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Clinical Science (1991)80,559-564 559 Glutathione depletion during experimental damage to rat skeletal muscle and its relevance to Duchenne muscular dystrophy M. J. JACKSON, M. H. BROOKE*t, K. KAISER* AND R. H. T. EDWARDS Muscle Research Centre, Department of Medicine, University of Liverpool, Liverpool, U.K. and *Departmentof Neurology, Washington University School of Medicine, St Louis, Missouri, U.S.A. (Received 13 August/l9 December 1990; accepted 4 January 1991) SUMMARY 1. The release of glutathione has been studied in comparison with the release of creatine kinase from isolated rat soleus muscles subjected to certain forms of experimental damage. 2. Excessive electrically stimulated contractile activity or treatment of muscles with the mitochondrial inhibitor, 2,4-dinitrophenol, induced a substantial release of both creatine kinase and glutathione and a reduction in the total glutathione content of the muscle. The time course of this release and depletion indicates that the efflux of the two molecules is not directly related and that a reduction in muscle glutathione content does not occur before cytosolic enzyme release. 3. 2,4-Dinitrophenol-stimulated release of creatine kinase was significantly reduced by the omission of external calcium from the incubation media, but glutathione release and depletion was relatively unaffected by this. Deliberate elevation of the muscle intracellular calcium content with the calcium ionophore, A23 187, induced a substantial loss of creatine kinase, but had no significant effect on the release of glutathione. 4. Muscle biopsies from patients with Duchenne muscular dystrophy were found to have an elevated content of glutathione and an equivalent protein-thiol content compared with control subjects. 5. We conclude that, although release of glutathione from skeletal muscle occurs after excessive contractile activity or inhibition of mitochondrial metabolism, this is not a key step in the damaging processes leading to cytosolic enzyme release, neither is it relevant to the onTPresent address: Department of Neurology, Mackenzie Health Science Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2B7. Correspondence:Dr M. J. Jackson, Muscle Research Centre, Department of Medicine, University of Liverpool, PO Box 147, Liverpool L69 3BX, U.K. going damage to skeletal muscle which occurs in patients with Duchenne muscular dystrophy. Key words: calcium, damage, free radicals, glutathione, skeletal muscle. Abbreviations: DNP, 2,4-dinitrophenol. INTRODUCTION Skeletal muscle damage occurs in a number of di€ferent physiological and pathological situations, such as after unaccustomed or excessive exercise or in patients suffering from the inherited muscular dystrophies. The mechanisms by which damage to skeletal muscle occurs have been studied in a number of different situations and two major theories have been presented. An accumulation of intracellular calcium with a consequent activation of calcium-sensitive degenerative pathways has been proposed to be important by some workers [l-51, while others have claimed that oxidizing free-radical species play a key role in the processes of damage [6-91. Both these processes have been suggested to be abnormal in muscle from patients with Duchenne and other muscular dystrophies [lo-151. Glutathione is the most abundant intracellular nonprotein thiol compound. It has a number of important cellular functions, mainly related to the thiol group of the cysteine residue [16]. In particular, it plays an essential role as a co-substrate for the enzyme glutathione peroxidase, which appears to act to detoxfy lipid peroxidase in the cell, thereby reducing the potential damaging effects of oxygen free-radical production. Many cells appear to have an active transport system to remove oxidized glutathione from the cell [ 171 and an increased release of glutathione from cells may therefore reflect an insult to the cell by oxidizing free-radical species. An increase in oxidized glutathione in the blood has been reported to 560 M. J. Jackson et al. occur after exercise [18] and has been attributed to increased oxidizing free-radical activity in this situation, although release of muscle glutathione may presumably also occur as a result of the changes in membrane permeability which lead to leakage of cytosolic proteins after damage to muscle cells (191. In order to clarify the role of glutathione in skeletal muscle damage, we have studied the release of glutathione by, and the glutathione content of, isolated rat muscles subjected to various forms of experimental damage. In addition, to clarify the relevance of this work to human patients we have analysed the glutathione content of muscle biopsy specimens from patients with Duchenne muscular dystrophy. Since one important consequence of a reduction in the muscle content of reduced glutathione could be the oxidation of protein-thiol groups, we have also measured these in the samples from patients with Duchenne muscular dystrophy and from control subjects. METHODS Experimental damage to normal rat muscles Female Wistar rats (100-200 g), fed on a standard laboratory diet, were killed by cervical dislocation and soleus muscles were carefully and rapidly removed. The muscles were mounted on special holders and incubated in 4 ml of an oxygenated, bicarbonate-buffered mammalian Ringer solution at 37°C as previously described [19]. After 30 min of incubation, the medium was removed and replaced by fresh Ringer solution. Damage to the muscle was then induced by either incorporation of various agents into the medium for 30 min or 30 min of repetitive electrically stimulated tetanii (for 0.5 s every 2 s at 100 Hz and 30 V ) under nitrogen. After a further 30 min in the oxygenated medium, it was again renewed and this was repeated every 30 min until the end of the experiment. All media were analysed immediately for creatine kinase activity (as a measure of the extent of the damage to the muscle) and for glutathione content. In all cases one of the pair of muscles from each animal provided the control tissue. At the end of the experiment muscles were rapidly frozen in liquid nitrogen and stored at - 70°C before analysis of the glutathione content. Human biopsy samples from control subjects and patients with Duchenne muscular dystrophy Samples were obtained from 11 patients with Duchenne muscular dystrophy (aged 4-1 1 years) and six control non-dystrophic subjects (aged 7-35 years), who were biopsied for investigational purposes and were subsequently found to have no biochemical evidence of muscle damage or degeneration on histological and histochemical analysis. All patients suffering from Duchenne muscular dystrophy showed no staining for dystrophin on immunocytochemical staining of muscle biopsies and had grossly elevated plasma creatine kinase activities (in the range 1000-20 000 unitsll), while control subjects all showed normal dystrophin staining and had normal plasma activities of creatine kinase ( < 100 units/l). Biopsies were obtained from the biceps muscle using the ‘open’-biopsy technique under local anaesthesia. A small piece of muscle (approximately 30 mg) was rapidly frozen in liquid nitrogen and stored at - 70°C for approximately 1 week, until analysed for glutathione and protein-thiol contents. Preliminary studies indicated that total glutathione, oxidized glutathione and protein-thiol contents were unchanged after storage at - 70°C for 2-3 weeks. The local ethical committee gave their approval for this study. Biochemical analyses The total glutathione content of all muscle samples and incubation media were analysed by using the glutathione reductase recycling method, as described by Anderson [ 201. Analyses of the oxidized glutathione content were also undertaken after derivitization using 2-vinylpyridine [20]. However, analyses of the oxidized glutathione content of the incubation media were found to be unreliable owing to oxidation of reduced glutathione in the incubation media during the 30 min incubation periods; therefore only the total glutathione contents of incubation media are presented. Muscle protein-thiol groups were analysed by titration of the sulphosalicylic acid precipitate from the glutathione assay with 5,5-dithiobis-2-nitrobenzoic acid essentially as described by Di Monte et al. (211. Creatine kinase activities of incubation fluids were analysed as previously described (191. All reagents used were of Analar grade or of the highest grade commercially available. The calcium ionophore, A23 187, was obtained from Sigma Chemical Co. (Poole, Dorset, U.K.). Statistical analysis The statistical significance of results was assessed by using Student’s t-test, a P value of less than 0.05 being considered significant. RESULTS Experimental damage to normal rat muscle The creatine kinase activity and total glutathione content of the incubation media surrounding the control muscles, the muscles treated with 2,4-dinitrophenol (DNP, 1 mmol/l) for 30 rnin and the muscles subjected to repetitive tetanic stimuli for 30 min are shown in Fig. 1. The release of both creatine kinase and glutathione was initially low and the release from control untreated muscles remained low throughout the experiment, but DNP treatment and stimulation induced substantial efflux of both creatine kinase and glutathione after the treatment period, accounting for the loss of approximately 70% of the muscle glutathione in both cases. The pattern of efflux of the two substances induced by the two damaging protocols differed in that a greater appearance of creatine kinase activity occurred after DNP treatment than after Glutathione and muscle damage 561 DNP or (a) DNP or stimulation / b* l** L I 150 180 30 60 , 90 120 0 . 23 Y 0 30 0 0 It DNP or 60 90 120 150 180 Time of incubation (min) Fig. 2. Total glutathione content of rat soleus muscles incubated in vitro. Muscles were either untreated (m), treated with 1 mmol/l DNP (A ) or subjected to electrically stimulated excessive contractile activity ( 0 ) during the period shown. Values are means, with bars indicating SEM. Statistical significance: *P<0.01, t P < 0.001 compared with untreated muscles at the same time point. ~ 30 60 90 120 150 180 Time of incubation (min) Fig. 1. Efflux of creatine b a s e (CK) ( a ) and total glutathione ( b ) from isolated rat soleus muscles. Muscles were either untreated (m), treated with 1 mmol/l DNP ( A) or subjected to electrically stimulated excessive contractile activity ).( during the period shown. Values are means, with bars indicating SEM. Statistical sigmficance: *P<0.05, **P<0.01, t P < 0.001 compared with untreated muscles at the same time point. 400 200 &+P 30 60 90 120 150 180 150 180 DNP stimulation, whereas the reverse was the case for glutathione efflux. Analysis of the total glutathione content of the muscles (Fig. 2) revealed a minor decrease in the control muscles during the course of the experiment and a much more substantial decrease in both the stimulated and DNPtreated tissues. Immediately at the end of DNP treatment the total glutathione content was not significantly different from the untreated controls (Fig. 2). The proportion of the glutathione in the oxidized form was unaffected by the various manipulations, remaining at 2-5% throughout. The glutathione lost from the muscles could be entirely accounted for by that detected in the incubation fluids (Fig. 1). Effect of manipulation of external and intracellular calcium on muscle glutathione content The calcium content of the external medium has previously been shown to have a marked influence on the damage to and release of cytosolic enzymes from skeletal muscles subjected to certain forms of experimental stress [ 5 ] . The effect of treatment of the muscles with DNP O' 30 60 90 120 Time of incubation (min) Fig. 3. Effect of DNP (200 pmol/l) on the efflux of creatine kinase (CK)( a ) and glutathione ( b )from isolated rat soleus muscles in the presence ( 0 )or absence (0) of extracellular calcium. Values are means, with bars indicating SEM. Statistical significance: *P<0.05, **P<0.01, tP< 0.001 compared with muscles in calciumcontaining media at the same time point. (200 pmol/l) in media with no added external calcium is shown in Fig. 3. This media (nominally calcium-free) produced a dramatic reduction in the creatine kinase efflux from DNP-treated muscles, the reduction being sigmfi- 562 M. J. Jackson et al. cant at all times from 30 min after the DNP treatment, but had a relatively minor effect on the glutathione efflux, the reduction in the release of glutathione being significant at only 30 min after DNP treatment with muscles from both calcium-containing and calcium-free media releasing identical amounts of glutathione at the end of the incubation. The effect of this manipulation of the extracellular calcium content of the muscle glutathione content is shown in Table 1 and demonstrates that the omission of external calcium had no protective effect on the reduction in muscle glutathione content. The effect of an elevation of intracellular calcium content by treatment of muscles with the calcium ionophore, A23187 is shown in Fig. 4.This induced a substantial efflux of creatine kinase from the muscles, but the release of glutathione was not significantly elevated compared with the control muscles, although the mean value was always higher than that of the controls. The muscle glutathione content was also not significantly influenced by the calcium ionophore treatment when compared with the control muscles (Table 1). Human muscle analyses The total glutathione content of the biopsy specimens from 11 patients with Duchenne muscular dystrophy are shown in Table 2, together with the protein-thiol content. Glutathione is expressed both as per g of muscle protein and p e r g of creatine in order to standardize for the amount of non-muscle material present in the analysed samples. Both ways of expressing the results show a significant increase in the glutathione content of the dystrophic muscle. Oxidized glutathione represented 2-5% of the total glutathione content in the muscles from both control subjects and patients with Duchenne muscular dystrophy. The protein-thiol content was identical in the patient and the control groups. DISCUSSION Little work has been undertaken on the glutathione status of skeletal muscle, although the glutathione content of cardiac muscle has been extensively studied as an index of Table 1. Total glutathione content of rat soleus muscle Results are presented as means k SEM ( n= 6). Statistical significance: *P< 0.002, **P< 0.01 compared with muscles which were incubated for 3 h but not treated. Values for muscles treated in the presence or absence of external calcium were not significantly different from each other. Total glutathione content (pmollg wet wt.) Fresh unincubated muscle Incubated, untreated muscle DNP-treated ( + Ca’+) muscle DNP-treated ( - CaZ+ ) muscle A23187-treated muscle 1.79 f0.18 1.37+0.14 0.79 If.0.02* 0.83 f0.06** 1.10 f0.10 .- 4001 -E zoo O ’ 30 60 90 150 180 O ’ 30 60 90 120 160 Time of incubation (min) 180 120 Fig. 4. Efflux of creatine kinase (CK) ( a ) and total glutathione ( b )from untreated ( 0 ) and A23187-treated ( w ) isolated rat soleus muscles. Values are means, with bars indicating SEM. Statistical significance: * P < 0.05, **P< 0.01, t P < 0.001 compared with untreated muscles at the same time point. Table 2. Total glutathione and protein-thiol contents of biopsy samples from patients with Duchenne muscular dystrophy and control subjects Results are presented as means k SEM. Abbreviation: DMD, Duchenne muscular dystrophy. Statistical significance: * P < 0.001 compared with control subjects. Control subjects (n=6) Patients with DMD ( n = 11) Glutathione content pmol/g of protein pmol/g of creatine 1 1.2 f0.8 523 k 54 20.3 f4.0* 1206 k 21 3* Protein-thiol content pmol/g of protein 118.2+8.1 103.3f 10.8 oxidative stress to the heart [22-241. This work has shown that, during ischaemia, there is an oxidation of intracellular glutathione and after reperfusion of ischaemic cardiac tissue there is a dramatic release of both oxidized and reduced glutathione [22]. These authors have suggested that the extent of the oxidation of glutathione during ischaemia is crucial in determining the vulnerability of the heart to damage due to oxygen radicals during the reperfusion process. Glutathione and muscle damage It has been suggested that in skeletal muscle substantial oxidation and release of glutathione may occur during severe exercise [25], reflecting an increased flux of oxidizing free-radicals in this situation. Oxidative soleus muscles were used for the present work because preliminary results indicated that the total glutathione content of rat soleus muscles (2.19 k 0.2pmol/g wet wt; mean+ SEM, n = 6 ) was much higher than that of fast, glycolytic extensor digitorum longus muscles (0.84 k 0.16 pmol/g wet wt.) or gastrocnemius muscles of mixed-fibre-type composition (0.97 k 0.06 pmol/g wet wt.). The results presented demonstrate that certain forms of damage to skeletal muscle can induce a substantial efflux of glutathione from the muscle cells. The extent of this release is not directly related to that of an indicator of damage to the muscle, release of creatine kinase (Fig. l), although it occurs with a similar time course and does not appear to precede the cytosolic enzyme efflux. Cytosolic enzyme efflux in this experimental system is dependent upon the presence of external calcium (Fig. 3), although the addition of calcium chelators (e.g. EGTA) to the incubation media causes damage to the muscle (results not presented in detail).However, removal of the external calcium had little effect on the release of glutathione by DNP-treated muscles (Fig. 3 ) or on the intracellular muscle glutathione content (Table 1).In a further attempt to study the calcium dependency of glutathione release from skeletal muscle we have treated soleus muscles with the calcium ionophore, A23187, in order to raise intracellular calcium levels. This compound precipitated a substantial efflux of creatine kinase (Fig. 4) from the muscles, but had only a minor effect on glutathione release (Fig. 4) or the total glutathione content of the muscle (Table 1).On balance, it therefore appears that the cytosolic enzyme creatine kinase and glutathione are released from damaged isolated skeletal muscles by different mechanisms, that leading to creatine kinase efflux involving a step dependent on the presence of external calcium which is not involved in glutathione release. In addition, neither the time courses of release (Fig. 1) nor the muscle glutathione content during the course of the experiment (Fig. 2) support a primary role for glutathione release or depletion in the processes leading to creatine kinase release. DNP is a known inhibitor of normal mitochondria1 function and we have previously shown that treatment of isolated soleus muscles with this compound leads to a total loss of cellular ATP and phosphocreatine within 6 min [26]. Likewise, measurements of the force produced by muscles subjected to the stimulation protocol used here suggests that these muscles suffer (at least) a transient failure of energy supply [19].It is therefore possible that a failure of energy supply causes the release of glutathione from the cell in both of these situations, although there is a time-lag between the failure of energy supply and the loss of glutathione from the muscles. Unfortunately, we were unable to determine the proportion of the glutathione released from the muscle which was present in the oxidized form. so we are unable to further consider the possibility that the efflux resulted from active transport of 563 oxidized glutathione [17]. However, since this would require consumption of scarce energy supplies in energydepleted cells, it seems an unlikely mechanism. Previous studies have suggested that although cytosolic enzyme efflux from DNP-treated or electricallystimulated muscle occurs via a mechanism that is dependent on the presence of external calcium, this may not be true of the ultrastructural damage which occurs due to these stresses [27]; it is therefore possible that the loss of cellular glutathione may be important in the initiation of these processes. Analysis of the glutathione and protein-thiol contents of muscle biopsy samples from patients with Duchenne muscular dystrophy have revealed an increase in the glutathione content when expressed on a protein or creatine basis and have demonstrated no s i m c a n t difference in the protein-thiol content compared with muscle from control subjects. This lack of evidence of substantial oxidation of muscle thiol groups essentially confirms the finding of other workers, who have examined animal models of muscular dystrophy [28], and is unsurprising in the light of the above results concerning the mechanisms of glutathione release from damaged skeletal muscle. Muscle from patients with Duchenne muscular dystrophy does not seem to be characterized by any sigruilcant depletion of energy supply [29], but current theories suggest that the lack of dystrophin, the protein thought to be the defective gene product in this disorder [30], may induce cellular damage by a failure of calcium homoeostasis [12,30,31]. We are unable to offer an explanation for the increase in the total glutathione content of dystrophic muscle. One possibility is that there is an increased release of arachidonic acid metabolites, such as prostaglandins, from the muscle of patients with Duchenne muscular dystrophy [32, 331, which may lead to an increased oxidative stress on the muscle. However, glutathione peroxidase activities are essentially normal in muscle from patients with this disorder [34], which argues against an increased glutathione requirement as a substate for this pathway. ACKNOWLEDGMENTS We are grateful for financial support from the Muscular Dystrophy Group of Great Britain and Northern Ireland, the Muscular Dystrophy Association of America and the NATO Scientific Programme. We thank Mr J. Planar, Washington University School of Medicine, and Mrs S. Page, Mrs N. Lowe, Miss A. McArdle and Miss A. Swift, University of Liverpool, for expert technical assistance. REFERENCES 1. Wrogemann, K. & Pena, SJ.G. Mitochondrial overload: a general mechanism for cell necrosis in muscle diseases. Lancet 1976; ii, 672-4. 2. 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