Journal of Molecular and Cellular Cardiology (1915) 7, 315-324
Ultrastructural
D. J. HEARSE,
Damage Associated with of the Anoxic Myocardium
S. M. HUMPHREY,
W. G. NAYLER,
Reoxygenation
A. SLADE
AND D. BORDER
Department of Biochemistry, Imperial College of Science and Technolopv, Kensington, London and the Cardiothoracic Institute, 2 Beaumont Street, London, U.K. (Received 7 January
1974, accepted in revisedform
28 May 1974)
D. J. J~EARSE,S. M. HUMPHREY, W. G. NAYLER, A. SLADE AND D. BORDER. Ultrastructural Damage Associated with Reoxygenation of the Anoxic Myocardium. Journal of Molecular and Cellular Cardiology (1975) 7, 3 15-324. Reoxygenation of the anoxic myocardium in the perfused rat heart following the onset of enzyme release causes an immediate and massive exacerbation of enzyme release. This effect appears to be directly attributable to the readmission of molecular oxygen. An electron microscopic investigation has revealed that reoxygenation induces sudden and major ultrastructural damage. Initially this damage involves some loss of the basement membrane and the physical disruption of the plasma membrane, the latter probably contributing to the sudden and large loss of cytoplasmic enzymes. The damage spreads rapidly throughout the cell causing disorganization of the myofibrils and mitochondrial damage. A possible biochemical basis of this effect is discussed. In addition, results are presented illustrating the metabolic protection afforded to the heart by exogenous supplies of glucose during anoxia. This is made possible by a glucose dependent preservation of myocardial ultrastructure and is reflected in a greatly reduced level of enzyme leakage. KEY WORDS: Anoxia; Reoxygenation; Enzyme release; Isolated perfused rat heart; Ultrastructural damage; Creatine phosphokinase; Oxygen toxicity; Electron microscopy; Glucose; Metabolic protection; Reperfusion.
1. Introduction In previous studies [S] of enzyme release from the isolated perfused anoxic rat heart we reported that abrupt reoxygenation of previously anoxic tissue can greatly exacerbate enzyme leakage from the myocardium. In these studies K+ arrested rat hearts were subjected to substrate free anoxic perfusion. Under these conditions, enzyme release commenced after less than 1 h and was maintained for at least 8 h. If the hearts were reoxygenated after 100 min anoxia an immediate and massive release of enzyme was observed, such that after 2 min, levels had increased by 100 to 200 fold. In these studies, due to the constancy of the perfusate ion concentration, the perfusion pressure and coronary flow, the exacerbation of release was attributed directly to the readmission of molecular oxygen. In view of the magnitude of release and the relative invariance of the ratio of protein to cytoplasmic enzyme release it was suggested that leakage probably resulted from
316
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ETAL.
extensive ultrastructural damage and not from simple changes in membrane permeability characteristics. In an attempt to explain the oxygen-induced exacerbation of enzyme release, an investigation of ultrastructural changes during reoxygenation has been undertaken. In additional studies [9] we have illustrated how glucose is able to protect the anoxic myocardium against enzyme leakage. This observation has also been characterized with electron microscopic investigations.
2. Materials Experimental
and Methods animals and chemicals
Male rats (280 to 320 g body wt) of the Sprague-Dawley strain, maintained on a standard diet, were used in these experiments. All substrates and enzymes used in the analysis of perfusion fluid were obtained from the Boehringer Corporation (London) Ltd.
Perfusion techniques Rats were lightly anaesthetized with diethyl ether, the left femoral vein was exposed and heparin (200 I U) was administered intravenously. One min after administration of heparin the heart was excised [14] and placed in ice-cold perfusion medium until contraction had ceased. The aorta was then cannulated and, after mounting, the arrested heart was restarted by perfusion (non-recirculating) as described by Langendorff 1131 with a perfusion pressure of 100 cm of water. During the anoxic perfusion period, atmospheric gas contamination of the heart was prevented by completely enclosing the heart in a waterjacketed (37 “C) chamber which was continuously gassed with a mixture of NZ + CO2 (95 : 5). In addition, to prevent diffusion of oxygen into the perfusion fluid, all silicone rubber tubing connecting reservoirs and the heart were replaced by glass delivery tubing.
Perfusion medium Krebs-Henseleit bicarbonate buffer [I,!?], pH 7.4 containing glucose (11.1 mM) was used in the initial pre-anoxic control perfusion period. During the anoxic perfusion and reoxygenation period the hearts were K+-arrested [8], under these conditions, the K+ concentration of the perfusion fluid was increased to 16.0 mM and the Na+ concentration was correspondingly decreased. In the glucose protec-
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317
tion studies, glucose (11.1 mM) was added to the anoxic perfusion fluid. All perfusion fluid was maintained at 37 “C. In aerobic studies the fluid was equilibrated with 0~ + CO2 (95 : 5, aortic 02 partial pressure was over 600 mm Hg) and in anoxic studies (high K+) the fluid was equilibrated with Na + CO2 (95 : 5, aortic 0s partial pressure was less than 5 mm, Hg). Precautions [20] were taken to prevent the precipitation of Ca 2+. Before use, the perfusion fluid was filtered through a cellulose acetate filter of pore size 5.0 pm (Millipore Ltd.).
Experimental
time course
Immediately after mounting, the hearts were perfused aerobically for a 5 min period. During this time the stability of the preparation could be confirmed. At the end of this period the hearts were perfused anoxically for a predetermined time period. At any time during this period the composition of the perfusion fluid could be changed by introducing another fluid from a second reservoir. For example, in reoxygenation studies a perfusion fluid (K+, 16.0 mM) gassed with 02 + COs (95 : 5) was introduced. During the entire experimental period coronary flow was monitored and samples could be taken for enzyme analysis. At any time the experiment could be terminated and fixative could be introduced into the coronary system via a side arm on the aortic cannula which was connected to an infusion pump. After fixing, the tissue could be taken for electron microscope studies.
Enzyme release analysis Creatine phosphokinase (ATP: creatine phosphotransferase E.C.2.7.3.2) assayed as described by Hearse et al. [8]. Enzyme activity in the perfusion was expressed as mIU released/ml.
was fluid
Tissue preparation and electron microscopy Hearts were fixed for electron microscopy at various times during the experimental time course (see results section for details of times and conditions). The fixative had the following composition: 5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 at 4 “C. At the end of the experimental period the heart was surrounded with ice-cold fixative. Using the side arm of the aortic cannula the heart was then perfused slowly (10 ml/min for 2 min) with ice-cold fixative. A left ventricular biopsy was taken close to a branch of the left anterior descending coronary artery. This was then cut into small pieces (approx. I mma)
318
D.
J.
HEARSE
ETAL.
and immersed in 5% glutaraldehyde at 4 “C for 2 h. The tissue was then washed buffer at 4 “C containing for 3 x 30 min periods in 0.1 M sodium cacodylate sucrose (4.5%). After a further 12 h in the buffer the tissue was post-fixed in osmium tetroxide (1 o/o in cacodylate buffer) for 2 h followed by 2 x 15 min washes in distilled water. After dehydration through a sequential series of ethanols the tissue was washed for 10 min in propylene oxide and then infiltrated with resin by leaving it for two consecutive I h periods in a mixture of propylene oxide and “araldite epoxy resin mixture” (50 : 50 and 20 : 80 respectively). The tissue was then placed in “araldite epoxy resin mixture” for 12 h (the “araldite” had the following composition: 10 ml araldite epoxy CY212 resin, 10 ml dodecenyl succinic anhydride, 0.4 ml dibutylphthalate). After this period the tissue was placed in fresh “araldite mixture” for 5 h. The tissue was then embedded (60 “C for 48 to 60 h) in the “araldite mixture” defined above but including 0.25 ml 2,4,6-tri(dimethylaminomethyl)phenol. Thin sections, cut on a LKB ultramicrotome, were stained with saturated aqueous uranyl acetate for 15 min followed by Reynolds [15] lead citrate (20% solution) and examined with an AEI 801 electron microscope.
3. Results Enzyme release studies Anoxia Hearts were subjected to anoxic perfusion for at least 7 h and enzyme release was measured at 15 min intervals. Previous studies [8, 91 have shown that the release of creatine phosphokinase is essentially representative of several enzymes (a-hydroxybutyrate dehydrogenase, glutamate oxaloacetate transaminase and adenylate kinase). The release profile for creatine phosphokinase is illustrated in Figure 1. Extensive enzyme release occurred over the 7 h period.
Reoxygenation Hearts were subjected to 150 min anoxia (perfusion fluid: Kf, 16.0 mM; (perfusion glucose free; gassed with Na + CO2 95 : 5) and then reoxygenated fluid: K+, 16.0 mM; glucose free; gassed with 02 + CO2 95 : 5). Immediately following reoxygenation (Figure 1) there was a massive release of enzyme activity such that after 2 min, levels had increased by approximately 100 fold. Due to the constancy of the per&sate ion concentration, perfusion pressure and coronary flow the exacerbation of release appears to be directly attributable to the readmission of molecular oxygen.
PLATE 3
PLATE 4
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P L A T E5
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DAMAGE
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319
4600
f $ P y % -: 0 P g E ‘2 0
600-
. .
500400300200100 0 em-*- .-.-. 0 50
.-’ 100
/--‘----I. .l /--• l- .l. I 250I 300i--r 350. 400I 0 200 Perfusion time (min)
FIGURE 1. Profile for the release of creatine phosphokinase (mIU/ml coronary flow) from the isolated perfused rat heart following the onset of anoxia (t = 5). (a), extended anoxia; (B), reoxygenation after 150 min. For details of perfusion see text.
Glucose protection Hearts were subjected to 7 h anoxia with glucose (11.1 mM) present in the perfusion fluid. Under these conditions (Figure 2), gIucose was shown to exert a marked protective effect. In contrast to anoxic perfusion in the absence of glucose, enzyme release was reduced by approximately 75% over the 7 h period. As a control, hearts were perfused aerobically in the presence of glucose (11.1 mM) for 7 h and under these conditions (Figure 2) enzyme release was negligible.
Ultrastructural The ultrastructure obtained from rat 150 min anoxic 150 min anoxic 180 min anoxic 150 min anoxic
studies
of ventricular myocardial cells was investigated using tissue hearts which had been subjected to the following conditions : perfusion perfusion plus 2 min reoxygenation perfusion perfusion plus 30 min reoxygenation
320
D. J. HEARSE
Perfusion
El-AL.
time (min)
FIGURE 2. Profile for the release of creatine phosphokinase (mIU/ml coronary flow) from the isolated perfused rat heart. (a), 420 min glucose free anoxic perfusion; (+), 420 min anoxic perfusion plus glucose (11.1 111~); (A), 420 min aerobic perfusion plus glucose (11.1 mM). For details of perfusion see text. Each point represents the mean for four hearts and the bars represent the S.E.M.
180 min anoxic perfusion in the presence of glucose (11.1 mM) 180 min aerobic perfusion in the presence of glucose (11.1 mM) Fresh tissue subjected to aerobic perfusion for 2 min.
Ultrastructural
changes induced by anoxia
In comparison to Plates 4(a) and 5(b) (fresh tissue and aerobically perfused tissue respectively) Plates 1,3(a) and 4(b) indicate damage that may be attributable to anoxia. After 150 min anoxic perfusion, enzyme release (Figure 1) has already commenced and some small ultrastructural changes are apparent (Plate 1). The T-tubules are slightly dilated and, while remaining intact, there is some evidence However, the basement membrane is of distortion of the plasma membrane. intact, the mitochondria appear dense and numerous with preserved cristae. The sarcoplasmic reticulum is intact, the Z lines, A and I bands are normal. Numerous, well defined electron dense granules are apparent in the mitochondria and cytoplasm.
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321
After 180 min anoxia [Plates 3(a) and 4(b)], when extensive enzyme release (Figure 1) has occurred, additional ultrastructural changes are apparent. While some Z lines remain well-defined others were distorted. There is evidence for the leaching of actin density and for the whisping of myofibrils. In addition there is evidence of dense, rounded, aggregated mitochondria, translocated towards the intact plasma membrane. Despite this, the mitochondria are intact, regular in outline and have well defined cristae. There is evidence of tissue oedema. The interstitial spaces are filled with electron dense material. Electron dense granules are not apparent in the cytoplasm or the mitochondria.
Ultrastructural
changes induced by reoxygenation
After 2 min reoxygenation (Figure 1) enzyme release has increased by approximately 100 fold. Examination of tissue at this time [Plate 2(a) and (b)] in comparison with tissue that has not been reoxygenated (Plate 1) reveals additional damage. There is evidence for some loss of basement membrane and fragmentation of the plasma membrane. There is a rapid loss of electron density of myofibrils, they appear whispy, the Z lines are distorted and there is a marked increase in interfibrillar space. In contrast with Plate 1 there are more interstitial spaces and they lack electron dense material. Some mitochondria are swollen and less electron dense. However, the majority of the mitochondrial membranes and cristae remain intact. Electron dense granules are not apparent. Thirty min reoxygenation [Plate 3(b)], in contrast with 180 min anoxia [Plate 3(a)], reveals the extensive damage associated with reoxygenation. There has been severe disruption of the myofibrils with the loss of Z lines. Some basement membrane is lost and the plasma membrane is extensively disrupted. The mitochondria are swollen and irregular [Plate 3(b)]. There is no mitochondrial alignment relative to the myofibrils and the T-tubules are distended. Electron dense granules are not apparent, in the cytoplasm or the mitochondria.
Glucose protection Inclusion of glucose (11.1 mM) in the anoxic perfusion fluid greatly reduces enzyme release (Figure 2) and has been shown to protect the myocardium against anoxic damage [7, 91. Examination of tissue obtained after 180 min anoxic perfusion plus glucose [Plate 5 (a)] reveals, in contrast to glucose free anoxic perfusion [Plate 4(b)], considerable ultrastructural preservation. Myofibrils are intact and electron dense. The Z lines are regular and show good continuity. The mitochondria are well preserved and aligned between the myofibrils. The T-tubules show some dilation but there are no interstitial spaces or electron dense granules.
3‘22
D. J. HEARSE
ETAL.
Plate 5(b) illustrates tissue obtained after 180 min aerobic perfusion in the presence of glucose. This shows very good preservation of myofibrils, mitochondria and T-tubules. Numerous electron dense granules are apparent in both the cytoplasm and the mitochondria.
4. Discussion The results described in this paper have revealed that the oxygen induced exacerbation of enzyme release from the anoxic rat heart occurs as a result of sudden and major ultrastructural damage. In the first few minutes of reoxygenation, this damage appears to be largely confined to the cell periphery with some loss of the basement membrane and the physical disruption of the plasma membrane. This fragmentation of the plasma membrane probably contributes to the rapid and large increase in the release of cytoplasmic enzymes and protein. In the course of the next 30 min of reoxygenation the damage spreads throughout the cell with disruption of the myofibrils and considerable mitochondrial damage. The mitochondria exhibit morphological changes including swelling, distortion, loss of cristae and translocation within the cell. The majority of the mitochondrial membranes do however remain intact, this is consistent with the fact that glutamate dehydrogenase and other mitochondrial enzymes are not released in high concentrations during the conditions of anoxia or reoxygenation used in this study. (D. J. Hearse and S.M. Humphrey, unpublished results.) The biochemical basis of this reoxygenation phenomenon is open to speculation. Controlled experiments (D. J. Hearse and S. M. Humphrey, unpublished results) with graded reoxygenation and with reoxygenation at earlier time periods have eliminated micro-emboli as possible causative agents and additional studies [9] would suggest that the results are not due to a loss of cellular anti-oxidants. Due to the maintenance of constant coronary flow and perfusion pressure, mechanical damage should not occur. Hyperoxia and consequent lipid peroxidation have been shown [I] to increase the permeability of lipoprotein membranes but there have been no reports of lipid peroxidation causing damage as rapidly or as extensively as that reported in this paper. A possible explanation for the reoxygenation damage to the plasma membrane may be that reoxygenation induces the sudden redistribution of ions, either lost from, or gained by, the cytoplasm during anoxia. This sudden ion flux may possibly cause damage to the plasma membrane. In this connection it has been reported [S, 18, 191 that reoxygenation or reperfusion of previously ischaemic tissue results in large and rapid ion shifts, particularly Ca2+. The presence or absence of electron dense granules (both cytoplasmic and mitochondrial) under various conditions in this study cannot be completely explained but may be related to ion fluxes. While some of the granules are un-
323
REOXYGENATIONDAMAGEINTHEANOXICMYOGARDIUM
doubtedly glycogen deposits [Plate 4(a)] others may possibly be calcium phosphate [IS] deposits. The existence of elevated concentrations of both Caa+ and inorganic phosphate in the cytoplasm during anoxia could be readily explained. The sudden loss of these granules observed upon reoxygenation [Plates Z(a) and (b)] may support the concept of membrane damage induced by sudden transmembrane ion fluxes. The findings reported in this paper, although obtained in reoxygenation studies in the isolated rat heart, may have some bearing on studies invoking reperfusion of ischaemic myocardial tissue. While many investigators have reported [for example 5, 171 reversal of damage and consequent tissue salvage following either the reperfusion or revascularization of ischaemic tissue, there have been a few reports [Z-4, 10, II] that reperfusion or revascularization can accelerate or extend tissue damage. In general these observations of increased tissue damage have been related to the mechanical consequences of a rapid re-establishment of flow. The possibility now exists however that the extension of damage may be explained in terms of the results presented in this paper. Investigating the possibility of protecting the myocardium by including glucose in the anoxic perfusion fluid our results have shown that the observed reduction in enzyme loss could be associated with a considerably improved preservation of myocardial ultrastructure. Sybers et al. [IS] have reported that glucose-insulinpotassium infusions had a protective effect on cardiac ultrastructure in dogs which had been subjected to experimental coronary occlusion. Despite the differences in experimental models, their results and ours both underline the potential value of metabolic interventions in anoxic or ischemic tissue.
Acknowledgement This work was carried out with aid of grants from the British and the Medical Research Council.
Heart
Foundation
REFERENCES ALLISON, A. C. Role of lysosomes in oxygen toxicity. nuture 205, 141-143 (1965). BANKA, V. S., CHADDA, K. D., MEISTER, S. G. & HELFANT, R. H. Limitations
of myocardial revascularization in restoration of regional contraction abnormalities produced by coronary occlusion. American Journal of Cardiology 31, 118 (1973). BELLER, G. A., SMITH, T. W., SNYDER, L. T. & HOOD, W. B. Accelerated injury in reperfused myocardium: Relation to duration of ischaemia. Circulation Suppl. IV to Vols VII and VIII, 143 (1973). BRESNAHAN, G. F., SHELL, W. E., Ross, J., ROBERTS, R. & SOBEL, B. E. Deleterious effects of reperfusion in evolving myocardial infarction. Circulation 46, Suppl. II, 1 I- 13 (1972).
324
9.
D. J. HEARSE ETAL. GINKS, W. R., SYBERS, H. D., MAROKO, P. R., CORELL, J. W., SOBEL, B. E. & ROSS, J. Coronary artery reperfusion. Journal of Clinical Investigation 51, 2717-2723 (1972). HAMILTON, D. G. & WHALEN, D. A. Early phase of irreversible myocardial cell injury. Federation Proceedings 31, 627 (1972). HEARSE, D. J. & CHAIN, E. B. The role of glucose in the survival and recovery of the anoxic isolated perfused rat heart. Biochemical Journal 128, 1125-l 133 (1972). HEARSE, D. J., HUMPHREY, S. M. & CHAIN, E. B. Abrupt reoxygenation of the anoxic potassium-arrested perfused rat heart: A study of myocardial enzyme release. Journal of Molecular and Cellular Cardiology 5, 395-407 (1973). HEARSE, D. J. & HUMPHREY, S. M. Enzyme release during myocardial anoxia: a study of metabolic protection. Journal of Molecular and Cellular CardioloQ (1975) (In
press). 10. HERDSON, P. B., SOMMERS, H. M. &JENNINGS, R. B. A comparative study of the fine structure of the normal and ischaemic dog myocardium with special reference to early changes following temporary occlusion of a coronary artery. American Journal of II.
Pathology 46, 367-377 (1965). JENNINGS, R. B., SOMMERS, H. M., SMYTH, G. A., FLACK, H. A. & LINN, H. Myo-
cardial necrosis induced by temporary 12. 13. 14. 15. 16.
occlusion of a coronary
artery in the dog.
Archives of Pathology 70, 82-92 (1960). KREBS, H. A. & HENSELEIT, K. Untersuchungen tiber die Harnstoffbildung im Tierkorper. EIo@e-Seyler’s zeitschrift fiir physiologische Chemie 210, 33-66 (1932). LANGENDORFF, 0. Untersuchungen am uberlebenden Saugertierherzen. Pjtigers Archiv ftir gesamte Physiologie de Men&en und der Tiere 61, 291-332 (1895). NEELY, J. R., LIEBERMEISTER, H., BATTERSBY, E. J. & MORGAN, H. E. Effect of pressure development on oxygen consumption by the isolated rat heart. American Journal of Physiology 212, 804-814 (1967). REYNOLDS, E. S. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. Journal of Cell Biology 17, 208-2 12 (1963). SYBERS, H. D., MAROKO, P. R., A~HRAP, M., LIBBY, P. & BRAUNWALD, E. The effect
of glucose-insulin-potassium on cardiac ultrastructure following acute experimental coronary occlusion. American Journal of Pathology 70, 401-420 (1973). 17. SYMES,J. F., ARNOLD, I. M. F. & BLUNDELL, P. E. Early revascularization of the acute myocardial infarction: The critical time factor. Canadian Journal of Surgery 16, 275-283 (1973). 18. SHEN, A. C. &JENNINGS, R. B. Myocardial calcium and magnesium in acute ischaemic injury. American Journal of Pathology 67, 417-440 (1972). 19. SHEN, A. C. &JENNINGS, R. B. Kinetics of calcium accumulation in acute myocardial ischaemic injury. American Journal of Pathology 67, 441-452 (1972). 20. UMBREIT, W. W., BURRIS, R. H. & STAUFFER, J. F. In Manometric Techniques, p. 132, Minneapolis : Burgess ( 1964).
Ultrastructural
D. J. HEARSE,
Damage Associated with of the Anoxic Myocardium
S. M. HUMPHREY,
W. G. NAYLER,
Reoxygenation
A. SLADE
AND D. BORDER
Department of Biochemistry, Imperial College of Science and Technolopv, Kensington, London and the Cardiothoracic Institute, 2 Beaumont Street, London, U.K. (Received 7 January
1974, accepted in revisedform
28 May 1974)
D. J. J~EARSE,S. M. HUMPHREY, W. G. NAYLER, A. SLADE AND D. BORDER. Ultrastructural Damage Associated with Reoxygenation of the Anoxic Myocardium. Journal of Molecular and Cellular Cardiology (1975) 7, 3 15-324. Reoxygenation of the anoxic myocardium in the perfused rat heart following the onset of enzyme release causes an immediate and massive exacerbation of enzyme release. This effect appears to be directly attributable to the readmission of molecular oxygen. An electron microscopic investigation has revealed that reoxygenation induces sudden and major ultrastructural damage. Initially this damage involves some loss of the basement membrane and the physical disruption of the plasma membrane, the latter probably contributing to the sudden and large loss of cytoplasmic enzymes. The damage spreads rapidly throughout the cell causing disorganization of the myofibrils and mitochondrial damage. A possible biochemical basis of this effect is discussed. In addition, results are presented illustrating the metabolic protection afforded to the heart by exogenous supplies of glucose during anoxia. This is made possible by a glucose dependent preservation of myocardial ultrastructure and is reflected in a greatly reduced level of enzyme leakage. KEY WORDS: Anoxia; Reoxygenation; Enzyme release; Isolated perfused rat heart; Ultrastructural damage; Creatine phosphokinase; Oxygen toxicity; Electron microscopy; Glucose; Metabolic protection; Reperfusion.
1. Introduction In previous studies [S] of enzyme release from the isolated perfused anoxic rat heart we reported that abrupt reoxygenation of previously anoxic tissue can greatly exacerbate enzyme leakage from the myocardium. In these studies K+ arrested rat hearts were subjected to substrate free anoxic perfusion. Under these conditions, enzyme release commenced after less than 1 h and was maintained for at least 8 h. If the hearts were reoxygenated after 100 min anoxia an immediate and massive release of enzyme was observed, such that after 2 min, levels had increased by 100 to 200 fold. In these studies, due to the constancy of the perfusate ion concentration, the perfusion pressure and coronary flow, the exacerbation of release was attributed directly to the readmission of molecular oxygen. In view of the magnitude of release and the relative invariance of the ratio of protein to cytoplasmic enzyme release it was suggested that leakage probably resulted from
316
D. J. HEARSE
ETAL.
extensive ultrastructural damage and not from simple changes in membrane permeability characteristics. In an attempt to explain the oxygen-induced exacerbation of enzyme release, an investigation of ultrastructural changes during reoxygenation has been undertaken. In additional studies [9] we have illustrated how glucose is able to protect the anoxic myocardium against enzyme leakage. This observation has also been characterized with electron microscopic investigations.
2. Materials Experimental
and Methods animals and chemicals
Male rats (280 to 320 g body wt) of the Sprague-Dawley strain, maintained on a standard diet, were used in these experiments. All substrates and enzymes used in the analysis of perfusion fluid were obtained from the Boehringer Corporation (London) Ltd.
Perfusion techniques Rats were lightly anaesthetized with diethyl ether, the left femoral vein was exposed and heparin (200 I U) was administered intravenously. One min after administration of heparin the heart was excised [14] and placed in ice-cold perfusion medium until contraction had ceased. The aorta was then cannulated and, after mounting, the arrested heart was restarted by perfusion (non-recirculating) as described by Langendorff 1131 with a perfusion pressure of 100 cm of water. During the anoxic perfusion period, atmospheric gas contamination of the heart was prevented by completely enclosing the heart in a waterjacketed (37 “C) chamber which was continuously gassed with a mixture of NZ + CO2 (95 : 5). In addition, to prevent diffusion of oxygen into the perfusion fluid, all silicone rubber tubing connecting reservoirs and the heart were replaced by glass delivery tubing.
Perfusion medium Krebs-Henseleit bicarbonate buffer [I,!?], pH 7.4 containing glucose (11.1 mM) was used in the initial pre-anoxic control perfusion period. During the anoxic perfusion and reoxygenation period the hearts were K+-arrested [8], under these conditions, the K+ concentration of the perfusion fluid was increased to 16.0 mM and the Na+ concentration was correspondingly decreased. In the glucose protec-
REOXYGENATION
DAMAGE
IN
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ANOXIC
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317
tion studies, glucose (11.1 mM) was added to the anoxic perfusion fluid. All perfusion fluid was maintained at 37 “C. In aerobic studies the fluid was equilibrated with 0~ + CO2 (95 : 5, aortic 02 partial pressure was over 600 mm Hg) and in anoxic studies (high K+) the fluid was equilibrated with Na + CO2 (95 : 5, aortic 0s partial pressure was less than 5 mm, Hg). Precautions [20] were taken to prevent the precipitation of Ca 2+. Before use, the perfusion fluid was filtered through a cellulose acetate filter of pore size 5.0 pm (Millipore Ltd.).
Experimental
time course
Immediately after mounting, the hearts were perfused aerobically for a 5 min period. During this time the stability of the preparation could be confirmed. At the end of this period the hearts were perfused anoxically for a predetermined time period. At any time during this period the composition of the perfusion fluid could be changed by introducing another fluid from a second reservoir. For example, in reoxygenation studies a perfusion fluid (K+, 16.0 mM) gassed with 02 + COs (95 : 5) was introduced. During the entire experimental period coronary flow was monitored and samples could be taken for enzyme analysis. At any time the experiment could be terminated and fixative could be introduced into the coronary system via a side arm on the aortic cannula which was connected to an infusion pump. After fixing, the tissue could be taken for electron microscope studies.
Enzyme release analysis Creatine phosphokinase (ATP: creatine phosphotransferase E.C.2.7.3.2) assayed as described by Hearse et al. [8]. Enzyme activity in the perfusion was expressed as mIU released/ml.
was fluid
Tissue preparation and electron microscopy Hearts were fixed for electron microscopy at various times during the experimental time course (see results section for details of times and conditions). The fixative had the following composition: 5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 at 4 “C. At the end of the experimental period the heart was surrounded with ice-cold fixative. Using the side arm of the aortic cannula the heart was then perfused slowly (10 ml/min for 2 min) with ice-cold fixative. A left ventricular biopsy was taken close to a branch of the left anterior descending coronary artery. This was then cut into small pieces (approx. I mma)
318
D.
J.
HEARSE
ETAL.
and immersed in 5% glutaraldehyde at 4 “C for 2 h. The tissue was then washed buffer at 4 “C containing for 3 x 30 min periods in 0.1 M sodium cacodylate sucrose (4.5%). After a further 12 h in the buffer the tissue was post-fixed in osmium tetroxide (1 o/o in cacodylate buffer) for 2 h followed by 2 x 15 min washes in distilled water. After dehydration through a sequential series of ethanols the tissue was washed for 10 min in propylene oxide and then infiltrated with resin by leaving it for two consecutive I h periods in a mixture of propylene oxide and “araldite epoxy resin mixture” (50 : 50 and 20 : 80 respectively). The tissue was then placed in “araldite epoxy resin mixture” for 12 h (the “araldite” had the following composition: 10 ml araldite epoxy CY212 resin, 10 ml dodecenyl succinic anhydride, 0.4 ml dibutylphthalate). After this period the tissue was placed in fresh “araldite mixture” for 5 h. The tissue was then embedded (60 “C for 48 to 60 h) in the “araldite mixture” defined above but including 0.25 ml 2,4,6-tri(dimethylaminomethyl)phenol. Thin sections, cut on a LKB ultramicrotome, were stained with saturated aqueous uranyl acetate for 15 min followed by Reynolds [15] lead citrate (20% solution) and examined with an AEI 801 electron microscope.
3. Results Enzyme release studies Anoxia Hearts were subjected to anoxic perfusion for at least 7 h and enzyme release was measured at 15 min intervals. Previous studies [8, 91 have shown that the release of creatine phosphokinase is essentially representative of several enzymes (a-hydroxybutyrate dehydrogenase, glutamate oxaloacetate transaminase and adenylate kinase). The release profile for creatine phosphokinase is illustrated in Figure 1. Extensive enzyme release occurred over the 7 h period.
Reoxygenation Hearts were subjected to 150 min anoxia (perfusion fluid: Kf, 16.0 mM; (perfusion glucose free; gassed with Na + CO2 95 : 5) and then reoxygenated fluid: K+, 16.0 mM; glucose free; gassed with 02 + CO2 95 : 5). Immediately following reoxygenation (Figure 1) there was a massive release of enzyme activity such that after 2 min, levels had increased by approximately 100 fold. Due to the constancy of the per&sate ion concentration, perfusion pressure and coronary flow the exacerbation of release appears to be directly attributable to the readmission of molecular oxygen.
PLATE 3
PLATE 4
-_,-
-
I
P L A T E5
-,._
--.-
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DAMAGE
IN THE
ANOXIC
MYOCARDIUM
319
4600
f $ P y % -: 0 P g E ‘2 0
600-
. .
500400300200100 0 em-*- .-.-. 0 50
.-’ 100
/--‘----I. .l /--• l- .l. I 250I 300i--r 350. 400I 0 200 Perfusion time (min)
FIGURE 1. Profile for the release of creatine phosphokinase (mIU/ml coronary flow) from the isolated perfused rat heart following the onset of anoxia (t = 5). (a), extended anoxia; (B), reoxygenation after 150 min. For details of perfusion see text.
Glucose protection Hearts were subjected to 7 h anoxia with glucose (11.1 mM) present in the perfusion fluid. Under these conditions (Figure 2), gIucose was shown to exert a marked protective effect. In contrast to anoxic perfusion in the absence of glucose, enzyme release was reduced by approximately 75% over the 7 h period. As a control, hearts were perfused aerobically in the presence of glucose (11.1 mM) for 7 h and under these conditions (Figure 2) enzyme release was negligible.
Ultrastructural The ultrastructure obtained from rat 150 min anoxic 150 min anoxic 180 min anoxic 150 min anoxic
studies
of ventricular myocardial cells was investigated using tissue hearts which had been subjected to the following conditions : perfusion perfusion plus 2 min reoxygenation perfusion perfusion plus 30 min reoxygenation
320
D. J. HEARSE
Perfusion
El-AL.
time (min)
FIGURE 2. Profile for the release of creatine phosphokinase (mIU/ml coronary flow) from the isolated perfused rat heart. (a), 420 min glucose free anoxic perfusion; (+), 420 min anoxic perfusion plus glucose (11.1 111~); (A), 420 min aerobic perfusion plus glucose (11.1 mM). For details of perfusion see text. Each point represents the mean for four hearts and the bars represent the S.E.M.
180 min anoxic perfusion in the presence of glucose (11.1 mM) 180 min aerobic perfusion in the presence of glucose (11.1 mM) Fresh tissue subjected to aerobic perfusion for 2 min.
Ultrastructural
changes induced by anoxia
In comparison to Plates 4(a) and 5(b) (fresh tissue and aerobically perfused tissue respectively) Plates 1,3(a) and 4(b) indicate damage that may be attributable to anoxia. After 150 min anoxic perfusion, enzyme release (Figure 1) has already commenced and some small ultrastructural changes are apparent (Plate 1). The T-tubules are slightly dilated and, while remaining intact, there is some evidence However, the basement membrane is of distortion of the plasma membrane. intact, the mitochondria appear dense and numerous with preserved cristae. The sarcoplasmic reticulum is intact, the Z lines, A and I bands are normal. Numerous, well defined electron dense granules are apparent in the mitochondria and cytoplasm.
REOXYGENATION
DAMAGE
IN
THE
ANOXIC
MYOCARDIUM
321
After 180 min anoxia [Plates 3(a) and 4(b)], when extensive enzyme release (Figure 1) has occurred, additional ultrastructural changes are apparent. While some Z lines remain well-defined others were distorted. There is evidence for the leaching of actin density and for the whisping of myofibrils. In addition there is evidence of dense, rounded, aggregated mitochondria, translocated towards the intact plasma membrane. Despite this, the mitochondria are intact, regular in outline and have well defined cristae. There is evidence of tissue oedema. The interstitial spaces are filled with electron dense material. Electron dense granules are not apparent in the cytoplasm or the mitochondria.
Ultrastructural
changes induced by reoxygenation
After 2 min reoxygenation (Figure 1) enzyme release has increased by approximately 100 fold. Examination of tissue at this time [Plate 2(a) and (b)] in comparison with tissue that has not been reoxygenated (Plate 1) reveals additional damage. There is evidence for some loss of basement membrane and fragmentation of the plasma membrane. There is a rapid loss of electron density of myofibrils, they appear whispy, the Z lines are distorted and there is a marked increase in interfibrillar space. In contrast with Plate 1 there are more interstitial spaces and they lack electron dense material. Some mitochondria are swollen and less electron dense. However, the majority of the mitochondrial membranes and cristae remain intact. Electron dense granules are not apparent. Thirty min reoxygenation [Plate 3(b)], in contrast with 180 min anoxia [Plate 3(a)], reveals the extensive damage associated with reoxygenation. There has been severe disruption of the myofibrils with the loss of Z lines. Some basement membrane is lost and the plasma membrane is extensively disrupted. The mitochondria are swollen and irregular [Plate 3(b)]. There is no mitochondrial alignment relative to the myofibrils and the T-tubules are distended. Electron dense granules are not apparent, in the cytoplasm or the mitochondria.
Glucose protection Inclusion of glucose (11.1 mM) in the anoxic perfusion fluid greatly reduces enzyme release (Figure 2) and has been shown to protect the myocardium against anoxic damage [7, 91. Examination of tissue obtained after 180 min anoxic perfusion plus glucose [Plate 5 (a)] reveals, in contrast to glucose free anoxic perfusion [Plate 4(b)], considerable ultrastructural preservation. Myofibrils are intact and electron dense. The Z lines are regular and show good continuity. The mitochondria are well preserved and aligned between the myofibrils. The T-tubules show some dilation but there are no interstitial spaces or electron dense granules.
3‘22
D. J. HEARSE
ETAL.
Plate 5(b) illustrates tissue obtained after 180 min aerobic perfusion in the presence of glucose. This shows very good preservation of myofibrils, mitochondria and T-tubules. Numerous electron dense granules are apparent in both the cytoplasm and the mitochondria.
4. Discussion The results described in this paper have revealed that the oxygen induced exacerbation of enzyme release from the anoxic rat heart occurs as a result of sudden and major ultrastructural damage. In the first few minutes of reoxygenation, this damage appears to be largely confined to the cell periphery with some loss of the basement membrane and the physical disruption of the plasma membrane. This fragmentation of the plasma membrane probably contributes to the rapid and large increase in the release of cytoplasmic enzymes and protein. In the course of the next 30 min of reoxygenation the damage spreads throughout the cell with disruption of the myofibrils and considerable mitochondrial damage. The mitochondria exhibit morphological changes including swelling, distortion, loss of cristae and translocation within the cell. The majority of the mitochondrial membranes do however remain intact, this is consistent with the fact that glutamate dehydrogenase and other mitochondrial enzymes are not released in high concentrations during the conditions of anoxia or reoxygenation used in this study. (D. J. Hearse and S.M. Humphrey, unpublished results.) The biochemical basis of this reoxygenation phenomenon is open to speculation. Controlled experiments (D. J. Hearse and S. M. Humphrey, unpublished results) with graded reoxygenation and with reoxygenation at earlier time periods have eliminated micro-emboli as possible causative agents and additional studies [9] would suggest that the results are not due to a loss of cellular anti-oxidants. Due to the maintenance of constant coronary flow and perfusion pressure, mechanical damage should not occur. Hyperoxia and consequent lipid peroxidation have been shown [I] to increase the permeability of lipoprotein membranes but there have been no reports of lipid peroxidation causing damage as rapidly or as extensively as that reported in this paper. A possible explanation for the reoxygenation damage to the plasma membrane may be that reoxygenation induces the sudden redistribution of ions, either lost from, or gained by, the cytoplasm during anoxia. This sudden ion flux may possibly cause damage to the plasma membrane. In this connection it has been reported [S, 18, 191 that reoxygenation or reperfusion of previously ischaemic tissue results in large and rapid ion shifts, particularly Ca2+. The presence or absence of electron dense granules (both cytoplasmic and mitochondrial) under various conditions in this study cannot be completely explained but may be related to ion fluxes. While some of the granules are un-
323
REOXYGENATIONDAMAGEINTHEANOXICMYOGARDIUM
doubtedly glycogen deposits [Plate 4(a)] others may possibly be calcium phosphate [IS] deposits. The existence of elevated concentrations of both Caa+ and inorganic phosphate in the cytoplasm during anoxia could be readily explained. The sudden loss of these granules observed upon reoxygenation [Plates Z(a) and (b)] may support the concept of membrane damage induced by sudden transmembrane ion fluxes. The findings reported in this paper, although obtained in reoxygenation studies in the isolated rat heart, may have some bearing on studies invoking reperfusion of ischaemic myocardial tissue. While many investigators have reported [for example 5, 171 reversal of damage and consequent tissue salvage following either the reperfusion or revascularization of ischaemic tissue, there have been a few reports [Z-4, 10, II] that reperfusion or revascularization can accelerate or extend tissue damage. In general these observations of increased tissue damage have been related to the mechanical consequences of a rapid re-establishment of flow. The possibility now exists however that the extension of damage may be explained in terms of the results presented in this paper. Investigating the possibility of protecting the myocardium by including glucose in the anoxic perfusion fluid our results have shown that the observed reduction in enzyme loss could be associated with a considerably improved preservation of myocardial ultrastructure. Sybers et al. [IS] have reported that glucose-insulinpotassium infusions had a protective effect on cardiac ultrastructure in dogs which had been subjected to experimental coronary occlusion. Despite the differences in experimental models, their results and ours both underline the potential value of metabolic interventions in anoxic or ischemic tissue.
Acknowledgement This work was carried out with aid of grants from the British and the Medical Research Council.
Heart
Foundation
REFERENCES ALLISON, A. C. Role of lysosomes in oxygen toxicity. nuture 205, 141-143 (1965). BANKA, V. S., CHADDA, K. D., MEISTER, S. G. & HELFANT, R. H. Limitations
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D. J. HEARSE ETAL. GINKS, W. R., SYBERS, H. D., MAROKO, P. R., CORELL, J. W., SOBEL, B. E. & ROSS, J. Coronary artery reperfusion. Journal of Clinical Investigation 51, 2717-2723 (1972). HAMILTON, D. G. & WHALEN, D. A. Early phase of irreversible myocardial cell injury. Federation Proceedings 31, 627 (1972). HEARSE, D. J. & CHAIN, E. B. The role of glucose in the survival and recovery of the anoxic isolated perfused rat heart. Biochemical Journal 128, 1125-l 133 (1972). HEARSE, D. J., HUMPHREY, S. M. & CHAIN, E. B. Abrupt reoxygenation of the anoxic potassium-arrested perfused rat heart: A study of myocardial enzyme release. Journal of Molecular and Cellular Cardiology 5, 395-407 (1973). HEARSE, D. J. & HUMPHREY, S. M. Enzyme release during myocardial anoxia: a study of metabolic protection. Journal of Molecular and Cellular CardioloQ (1975) (In
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Pathology 46, 367-377 (1965). JENNINGS, R. B., SOMMERS, H. M., SMYTH, G. A., FLACK, H. A. & LINN, H. Myo-
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Archives of Pathology 70, 82-92 (1960). KREBS, H. A. & HENSELEIT, K. Untersuchungen tiber die Harnstoffbildung im Tierkorper. EIo@e-Seyler’s zeitschrift fiir physiologische Chemie 210, 33-66 (1932). LANGENDORFF, 0. Untersuchungen am uberlebenden Saugertierherzen. Pjtigers Archiv ftir gesamte Physiologie de Men&en und der Tiere 61, 291-332 (1895). NEELY, J. R., LIEBERMEISTER, H., BATTERSBY, E. J. & MORGAN, H. E. Effect of pressure development on oxygen consumption by the isolated rat heart. American Journal of Physiology 212, 804-814 (1967). REYNOLDS, E. S. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. Journal of Cell Biology 17, 208-2 12 (1963). SYBERS, H. D., MAROKO, P. R., A~HRAP, M., LIBBY, P. & BRAUNWALD, E. The effect
of glucose-insulin-potassium on cardiac ultrastructure following acute experimental coronary occlusion. American Journal of Pathology 70, 401-420 (1973). 17. SYMES,J. F., ARNOLD, I. M. F. & BLUNDELL, P. E. Early revascularization of the acute myocardial infarction: The critical time factor. Canadian Journal of Surgery 16, 275-283 (1973). 18. SHEN, A. C. &JENNINGS, R. B. Myocardial calcium and magnesium in acute ischaemic injury. American Journal of Pathology 67, 417-440 (1972). 19. SHEN, A. C. &JENNINGS, R. B. Kinetics of calcium accumulation in acute myocardial ischaemic injury. American Journal of Pathology 67, 441-452 (1972). 20. UMBREIT, W. W., BURRIS, R. H. & STAUFFER, J. F. In Manometric Techniques, p. 132, Minneapolis : Burgess ( 1964).
