (X 100.) PLATE 3. Lactic and succinic dehydrogenase activity in the myocardium of normal and epinephrine-treated rats. (a) Normal heart. Lactic dehydrogenase activity. The distribution of the staining reaction is eimilar to that of succinic dehydrogenase. ( x 200.) (b) Twenty hours aft’r s epinephrine. Succinic dehydrogenase activity. High-power view of an area of hyaline necrr!,:is showing a decrease in the intensity of the staining reaction and mitochondrial swelling. Fatty change is absent. ( x 400.) (c) Twenty-four hours after epinephrine. Succinic dehydrogenase activity. Area of necrosis and severe fatty change showing relaxation of the myofibrils and irregularly distributed, granular formazan deposits, some of which outline the periphery of large lipid droplets. ( x 600.) (d) Section similar to that of (c), stained for succinic dehydrogenase activity after extraction of neutral lipids with cold acetone. Note the disappearance of the granular formazan deposits. ( x 600.) (e) Normal heart. Section treated with cold acetone as in (d) prior to staining for succinic dehydrogenase activity. Comparison with Plate 2(a) shows that the normal staining pattern remains unchanged. ( x 800.) (f) Seventy-two hours after epinephrine. Succinic dehydrogenase activity. Nuclei counterstained with Methyl Green. Cross-section of myocardial fibres showing a normal staining reaction. Note the surroundi.lg inflammatory reaction. ( x 100.) PLATE 4. Myocardial glycogen in normal and epinephrine-treated rats. (a.) Normal rat myooardium. Electron micrograph of an oblique section showing dense glycogen particles (GL) between the myofibrils. (M) Mitochondria; (SR) sarooplasmic reticulum; (Z) Z line filaments; (A) A band filaments; (I) I band filaments. (X 30,000.) (b) Rat myocardium 12 h after epinephrine. Note the absence of glycogen particles. (M) Mitochondria; (SR) sarcoplasmic reticulum; (N) nucleus. ( x 30,000.) [faciv,g page 141
PLATE 5. Mitochondrial alterations in the myocardium of epinephrine-treated rats. (a) Twenty-four hours after epinephrine. Longitudinal section through a cardiac muscle fibre showing mitochondrial damage and disruption of the cristae (M), and the deposition of numerous lipid drnulets (L). (2) Z lines. ( x 20,000.) (b) Sixteen hoc+ts after epinephrine. Longitudinal section through the edge of a cardiac muscle fibre. Note the sharp angulations of the mitochondrial cristae. (M) Mitochondria; (L) lipid droplet; (S) sarcolemma. ( x 50,000.) PLATE 6. Alterations of the mitochondria and sarcoplasmic reticulum in the myocardium of epinephrine-treated rats. (a) Forty-eight hours after epinephrine. Swollen mitoohondria (M,) that have lost many of their cristae are seen in the lower part of the field. The mitochondria (M,) in the upper part of the field are normal. The sarcoplasmic reticulum (SR) is dilated. (L) Lipid droplet. ( x 50,000.) (b) Seventy-two hours after epinephrine. Section through an area of myooardial necrosis showing marked degenerative changes in the mitochondria (M), swelling of the sarcoplasmic reticulum (SR), and the formation of large vesicles (V) that contain amorphous debris. ( x 30,000.) PLATE 7. Seventy-two hours after epinephrine. Longitudinal section through a degenerating cardiac muscle cell. Note the swelling of the sarcoplasmic reticulum (SR), the absence of striations in the myofibrils (MF), and the areas of cytoplasmic degeneration (CD), some of which take the form of myelin figures (MY), in which fragments of mitochondria are recognizable. Some of the mitochondria (M) show swelling and partial loss of their oristae. (x 30,000.)
PLA
PLATE
Y
t’l..\‘l’t~
4
PLATE
5
EPINEPHRINE-INDUCED
MYOCARDIAI,
NECROSIS
15
During the first few hours there was a noticeable increase in the volume of mitochondria within the cells. Most of this increase was apparently due to an increase in number; however, this change may have been related to the fact that the myofibrils often appeared strongly contracted. Nevertheless, the average size of the mitochondria, especially in the perinuolear region, was larger than in the controls. The mitochondrial cristae were more closely packed, and often showed an increase in area, with marked angulations in their ordinarily straight, parallel surfaces. This resulted in a “crinkling” so that the cristae appeared to follow zigzag courses across the mitochondria. This alteration was best demonstrated in sections perpendicular to the plane of the surface of the cristae [Plate 5(b)]. By 12 h, the mitochondria in many regions showed a slight reduction in the number of their cristae, and occasional mitochondria were encountered that appeared swollen and almost devoid of cristae. In later stages (16 to 48 h), small areas of marked mitochondrial degeneration were present [Plate 6(a) and (b)]. These changes were rather spotty in distribution, so that areas of severe mitochondrial damage were found immediately adjacent to otherwise normal tissue. At 72 h plate 71 the regions of mitochondrial degeneration were necrotic ; all of the celhdar elements were disrupted and the tissue was scarcely recognizable as myocardium. In the remainder of the heart the mitochondria appeared essentially normal, except for some areas in which the zigzag arrangement of the cristae was still prominent. Slight to moderate swelling of the sarooplasmic reticulum and the T-system was encountered in all stages from 1 h on. This did not seem to be a progressive change, since the degree of swelling was about the same in all stages up to 24 h, after which the swelling regressed. Only in the areas of necrosis were the reticulum and T-system greatly disorganized and vesiculated [Plate 6(b)]. During the first 12 h the myofibrils showed relatively little change, which consisted of the formation of a few, scattered contraction bands. At 20 h some areas were encountered in which the striations were faint or absent, with indistinct myofllaments that appeared to have fused into homogeneous masses. These changes occurred in the areas of persistent mitochondrial damage and were seen most frequently after 48 h (Plate 7). However, areas of myofibrillar alterations made up only a small percentage of the total volume of the myocardium, and at 48 and 72 h the greatest part of the contractile elements appeared normal. 4. Discussion A number of different biochemical and hemodynamic changes may contribute to the pathogenesis of the cardiac lesions produced by overdoses of catecholamines [46, 47, 481. It is not surprising, therefore, that some distinctive features emerge on comparison of the histochemical and electron microscopic characteristics of the 2
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myocardial damage produced by epinephrine [25,49,57], norepinephrine [S, 13,311 and isoproterenol[7, 12, 19, 311. An analysis of these data is presented below. Ch~e.s
in glycogen and phosphxylade
The marked decrease in myocardial glycogen observed after the administration of epinephrine is related to the activation of phosphorylase and acceleration of glycogenolysis that this drug produces in cardiac muscle [9, lo]. Nevertheless, rapid loss of glycogen from cardiac muscle is a non-specific change common to various forms of myocardial damage, particularly that due to ischaemia, hypoxia and infarction [Z, 4, 14, 24, 33, 611. The accumulation of glycogen into membraneenclosed bodies, such as that reported by David et al. [8] in norepinephrine-treated animals, was not observed in this study. In the early stages after the administration of epinephrine there was an increase in the activity of phosphorylase, which may be attributed to the pharmacological effect of the drug. Although the reason for the subsequent decrease in phosphorylase activity is less clear, rapid loss of phosphorylase activity from myocardium has been shown to occur in ischemia and early infarction [ZO, 21, 281 and after the administration of methoxamine and metaraminol [l]. Biochemical studies have shown also that epinephrine produces first an increase and then a decrease in the activity of phosphorylase in heart [60], and liver [273. These observations, and the fact that epinephrine continues to stimulate oxidative metabolism in myocardium after the supply of cardiac muscle glycogen is depleted, are in agreement with the concept [Sl] that the glycogenolytic and metabolic stimulatory effects of epinephrine represent separate phenomena. Lipid accumuluhm The coexistence of cellular damage and lipid accumulation has been observed frequently in cardiac muscle [7, 12,13,14, 241. Conditions of hypoxia, with ensuing acidosis, accumulation of metabolites, and depletion of high energy phosphates, depress the myocardial oxidation of fatty acids. Furthermore, direct effects of epinephrine on lipid metabolism also contribute to the marked elevation of myocardial triglycerides that occurs [373 after the administration of this agent. Epinephrine causes an elevation in the serum level of free fatty aoids (FFA) as the result of an increase in lipid mobilization, which is mediated through the activation of a lipase in adipose tissue [39, 581. Regan [44] has suggested that, in addition to its lipid mobilizing effect, epinephrine has a direct effect in stimulating the myocardial uptake of triglycerides. However, Kreisberg [34] concluded that epinephrine stimulates the utilization of endogenous lipid by myocardium, and that it influences indirectly the myocardial FFA uptake by virtue of its ability to elevate circulating levels of FFA rather than by a direct effect on myocardial extraction. Therefore, the accumulation of lipids observed in cardiac muscle cells after the administration
EPINEPHRINE-INDUCED
MYOCARDJAL
NECROSIS
17
of epinephrine results from the combined effects of increased mobilization of lipid from adipose tissue, and alterations in uptake and utilization of lipids by myocardium. Changes in the sarwplasmic reticulum, T-system and myojbrils Our data, which are in agreement with those obtained by Hiramoto [25] and Shimamoto and Hiramoto [49] in their studies of the effects of epinephrine on rabbit myocardium, ahow that this drug produces much more transient and much less pronounced swelling of the sarcoplasmio reticulum and T-system than do either isoproterenol[7,12,19,32] or norepinephrine [8,13]. In epinephrine-treated animals the sarcoplasmic reticulum and T-system were severely swollen and disrupted only in obviously necrotic cells. Overdoses of isoproterenol and norepinephrine also result in the formation of marked contraction bands; these are so prominent that David et al. [8] have postulated that the myofibrils are the primary site of action of norepinephrine. However, after the administration of epinephrine these changes were found in a relatively small percentage of the muscle cells, usually in those that showed other evidence of necrosis. There is no clear explanation for the preceding differences. They may be the morphological counterparts of specific pharmacological effects of these three agents on cardiac muscle, as suggested by the fact that norepinephrine, like epinephrine, produces only small, patchy foci of necrosis,while isoproterenol, even in low doses, causeslarge, infarct-like lesions that differ histologically from those induced by other sympathomimetic amines. Unfortunately, the possibility that these changes represent dose-related phenomena could not be investigated since the administration of larger dosesof epinephrine caused immediate death from ventricular fibrillation.
Histochemical tests for the activity of various oxidative enzymes are considered to be sensitive indicators of myooardial damage, and can demonstrate mitochondrial alterations that occur prior to the development of changes visible by ordinary histological techniques [2, 30, 50, 591. The histochemical changesobserved in the mitochondria in this study resemble closely those produced by isoproterenol [12] and by norepinephrine [12, 311.The initial rise in the activity of oxidative enzymea paralleled metabolic changesin myocardium, with oxidative metabolism at a peak as the result of the stimulatory effect of epinephrine. The formation of the dark formazan granules observed histochemically may be related to an increase in the permeability of the mitochondria to the tetrazolium salts. These changesreverted to normal except in the areasof necrosis,which showedthe lossof oxidative enzymes associatedwith cellular death. As in the caseof isoproterenol- and norepinephrine-induced myocardial necrosis, electron microscopy showedthat the mitoohondrial damageproduced by epinephrine
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developed more slowly than that due to myocardial infarction [3,5,22,29,33,52]. Of particular interest is the fact that angular configurations of the cristae, such aa those observed in our epiuephrine-treated animals, have not been described previously in the rat heart. However, we have found this type of configuration in a small percentage of the mitochondria in the myocardium of some apparently normal animals. This configuration has been observed also in the ventricle [11] and atrium [38] of the cat, as well as in the small portion of the mitochondria population of a variety of cell types [45]. According to Slautterback [51], such a configuration is very prominent in the mitochondria of cardiac muscle cells of the canary; however, it does not appear to be directly related to the rapid cardiac rate of this species, since it was not observed in the cardiac muscle of other birds with equally fast rates. Angulations of the mitochondrial cristae have been described in aasoaiation with only one pathological state in the human. This change, along with the formation of giant mitochondria, as found by Luft and co-workers [35,36] in the skeletal muscle cells of a patient with an unexplained hypermetabolic state characterized by loss of the control of mitochondrial respiration and partial uncoupling of oxidative phosphorylation. Similar angulations have been observed in the skeletal muscle of rats treated with thyroxin [17], which uncouples oxidative phosphorylation and also shares some of the properties of epinephrine, including the activation of myocardial phosphorylase [23] and the marked increase in oxygen consumption by heart muscle [26]. However, changes of this type were not observed in the mitochondrial cristae either by Poche [40] or by Susin and Her&on [54] in the myocardium of thyroxin-treated rats; furthermore, they are not produced by iaoproterenol[7,12,19,32], and are seen infrequently after the administration of norepinephrine [13]. The angulations of the mitochondrial cristae found in this study correspond to the “energized-twisted” configurations observed in normal rat myocardium by Harris et al. [18] after perfusion with an oxygenated medium that contained substrate for electron transfer aa well as inhibitors (rotenone and rutamycin) which prevented the discharge of the energized state. These authors emphasized the fact that optimal oonditiona of tissue fixation are necessary to demonstrate such changes. The work of Sordahl et al. [53] suggeststhat conformational changes in the inner mitoohondrial membranesmay be more closely related to energy-linked functions, such as ion or substrate transport or to respiratory control. Studies of the combined effects of epinephrine and a- and /%adrenergenicblocking agents on the ultraatructure and biochemistry of mitochondria in cardiac muscle may help to elucidate the nature of these changes. They are of special interest in view of numerous recent studies which have shown that the morphology of mitochondria in a number of tissuescan be modified in specific ways by physiological change+ pathological states and pharmacological agents. It is possible, therefore, that the recognition of alterations in the mitochondria of cardiac muscle will be of help in the classification of various types of myocardial lesions.
EPINEPHRINE-INDUCED
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NECROSIS
AckrwwlecEgments This work was supported by Research Grants No. HE-06100, HE-86769 and Career Development Award 5-K3-GM-15-281-05 the United States Public Health Service. REFERENCES 1. BAJUSZ, E. & JASMIN, G. Observations on histochemical
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GUSTAKSSON, R., TATA, J. R., LINDBERO, 0. & ERNSTER, L. The relationship between the structure and activity of rat skeletal muscle mitochondria after thyroidectomy and thyroid hormone treatment. Journal of Cell Biology 26, 555-578 (1965). HARRIS, R. A., WILLIES, C. H., CALDWELL, M., GREEN, D. E. & VALDIXA, E. Energized configurations of heart mitochondria in titu. Science, New York 165, 700-702 (1969). HAUSAMEN, T. U. & POCHE, R. Elektronenmikroskopisohe Untersuchungen tiber die Wirkung von Alupent auf die Ultrastruktur des Herzmuskels der Ratte. Virchows Arc?& fiir pathologbche Anatomic und Physiologic und fiir klinische Medizin 339, 226-233. (1965). Hxcrrr, A. Zum problem der fermenthistochemischen Erfassung unterschiedlicher Schiidigungsmechanismen am Rattenherzmuskel am Beispiel der Phosphorylaseaktivitat. Beitr&ge zur pathologischelt Anatomic und zur allgemeinen Pathologic 135,322-332 (1967). HEOET, A. Das Verhalten der histochemisch nachweisbaren Phosphorylaseaktivitiit am anoxisch ruhenden und anoxisch ttitigen (ischiimischen) Rattenherzmuskel. Histochemie 9, 48-57 (1967). HECHT, A., KORB, C. & DAVID, H. Vergleichende histochemische, fluoreszenzmikroskopische und elektronenoptische Untersuchungen zur Fruhdiagnose des Herzinfarktes bei der Ratte. V’irchows Archiv fiir pathologische Anatomic und physiologic klinische Me&i&a 334,267-284 (1961). HESS, M. E. & SHANFELD, J. Cardiovascular and metabolic interrelationships between thyroxine and the sympathetic nervous system. Journal of Pharmacology 148,290-297 (1965). HIBBS, R. G., FERRANS, V. J., BLACK, W. C., WEILBAECHER, D. G., WALSH, J. J. & BURCH, G. E. Alcoholic cardiomyopathy. An electron microscopic study. American Heart Journal 69, 766-779 (1965). HIRAMOTO, Y. Fine structure of rabbit cardiac muscle with special reference to myofibril and sarcoplasmic reticulum. Japanese Circulation Journal 31, 1543-l 550 (1967). HOCE, F. L. Thyrotoxicosis as a disease of mitochondria. New England Journal of Medicine 266, 446-454 (1962). Huo, S., SCHUBERT, W. R., CHUCK, G. & GARANCIS, J. C. Liver phosphorylsse. Deactivation in a child with progressive brain disease, increased hepatic glycogen and increased urinary catecholamines. American Journal of Medicine 42, 139-145 (1967). JGSKELXINEN, A. J. Phosphorylase activity in myocardial infarction and its postmortem demonstrability in the rat. Annales medki?zae experiment&s et biologiizle Fenniae 44,541-543. JENNINIX, R. B., SOWERS, H. M., HERDSON, P. B. & KALTENBACH, J. P. Ischemic injury of myocardium. Annals of the New York Acddemyof Science 156,61-78 (1969). KOLIN, A. A morphological indicator of reversible myocardial damage. Histochemical demonstration of succinodehydrogenase (SDH) activity of the muscle fibres. Cor et wma 7, 30-35 (1965). KOLIN, A. & KVASNICKA, J. Pseudoinfarction of the QRS complex in experimental card&o hypoxia induced by noradrenalin. Cardiologia 43, 362-370 (1963). KORB, G. Elektronenmikroskopische Untersuchungen zur Aludrin (Isoproterenol&fat)-schadigung des Herzmuskels. Virchows Archiw fiir pathologiache Anatomic und Physiologie und fiir klinische Medizin 339, 136-150 (1965).
undfiir
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25.
26. 27.
28.
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30.
31.
32.
ET AL.
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MYOCARDIALNECROSIS
21
33. KORB, G. & TOTOVIC, V. Electron microscopical studies of experimental ischemic lesions of the heart. Annals of the New York Academy of Science1!56,4&60 (1969). 34. KREISBERG. R. A. Effects of epinephrine on myocardial triglyceride and free fatty acid utilization. American JournaZ of PhyeioZogy 210, 385-389 (1966). 35. LUFT, K. & ERNSTER, L. Further studies on a population of human skeletal muscle mitochondrialaokingrespiratorycontrol. Ex~erimentalCellR~rch32,26-35 (1963). 36. LuFT,R., IKKOS, D., PALMIERI, G.,ERNSTER, L. t ARZELIUS, B. A case ofsevere hypermetabolism of non-thyroid origin with a defect in the maintenance of mitoohondrial respiratory control: a correlated clinical, biochemical and morphological study. Journal of Clinical Inveetigation 41, 1776-1804 (1962). 37. -a, H. M., HIGHMAN, B. & !bIOIKPSON, E. C. Some similar effects after large doses of catecholamines and myocardial infarction in dogs. Arneriml Jmwnal of Cardiology 5, 628-633 (1960). 38. MoNm, M. S. & FAWCETT, D. W. The ultrastructure of the cat myooardium. II. Atria1 muscle. Journal of Cell Biology 42, 46-67 (1969). 39. MUELLER, P. 5. & HOR-Z, D. Plasma free fatty acid and blood glucose responses to analogues of norepinephrine in man. Journal of Lipid Research 3,251-255 (1962). 40. POOEE, R. Das submikroakopische Bild der HerzmuskelverLtnderungen naoh ~berdosierung von Schilddriisenhormon. Beitr&ge zur pathologischm Anatomic tind ZUT allgerne~nen Path&@ 118, 407-420 (1957). 41. POUHE, R. & OHM, H. G. Lichtmikroskopische, histochemische und elektronenmikroskopische Untersuchungen des Herzmuskels vom Menschen nach induziertem Herzstillstand. Archiv ftXr Kreislaufforechung 41, 86-135 (1963). 42. RN, W. The sympathogenic biochemical trigger mechanism of angina pectoris. American Journal of Cardiology 9, 576-584 (1962). 43. RAAB, W., VAN LITH, P., LOPESOHEIN, B. & HERRLIOH, H. C. Catecholamineinduced myocardial hypoxia in the presence of impaired coronary dilatability independent of external cardiac work. American Journal of Cardiology 9, 455-470 (1962). 44. REGAN, T. J. Vasopressin, catecholamines and serotonin as possible causes of myocardial &hernia. In The Etiology of Myocardial Infarction, pp. 375-387. (T. N. James & J. W. Keyes, Eds). Boston: Little, Brown and Co. (1963). 45. REVEL, J. P., FAWCETT, D. W. & PEULPOTT, C. W. Observations on mitochondrial structure. Angular configurations of the cristae. JournaZ of CeU Biology 16, 187-195 (1963). 46. RONA, 0. & KAHN, D. S. The healing of cardiac necrosis as reflected by experimental studies. In Methods and Achievenzenta in Experimenti Pathology, Vol. III, pp. 200249. (E. Bajusz and G. Jasmin, Eds). Basel, New York: Karger (1967). 47. ROSENEZL~, I., Worn, A. & STEIN, A. A. Studies in cardiac necrosis. I. Production of cardiac lesions with sympathomimetic amines. Toxicology and Applied P?wwm.acobgy 7, l-8 (1965). 48. ROSENBLUM, I., WOHL, A. & STEIN, A. A. Studies in cardiac necrosis. II. Cardiovascular effects of sympathomimetic amines producing cardiac lesions. To&cology and Applied Pharmwology 7, 9-17 (1965). 49. SHIMAMOTO, T. & HIRAMOTO, Y. Fine structure of rabbit cardiac muscle and changes induced by adrenaline. In Electrophyeiology and Ultrastructure of the Heart, pp. 5564. (T. Sano, V. Mizuhira t K. Matsuda, Eds). New York: Grune & Stratton (1967). 50. SHNITKA, T. K. & NACELAS, M. M. Histochemical alterations in ischemio heart muscle and early myocardial infarction. Am&can Journal of Pathology 42, 507-527 (1963).
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D. B. Mitochondria in cardiac muscle cells of the canazy and some other birds. Journal of Cell Biology 24, 1-22 (1965). SONBIERS, H. M. & JENNINGS, R. B. Experimental acute myoosrdial infarction. Histologic and histochemioal studies of early myocardial infarcts induced by temporary or permanent ooclusion of & coronary artery. Laboratory Invedga$ion 13,1491-1603(1964). SORDAHL, L. A., BLAILOCK, Z. R., KRAFT, G. H. & SCHWARTZ, A. The possible relationship between ultrastructura and biochemical state of heart mitochondria. Arohivee of Biochmktry and Biophy& 132, 404-415 (1969). SUSIN, M. & HERDSON, P. B. Fine structural changes in rat myocardium induced by thyroxine and by magnesium deficiency. Archives of Pa&logy (Chicugo) 83, 86-98 (1967). SZ~KACS, J. E., DIMMETTE, R. M. & COWART, E. C. Pathologic implications of the catecholsmines, epinephrine, and norepinephrine. U.S. Arm& Forces Medical Journal 10,908-925 (1959). TAJZEUOHI, T. & KURIAKI, H. Histochemical detection of phosphorylsse in animal tissues. Journal of Histochemiet~ and Cytochemietry 3, 153-160 (1955). TSELLARIUS, Y. G. Changes in muscle fibers of the heart in adrenalin injuries. Arkhiv pa&dog& 29, 34-39 (1967). VAUGHAN, M., BERUER, J. B. & STEINBERG, D. Hormone-sensitive lipase and monoglyceride lipase activities in adipose tissue. Journal of Biological Chedetry 239, 401-409 (1964). WAOHSTEIN, M. & MEISEL, H. Succinic dehydrogenase activity in myocardisl infarction and induced myocardial necrosis. American Journal of Pathology 31, 363-363( 1955). WILLIAMSON, J. R. Metabolic effects of epinephrine in the isolated, perfused rat heart. I. Dissociation of the glycogenolytic from the metabolic stimulatory effect. Journd of Biologic& Ckmktry 239, 2721-2729 (1964). YOKOYAMA, H. O., JENNINGS, R. B., CLABAUGH, G. F. & WARTMAN, W. B. Histochemical studies of early experimental myocardial infarction. Periodic acid-Schiff method. Archive.9 of Pathology 59, 347-354 (1955). SLA~TERBAOK,
and
Cellu.!ar Cardiology (1970)
1, 1 l-22
A Histochemical and Electron of Epinephrine-induced 1’. J. FERRANS,*
R. G. HIBBS, J. J. WALSH
H. AND
Microscopic Myocardial
Study of Necrosis
S. WEILY, D. G. WEILBAECHER, G. E. BURCH
of Medicine and Anatomy of the Tulane University School of Medicine the Seamen’s Memorial Reaeurch Laboratories of the United States Public Health ServiceHospital, New Orleans,Louisiana, U.S.A.
Departments
and
V. J. FERRANS, R. G. HIBBS, H. S. WEILY, D. G. WEILBAECEER, J. J. WALEX AND G. E. Brmca. AHistochemicalend ElectronMicroscopic Studyof Epinephrine-induced Myocardiel Necrosis, Journal of Molecular and CeUulur Cardiology (1970) 1, 11-22. A histochemical rmd electron microscopic study wes made of the effects oflarge doses of epinephrine on the rat heart. Diffuse changes observed in the initial phases of epinephrine toxicity consisted of the deposition of lipid droplets in the myocardiel fibres, an increase in the activity of oxidative enzymes, and 8 rapid fall in myocardial glycogen. These alterations gradually subsided except in certtrin areas, mostly in the subendocardium, in which foci of myocardiml necrosis developed with marked fatty degeneration and total loss of oxidative enzyme activity. Electron microscopy revealed swelling of the sarcopleamio reticulum, the T-system, tmd the mitochondria. The mitochondrial cristae often developed angulations in their ordinr&ly straight, parallel surfties. These clnmges appeared to be largely reversible. Alter&ions of the contractile elements were found only in association with myocardisl necrosis and were characterized by hyalinizcttion and loss of the stristions.
1. Introduction Catecholaminesplay a crucial role in the physiologic regulation of cardiovascular functions. However, the excessive secretion of catecholamines or the potentiation of their inotropic and vasomotor actions can produce or aggravate various manifestations of cardiovascular disease.These effects have been documented clinically in pheochromocytomas [55], in hyperthyroidism [16] and in angina pectoris [42, 431. Overdosesof epinephrine and other cateoholamines produce focal necrosesin the heart and other organs and sensitize the myocardium to the necrotizing effect of other agents [6, 43, 46, 55, 571. Since little is known of the histochemical and ultrastructural changesinduced in the myocardium by epinephrine, the present studies were undertaken as part of an investigationof the responsesofthe heart muscleto various sympathomimetic agents.
2. Materials
and Methods
Adult male rats of the Holtzmann strain were given one subcutaneousinjection of 1 mg/kg of epinephrine in the form of a 1 : 1000 aqueous solution. The animals *Requests for reprints National Heert end Lung
should be addressed to Dr Victor J. Ferrets, Section Institute, Nation81 Institutes of Health, Bethesda, Md. 11
of Pathology, 20014, U.S.A.
12
V. J. FERRANS
ET
AL.
were sacrificed by decapitation at 30 min, 1, 2, 4, 8, 12, 16, 20, 24, 48 and 72 h after injection. Tissues samples were collected for electron microscopy from the free wall and the papillary muscles of the left ventricle. The remainder of the heart was used for the histochemical studies. The following histochemical techniques were performed on cryostat sections: benzpyrene-caffeine was used for the fluorescence microscopy of lipids; the PAS reaction, for the localization of carbohydrates, and a series of methods for the demonstration of the activity of the following enzymes: succinic dehydrogenase, DPN-diaphorase, isocitric dehydrogenase, malic dehydrogenase, lactic dehydrogenase, glutamic dehydrogenase, ethanol dehydrogenase and cytochrome oxidase. Details of these methods have been given elsewhere [la]. Phosphorylase was studied by the procedure of Takeuchi and Kuriaki [56]. For electron microscopy, the tissues were cut into l/2 mm cubes, fixed in phosphate-buffered osmium tetroxide, dehydrated in a graded series of alcohols and propylene oxide, and embedded in Maraglas [15]. Sections were cut with an LKB Ultrotome, stained with uranyl acetate and lead citrate, and examined with an RCA EMU 3C electron microscope. 3. Results Histochemical observations Clywgen As visualized by the PAS reaction, cardiac muscle glycogen was fairly evenly distributed throughout the cytoplasm of the cardiac muscle fibers. A pronounced, sustained fall in glycogen was noted following the administration of epinephrine. This change was evident, particularly in the subendocardial fibers, 30 mm after injection and persisted for 24 h, after which a gradual reappearance of glycogen was noted in all areas except in those that were necrotic.
Study of benzpyrene-stained sections of normal rat heart [Plate l(a)] revealed a pale blue fluorescence, due to the presence of phospholipids, in the muscle fibers, in the capillary endothelium, and in the fibroblasts and macrophages of the valvular and subendocardial connective tissues. In the muscle cells the fluorescence was localized to the intercalated discs, the sarcoplasm, and the mitochondria. Neutral lipid droplets, present only in an occasional cell, measured 0.1 to 1~ in diameter and exhibited a brighter fluorescence than the remaining cellular elements. In some cells these droplets formed parallel, longitudinally oriented rows in the interfibrillary spaces. As early as 30 min after the injection of epinephrine, large numbers of small lipid droplets were observed in some groups of fibers in the subendocardium and immediately adjacent areas. By 4 h the degree of fatty change [Plate l(b)] had
EPINEPHRINE-INDUCED
MYOCARDIAL
NECROSIS
13
increased and the intracellular distribution of the droplets had become more diffuse. The number and size of these droplets increased up to about 20 h [Plate l(c), (d), (e)], at which time some of the areas of lipid deposition were cotiuent and contained necrotic fibers laden with very large lipid droplets. After 24 h, the amount of neutral lipid gradually diminished [Plate l(f)], and by 72 h it was normal throughout most of the myocardium.
S,u.ccinic dehydrogenase
(SDH)
The staining reaction for this enzyme was localized in the mitochondria [Plate 2(a)], A particularly intense reaction was observed in the perinuclear areas, due to the large numbers of mitochondria present in these sites. The nuclei and intercalated discs remained unstained. A change in SDH activity was noted 30 min after the administration of epinephrine. At that time, and up to 4 h, an increase in staining was observed throughout the myocardium in association with very dark, granular formazan deposits [Plate 2(b)]. After 6 h the increased staining was no longer prominent; instead, there were spotty areas of diminished staining and disruption of the orderly mitochondrial pattern. At 24 h, foci of necrosis [Plate 2(c) and (d)] with grossmitochondrial swelling [Plate 3(b), (c) and (d)] and loss of the normal banding pattern were evident in the subendocardial fibres. Some of these cells showed hyaline necrosiswith little or no accumulation of fat [Plates 2(d) and 3(b)]. However, a great deal of fat was found in most of the necrotic cells. Inflammatory cells showed faint SDH activity in the form of a few perinuclear formazan deposits. After 48 h most fibers appeared normal [Plate 3(f)] except those in the areas of necrosis,which showed very little staining. Formazan depositswere frequently encountered at the lipid-water interface of the fat droplets [Plate 3(c)]. This artifactual pattern, which simulated closely that of mitochondrial swelling, was eliminated [Plate 3(d) and (e)] by extracting the sectionswith acetone at -20°C prior to incubation in the SDH medium.
DPN-diaphorme
and DPN-
and TPN-linked
dehydrogenases
In the normal rat heart, the intensity of the reactions for theseenzymes, on a scale from 0 to 4+, was estimated as follows : DPN-diaphorase, 4+ ; isocitric dehydrogenase,3+ ; lactic dehydrogenase,3+ ; malic dehydrogenase,2+ ; ethanol dehydrogenase,trace, and glutamic dehydrogenase, 1+. DPN-diaphorase, lactic dehydrogenase[Plate 3(a)], and isocitric dehydrogenasewere localized in the mitochondria of the cardiac muscle fibres and in the cytoplasm of endocardial and connective tissuecells. The reaction for malic dehydrogenasewas fainter, and its mitochondrial localization was not as sharp as that of the other enzymes; macrophages and connective tissue cells showed only a weak reaction for malic dehydrogenase. The reactions for glutamic and ethanol dehydrogenase showed faint staining in the
14
V. J. FRRRANS
XT
AL.
cytoplasm of muscle and connective tissue cells, but no definite intracellular localization. The changes observed in the activity of these enzymes after the administration of epinephrine were very similar to those described for SDH, i.e. areas of increased staining with a precipitate of dark formazan granules, followed by the development of necrotic foci with little or no activity. Cytochrome
oxidme
The distribution of this enzyme in normalmyocardium was similar to that of SDH ; however, its histochemical reaction product was much more granular in character. In contrast to the pyridine nucleotide-linked enzymes, no initial rise in the activity of cytochrome oxidase was observed following treatment with epinephrine. A decreasein activity was observed as musclenecrosisdeveloped, but interpretation of results was rendered somewhat difficult by the affinity of the reaction product for the lipid droplets in the tissue.
The intensity of the staining reaction for phosphorylase showedpatchy, irregular variations in the normal rat heart. A definite but transient increasein this reaction was observed after the administration of epinephrine. This was followed by a gradual decreaseuntil 24 h, at which time a subnormal level of staining was evident throughout the heart. Areas of myocardial necrosisshowed very little or no staining. A progressive return of enzymatic activity occurred in non-necrotic areas at 48 and 72 h.
Comparison with control animals [Plate 4(a)] showed that the earliest alteration consistently observed in the myocardium was a reduction in the number of glycogen granules. This change was first noted 30 min after the administration of epinephrine and persisted for 24 h [Plate 4(b)]. Throughout that period the muscle fibres were almost completely devoid of stainable glycogen, and the small amount that remained was found between the myofibrils in the form of widely spaced clusters of dark granules. After 24 h, myocardial glyoogen gradually increased, and at 72 h it was roughly normal. Necrotic areasusually contained very little or no glyoogen. During the period of glycogen depletion, numerous small lipid droplets were found between the myofibrils and in the perinuclear sarcoplasm [Plate 5(a)]. These droplets reached an average size of 0.5 ,X in 24 h. A few of the droplets were enclosedby single layered membranesclosely resembling those of the sarooplasmic reticulum; most of them, however, appeared to be free in the sarooplasm,without discernible membranes. The largest number of lipid droplets was found at 24 h; somewhat fewer were encountered at 48 h, and at 72 h the number of lipid droplets was only slightly greater than in the controls.
PLATE 1. Fluorescence photomicrographs of the myooardium of normal and epinephrinetreated rats. Sections stained with benzpyrene for the fluorescence microscopy of lipids. (a) Normal heart. Longitudinal section showing the distribution of lipids in normal cardiac muscle fibres. The mitochondria form parallel rows in the interflbrillary spaces, and small clumps in the perinuclear area. Note the intercalated discs and the unstained nuclei. ( x 400.) (b) One hour after epinephrine. Note the patchy areas of fatty change in the cytoplasm of the cardiac muscle fibres (X 400.) (c) Sixteen hours after epinephrine. The lipid droplets are now much larger and numerous, and their distribution is now more diffuse. ( x 400.) (d) Twenty hours after epinephrine. Cross-se&ion of myocardial fibres showing edema and lipid deposits. Note the lipid droplets immediately under the cell membrane. ( x 1000.) (e) Twenty hours after epinephrine. Cross-section through subendocardial fibres laden with large numbers of lipid droplets. ( x 100.) (f) Forty-eight hours after epinephrine. Comparison with (e) shows a marked decrease in the amount of lipid present in the muscle fibres. ( x 100.) PLAT'E 2. Succinic dehydrogenase activity in the myocardium of normal and epinephrinetreat& rats. (a) Normal heart. The staining reaction is localized in the mitochondria. ( x 400.) (b) Four hours after epinephrine. There is a marked increase in the granularity of the reation. ( X 400.) (c) Twenty hours after epinephrine. Necrotic fibres with a marked decrease in succinic dehydrogenase activity are seen adjacent to fibres that show increased staining. ( x 35.) (d) Twenty hours after epinephrine. Area of hyaline necrosis showing varying degrees of cellular damage and partial loss of enzymic activity. (X 100.) PLATE 3. Lactic and succinic dehydrogenase activity in the myocardium of normal and epinephrine-treated rats. (a) Normal heart. Lactic dehydrogenase activity. The distribution of the staining reaction is eimilar to that of succinic dehydrogenase. ( x 200.) (b) Twenty hours aft’r s epinephrine. Succinic dehydrogenase activity. High-power view of an area of hyaline necrr!,:is showing a decrease in the intensity of the staining reaction and mitochondrial swelling. Fatty change is absent. ( x 400.) (c) Twenty-four hours after epinephrine. Succinic dehydrogenase activity. Area of necrosis and severe fatty change showing relaxation of the myofibrils and irregularly distributed, granular formazan deposits, some of which outline the periphery of large lipid droplets. ( x 600.) (d) Section similar to that of (c), stained for succinic dehydrogenase activity after extraction of neutral lipids with cold acetone. Note the disappearance of the granular formazan deposits. ( x 600.) (e) Normal heart. Section treated with cold acetone as in (d) prior to staining for succinic dehydrogenase activity. Comparison with Plate 2(a) shows that the normal staining pattern remains unchanged. ( x 800.) (f) Seventy-two hours after epinephrine. Succinic dehydrogenase activity. Nuclei counterstained with Methyl Green. Cross-section of myocardial fibres showing a normal staining reaction. Note the surroundi.lg inflammatory reaction. ( x 100.) PLATE 4. Myocardial glycogen in normal and epinephrine-treated rats. (a.) Normal rat myooardium. Electron micrograph of an oblique section showing dense glycogen particles (GL) between the myofibrils. (M) Mitochondria; (SR) sarooplasmic reticulum; (Z) Z line filaments; (A) A band filaments; (I) I band filaments. (X 30,000.) (b) Rat myocardium 12 h after epinephrine. Note the absence of glycogen particles. (M) Mitochondria; (SR) sarcoplasmic reticulum; (N) nucleus. ( x 30,000.) [faciv,g page 141
PLATE 5. Mitochondrial alterations in the myocardium of epinephrine-treated rats. (a) Twenty-four hours after epinephrine. Longitudinal section through a cardiac muscle fibre showing mitochondrial damage and disruption of the cristae (M), and the deposition of numerous lipid drnulets (L). (2) Z lines. ( x 20,000.) (b) Sixteen hoc+ts after epinephrine. Longitudinal section through the edge of a cardiac muscle fibre. Note the sharp angulations of the mitochondrial cristae. (M) Mitochondria; (L) lipid droplet; (S) sarcolemma. ( x 50,000.) PLATE 6. Alterations of the mitochondria and sarcoplasmic reticulum in the myocardium of epinephrine-treated rats. (a) Forty-eight hours after epinephrine. Swollen mitoohondria (M,) that have lost many of their cristae are seen in the lower part of the field. The mitochondria (M,) in the upper part of the field are normal. The sarcoplasmic reticulum (SR) is dilated. (L) Lipid droplet. ( x 50,000.) (b) Seventy-two hours after epinephrine. Section through an area of myooardial necrosis showing marked degenerative changes in the mitochondria (M), swelling of the sarcoplasmic reticulum (SR), and the formation of large vesicles (V) that contain amorphous debris. ( x 30,000.) PLATE 7. Seventy-two hours after epinephrine. Longitudinal section through a degenerating cardiac muscle cell. Note the swelling of the sarcoplasmic reticulum (SR), the absence of striations in the myofibrils (MF), and the areas of cytoplasmic degeneration (CD), some of which take the form of myelin figures (MY), in which fragments of mitochondria are recognizable. Some of the mitochondria (M) show swelling and partial loss of their oristae. (x 30,000.)
PLA
PLATE
Y
t’l..\‘l’t~
4
PLATE
5
EPINEPHRINE-INDUCED
MYOCARDIAI,
NECROSIS
15
During the first few hours there was a noticeable increase in the volume of mitochondria within the cells. Most of this increase was apparently due to an increase in number; however, this change may have been related to the fact that the myofibrils often appeared strongly contracted. Nevertheless, the average size of the mitochondria, especially in the perinuolear region, was larger than in the controls. The mitochondrial cristae were more closely packed, and often showed an increase in area, with marked angulations in their ordinarily straight, parallel surfaces. This resulted in a “crinkling” so that the cristae appeared to follow zigzag courses across the mitochondria. This alteration was best demonstrated in sections perpendicular to the plane of the surface of the cristae [Plate 5(b)]. By 12 h, the mitochondria in many regions showed a slight reduction in the number of their cristae, and occasional mitochondria were encountered that appeared swollen and almost devoid of cristae. In later stages (16 to 48 h), small areas of marked mitochondrial degeneration were present [Plate 6(a) and (b)]. These changes were rather spotty in distribution, so that areas of severe mitochondrial damage were found immediately adjacent to otherwise normal tissue. At 72 h plate 71 the regions of mitochondrial degeneration were necrotic ; all of the celhdar elements were disrupted and the tissue was scarcely recognizable as myocardium. In the remainder of the heart the mitochondria appeared essentially normal, except for some areas in which the zigzag arrangement of the cristae was still prominent. Slight to moderate swelling of the sarooplasmic reticulum and the T-system was encountered in all stages from 1 h on. This did not seem to be a progressive change, since the degree of swelling was about the same in all stages up to 24 h, after which the swelling regressed. Only in the areas of necrosis were the reticulum and T-system greatly disorganized and vesiculated [Plate 6(b)]. During the first 12 h the myofibrils showed relatively little change, which consisted of the formation of a few, scattered contraction bands. At 20 h some areas were encountered in which the striations were faint or absent, with indistinct myofllaments that appeared to have fused into homogeneous masses. These changes occurred in the areas of persistent mitochondrial damage and were seen most frequently after 48 h (Plate 7). However, areas of myofibrillar alterations made up only a small percentage of the total volume of the myocardium, and at 48 and 72 h the greatest part of the contractile elements appeared normal. 4. Discussion A number of different biochemical and hemodynamic changes may contribute to the pathogenesis of the cardiac lesions produced by overdoses of catecholamines [46, 47, 481. It is not surprising, therefore, that some distinctive features emerge on comparison of the histochemical and electron microscopic characteristics of the 2
16
V. J. FRRRANS
ET
AL.
myocardial damage produced by epinephrine [25,49,57], norepinephrine [S, 13,311 and isoproterenol[7, 12, 19, 311. An analysis of these data is presented below. Ch~e.s
in glycogen and phosphxylade
The marked decrease in myocardial glycogen observed after the administration of epinephrine is related to the activation of phosphorylase and acceleration of glycogenolysis that this drug produces in cardiac muscle [9, lo]. Nevertheless, rapid loss of glycogen from cardiac muscle is a non-specific change common to various forms of myocardial damage, particularly that due to ischaemia, hypoxia and infarction [Z, 4, 14, 24, 33, 611. The accumulation of glycogen into membraneenclosed bodies, such as that reported by David et al. [8] in norepinephrine-treated animals, was not observed in this study. In the early stages after the administration of epinephrine there was an increase in the activity of phosphorylase, which may be attributed to the pharmacological effect of the drug. Although the reason for the subsequent decrease in phosphorylase activity is less clear, rapid loss of phosphorylase activity from myocardium has been shown to occur in ischemia and early infarction [ZO, 21, 281 and after the administration of methoxamine and metaraminol [l]. Biochemical studies have shown also that epinephrine produces first an increase and then a decrease in the activity of phosphorylase in heart [60], and liver [273. These observations, and the fact that epinephrine continues to stimulate oxidative metabolism in myocardium after the supply of cardiac muscle glycogen is depleted, are in agreement with the concept [Sl] that the glycogenolytic and metabolic stimulatory effects of epinephrine represent separate phenomena. Lipid accumuluhm The coexistence of cellular damage and lipid accumulation has been observed frequently in cardiac muscle [7, 12,13,14, 241. Conditions of hypoxia, with ensuing acidosis, accumulation of metabolites, and depletion of high energy phosphates, depress the myocardial oxidation of fatty acids. Furthermore, direct effects of epinephrine on lipid metabolism also contribute to the marked elevation of myocardial triglycerides that occurs [373 after the administration of this agent. Epinephrine causes an elevation in the serum level of free fatty aoids (FFA) as the result of an increase in lipid mobilization, which is mediated through the activation of a lipase in adipose tissue [39, 581. Regan [44] has suggested that, in addition to its lipid mobilizing effect, epinephrine has a direct effect in stimulating the myocardial uptake of triglycerides. However, Kreisberg [34] concluded that epinephrine stimulates the utilization of endogenous lipid by myocardium, and that it influences indirectly the myocardial FFA uptake by virtue of its ability to elevate circulating levels of FFA rather than by a direct effect on myocardial extraction. Therefore, the accumulation of lipids observed in cardiac muscle cells after the administration
EPINEPHRINE-INDUCED
MYOCARDJAL
NECROSIS
17
of epinephrine results from the combined effects of increased mobilization of lipid from adipose tissue, and alterations in uptake and utilization of lipids by myocardium. Changes in the sarwplasmic reticulum, T-system and myojbrils Our data, which are in agreement with those obtained by Hiramoto [25] and Shimamoto and Hiramoto [49] in their studies of the effects of epinephrine on rabbit myocardium, ahow that this drug produces much more transient and much less pronounced swelling of the sarcoplasmio reticulum and T-system than do either isoproterenol[7,12,19,32] or norepinephrine [8,13]. In epinephrine-treated animals the sarcoplasmic reticulum and T-system were severely swollen and disrupted only in obviously necrotic cells. Overdoses of isoproterenol and norepinephrine also result in the formation of marked contraction bands; these are so prominent that David et al. [8] have postulated that the myofibrils are the primary site of action of norepinephrine. However, after the administration of epinephrine these changes were found in a relatively small percentage of the muscle cells, usually in those that showed other evidence of necrosis. There is no clear explanation for the preceding differences. They may be the morphological counterparts of specific pharmacological effects of these three agents on cardiac muscle, as suggested by the fact that norepinephrine, like epinephrine, produces only small, patchy foci of necrosis,while isoproterenol, even in low doses, causeslarge, infarct-like lesions that differ histologically from those induced by other sympathomimetic amines. Unfortunately, the possibility that these changes represent dose-related phenomena could not be investigated since the administration of larger dosesof epinephrine caused immediate death from ventricular fibrillation.
Histochemical tests for the activity of various oxidative enzymes are considered to be sensitive indicators of myooardial damage, and can demonstrate mitochondrial alterations that occur prior to the development of changes visible by ordinary histological techniques [2, 30, 50, 591. The histochemical changesobserved in the mitochondria in this study resemble closely those produced by isoproterenol [12] and by norepinephrine [12, 311.The initial rise in the activity of oxidative enzymea paralleled metabolic changesin myocardium, with oxidative metabolism at a peak as the result of the stimulatory effect of epinephrine. The formation of the dark formazan granules observed histochemically may be related to an increase in the permeability of the mitochondria to the tetrazolium salts. These changesreverted to normal except in the areasof necrosis,which showedthe lossof oxidative enzymes associatedwith cellular death. As in the caseof isoproterenol- and norepinephrine-induced myocardial necrosis, electron microscopy showedthat the mitoohondrial damageproduced by epinephrine
18
V. J. FEBRANS
ET AL.
developed more slowly than that due to myocardial infarction [3,5,22,29,33,52]. Of particular interest is the fact that angular configurations of the cristae, such aa those observed in our epiuephrine-treated animals, have not been described previously in the rat heart. However, we have found this type of configuration in a small percentage of the mitochondria in the myocardium of some apparently normal animals. This configuration has been observed also in the ventricle [11] and atrium [38] of the cat, as well as in the small portion of the mitochondria population of a variety of cell types [45]. According to Slautterback [51], such a configuration is very prominent in the mitochondria of cardiac muscle cells of the canary; however, it does not appear to be directly related to the rapid cardiac rate of this species, since it was not observed in the cardiac muscle of other birds with equally fast rates. Angulations of the mitochondrial cristae have been described in aasoaiation with only one pathological state in the human. This change, along with the formation of giant mitochondria, as found by Luft and co-workers [35,36] in the skeletal muscle cells of a patient with an unexplained hypermetabolic state characterized by loss of the control of mitochondrial respiration and partial uncoupling of oxidative phosphorylation. Similar angulations have been observed in the skeletal muscle of rats treated with thyroxin [17], which uncouples oxidative phosphorylation and also shares some of the properties of epinephrine, including the activation of myocardial phosphorylase [23] and the marked increase in oxygen consumption by heart muscle [26]. However, changes of this type were not observed in the mitochondrial cristae either by Poche [40] or by Susin and Her&on [54] in the myocardium of thyroxin-treated rats; furthermore, they are not produced by iaoproterenol[7,12,19,32], and are seen infrequently after the administration of norepinephrine [13]. The angulations of the mitochondrial cristae found in this study correspond to the “energized-twisted” configurations observed in normal rat myocardium by Harris et al. [18] after perfusion with an oxygenated medium that contained substrate for electron transfer aa well as inhibitors (rotenone and rutamycin) which prevented the discharge of the energized state. These authors emphasized the fact that optimal oonditiona of tissue fixation are necessary to demonstrate such changes. The work of Sordahl et al. [53] suggeststhat conformational changes in the inner mitoohondrial membranesmay be more closely related to energy-linked functions, such as ion or substrate transport or to respiratory control. Studies of the combined effects of epinephrine and a- and /%adrenergenicblocking agents on the ultraatructure and biochemistry of mitochondria in cardiac muscle may help to elucidate the nature of these changes. They are of special interest in view of numerous recent studies which have shown that the morphology of mitochondria in a number of tissuescan be modified in specific ways by physiological change+ pathological states and pharmacological agents. It is possible, therefore, that the recognition of alterations in the mitochondria of cardiac muscle will be of help in the classification of various types of myocardial lesions.
EPINEPHRINE-INDUCED
MYOCARDIAL
19
NECROSIS
AckrwwlecEgments This work was supported by Research Grants No. HE-06100, HE-86769 and Career Development Award 5-K3-GM-15-281-05 the United States Public Health Service. REFERENCES 1. BAJUSZ, E. & JASMIN, G. Observations on histochemical
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