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Structural changes in the human myocardium following hypoxia
Structural changes in the human myocardium following hypoxia Walter J. Burdette, Ph.D., M.D., and Thomas P. Ashford,
M.D.,
Salt Lake City, Utah
J_ylectron microscopy offers a medium for localizing damage in the myocardium pre cisely. Since earlier studies of the canine myocardium 13 · 14 delineated a number of changes brought about by hypoxia, samples of myocardium were taken from patients during the course of surgery within the thorax at various times after occlusion of the circulation locally and were examined by means of electron microscopy. Methods Samples of myocardium from the left atrium were removed from 17 patients and left ventricular myocardium from 1. Two patients undergoing pulmonary surgery had biopsies of the atrium taken to compare to the myocardium from 15 patients with rheumatic heart disease. Control samples (in 10 cases) were taken before an oc cluding clamp was placed on the base of the auricular appendages, and subsequent samples were taken at intervals after the clamp was applied. In this way the fine structure of the muscle could be studied after varying periods of hypoxia. Twelve of the patients were males and 6 were females, with ages ranging from 21 to 72
From the Department of Surgery, University of Utah Col lege of Medicine, Salt Lake City, Utah. Aided by grants from the National Institutes of Health, Department of Health, Education and Welfare. Received for publication Feb. 15, 1965.
210
years. In 8 of the cases of rheumatic heart disease the heart was fibrillating; 7 patients required digitalis for control of cardiac failure before surgery. The period of occlusion of blood supply to the muscle varied from 4 to 45 minutes. Specimens were fixed immediately (in the operating suite) either with 1.0 per cent osmium tetroxide (pH 7.4) buffered with potassium phosphate 28 or doubly fixed in glutaraldehyde and osmic acid42 in the same buffer. Following dehydration, the samples were embedded in Epon 812. 25 Lead stain30 was applied to thin sections and studied with a Siemens electron microscope. Conventional paraffin sections were stained with hematoxylin and eosin. Results Light microscope. The changes seen in paraffin sections were compatible with those ordinarily encountered in patients of the respective age and with rheumatic fever when it was present. No change was enTable I. Source of atrial appendage Sex
Control Rheumatic heart di sease Total
No.
M
F
Fibril lating
Digitalized
2
2
0
0
0
15 17
9 11
6 6
8 8
7 7
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Changes in myocardium following hypoxia
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countered that could be attributed to or correlated with the duration or presence of hypoxia. Electron microscope. Changes noted in studies of specimens with the electron microscope were correlated with hypoxia. No difference was found when samples from males were compared to those from females, and the material taken from patients early was compared to that taken later in life. When the morphologic features of various organelles were reviewed in temporal sequence related to the duration of hypoxia, no decisive change was noted until after 15 minutes of occlusion. A number of alterations that appeared to progress as the time of exposure to hypoxia increased were then perceived. The changes noted appeared in the nucleus, the mitochondria, the spatial re lationship of the myofilaments, and in the amount of hydration of the tissue. The human myocardium is abundantly supplied with row after row of mitochondria, im pressive evidence of adaptation to aerobic metabolism. Some of these attain very large size. Early changes in the appearance of the mitochondria subjected to hypoxia con sisted of diminution in number and size of the granules and density of the matrix (Fig. 4ft). Later the matrix became dense and disruption of the organelle occurred (Fig. 5 b). The integrity of the structure of the cristae was maintained (Fig. 5a) in most of the mitochondria seen. Slight condensation of the membrane was seen at times (Fig. Ab), as well as swelling of the entire struc ture. Marginal clumping of nuclear chromatin appeared in 15 to 30 minutes and was quite pronounced at the end of 45 minutes. The heavy granules aggregated at the periphery of the nucleus, but the double membrane appeared intact except for the normal communications with the cytoplasm (Fig. 5a). After half an hour of hypoxia, intracellular fluid had increased in all specimens and was a consistent finding in the entire study (Fig. Ab). With the passage of time, intracellular edema progressed dramatically with spreading of the cellular
components. After clamping of the atrium from 20 to 30 minutes, the myofibrils were separated in bundles when viewed in either longitudinal or cross-section, with retention of the striated fascicular pattern in the former and the stellate arrangement of myo fibrils in the latter view of the portion of the muscle fiber not disrupted (Fig. 5b). Thus the changes were consistent, progres sive, and easily identified, affecting nucleus, mitochondria, myofibrils, and spatial rela tionships of all organelles. A large number of heterogeneous dark bodies were noted in all preparations (Fig. 2b). The amount of glycogen present did not seem related to any of the variables introduced by the de sign of this experiment. The endoplasmic reticulum was easily identified, but its ap pearance in relation to deprivation of oxy gen was not altered consistently. Possible changes in the functional state of mem branes similarly were not detectable in the study by any structural alteration but were suggested by the edema and other changes encountered within the cell. The one sample of left ventricular myocardium studied ex hibited the same changes described for the atrial muscle as a result of hypoxia. Discussion Investigation of the fine structure of cardiac muscle has included examination of samples from dog, cat, rat, guinea pig, frog, turtle, chick, rabbit, mouse, sheep, cow, axolotl, bat, snake, snail, lobster, cockroach, fish, and cyclostome. Fifteen years ago, Burdette7 was the first to use human cardiac muscle taken at operation in a laboratory experiment and followed this with a series of investigations on the living muscle.8-13 Subsequently the general features of myocardial fine structure in the human heart have been noted by a number of investi gators.4' 22> -1' 35>3G'41- 48 In addition to surgi cal excision, sufficient muscle for study has been obtained by the use of a biopsy needle in patients 45 as well as in dogs.39 Changes in structure with failure,23 age, hyper trophy,41 and following arrest36 have been described. Examination of electron micro-
Fig. 1. This electron micrograph shows a portion of two myocardial cells from the auricle of a patient with a normal cardiovascular system. The cells are divided by the extracellular space stretching from lower left to upper right. The sarcolemma (S) contains numerous pits (P), probably representing pinocytosis, the process of engulfing larger molecular aggregations. The myofibrils (myo) are parallel in this longitudinal section and display the longitudinal striations of individual myofilaments and the transverse markings of the various bands. The most prominent recurring band is the Z band (Z). The space between the myofibrils con tains mitochondria (m) glycogen granules (gly), and smooth endoplasmic reticulum (er). Some glycogen is interspersed with the myofilaments (A). The reticulum at the Z line is a portion of transverse system (T) that is recognized as an invagination of the sarcolemma in certain types of striated muscle. The small dark bodies (L) probably represent lipid bodies. The bar in the lower right hand corner of each micrograph represents one micron. (xl5,000; reduced }4o·)
Volume 50 Number 2 August, 1965
Changes in myocardium following hypoxia 2 13
Fig. 2a= Ihis puiliun u! a ruit.luus from a rheumatic myocardial cell before occlusion demon strates finely divided, well-distributed chromatin, a usual finding using current methods of fixation. The nucleolus (Nu) is seen as an area of denser material and the nuclear envelope (e) as a double membrane perforated by pores (g). (xl9,000; reduced YJ.)
Fig. 2b. This section represents a portion of a normal myocardial cell from the human auricle under higher power. The collection of heterogeneous dark bodies (S) seen is characteristic of all human auricular tissue examined. These are more prominent in human myocardial tissue than in others reported or examined. They possibly represent the end product of the action of acid hydrolytic enzymes and may represent a group of myocardial lysosomes (sarcosomes). A nucleus (N) is also seen. (xl5,000; reduced %.)
2 14
Burdette and Ashjord
Journal of Thoracic and Cardiovascular Surgery
Fig. 3a. This normal myocardial cell demonstrates features described in Fig. 1 at a higher power. Note the basement membrane (bm) of the sarcolemma. (x24,000; reduced Vr·)
Fig. 3b. This represents a myocardial cell from a patient with rheumatic heart disease before occlusion of the blood supply. It is similar to the normal myocardial cell (Figs. 1 and 3a) in every respect. A capillary endothelial cell (end) is present in the upper left hand corner of the photograph. (x27,000; reduced ¥,.)
Volume 50
Changes in myocardium following hypoxia 2 15
Number 2 August, 1965
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Fig. 4a. This sample taken before occlusion of a rheumatic auricle exhibits the same features seen normally. It should be compared to Fig. 4b. (χ24,000; reduced Vi-)
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Fig. 4b. This electron micrograph represents moderate change after hypoxia. The mito chondria (m) have lost the characteristic dense granules that probably represent collections of divalent cations (Mg, Mn, Ca), although they demonstrate little other change of form. The uneven mitochondrial matrix seen here probably represents an artifact of sectioning. The cristae show slight condensation. Although the membranous system is generally intact, there is an obvious increase in intracellular edema with myoflbrils (myo) still well organized. The sarcolemma remains normal with well-developed pits (p). (x23,000; reduced ty.)
Journal of
2 16
Burdette and Ashford
Thoracic and Cardiovascular Surgery
Fig. 5a. The nucleus (N) of a myocardial cell after lengthy occlusion displays margination and clumping of chromatin (compare with Fig. 2 a ) . Although the envelope is obscured, it is apparently intact (e). The mitochondria (in) are denser than normal, and fibrin is precipi tated in the intercellular space. Pits are still present. (x24,000; reduced \<[.)
Fig. 5b. This is a cross section of a human myocardial cell after lengthy occlusion of the blood supply. The thick and thin filaments of the myofibrils can be seen to be interspersed in a geometric pattern. Although those filaments in contact have maintained the same spatial relationships with other myofilaments, groups of filaments are widely dispersed and in other sections are rarely parallel for any significant distance. The extreme amount of intracellular edema is very striking. In these cells, the organization of the endoplasmic reticulum is dis rupted (er), and the mitochondria are damaged to an advanced degree. (x29,000; reduced %.)
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Changes in myocardium following hypoxia
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August, 1905
graphs of human material taken at opera tion or autopsy suggest that specimens not expected to be unusual do not always yield a valid picture of normal myocardium, probably because of technical problems and delay in fixing the specimens, in addition to the state of the myocardium due to disease. Care, therefore, is necessary to distinguish real from spurious appearances of human material. With the possible ex ception of more abundant lipid droplets, nothing exceptional has been pointed out about the fine structure of human myocar dium not observed in cardiac tissue from other sources. The appearance of cardiac muscle 14 '19 47,49 j s v e r v m u c n like that of skeletal muscle5 in general features, and some of the original ideas concerning its syncytial nature have been abandoned as the signifi cance of the intercalated disc as a cellular boundary has been recognized. 31 - 33 · 38 - 44 · 51 The muscle constituting atrium and ventri cle is quite similar, with only minor possible differences.21 The structure of the nucleus with its double membrane and pores and contained nucleolus or nucleoli is the same granular enigma featured in other tissues. The festooned contour sometimes observed is probably associated with contraction of myofibrils. The same striations and relation ships of thick and thin fibrils are seen in cardiac and skeletal muscle. The sliding1 s and folding32 theories of contraction, or a combination of the two, have not been en tirely resolved by current studies. Three dimensional reconstructions of endoplasmic reticulum reveal a remarkable branching system of conduits connecting the sarcolemma to the interior of the cell,37 - 43 and studies on prodigiously quick-acting skeletal muscles, such as the cricothyroid of the bat responsible for emitting radar-like signals40 and fish,15 have been useful in determining the ramifications of this system. Although human cardiac muscle contains endoplasmic reticulum in the usual locations, it is less prominent than in specimens from other sites and species. Possibly the T-system ex tending inward from the sarcolemma at the Z line synchronizes activity of the inner
myofibrils with those more peripheral in human myocardium, as in other muscular tissue. Although the neural elements have been described,14' 20>2C· 34- 47 - 48 more exten sive investigation is needed, as well as additional study of the possible role of en doplasmic reticulum in the transmission of the action potential. Equally desirable is a better understanding of the functions of the sarcolemma and other membranes, in cluding those of the mitochondria. The abundant pinocytotic globules in the sarco lemma of human muscle suggest a very active membranous structure. The mito chondria with contained cristae continuing the double outer membrane are exception ally numerous in cardiac muscle, but we have not found a strict ratio of numbers of mitochondria to sarcomeres in the human myocardium. Nowhere is the relationship of morphology to function better illustrated than in the myocardium, and studies of bio chemical events should be related to the information available from the fine struc ture. 13 Recent advances in technology that permit localization of labeled compounds, coupled with other cytochemical techniques in electron micrographs, 3 - 36 - 4G - 50 should be exceedingly valuable in future additional studies. Brief asphyxia of the canine heart 3 to 4 minutes in duration has been used by Bahr and Jennings2 to relax the myocardium for studies of fine structure. They found no other changes in the dog after this brief period. When Bryant and associates6 ligated the anterior descending branch of the left coronary in the rat, the mitochondria and sarcoplasmic reticulum became swollen, followed by the appearance of an increasing number of lipid droplets and myolysis. These changes occurred 1 hour after ligation and became more severe in the infarct as the interval following ligation increased. Others16 have also recorded changes in the murine myocardium postmortem. When cardiac arrest was induced in rabbits and dogs by Hoelscher and his co-workers,17 the mitochondria of the ventricular myocar dium became swollen and vacuolated with clumping of the cristae, and empty areas
Journal of
2 18
Burdette and Ashford
Thoracic and Cardiovascular Surgery
were seen in the myofibrils along with more numerous lipid bodies. These changes were inhibited by hypothermia. Potassium and sodium citrate produced ballooning of the endoplasmic reticulum. Zonal lesions were noted when the myocardium was studied during shock by Martin and associates,27 and Miller and co-workers29 presented evidence suggesting residual damage to canine mitochondria 24 to 72 hours. after recovery from myocardial hypoxia, although the cells appeared and performed as usual otherwise. In our laboratory the effect of hypoxia on the fine structure of the canine myo cardium 13 was determined before the in vestigation on human myocardium being reported was completed. Condensation of the mitochondrial envelope occurred along with clearing and subsequent general and focal condensation of the matrix and less pronounced changes in the cristae. Periph eral condensation of nucleus and intracellular edema were also noted. Hypothermia inhibited these changes. It is apparent that the changes noted in human myocardium are not so far advanced and complete as in the normothermic canine myocardium, but they are very similar in nature. During exposure and occlusion of the circulation to the atrial appendage at the time of surgery, the temperature of the muscle was doubtless not maintained at 37.5° C. Since hypothermia protects the myocardium against these changes in fine structure, the difference in extent of damage in human and canine myocardium may be more apparent than real. In a recent study of morphologic changes in hepatic cells related to cellular death, 1 disorganization of endo plasmic reticulum, disruption and flattening of the cellular membrane, and vacuolization of the cytoplasm were found to be corre lated. None of these alterations obviously accompany hypoxia of the myocardium in the cases studied. On the other hand the structure of hepatic mitochondria in the liver of rats perfused seemed more resistant to anaerobic conditions than either canine or human cardiac mitochondria. The signif-
icance of sarcosomes in relation to hypoxia of human myocardium is not apparent from the samples available (Fig. 2b). No attempt was made to study the rheumatic lesions in the auricles. (On the basis of examinations with the electron microscope, Lannigan and Zaki 24 believe they follow damage to con nective tissue primarily.) The changes in structure of the human myocardium reported suggest that anaerobic conditions alter cardiac function by dis rupting the structural integrity of the inotropic unit, the transport of fluid across membranes, and the utilization of substrate via the Krebs cycle and oxidative phosphorylation as functions of the mitochon dria. Attempts to remedy early metabolic and structural derangements resulting from anaerobic conditions should be directed toward these defects. Summary Several consistent and progressive changes in the fine structure of the human myocardium obtained at operation were easily identified after hypoxia was induced by clamping the auricular appendage for varying periods of time. They appeared in the nucleus and mitochondria and affected the spatial relationships of myofilaments and amount of hydration of the tissues. Therefore, attempts to remedy early meta bolic and structural derangements resulting from anoxia should be directed toward membranous transport of fluid and ions, aerobic utilization of substrate, oxidative phosphorylation, and restoration of the structural integrity of the inotropic unit. The technical assistance of Jo Ann Kolb is grate fully acknowledged. REFERENCES 1 Ashford, T. P., and Burdette, W. J.: Re sponses of the Isolated Perfused Hepatic Parenchyma to Hypoxia. Ann. Surg. In press. 2 Bahr, G. F., and Jennings, R. B.: Ultrastruc ture of Normal and Asphyxie Myocardium of the Dog, Lab. Invest. 10: 548, 1961. 3 Barnett, R. J., editor: Symposium Applica tion of Cytochemistry to Electron Micros copy, J. Histochem. & Cytochem. 12: 1, 1964.
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4 Battig, C. G., and Low, F . N.: The Ultrastructure of Human Cardiac Muscle and Its Associated Tissue Space, Am. J. Anat. 108: 199, 1961. 5 Bennett, H. S.: Modern Concepts of Struc ture of Striated Muscle, Am. J. Phys. Med. 34: 46, 1955. 6 Bryant, R. E., Thomas, W. A., and O'Neal, R. M.: An Electron Microscopic Study of Myocardial Ischemia in the Rat, Circulation Res. 6: 699, 1958. 7 Burdette, W. J.: Studies on the Metabolism of Human Cardiac Muscle Obtained by Auric ular Appendectomy, Yale J. Biol. & Med. 24: 9, 1951. 8 Burdette, W. J.: Increase in Oxygen Consump tion of Human Cardiac Muscle Incubated With Lanatoside C, J. Lab. & Clin. Med. 40: 867, 1952. 9 Burdette, W. J.: The Krebs Cycle in Human Cardiac Muscle, Am. Heart J. 44: 823, 1952. 10 Burdette, W. J.: Exposure of Human Cardiac Muscle to Radioactive Digitoxin In Vitro, Am. Heart J. 46: 602, 1953. 11 Burdette, W. J.: Nucleotide Levels in Human Cardiac Muscle, Proc. Soc. Clin. Res. 9: 8, 1954. 12 Burdette, W. J., and Al-Shamma, A.: Changes in High-Energy Phosphates During Cardiac Arrest, Arch. Surg. 85: 4, 1962. 13 Burdette, W. J., and Ashford, T. P.: Responses of Myocardial Fine Structure to Cardiac Ar rest and Hypothermia, Ann. Surg. 158: 513, 1963. 14 Fawcett, D. W., and Selby, C. C : Observa tions on the Fine Structure of the Turtle Atrium, J. Biophys. & Biochem. Cytol. 4: 63, 1958. 15 Fawcett, D . W., and Revel, J. P.: The Sarcoplasmic Reticulum of a Fast-acting Fish Muscle, J. Biophys. & Biochem. Cytol. 10: Suppl. 89, 1961. 16 Hibbs, R. G., and Black, W. C : Electron Microscopy of Post Mortem Changes in the Rat Myocardium, Anat. Rec. 147: 261, 1963. 17 Hoelscher, B., Just, O. H., and Merker, H. J.: Studies on Electron Microscope on Various Forms of Induced Cardiac Arrest in Dog and Rabbit, Surgery 49: 492, 1964. 18 Huxley, H. E., and Hanson, J.: The Struc tural Basis of the Contraction Mechanism in Striated Muscle, Ann. New York Acad. Sc. 81: 403, 1959. 19 Khristolyubova, N . B.: Study of the Structure of Heart Muscle With the Electron Micro scope, Rep. Acad. Se. U.S.S.R. 119: 168, 1958. 20 Kisch, B.: Electron Microscopy of Cardiac Nerves, Am. J. Cardiol. 2: 475, 1958. 21 Kisch, B.: A Significant Electron Microscopic Difference Between the Atria and the Ven
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23
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28 29
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33
34
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tricles of the Mammalian Heart, Exper. Med. & Surg. 2 1 : 193, 1963. Kisch, B., and Cavusoglu, M.: The Ultrastruc ture of the Human Heart, Exper. Med. & Surg. 18: 70, 1960. Kisch, B., Cavusoglu, M., and Marangoni, B. A.: Electron Microscopic Changes in the Human Heart in Cardiac Failure, Exper. Med. & Surg. 17: 85, 1959. Lannigan, R., and Zaki, S.: Electron Micro scopic Appearance of Rheumatic Lesions in the Left Auricular Appendage in Mitral Ste nosis, Nature 198: 898, 1963. Luft, J. H.: Improvements in Epoxy Embed ding Methods, J. Biophys. & Biochem. Cytol. 9: 409, 1961. Mandelmann, N., Stucky, J. H., Hoffman, B. F., and Herman, L.: Comparative Electron Microscopy of Purkinje Fibers and Ventricu lar Muscle of Dog Heart, S. Forum 13: 202, 1962. Martin, A. M., Jr., Hackel, D . B., and Kurtz, S. M.: The Ultrastructure of Zonal Lesions of the Myocardium in Hemorrhagic Shock, Am. J. Path. 44: 127, 1964. Miller, F.: Universität München. Personal communication. Miller, D . R., Rasmussen, P., and Klinosky, B.: Reversibility of Morphologic Changes Fol lowing Elective Cardiac Arrest, Ann. Surg. 159: 208, 1964. Millonig, G.: A Modified Procedure for Lead Staining of Thin Sections, J. Biophys. & Bio chem. Cytol. 11: 736, 1961. Moore, D . H., and Ruska, H.: Electron Mi croscope Studies of Mammalian Cardiac Mus cle Cells, J. Biophys. & Biochem. Cytol. 3: 261, 1957. Morales, M. F., and Botts, J.: A Model for the Elementary Process in Muscle Action, Arch. Biochem. 37: 283, 1952. Muir, A. R.: An Electron Microscope Study of the Embryology of the Intercalated Disc in the Heart of the Rabbit, J. Biophys. & Biochem. Cytol. 3: 193, 1957. Napolitano, L., Cooper, T., Williams, V. L., and Hanlon, C. R.: Fine Structure of the Heart After Transplantation With Special Ref erence to the Neural Elements, Circulation 29: 8 1 , 1964. Nelson, D. A., and Benson, E. S.: On the Structural Continuities of the Transverse Tubu lar System of Rabbit and Human Myocardial Cells, J. Cell Biol. 16: 297, 1963. Poche, R., and Ohm, H. G.: Photomicroscopic, Histochemical, and Electron Micro scopic Studies of the Human Heart Muscle After Induced Heart Arrest, Arch. Kreislauf forsch. 41: 86, 1963. Porter, K. R., and Paladi, G. E.: Studies on
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the Endoplasmic Reticulum. III. Its Form and Distribution in Striated Muscle Cells, J. Biochem. & Biophys. Cytol. 3: 269, 1957. Price, A., Eide, B., Prinzmetal, M., and Car penter, C : Ultrastructure of the Dog Cardiac Muscle Cell, Circulation Res. 7: 858, 1959. Price, K., Weiss, J. M., Daikichi, H., and Smith, J. R.: Experimental Needle Biopsy of the Myocardium of Dogs With Particular Reference to Histologie Study by Electron Microscope, J. Exper. Med. 101: 687, 1955. Revel, J. P.: The Sarcoplasmic Reticulum of the Bat Cricothyroid Muscle, J. Cell. Biol. 12: 571, 1962. Richter, G. W., and Kellner, A.: Hypertrophy of the H u m a n Heart at the Level of Fine Structure, J. Cell Biol. 18: 195, 1963. Sabatini, D . D., Bensch, K., and Barrnett, R. J.: Cytochemistry and Electron Micros copy. The Preservation of Cellular Ultrastructure and Enzymatic Activity by Alde hyde Fixation, J. Cell. Biol. 17: 19, 1963. Simpson, F . O., and Oertelis, S. J.: The Fine Structure of Sheep Myocardial Cells; Sarcolemmal Invaginations and the Transverse Tu bular System, J. Cell Biol. 12: 91, 1962. Sjöstrand, F . S.: Andersson-Cedergren, E., and Dewey, M. M.: The Ultrastructure of the
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Intercalated Discs of Frog, Mouse, Guinea Pig Cardiac Muscle, J. Ultrastructure Res. 1: 271, 1958. Smith, J. R., Burford, T. H., and Chiquoine, A. D.: Electron Microscopic Observations of the Ventricular Heart Muscle of Man Ob tained by Surgical Biopsy During Thoracotomy, Exper. Cell. Res. 20: 228, 1960. Smith, J. R., and Fozzard, H. A.: Localiza tion of Tritriated Digoxin in the Myocardial Cell of Frogs and Dogs by Autoradiography Combined With Electron Microscopy, Nature 197: 562, 1963. Spiro, D.: The Ultrastructure of Heart Mus cle, Tr. New York Acad. Sc. 24: 879, 1962. Stein, A. A., Thibodeau, F., and Stranahan, A.: Electron Microscope Studies of Human Myocardium, J. A. M. A. 182: 537, 1962. Stenger, R. J., and Spiro, D.: Structure of Cardiac Muscle Cell, Am. J. Med. 30: 653, 1961. Tubbs, F . E., Crevasse, L., and Wheat, M. W.: Localization of Tritiated Digoxin in Dog Myocardium by Electron Microscopic Auto radiography, Circulation Res. 14: 236, 1964. Van Breeman, V. L.: Intercalated Discs in Heart Muscle Studies With the Electron Mi croscope, Anat. Rec. 117: 49, 1953.
M.D.,
Salt Lake City, Utah
J_ylectron microscopy offers a medium for localizing damage in the myocardium pre cisely. Since earlier studies of the canine myocardium 13 · 14 delineated a number of changes brought about by hypoxia, samples of myocardium were taken from patients during the course of surgery within the thorax at various times after occlusion of the circulation locally and were examined by means of electron microscopy. Methods Samples of myocardium from the left atrium were removed from 17 patients and left ventricular myocardium from 1. Two patients undergoing pulmonary surgery had biopsies of the atrium taken to compare to the myocardium from 15 patients with rheumatic heart disease. Control samples (in 10 cases) were taken before an oc cluding clamp was placed on the base of the auricular appendages, and subsequent samples were taken at intervals after the clamp was applied. In this way the fine structure of the muscle could be studied after varying periods of hypoxia. Twelve of the patients were males and 6 were females, with ages ranging from 21 to 72
From the Department of Surgery, University of Utah Col lege of Medicine, Salt Lake City, Utah. Aided by grants from the National Institutes of Health, Department of Health, Education and Welfare. Received for publication Feb. 15, 1965.
210
years. In 8 of the cases of rheumatic heart disease the heart was fibrillating; 7 patients required digitalis for control of cardiac failure before surgery. The period of occlusion of blood supply to the muscle varied from 4 to 45 minutes. Specimens were fixed immediately (in the operating suite) either with 1.0 per cent osmium tetroxide (pH 7.4) buffered with potassium phosphate 28 or doubly fixed in glutaraldehyde and osmic acid42 in the same buffer. Following dehydration, the samples were embedded in Epon 812. 25 Lead stain30 was applied to thin sections and studied with a Siemens electron microscope. Conventional paraffin sections were stained with hematoxylin and eosin. Results Light microscope. The changes seen in paraffin sections were compatible with those ordinarily encountered in patients of the respective age and with rheumatic fever when it was present. No change was enTable I. Source of atrial appendage Sex
Control Rheumatic heart di sease Total
No.
M
F
Fibril lating
Digitalized
2
2
0
0
0
15 17
9 11
6 6
8 8
7 7
Volume 50
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Changes in myocardium following hypoxia
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August, 1965
countered that could be attributed to or correlated with the duration or presence of hypoxia. Electron microscope. Changes noted in studies of specimens with the electron microscope were correlated with hypoxia. No difference was found when samples from males were compared to those from females, and the material taken from patients early was compared to that taken later in life. When the morphologic features of various organelles were reviewed in temporal sequence related to the duration of hypoxia, no decisive change was noted until after 15 minutes of occlusion. A number of alterations that appeared to progress as the time of exposure to hypoxia increased were then perceived. The changes noted appeared in the nucleus, the mitochondria, the spatial re lationship of the myofilaments, and in the amount of hydration of the tissue. The human myocardium is abundantly supplied with row after row of mitochondria, im pressive evidence of adaptation to aerobic metabolism. Some of these attain very large size. Early changes in the appearance of the mitochondria subjected to hypoxia con sisted of diminution in number and size of the granules and density of the matrix (Fig. 4ft). Later the matrix became dense and disruption of the organelle occurred (Fig. 5 b). The integrity of the structure of the cristae was maintained (Fig. 5a) in most of the mitochondria seen. Slight condensation of the membrane was seen at times (Fig. Ab), as well as swelling of the entire struc ture. Marginal clumping of nuclear chromatin appeared in 15 to 30 minutes and was quite pronounced at the end of 45 minutes. The heavy granules aggregated at the periphery of the nucleus, but the double membrane appeared intact except for the normal communications with the cytoplasm (Fig. 5a). After half an hour of hypoxia, intracellular fluid had increased in all specimens and was a consistent finding in the entire study (Fig. Ab). With the passage of time, intracellular edema progressed dramatically with spreading of the cellular
components. After clamping of the atrium from 20 to 30 minutes, the myofibrils were separated in bundles when viewed in either longitudinal or cross-section, with retention of the striated fascicular pattern in the former and the stellate arrangement of myo fibrils in the latter view of the portion of the muscle fiber not disrupted (Fig. 5b). Thus the changes were consistent, progres sive, and easily identified, affecting nucleus, mitochondria, myofibrils, and spatial rela tionships of all organelles. A large number of heterogeneous dark bodies were noted in all preparations (Fig. 2b). The amount of glycogen present did not seem related to any of the variables introduced by the de sign of this experiment. The endoplasmic reticulum was easily identified, but its ap pearance in relation to deprivation of oxy gen was not altered consistently. Possible changes in the functional state of mem branes similarly were not detectable in the study by any structural alteration but were suggested by the edema and other changes encountered within the cell. The one sample of left ventricular myocardium studied ex hibited the same changes described for the atrial muscle as a result of hypoxia. Discussion Investigation of the fine structure of cardiac muscle has included examination of samples from dog, cat, rat, guinea pig, frog, turtle, chick, rabbit, mouse, sheep, cow, axolotl, bat, snake, snail, lobster, cockroach, fish, and cyclostome. Fifteen years ago, Burdette7 was the first to use human cardiac muscle taken at operation in a laboratory experiment and followed this with a series of investigations on the living muscle.8-13 Subsequently the general features of myocardial fine structure in the human heart have been noted by a number of investi gators.4' 22> -1' 35>3G'41- 48 In addition to surgi cal excision, sufficient muscle for study has been obtained by the use of a biopsy needle in patients 45 as well as in dogs.39 Changes in structure with failure,23 age, hyper trophy,41 and following arrest36 have been described. Examination of electron micro-
Fig. 1. This electron micrograph shows a portion of two myocardial cells from the auricle of a patient with a normal cardiovascular system. The cells are divided by the extracellular space stretching from lower left to upper right. The sarcolemma (S) contains numerous pits (P), probably representing pinocytosis, the process of engulfing larger molecular aggregations. The myofibrils (myo) are parallel in this longitudinal section and display the longitudinal striations of individual myofilaments and the transverse markings of the various bands. The most prominent recurring band is the Z band (Z). The space between the myofibrils con tains mitochondria (m) glycogen granules (gly), and smooth endoplasmic reticulum (er). Some glycogen is interspersed with the myofilaments (A). The reticulum at the Z line is a portion of transverse system (T) that is recognized as an invagination of the sarcolemma in certain types of striated muscle. The small dark bodies (L) probably represent lipid bodies. The bar in the lower right hand corner of each micrograph represents one micron. (xl5,000; reduced }4o·)
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Changes in myocardium following hypoxia 2 13
Fig. 2a= Ihis puiliun u! a ruit.luus from a rheumatic myocardial cell before occlusion demon strates finely divided, well-distributed chromatin, a usual finding using current methods of fixation. The nucleolus (Nu) is seen as an area of denser material and the nuclear envelope (e) as a double membrane perforated by pores (g). (xl9,000; reduced YJ.)
Fig. 2b. This section represents a portion of a normal myocardial cell from the human auricle under higher power. The collection of heterogeneous dark bodies (S) seen is characteristic of all human auricular tissue examined. These are more prominent in human myocardial tissue than in others reported or examined. They possibly represent the end product of the action of acid hydrolytic enzymes and may represent a group of myocardial lysosomes (sarcosomes). A nucleus (N) is also seen. (xl5,000; reduced %.)
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Fig. 3a. This normal myocardial cell demonstrates features described in Fig. 1 at a higher power. Note the basement membrane (bm) of the sarcolemma. (x24,000; reduced Vr·)
Fig. 3b. This represents a myocardial cell from a patient with rheumatic heart disease before occlusion of the blood supply. It is similar to the normal myocardial cell (Figs. 1 and 3a) in every respect. A capillary endothelial cell (end) is present in the upper left hand corner of the photograph. (x27,000; reduced ¥,.)
Volume 50
Changes in myocardium following hypoxia 2 15
Number 2 August, 1965
toœ***^ '"
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Fig. 4a. This sample taken before occlusion of a rheumatic auricle exhibits the same features seen normally. It should be compared to Fig. 4b. (χ24,000; reduced Vi-)
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Fig. 4b. This electron micrograph represents moderate change after hypoxia. The mito chondria (m) have lost the characteristic dense granules that probably represent collections of divalent cations (Mg, Mn, Ca), although they demonstrate little other change of form. The uneven mitochondrial matrix seen here probably represents an artifact of sectioning. The cristae show slight condensation. Although the membranous system is generally intact, there is an obvious increase in intracellular edema with myoflbrils (myo) still well organized. The sarcolemma remains normal with well-developed pits (p). (x23,000; reduced ty.)
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Fig. 5a. The nucleus (N) of a myocardial cell after lengthy occlusion displays margination and clumping of chromatin (compare with Fig. 2 a ) . Although the envelope is obscured, it is apparently intact (e). The mitochondria (in) are denser than normal, and fibrin is precipi tated in the intercellular space. Pits are still present. (x24,000; reduced \<[.)
Fig. 5b. This is a cross section of a human myocardial cell after lengthy occlusion of the blood supply. The thick and thin filaments of the myofibrils can be seen to be interspersed in a geometric pattern. Although those filaments in contact have maintained the same spatial relationships with other myofilaments, groups of filaments are widely dispersed and in other sections are rarely parallel for any significant distance. The extreme amount of intracellular edema is very striking. In these cells, the organization of the endoplasmic reticulum is dis rupted (er), and the mitochondria are damaged to an advanced degree. (x29,000; reduced %.)
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Changes in myocardium following hypoxia
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graphs of human material taken at opera tion or autopsy suggest that specimens not expected to be unusual do not always yield a valid picture of normal myocardium, probably because of technical problems and delay in fixing the specimens, in addition to the state of the myocardium due to disease. Care, therefore, is necessary to distinguish real from spurious appearances of human material. With the possible ex ception of more abundant lipid droplets, nothing exceptional has been pointed out about the fine structure of human myocar dium not observed in cardiac tissue from other sources. The appearance of cardiac muscle 14 '19 47,49 j s v e r v m u c n like that of skeletal muscle5 in general features, and some of the original ideas concerning its syncytial nature have been abandoned as the signifi cance of the intercalated disc as a cellular boundary has been recognized. 31 - 33 · 38 - 44 · 51 The muscle constituting atrium and ventri cle is quite similar, with only minor possible differences.21 The structure of the nucleus with its double membrane and pores and contained nucleolus or nucleoli is the same granular enigma featured in other tissues. The festooned contour sometimes observed is probably associated with contraction of myofibrils. The same striations and relation ships of thick and thin fibrils are seen in cardiac and skeletal muscle. The sliding1 s and folding32 theories of contraction, or a combination of the two, have not been en tirely resolved by current studies. Three dimensional reconstructions of endoplasmic reticulum reveal a remarkable branching system of conduits connecting the sarcolemma to the interior of the cell,37 - 43 and studies on prodigiously quick-acting skeletal muscles, such as the cricothyroid of the bat responsible for emitting radar-like signals40 and fish,15 have been useful in determining the ramifications of this system. Although human cardiac muscle contains endoplasmic reticulum in the usual locations, it is less prominent than in specimens from other sites and species. Possibly the T-system ex tending inward from the sarcolemma at the Z line synchronizes activity of the inner
myofibrils with those more peripheral in human myocardium, as in other muscular tissue. Although the neural elements have been described,14' 20>2C· 34- 47 - 48 more exten sive investigation is needed, as well as additional study of the possible role of en doplasmic reticulum in the transmission of the action potential. Equally desirable is a better understanding of the functions of the sarcolemma and other membranes, in cluding those of the mitochondria. The abundant pinocytotic globules in the sarco lemma of human muscle suggest a very active membranous structure. The mito chondria with contained cristae continuing the double outer membrane are exception ally numerous in cardiac muscle, but we have not found a strict ratio of numbers of mitochondria to sarcomeres in the human myocardium. Nowhere is the relationship of morphology to function better illustrated than in the myocardium, and studies of bio chemical events should be related to the information available from the fine struc ture. 13 Recent advances in technology that permit localization of labeled compounds, coupled with other cytochemical techniques in electron micrographs, 3 - 36 - 4G - 50 should be exceedingly valuable in future additional studies. Brief asphyxia of the canine heart 3 to 4 minutes in duration has been used by Bahr and Jennings2 to relax the myocardium for studies of fine structure. They found no other changes in the dog after this brief period. When Bryant and associates6 ligated the anterior descending branch of the left coronary in the rat, the mitochondria and sarcoplasmic reticulum became swollen, followed by the appearance of an increasing number of lipid droplets and myolysis. These changes occurred 1 hour after ligation and became more severe in the infarct as the interval following ligation increased. Others16 have also recorded changes in the murine myocardium postmortem. When cardiac arrest was induced in rabbits and dogs by Hoelscher and his co-workers,17 the mitochondria of the ventricular myocar dium became swollen and vacuolated with clumping of the cristae, and empty areas
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were seen in the myofibrils along with more numerous lipid bodies. These changes were inhibited by hypothermia. Potassium and sodium citrate produced ballooning of the endoplasmic reticulum. Zonal lesions were noted when the myocardium was studied during shock by Martin and associates,27 and Miller and co-workers29 presented evidence suggesting residual damage to canine mitochondria 24 to 72 hours. after recovery from myocardial hypoxia, although the cells appeared and performed as usual otherwise. In our laboratory the effect of hypoxia on the fine structure of the canine myo cardium 13 was determined before the in vestigation on human myocardium being reported was completed. Condensation of the mitochondrial envelope occurred along with clearing and subsequent general and focal condensation of the matrix and less pronounced changes in the cristae. Periph eral condensation of nucleus and intracellular edema were also noted. Hypothermia inhibited these changes. It is apparent that the changes noted in human myocardium are not so far advanced and complete as in the normothermic canine myocardium, but they are very similar in nature. During exposure and occlusion of the circulation to the atrial appendage at the time of surgery, the temperature of the muscle was doubtless not maintained at 37.5° C. Since hypothermia protects the myocardium against these changes in fine structure, the difference in extent of damage in human and canine myocardium may be more apparent than real. In a recent study of morphologic changes in hepatic cells related to cellular death, 1 disorganization of endo plasmic reticulum, disruption and flattening of the cellular membrane, and vacuolization of the cytoplasm were found to be corre lated. None of these alterations obviously accompany hypoxia of the myocardium in the cases studied. On the other hand the structure of hepatic mitochondria in the liver of rats perfused seemed more resistant to anaerobic conditions than either canine or human cardiac mitochondria. The signif-
icance of sarcosomes in relation to hypoxia of human myocardium is not apparent from the samples available (Fig. 2b). No attempt was made to study the rheumatic lesions in the auricles. (On the basis of examinations with the electron microscope, Lannigan and Zaki 24 believe they follow damage to con nective tissue primarily.) The changes in structure of the human myocardium reported suggest that anaerobic conditions alter cardiac function by dis rupting the structural integrity of the inotropic unit, the transport of fluid across membranes, and the utilization of substrate via the Krebs cycle and oxidative phosphorylation as functions of the mitochon dria. Attempts to remedy early metabolic and structural derangements resulting from anaerobic conditions should be directed toward these defects. Summary Several consistent and progressive changes in the fine structure of the human myocardium obtained at operation were easily identified after hypoxia was induced by clamping the auricular appendage for varying periods of time. They appeared in the nucleus and mitochondria and affected the spatial relationships of myofilaments and amount of hydration of the tissues. Therefore, attempts to remedy early meta bolic and structural derangements resulting from anoxia should be directed toward membranous transport of fluid and ions, aerobic utilization of substrate, oxidative phosphorylation, and restoration of the structural integrity of the inotropic unit. The technical assistance of Jo Ann Kolb is grate fully acknowledged. REFERENCES 1 Ashford, T. P., and Burdette, W. J.: Re sponses of the Isolated Perfused Hepatic Parenchyma to Hypoxia. Ann. Surg. In press. 2 Bahr, G. F., and Jennings, R. B.: Ultrastruc ture of Normal and Asphyxie Myocardium of the Dog, Lab. Invest. 10: 548, 1961. 3 Barnett, R. J., editor: Symposium Applica tion of Cytochemistry to Electron Micros copy, J. Histochem. & Cytochem. 12: 1, 1964.
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4 Battig, C. G., and Low, F . N.: The Ultrastructure of Human Cardiac Muscle and Its Associated Tissue Space, Am. J. Anat. 108: 199, 1961. 5 Bennett, H. S.: Modern Concepts of Struc ture of Striated Muscle, Am. J. Phys. Med. 34: 46, 1955. 6 Bryant, R. E., Thomas, W. A., and O'Neal, R. M.: An Electron Microscopic Study of Myocardial Ischemia in the Rat, Circulation Res. 6: 699, 1958. 7 Burdette, W. J.: Studies on the Metabolism of Human Cardiac Muscle Obtained by Auric ular Appendectomy, Yale J. Biol. & Med. 24: 9, 1951. 8 Burdette, W. J.: Increase in Oxygen Consump tion of Human Cardiac Muscle Incubated With Lanatoside C, J. Lab. & Clin. Med. 40: 867, 1952. 9 Burdette, W. J.: The Krebs Cycle in Human Cardiac Muscle, Am. Heart J. 44: 823, 1952. 10 Burdette, W. J.: Exposure of Human Cardiac Muscle to Radioactive Digitoxin In Vitro, Am. Heart J. 46: 602, 1953. 11 Burdette, W. J.: Nucleotide Levels in Human Cardiac Muscle, Proc. Soc. Clin. Res. 9: 8, 1954. 12 Burdette, W. J., and Al-Shamma, A.: Changes in High-Energy Phosphates During Cardiac Arrest, Arch. Surg. 85: 4, 1962. 13 Burdette, W. J., and Ashford, T. P.: Responses of Myocardial Fine Structure to Cardiac Ar rest and Hypothermia, Ann. Surg. 158: 513, 1963. 14 Fawcett, D. W., and Selby, C. C : Observa tions on the Fine Structure of the Turtle Atrium, J. Biophys. & Biochem. Cytol. 4: 63, 1958. 15 Fawcett, D . W., and Revel, J. P.: The Sarcoplasmic Reticulum of a Fast-acting Fish Muscle, J. Biophys. & Biochem. Cytol. 10: Suppl. 89, 1961. 16 Hibbs, R. G., and Black, W. C : Electron Microscopy of Post Mortem Changes in the Rat Myocardium, Anat. Rec. 147: 261, 1963. 17 Hoelscher, B., Just, O. H., and Merker, H. J.: Studies on Electron Microscope on Various Forms of Induced Cardiac Arrest in Dog and Rabbit, Surgery 49: 492, 1964. 18 Huxley, H. E., and Hanson, J.: The Struc tural Basis of the Contraction Mechanism in Striated Muscle, Ann. New York Acad. Sc. 81: 403, 1959. 19 Khristolyubova, N . B.: Study of the Structure of Heart Muscle With the Electron Micro scope, Rep. Acad. Se. U.S.S.R. 119: 168, 1958. 20 Kisch, B.: Electron Microscopy of Cardiac Nerves, Am. J. Cardiol. 2: 475, 1958. 21 Kisch, B.: A Significant Electron Microscopic Difference Between the Atria and the Ven
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tricles of the Mammalian Heart, Exper. Med. & Surg. 2 1 : 193, 1963. Kisch, B., and Cavusoglu, M.: The Ultrastruc ture of the Human Heart, Exper. Med. & Surg. 18: 70, 1960. Kisch, B., Cavusoglu, M., and Marangoni, B. A.: Electron Microscopic Changes in the Human Heart in Cardiac Failure, Exper. Med. & Surg. 17: 85, 1959. Lannigan, R., and Zaki, S.: Electron Micro scopic Appearance of Rheumatic Lesions in the Left Auricular Appendage in Mitral Ste nosis, Nature 198: 898, 1963. Luft, J. H.: Improvements in Epoxy Embed ding Methods, J. Biophys. & Biochem. Cytol. 9: 409, 1961. Mandelmann, N., Stucky, J. H., Hoffman, B. F., and Herman, L.: Comparative Electron Microscopy of Purkinje Fibers and Ventricu lar Muscle of Dog Heart, S. Forum 13: 202, 1962. Martin, A. M., Jr., Hackel, D . B., and Kurtz, S. M.: The Ultrastructure of Zonal Lesions of the Myocardium in Hemorrhagic Shock, Am. J. Path. 44: 127, 1964. Miller, F.: Universität München. Personal communication. Miller, D . R., Rasmussen, P., and Klinosky, B.: Reversibility of Morphologic Changes Fol lowing Elective Cardiac Arrest, Ann. Surg. 159: 208, 1964. Millonig, G.: A Modified Procedure for Lead Staining of Thin Sections, J. Biophys. & Bio chem. Cytol. 11: 736, 1961. Moore, D . H., and Ruska, H.: Electron Mi croscope Studies of Mammalian Cardiac Mus cle Cells, J. Biophys. & Biochem. Cytol. 3: 261, 1957. Morales, M. F., and Botts, J.: A Model for the Elementary Process in Muscle Action, Arch. Biochem. 37: 283, 1952. Muir, A. R.: An Electron Microscope Study of the Embryology of the Intercalated Disc in the Heart of the Rabbit, J. Biophys. & Biochem. Cytol. 3: 193, 1957. Napolitano, L., Cooper, T., Williams, V. L., and Hanlon, C. R.: Fine Structure of the Heart After Transplantation With Special Ref erence to the Neural Elements, Circulation 29: 8 1 , 1964. Nelson, D. A., and Benson, E. S.: On the Structural Continuities of the Transverse Tubu lar System of Rabbit and Human Myocardial Cells, J. Cell Biol. 16: 297, 1963. Poche, R., and Ohm, H. G.: Photomicroscopic, Histochemical, and Electron Micro scopic Studies of the Human Heart Muscle After Induced Heart Arrest, Arch. Kreislauf forsch. 41: 86, 1963. Porter, K. R., and Paladi, G. E.: Studies on
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the Endoplasmic Reticulum. III. Its Form and Distribution in Striated Muscle Cells, J. Biochem. & Biophys. Cytol. 3: 269, 1957. Price, A., Eide, B., Prinzmetal, M., and Car penter, C : Ultrastructure of the Dog Cardiac Muscle Cell, Circulation Res. 7: 858, 1959. Price, K., Weiss, J. M., Daikichi, H., and Smith, J. R.: Experimental Needle Biopsy of the Myocardium of Dogs With Particular Reference to Histologie Study by Electron Microscope, J. Exper. Med. 101: 687, 1955. Revel, J. P.: The Sarcoplasmic Reticulum of the Bat Cricothyroid Muscle, J. Cell. Biol. 12: 571, 1962. Richter, G. W., and Kellner, A.: Hypertrophy of the H u m a n Heart at the Level of Fine Structure, J. Cell Biol. 18: 195, 1963. Sabatini, D . D., Bensch, K., and Barrnett, R. J.: Cytochemistry and Electron Micros copy. The Preservation of Cellular Ultrastructure and Enzymatic Activity by Alde hyde Fixation, J. Cell. Biol. 17: 19, 1963. Simpson, F . O., and Oertelis, S. J.: The Fine Structure of Sheep Myocardial Cells; Sarcolemmal Invaginations and the Transverse Tu bular System, J. Cell Biol. 12: 91, 1962. Sjöstrand, F . S.: Andersson-Cedergren, E., and Dewey, M. M.: The Ultrastructure of the
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Intercalated Discs of Frog, Mouse, Guinea Pig Cardiac Muscle, J. Ultrastructure Res. 1: 271, 1958. Smith, J. R., Burford, T. H., and Chiquoine, A. D.: Electron Microscopic Observations of the Ventricular Heart Muscle of Man Ob tained by Surgical Biopsy During Thoracotomy, Exper. Cell. Res. 20: 228, 1960. Smith, J. R., and Fozzard, H. A.: Localiza tion of Tritriated Digoxin in the Myocardial Cell of Frogs and Dogs by Autoradiography Combined With Electron Microscopy, Nature 197: 562, 1963. Spiro, D.: The Ultrastructure of Heart Mus cle, Tr. New York Acad. Sc. 24: 879, 1962. Stein, A. A., Thibodeau, F., and Stranahan, A.: Electron Microscope Studies of Human Myocardium, J. A. M. A. 182: 537, 1962. Stenger, R. J., and Spiro, D.: Structure of Cardiac Muscle Cell, Am. J. Med. 30: 653, 1961. Tubbs, F . E., Crevasse, L., and Wheat, M. W.: Localization of Tritiated Digoxin in Dog Myocardium by Electron Microscopic Auto radiography, Circulation Res. 14: 236, 1964. Van Breeman, V. L.: Intercalated Discs in Heart Muscle Studies With the Electron Mi croscope, Anat. Rec. 117: 49, 1953.