The Relationship of Actin and Myosin Filaments Within Myocardial Zonal Lesions Stephen W. Unger, BA, and Norman B. Ratliff, MD
Cross sections, including serial sections, of myocardial zonal lesions in right vetricular papillary muscles of cats subjected to hypovolemic shock were examined by electron microscopy. It was determined that the basic structure of A and I bands was retained even in severe zonal lesions with herniation of A and I bands through the intercalated disc into adjacent myocytes. The herniated A and I bands, together with accompanying mitochondria, were enclosed in membranes, apparently the unspecialized portion of the intercalated disc. Since the longitudinal orientation of myofilaments is retained in zonal lesions, this study indicates that intact A and I bands must slide past one another during the supercontraction of sarcomeres which is characteristic of zonal lesions. This study also presents evidence that the nexus may become involved in zonal lesions. (Am J Pathol 80:471-480, 1975)
MYOCARDIAL ZON-AL LESION-S have, to date. been fouind only in hvpovolemic shock. and are characterized bv stupercontraction of a contiguouis group of sarcomeres adjacent to an intercalated disc with loss of recognizable Z bands, displacement of mitochondria away from the suipercontracted portion of the lesion, and, in severe lesions. herniation of the affected mvocvte through the intercalated disc into an adJacent. otherwise uininvolved, mvocvte.'"6 The other lesions of hvpovolemic shock (suibendocardial hemorrhage and myofibrillar degeneration and necrosis) are not uiniquie and are both struetturallv and etiologicallv distinct from myocardial zonal lesions. 1-2 The uiltrastruicture of zonal lesions has been stuidied primarily in longituidinal sections.3 While those stuidies revealed stages in the formation and reversal of zonal lesions, the relationship between actin and mvosin filaments within the lesions remained uinclear. The inference from earlier work was that this relationship was significantlv altered, but suibsequient stuidies cast douibt on this inference.5 This paper will describe the relationship of actin and myosin filaments within myocardial zonal lesions as revealed bv electron microscopic examination of serial cross sections throuigh zonal lesions. In addition. evidence that the nexuis mav be involved in zonal lesions will be presented. From the Department of Pathology. Duike Universitv Medical Center- Duirham. North Carolina Suipported bv a grant from the American Heart Association with funds contribuited in part b%- the
North Carolina Heart Association. Accepted for puiblication May 1. 1973 Address reprint requiests to Dr. Norman B. Ratliff. Department of Laborator- Medicine and Pathology. University of Minnesota, Box 198 Mayo Memorial Building, Minneapolis. MN 55455. 471
472
UNGER AND RATLIFF
American Journal of Pathology
Materials and Methods Fuill details of the experimental design have been described elsewhere.6 Briefly 10 healthy adult mongrel cats were anesthetized with an intraperitoneal injection of a 1: 1 mixture, by voluime, of sodium pentabarbital and allobarbital urethane. Intravenous heparin and intramuscuilar streptomycin and penicillin were administered. Body temperatture was maintained with a heating pad at 37 C throughout the experiment, and electrocardiograms were recorded intermittently throuighouit the proceduire. Femoral arteries and veins were cannuilated. In 5 of the cats, the mean arterial pressulre was lowered, over a period of 30 minuites, from a control mean of 125 mmHg to a mean of 50 mmHg by bleeding from a femoral artery into a plastic reservoir. Pressuire was maintained at this level for 1 to 2 houirs by adjuisting the height of the reservoir. Cats were sacrificed by rapid thoracotomy and removal of the heart. In 2 of these cats, the right ventricle was opened in Karnovsky's fixative, buffered with phosphate to pH 7.4, and three right ventricuilar papillary muiscles from each were immediately fixed, uinder gentle tension, in Karnovsky,s fixative followed by osmium tetroxide postfixation.'3 1l In 3 cats, the right ventricle was opened in modified Krebs-Henseleit soluition gassed with 95% 02 and 5% CO26'l5 One papillary muiscle from each of these cats was immediately fixed, tinder gentle tension, in either Karnovsky's fixative or in a freshly prepared mixtuire of glutaraldehyde and hydrogen peroxide."6 The other papillary muscle was mouinted in a papillary mluscle chamber, stimuilated for 2 to 3 hours at the apex of its length-tension cuirve, and then fixed in the chamber in Karnovsky's fixative or the gltitaraldehyde-hydrogen peroxide mixtulre as previouisly described.6 Five control cats were treated in the same manner as the latter 3 cats except that they were not bled.'6 Their papillary muscles were fixed in Karnovsky's fixative, the glutaraldehyde-hydrogen peroxide mixture, or half-strength Karnovsky's fixative. All of the above muiscles were postfixed in 2% S-collidine-buffered osmiuim tetroxide, and all fixation was carried otit at room temparatuire. Two additional control cats were sacrificed immediately after anesthesia, and two right venticular papillary mtiscles from each were fixed, tinder gentle tension, in a freshly prepared mixtuire of 8% cacodylate-buiffered gltitaraldehyde and 2% S-collidine-buffered osmitim tetroxide witholut postfixation.'7 All mtiscles were dehydrated in graded ethanols followed by propylene oxide, and all were embedded in toto in Epon 812. Dtiring embedding, six nonstimtilated shock muiscles were oriented for cross sections, while the remainder of the muiscles were oriented for longittidinal section. Thick sections (0.5 to 1.0 u) were ctit on a Porter-Bluim microtome and stained with toltiidine bltie. Thin sections (25 to 50 nm) were cutt with a diamond knife on a Porter-Bltim microtome, picked tip on copper grids, and briefly, seqtlentially stained with 2% tiranyl magnesitim acetate and lead citrate. From the blocks originally embedded for cross sections, two were selected on the basis of orientation for serial sectioning. Cross sections were ctit serially throtigh a 10 thickness. Half of these sections were stained with lead citrate only, and half were seqtientially stained with 2% tiranyl magnesitim acetate and lead citrate. Twenty-seven additional blocks were reoriented for cross sections after examination of longittidinal sections, btit were not serially sectioned. Restilts of the stuidy of these longittidinal sections have already been reported. 5,6,11,12
Results
Controls, as previously reported, appeared normal and conformed to descriptions of normal cat right ventricular papillary muiscle.5'6"l8"l9 Zonal lesions which had not reached the stage of mitochondrial displacement or herniation were not identified on cross sections, buit muiltiple zonal lesions were characterized by cross sections of myocytes virtuially devoid of
Vol. 80, No. 3 September 1975
MYOCARDIAL ZONAL LESIONS
473
mitochondria (Figures 1 and 2) or by hemiations throuigh the intercalated disc (Figures 3-6). In the majority of these lesions, the section was slightlv tangential or the plane of the section was through the peripherv of the lesions, and orientation was such that serial sections wouild have moved away from the lesion. One severe zonal lesion with hemiation and appropriate orientation was identified and the block serially sectioned. The results to follow are based primarily on those serial sections, reinforced by observations of other lesions in serial and individuial sections. The relationships of A band and I band myofilament remained basically intact (Figures 3-6). I bands, identified as grouips of thin (actin) filaments (Figure 6), and A bands, identified as thick (myosin) filaments suirrouinded by the normal configuration of thin filaments (Figuires 4 and 5), passed through the hemiations in the intercalated disc. A bands and I bands lay side by side in the section, but each band was uisuially enclosed bv membranes, presumably the unspecialized portion of the intercalated disc, since desmosomes were not seen on those membranes. No thick filaments were seen which were not accompanied by thin filaments in the normal configuration of A bands. Thus, distinct A bands and I bands, individually enclosed by membranes, abutted each other in the herniation without distortion of their basic struictural relationships. Mitochondria were also present, both among the filaments and alone in herniations, and were enclosed by membranes. The mitochondria within these herniations appeared normal (Figures 5 and 6). In one section, a portion of a herniated A band was enclosed by a portion of nexuis (Figuires 3 and 4). In contrast to our previous report, this is the first evidence of involvement of the nexus in zonal lesions.5
Previous ultrastructural studies have resulted in the concept of the sequential steps involved in the formation and reversal of myocardial zonal lesions.56 The present study, through the use of serial cross sections, has disclosed that the basic strtcture of the A and I bands is preserved in herniations through the intercalated disc even though suich herniation is a late event in the formation of zonal lesions and is a featuire of severe zonal lesions.5 Zonal lesions are potentially reversible, and this demonstration of the retention of A and I band strueture, together with the knowledge that displaced mitochondria retain normal structure, makes it easier to uinderstand the reversibility of zonal lesions, since the basic stnrcture of the elements of force and energy generation remains intact.4'5 It is also clear that if the A and I band stnrctuire and the longituidinal orientation of the filaments are both retained, then stupercontraction of
474
UNGER AND RATLIFF
American Journal of Pathology
these myocytes can occur only if entire A and I bands slide past one another during the formation of zonal lesions. Figuire 6 may be an illuistration of this. Space for the resultant increased density of myofilaments within the lesion may be created by the displacement of mitochondria and herniation of the affected myocytes into adjacent, otherwise lunaffected myocytes. In spite of this additional information, the pathogenesis of the loss of Z band continuity and of the separation of actin filaments from the intercalated disc remains unknown, as does the role of myocardial zonal lesions in the loss of ventricular contractility and cardiac failuire which often accompanies hypovolemic shock. 15,20-23 References 1. Hackel DB, Ratliff NB, Mikat E: The heart in shock. Circ Res 35:805-811, 1974 2. Hackel DB, Ratliff NB, Mikat E, Graham T: The effects of hemorrhagic shock on the heart. Comparative Pathophysiology of Circutlatory Distturbances. Edited by CM Bloor. New York, Plenuim Puiblishing, 1972 3. Martin AM Jr Hackel DB Kuirtz SM, The uiltrastruictuire of zonal lesions of the myocardiuim in hemorrhagic shock. Am J Pathol 44:127-140, 1964 4. Martin AM Jr Hackel DB: An electron microscopic stuidy of the progression of myocardial lesions in the dog after hemorrhagic shock. Lab Invest 15:243-260, 1966 5. Ratliff NB, Kopelman RI, Goldner RD, Crtiz PT, Hackel DB: Formation of myocardial zonal lesions. Am J Pathol 79:321-384, 1975 6. Goldner RD, Ratliff NB, Kopelman RI, Hackel DB: Ultrastruictuiral effects of in vitro experimentation on right ventrictular papillary mtuscle from cats in hypovolemic shock. Proc Soc Exp Biol Med 148:113-117, 1975 7. Hackel DB, Goodale WT: Effects of hemorrhagic shock on the heart and circtulation of intact dogs. Circulation 11:628-634, 1955 8. Ratliff NB, Hackel DB, Mikat E: The effect of hyperbaric oxygen on the myocardial lesions of hemorrhagic shock in dogs. Am J Pathol 51:341-349 1967 9. Ratliff NB, Hackel DB, Mikat E: Myocardial oxygen metabolism and myocardial blood flow in dogs in hemorrhagic shock: Effects of Hyperbaric oxygen. Circ Res 24:901-909 1969 10. Martin AM Jr, Hackel DB: The myocardiuim of the dog in hemorrhagic shock, a histochemical stuidy. Lab Invest 12:77-91, 1963 11. Ratliff NB: Moyeardial zonal lesions and myofibrillar degeneration: The need for definition. J Mol Cell Cardiol 7:225-226, 1975 12. Ratliff NB: Ultrastruictuiral comparison of myocardial zonal lesions and myofibrillar degeneration in cats suibjected to hemorrhagic shock. Circuilatory Shock (In press) 13. Karnovsky MJ: A formaldehyde-gluitaraldehyde fixative of high osmolality for uise in electron microscopy. J Cell Biol 27:137A, 1965 (Abstr) 14. Graham RC, Karnovsky MJ: The early stages of absorption of injected horseradish peroxidase in the proximal tubuiles of mouise kidney: Ultrastruictuiral cytochemistry by a new techniquie. J Histochem Cytochem 14:291-302, 1966 15. Lefer AM: Corticosteroid antagonism of the positive inotropic effect of ollabain. J Pharmacol Exp Ther 151:294-299, 1966 16. Peracchia C, Mittler BS: Fixation by means of glhitaraldehyde-hydrogen peroxide reaction products. J Cell Biol 53:234-238, 1972
Vol. 80, No. 3 September 1975
MYOCARDIAL ZONAL LESIONS
475
17. Trump BF, Bulger R: New ultrastructural characteristics of cells fixed in a glitaraldehvde-osmiuim tetroxide mixtuire. Lab Invest 15:368-379. 1966 18. Fawcett DW. McNtitt NS: The ultrastrtctutre of the cat myocardiuim I Ventricullar papillarv muscle. J Cell Biol 42:1-453 1969 19. McNutt NS, Fawcett DW: Mvocardial uiltrastnrctuire. The Mammalian Mvocardium. Edited bv GA Langer. AJ Bradv. New York. John Wiley and Sons. 1974 20. Crowell JW, Gtuvton AC: Fuirther evidence favoring a cardiac mechanism in irreversible hemorrhagic shock. Am J Phvsiol 203:248-252. 1962 21. Gomez OA. Hamilton WF: Functional cardiac deterioration duiring development of hemorrhagic circtulatorv deficienev. Circ Res 14:327-336. 1964 22. Siegel HW. Downing SE: Reduiction of left ventricuilar contractility duiring acuite hemorrhagic shock. Am J Phvsiol 218:772-779. 1970 23. Kajihara H. Hara H. Sevama S. lijima S. Yoshidoa M: Light and electron microscopic observations of the myocardium of dogs in hemorrhagic shock Acta Pathol Jap 23:315-333. 1973
[Illustrations follow]
476
UNGER AND RATLIFF
American Journal of Pathology
Legends for Figures All illustrations are cross sections of right ventricular papillary muscles which were taken from cats subjected to hemorrhagic shock and were then fixed in Karnovsky's fixative.
Figure 1-Portions of three myocytes are illustrated for comparison. The myocyte at the top contains a convoluted intercalated disc. The myocyte at the top contains a convoluted intercalated disc. The myocyte to the right is essentially normal, with mitochondria and myofilaments in the plane of section. The lower myocyte contains a zonal lesion (*) identified by the absence of mitochondria among the myofilaments. In addition, electron-dense material compatible with fragmented Z lines is scattered through the myocyte. (Uranyl magnesium acetate and lead citrate, x 8300)
Figure 2-Portions of two myocytes are illustrated. Both contain zonal lesions(*) identified by the same criteria as in Figure 1. The proximity of the zonal lesion to the intercalated disc (arrow) is evident in the myocyte to the right, which is cut on a slightly oblique plane. (Uranyl magnesium acetate and lead citrate, x 12,000)
2
3 V
Figure 3-This illustrates a zonal lesion in the plane of the intercalated disc. Several herniations (arrows) through the intercalated disc are evident and are surrounded by membranes of the unspecialized portion of the intercalated disc. One bundle of myofilaments is enclosed by a portion of Figure 4-This is a nexus (double arrows). (Uranyl magnesium acetate and lead citrate, x 15,700) higher magnification of part of Figure 3. This demonstrates portions of A bands enclosed by the unspecialized membranes of the intercalated disc (arrows) and by nexus (double arrows). (Uranyl magnesium acetate and lead citrate, X 53,000)
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Figure 5-At the bottom left is a portion of a flattened intercalated disc. One bundle of herniated thin filaments, an I band, is enclosed by membranes (double arrows). Numerous herniated A bands enclosed by membranes are also evident. One of these contains a mitochondrion in the center of the filaments (single arrow). These herniated portions of the myocyte containing myofilaments from a zonal lesion are all enclosed by membranes and are intermingled with the myofilaments of the myocyte into which the herniation has occurred. (Uranyl magnesium acetate and lead citrate, x 45,000)
.:: d.
Figure 6-Portions of three myocytes are illustrated. The myocyte at the top of the picture contains a zonal lesion as does the one at the bottom (*). Both are separated by intercalated discs from the central myocyte into which the herniations have occurred. The slightly oblique nature of the plane of section, especially notable in the myocyte at the bottom, permits this unusual view. Herniations through both intercalated discs are evident (long single arrows). The center myocyte, which is cut closer to a perfect cross section than is the bottom myocyte, contains several bands which are enclosed by membranes and which have herniated into this myocyte (short double arrows). A mitochondrion is present in one of these I bands (long double arrows). Toward the center of the picture, portions of herniated A and bands lie adjacent to each other enclosed by the same membrane (short single arrow). (Uranyl magnesium acetate and lead citrate, X 37,000)
Cross sections, including serial sections, of myocardial zonal lesions in right vetricular papillary muscles of cats subjected to hypovolemic shock were examined by electron microscopy. It was determined that the basic structure of A and I bands was retained even in severe zonal lesions with herniation of A and I bands through the intercalated disc into adjacent myocytes. The herniated A and I bands, together with accompanying mitochondria, were enclosed in membranes, apparently the unspecialized portion of the intercalated disc. Since the longitudinal orientation of myofilaments is retained in zonal lesions, this study indicates that intact A and I bands must slide past one another during the supercontraction of sarcomeres which is characteristic of zonal lesions. This study also presents evidence that the nexus may become involved in zonal lesions. (Am J Pathol 80:471-480, 1975)
MYOCARDIAL ZON-AL LESION-S have, to date. been fouind only in hvpovolemic shock. and are characterized bv stupercontraction of a contiguouis group of sarcomeres adjacent to an intercalated disc with loss of recognizable Z bands, displacement of mitochondria away from the suipercontracted portion of the lesion, and, in severe lesions. herniation of the affected mvocvte through the intercalated disc into an adJacent. otherwise uininvolved, mvocvte.'"6 The other lesions of hvpovolemic shock (suibendocardial hemorrhage and myofibrillar degeneration and necrosis) are not uiniquie and are both struetturallv and etiologicallv distinct from myocardial zonal lesions. 1-2 The uiltrastruicture of zonal lesions has been stuidied primarily in longituidinal sections.3 While those stuidies revealed stages in the formation and reversal of zonal lesions, the relationship between actin and mvosin filaments within the lesions remained uinclear. The inference from earlier work was that this relationship was significantlv altered, but suibsequient stuidies cast douibt on this inference.5 This paper will describe the relationship of actin and myosin filaments within myocardial zonal lesions as revealed bv electron microscopic examination of serial cross sections throuigh zonal lesions. In addition. evidence that the nexuis mav be involved in zonal lesions will be presented. From the Department of Pathology. Duike Universitv Medical Center- Duirham. North Carolina Suipported bv a grant from the American Heart Association with funds contribuited in part b%- the
North Carolina Heart Association. Accepted for puiblication May 1. 1973 Address reprint requiests to Dr. Norman B. Ratliff. Department of Laborator- Medicine and Pathology. University of Minnesota, Box 198 Mayo Memorial Building, Minneapolis. MN 55455. 471
472
UNGER AND RATLIFF
American Journal of Pathology
Materials and Methods Fuill details of the experimental design have been described elsewhere.6 Briefly 10 healthy adult mongrel cats were anesthetized with an intraperitoneal injection of a 1: 1 mixture, by voluime, of sodium pentabarbital and allobarbital urethane. Intravenous heparin and intramuscuilar streptomycin and penicillin were administered. Body temperatture was maintained with a heating pad at 37 C throughout the experiment, and electrocardiograms were recorded intermittently throuighouit the proceduire. Femoral arteries and veins were cannuilated. In 5 of the cats, the mean arterial pressulre was lowered, over a period of 30 minuites, from a control mean of 125 mmHg to a mean of 50 mmHg by bleeding from a femoral artery into a plastic reservoir. Pressuire was maintained at this level for 1 to 2 houirs by adjuisting the height of the reservoir. Cats were sacrificed by rapid thoracotomy and removal of the heart. In 2 of these cats, the right ventricle was opened in Karnovsky's fixative, buffered with phosphate to pH 7.4, and three right ventricuilar papillary muiscles from each were immediately fixed, uinder gentle tension, in Karnovsky,s fixative followed by osmium tetroxide postfixation.'3 1l In 3 cats, the right ventricle was opened in modified Krebs-Henseleit soluition gassed with 95% 02 and 5% CO26'l5 One papillary muiscle from each of these cats was immediately fixed, tinder gentle tension, in either Karnovsky's fixative or in a freshly prepared mixtuire of glutaraldehyde and hydrogen peroxide."6 The other papillary muscle was mouinted in a papillary mluscle chamber, stimuilated for 2 to 3 hours at the apex of its length-tension cuirve, and then fixed in the chamber in Karnovsky's fixative or the gltitaraldehyde-hydrogen peroxide mixtulre as previouisly described.6 Five control cats were treated in the same manner as the latter 3 cats except that they were not bled.'6 Their papillary muscles were fixed in Karnovsky's fixative, the glutaraldehyde-hydrogen peroxide mixture, or half-strength Karnovsky's fixative. All of the above muiscles were postfixed in 2% S-collidine-buffered osmiuim tetroxide, and all fixation was carried otit at room temparatuire. Two additional control cats were sacrificed immediately after anesthesia, and two right venticular papillary mtiscles from each were fixed, tinder gentle tension, in a freshly prepared mixtuire of 8% cacodylate-buiffered gltitaraldehyde and 2% S-collidine-buffered osmitim tetroxide witholut postfixation.'7 All mtiscles were dehydrated in graded ethanols followed by propylene oxide, and all were embedded in toto in Epon 812. Dtiring embedding, six nonstimtilated shock muiscles were oriented for cross sections, while the remainder of the muiscles were oriented for longittidinal section. Thick sections (0.5 to 1.0 u) were ctit on a Porter-Bluim microtome and stained with toltiidine bltie. Thin sections (25 to 50 nm) were cutt with a diamond knife on a Porter-Bltim microtome, picked tip on copper grids, and briefly, seqtlentially stained with 2% tiranyl magnesitim acetate and lead citrate. From the blocks originally embedded for cross sections, two were selected on the basis of orientation for serial sectioning. Cross sections were ctit serially throtigh a 10 thickness. Half of these sections were stained with lead citrate only, and half were seqtientially stained with 2% tiranyl magnesitim acetate and lead citrate. Twenty-seven additional blocks were reoriented for cross sections after examination of longittidinal sections, btit were not serially sectioned. Restilts of the stuidy of these longittidinal sections have already been reported. 5,6,11,12
Results
Controls, as previously reported, appeared normal and conformed to descriptions of normal cat right ventricular papillary muiscle.5'6"l8"l9 Zonal lesions which had not reached the stage of mitochondrial displacement or herniation were not identified on cross sections, buit muiltiple zonal lesions were characterized by cross sections of myocytes virtuially devoid of
Vol. 80, No. 3 September 1975
MYOCARDIAL ZONAL LESIONS
473
mitochondria (Figures 1 and 2) or by hemiations throuigh the intercalated disc (Figures 3-6). In the majority of these lesions, the section was slightlv tangential or the plane of the section was through the peripherv of the lesions, and orientation was such that serial sections wouild have moved away from the lesion. One severe zonal lesion with hemiation and appropriate orientation was identified and the block serially sectioned. The results to follow are based primarily on those serial sections, reinforced by observations of other lesions in serial and individuial sections. The relationships of A band and I band myofilament remained basically intact (Figures 3-6). I bands, identified as grouips of thin (actin) filaments (Figure 6), and A bands, identified as thick (myosin) filaments suirrouinded by the normal configuration of thin filaments (Figuires 4 and 5), passed through the hemiations in the intercalated disc. A bands and I bands lay side by side in the section, but each band was uisuially enclosed bv membranes, presumably the unspecialized portion of the intercalated disc, since desmosomes were not seen on those membranes. No thick filaments were seen which were not accompanied by thin filaments in the normal configuration of A bands. Thus, distinct A bands and I bands, individually enclosed by membranes, abutted each other in the herniation without distortion of their basic struictural relationships. Mitochondria were also present, both among the filaments and alone in herniations, and were enclosed by membranes. The mitochondria within these herniations appeared normal (Figures 5 and 6). In one section, a portion of a herniated A band was enclosed by a portion of nexuis (Figuires 3 and 4). In contrast to our previous report, this is the first evidence of involvement of the nexus in zonal lesions.5
Previous ultrastructural studies have resulted in the concept of the sequential steps involved in the formation and reversal of myocardial zonal lesions.56 The present study, through the use of serial cross sections, has disclosed that the basic strtcture of the A and I bands is preserved in herniations through the intercalated disc even though suich herniation is a late event in the formation of zonal lesions and is a featuire of severe zonal lesions.5 Zonal lesions are potentially reversible, and this demonstration of the retention of A and I band strueture, together with the knowledge that displaced mitochondria retain normal structure, makes it easier to uinderstand the reversibility of zonal lesions, since the basic stnrcture of the elements of force and energy generation remains intact.4'5 It is also clear that if the A and I band stnrctuire and the longituidinal orientation of the filaments are both retained, then stupercontraction of
474
UNGER AND RATLIFF
American Journal of Pathology
these myocytes can occur only if entire A and I bands slide past one another during the formation of zonal lesions. Figuire 6 may be an illuistration of this. Space for the resultant increased density of myofilaments within the lesion may be created by the displacement of mitochondria and herniation of the affected myocytes into adjacent, otherwise lunaffected myocytes. In spite of this additional information, the pathogenesis of the loss of Z band continuity and of the separation of actin filaments from the intercalated disc remains unknown, as does the role of myocardial zonal lesions in the loss of ventricular contractility and cardiac failuire which often accompanies hypovolemic shock. 15,20-23 References 1. Hackel DB, Ratliff NB, Mikat E: The heart in shock. Circ Res 35:805-811, 1974 2. Hackel DB, Ratliff NB, Mikat E, Graham T: The effects of hemorrhagic shock on the heart. Comparative Pathophysiology of Circutlatory Distturbances. Edited by CM Bloor. New York, Plenuim Puiblishing, 1972 3. Martin AM Jr Hackel DB Kuirtz SM, The uiltrastruictuire of zonal lesions of the myocardiuim in hemorrhagic shock. Am J Pathol 44:127-140, 1964 4. Martin AM Jr Hackel DB: An electron microscopic stuidy of the progression of myocardial lesions in the dog after hemorrhagic shock. Lab Invest 15:243-260, 1966 5. Ratliff NB, Kopelman RI, Goldner RD, Crtiz PT, Hackel DB: Formation of myocardial zonal lesions. Am J Pathol 79:321-384, 1975 6. Goldner RD, Ratliff NB, Kopelman RI, Hackel DB: Ultrastruictuiral effects of in vitro experimentation on right ventrictular papillary mtuscle from cats in hypovolemic shock. Proc Soc Exp Biol Med 148:113-117, 1975 7. Hackel DB, Goodale WT: Effects of hemorrhagic shock on the heart and circtulation of intact dogs. Circulation 11:628-634, 1955 8. Ratliff NB, Hackel DB, Mikat E: The effect of hyperbaric oxygen on the myocardial lesions of hemorrhagic shock in dogs. Am J Pathol 51:341-349 1967 9. Ratliff NB, Hackel DB, Mikat E: Myocardial oxygen metabolism and myocardial blood flow in dogs in hemorrhagic shock: Effects of Hyperbaric oxygen. Circ Res 24:901-909 1969 10. Martin AM Jr, Hackel DB: The myocardiuim of the dog in hemorrhagic shock, a histochemical stuidy. Lab Invest 12:77-91, 1963 11. Ratliff NB: Moyeardial zonal lesions and myofibrillar degeneration: The need for definition. J Mol Cell Cardiol 7:225-226, 1975 12. Ratliff NB: Ultrastruictuiral comparison of myocardial zonal lesions and myofibrillar degeneration in cats suibjected to hemorrhagic shock. Circuilatory Shock (In press) 13. Karnovsky MJ: A formaldehyde-gluitaraldehyde fixative of high osmolality for uise in electron microscopy. J Cell Biol 27:137A, 1965 (Abstr) 14. Graham RC, Karnovsky MJ: The early stages of absorption of injected horseradish peroxidase in the proximal tubuiles of mouise kidney: Ultrastruictuiral cytochemistry by a new techniquie. J Histochem Cytochem 14:291-302, 1966 15. Lefer AM: Corticosteroid antagonism of the positive inotropic effect of ollabain. J Pharmacol Exp Ther 151:294-299, 1966 16. Peracchia C, Mittler BS: Fixation by means of glhitaraldehyde-hydrogen peroxide reaction products. J Cell Biol 53:234-238, 1972
Vol. 80, No. 3 September 1975
MYOCARDIAL ZONAL LESIONS
475
17. Trump BF, Bulger R: New ultrastructural characteristics of cells fixed in a glitaraldehvde-osmiuim tetroxide mixtuire. Lab Invest 15:368-379. 1966 18. Fawcett DW. McNtitt NS: The ultrastrtctutre of the cat myocardiuim I Ventricullar papillarv muscle. J Cell Biol 42:1-453 1969 19. McNutt NS, Fawcett DW: Mvocardial uiltrastnrctuire. The Mammalian Mvocardium. Edited bv GA Langer. AJ Bradv. New York. John Wiley and Sons. 1974 20. Crowell JW, Gtuvton AC: Fuirther evidence favoring a cardiac mechanism in irreversible hemorrhagic shock. Am J Phvsiol 203:248-252. 1962 21. Gomez OA. Hamilton WF: Functional cardiac deterioration duiring development of hemorrhagic circtulatorv deficienev. Circ Res 14:327-336. 1964 22. Siegel HW. Downing SE: Reduiction of left ventricuilar contractility duiring acuite hemorrhagic shock. Am J Phvsiol 218:772-779. 1970 23. Kajihara H. Hara H. Sevama S. lijima S. Yoshidoa M: Light and electron microscopic observations of the myocardium of dogs in hemorrhagic shock Acta Pathol Jap 23:315-333. 1973
[Illustrations follow]
476
UNGER AND RATLIFF
American Journal of Pathology
Legends for Figures All illustrations are cross sections of right ventricular papillary muscles which were taken from cats subjected to hemorrhagic shock and were then fixed in Karnovsky's fixative.
Figure 1-Portions of three myocytes are illustrated for comparison. The myocyte at the top contains a convoluted intercalated disc. The myocyte at the top contains a convoluted intercalated disc. The myocyte to the right is essentially normal, with mitochondria and myofilaments in the plane of section. The lower myocyte contains a zonal lesion (*) identified by the absence of mitochondria among the myofilaments. In addition, electron-dense material compatible with fragmented Z lines is scattered through the myocyte. (Uranyl magnesium acetate and lead citrate, x 8300)
Figure 2-Portions of two myocytes are illustrated. Both contain zonal lesions(*) identified by the same criteria as in Figure 1. The proximity of the zonal lesion to the intercalated disc (arrow) is evident in the myocyte to the right, which is cut on a slightly oblique plane. (Uranyl magnesium acetate and lead citrate, x 12,000)
2
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Figure 3-This illustrates a zonal lesion in the plane of the intercalated disc. Several herniations (arrows) through the intercalated disc are evident and are surrounded by membranes of the unspecialized portion of the intercalated disc. One bundle of myofilaments is enclosed by a portion of Figure 4-This is a nexus (double arrows). (Uranyl magnesium acetate and lead citrate, x 15,700) higher magnification of part of Figure 3. This demonstrates portions of A bands enclosed by the unspecialized membranes of the intercalated disc (arrows) and by nexus (double arrows). (Uranyl magnesium acetate and lead citrate, X 53,000)
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Figure 5-At the bottom left is a portion of a flattened intercalated disc. One bundle of herniated thin filaments, an I band, is enclosed by membranes (double arrows). Numerous herniated A bands enclosed by membranes are also evident. One of these contains a mitochondrion in the center of the filaments (single arrow). These herniated portions of the myocyte containing myofilaments from a zonal lesion are all enclosed by membranes and are intermingled with the myofilaments of the myocyte into which the herniation has occurred. (Uranyl magnesium acetate and lead citrate, x 45,000)
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Figure 6-Portions of three myocytes are illustrated. The myocyte at the top of the picture contains a zonal lesion as does the one at the bottom (*). Both are separated by intercalated discs from the central myocyte into which the herniations have occurred. The slightly oblique nature of the plane of section, especially notable in the myocyte at the bottom, permits this unusual view. Herniations through both intercalated discs are evident (long single arrows). The center myocyte, which is cut closer to a perfect cross section than is the bottom myocyte, contains several bands which are enclosed by membranes and which have herniated into this myocyte (short double arrows). A mitochondrion is present in one of these I bands (long double arrows). Toward the center of the picture, portions of herniated A and bands lie adjacent to each other enclosed by the same membrane (short single arrow). (Uranyl magnesium acetate and lead citrate, X 37,000)
