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Effects of anoxia on cellular damage in the incubated mouse diaphragm
TISSUE & CELL 1989 21 (6) 857-861 @ 1989 Longman Group UK Ltd
NASRIN
M. SHAMSADEEN
and C. J. DUNCAN*
EFFECTS OF ANOXIA ON CELLULAR DAMAGE IN THE INCUBATED MOUSE DIAPHRAGM Keywords:
Muscle, cellular damage,
calcium, oxygen, anoxia
ABSTRACT. Satisfactory ultrastructural integrity of the mouse diaphragm was maintained in vitro in modified Krebs-Hens&it saline for 3h when the rate of oxygenation was 2.8ml set-’ (95% 02 + 5% CO*). Hypoxic (0, = 1.62.0 ml see-‘) or anoxic (95% N2 + 5% CO*) conditions triggered typical Ca-triggered myotilament damage, believed to be induced by a rise in [Cal,. It was unaffected by omission of Ca from the saline, but the muscle was protected at 7.8”C. ‘High-Oj gassing (lOmlsec_‘) also caused a characteristic, but different. damage with swollen sarcoplasmic reticulum and spacing of the myotibrils.
Introduction
Materials and Methods
The mouse diaphragm appears to be particularly suitable for in vitro studies of protein
BALB/c mice (12-20g) were killed by cervical dislocation and their diaphragms were quickly removed and washed in ice-cold Krebs-Henseleit saline. The diaphragm was left intact in its ring of bone in some preparations, whereas in others it was separated into two hemidiaphragms. Additionally, preparations were made by completely separating the diaphragm, either by dissection or by electrocautery. Preparations were (i) secured by a stainless steel clip and suspended by surgical thread or (ii) pinned with entomological pins to dental wax or (iii) left free in the incubation medium. The diaphragms were then either (i) incubated in conical flasks where 65mI of the incubation medium was directly gassed with a fine stream of bubbles via a hypodermic needle and the rate of gassing was continuously monitored or (ii) suspended in small muscle baths (volume = 45 ml) throughwhich the pregassed (via a diffuser stone) medium was pumped (8 ml min-I). Preparations were normally maintained at 37°C * 0.2”C in a water bath. The standard incubation medium was based on Krebs-Henseleit solution and contained (mM): NaCl 117, KC1 5, CaCl, 2.55, NaHCO, 25, MgSO, 0.5, Hepes 1, Glucose 5, pH 7.35 after gassing with mixtures
turnover and of the biochemical and ultrastructural changes that occur during cellular damage in skeletal muscle. It has also been used for longer-term culture studies (Trowell, 1959). Diffusion is presumably relatively rapid in this sheet of muscle and its organization makes it ideal for ultrastructural study. Nevertheless, electron micrographs of preparations that had been fixed after incubation for 60min frequently showed that cellular damage had occurred. It has been shown (Duncan and Jackson, 1987) that at least two separate damage pathways exist in mammalian skeletal muscle, both activated by a rise in intracellular calcium concentration. We report here that one of these pathways can be activated by changes in the oxygen tension of the incubation medium. The different factors affecting the ultrastructural integrity of the mouse diaphragm in vitro are explored; it is peculiarly sensitive to oxygen tension and an optimum rate of gassing for this preparation is suggested. Department of Zoology, University of Liverpool, P.O. Box 147, Liverpool L69 3BX. ‘To whom requests for reprints should be addressed. Received
18 Apri11988.
857
SHAMSADEEN
containing 5% CO,. Diaphragms were also incubated in Earle’s medium. Incubation media were gassed with one of the following gas mixtures (i) 95% 0, + 5% CO, (ii) 95% N, + 5% CO* (iii) 75% N, + 20% 0, + 5% COz (BOC, Special Gases). Muscles were prepared for electron microscopy by fixation in ice-cold 3% glutaraldehyde in cacodylate buffer, pH 7.2, for 15 hr. Specimens were washed in 0.1 M sodium cacodylate, pH 7.2, for 30 min (two changes). Tissues were then cut into smaller pieces and post-fixed in 0~0, for 2 hr at room temperature; small blocks were subsequently washed in cacodylate buffer (two changes), dehydrated through the alcohol series and embedded in Spurr’s resin. Sections were cut at 60-90 nm, stained with uranyl acetate and lead citrate and examined on a Leitz EM 10CR. Cellular damage and other ultrastructural changes were assessed semi-quantitatively by scoring the electron microscope picture on a standardised check-list and the electronmicrographs were then reassessed independently. All reagents were AnalaR grade. Hepes (N-2-Hydroxyethyl-piperazine-N’-2-ethanesulfonic acid) was obtained from Sigma Chemical Co., St. Louis, MO, USA. Earle’s medium was obtained from Flow Laboratories, Rickmansworth, Herts, (Medium E199, No. 14-200) and 2.2g I-’ NaHCO, was added before gassing with 95% 0, + 5% COz ([Cal = 1.8 mM).
Mouse diaphragm
incubated
AND DUNCAN
Results
Incubation of diaphragm preparations for less than 20 min in Nz-gassed medium produced muscle with a normal ultrastructure; preparations incubated for 20-35 min were found to be progressively more damaged. Early changes were the breakdown of relaxed sarcomeres with spacing of the myofibrils (Fig. 3), sometimes accompanied with swollen and damaged mitochondria. However, later changes at 35-40min were the marked contraction of the myofibrils with characteristic, blurred Z-lines and the myofilaments were clearly damaged (Figs l-3). The sarcoplasmic reticulum was usually swollen and appeared as a sequence of vesicles (Fig. 2). Mitochondria were frequently swollen (Fig. 1) with lipid droplets (Fig. 2) and sometimes exhibited the septation and sub-division described previously that is characteristic of cellular damage in muscle cells (Duncan, Greenaway, Publicover, Rudge and Smith, 1980). After 40min, considerable portions of the muscle sheet were severely damaged. Identical ultrastructural changes (Fig. l), namely contracted sarcomeres, blurred Zlines, damaged myofilaments and swollen mitochondria were also found after 2 hr when the preparation was gassed with 95% 0, + 5% COz, but at a low rate (1.62.0ml set-‘). Contraction damage was also found after gassing with 75% N, + 20% O2 + 5% CO, for 35,40 or 60 min.
at 37°C in Krebs-Henseleit
saline.
Fig. 1. Hypoxic conditions. Gassed with 95% 0, + 5% CO2 at 1~6mlsec~ Blurred Z-lines, contracted myolibrils, myofilament damage and greatly-swollen x 16,OGQ.
for 120min. mitochondria
Fig. 2. Anoxia; 95% Nz + 5% CO* for 40min. Blurred Z-lines, contracted myofibrils an increase in the numbers of mitochondria, together with many lipid droplets X8m.
and
Fig. 3. Anoxia, 95% N, + 5% CO2 for 20 min. Damage and dissolution of the myofilaments. Z-lines are blurred but the sarcomeres are less severely contracted x 16,000. Fig. 4. 95% O2 + 5% CO2 at 10mlsec~’ for 10 min. Characteristic with limited damage to the myolilaments x 16,000. Fig. 5. 95% O2 + 5% COz at 2.8 ml set-’ for 60 min x 16,000
separation
of the fibrils
This ‘contraction-type’ damage under conditions of anoxia or hypoxia was not protected by the omission of Ca from the medium, the same cellular damage occurring after 40 min gassing with 95% N2 + 5% C02. However, when this anoxic incubation was repeated at 78°C for 40 min, the fibres were almost completely protected; the myofilaments were undamaged, although some mitochondria were swollen and damaged. Hypoxic conditions would reduce the aerobic production of high-energy phosphates, thereby potentially reducing active transport of Ca. However, omission of glucose from the incubation medium did not exacerbate or accelerate the effects of anoxia. For example, normal ultrastructure of the myofilaments was found following 15 or 20min incubation without glucose when the medium was gassed with 95% N2 + 5% CO,. However, neither did gassing the medium with oxygen (95% Oz + 5% CO, at 10 ml set-’ at 37°C) provide satisfactory conditions for incubation. Different but characteristic damage was found after 10, 15 or 20min; the sarcomeres were relaxed but clearly separated and the T-tubules and sarcoplasmic reticulum were slightly swollen. Some dissolution of the myofilaments was found (Fig. 4). The same pattern of damage was found when the gassed medium (95% 0, + 5% CO,; 37°C) was pumped through the incubation flask instead of gassing directly. Identical damage was also found when the experiments with 95% 0, + 5% CO, at were repeated using Earle’s lOmlsec_’ medium, so that the amino acids added thereby provided no protection. Finally, diaphragms were isolated at different rates of gassing in Krebs-Henseleit saline in a series of experiments. Again, 10 ml set-’ for 10 or 20 min produced spacing and separation of the myofibrils, but gassing at 2.3 or 3 ml set-’ for 20, 60, 90, 120 or 150min all yielded muscles with excellent ultrastructural integrity (Fig. 5). Isolated diaphragm preparations were then routinely gassed at 2.8mlsec-’ when incubated in vitro for 34hr.
Discussion Previous studies ragm (Mulligan
with isolated mouse diaphand Beynon, 1985) have
emphasized the importance of supplementation of the medium with amino acids and of a high rate of oxygenation. However, in experiments in which the medium was saturated with 95% O2 + 5% COz, using any of the methods described above, limited, but was specialised ultrastructural damage invariably found after 10 min. These cellular changes were a swelling of the sarcotubular system accompanied by a characteristic separation of the myofibrils. This ‘high-O,’ damage was not prevented or ameliorated by substituting Earle’s medium for incubation (which is supplemented with 22 amino acids, vitamins, ATP and 5’-AMP). It appears that this type of damage is directly triggered by high rates of oxygenation (10 ml see-’ of 95% 0, + 5% CO, or greater) whilst good ultrastructural integrity could be maintained for 3 hr at a gassing rate of 2.8 ml set-’ 95% O? + cop. The standard cellular damage of mammalian skeletal muscle was observed when the diaphragm preparations were incubated under conditions of anoxia or hypoxia (gassing with N, or low rates of 0,). This damage was predominantly associated with contraction of the sarcomeres and heavily blurred Z-lines. These patterns of damage are typical of those described previously in mouse diaphragm (Publicover, Duncan and Smith, 1978; Duncan, Greenaway and Smith, 1980) and mouse soleus muscle (Duncan and Jackson, 1987) when these have been treated with agents that are believed to raise intracellular Ca concentration. The ultrastructural damage also corresponds closely with that described in saponin- skinned amphibian skeletal muscle and which is rapidly and specifically triggered by EGTA-buffered [Cal at 10m6M to 8 x lo-‘M (Duncan, 1987). We conclude that anoxia or hypoxia rapidly terminate or reduce oxidative phosphorylation. thereby reducing the supply of high-energy phosphates (Andersson, Aw and Jones, 1987) so that active intracellular uptake of Ca and extrusion of Ca at the sarcolemma are impaired, and consequently intracellular Ca concentration rises and initiates the damage mechanism. However, a further depletion of high-energy phosphates produced by incubating in the absence of glucose did not accelerate or potentiate damage. Hepatocytes exposed to anoxia for 30 min showed a marked decrease in mitochondrial
ANOXIA AND MUSCLE DAMAGE
Ca (Aw, Andersson and Jones, 1987) so that anoxia may also cause the release of stored mitochondrial Ca, thereby directly raising [Cali. Since removal of extracellular Ca did not protect against anoxia-induced damage, intracellularly-stored Ca is sufficient to initiate widespread and severe cellular damage in the diaphragm in vitro. No ultrastructural damage was found when the diaphragm was gassed with 95% N, + 5% CO, at 7.8”C, which contrasts with the findings of the Ca-paradox or Oz-paradox of rat heart where little protection is provided by low temperatures against the release of creatine kinase via the damaged sarcolemma which is directly triggered by Ca-entry (Hearse, Humphrey and Bullock, 1978). The two separate pathways producing myofilament breakdown and sarcolemma damage (Duncan and Jackson, 1987) may have different temperature sensitivities or the factors leading to the rise in [Cal, in anoxia may be inhibited at low temperature.
861
High 0, tensions also appear to have a deleterious effect on the ultrastructural integrity of the mouse diaphragm in vitro, although the type of damage is different. The myofibrils are relaxed and so there may be no localized rises in [Cali. High O2 tension appears to cause rapidly marked separation of the myofibrils, together with swelling of the sarcotubular system and some limited myofilament degradation in this specialised muscle preparation which is composed of red fibres with an abundance of mitochondria (Gauthier and Padykula, 1966; Bass, Gutmann and Hanikova, 1971).
Acknowledgements
We thank Mr J. L. Smith for assistance with electron microscopy and Miss S. Scott for preparation of the manuscript.
Andersson, B. S., Aw, T. Y. and Jones, D. P. 1987. Mitochondrial transmembrane potential and pH gradient during anoxia. Am. J. Physiol., 252, C349-C355. Aw, T. Y., Andersson, B. S. and Jones, D. P. 1987. Suppression of mitochondrial respiratory function after short-term anoxia. Am. .I. Physiol., 252, C362-C368. Bass, A.. Gutmann, E. and Hanikova, M. 1971. Contraction properties and enzyme pattern of the mouse and rat diaphragm. Physiol. Eohemoslov, 20,5-10. Duncan, C. J. 1987. Calcium and rapid myofilament damage. Biochem. Sot. Tram., 15,957-958. Duncan, C. J., Greenaway, H. C., Publicover, S. J., Rudge, M. F. and Smith, J. L. 1980. Experimental production of “septa” and apparent subdivision of muscle mitochondria. J. Bioenerg. Biomembr. 12,13-33. Duncan, C. J., Greenaway, H. C. and Smith, J. L. 1980. 2,4-Dinitrophenol, lysosomal breakdown and rapid myofilament degradation in vertebrate skeletal muscle. Naunyn-Schmiedebergs Arch.exp. P&h. Phannac. 315,77-82. Duncan, C. J. and Jackson, M. J. 1987. Different mechanisms mediate structural changes and intracellular enzyme eftlux following damage to skeletal muscle. J. Cell Sci., 87,183-188. Gauthier, G. F. and Padykula, H. A. 1966. Cytological studies of fiber types in skeletal muscle. A comparative study of the mammalian diaphragm. J. Cell Bid., 28,333-354. Mulligan, M. T. and Beynon, R. J. 1985. The isolated mouse diaphragm as a test system for new inhibitors of muscle protein breakdown. Biochem. Sot. Trans., 13,1167-1168. Publicover, S. J., Duncan, C. J. and Smith, J. L. 1978. The use of A23187 to demonstrate the role of intracellular calcium in causingultrastructural damage in mammalian muscle. .I. Neuroparh. exp. Neural., 37,544-557. Trowel& 0. A. 1959. The culture of mature organs in a synthetic medium. Exper. Cell Res., 16,118-147.
NASRIN
M. SHAMSADEEN
and C. J. DUNCAN*
EFFECTS OF ANOXIA ON CELLULAR DAMAGE IN THE INCUBATED MOUSE DIAPHRAGM Keywords:
Muscle, cellular damage,
calcium, oxygen, anoxia
ABSTRACT. Satisfactory ultrastructural integrity of the mouse diaphragm was maintained in vitro in modified Krebs-Hens&it saline for 3h when the rate of oxygenation was 2.8ml set-’ (95% 02 + 5% CO*). Hypoxic (0, = 1.62.0 ml see-‘) or anoxic (95% N2 + 5% CO*) conditions triggered typical Ca-triggered myotilament damage, believed to be induced by a rise in [Cal,. It was unaffected by omission of Ca from the saline, but the muscle was protected at 7.8”C. ‘High-Oj gassing (lOmlsec_‘) also caused a characteristic, but different. damage with swollen sarcoplasmic reticulum and spacing of the myotibrils.
Introduction
Materials and Methods
The mouse diaphragm appears to be particularly suitable for in vitro studies of protein
BALB/c mice (12-20g) were killed by cervical dislocation and their diaphragms were quickly removed and washed in ice-cold Krebs-Henseleit saline. The diaphragm was left intact in its ring of bone in some preparations, whereas in others it was separated into two hemidiaphragms. Additionally, preparations were made by completely separating the diaphragm, either by dissection or by electrocautery. Preparations were (i) secured by a stainless steel clip and suspended by surgical thread or (ii) pinned with entomological pins to dental wax or (iii) left free in the incubation medium. The diaphragms were then either (i) incubated in conical flasks where 65mI of the incubation medium was directly gassed with a fine stream of bubbles via a hypodermic needle and the rate of gassing was continuously monitored or (ii) suspended in small muscle baths (volume = 45 ml) throughwhich the pregassed (via a diffuser stone) medium was pumped (8 ml min-I). Preparations were normally maintained at 37°C * 0.2”C in a water bath. The standard incubation medium was based on Krebs-Henseleit solution and contained (mM): NaCl 117, KC1 5, CaCl, 2.55, NaHCO, 25, MgSO, 0.5, Hepes 1, Glucose 5, pH 7.35 after gassing with mixtures
turnover and of the biochemical and ultrastructural changes that occur during cellular damage in skeletal muscle. It has also been used for longer-term culture studies (Trowell, 1959). Diffusion is presumably relatively rapid in this sheet of muscle and its organization makes it ideal for ultrastructural study. Nevertheless, electron micrographs of preparations that had been fixed after incubation for 60min frequently showed that cellular damage had occurred. It has been shown (Duncan and Jackson, 1987) that at least two separate damage pathways exist in mammalian skeletal muscle, both activated by a rise in intracellular calcium concentration. We report here that one of these pathways can be activated by changes in the oxygen tension of the incubation medium. The different factors affecting the ultrastructural integrity of the mouse diaphragm in vitro are explored; it is peculiarly sensitive to oxygen tension and an optimum rate of gassing for this preparation is suggested. Department of Zoology, University of Liverpool, P.O. Box 147, Liverpool L69 3BX. ‘To whom requests for reprints should be addressed. Received
18 Apri11988.
857
SHAMSADEEN
containing 5% CO,. Diaphragms were also incubated in Earle’s medium. Incubation media were gassed with one of the following gas mixtures (i) 95% 0, + 5% CO, (ii) 95% N, + 5% CO* (iii) 75% N, + 20% 0, + 5% COz (BOC, Special Gases). Muscles were prepared for electron microscopy by fixation in ice-cold 3% glutaraldehyde in cacodylate buffer, pH 7.2, for 15 hr. Specimens were washed in 0.1 M sodium cacodylate, pH 7.2, for 30 min (two changes). Tissues were then cut into smaller pieces and post-fixed in 0~0, for 2 hr at room temperature; small blocks were subsequently washed in cacodylate buffer (two changes), dehydrated through the alcohol series and embedded in Spurr’s resin. Sections were cut at 60-90 nm, stained with uranyl acetate and lead citrate and examined on a Leitz EM 10CR. Cellular damage and other ultrastructural changes were assessed semi-quantitatively by scoring the electron microscope picture on a standardised check-list and the electronmicrographs were then reassessed independently. All reagents were AnalaR grade. Hepes (N-2-Hydroxyethyl-piperazine-N’-2-ethanesulfonic acid) was obtained from Sigma Chemical Co., St. Louis, MO, USA. Earle’s medium was obtained from Flow Laboratories, Rickmansworth, Herts, (Medium E199, No. 14-200) and 2.2g I-’ NaHCO, was added before gassing with 95% 0, + 5% COz ([Cal = 1.8 mM).
Mouse diaphragm
incubated
AND DUNCAN
Results
Incubation of diaphragm preparations for less than 20 min in Nz-gassed medium produced muscle with a normal ultrastructure; preparations incubated for 20-35 min were found to be progressively more damaged. Early changes were the breakdown of relaxed sarcomeres with spacing of the myofibrils (Fig. 3), sometimes accompanied with swollen and damaged mitochondria. However, later changes at 35-40min were the marked contraction of the myofibrils with characteristic, blurred Z-lines and the myofilaments were clearly damaged (Figs l-3). The sarcoplasmic reticulum was usually swollen and appeared as a sequence of vesicles (Fig. 2). Mitochondria were frequently swollen (Fig. 1) with lipid droplets (Fig. 2) and sometimes exhibited the septation and sub-division described previously that is characteristic of cellular damage in muscle cells (Duncan, Greenaway, Publicover, Rudge and Smith, 1980). After 40min, considerable portions of the muscle sheet were severely damaged. Identical ultrastructural changes (Fig. l), namely contracted sarcomeres, blurred Zlines, damaged myofilaments and swollen mitochondria were also found after 2 hr when the preparation was gassed with 95% 0, + 5% COz, but at a low rate (1.62.0ml set-‘). Contraction damage was also found after gassing with 75% N, + 20% O2 + 5% CO, for 35,40 or 60 min.
at 37°C in Krebs-Henseleit
saline.
Fig. 1. Hypoxic conditions. Gassed with 95% 0, + 5% CO2 at 1~6mlsec~ Blurred Z-lines, contracted myolibrils, myofilament damage and greatly-swollen x 16,OGQ.
for 120min. mitochondria
Fig. 2. Anoxia; 95% Nz + 5% CO* for 40min. Blurred Z-lines, contracted myofibrils an increase in the numbers of mitochondria, together with many lipid droplets X8m.
and
Fig. 3. Anoxia, 95% N, + 5% CO2 for 20 min. Damage and dissolution of the myofilaments. Z-lines are blurred but the sarcomeres are less severely contracted x 16,000. Fig. 4. 95% O2 + 5% CO2 at 10mlsec~’ for 10 min. Characteristic with limited damage to the myolilaments x 16,000. Fig. 5. 95% O2 + 5% COz at 2.8 ml set-’ for 60 min x 16,000
separation
of the fibrils
This ‘contraction-type’ damage under conditions of anoxia or hypoxia was not protected by the omission of Ca from the medium, the same cellular damage occurring after 40 min gassing with 95% N2 + 5% C02. However, when this anoxic incubation was repeated at 78°C for 40 min, the fibres were almost completely protected; the myofilaments were undamaged, although some mitochondria were swollen and damaged. Hypoxic conditions would reduce the aerobic production of high-energy phosphates, thereby potentially reducing active transport of Ca. However, omission of glucose from the incubation medium did not exacerbate or accelerate the effects of anoxia. For example, normal ultrastructure of the myofilaments was found following 15 or 20min incubation without glucose when the medium was gassed with 95% N2 + 5% CO,. However, neither did gassing the medium with oxygen (95% Oz + 5% CO, at 10 ml set-’ at 37°C) provide satisfactory conditions for incubation. Different but characteristic damage was found after 10, 15 or 20min; the sarcomeres were relaxed but clearly separated and the T-tubules and sarcoplasmic reticulum were slightly swollen. Some dissolution of the myofilaments was found (Fig. 4). The same pattern of damage was found when the gassed medium (95% 0, + 5% CO,; 37°C) was pumped through the incubation flask instead of gassing directly. Identical damage was also found when the experiments with 95% 0, + 5% CO, at were repeated using Earle’s lOmlsec_’ medium, so that the amino acids added thereby provided no protection. Finally, diaphragms were isolated at different rates of gassing in Krebs-Henseleit saline in a series of experiments. Again, 10 ml set-’ for 10 or 20 min produced spacing and separation of the myofibrils, but gassing at 2.3 or 3 ml set-’ for 20, 60, 90, 120 or 150min all yielded muscles with excellent ultrastructural integrity (Fig. 5). Isolated diaphragm preparations were then routinely gassed at 2.8mlsec-’ when incubated in vitro for 34hr.
Discussion Previous studies ragm (Mulligan
with isolated mouse diaphand Beynon, 1985) have
emphasized the importance of supplementation of the medium with amino acids and of a high rate of oxygenation. However, in experiments in which the medium was saturated with 95% O2 + 5% COz, using any of the methods described above, limited, but was specialised ultrastructural damage invariably found after 10 min. These cellular changes were a swelling of the sarcotubular system accompanied by a characteristic separation of the myofibrils. This ‘high-O,’ damage was not prevented or ameliorated by substituting Earle’s medium for incubation (which is supplemented with 22 amino acids, vitamins, ATP and 5’-AMP). It appears that this type of damage is directly triggered by high rates of oxygenation (10 ml see-’ of 95% 0, + 5% CO, or greater) whilst good ultrastructural integrity could be maintained for 3 hr at a gassing rate of 2.8 ml set-’ 95% O? + cop. The standard cellular damage of mammalian skeletal muscle was observed when the diaphragm preparations were incubated under conditions of anoxia or hypoxia (gassing with N, or low rates of 0,). This damage was predominantly associated with contraction of the sarcomeres and heavily blurred Z-lines. These patterns of damage are typical of those described previously in mouse diaphragm (Publicover, Duncan and Smith, 1978; Duncan, Greenaway and Smith, 1980) and mouse soleus muscle (Duncan and Jackson, 1987) when these have been treated with agents that are believed to raise intracellular Ca concentration. The ultrastructural damage also corresponds closely with that described in saponin- skinned amphibian skeletal muscle and which is rapidly and specifically triggered by EGTA-buffered [Cal at 10m6M to 8 x lo-‘M (Duncan, 1987). We conclude that anoxia or hypoxia rapidly terminate or reduce oxidative phosphorylation. thereby reducing the supply of high-energy phosphates (Andersson, Aw and Jones, 1987) so that active intracellular uptake of Ca and extrusion of Ca at the sarcolemma are impaired, and consequently intracellular Ca concentration rises and initiates the damage mechanism. However, a further depletion of high-energy phosphates produced by incubating in the absence of glucose did not accelerate or potentiate damage. Hepatocytes exposed to anoxia for 30 min showed a marked decrease in mitochondrial
ANOXIA AND MUSCLE DAMAGE
Ca (Aw, Andersson and Jones, 1987) so that anoxia may also cause the release of stored mitochondrial Ca, thereby directly raising [Cali. Since removal of extracellular Ca did not protect against anoxia-induced damage, intracellularly-stored Ca is sufficient to initiate widespread and severe cellular damage in the diaphragm in vitro. No ultrastructural damage was found when the diaphragm was gassed with 95% N, + 5% CO, at 7.8”C, which contrasts with the findings of the Ca-paradox or Oz-paradox of rat heart where little protection is provided by low temperatures against the release of creatine kinase via the damaged sarcolemma which is directly triggered by Ca-entry (Hearse, Humphrey and Bullock, 1978). The two separate pathways producing myofilament breakdown and sarcolemma damage (Duncan and Jackson, 1987) may have different temperature sensitivities or the factors leading to the rise in [Cal, in anoxia may be inhibited at low temperature.
861
High 0, tensions also appear to have a deleterious effect on the ultrastructural integrity of the mouse diaphragm in vitro, although the type of damage is different. The myofibrils are relaxed and so there may be no localized rises in [Cali. High O2 tension appears to cause rapidly marked separation of the myofibrils, together with swelling of the sarcotubular system and some limited myofilament degradation in this specialised muscle preparation which is composed of red fibres with an abundance of mitochondria (Gauthier and Padykula, 1966; Bass, Gutmann and Hanikova, 1971).
Acknowledgements
We thank Mr J. L. Smith for assistance with electron microscopy and Miss S. Scott for preparation of the manuscript.
Andersson, B. S., Aw, T. Y. and Jones, D. P. 1987. Mitochondrial transmembrane potential and pH gradient during anoxia. Am. J. Physiol., 252, C349-C355. Aw, T. Y., Andersson, B. S. and Jones, D. P. 1987. Suppression of mitochondrial respiratory function after short-term anoxia. Am. .I. Physiol., 252, C362-C368. Bass, A.. Gutmann, E. and Hanikova, M. 1971. Contraction properties and enzyme pattern of the mouse and rat diaphragm. Physiol. Eohemoslov, 20,5-10. Duncan, C. J. 1987. Calcium and rapid myofilament damage. Biochem. Sot. Tram., 15,957-958. Duncan, C. J., Greenaway, H. C., Publicover, S. J., Rudge, M. F. and Smith, J. L. 1980. Experimental production of “septa” and apparent subdivision of muscle mitochondria. J. Bioenerg. Biomembr. 12,13-33. Duncan, C. J., Greenaway, H. C. and Smith, J. L. 1980. 2,4-Dinitrophenol, lysosomal breakdown and rapid myofilament degradation in vertebrate skeletal muscle. Naunyn-Schmiedebergs Arch.exp. P&h. Phannac. 315,77-82. Duncan, C. J. and Jackson, M. J. 1987. Different mechanisms mediate structural changes and intracellular enzyme eftlux following damage to skeletal muscle. J. Cell Sci., 87,183-188. Gauthier, G. F. and Padykula, H. A. 1966. Cytological studies of fiber types in skeletal muscle. A comparative study of the mammalian diaphragm. J. Cell Bid., 28,333-354. Mulligan, M. T. and Beynon, R. J. 1985. The isolated mouse diaphragm as a test system for new inhibitors of muscle protein breakdown. Biochem. Sot. Trans., 13,1167-1168. Publicover, S. J., Duncan, C. J. and Smith, J. L. 1978. The use of A23187 to demonstrate the role of intracellular calcium in causingultrastructural damage in mammalian muscle. .I. Neuroparh. exp. Neural., 37,544-557. Trowel& 0. A. 1959. The culture of mature organs in a synthetic medium. Exper. Cell Res., 16,118-147.