How does dystrophin deficiency lead to muscle degeneration? — Evidence from the MDX mouse

Neuromusc. Disord., Vol. 5. No. 6, pp. 445-456, 1995 Copyright © Elsevier Science Ltd Printed in Great Britain. All rights reserved 0960~8966/95 $29.00 + .00

Pergamon

o96o-s966(95)oooo1-1 REVIEW ARTICLE

HOW DOES DYSTROPHIN DEGENERATION?

DEFICIENCY

-- EVIDENCE

FROM

LEAD TO MUSCLE THE MDX MOUSE

A. McARDLE, R. H. T. EDWARDS and M. J. JACKSON* Muscle Research Centre, Department of Medicine, University of Liverpool, Liverpool L69 3BX, U.K.

(Received 5 September 1994; revised 10 January 1995; accepted 12 January 1995) Abstract--The mdx mouse has a defect in the same gene as boys with Duchenne muscular

dystrophy, which results in the absence of the protein product, dystrophin. A large number of recent studies have used the mdx mouse model to examine the potential role of dystrophy in normal muscle and the mechanisms by which dystrophin-deficiency leads to myopathy. This review discusses critically the results of these studies and their relevance to understanding the mechanisms by which dystrophin-deficiency leads to muscle necrosis. Key Words: Duchenne muscular dystrophy, dystrophin, mdx mouse, calcium.

INTRODUCTION

CELL BIOLOGY OF DYSTROPHIN

The genetic defect responsible for Duchenne and Becker muscular dystrophies (DMD and BMD) has been identified [1] and localized to band Xp21 on the human X chromosome. The cloned gene was used to identify the protein product, named dystrophin [2], which was shown to be absent or greatly diminished (< 3%) in muscle from D M D patients [2]. It has subsequently been shown that dystrophin is absent in three animal models of muscular dystrophy, the mdx mouse, the XMD dog and the dystrophic cat [2-4]. The elucidation of these animal models has had a dramatic effect on studies of the pathophysiology of DMD. In particular, the short breeding time, low relative cost and availability of the mdx mouse model has facilitated a large number of recent studies of the potential role of dystrophin in normal muscle and of the means by which a lack of this protein leads to the development of the dystrophic process. The aim of this review is to examine critically the relevance of these studies to the pathophysiology of D M D and the conclusions which can be drawn from them.

Dystrophin is found in skeletal, cardiac and smooth muscle was well as brain [5] and proteins from the small C-terminal transcripts of the same gene are found in other tissues [6]. Immunohistological staining using antibodies raised against dystrophin, located the protein on the cytoplasmic side of the plasma membrane [7, 8]. Karpati and Carpenter [5] initially proposed that dystrophin was orientated with its N terminal attached to actin of the cytoskeleton, and the C terminal anchored through the plasma membrane. Research groups in the U.S.A. [9] and Japan [10] have proposed that dystrophin is a cytoplasmic protein which exists in association with a large, plasma membrane spanning, oligomeric glycoprotein complex, which is now thought to be attached to the merosin (laminin M) component of the muscle extracellular matrix [11], although the precise molecular organisation of the glycoprotein-dystrophin complex remains unclear [9, 12]. Lack of dystrophin appears to lead to the loss of the associated proteins in mdx mouse and D M D muscle [13, 14]. It has been suggested that it may be the loss of one or more of these associated proteins, or the whole

*Author to whom correspondence should be addressed.

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complex, that leads to the degeneration seen in dystrophin-deficient muscle since the specific loss of one of the associated glycoproteins in the presence of apparently normal expression and localisation of dystrophin, results in a myopathy similar to DMD [15, 16]. Furthermore, recent data also indicates that patients with congenital muscular dystrophy have a specific reduction in merosin in muscle extracellular matrix [17], further supporting the idea that defects in various parts of this protein/glycoprotein structure lead to myopathy. ANIMAL MODELS OF MUSCULAR DYSTROPHY

Large numbers of animal models of myopathies have previously been reported and used in research without any information about the primary lesion in the individual model. These included the dystrophic mouse, chicken, mink, turkey, dog and sheep [18]. This has made it difficult to relate the results of studies in one species to others, since different primary lesions may produce similar clinical presentations. Thus, these models have been useful in studying general aspects of the process of muscle damage or wasting, but may not have been useful in studying the specific pathological mechanisms in myopathies such as DMD. Recognition of the exact phenotypic relationship between the mdx mouse, XMD dog, the dystrophic cat and patients with D M D has, therefore, facilitated investigation of the processes of muscle degeneration in DMD, but has not been accompanied by a recognition of the highly relevant differences in the time course of expression of the dystrophic process in these models, all lack dystrophin in muscle, but present with very different levels of expression of the disease. It has recently been shown that the dystrophic hamster has a reduced expression of the 50-KD and 35-KDdystrophin-associated glycoproteins [19], supporting the proposal that loss of one, or both, of these associated proteins, rather than dystrophin can lead to the degeneration seen in dystrophic muscle. However, although cardiac muscle fibres of the dystrophic hamster display focal necrosis with calcium deposition, skeletal muscle weakness is less prominent [19]. Several groups have recently indicated that a further animal model (the mutant dy/dy mouse)

may be of relevance in this area. They have reported that these animals have a reduction in merosin in a manner similar to patients with congenital muscular dystrophy [20, 21]. THE M D X MOUSE

The mdx mouse was first described by Bulfield et al. in 1984 [22] in a colony of C57BL/10 Scn/Scn mice. The genetic defect is a point mutation introducing a premature stop codon into an exon in the gene coding for dystrophin [23]. This results in the absence of dystrophin in mdx mouse muscle [2]. A relative large number of experimental studies have utilised the mdx mouse, but few have acknowledged that these mice appear to have a multi-staged disorder (Fig. 1A). In the immediate postnatal period, muscle appears histologically normal, but an acute phase of muscle necrosis occurs at three to four weeks of age and is followed by apparent stability of the myopathy with increasing age [24]. Skeletal muscle recovery is seen after this period of necrosis. This recovery can be explained, in part, by the occurrence of substantial muscle regeneration in mice of 30-40 days old [24], at which age muscle protein turnover was also elevated [25]. It has been reported that occasional regenerating fibres are found in muscles of mice of 120-270 days old although other workers claim that skeletal muscle have completed regeneration by three to four months [24, 26]. This apparent stabilisation of the muscle in older mice is associated with hypertrophy of the limb muscles and with some evidence of ongoing dystrophic degeneration. Although few fibres show necrosis at this age, the serum activity of creatine kinase remains grossly elevated (Fig. IB) and accumulation of extracellular 45Ca is elevated (Fig. 1C), indicating chronic degenerative changes in the muscle fibre membrane [27]. Muscles of marx mice from 15 months old are then reported to atrophy progressively, and some fibrosis is evident [28]. The apparent ability of mdx muscle to compensate for chronic degeneration by increasing regenerative activity may have important implications for therapy in patients with D M D and BMD. Very little is known about the mechanisms responsible for initiating muscle regeneration in normal muscle and further elucidation of these pathways may provide an understanding of how the mdx

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mouse can initiate and maintain muscle bulk for a significant period of their lifespan, in comparison with patients with D M D who show a progressive reduction in muscle mass. Comparison of the relative regenerative abilities of m d x mouse muscle and D M D muscle is complicated by the differences in lifespan of the two species although some studies have suggested that murine muscle has a greater regenerative capacity [29]. In contrast to the pattern of the disease seen in the limb muscles, Stedman et al. [30] have reported that after 25 days of age, the mdx mouse diaphragm exhibits a pattern of degeneration, loss of tissue compliance, loss of regenerative capacity and increased fibrosis similar to that seen in patients with DMD. The mdx diaphragm also shows a reduction in maximal force production, elasticity and twitch speed and the collagen density is significantly higher than in control muscle.

There is an apparent contradiction between the information on disease activity obtained by histological examination of muscles from older m d x mice and that provided by serum CK activity and muscle calcium uptake (Fig. 1), i.e. although there is little histological evidence of muscle degeneration at six months of age, serum CK activities are not significantly reduced in comparison to those at 21 days of age when substantial numbers of necrotic fibres are seen. Elevated release of cytosolic proteins such as CK from muscle is generally assumed to be associated with a loss of membrane viability and to be a stage in the progression of cells to necrosis [31]. Clearly this is not true in the older m d x mouse, these animals appear to be able to maintain relatively normal muscle function for a significant period of time in the presence of apparent chronic loss of cytosolic components. It is therefore clear that there is considerable heterogeneity in the time course and extent of

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expression of the disease process in individual muscles in the mdx mouse (Fig. 1), however, this has not been considered in many of the investigations which have used this model to examine potential pathogenic mechanisms. PROPOSED FUNCTIONS OF DYSTROPHIN

The precise function of dystrophin remains to be elucidated, although various theories have been proposed concerning the function of the protein in skeletal muscle. There is considerable overlap between the theories and various authors have attempted to produce integrated hypotheses. However, for convenience we will consider the three main theories separately. 1. The mechanical damage hypothesis

Following the identification of the genetic and phenotypic defect responsible for DMD, Karpati and Carpenter [5] have adapted the initial Mechanical Damage Hypothesis of Edwards et al. [32] to propose a possible function for dystrophin. Based on the structure and location of dystrophin within the muscle fibres, Karpati and Carpenter [5] proposed that dystrophin provides mechanical stability to the muscle plasma membrane against the substantial stresses placed on it during normal muscle contraction. 2. The leaky membrane theory

Prior to the identification of the genetic defect responsible for DMD, Rowland [33] proposed that the muscle damage observed was due to a defective/absent membrane protein which resulted in abnormal function of the muscle surface membrane. This has since been supported by Hoffman and Gorospe [34], who have proposed that a lack of dystrophin results in muscle plasma membrane instability and increased permeability with a resultant chronic myofiber leakage. 3. The calcium theory

Various workers have provided evidence for a role of dystrophin in preventing loss of muscle calcium homeostasis which possibly results from abnormalities in specific and novel ion channels in dystrophic tissue [35-37].

EXAMINATION OF THESE THEORIES USING THE M D X MOUSE MODEL

1. The mechanical damage theory

The theoretical role of dystrophin in mechanical stability proposed by Karpati and Carpenter [5] was supported by experimental work from the same group [38]. These authors argued that the use of muscles in lengthening (eccentric) contractions was particularly damaging to normal muscle by increasing the mechanical stresses on the muscle fibre [39] and that dystrophin-deficient muscle would be more susceptible to this form of damage. Weller et al. [38] reported an increased susceptibility of anterior tibialis (AT) muscles form 100-day-old mdx mice to lengthening contractions in vivo. Recent loss of viability of msucle fibres was assessed by positive staining of individual fibres for IgG. This is a relatively novel indication of muscle damage and it is unclear whether demonstration of IgG in muscle provides a valid quantitative indicator of recent muscle damage in this situation, since the authors do not report relative circulating levels of IgG in mdx and control mice. More fundamentally it will be clear from previous comments that muscles from mice of the age studies by this group show signs of on-going degeneration and deformed fibres [24, 40]. Thus, the increased loss of viability observed with eccentric contractions may only be occurring in fibres which are already compromised by the underlying degenerative process. Head et al. [40] have shown that between 5 and 40% of fibres in muscles from mdx mice of this age are abnormal and suggest that it is these fibres that have an increased susceptibility to eccentric contraction-induced damage. Recent data examining the effect of eccentric contractions on procion orange exclusion by dystrphic muscle fibres supports the studies of Weller et al. [38]. Petrof et al. [41] demonstrated that fibres from 90-100-day-old mdx mice exhibited an increased susceptibility to contraction-induced sarcolemmal rupture. A specific susceptibility of extnesor digitorum longus (EDL) muscles from adult mdx mice to eccentric activity-induced damage was claimed by Moens et al. [42], although soleus msucles subjected to eccentric activity and either soleus or EDL muscles subjected to isometric activity displayed force losses and accumulation of procion red comparable with control muscles.

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Fig. 3. Uptake of external calcium by resting isolated EDL muscles of mdx ([3) and control ((3) mice and musdes of mdx (1) and control (@) mice following repetitive isometric contractile activ/ty. Data derived from McArdle et al., 1992 [48]. Calcium uptake was measured by following the accumulation of extracellular 45Ca. * P < 0.05.

However, the mice studied by both Petrof et al. [40] and Moens et al. [42] would have muscle swhich had undergone previous periods of degeneration and regeneration and the changes may represent a precipitation of losss of viability in already degenerating cells or an increased susceptibility of regenerated but deformed cells to contraction induced damage, rather tyhan a true increased susceptibility of all dystrophindeficient fibres to this form of damage. Directly contradictory data were presented by Sacco et al. [43] who studied an alternative measure of damage, the loss of force generation, by muscles from 16-26-week-old mdx mice subjected to repetitive eccentric contractile activity in vivo. They observed no significant difference between m d x and age-matched control muscles.

449

The work of Menke and J0ckusch [44] appeared to support the proposed function of dystrophin in maintaining mechanical stability of the muscle membrane. These workers used enzymatically isolated muscle fibres from 50-500-day-old m d x mice, and cultured myotubes to study the susceptibility of dystrophin sufficient and deficient fibres to hypo-osmotic shock. Results indicated that isolated fibres and myotubes from dystrophindeficient mdx mice were more susceptible to hypo-osmotic shock. Parallel studies using regernated fibres from control muscles revealed that the increased susceptibility of m d x fibres was not due to the presence of regenerating fibres in the model. However, the system may lack physiological relevance [45], since osmotic stress not only cuases mechanical damage, but alters ion fluxes in intact muscles and thus the authors were unable to propose a mechanism by which the damage might occur. An alternative experiment to determine the ability of the muscles plasma membrane to withstand sectional forces applied through a patch clamp pipette does not support these data. This study demonstrated no difference between isolated muscle fibres, or membrane vesicles, from m d x and control mice [46]. Work by our group [47, 48] was also not supportive of the mechanical damage hypothesis in that we could not demonstrate an increased susceptibility of isolated (EDL) muscles from 40-day-old m d x mice to damage cuased by both isometric (Figs 2 and 3) and eccentric activity in vitro in comparison with muscles from control animals. Two alternative indicators of damage to muscles were examined for these studies; the release of intracellular creatine kinase (Fig. 2) and accumulation of extracellular 45Ca (Fig. 3). These results did not appear to be complicated by the presence of regenerating fibres in the m d x muscles since control studies with regenerating muscle in the same system under the same conditions demonstrated a similar susceptibility to contractioninduced damage [49]. Work from other species is also relevant although also inconclusive in indicating the relative susceptibility of dystrophin-deficient muscle to mechanical damage. Studies of the effect of eccentric exercise on patients with DMD do not indicate an increased susceptibility to this form of damage [50] in contrast to the theoretical calculations undertaken by

450

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Edwards et al. [32]. However, it is also clear malities in maintenance of intracellular compothat the XMD dog shows substnatial increased sition. Subsequently, with knowledge of the susceptibility to exercise-induced muscle genetic defect in DMD, Hoffman and Gorospe damage [51]. [34] have suggested that dystrophin deficiency It is therefore apparent that there is consider- leads to increased permeability of the plasma able discrepancy between the results of studies membrane (possibly through transient tears), to examine the possibility of dystrophin provid- allowing leakage of intracellular contents which ing mechanical stability to the muscle plasma is manifested by elevated serum CK activity. membrane. An almost universal problem with These authors suggested that if CK can leak all of the experiments cited in this section is the out of fibres, calcium must be able to leak in age of mice studied. Most workers appear to down its very large concentration gradient and have assumed that mdx muscle is essentially when this calcium influx exceeds the calcium normal muscle, lacking dystrophin. However, regulatory capacity of the cell, necrosis ensues. changes such as the persistent abnormalities in Evidence for this comes from the elevated levels muscle permeability (Fig. 1) and the structural of serum CK activity in all animal models of deformities elegantly demonstrated by Head et the disorder as well as patients with DMD [34], al. [40] are likely to influence the data obtained. and elevated levels of both free intracellular in addition, the optimal experimental design to and total cellular calcium found in dystrophic study the effects of eccentric contractions in fibres from mdx mice, dystrophic cats and mice is not immediately apparent. If a physio- Duchenne patients [4, 35, 37, 52, 53]. Cardiac logical mechanical stress is the initiator of muscle in the dystrophic dog and cat also damage to mdx muscle in vivo, then studies shows deposition of calcium [4, 51]. should be aimed at experimental reproduction It is conceptually difficult to evisage how any of similar stresses. Authors do not appear to cell could remain viable with the type of defect have attempted this type of physiological suggested by Hoffman and Gorospe [34] unless approach. the inability to maintain intracellular:extracelIt can also be argued that the multiple stages lular ion gradients were only disturbed to a of degeneration in the mdx mouse do not very minor extent. It has been adequately support a fundamental role for dystrophin in demonstrated that any cell unable to maintain providing mechanical stability to the muscle an adequate calcium potential will rapidly lose fibre. Thus the rapid onset of degeneration in viability [54-56] and hence an inability to mainthis model at 14-21 days old does not appear to tain the calcium potential in all cells (as enbe related to a sudden increase in activity by visaged by Hoffman and Gorospe) would lead the animals or an increase in grwoth rate, but to a more or less simultaneous loss of cell argues more for the involvement of develop- viability, but this does not occur. However, mental factors in the degenerative process. Also these authors suggest that the myofibre leakage the relatively severe pattern of degeneration in does not necessarily lead to myofibre death, but the diaphragm compared to limb muscles of the that the muscle fibre has a threshold below mouse would be unlikely according to the orig- which it can sustain the leakage, yet survive. inal mechanical damage hypothesis of Edwards There is also evidence from studies of normal et al. [32]. muscle that the process of release of CK from muscle may not occur by simple leakage. 2. The leaky membrane theory Muscles retaining normal high energy phosA related theory which has been proposed for phate content can release CK [57] and the the function of dystrophin is the membrane, or process appears to be stimulated and controlled leaky membrane theory. The original theory by an elevation of cytosolic calcium [48, 56, 58]. Relatively few investigations have directly pre-dates the recognition of dystrophin since, in the absence of knowledge of the genetic defect, examined the possibility of increased chronic Rowland [33] proposed the membrane theory myofibre leakage as a consequence of a lack of in an attempt to explain the mechanism by dystrophin, but the pattern of serum musclewhich muscle damage was occurring in DMD. derived enzyme activity in the mdx mouse Rowland envisaged that the genetic defect argues against this possibility. Thus, although resulted in the absence or abnormal function of adult mdx mice have elevated serum levels of a surface membrane protein, resulting in abnor- CK activity (Fig. 1B), pre-necrotic mdx mice

451

How does Dystrophin Deficiency lead to Muscle Degeneration? Table 1. Calcium content of dyltrophic muscle.

Total calcium

Mmw.leexamined ~ . d S n ~ , 197S [6o] Mmmder-Sewry et al. 1980 [62] Bmmbali #t a/. 1980 [63] Bartoriai eta/. 1982 [521 J~,kmn et ai. 1985 [53] Mongini el al. 1988 [37] Turn~ et al. 1988. 1991 [35. 67] Fong eta/. 1990 [64] Me,Ardiz et am/.1992 [48] Rivet-Bagide eta/. 1993 [71] Sod~nar

1993 [68]

P r m m ~ r et al. 1993 [70] I~ge ¢ta/. 1994 [69]

D M D biopaim D M D biopsies Pre.n¢crotic: fetal D M D muscle D M D biopsies D M D l~opsles M y o t u b c s collamsl from D M D muscle Isolated malk m o u s e muscle fibres M y o t u b e s cultured from n u ~ m o u s e and D M D muscle mdx morn EDL mm~les

Myobla~ cultured from D M D

muscle

--

Myotubuc o l m r e d from D M D muscle lmhted mdzm o u s e mugzl¢ fibres

Myotubes cultured from mdx and D M D Isolated mdx mouse m u ~ l e fibres

(14 days old) have normal levels of serum CK and PK activity (Fig. 1B) [27]. In addition, muscles from 40-day-old [27] (Fig. 4) and 90-110-day-old [41] mdx mice show some fibres which have increased permeability to the vital stain--procion orange (Mr 631) on incubation in vitro, but, even in the 40-day-old marx mouse, approximately 85% of these fibres remain impermeable to the stain. It is also relevant that in isolated muscles from both pre-necrotic mdx and control mice (14 days old), only a very small number of fibres accumulate procion orange [27]. Thus, it is clear that, although all mdx muscle fibres are dystrophin-deficient, all fibres from pre-necrotic mdx mice and at least 85% of fibres in the 40-day-old mdx mice show no evidence of an enhanced permeability. Studies which we have undertaken into the influx of external calcium into mdx muscle also argue against a chronic membrane leakage in dystrophin-deficient muscle. These will be considered in more detail in the next section, but essentially they demonstrated no elevation in calcium influx into mdx fibres in vitro [48]

I n ~ laomamd Inea'cued Increased Increased ---Increased

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and no increase in uptake of external calcium by muscles of the 14-day-old (pre-necrotic) mdx mouse in vivo although once the degenerative process has commenced, elevated uptake of external calcium was observed in vivo [27]. In summary, it appears that dystrophin-deficient mdx muscle has no inherent increased permeability to external calcium or cytosolic proteins, but that once degeneration has been initiated both influx of external calcium and release of cytoplasmic proteins occurs. However, this cannot be viewed as a primary effect of the lack of dystrophin although the influx of calcium may propagate the ensuing degenerative process in dystrophic muscle fibres. 3. The calcium theory

Abnormal calcium accumulation has been implicated for some time in the degenerative processes seen in D M D and BMD [59, 60]. Although the observed elevation in intracellular calcium may be secondary to the basic genetic,

Fig. 4. Transverse sectionsof EDL musclesfrom 40--day-old md.'¢and control mice following incubation in the vital stain, procion orange.

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and resulting biochemical, defect in muscular dystrophy, it could account for many of the degenerative features of the disease. Due to the well documented elevation in total muscle calcium levels in DMD (see Table 1), it was previously proposed that calcium channel blockers may have beneficial effects in these patients. Emery and Skinner [61] used verapamil, and although results suggested that the drug may be of benefit, this was outweighed by its cardiac-related side effects. A large number of studies have reported elevated levels of free and total calcium in dystrophic muscle (Table 1). A significant homology has been reported between dystrophin and a calcium regulating protein, Troponin I [65]. Abnormalities in ion channels regulating cellular calcium uptake by dystrophic muscle have also been reported. Franco and Lansman [36] have provided evidence for abnormalities in stretch-inactivated ion channels in mdx mouse myotubes although the same group have shown that these channels are absent in mature dystrophic mdx mouse muscle [66] and their relevance to the pathophysiology of mdx mice is not apparent. Free intracellular calcium levels were initially reported to be increased in dystrophin-deficient muscle fibres from l--4-month-old mdx mice by Turner and co-workers [35] and in cultures of Duchenne muscle by Mongini et al. [37]. Turner et al. [67] subsequently reported that the calcium leak channel opening probability was significantly higher in mdx myotubes and that agents which increased the activity of these channels caused elevations in resting free intracellular calcium levels. Thus, they proposed that an increase in leak channel activity in dystrophic muscle could account for the elevated free intracellular calcium levels they had previously observed in dystrophindeficient mdx muscle [67]. In contrast, other groups have subsequently reported normal levels of free intracellular calcium in viable, intact, isolated muscle fibres from three- to nine-week-old marx mice [68, 69] and Pressmar et al. [70] found normal resting levels of free intracellular calcium in myotubes cultured from human control, Duchenne, mdx and control mouse muscle satellite cells. Our group has examined the free intracellular calcium content of isolated fibres from muscles of 60-day-old mdx and control mice and have observed that morphologically normal fibres from both mdx

Table 2, Possible developmental changes intiueadng precipitation of damase in the mdx mouse. 1. 2. 3. 4.

5. 6. 7.

Dowaregulation of u~ophin Endocrine maturation e.g. Thyroid hormone Growth hormones Intrigued mobility Weaning Inct~__a~_ 8rov~h rate Maturation of mmcle Increased size of muscle fibrin

and control muscles had an equivalent intracellular calcium content [69]. However, both control and mdx fibres showing evidence of degeneration had an elevated free intracellular calcium content. The balance of the data therefore appears to indicate that the free intracellular calcium content of morphologically normal mdx muscle fibres is unchanged compared with control fibres. The apparent discrepancies between the studies of Turner et al. [35, 67] and other workers is therefore likely to be explained by the degenerative state of the fibres examined by the different groups. Again, examination of fibres from pre-necrotic mdx may help to clarify the role of calcium in the muscle degeneration observed. The results of Rivet-Bastide et al. [71] are difficult to interpret since they reported a greater than doubling of the free intracellular calcium concentrations in myoblasts isolated from DMD muscle, i.e. in cells prior to the developmental expression of dystrophin. However, they demonstrated no significant difference between DMD and control myotubes. A similar pattern was seen in myoblasts cultured from patients with facioscapulohumeral (FSH) muscular dystrophy. Our group has also demonstrated no increased accumulation of extracellular calcium by either resting mdx mouse muscles or mdx muscles subjected to isometric contractile activity in vitro compared with control muscles [48]. However, in an alternative approach, we have also measured the flux of 45Ca into both postnecrotic (40-day-old) and pre-necrotic (14-dayold) muscles of mdx mice in vivo [27]. These studies demonstrated that although a range of muscles of 40-day-old mdx mice had an abnormally high level of 45Ca accumulation, this appeared to be secondary to the initiation of the dystrophic degeneration, since muscles from 14-day-old mdx mice accumulated normal levels of 45Ca (Fig. 1C).

How does DystrophinDeficiencylead to MuscleDegeneration? Thus, abnormalities in calcium handling reported in dystrophic muscle do not appear to be an inherent property of dystrophic muscle, although once degeneration is initiated, resulting in increased membrane permeability, calcium will accumulate within the cell and the well known pathological effects of abnormal calcium fluxes may mediate some aspects of the degenerative process.

degeneration in the mdx mouse model. The pattern of appearance of the degeneration suggests that some developmental change(s) or factor(s) in mice may initiate the degeneration. The current data suggest that this occurs at 14-21 days old in the C57B1/10 mouse and that this directly or indirectly triggers the phase of myodegeneration and increased permeability of the plasma membrane observed in the mdx mouse. A coordinated pathophysiological response to these putative developmental factors may also explain the grouped fibre necrosis which is seen in mdx muscle [34]. Table 2 shows a number of possible changes during the post-natal development of the mouse which might be involved in the precipitation of the degeneration in mdx animals. One of the possible factors, utrophin, appears to be most abundant during embryonic and early post-natal development in the C57B1/10 mouse [72, 73]. Utrophin is a similar size to dystrophin, with substantial sequence homology [74]. Expression of utrophin is down regulated when the mouse is approximately two weeks old, i.e. at about the time of initiation of the nectroic phase in the mdx mouse, it has been proposed that utrophin may compensate for dystrophin by anchoring the associated

CONCLUSIONS It is difficult to differentiate primary pathogenic from secondary events in chronic degenerative disorders, but we have assumed that a comparative examination of the reported abnormalities with the time course of development of the disorder in mdx mice will allow us to begin to differentiate these processes. Studies of the mechanisms of myodegeneration in the mdx mouse do not provide universal support for any of the three main theories concerning the function of dystrophin in normal muscle and the manner in which its absence leads to muscle degeneration, but it is apparent that dystrophin-deficiency and the concomitant loss of the membrane glycoprotein complex alone do not appear sufficient to precipitate myolack of dystrophin

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spanning glycoproteins to the plasma membrane since the protein co-purifies with the dystrophin-associated complex or an antigenically similar complex [75]. However, isolated plasma membrane from l-week-old mdx mice has a large reduction in the dystrophin-associated glycoprotein complex, comparable to the reduction seen in adult muscle [76], even though utrophin expression is abnormally abundant at this age [73]. Thus, utrophin could not be protecting the mdx mouse muscle against damage via this mechanism. A similar unidentified factor may be responsible for the rapid normalisation of certain aspects of dystrophic degeneration in vitro compared with in vivo [27, 47, 48, 77, 78] where abnormalities observed in vivo are rapidly lost

4.

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