Journal of Molecular and Cellular Cardiology (1975) 7, 307-314
Ultrastructural
Modifications Induced by Reoxygenation in the Anoxic Isolated Rat Heart Perfused Without Exogenous Substrate DANIELLE
Laboratoire
FEUVRAY
AND
JOEL
DE
LEIRIS
de Physiologic Cornparkeet de Physiologic, Cellulaire associe’ au C.N.R.S. Uniuersite’ Paris XI-Centre d’orsay, 91405 Orsay, France
(Received 15 December 1973, accepted in revisedform
9 May 1974)
D. FEUVRAY AND J. DE LEIRIS. Ultrastructural Modifications Induced by Reoxygenation in the Anoxic Isolated Rat Heart Perfused Without Exogenous Substrate. Journal of Molecular and Cellular Cardiology (1975) 7, 307-314. Isolated potassium-arrested rat hearts were submitted to anoxic perfusions (30 or 100 min) at 37°C. After the period of anoxia, oxygen was re-introduced for 5 or 20 min. The ultrastructure of ventricular myocardium was studied: (1) in control experiments after 20 min stabilization period (standard perf&sate, normal potassium concentration, with glucose); (2) after 35 or 105 min anoxia following the stabilization period; (3) after 5 min reoxygenation following 30 or 100 min anoxia; and (4) after 20 min reoxygenation following 100 min anoxia. During both anoxia and reoxygenation the perfusion fluid contained a high potassium concentration (17 mM) and no glucose of other substrate. After 35 min of anoxic perfusion, or 30 min of anoxic perfusion and reoxygenation for 5 min, very slight ultrastructural modifications were observed. On the other hand, marked ultrastructural modifications in myofibrils and sarcoplasmic reticulum were encountered after 105 min anoxia. But most striking morphologic changes concerning the mitochondria were observed after 5 min of reoxygenation following 100 min of anoxia. However, after 20 min reoxygenation, these mitochondrial alterations were less marked. Our results indicate that in potassium-arrested rat hearts perfused without substrate, ultrastructural alterations appeared which were dependent on the duration of the anoxic period and were greatly enhanced by reoxygenation. It is concluded that enzyme release occurring in such experimental conditions may be related to these ultrastructural alterations. KEY WORDS: Anoxia;
chondria;
Longitudinal
Reoxygenation; Ultrastructure; tubules; Substrate-free.
Isolated perfused rat heart; Mito-
1. Introduction Previous investigations have shown that anoxia or severe hypoxia were able to induce the release of several enzymes from the isolated rat heart [Z, 8, 131. Hearse et al. [8] have indicated that such a release develops in two phases and, in particular, the second phase is greatly increased when reoxygenated simultaneously. On the other hand it has been shown that perfusion of isolated rat hearts by solutions with altered ionic composition [ 23, 15, 211 or containing various pharmacological substances [S, 141 could induce a release of intracellular enzymes. It
308
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has also been suggested that contratile activity was a major determinant in the tissue ejection of released enzymes, particularly when contraction recovered after being temporarily stopped. But in Hearse’s experiments [8] the enzyme release described after anoxia (with or without subsequent reoxygenation) could not be related to a recovery of contractile activity since the hearts were arrested by a high potassium concentration during all the perfusion. The purpose of the present work was to elucidate whether enzyme release induced by anoxic perfusions could be related to ultrastructural damage and whether or not such damage was exaggerated by the reoxygenation and therefore might have been responsible for the increased enzyme release [8]. We especially searched the modifications of the mitochondria and the T-tubules because reIease of enzymes and such ultrastructural modifications were associated in the case of perfusion with dimethylsulfoxide [S, 71.
2. Material
and Methods
Male rats of the Wistar strain with an average weight of 200 g and fed on standard laboratory diet were used. The heart was rapidly excised from the animal which was lightly anaesthetized with diethylether and then placed in ice-cold perfusion medium until contraction ceased. The heart was then mounted on the perfusion apparatus and perfused with a constant hydrostatic perfusion pressure (65 cm HzO) through the coronary vessels as described by Langendorff [II]. The temperature of the perfusate was maintained at 37°C 4 0.5”C. Once the heart was spontaneously beating, it was enclosed in a thick wall perspex container which was also kept at 37°C & 0.5”C and was continuously gassed either with 02 : COZ (95 : 5) or Na : COa (95 : 5). Two perfusion fluids were used in these experiments: (1) A modified KrebsHenseleit bicarbonate buffer [IO] containing the following (expressed in mM) : NaCI, 131; KCl, 5.6; CaCla, 2.16; MgC12, 1.Og; NaHsPOd, 0.6; NaHCOs, 25; glucose 11.1. This solution (295 mOsm, pH 7.4) was equilibrated with 95% 02 and 5% COz. The aortic 02 partial pressure was over 600 mmHg. Immediately after mounting the heart was initially perfused with this solution for a 20 min stabilization period. In experiments with enzyme release studies [S, 13-151 this stabilization time was chosen to avoid artefacts caused by the initial enzyme release following the mounting of the isolated heart. Some control preparations were fixed for ultrastructural studies at the end of this stabilization period. (2) Then a second perfusion fluid was used for the remainder of the experiment, In this solution there was no substrate; moreover the concentration of potassium was three times the normal concentration (17.0 mM) to induce membrane depolarization and cardiac arrest as described by Hearse et al. [8]. In this solution the concentration of sodium was decreased (119 mM) to avoid hyperosmotic effect of increased
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potassium. This perfusion fluid was equilibrated either with 95% Nz and 5% CO2 (anoxic perfusions-aortic 0s partial pressure less than 5 mmHg) or with 95% 0s and 5% COz (reoxygenation period-aortic 02 partial pressure as previously). Two different durations of anoxic perfusions were performed (30 and 100 min) . At the end of these anoxic periods the preparation was reoxygenated for 5 or 20 min and then fixed for ultrastructural studies. In control experiments without reoxygenation, hearts were fixed for electron microscopic studies after respectively 35 and 105 min of anoxic perfusions. The fixation was made up by perfusion via the aortic cannula with cold (4°C) phosphate buffer at pH 7.4 (410 mOsm) glutaraldehyde 2.5% in 0.1 M Millonig’s previously aerated either with N2 : CO2 (fixation during anoxia) or with 02 : CO2 (fixation after reoxygenation). After a 5 min perfusion the whole heart was immersed in the same fixative; a portion of the right ventricular wall was cut into small pieces and fixed at 4°C for 2 h. The tissue pieces were then rinsed in phosphate buffer overnight, washed several times in the same buffer and post fixed in buffered osmic acid 1 y0 for 1 h. After dehydrating in ethanol and embedding in Epon [IS], blocks were sectioned on a LKB ultramicrotome, doubly stained with uranyl acetate and lead citrate [17] and examined under a Siemens Elmiskop 1A electronmicroscope.
3. Results
Control sjecimens The ultrastructure of ventricular myocardial cells of control specimens after 20 min stabilization period is illustrated in Plate 1. This ultrastructural appearance was very similar to that which we have previously observed in hearts perfused at constant coronary flow [7]. Mitochondria, sarcoplasmic reticulum, T-tubules and glycogen granules all appeared to be normal. In the sarcomeres, the characteristic A-I banding pattern of myofibrils was visible.
After a 35 min anoxic period As shown in Plate 2 the ultrastructure of ventricular myocardial cells was practically unchanged after 35 min of anoxic perfusion (high-potassium and no glucose). The morphological appearance was normal in all the specimens examined, especially the mitochondria and the size of tubular systems. However, a very slight contraction of the sarcomeres was generally present.
310
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After 5 min reoxygenation following
30 min anoxia
There was no morphological change in mitochondria and sarcoplasmic reticulum. Intramitochondrial bodies were still observed. Contracted sarcomeres were consistently visible. Glycogen was still abundant.
After a 105 min anoxic period Plate 4 depicts pronounced changes in the ultrastructure of the ventricular myocardial cell. Myofibrils were markedly contracted with indistinct I-bands and sometimes distorted and thickened Z-lines. The sarcoplasmic reticulum appeared distinctly enlarged and vesiculated. Glycogen and intramitochondrial bodies had disappeared. The mitochondrial matrix was always dense.
After 5 min reoxygenation following
100 mi?l anoxia
In addition to the contraction of sarcomeres and dilated tubules, drastic changes were observed in mitochondria (Plate 5). In all the observed cells mitochondria were large and swollen. Some mitochondria appeared vesiculated, but in most of them there was a loss of cristae.
After 20 min reoxygenation following
100 min anoxia
Contracted sarcomeres and dilated sarcoplasmic reticulum were still visible (Plate 6). But the most important observation was that while some mitochondria with separation of cristae were observed as in Plate 5, after this extended reoxygenation other mitochondria in the vicinity showed regular cristae and therefore had a normal appearance.
4. Discussion Observations of control specimens indicate that our experimental conditions of perfusion were able satisfactorily to maintain ultrastructural integrity of the myocardium. The fixative used, resulted in T-tubules which were normal in size and configuration and mitochondria which showed electron density of the matrix. The same fixative was used throughout the different steps of experiment. Though
PLATE 1. Electron micrograph of right ventricular myocardium after 20 min aerobic stabilization period. Fine structure of myofibrils (Myo) is well preserved and mitochondria (M) have an electron-opaque matrix. This longitudinal section illustrates also T-tubules (T), sarcoplasmir reticulum (SR) and glycogen granules (G). bm: basement membrane. x 16 000. PLATE 2. Electron micrograph of right ventricular myocardium after 35 min anoxia. No detectable changes appear neither in longitudinal and transverse tubules, nor in the mitochondria. Note the slight contracture of sarcomeres. N nucleus; Go Golgi apparatus; C adjacent cistern of sarcoplasmic reticulum. x 16 000. [./ici~/g page 3 1O]
PLATE 3. Electron micrograph of right ventricular myocardium after 5 min rcoxygenation following 30 min anoxia. Contracted sarcomerca arc observed while well preserved mitochondria (M) are always visible. Glycogcn granules (G) arc still present. x 16 000. PLATE 4. Electron micrograph of right ventricular myocardium after 105 min anoxia. Dilatation and vesiculation (arrows) of sarcoplasmic reticulum are observed. Contracted sarcomeres with distorted Z-lines are visible. x 16 000.
PLATE 5. Electron micrograph of right ventricular myocardium after 5 min reoxygenation following 100 min anoxia. (a)-here is a striking example of the large swelling of mitochondria, with loss of cristae; contracted sarcomeres are always visible. (b)-this view shows mitochondria with vesiculated cristae. x 16 000. PLATE 6. Electron micrograph of right ventricular myocardium after 20 min reoxygenation following 100 min anoxia. Note the two sorts of mitochondria: with vesiculated cristae (black arrow) or with parallel and regular cristae (white arrow). x 16 000.
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the fixative osmolarity was high (410 mOsm) compared to the perfusion solutions, solutions having an osmolarity lower than 700 mOsm do not markedly distend T-tubules [19]. In cat cardiac muscle mitochondria shrank in hypertonic solution [18] but this change was not observed here. We should emphasize that these observations were made using hearts perfused without exogenous substrate and with a high-potassium concentration. Furthermore the changes in oxygen tension were major. Thus our experiments do not necessarily reflect conditions likely to be found in the body in physiological or pathological conditions. After 100 min anoxic perfusion without reoxygenation, small ultrastructural changes appeared, which could be related to the slow enzyme release previously described [8]. On the other hand marked ultrastructural modifications were observed after an abrupt reoxygenation following anoxic perfusions. Moreover, the longer the anoxic perfusion, the larger were the alterations induced at the time of reoxygenation. Such an observation can also be directly related to the results of Hearse et al. [S] who described an exaggeration of enzyme release induced by anoxia when reoxygenation occurred after 100 min of anoxic perfusion with a substrate-free medium. In the present experiments, during all the periods of anoxia and reoxygenation, we used a high potassium concentration (17 mM) in the perfusate as did Hearse et al. [8]. It could be pointed out that such an abnormal perfusion medium could be responsible for the observed ultrastructural changes. Emberson and Muir [4] found distended T-tubules in hearts perfused with I2 or 20 mM potassium. This distension appeared after only 1 min and was very marked in the case of 20 mM KCl. However, we never observed such a dilatation of T-tubules after 35 or 105 min perfusion with high potassium concentration. At the end of 35 min and 105 min anaerobic periods, contraction was present in all sarcomeres but was greater at 105 min. These results agree with those by Weissler et al. [ZO], who detected a few contracted sarcomeres after 30 min anoxia. In our experiments the contraction intensity increased with time, in agreement with the observations of Dhalla et al. [3] who described hypercontractions of sarcomeres and distorted Z-lines after 2 h of perfusion without substrate. But Weissler et al. [ZO] observed also marked changes in mitochondria after 30 min anaerobic glucose-free perfusion. In our experimental conditions, we have never encountered such alterations, even after 100 min of anoxia without glucose. At the end of such a period of anoxia the ultrastructural modifications appeared only in myofibrils and tubular systems. On the other hand, mitochondria were markedly altered after 5 min reoxygenation following a 100 min period of anoxia. Therefore, readmission of oxygen appears to be the major determinant of mitochondrial alterations. It has been previously described that a slow and progressive release of enzyme occurred during the period of anoxia in both beating [13] or KC&arrested EC!?]
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hearts. Since longitudinal tubules were demonstrated to be the major sites for intracellular glycolytic dehydrogenase either in heart muscle [1] or in white skeletal muscle [5], it is possible that enzyme release could be due to a progressive alteration of sarcoplasmic reticulum in the course of anoxia such as swelling of longitudinal tubules (Plates 4 and 5). But numerous enzymes are also located partly or totally in mitochondria. For example, glutamic oxalacetic transaminase or lactate dehydrogenase have been detected histochemically on the cristae [I, 121. Therefore it is likely that the major damage occurring in cristae after 5 min reoxygenation could also be closely related to the massive enzyme release appearing at this time [8]. Increased leakage of enzymes induced by the reoxygenation could be due, to a large extent, to the disruption and displacement of mitochondrial cristae, and sometimes, to the disrupted lining membranes of these organelles. Thus, in accordance with the proposals of Hearse and Chain [9], after an anaerobic period lasting 100 min, membrane structures have no longer sufficient energy stores to maintain morphological integrity. These membranes could easily be disrupted when oxygen is supplied. In other words, the fact that oxygen is abruptly readmitted without an exogenous substrate seems to have drastic effects on myocardial cells and particularly on mitochondria. After 20 min reoxygenation many mitochondria show minimal swelling and regular cristae, but others still show vesiculated cristae. Such an observation could therefore indicate that it would be a normalization process developing progressively after a longer period of reoxygenation. In conclusion the most important result of the present work is the observation that in anoxic isolated rat heart sudden reoxygenation without substrate produces marked alteration of mitochondrial membranes provided that these membranes have been previously deprived of exogenous substrate. It is likely that such ultrastructural alterations are responsible for the major release of enzymes induced by reoxygenation [ 81.
Acknowledgements This work was supported in part by a grant from D.G.R.S.T. (France). We thank Mr J. C. Ronceray, Mrs J. Tansini and Mrs J. Wicks for excellent technical assistance.
REFERENCES 1.
BABA, N. & SHARMA, H. M. Histochemistry of lactic dehydrogenase pectoralis muscle of rat. Journal of Cell Biology 51, 62 l-635 (1971).
in heart and
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2. BUTTERWORTH,P. J., PLUMMER, D. T., ROCHMAN, H. & WEBER, W. V. Factors affecting
3.
8.
9.
10. 11. 12.
13.
14.
15.
16. 17.
18. 19.
20.
the release of enzymes from the isolated perfused rat heart. Journal of PhysioloD 209, 3P (1970). DHALLA, N. S., YATES, J, C. & OLSON, R. E. Energy state and ultrastructure of the substrate-depleted heart. In Myocardiology : Recent Advances in Studies on Cardiac Structure and Metabolism 81-94 1, E. Bajusz and G. Rona, Eds. Baltimore: University Park Press (1972). EMBERSON, J. W. & MUIR, A. R. Changes in the ultrastructure of rat myocardium induced by hyperkalaemia 3ownul of Anatomy 104, 41 l-42 1 (1969). FAHIMI, H. D. & KARNOVSKY, M. J. Cytochemical localization of two glycolytic dehydrogenases in white skeletal muscle. Journal of Cell Biology 29, 113-128 (1966). FEUVRAY, D. & DE LEIRIS, J. Effect of dimethylsulfoxide on isolated rat heart and lacticodehydrogenase release. Euroljean Journal of Pharmacologv 16, 8-13 (1971). FEUVRAY, D. & DE LEIRIS, J. Effect of short dimethylsulfoxide perfusions on ultrastructure of the isolated rat heart. Journal of Molecular and Cellular Cardiology 5, 63-69 (1973). 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. & CHAIN, E. B. The effect of glucose on enzyme release from, and recovery of, the anoxic myocardium. In Myocardial Metabolism: Recent Advances in Studies on Cardiac Structure and Metabolism 3, E. Bajusz and G. Rona, Eds. Baltimore: University Park Press. (1973). KREBS, H. A. & HENSELEIT, K. Untersuchungen iiber die Harnstoffbilding im Tierkorper.
Ultrastructural
Modifications Induced by Reoxygenation in the Anoxic Isolated Rat Heart Perfused Without Exogenous Substrate DANIELLE
Laboratoire
FEUVRAY
AND
JOEL
DE
LEIRIS
de Physiologic Cornparkeet de Physiologic, Cellulaire associe’ au C.N.R.S. Uniuersite’ Paris XI-Centre d’orsay, 91405 Orsay, France
(Received 15 December 1973, accepted in revisedform
9 May 1974)
D. FEUVRAY AND J. DE LEIRIS. Ultrastructural Modifications Induced by Reoxygenation in the Anoxic Isolated Rat Heart Perfused Without Exogenous Substrate. Journal of Molecular and Cellular Cardiology (1975) 7, 307-314. Isolated potassium-arrested rat hearts were submitted to anoxic perfusions (30 or 100 min) at 37°C. After the period of anoxia, oxygen was re-introduced for 5 or 20 min. The ultrastructure of ventricular myocardium was studied: (1) in control experiments after 20 min stabilization period (standard perf&sate, normal potassium concentration, with glucose); (2) after 35 or 105 min anoxia following the stabilization period; (3) after 5 min reoxygenation following 30 or 100 min anoxia; and (4) after 20 min reoxygenation following 100 min anoxia. During both anoxia and reoxygenation the perfusion fluid contained a high potassium concentration (17 mM) and no glucose of other substrate. After 35 min of anoxic perfusion, or 30 min of anoxic perfusion and reoxygenation for 5 min, very slight ultrastructural modifications were observed. On the other hand, marked ultrastructural modifications in myofibrils and sarcoplasmic reticulum were encountered after 105 min anoxia. But most striking morphologic changes concerning the mitochondria were observed after 5 min of reoxygenation following 100 min of anoxia. However, after 20 min reoxygenation, these mitochondrial alterations were less marked. Our results indicate that in potassium-arrested rat hearts perfused without substrate, ultrastructural alterations appeared which were dependent on the duration of the anoxic period and were greatly enhanced by reoxygenation. It is concluded that enzyme release occurring in such experimental conditions may be related to these ultrastructural alterations. KEY WORDS: Anoxia;
chondria;
Longitudinal
Reoxygenation; Ultrastructure; tubules; Substrate-free.
Isolated perfused rat heart; Mito-
1. Introduction Previous investigations have shown that anoxia or severe hypoxia were able to induce the release of several enzymes from the isolated rat heart [Z, 8, 131. Hearse et al. [8] have indicated that such a release develops in two phases and, in particular, the second phase is greatly increased when reoxygenated simultaneously. On the other hand it has been shown that perfusion of isolated rat hearts by solutions with altered ionic composition [ 23, 15, 211 or containing various pharmacological substances [S, 141 could induce a release of intracellular enzymes. It
308
D.
FEUVRAY
AND
J.
DE
LEIRIS
has also been suggested that contratile activity was a major determinant in the tissue ejection of released enzymes, particularly when contraction recovered after being temporarily stopped. But in Hearse’s experiments [8] the enzyme release described after anoxia (with or without subsequent reoxygenation) could not be related to a recovery of contractile activity since the hearts were arrested by a high potassium concentration during all the perfusion. The purpose of the present work was to elucidate whether enzyme release induced by anoxic perfusions could be related to ultrastructural damage and whether or not such damage was exaggerated by the reoxygenation and therefore might have been responsible for the increased enzyme release [8]. We especially searched the modifications of the mitochondria and the T-tubules because reIease of enzymes and such ultrastructural modifications were associated in the case of perfusion with dimethylsulfoxide [S, 71.
2. Material
and Methods
Male rats of the Wistar strain with an average weight of 200 g and fed on standard laboratory diet were used. The heart was rapidly excised from the animal which was lightly anaesthetized with diethylether and then placed in ice-cold perfusion medium until contraction ceased. The heart was then mounted on the perfusion apparatus and perfused with a constant hydrostatic perfusion pressure (65 cm HzO) through the coronary vessels as described by Langendorff [II]. The temperature of the perfusate was maintained at 37°C 4 0.5”C. Once the heart was spontaneously beating, it was enclosed in a thick wall perspex container which was also kept at 37°C & 0.5”C and was continuously gassed either with 02 : COZ (95 : 5) or Na : COa (95 : 5). Two perfusion fluids were used in these experiments: (1) A modified KrebsHenseleit bicarbonate buffer [IO] containing the following (expressed in mM) : NaCI, 131; KCl, 5.6; CaCla, 2.16; MgC12, 1.Og; NaHsPOd, 0.6; NaHCOs, 25; glucose 11.1. This solution (295 mOsm, pH 7.4) was equilibrated with 95% 02 and 5% COz. The aortic 02 partial pressure was over 600 mmHg. Immediately after mounting the heart was initially perfused with this solution for a 20 min stabilization period. In experiments with enzyme release studies [S, 13-151 this stabilization time was chosen to avoid artefacts caused by the initial enzyme release following the mounting of the isolated heart. Some control preparations were fixed for ultrastructural studies at the end of this stabilization period. (2) Then a second perfusion fluid was used for the remainder of the experiment, In this solution there was no substrate; moreover the concentration of potassium was three times the normal concentration (17.0 mM) to induce membrane depolarization and cardiac arrest as described by Hearse et al. [8]. In this solution the concentration of sodium was decreased (119 mM) to avoid hyperosmotic effect of increased
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potassium. This perfusion fluid was equilibrated either with 95% Nz and 5% CO2 (anoxic perfusions-aortic 0s partial pressure less than 5 mmHg) or with 95% 0s and 5% COz (reoxygenation period-aortic 02 partial pressure as previously). Two different durations of anoxic perfusions were performed (30 and 100 min) . At the end of these anoxic periods the preparation was reoxygenated for 5 or 20 min and then fixed for ultrastructural studies. In control experiments without reoxygenation, hearts were fixed for electron microscopic studies after respectively 35 and 105 min of anoxic perfusions. The fixation was made up by perfusion via the aortic cannula with cold (4°C) phosphate buffer at pH 7.4 (410 mOsm) glutaraldehyde 2.5% in 0.1 M Millonig’s previously aerated either with N2 : CO2 (fixation during anoxia) or with 02 : CO2 (fixation after reoxygenation). After a 5 min perfusion the whole heart was immersed in the same fixative; a portion of the right ventricular wall was cut into small pieces and fixed at 4°C for 2 h. The tissue pieces were then rinsed in phosphate buffer overnight, washed several times in the same buffer and post fixed in buffered osmic acid 1 y0 for 1 h. After dehydrating in ethanol and embedding in Epon [IS], blocks were sectioned on a LKB ultramicrotome, doubly stained with uranyl acetate and lead citrate [17] and examined under a Siemens Elmiskop 1A electronmicroscope.
3. Results
Control sjecimens The ultrastructure of ventricular myocardial cells of control specimens after 20 min stabilization period is illustrated in Plate 1. This ultrastructural appearance was very similar to that which we have previously observed in hearts perfused at constant coronary flow [7]. Mitochondria, sarcoplasmic reticulum, T-tubules and glycogen granules all appeared to be normal. In the sarcomeres, the characteristic A-I banding pattern of myofibrils was visible.
After a 35 min anoxic period As shown in Plate 2 the ultrastructure of ventricular myocardial cells was practically unchanged after 35 min of anoxic perfusion (high-potassium and no glucose). The morphological appearance was normal in all the specimens examined, especially the mitochondria and the size of tubular systems. However, a very slight contraction of the sarcomeres was generally present.
310
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After 5 min reoxygenation following
30 min anoxia
There was no morphological change in mitochondria and sarcoplasmic reticulum. Intramitochondrial bodies were still observed. Contracted sarcomeres were consistently visible. Glycogen was still abundant.
After a 105 min anoxic period Plate 4 depicts pronounced changes in the ultrastructure of the ventricular myocardial cell. Myofibrils were markedly contracted with indistinct I-bands and sometimes distorted and thickened Z-lines. The sarcoplasmic reticulum appeared distinctly enlarged and vesiculated. Glycogen and intramitochondrial bodies had disappeared. The mitochondrial matrix was always dense.
After 5 min reoxygenation following
100 mi?l anoxia
In addition to the contraction of sarcomeres and dilated tubules, drastic changes were observed in mitochondria (Plate 5). In all the observed cells mitochondria were large and swollen. Some mitochondria appeared vesiculated, but in most of them there was a loss of cristae.
After 20 min reoxygenation following
100 min anoxia
Contracted sarcomeres and dilated sarcoplasmic reticulum were still visible (Plate 6). But the most important observation was that while some mitochondria with separation of cristae were observed as in Plate 5, after this extended reoxygenation other mitochondria in the vicinity showed regular cristae and therefore had a normal appearance.
4. Discussion Observations of control specimens indicate that our experimental conditions of perfusion were able satisfactorily to maintain ultrastructural integrity of the myocardium. The fixative used, resulted in T-tubules which were normal in size and configuration and mitochondria which showed electron density of the matrix. The same fixative was used throughout the different steps of experiment. Though
PLATE 1. Electron micrograph of right ventricular myocardium after 20 min aerobic stabilization period. Fine structure of myofibrils (Myo) is well preserved and mitochondria (M) have an electron-opaque matrix. This longitudinal section illustrates also T-tubules (T), sarcoplasmir reticulum (SR) and glycogen granules (G). bm: basement membrane. x 16 000. PLATE 2. Electron micrograph of right ventricular myocardium after 35 min anoxia. No detectable changes appear neither in longitudinal and transverse tubules, nor in the mitochondria. Note the slight contracture of sarcomeres. N nucleus; Go Golgi apparatus; C adjacent cistern of sarcoplasmic reticulum. x 16 000. [./ici~/g page 3 1O]
PLATE 3. Electron micrograph of right ventricular myocardium after 5 min rcoxygenation following 30 min anoxia. Contracted sarcomerca arc observed while well preserved mitochondria (M) are always visible. Glycogcn granules (G) arc still present. x 16 000. PLATE 4. Electron micrograph of right ventricular myocardium after 105 min anoxia. Dilatation and vesiculation (arrows) of sarcoplasmic reticulum are observed. Contracted sarcomeres with distorted Z-lines are visible. x 16 000.
PLATE 5. Electron micrograph of right ventricular myocardium after 5 min reoxygenation following 100 min anoxia. (a)-here is a striking example of the large swelling of mitochondria, with loss of cristae; contracted sarcomeres are always visible. (b)-this view shows mitochondria with vesiculated cristae. x 16 000. PLATE 6. Electron micrograph of right ventricular myocardium after 20 min reoxygenation following 100 min anoxia. Note the two sorts of mitochondria: with vesiculated cristae (black arrow) or with parallel and regular cristae (white arrow). x 16 000.
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the fixative osmolarity was high (410 mOsm) compared to the perfusion solutions, solutions having an osmolarity lower than 700 mOsm do not markedly distend T-tubules [19]. In cat cardiac muscle mitochondria shrank in hypertonic solution [18] but this change was not observed here. We should emphasize that these observations were made using hearts perfused without exogenous substrate and with a high-potassium concentration. Furthermore the changes in oxygen tension were major. Thus our experiments do not necessarily reflect conditions likely to be found in the body in physiological or pathological conditions. After 100 min anoxic perfusion without reoxygenation, small ultrastructural changes appeared, which could be related to the slow enzyme release previously described [8]. On the other hand marked ultrastructural modifications were observed after an abrupt reoxygenation following anoxic perfusions. Moreover, the longer the anoxic perfusion, the larger were the alterations induced at the time of reoxygenation. Such an observation can also be directly related to the results of Hearse et al. [S] who described an exaggeration of enzyme release induced by anoxia when reoxygenation occurred after 100 min of anoxic perfusion with a substrate-free medium. In the present experiments, during all the periods of anoxia and reoxygenation, we used a high potassium concentration (17 mM) in the perfusate as did Hearse et al. [8]. It could be pointed out that such an abnormal perfusion medium could be responsible for the observed ultrastructural changes. Emberson and Muir [4] found distended T-tubules in hearts perfused with I2 or 20 mM potassium. This distension appeared after only 1 min and was very marked in the case of 20 mM KCl. However, we never observed such a dilatation of T-tubules after 35 or 105 min perfusion with high potassium concentration. At the end of 35 min and 105 min anaerobic periods, contraction was present in all sarcomeres but was greater at 105 min. These results agree with those by Weissler et al. [ZO], who detected a few contracted sarcomeres after 30 min anoxia. In our experiments the contraction intensity increased with time, in agreement with the observations of Dhalla et al. [3] who described hypercontractions of sarcomeres and distorted Z-lines after 2 h of perfusion without substrate. But Weissler et al. [ZO] observed also marked changes in mitochondria after 30 min anaerobic glucose-free perfusion. In our experimental conditions, we have never encountered such alterations, even after 100 min of anoxia without glucose. At the end of such a period of anoxia the ultrastructural modifications appeared only in myofibrils and tubular systems. On the other hand, mitochondria were markedly altered after 5 min reoxygenation following a 100 min period of anoxia. Therefore, readmission of oxygen appears to be the major determinant of mitochondrial alterations. It has been previously described that a slow and progressive release of enzyme occurred during the period of anoxia in both beating [13] or KC&arrested EC!?]
312
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FEUVRAY
AND
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DE
LEIRIS
hearts. Since longitudinal tubules were demonstrated to be the major sites for intracellular glycolytic dehydrogenase either in heart muscle [1] or in white skeletal muscle [5], it is possible that enzyme release could be due to a progressive alteration of sarcoplasmic reticulum in the course of anoxia such as swelling of longitudinal tubules (Plates 4 and 5). But numerous enzymes are also located partly or totally in mitochondria. For example, glutamic oxalacetic transaminase or lactate dehydrogenase have been detected histochemically on the cristae [I, 121. Therefore it is likely that the major damage occurring in cristae after 5 min reoxygenation could also be closely related to the massive enzyme release appearing at this time [8]. Increased leakage of enzymes induced by the reoxygenation could be due, to a large extent, to the disruption and displacement of mitochondrial cristae, and sometimes, to the disrupted lining membranes of these organelles. Thus, in accordance with the proposals of Hearse and Chain [9], after an anaerobic period lasting 100 min, membrane structures have no longer sufficient energy stores to maintain morphological integrity. These membranes could easily be disrupted when oxygen is supplied. In other words, the fact that oxygen is abruptly readmitted without an exogenous substrate seems to have drastic effects on myocardial cells and particularly on mitochondria. After 20 min reoxygenation many mitochondria show minimal swelling and regular cristae, but others still show vesiculated cristae. Such an observation could therefore indicate that it would be a normalization process developing progressively after a longer period of reoxygenation. In conclusion the most important result of the present work is the observation that in anoxic isolated rat heart sudden reoxygenation without substrate produces marked alteration of mitochondrial membranes provided that these membranes have been previously deprived of exogenous substrate. It is likely that such ultrastructural alterations are responsible for the major release of enzymes induced by reoxygenation [ 81.
Acknowledgements This work was supported in part by a grant from D.G.R.S.T. (France). We thank Mr J. C. Ronceray, Mrs J. Tansini and Mrs J. Wicks for excellent technical assistance.
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