3mrnal of Molecular
and Cellular
Cardiology
Autophagic
(1979)
11, 331-338
Response to Sublethal iu Cardiac Myocytes
H. D. SYBERS,*
J. INGWALL,t
AND M.
* Department of Pathology, Baylor College of Medicine, t Department of Medicine and Biochemistry, University La Jolla, California, U.S.A.
(Received 15 August
1977,
Injury
DELUCAt
Houston, Texas, and of Califoornia, San Diego,
accepted in revised&m
5
3u& 1978)
H. D. SYBERS, J. INGWALL AND M. DaLuo~. Autophagic Response to Sublethal Injury in Cardiac Myocytes. jkwnal of Molecular and Cellular Cardiology (1979) 11, 331-338. Fetal mouse hearts maintained in oxygenated organ culture media continue to beat for a period of weeks. Study of the ultrastructure of the myocytes after the first day in culture revealed that most of the cells were normal, however an occasional cell had autophagic vacuoles within the cytoplasm which contained degenerating organelles indicating that focal cytcplasmic injury had occurred. Deprivation of oxygen and glucose for a period of time followed by resupply resulted in increased numbem of autophagic vacuoles. In this study,
fetal mouse hearts were maintained from 1 to 4 h in glucose-free media in an atmosphere of 95% N-5% COs after which resupply of 0s and glucose was maintained for as long aa 24 h. Many cells recovered without apparent residual injury while others contained autophagic vacuoles in an otherwise normal cytoplasm. The contents of the vacuoles ranged
from those in which organelles were readily identified to those characteristic of residual bodies. Mitochondria, glycogen and tubular structureS were seen most frequently in the vacuoles but occasionally myofilaments were identified. It is suggested that focal cytoplasmic injury which occurred during oxygen and glucose deprivation stimulates the formation of membrane to enclose the irreversibly damaged components permitting localized lysosomal digestion without causing further injury to the cell. The fetal mouse heart organ culture provides an excellent model for studying the sequential autophagic changes
which
occur in response
KEY WORDS: Autophagy; &hernia; Sublethal injury.
Fetal
to sublethal mouse
injury. Focal
heart;
cytoplasmic
injury;
Myocardial
1. Introduction The ultrastructural changes which occur following myocardial ischemia have been studied extensively in adult animals. The early changes which occur consist of swelling of organelles and depletion of glycogen granules [Z, 3, 7-91. These alterations progress to a point of irreversible injury. By this time there is margination of nuclear chromatin, mitochondria are swollen, and they often contain large dense precipitates [I#]. Eventually disruption of membranes and loss of structural Supported
by NHLI
0022-2828/79/040331+
grant
19147-02
14 $02.00/O
and NHLI
Contract 0
L-HL-81332.
1979 Academic
Press Inc.
(London)
Limited
332
H. D. SYBERS
ET AL.
integrity occurs. If blood is reperfused into the ischemic region there is an acceleration of structural alterations and calcium uptake in those cells destined to undergo necrosis, while the cell cytoplasm reverts to normal in those which will recover [5, 151. The question of whether or not focal irreversible cytoplasmic injury has occurred in the cells which recover and, if so, in what manner these injured orgenellesare handled, has not been well studied in cardiac myocytes. Studies in which we employed fetal mouse hearts (FMH) maintained in organ culture under conditions of oxygen and glucosedeprivation to simulate someof the conditions of ischemia revealed many cellular alterations which are similar to those seenin coronary induced ischemia of mature cardiac cells [6, 16, 171. There was swelling of organelles, reduction in glycogen, increase in lipid vacuoles and margination of nuclear chromatin followed by membrane destruction. These alterations did not occur as rapidly in the FMH organ culture as those seen in adult ischemia, and many of them could be reversed in some of the cells after as long as4 h of deprivation. Many of the cellswhich returned to an otherwise normal ultrastructural appearance, revealed the presenceof autophagic vacuoles containing degenerating organellesin their cytoplasm. Since similar findings have not been reported in sublethal adult ischemic cardiac injury, it would appear that fetal cells are better able to isolate focally injured cytoplasmic components for lysosomal degradation. The present report details the sequenceof morphologic changeswhich is seen in the development of the autophagic vacuoles and offers a speculation as to why similar findings are not commonly seen in adult myocytes following ischemic injury. 2. Materials
and Methods
Intact beating hearts obtained from 15 to 20 day fetal mice were maintained with Minimum Essential Medium (MEM), on stainless steel grids in organ culture dishes at an air (95% Os-5’$$ COa)-medium interface at 37°C as previously described [16l. Hearts were maintained in culture for at least 18 h to allow stabilization before the experimental conditions were imposed. Controls The control hearts were maintained in culture medium at 37°C in an environment of 95% Oa-5°/o CO2 for periods of time which corresponded with those of the experimental groups. Hearts from matched litter mates were used in the experimental groups. .?G#whntal:
deprivation of glucose and oxygen
The hearts were deprived of oxygen and glucosefor periods ranging from 1 to 4 h
AUTOPHAGY
IN
CARDIAC
MYOCYTES
333
at 37°C by replacing the culture medium with argon or nitrogen saturated, glucose-free MEM and incubating them in sealed culture jars continuously, In some cases they were periodically flushed with 95% Ns-5% COs. Oxygen content of the media, as determined with an Instrumentation Laboratories gas analyzer, was less than 5 mmHg. At the end of the experimental period the hearts were rapidly fixed by immersion in 5% gluteraldehyde in phosphate buffer, pH 7.3 at 4°C. The atria were removed under a dissecting microscope and tissue from the ventricles was minced and allowed to fix for 4 h. After postfixation in 1o/o osmium tetroxide and dehydration in acetone, the ventricular tissue was embedded in Araldite for electron microscopy. At least two blocks of tissue were selected randomly from each heart for examination. Thick sections (1 pm) were prepared and stained with toluidine blue for orientation. Thin sections were stained with uranyl acetate and lead citrate and examined in a Zeiss 9A electron microscope. Experimental:
resupply of glucose and oxygen
In several of the hearts, oxygen and glucosewere resupplied for 1 to 24 h at 37°C after periods of deprivation of 1 to 4 h duration and were prepared for and examined with the electron microscope as described. In four hearts resupply of glucoseand oxygen was begun after 1 h of deprivation and in another four hearts after 2 h of deprivation. Two from each group were processedfor electron microscopy after 1 and 2 h of resupply. An additional two hearts were subjected to 3 h and two hearts to 4 h of deprivation followed by a period of 24 h of resupply of oxygen and glucosebefore preparing them for electron microscopy. 3. Results
Myocytes from the control hearts which had been incubated in oxygenated media retained a normal ultrastructure in most of the cells (Plate 1)) however an occasional cell showed evidence of degenerative changes as previously described [I6]. The cells were oval to slightly elongated and the extent of development of the myofibrils was variable. In those cells with poorly developed myofibrils the thin filaments often appeared to arise from or to be attached to desmosomes.Round to oval nuclei occupied a large proportion of the cell volume. Mitochondria were frequently elongated with irregular shapes.Glycogen granules were abundant and numerous ribosomeswere present. A prominent Golgi apparatus was often seenin the perinuclear region. Tubular structures, thought to be sarcoplasmicreticulum, were found throughout the cytoplasm but transverse tubules were identified only when the myofibrils were well developed. The sarcolemma had specialized regions with desmosomes and occasionalgap junctions. Pinocytotic vesicleswere frequently seenalong the sarcolemma.
334
H.
D. SYBERS
ET AL.
The hearts subjected to oxygen and glucose deprivation revealed ultrastructural alterations which became more apparent as the duration of deprivation was increased. After 1 h most cells showed only minimal to moderate alterations, with mild swelling of mitochondria, a slight reduction in glycogen granules, and often an increased number of lipid vacuoles. There was a slight increase in the number of cells containing autophagic vacuoles, however, many cells remained quite normal in appearance. Interstitial space often appeared to be increased. After 2 to 3 h of deprivation, mitochondrial swelling with decreased matrix density was more pronounced in most of the cells, but the presence of dense intramitochondrial granules was rarely seen and, except for occasional cells, nuclear alterations were not severe at 2 h. Structurally normal or nearly normal cells were still seen occasionally after 3 h of glucose and oxygen deprivation (Plate 2). Four h of deprivation resulted in moderate to severe ultrastructural alterations in virtually all of the myocytes with a marked decrease in glycogen granules, swelling and decreased matrix density of motochondria, and myofibrillar disorganization. Margination of nuclear chromatin was present in most of the cells. Sarcolemmal disruption was frequently seen and cellular debris was common in the interstitial spaces (Plate 3). Phagocytic cells ingesting remnants of myocytes were often seen. The hearts in which oxygen and glucose were resupplied following a period of deprivation resumed beating and structural recovery occurred in many of the cells following periods of up to 4 h of deprivation. Numerous cells, however, had vacuoles containing degenerating organelles in an otherwise normal cytoplasm which suggested that the period of deprivation caused the occurrence of focal irreversible injury in cells which ultimately recovered. Resupply after 1 h resulted in a return toward normal structure in the majority of the cells, however, a marked increase in the number of cells containing autophagic vacuoles was observed after the first hour of resupply. These vacuoles contained structurally altered cellular components which were easily identified. Degenerating mitochondria, myofibrils, and glycogen granules were often seen surrounded by a single or double layered membrane which at times was incomplete. The vacuoles were most commonly located in the perinuclear region and the nuclear envelope frequently had a concavity adjacent to the vacuoles as if indented by pressure from the vacuole (Plate 4). Vacuoles were also often found in the periphery of the cell. In these the organelles frequently appeared to have undergone a greater degree of degradation than those in the perinuclear region and they occasionally had densely stained osmiophilic contents (Plate 5). The membranes surrounding the vacuoles did not have desmosomes or gap junctions. Furthermore, the adjacent cells were usually normal in appearance. This suggests an intracellular origin hence a true autophagic vacuole, rather than a protrusion of an adjacent cell into the cytoplasm. When the hearts were exposed to 3 to 4 h of oxygen and glucose deprivation,
PLATE 1. Normal control. The nuclei (N) are proportionally large and have evenly dispersed (m) are chromatin. The Golgi apparatus (g) is p rominent in the perinuclear region. Mitochondria normal. Myofilaments are moderately developed. x 11 500. PLATE 2. Three h of oxygen and glucose deprivation. Margination of nuclear chromatin is apparent. Mitochondria (m) are swollen and the number of christae appears to be reduced. Lipid vacuoles (L) are frequently seen and the sarcoplasmic reticulum (SR) is dilated. Myofibrillar disorganization and reduction in myofibrils is apparent. Nucleus (N). x 10 ZOO. PLATE 3. Four h of deprivation. Margination of nuclear chromatin and mitochondrial swelling is more pronounced than in Plate 2. Many cells are frankly necrotic (arrows) with ruptured sarcolemma and mitorhondrial (m) membranes. x 7300. PLATE 4. Early autophagic vacuole. An early stage of vacuole formation is shown which contains mitochondria (m) with decreased matrix density and dense precipitates, tubules, and filaments. A double-membrane surrounds most of the vacuole (arrows). The nucleus (N) has a concave indentation as if caused by pressure from the autophagic vacuole (A). Desmosomes (d). x 11 700. PLATE 5. Intermediate stage. Three autophagic vacuoles (A) are seen in the periphery of a cell. Degenerating mitochondria containing dense granules and myofilaments (mf) can be seen. The remaining cytoplasm is normal appearing and contains numerous ribosomes (arrow) along the myofilaments. Mitochondria (m). x 23 000. PLATE 6. Four h oxygen and glucose deprivation followed by 24 h of resupply. Many cells have returned to a normal appearance. Mitochondria (M) have well-defined cristae. There is a normal dispersion of nuclear chromatin (N). Dense autophagic vacuoles (A) are frequently found. Many crlls have undergone necrosis (NE). x 9240. PLATE 7. Intermediate stage of autophagic vacuoles. Some organelles are still recognized in these coalsescing vacuoles (A) which contain myofilaments (mf) and mitochondria with dense intramitochondrial granules. The surrounding membrane can be seen in some areas (arrows). Note the normal appearance of the remaining cell cytoplasm. Glycogen (G). Interstitial space (IS). x 18 400. PLATE 8. Late stage. Dense osmiophilic residues (A) make up the end stages of autophagic digestion. Myelin figures (MY) are seen on the periphery of the vacuole. x 17 480. PLATE 9. Phagocytosis. When extensive cell breakdown occurs, phagocytic cells (P) containing an abundance of rough endoplasmic reticulum (ER) engulf the necrotic debris from the degenerating myocytes. The myofilaments and degenerating mitochondria within this vacuole are readily identified. A thin rim of cytoplasm from the phagocyte encircles the vacuole. Phagocyte nucleus i?j). A fibril from an adjacent normal myocyte (M) is seen on the left. x 11 500.
AUTOPHAGY
IN
CARDIAC
bfYOCYTES
335
resupply for 24 h resulted in a return to normal structure in many of the cells but residual bodies or lamellar bodies were frequently found within their cytoplasm. Discrete organelles were less frequently seen within the autophagic vacuoles suggesting that by this time extensive lysosomal degradation had occurred. The remainder of the cell cytoplasm and nucleus had returned to a normal ultrastructure except for an apparent decrease in myofilaments. As indicated previously, many cells had undergone necrosis when the period of injury was of 3 or 4 h duration and no attempt was made in this study to determine the relative proportion of cells which suffered irreversible damage as compared to those which sustained sublethal injury and subsequently recovered (Plate 6). The structural appearance of autophagic vacuoles from the early stages of sequestration of focally injured cytoplasm to degradation and residual body formation is shown in Plates 4, 5, 7 and 8. In addition to the autophagic response described above, phagocytosis of necrotic myocyte debris by phagocytic cells was frequently seen when lethally injured cells were present (Plate 9). These phagocytic cells are characterized by an abundance of rough endoplasmic reticulum in their cytoplasm. 4. Discussion
The presenceof autophagic vacuoles in adult cells following transient ischemia has not been emphasized [7, 91 but has been reported in fetal cells in organ culture [S, 16, 171. This suggeststhat autophagy is not a common occurrence in sublethal ischemic injury or that the vacuoles are not conspicuousin adult cells. While it is recognized that ischemia in the experimental animal is not identical with the conditions of glucose and oxygen deprivation in an organ culture system, the structural alterations which occur are similar and it is reasonable to suspect that many of the same stimuli for initiation of focal repair processesoccur in both systems. The formation of autophagic vacuoles in cardiac myocytes for the sequestration, isolation, and degradation of focally damaged cytoplasmic components appears to be a common reaction to sublethal injury in fetal mouse hearts (FMH) in organ culture. While they are seenoccasionally in the absenceof interventions designed specifically to produce cell injury [16], their occurrence is probably a result of transient anoxia during removal of the heart from the fetus. This period may be of 30 to 40 min duration before dissection of the last fetus in the litter has been completed. Deprivation of oxygen and glucose from the organ culture results in cell alterations which are structurally similar to thoseof ischemic injury in adult myocytes. Its resupply prior to development of irreversible injury elicits a marked increase in the number of cells containing autophagic vacuoles. The rapid increase in vacuole formation following resupply is an indication of the need for adequate energy
336
Ii.
D. SYBERS
ET
AL.
supplies for these processes to occur. Shelburne et al. [I.?] have shown that glucagon induced autophagy in liver cells is dependent upon adequate ATP levels. The membrane convolutional changes associated with formation of autophagic vacuoles could be blocked by lowering intracellular ATP levels while inhibition of protein synthesis did not prevent their formation. This suggested that pre-existing enzyme and membrane pools were utilized. In previous studies with the FMH organ culture model we have shown that ATP levels rapidly decrease following glucose and oxygen deprivation and return rapidly following resupply [q. The observation that occasional vacuole formation occurs during the period of deprivation indicates that sufficient energy is available in this system to initiate the process although it does not progress to the same extent as occurs in the presence of oxygen as is indicated by the rapid increase following reoxygenation. The specific factors which stimulate formation of an autophagic vacuole and the membrane from which it is derived were not determined in this study, however, their location in the periphery of the cell as well as in the perinuclear region indicates that the endoplasmic reticulum which is found in both locations is a likely contributor. This is consistent with the study of Arstila and Trump [I] in liver cells where it was shown that enzymes which typi@ endoplasmic reticulum were found in the double-membrane system enclosing the autophagic vacuoles. Whether the membranes involved are Golgi or the Golgi associated endoplasmic reticulum (GERL) of Novikoff et al. [JO] is not clear, however, and no detailed attempt was made in the present study to determine the origin of the vacuolar membrane. A preliminary study of FMH organ culture using the Gomori method for acid phosphatase revealed the presence of reaction product in the contents of some of the vacuoles [17]. One might speculate that in the early stages secretion of materials, possibly hydrolytic enzymes, into the vacuoles causes their distension with subsequent compression on the adjacent nuclear envelope resulting in the concave depression so frequently seen. It might be argued that the vacuoles seen in these cells represent a stage in progressive degeneration of the cell. However, the frequent occurrence of residual bodies, the end stage of autophagic digestion, in normal appearing myocytes in hearts resupplied with oxygen and glucose for 24 h is evidence that only focal irreversible injury occurred. If autophagic vacuoles in these cells were an indication of a continuing or accelerated generalized degenerative process, one would not expect the degree of recovery of the organelles which is seen. Instead a more generalized indication of metabolic dysfunction such as swollen mitochondria or altered distribution of nuclear chromatin should be expected. The possibility that the vacuoles are heterophagocytic rather than autophagic should be considered since it is known that muscle cells in cell culture have phagocytic capabilities [4]. Myocytes containing autophagic vacuoles were usually surrounded by structurally normal cells suggesting that the damaged cellular contents of the vacuole originated within the cell rather than from adjacent
AUTOPHAGY
IN
CARDIAC
MYOCYTES
337
necrotic debris. Furthermore, in organ culture the myocytes appear to maintain the normal contact regions with one another which would limit their mobility and inhibit migration of myocytes to a site of necrotic cells for participation in phagocytic activity. Phagocytosis of necrotic debris by cardiac myocytes may also be possible and in fact, some cells suggest that this is occurring. In these instances, however, the ingesting cell was adjacent to cells which were obviously severely damaged. It has been suggested that autophagy plays a role in the catabolic events associated with aging in mature cardiac cells. Studies in aging rats [18, 191 revealed double and single membrane-bound vacuoles containing degenerating organelles which are similar in appearance to some of the vacuoles seen in our study; a major difference being the presence of myofilaments in some vacuoles in this study. The presence of myofilaments in vacuoles in fetal cells and their absence in adult cells may be related to the structural differences between developing and mature cells. The fetal myocyte has incompletely developed myofibrils which have not yet assumed the closely packed parallel arrangements of fibrils that is seen in the adult myocyte. As such, the mitochondria and other membrane systems are often present as aggregates of organelles in close proximity to one another [13]. Events which cause focal injury to cytoplasmic components such as sublethal X-ray injury in cultured embryonic chicken heart cells [lrj or simulated ischemia in this study result in the formation of a membranous vacuole which surrounds and entraps adjacent injured mitochondria and cytoplasmic components. Occasionally short segments of myofibrils may become entrapped. The adult cell, however, with its parallel array of myofibrils tends to isolate the organelles from each other except in the relatively small perinuclear region. Consequently, the development of large vacuoles may be impeded mechanically by the fibrils. Myofibrillar engulfment is not readily achieved because of their length and because the fibrils remain relatively intact during the early stages of ischemic injury. Short isolated segments are not found unless irreversible injury has occurred. Consequently, the structural differences between adult and fetal cells may be important in the apparent differences in response to sublethal injury.
REFERENCES 1.
B. F. Studies on cellular autophagocytosis. Atian Journal (1968). BRYANT, R., THOMAS, W. A. & O’NEAL, R. M. An electron microscopic study of myocardial ischemia in the rat. Circulation Research 6, 699-709 (1958). CAULFIELD, J. & KLIONSKY, B. Myocardial ischemia and early infarction: an electron microscopic study. American 3oumalof Pathology 35,489-523 (1959). GARFIELD, R. E., CHACKO, S. & BLOSE, S. Phagocytosis by muscle cells. Luboratory Investigation 33, 418-427 (1975). HERDSON, P. B., SOMMERS, H. M. & JENNINGS, R. B. A comparative study of the fine ARSTILA,
A.
U.
& TRUMP,
of Pathology 53,687-733
2. 3. 4. 5.
338
6.
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
18. 19.
H.D.
SYBERSEl AL.
structure of normal and ischemic dog myocardium with special reference to early changes following temporary occlusion of a coronary artery. American Journal of Pathology 46,367-386 (1965). INOWALL, J. S., DELUCA, M. A., SYBERS, H. D. & WILDENTHAL, K. Fetal mouse hearts: a model for studying ischemia (ATP content/lysosomal enzymes/cardiac ultrastructure). Proceedings of the National Academy of Science, U.S.A., 72 2809-2813 (1975). JENNINGS, R. B. & GANOTE, C. E. Structural changes in myocardium during acute ischemia. CimuZation Research 35, 156-172 (1974). JENNINGS, R. B., BAUM, J. H. & HERDSON, P. B. Fine structural changes in myocardial ischemic injury. Archives of Pathology 79, 135-143 ( 1965). JENNINGS, R. B., SOMMERS, H. M., HBRDSON, P. B. & KALTENBACH, J. P. Ischemic injury of myocardium. Annals of the .New York Academy of Science 156,6 1-78 ( 1969). NOVIKOFF, A. B., ESSNER, E. & QUINTANA, N. Golgi apparatus and lysosomes. Federation Proceedings 23, 1010-1022 (1964). SCHAFER, D. Autophagosomen in gezuchteten Huhnerherzzellen.
and Cellular
Cardiology
Autophagic
(1979)
11, 331-338
Response to Sublethal iu Cardiac Myocytes
H. D. SYBERS,*
J. INGWALL,t
AND M.
* Department of Pathology, Baylor College of Medicine, t Department of Medicine and Biochemistry, University La Jolla, California, U.S.A.
(Received 15 August
1977,
Injury
DELUCAt
Houston, Texas, and of Califoornia, San Diego,
accepted in revised&m
5
3u& 1978)
H. D. SYBERS, J. INGWALL AND M. DaLuo~. Autophagic Response to Sublethal Injury in Cardiac Myocytes. jkwnal of Molecular and Cellular Cardiology (1979) 11, 331-338. Fetal mouse hearts maintained in oxygenated organ culture media continue to beat for a period of weeks. Study of the ultrastructure of the myocytes after the first day in culture revealed that most of the cells were normal, however an occasional cell had autophagic vacuoles within the cytoplasm which contained degenerating organelles indicating that focal cytcplasmic injury had occurred. Deprivation of oxygen and glucose for a period of time followed by resupply resulted in increased numbem of autophagic vacuoles. In this study,
fetal mouse hearts were maintained from 1 to 4 h in glucose-free media in an atmosphere of 95% N-5% COs after which resupply of 0s and glucose was maintained for as long aa 24 h. Many cells recovered without apparent residual injury while others contained autophagic vacuoles in an otherwise normal cytoplasm. The contents of the vacuoles ranged
from those in which organelles were readily identified to those characteristic of residual bodies. Mitochondria, glycogen and tubular structureS were seen most frequently in the vacuoles but occasionally myofilaments were identified. It is suggested that focal cytoplasmic injury which occurred during oxygen and glucose deprivation stimulates the formation of membrane to enclose the irreversibly damaged components permitting localized lysosomal digestion without causing further injury to the cell. The fetal mouse heart organ culture provides an excellent model for studying the sequential autophagic changes
which
occur in response
KEY WORDS: Autophagy; &hernia; Sublethal injury.
Fetal
to sublethal mouse
injury. Focal
heart;
cytoplasmic
injury;
Myocardial
1. Introduction The ultrastructural changes which occur following myocardial ischemia have been studied extensively in adult animals. The early changes which occur consist of swelling of organelles and depletion of glycogen granules [Z, 3, 7-91. These alterations progress to a point of irreversible injury. By this time there is margination of nuclear chromatin, mitochondria are swollen, and they often contain large dense precipitates [I#]. Eventually disruption of membranes and loss of structural Supported
by NHLI
0022-2828/79/040331+
grant
19147-02
14 $02.00/O
and NHLI
Contract 0
L-HL-81332.
1979 Academic
Press Inc.
(London)
Limited
332
H. D. SYBERS
ET AL.
integrity occurs. If blood is reperfused into the ischemic region there is an acceleration of structural alterations and calcium uptake in those cells destined to undergo necrosis, while the cell cytoplasm reverts to normal in those which will recover [5, 151. The question of whether or not focal irreversible cytoplasmic injury has occurred in the cells which recover and, if so, in what manner these injured orgenellesare handled, has not been well studied in cardiac myocytes. Studies in which we employed fetal mouse hearts (FMH) maintained in organ culture under conditions of oxygen and glucosedeprivation to simulate someof the conditions of ischemia revealed many cellular alterations which are similar to those seenin coronary induced ischemia of mature cardiac cells [6, 16, 171. There was swelling of organelles, reduction in glycogen, increase in lipid vacuoles and margination of nuclear chromatin followed by membrane destruction. These alterations did not occur as rapidly in the FMH organ culture as those seen in adult ischemia, and many of them could be reversed in some of the cells after as long as4 h of deprivation. Many of the cellswhich returned to an otherwise normal ultrastructural appearance, revealed the presenceof autophagic vacuoles containing degenerating organellesin their cytoplasm. Since similar findings have not been reported in sublethal adult ischemic cardiac injury, it would appear that fetal cells are better able to isolate focally injured cytoplasmic components for lysosomal degradation. The present report details the sequenceof morphologic changeswhich is seen in the development of the autophagic vacuoles and offers a speculation as to why similar findings are not commonly seen in adult myocytes following ischemic injury. 2. Materials
and Methods
Intact beating hearts obtained from 15 to 20 day fetal mice were maintained with Minimum Essential Medium (MEM), on stainless steel grids in organ culture dishes at an air (95% Os-5’$$ COa)-medium interface at 37°C as previously described [16l. Hearts were maintained in culture for at least 18 h to allow stabilization before the experimental conditions were imposed. Controls The control hearts were maintained in culture medium at 37°C in an environment of 95% Oa-5°/o CO2 for periods of time which corresponded with those of the experimental groups. Hearts from matched litter mates were used in the experimental groups. .?G#whntal:
deprivation of glucose and oxygen
The hearts were deprived of oxygen and glucosefor periods ranging from 1 to 4 h
AUTOPHAGY
IN
CARDIAC
MYOCYTES
333
at 37°C by replacing the culture medium with argon or nitrogen saturated, glucose-free MEM and incubating them in sealed culture jars continuously, In some cases they were periodically flushed with 95% Ns-5% COs. Oxygen content of the media, as determined with an Instrumentation Laboratories gas analyzer, was less than 5 mmHg. At the end of the experimental period the hearts were rapidly fixed by immersion in 5% gluteraldehyde in phosphate buffer, pH 7.3 at 4°C. The atria were removed under a dissecting microscope and tissue from the ventricles was minced and allowed to fix for 4 h. After postfixation in 1o/o osmium tetroxide and dehydration in acetone, the ventricular tissue was embedded in Araldite for electron microscopy. At least two blocks of tissue were selected randomly from each heart for examination. Thick sections (1 pm) were prepared and stained with toluidine blue for orientation. Thin sections were stained with uranyl acetate and lead citrate and examined in a Zeiss 9A electron microscope. Experimental:
resupply of glucose and oxygen
In several of the hearts, oxygen and glucosewere resupplied for 1 to 24 h at 37°C after periods of deprivation of 1 to 4 h duration and were prepared for and examined with the electron microscope as described. In four hearts resupply of glucoseand oxygen was begun after 1 h of deprivation and in another four hearts after 2 h of deprivation. Two from each group were processedfor electron microscopy after 1 and 2 h of resupply. An additional two hearts were subjected to 3 h and two hearts to 4 h of deprivation followed by a period of 24 h of resupply of oxygen and glucosebefore preparing them for electron microscopy. 3. Results
Myocytes from the control hearts which had been incubated in oxygenated media retained a normal ultrastructure in most of the cells (Plate 1)) however an occasional cell showed evidence of degenerative changes as previously described [I6]. The cells were oval to slightly elongated and the extent of development of the myofibrils was variable. In those cells with poorly developed myofibrils the thin filaments often appeared to arise from or to be attached to desmosomes.Round to oval nuclei occupied a large proportion of the cell volume. Mitochondria were frequently elongated with irregular shapes.Glycogen granules were abundant and numerous ribosomeswere present. A prominent Golgi apparatus was often seenin the perinuclear region. Tubular structures, thought to be sarcoplasmicreticulum, were found throughout the cytoplasm but transverse tubules were identified only when the myofibrils were well developed. The sarcolemma had specialized regions with desmosomes and occasionalgap junctions. Pinocytotic vesicleswere frequently seenalong the sarcolemma.
334
H.
D. SYBERS
ET AL.
The hearts subjected to oxygen and glucose deprivation revealed ultrastructural alterations which became more apparent as the duration of deprivation was increased. After 1 h most cells showed only minimal to moderate alterations, with mild swelling of mitochondria, a slight reduction in glycogen granules, and often an increased number of lipid vacuoles. There was a slight increase in the number of cells containing autophagic vacuoles, however, many cells remained quite normal in appearance. Interstitial space often appeared to be increased. After 2 to 3 h of deprivation, mitochondrial swelling with decreased matrix density was more pronounced in most of the cells, but the presence of dense intramitochondrial granules was rarely seen and, except for occasional cells, nuclear alterations were not severe at 2 h. Structurally normal or nearly normal cells were still seen occasionally after 3 h of glucose and oxygen deprivation (Plate 2). Four h of deprivation resulted in moderate to severe ultrastructural alterations in virtually all of the myocytes with a marked decrease in glycogen granules, swelling and decreased matrix density of motochondria, and myofibrillar disorganization. Margination of nuclear chromatin was present in most of the cells. Sarcolemmal disruption was frequently seen and cellular debris was common in the interstitial spaces (Plate 3). Phagocytic cells ingesting remnants of myocytes were often seen. The hearts in which oxygen and glucose were resupplied following a period of deprivation resumed beating and structural recovery occurred in many of the cells following periods of up to 4 h of deprivation. Numerous cells, however, had vacuoles containing degenerating organelles in an otherwise normal cytoplasm which suggested that the period of deprivation caused the occurrence of focal irreversible injury in cells which ultimately recovered. Resupply after 1 h resulted in a return toward normal structure in the majority of the cells, however, a marked increase in the number of cells containing autophagic vacuoles was observed after the first hour of resupply. These vacuoles contained structurally altered cellular components which were easily identified. Degenerating mitochondria, myofibrils, and glycogen granules were often seen surrounded by a single or double layered membrane which at times was incomplete. The vacuoles were most commonly located in the perinuclear region and the nuclear envelope frequently had a concavity adjacent to the vacuoles as if indented by pressure from the vacuole (Plate 4). Vacuoles were also often found in the periphery of the cell. In these the organelles frequently appeared to have undergone a greater degree of degradation than those in the perinuclear region and they occasionally had densely stained osmiophilic contents (Plate 5). The membranes surrounding the vacuoles did not have desmosomes or gap junctions. Furthermore, the adjacent cells were usually normal in appearance. This suggests an intracellular origin hence a true autophagic vacuole, rather than a protrusion of an adjacent cell into the cytoplasm. When the hearts were exposed to 3 to 4 h of oxygen and glucose deprivation,
PLATE 1. Normal control. The nuclei (N) are proportionally large and have evenly dispersed (m) are chromatin. The Golgi apparatus (g) is p rominent in the perinuclear region. Mitochondria normal. Myofilaments are moderately developed. x 11 500. PLATE 2. Three h of oxygen and glucose deprivation. Margination of nuclear chromatin is apparent. Mitochondria (m) are swollen and the number of christae appears to be reduced. Lipid vacuoles (L) are frequently seen and the sarcoplasmic reticulum (SR) is dilated. Myofibrillar disorganization and reduction in myofibrils is apparent. Nucleus (N). x 10 ZOO. PLATE 3. Four h of deprivation. Margination of nuclear chromatin and mitochondrial swelling is more pronounced than in Plate 2. Many cells are frankly necrotic (arrows) with ruptured sarcolemma and mitorhondrial (m) membranes. x 7300. PLATE 4. Early autophagic vacuole. An early stage of vacuole formation is shown which contains mitochondria (m) with decreased matrix density and dense precipitates, tubules, and filaments. A double-membrane surrounds most of the vacuole (arrows). The nucleus (N) has a concave indentation as if caused by pressure from the autophagic vacuole (A). Desmosomes (d). x 11 700. PLATE 5. Intermediate stage. Three autophagic vacuoles (A) are seen in the periphery of a cell. Degenerating mitochondria containing dense granules and myofilaments (mf) can be seen. The remaining cytoplasm is normal appearing and contains numerous ribosomes (arrow) along the myofilaments. Mitochondria (m). x 23 000. PLATE 6. Four h oxygen and glucose deprivation followed by 24 h of resupply. Many cells have returned to a normal appearance. Mitochondria (M) have well-defined cristae. There is a normal dispersion of nuclear chromatin (N). Dense autophagic vacuoles (A) are frequently found. Many crlls have undergone necrosis (NE). x 9240. PLATE 7. Intermediate stage of autophagic vacuoles. Some organelles are still recognized in these coalsescing vacuoles (A) which contain myofilaments (mf) and mitochondria with dense intramitochondrial granules. The surrounding membrane can be seen in some areas (arrows). Note the normal appearance of the remaining cell cytoplasm. Glycogen (G). Interstitial space (IS). x 18 400. PLATE 8. Late stage. Dense osmiophilic residues (A) make up the end stages of autophagic digestion. Myelin figures (MY) are seen on the periphery of the vacuole. x 17 480. PLATE 9. Phagocytosis. When extensive cell breakdown occurs, phagocytic cells (P) containing an abundance of rough endoplasmic reticulum (ER) engulf the necrotic debris from the degenerating myocytes. The myofilaments and degenerating mitochondria within this vacuole are readily identified. A thin rim of cytoplasm from the phagocyte encircles the vacuole. Phagocyte nucleus i?j). A fibril from an adjacent normal myocyte (M) is seen on the left. x 11 500.
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resupply for 24 h resulted in a return to normal structure in many of the cells but residual bodies or lamellar bodies were frequently found within their cytoplasm. Discrete organelles were less frequently seen within the autophagic vacuoles suggesting that by this time extensive lysosomal degradation had occurred. The remainder of the cell cytoplasm and nucleus had returned to a normal ultrastructure except for an apparent decrease in myofilaments. As indicated previously, many cells had undergone necrosis when the period of injury was of 3 or 4 h duration and no attempt was made in this study to determine the relative proportion of cells which suffered irreversible damage as compared to those which sustained sublethal injury and subsequently recovered (Plate 6). The structural appearance of autophagic vacuoles from the early stages of sequestration of focally injured cytoplasm to degradation and residual body formation is shown in Plates 4, 5, 7 and 8. In addition to the autophagic response described above, phagocytosis of necrotic myocyte debris by phagocytic cells was frequently seen when lethally injured cells were present (Plate 9). These phagocytic cells are characterized by an abundance of rough endoplasmic reticulum in their cytoplasm. 4. Discussion
The presenceof autophagic vacuoles in adult cells following transient ischemia has not been emphasized [7, 91 but has been reported in fetal cells in organ culture [S, 16, 171. This suggeststhat autophagy is not a common occurrence in sublethal ischemic injury or that the vacuoles are not conspicuousin adult cells. While it is recognized that ischemia in the experimental animal is not identical with the conditions of glucose and oxygen deprivation in an organ culture system, the structural alterations which occur are similar and it is reasonable to suspect that many of the same stimuli for initiation of focal repair processesoccur in both systems. The formation of autophagic vacuoles in cardiac myocytes for the sequestration, isolation, and degradation of focally damaged cytoplasmic components appears to be a common reaction to sublethal injury in fetal mouse hearts (FMH) in organ culture. While they are seenoccasionally in the absenceof interventions designed specifically to produce cell injury [16], their occurrence is probably a result of transient anoxia during removal of the heart from the fetus. This period may be of 30 to 40 min duration before dissection of the last fetus in the litter has been completed. Deprivation of oxygen and glucose from the organ culture results in cell alterations which are structurally similar to thoseof ischemic injury in adult myocytes. Its resupply prior to development of irreversible injury elicits a marked increase in the number of cells containing autophagic vacuoles. The rapid increase in vacuole formation following resupply is an indication of the need for adequate energy
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supplies for these processes to occur. Shelburne et al. [I.?] have shown that glucagon induced autophagy in liver cells is dependent upon adequate ATP levels. The membrane convolutional changes associated with formation of autophagic vacuoles could be blocked by lowering intracellular ATP levels while inhibition of protein synthesis did not prevent their formation. This suggested that pre-existing enzyme and membrane pools were utilized. In previous studies with the FMH organ culture model we have shown that ATP levels rapidly decrease following glucose and oxygen deprivation and return rapidly following resupply [q. The observation that occasional vacuole formation occurs during the period of deprivation indicates that sufficient energy is available in this system to initiate the process although it does not progress to the same extent as occurs in the presence of oxygen as is indicated by the rapid increase following reoxygenation. The specific factors which stimulate formation of an autophagic vacuole and the membrane from which it is derived were not determined in this study, however, their location in the periphery of the cell as well as in the perinuclear region indicates that the endoplasmic reticulum which is found in both locations is a likely contributor. This is consistent with the study of Arstila and Trump [I] in liver cells where it was shown that enzymes which typi@ endoplasmic reticulum were found in the double-membrane system enclosing the autophagic vacuoles. Whether the membranes involved are Golgi or the Golgi associated endoplasmic reticulum (GERL) of Novikoff et al. [JO] is not clear, however, and no detailed attempt was made in the present study to determine the origin of the vacuolar membrane. A preliminary study of FMH organ culture using the Gomori method for acid phosphatase revealed the presence of reaction product in the contents of some of the vacuoles [17]. One might speculate that in the early stages secretion of materials, possibly hydrolytic enzymes, into the vacuoles causes their distension with subsequent compression on the adjacent nuclear envelope resulting in the concave depression so frequently seen. It might be argued that the vacuoles seen in these cells represent a stage in progressive degeneration of the cell. However, the frequent occurrence of residual bodies, the end stage of autophagic digestion, in normal appearing myocytes in hearts resupplied with oxygen and glucose for 24 h is evidence that only focal irreversible injury occurred. If autophagic vacuoles in these cells were an indication of a continuing or accelerated generalized degenerative process, one would not expect the degree of recovery of the organelles which is seen. Instead a more generalized indication of metabolic dysfunction such as swollen mitochondria or altered distribution of nuclear chromatin should be expected. The possibility that the vacuoles are heterophagocytic rather than autophagic should be considered since it is known that muscle cells in cell culture have phagocytic capabilities [4]. Myocytes containing autophagic vacuoles were usually surrounded by structurally normal cells suggesting that the damaged cellular contents of the vacuole originated within the cell rather than from adjacent
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necrotic debris. Furthermore, in organ culture the myocytes appear to maintain the normal contact regions with one another which would limit their mobility and inhibit migration of myocytes to a site of necrotic cells for participation in phagocytic activity. Phagocytosis of necrotic debris by cardiac myocytes may also be possible and in fact, some cells suggest that this is occurring. In these instances, however, the ingesting cell was adjacent to cells which were obviously severely damaged. It has been suggested that autophagy plays a role in the catabolic events associated with aging in mature cardiac cells. Studies in aging rats [18, 191 revealed double and single membrane-bound vacuoles containing degenerating organelles which are similar in appearance to some of the vacuoles seen in our study; a major difference being the presence of myofilaments in some vacuoles in this study. The presence of myofilaments in vacuoles in fetal cells and their absence in adult cells may be related to the structural differences between developing and mature cells. The fetal myocyte has incompletely developed myofibrils which have not yet assumed the closely packed parallel arrangements of fibrils that is seen in the adult myocyte. As such, the mitochondria and other membrane systems are often present as aggregates of organelles in close proximity to one another [13]. Events which cause focal injury to cytoplasmic components such as sublethal X-ray injury in cultured embryonic chicken heart cells [lrj or simulated ischemia in this study result in the formation of a membranous vacuole which surrounds and entraps adjacent injured mitochondria and cytoplasmic components. Occasionally short segments of myofibrils may become entrapped. The adult cell, however, with its parallel array of myofibrils tends to isolate the organelles from each other except in the relatively small perinuclear region. Consequently, the development of large vacuoles may be impeded mechanically by the fibrils. Myofibrillar engulfment is not readily achieved because of their length and because the fibrils remain relatively intact during the early stages of ischemic injury. Short isolated segments are not found unless irreversible injury has occurred. Consequently, the structural differences between adult and fetal cells may be important in the apparent differences in response to sublethal injury.
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