Exp Toxic Pathol 1992; 44: 70-73 Gustav Fischer Verlag Jena
La Trobe University, Department of Human Biosciences, Bundoora Victoria, Australia
Induction of mitochondrial alterations ex vivo in skeletal muscle M . A.
KHAN
With 5 figures Received : June 28, 1990; Accepted: July 13, 1990 Address for correspondence: Dr. Dr. M. A. 3083, Australia
KHAN,
La Trobe University, Department of Human Biosciences, Bundoora, Victoria
Key words: mitochondria ; skeletal muscle; muscle pathology
Abstract
Materials aJld Methods
The ultrastructural alterations of mitochondria were studied, following a fixation delay of 45 min, in the normal type II red muscle fibres of pigeon semimetapatagialis muscle . These muscle fibres, were devoid of myofibrillar degeneration; however, their mitochondria showed derangement. The latter included irregular orientation and loss of cristae, and clearing of the matrix. Some mitochondria showed myelin figures of varying complexity in the subsarcolemmal areas as well as in the interior of muscle fibres. These alterations bear striking resemblance to that seen during some lys~somal formation . The significance of these observations is discussed.
Adult pigeons (Columbia Livia) weighing about 285 g were used in this study. The birds were anaesthetised with ether and the semimetapatagialis (SMP) muscle was exposed (14). Following decapitation 2 sets of small pieces (4 mm X 2 mm) of the SMP muscle were excised. One set of tissue (Controls) was fixed immediately, whereas pieces of the 2nd set were left at room temperature for 45 min. All tissue blocks were fixed in 2.5% glutaraldehyde in O. IM cacodylate buffer, pH 7.4 and post-fixed in I % osmium tetroxide buffered with 0.1 M cacodylate buffer, pH 7.4. Following fixation, tissues were dehydrated in graded ethanol solutions and then embedded in araldite . Ultrathin sections were stained with uranyl acetate and lead citrate and were examined on a Zeiss EM9S electron microscope.
Introduction Histochemical and ultrastructural studies have shown that the pigeon semimetapatagialis (SMP) muscle is unique, in that it is comprised of an outer thin and pale band of Type I White (or slowtwitch glycolytic) muscle fibres ; however, rest of the muscle possesses Type II Red (or fast-twitch oxidative glycolytic) muscle fibres (15). Ultrastructurally these Type II Red muscle fibres are characterised by high oxidative enzymes, high myofibrillar ATPase activity, subsarcolemmal mitochondrial aggregates , many interfibrillar mitochondria, distinct sarcoplasmic reticulum, single M bands, and smooth but thin Z bands (14). Two different configurations of mitochondrial cristae are: (i) orthodox, and (ii) energized (i. e. aggregate or condensed configurations) (l,2l). The above configurations are related to the metabolic energy states; mitochondria in the energized configuration are essentially uncoupled , whereas mitochondria in the energized configuration are capable of coupled oxidative phosphorylation (1). Although there have been many electron microscopic investigations, the occurrence of derangement of mitochondria in normal avian striated muscle has not been described. In the present study , ultrastructural alterations in the mitochondria of normal Type II Red fibres of the pigeon SMP muscle, formed ex vivo, are presented. These morphological changes are similare to those seen in the origin of Iysosomes from mitochondria in the-parietal cells or during myocytolysis (4, 6, 11, 22). Furthermore, the mitochondrial alterations illustrated here differ from those of ischemic skeletal muscle (7). 70
Exp Toxic Pathol 44 (1992) 2
Results The myofibrillar apparatus , sarcoplasmic reticulum, mitochondria and transverse tubules appeared normal and were devoid of any derangement in the control tissue blocks. The mitochondria in the control Type II Red fibres of the pigeon SMP muscle showed an orthodox arrangement of the mitochondrial cllstae and were distributed in closely packed groups beneath the sarcolemma and in chains between the myofibrils (fig. 1). However, pronounced ultrastructural alterations were present in many mitochondria after delayed fixation ; this deranged organelle was located in the interior as well as in the subsarcolemmal areas. These mitochondria displayed minimal to severe degenerative changes. The' affected mitochondria were markedly swollen and some were of "giant" size in comparison with the obviously normal looking ones (figs . 2,3) . The anomalous mitochondria displayed separation, irregular orientation and loss of cristae , and clearing of the matrix; many were reduced to vacuoles possessing remnants of only a few cristae. Some deranged mitochondria displayed lamellar membranes or "myelin" figures of varying complexity and size. The concentric whorls were mostly found in the centre of the organelle. These mitochondria lacked energized cristal configuration, as well as any flocculent densities as seen in the mitochondrial matrix of ischemic muscle cells.
Fig. 1. Pigeon SMP muscle. Type II Red muscle fibres . Normal mitochondria show an even (orthodox) arrangement of the mitochondrial cristae; x 24,300.
Discussion The mitochondrial morphology was normal in the control tissue. The mitochondrial alterations observed in the experimental tissue blocks encompassed swelling, clearing of the matrix, a focal loss of cristae and myelin figure formation throughout the Type II Red muscle fibres, unlike the giant mitochondria of ischemic muscle which were subsarcolemmal only (7). The causes of these mitochondrial derangement remain speculative. Fixation artefact may not be the sole reason since mitochondria of the 2 sets of blocks were subjected to identical fixation and subsequent procedure. Myelin figure formation and mitochondrial degeneration have been shown to arise quite rapidly following breakdown of oxidative phosphorylation , diminution of available energy and cellular functions (6). The reason as to why only some mitochondria are susceptible could be due to an inherent mitochondrial heterogeneity (11 , 13). Studies on lysosomal formation in the parietal cells of the hamster stomach demonstrated the occurrence of myelin figures in the mitochondria (22). The myelin figures, considered to be lysosomes and reflected age-related changes, were present in some large fibroblasts also (23). The formation of myelin figures in the SMP
muscle here, initiated by degradative changes in mitochondria, possibly reflects an autophagic process existing in metabolically highly active "Type II Red" muscle fibres . The mitochondrial myelin figures were present together with a loss in the integrity of cristae and walls following a 3 min exposure to high oxygen tension (10) . Most mitochondria develop myelin figures in hamster muscle when exposed to 99 % oxygen (4). However, myelin figures were absent in muscle tissue following even prolonged ischemic injury (3, 7, 10). Thus myelin-figure formation in the S¥P muscle fibres, which are virtually anoxic, may be due to rigor toxicity and lowering of pH (9). Myelin figures are probably non-specific structures since they are found in cardiomyopathy (radiation-induced) (16) and smooth muscle (12) also. 72h after epinephrine administration , mitochondria of the degenerating myocardial cells showed distinct myelin figures (5). The latter formed around the periphery of the mitochondria as compared to the mainly centrally placed myelin figures seen here in the SMP muscle fibres . An activation of endogenous phospholipase A may play an important role in the control of mitochondrial permeability and swelling (19). Further, the degree of mitochondrial swelling is, however, proportional to permeability, which in turn is stimuExp Toxic Pathol 44 (1992) 2
71
Fig. 2. Two mitochondria are swollen; there is clearing of the matrix and a focal loss of the cristae; x 13,500.
Fig. 3. Myelin figure is present in a giant mitochondrium. Note one elongated interfibrillar and 2 degenerating mitochondria in which cristae are recognizable; x 11 ,500. 72
Exp Toxic Pathol 44 (1992) 2
lated by Ca + + ions (18). Finally, the mitochondrial alterations in these' highly active muscle fibres may be in some way associated with the above phenomena during acute catabolism (2, 18). Although the pathogenesis of mitochondrial abnormal morphology is not clear, it may result due to peroxidation, cytochrome perturbation and a compensatory change of mitochondria to overcome acute insult. The precise mechanism, however, requires further study.
12.
References
13.
I. ALLMAN, D.W., MUNROE, l., WAKABAYASHI, T., HARRIS, R.A., GREEN, D. E.: Studies on the transition of the cristal membrane from the orthodox to the aggregated configuration. II. Determinants of the orthodox-aggregated transition in adrenal cortex mitochondria. Bioenergetics 1970; 1: 87-107. 2. AMENTA, l.S., SARGUS, M.l., VENKATESAN, S., SCHIMOZUKA, H.: Role of the vacuolar apparatus in augmented protein degradation in cultured fibroblasts. l. Cell. Physiol. 1978, 94: 77-86. 3. CAULFIELD, l., KLIONSKY, B.: Myocardial ischemia and early infarction. An early electron microscopic study. AM. l. Pathol. 1959; 35: 489-497. 4. CAULFIELD, l.B., SHELTON, W., BURKE, l.F.: Cytotoxic effects of oxygen on striated muscle. Arch. Pathol. 1972; 94: 127-132. 5. FERRANS, V.l., HIBBS, R.G., WElLY, H.S., WEILBAECHER, D.G., WALSH, l. l., BURCH, G. E.: A histochemical and electron microscopic study of epinephrine-induced myocardial necrosis. l. Mol. Cell. Cardiol. 1970; 1: 11-22. 6. GRODUMS, E. I.: Ultrastructural changes in the mitochondria of brown adipose cells during the hibernation cycle of eitel/us lateralis. Cell. Tiss. Res. 1977; 185: 231-237. 7. HANZLlKOVA, V., SCHIAFFINO, S.: Mitochondrial changes in ischemic skeletal muscle. l. Ultrastruct. Res. 1977; 60: 121-133. 8. HEGARTY, P. V.l., DAHLIN, K.l., BENSON, E.S.: Ultrastructural differences in mitochondria of skeletal muscle in the prerigor and rigor states. Experientia 1978; 34: 1070-1071. 9. HERDSON, P.B., SOMMERS, H.M., lENNINGS, R.B.: A comparative study of the fine structure of normal and ischemic dog myocardium
10.
11.
14. 15. 16.
17. 18.
19.
20.
21.
22.
with special reference to early changes following temporary exclusion of a coronary artery. Am. l. Pathol. 1965; 46: 367-375. HUGHSON, M., BALENTINE, l., DANIELL, H.: The ultrastructural pathology of hyperbaric oxygen exposure. Observations on the heart. Lab. Invest. 1977; 37: 516-525. HULSMANN, W. C, DE lONG, l. W., VAN TOL, A.: Mitochondria with loosely and tightly coupled oxidative phosphorylation in skeletal muscle. Biochem. Biophys. Acta 1968; 162: 292-293. lORIS, L, MAJNO, G.: Cell-to-cell herniae in the arterial wall. L The pathogenesis of vacuoles in the normal media. Am. 1. Pathol. 1977; 87: 375-398. KHAN, M. A.: On the subsarcolemmal localization of phenazine ethosulfate-linked a-glycerophosphate dehydrogenase activity in pigeon pectoralis white muscle fibres. Histochemistry 1976; 50: 103-110. - Histochemical and ultrastructural characteristics of a new muscle tibre type in avian striated muscle. Histochem. l. 1979; 11: 321-335. - Ultrastructural study of mitochondria in the normal skeletal muscle. Cell. Molec. BioI. 1979; 24: 81-87. KHAN, M. Y.: Radiation induced cardiomyopathy. L An electron microscopic study of cardiac muscle cells. Am. l. Pathol. 1973; 73: 131-146. NAPOLITANO, L.: Cytolysomes in metabolically active cells. l. Cell. BioI. 1963; 18: 478-481. PUBLICOVER, S.J., DUNCAN, CJ., SMITH, l.L.: Ultrastructural changes in muscle mitochondria in situ, including the apparent development of internal septa, associated with the uptake and release of calcium. Cell Tissue Res. 1977; 185: 373-385. WAITE, M., VAN DEENEN, L.L.M., RUIGROK, T.J.C., ALBERS, P. F.: Relation of mitochondrial phospholipase A activity to mitochondrial swelling. J. Lipid Res. 1969a; 10: 599-608. SCHERPHOF, G.L., BOSHOUWRS, F.M.G. and VAN DEENEN, L.L.M.: Differentiation of phospholiphase A in mitochondria and 1ysosomes of rat livers. l. Lipid Res. 1969b; 10: 411-420. WILLIAMS, C.H., VAIL, W.l.: Ultrastructural transitions in energized and de-energized mitochondria. In: Advances in Cellular and Molecular Biology, vol. 2, (ed. PURNOW, E.l.). 1972,385-406. WINBORN, W. B., BOCKMAN, D. E.: Origin of Iysosomes in parietal cells. Lab. Invest. 1968, 19: 256-264.
Exp Toxic Pathol 44 (1992) 2
73
La Trobe University, Department of Human Biosciences, Bundoora Victoria, Australia
Induction of mitochondrial alterations ex vivo in skeletal muscle M . A.
KHAN
With 5 figures Received : June 28, 1990; Accepted: July 13, 1990 Address for correspondence: Dr. Dr. M. A. 3083, Australia
KHAN,
La Trobe University, Department of Human Biosciences, Bundoora, Victoria
Key words: mitochondria ; skeletal muscle; muscle pathology
Abstract
Materials aJld Methods
The ultrastructural alterations of mitochondria were studied, following a fixation delay of 45 min, in the normal type II red muscle fibres of pigeon semimetapatagialis muscle . These muscle fibres, were devoid of myofibrillar degeneration; however, their mitochondria showed derangement. The latter included irregular orientation and loss of cristae, and clearing of the matrix. Some mitochondria showed myelin figures of varying complexity in the subsarcolemmal areas as well as in the interior of muscle fibres. These alterations bear striking resemblance to that seen during some lys~somal formation . The significance of these observations is discussed.
Adult pigeons (Columbia Livia) weighing about 285 g were used in this study. The birds were anaesthetised with ether and the semimetapatagialis (SMP) muscle was exposed (14). Following decapitation 2 sets of small pieces (4 mm X 2 mm) of the SMP muscle were excised. One set of tissue (Controls) was fixed immediately, whereas pieces of the 2nd set were left at room temperature for 45 min. All tissue blocks were fixed in 2.5% glutaraldehyde in O. IM cacodylate buffer, pH 7.4 and post-fixed in I % osmium tetroxide buffered with 0.1 M cacodylate buffer, pH 7.4. Following fixation, tissues were dehydrated in graded ethanol solutions and then embedded in araldite . Ultrathin sections were stained with uranyl acetate and lead citrate and were examined on a Zeiss EM9S electron microscope.
Introduction Histochemical and ultrastructural studies have shown that the pigeon semimetapatagialis (SMP) muscle is unique, in that it is comprised of an outer thin and pale band of Type I White (or slowtwitch glycolytic) muscle fibres ; however, rest of the muscle possesses Type II Red (or fast-twitch oxidative glycolytic) muscle fibres (15). Ultrastructurally these Type II Red muscle fibres are characterised by high oxidative enzymes, high myofibrillar ATPase activity, subsarcolemmal mitochondrial aggregates , many interfibrillar mitochondria, distinct sarcoplasmic reticulum, single M bands, and smooth but thin Z bands (14). Two different configurations of mitochondrial cristae are: (i) orthodox, and (ii) energized (i. e. aggregate or condensed configurations) (l,2l). The above configurations are related to the metabolic energy states; mitochondria in the energized configuration are essentially uncoupled , whereas mitochondria in the energized configuration are capable of coupled oxidative phosphorylation (1). Although there have been many electron microscopic investigations, the occurrence of derangement of mitochondria in normal avian striated muscle has not been described. In the present study , ultrastructural alterations in the mitochondria of normal Type II Red fibres of the pigeon SMP muscle, formed ex vivo, are presented. These morphological changes are similare to those seen in the origin of Iysosomes from mitochondria in the-parietal cells or during myocytolysis (4, 6, 11, 22). Furthermore, the mitochondrial alterations illustrated here differ from those of ischemic skeletal muscle (7). 70
Exp Toxic Pathol 44 (1992) 2
Results The myofibrillar apparatus , sarcoplasmic reticulum, mitochondria and transverse tubules appeared normal and were devoid of any derangement in the control tissue blocks. The mitochondria in the control Type II Red fibres of the pigeon SMP muscle showed an orthodox arrangement of the mitochondrial cllstae and were distributed in closely packed groups beneath the sarcolemma and in chains between the myofibrils (fig. 1). However, pronounced ultrastructural alterations were present in many mitochondria after delayed fixation ; this deranged organelle was located in the interior as well as in the subsarcolemmal areas. These mitochondria displayed minimal to severe degenerative changes. The' affected mitochondria were markedly swollen and some were of "giant" size in comparison with the obviously normal looking ones (figs . 2,3) . The anomalous mitochondria displayed separation, irregular orientation and loss of cristae , and clearing of the matrix; many were reduced to vacuoles possessing remnants of only a few cristae. Some deranged mitochondria displayed lamellar membranes or "myelin" figures of varying complexity and size. The concentric whorls were mostly found in the centre of the organelle. These mitochondria lacked energized cristal configuration, as well as any flocculent densities as seen in the mitochondrial matrix of ischemic muscle cells.
Fig. 1. Pigeon SMP muscle. Type II Red muscle fibres . Normal mitochondria show an even (orthodox) arrangement of the mitochondrial cristae; x 24,300.
Discussion The mitochondrial morphology was normal in the control tissue. The mitochondrial alterations observed in the experimental tissue blocks encompassed swelling, clearing of the matrix, a focal loss of cristae and myelin figure formation throughout the Type II Red muscle fibres, unlike the giant mitochondria of ischemic muscle which were subsarcolemmal only (7). The causes of these mitochondrial derangement remain speculative. Fixation artefact may not be the sole reason since mitochondria of the 2 sets of blocks were subjected to identical fixation and subsequent procedure. Myelin figure formation and mitochondrial degeneration have been shown to arise quite rapidly following breakdown of oxidative phosphorylation , diminution of available energy and cellular functions (6). The reason as to why only some mitochondria are susceptible could be due to an inherent mitochondrial heterogeneity (11 , 13). Studies on lysosomal formation in the parietal cells of the hamster stomach demonstrated the occurrence of myelin figures in the mitochondria (22). The myelin figures, considered to be lysosomes and reflected age-related changes, were present in some large fibroblasts also (23). The formation of myelin figures in the SMP
muscle here, initiated by degradative changes in mitochondria, possibly reflects an autophagic process existing in metabolically highly active "Type II Red" muscle fibres . The mitochondrial myelin figures were present together with a loss in the integrity of cristae and walls following a 3 min exposure to high oxygen tension (10) . Most mitochondria develop myelin figures in hamster muscle when exposed to 99 % oxygen (4). However, myelin figures were absent in muscle tissue following even prolonged ischemic injury (3, 7, 10). Thus myelin-figure formation in the S¥P muscle fibres, which are virtually anoxic, may be due to rigor toxicity and lowering of pH (9). Myelin figures are probably non-specific structures since they are found in cardiomyopathy (radiation-induced) (16) and smooth muscle (12) also. 72h after epinephrine administration , mitochondria of the degenerating myocardial cells showed distinct myelin figures (5). The latter formed around the periphery of the mitochondria as compared to the mainly centrally placed myelin figures seen here in the SMP muscle fibres . An activation of endogenous phospholipase A may play an important role in the control of mitochondrial permeability and swelling (19). Further, the degree of mitochondrial swelling is, however, proportional to permeability, which in turn is stimuExp Toxic Pathol 44 (1992) 2
71
Fig. 2. Two mitochondria are swollen; there is clearing of the matrix and a focal loss of the cristae; x 13,500.
Fig. 3. Myelin figure is present in a giant mitochondrium. Note one elongated interfibrillar and 2 degenerating mitochondria in which cristae are recognizable; x 11 ,500. 72
Exp Toxic Pathol 44 (1992) 2
lated by Ca + + ions (18). Finally, the mitochondrial alterations in these' highly active muscle fibres may be in some way associated with the above phenomena during acute catabolism (2, 18). Although the pathogenesis of mitochondrial abnormal morphology is not clear, it may result due to peroxidation, cytochrome perturbation and a compensatory change of mitochondria to overcome acute insult. The precise mechanism, however, requires further study.
12.
References
13.
I. ALLMAN, D.W., MUNROE, l., WAKABAYASHI, T., HARRIS, R.A., GREEN, D. E.: Studies on the transition of the cristal membrane from the orthodox to the aggregated configuration. II. Determinants of the orthodox-aggregated transition in adrenal cortex mitochondria. Bioenergetics 1970; 1: 87-107. 2. AMENTA, l.S., SARGUS, M.l., VENKATESAN, S., SCHIMOZUKA, H.: Role of the vacuolar apparatus in augmented protein degradation in cultured fibroblasts. l. Cell. Physiol. 1978, 94: 77-86. 3. CAULFIELD, l., KLIONSKY, B.: Myocardial ischemia and early infarction. An early electron microscopic study. AM. l. Pathol. 1959; 35: 489-497. 4. CAULFIELD, l.B., SHELTON, W., BURKE, l.F.: Cytotoxic effects of oxygen on striated muscle. Arch. Pathol. 1972; 94: 127-132. 5. FERRANS, V.l., HIBBS, R.G., WElLY, H.S., WEILBAECHER, D.G., WALSH, l. l., BURCH, G. E.: A histochemical and electron microscopic study of epinephrine-induced myocardial necrosis. l. Mol. Cell. Cardiol. 1970; 1: 11-22. 6. GRODUMS, E. I.: Ultrastructural changes in the mitochondria of brown adipose cells during the hibernation cycle of eitel/us lateralis. Cell. Tiss. Res. 1977; 185: 231-237. 7. HANZLlKOVA, V., SCHIAFFINO, S.: Mitochondrial changes in ischemic skeletal muscle. l. Ultrastruct. Res. 1977; 60: 121-133. 8. HEGARTY, P. V.l., DAHLIN, K.l., BENSON, E.S.: Ultrastructural differences in mitochondria of skeletal muscle in the prerigor and rigor states. Experientia 1978; 34: 1070-1071. 9. HERDSON, P.B., SOMMERS, H.M., lENNINGS, R.B.: A comparative study of the fine structure of normal and ischemic dog myocardium
10.
11.
14. 15. 16.
17. 18.
19.
20.
21.
22.
with special reference to early changes following temporary exclusion of a coronary artery. Am. l. Pathol. 1965; 46: 367-375. HUGHSON, M., BALENTINE, l., DANIELL, H.: The ultrastructural pathology of hyperbaric oxygen exposure. Observations on the heart. Lab. Invest. 1977; 37: 516-525. HULSMANN, W. C, DE lONG, l. W., VAN TOL, A.: Mitochondria with loosely and tightly coupled oxidative phosphorylation in skeletal muscle. Biochem. Biophys. Acta 1968; 162: 292-293. lORIS, L, MAJNO, G.: Cell-to-cell herniae in the arterial wall. L The pathogenesis of vacuoles in the normal media. Am. 1. Pathol. 1977; 87: 375-398. KHAN, M. A.: On the subsarcolemmal localization of phenazine ethosulfate-linked a-glycerophosphate dehydrogenase activity in pigeon pectoralis white muscle fibres. Histochemistry 1976; 50: 103-110. - Histochemical and ultrastructural characteristics of a new muscle tibre type in avian striated muscle. Histochem. l. 1979; 11: 321-335. - Ultrastructural study of mitochondria in the normal skeletal muscle. Cell. Molec. BioI. 1979; 24: 81-87. KHAN, M. Y.: Radiation induced cardiomyopathy. L An electron microscopic study of cardiac muscle cells. Am. l. Pathol. 1973; 73: 131-146. NAPOLITANO, L.: Cytolysomes in metabolically active cells. l. Cell. BioI. 1963; 18: 478-481. PUBLICOVER, S.J., DUNCAN, CJ., SMITH, l.L.: Ultrastructural changes in muscle mitochondria in situ, including the apparent development of internal septa, associated with the uptake and release of calcium. Cell Tissue Res. 1977; 185: 373-385. WAITE, M., VAN DEENEN, L.L.M., RUIGROK, T.J.C., ALBERS, P. F.: Relation of mitochondrial phospholipase A activity to mitochondrial swelling. J. Lipid Res. 1969a; 10: 599-608. SCHERPHOF, G.L., BOSHOUWRS, F.M.G. and VAN DEENEN, L.L.M.: Differentiation of phospholiphase A in mitochondria and 1ysosomes of rat livers. l. Lipid Res. 1969b; 10: 411-420. WILLIAMS, C.H., VAIL, W.l.: Ultrastructural transitions in energized and de-energized mitochondria. In: Advances in Cellular and Molecular Biology, vol. 2, (ed. PURNOW, E.l.). 1972,385-406. WINBORN, W. B., BOCKMAN, D. E.: Origin of Iysosomes in parietal cells. Lab. Invest. 1968, 19: 256-264.
Exp Toxic Pathol 44 (1992) 2
73
