Dystrophin deficiency, altered cell signalling and fibre hypertrophy

Neuromusc. Disord..Vol. 4, No. 4, pp. 30%315, 1994 Copyright © ElsevierScienceLtd Printed in Great Britain. All rights reserved 0960-8966/94 $7.00+ .00

Pergamon

960-8966(93)E0011-I REVIEW DYSTROPHIN

DEFICIENCY, FIBRE

ARTICLE

ALTERED

CELL

SIGNALLING

AND

HYPERTROPHY

O. HARDIMAN D e p a r t m e n t o f H u m a n A n a t o m y a n d Physiology, U n i v e r s i t y College D u b l i n , E a r l s f o r t Terrace, D u b l i n 2, I r e l a n d

(Received 24 June 1993, revised 4 October 1993, accepted 18 November 1993)

Abstraet--Dystrophin is a subsarcolemmal protein which is defective in Duchenne and Becker muscular dystrophy (DMD/BMD), and in three animal models. Clinical manifestations of dystrophin deficiency in humans range from a mild calf muscle hypertrophy with cramps to the classical progressive degenerative hypertrophic myopathy of Duchenne. A common feature in the clinical presentation of dystrophin deficiency in humans and in the three documented animal models is the presence of muscle fibre hypertrophy. This paper explores the hypothesis that membrane-bound signalling processes are disrupted in the absence of dystrophin, and suggests that these abnormalities may contribute to both the hypertrophic and degenerative changes of dystrophin deficiency.

Key words: Duchenne muscular dystrophy, dystrophin, muscle hypertrophy, cell signalling.

CLINICAL SPECTRUM

OF DYSTROPHIN

DEFICIENCY

The identification of dystrophin as the gene product of the D M D / B M D alleles has provoked a minor revolution in the classification of muscle disease in humans and in animals [1]. Although a large percentage of dystrophin-deficient individuals present clinically with "classical" Duchenne/Becker phenotypes [2], an increasing number of clinical variations are being recognized, including a syndrome of cramps and myoglobinuria [3-5], isolated quadriceps myopathy [6], subgroups of limb girdle muscular dystrophy [7-9], and a condition clinically resembling spinal muscle atrophy [10, 11]. Some of the more coommonly studied animal models, such as the dy/dy mouse, the dystrophic chick, and the dystrophic hamster, express normal levels of dystrophin, and thus cannot serve as reliable models of the dystrophin-deficient state [12-14]. Three animal models of dystrophin deficiency have been characterized: mdx mouse [15, 16], canine X-linked muscular dystrophy [17], and hypertrophic feline muscular dystrophy [18]. Of these, canine muscular dystrophy is clincially closest to D M D [17, 19]. However, for reasons of 305

availability, investigations of dystrophin function in humans has relied heavily on the mdx mouse model [15, 16]. Common features shared by all affected species include the absence or abnormality of dystrophin, the presence of clinical hypertrophy, histological evidence of individual fibre hypertrophy, myopathic features on muscle biopsy, and the presence of very high serum levels of muscle-specific enzymes [2, 13, 15-18]. (Elevations in serum muscle-specific enzymes are not exclusive to dystrophin deficiency; other conditions as diverse as denervation and inflammation can also induce enzyme leakage from muscle [20]. Therefore, this finding itself does not localize the pathological process to the membrane, as it may merely reflect the ongoing process of degeneration/regeneration.) Significant differences exist between species in the clinical severity of the disease. For example, mdx mice undergo a "crisis" of fibre necrosis at 4-6 weeks, from which they recover, and subsequently have a clinical course and life expectancy similar to normal mice. This crisis occurs in all mice, and may involve necrosis of hundreds of adjacent fibres. The pathogenesis and reasons for the specific timing of this process are not well understood at present [13, 15, 16], but may relate to down-regulation of the

306

O. HARDIMAN

Table 1. Clinical and pathological features of the c o m m o n forms of dystrophin deficiency in h u m a n s and animal models Human

Dog

Cat

Mouse

X-linked

X-linked

X-linked

X-linked

Yes Yes

Yes Yes

Yes Minimal

Progression Muscle Hypertrophy

Yes Yes

Yes Yes

No Yes (marked)

Yes No (except during "crisis") No Yes

Biopsy Necrosis Fibrosis Calcinosis Cardiomyopathy

Yes Yes No Yes

Yes Yes Yes Yes

Yes No Yes (marked) Yes

Yes No Yes Yes

Genetic Clinical Elevated CK Weakness

expression of dystrophin-related protein at the onset of the crisis [21]. Themuscles of "recovered" mdx mice are hypertrophied, both clinically and by morphometric analysis of individual fibres [15]. Cats with hypertrophic feline muscular dystrophy have presented with an unusual distribution of clinical muscle hypertrophy which may be fatal [18]. These animals do not appear to exhibit any significant clinical weakness. However, the range of presentations may be limited by the small number of cats that have come to clinical attention [18, 22]. The canine form of the condition is characterized by progressive disease with weakness, muscle atrophy and extensive fibrosis, similar to Duchenne muscular dystrophy in humans [2, 17]. Fibroblast proliferation is not a marked feature of either the cat or the mouse models (although extensive fibrosis has been described in the mdx diaphragm [23]), whereas it is a significant feature of the disease in dogs and humans. The reasons for this difference across species are also poorly understood. Comparing the clinical presentations of disease across species (Table 1) it is clear that muscle hypertrophy is a common feature. This suggests that hypertrophy represents an integral part of the disease, and is either caused by, or is intimately associated with the functional abnormality of dystrophin. Classical calf hypertrophy in Duchenne and Becker muscular dystrophy has been attributed in the past to a "pseudohypertrophy'" associated with extensive endomysial fibrosis [24]. However, it is clear, both from the animal models and the analysis of biopsies from fetal and infantile dystrophin-deficient muscles, that the enlargement which characterizes the early course of these diseases is due to true fibre hypertrophy (Fig. 1). [13, 25]. This hypertrophy cannot be attributed entirely to a compensatory

"work" response, as in animal models tt occurs in muscles where there is little or no fibre loss or weakness, as is evident by the involvement of tongue and jaw muscles in hypertrophic feline muscular dystrophy [18, 22]. Furthermore, in humans, it is noteworthy that the "atypical" reported cases of dystrophin deficiency have also been associated with at least some degree of clinical muscle hypertrophy, usually involving the calf muscles (Table 2). The significance of muscle fibre hypertrophy is unclear in the genesis of fibre necrosis. It has been suggested that a critical myofibre size or load exists, beyond which muscle fibre viability is reduced in the absence of dystrophin [26]. The presence of excessively enlarged fibres may, thus, contribute to the genesis of necrosis and wasting in the larger species (i.e. dogs and humans). The correlation between myofibre size and necrosis may also reflect in part the ability of dystrophinlike cytoskeletal proteins (e.g. DRP or "utrophin") [27] to substitute for normal dystrophin in small but not large fibres. The degree of fibre enlargement may, therefore, contribute to the species differences, and produce a spectrum of clinical phenotypes ranging from the mildest in mdx mouse, to the most severe in humans [28]. Although this hypothesis is attractive, it does not strictly apply in the dog [17], nor does it account for all of the variations in the severity of the disease in humans, particularly the rare forms of dystrophin deficiency with atypical or minimal weakness [3--11]. It is perhaps more likely that the fibre hypertrophy represents a clue to the direct cellular effects of dystrophin deficiency, which may also lead ultimately to fibre necrosis. This review will attempt to correlate the prevailing theories regarding the dysfunction of dystrophin with reports of alterations in signalling processes in dystrophic muscle with a view to

Cell Signalling in DMD

307

Fig. 1. Biopsy from 2-yr-old patient with Duchenne muscular distrophy. Note the enlarged eosinophilic fibres.

Table 2. Reported clinical features of atypical dystrophin deficiency in humans

Gold et al. [4] (4 affected males in 2 families) McDonald et al. [10] (1 affected male) Sunohara et al. [6] (5 affected males in 3 families) Gospe et aL [3] (9 affected in 1 family) Thakker and Sharma [5] (1 atypical male: maternal uncle with typical BMD) Lunt et al. [11] (4 affected males with features of spinal muscle atrophy) Norman et aL [9] (4 affected males with features of limb girdle muscular dystrophy)

Distribution

Weakness

Hypertrophy

Progression

Cardiomyopathy (2 patients) Limb myalgias Pelvic girdle

None

Yes

No

Mild

Yes

No

Quadriceps only

Mild

Yes

No

Limb myalgias

None

Yes

No

Limb myalgia

None

Yes

No

Limb girdle

Yes

Yes

Yes

Limb girdle

Yes

Yes

Yes

u n d e r s t a n d i n g the fibre h y p e r t r o p h y o f d y s t r o p h i n deficiency. It p r o p o s e s t h a t the presence o f muscle fibre h y p e r t r o p h y in d y s t r o p h i n deficiency m a y represent a key feature in u n d e r standing some o f the n o r m a l functions o f d y s t r o p h i n [21], a n d suggests that the absence o f

d y s t r o p h i n m a y result in specific signalling defects at the s a r c o l e m m a l m e m b r a n e . This in turn m a y lead to muscle fibre h y p e r t r o p h y , a n d c o n t r i b u t e further to the cycles o f necrosis a n d regeneration that characterize classical D u c h e n n e a n d Becker m u s c u l a r d y s t r o p h y in humans.

308

O. HARDIMAN

MEMBRANE BOUND COMPLEX RECEPTORS

RECEPTOR COMPLEX

ALPHA RECEPTOR

BETA

a l t e r e d sensitivity/activity?

4~ DYSTROPHIN DEFICIENCY MEMBRANE

BOUND

G-PROTEINS:

RECEPTOR-EFFECTOR

COUPLING

a l t e r e d activity?

4,

I

4,

PHOSPHOLIPASE C

i

A D E N Y L Y L CYCLASE

I

CALCIUM FLUX

PHOSPHO-INOSITIDES; DAG

• • m•

~

.

[SARCOPLASMIC RETICULUM

I

.

.

.

.

.

.

.

.

.

.

.

i

"• t

~

t.J I N T R"A C E L L U L A R CALCIUM i

MITOCHONDRION

•

•

•"

•

..

II

CALMODULINS

,

PROTEIN

KINASES

.

IGFs MYOGENIC REGULATORS

PROTEASES

LIPID-OXIDATION

1 NECROSIS]

~

• • • • • • • -•

IHYPERTROPHYI

Fig. 2. Schematic diagram of possible signalling abnormalities in dystrophin deficiency.

Cell Signallingin DMD DYSTROPHIN DEFICIENCY AND MUSCLE DEGENERATION

Mechanical hypothesis

The mechanical hypothesis (reviewed by Hutter [29]) is based on the following observations: (a) dystrophin is structurally similar to other known cytoskeletal proteins [21, 30]; (b) it is present at the surface membrane of muscle [31 ]; and (c) it is tightly bound to membraneassociated glycoproteins which interact with the extracellular matrix [21, 32]. This hypothesis, which predicts that the deficiency of dystrophin results in mechanical "wear and tear" of the sarcolemma, is supported by the observation that the sarcolemmal membrane may be "leaky" to proteins [33], and by the presence on electron microscopy of disruptions ("delta lesions") in the sarcolemmal membrane [34]. It proposes that a deficiency of dystrophin results in reduced membrane folding [35], thus predisposing to increased rupture following the stress of eccentric contraction with consequent shedding of membrane, and to increased osmotic fragility because of a lower ratio of membrane surface to cell volume [29]. Attempts to prove the relationship between dystrophin deficiency, mechanical fragility and the pathophysiology of fibre necrosis in animal models of DMD have been complicated by the limitations in understanding the factors that confer tensile strength on muscle fibres. Experiments using models of eccentric contraction and by measurements of tensile strength of myotubes have been inconclusive in demonstrating significant differences between normal and dystrophindeficient fibres [29]. Similarly, studies indicating an increased osmotic fragility of dystrophic myotubes and isolated fibres await confirmation [29, 36]. Thus, although there is evidence in favour of mechanical fragility of dystrophindeficient muscle, there has been no conclusive evidence to indicate that this is the primary or exclusive mechanism by which DMD fibres undergo degeneration, nor has a pathway been unequivocally identified by which increased membrane fragility leads to fibre necrosis. Calcium influx hypothesis

The calcium influx theory suggests that the absence of dystrophin may interfere with the proper anchoring of integral membrane proteins and thus disrupt their function, leading directly to abnormal accumulations of intracellular free

309

calcium [32, 37, 38]. Evidence cited from in vivo models in favour of this hypothesis would, therefore, indirectly support the "mechanical" hypothesis. Evidence of abnormal accumulations of calcium in vitro would indirectly undermine the mechanical hypothesis, as it would suggest that muscle contraction is not essential to the genesis of the degenerative process. Although there is experimental evidence to suggest direct movement of calcium and other ions into the cytoplasm of dystrophin-deficient tissue [29, 37, 38] the circumstances under which this might occur are unclear. Calcium levels are reproducibly elevated in dystrophin-deficient muscle in vivo [37, 39] leading to an assumption that increased transmembrane calcium flux is the primary event in the degenerative process. If increased calcium flux is the primary event, it should be possible to consistently demonstrate elevated levels of calcium in isolated dystrophindeficient fibres, and in cultured dystrophindeficient myotubes. However, in vitro studies of sodium and calcium levels in intact fibres and cultured myotubes have been conflicting [29]. Dunn et al. [40] showed that free Na ÷ is elevated in the cytoplasm of m d x mice, and have suggested that this may reflect a reduced flux through the Na/K ATPase. However, elevations in free Nai have also been observed in other nondystrophin-related animal models, and may not be a specific effect of dystrophin deficiency [41]. Furthermore, abnormal movements of calcium across the membrane might be expected to alter the resting membrane potential; electrophysiological studies of membrane potential have been normal in muscle from m d x mice [40], and DMD patients [40, 42]. Consistent elevations in intracellular free calcium at rest and following acetylcholine stimulation have been reported in dystrophic myotube cultures [43]. The significance of these findings is unclear in the context of fibre degeneration, as similar elevations of intracellular free calcium have been recorded in intact fibres from patients with malignant hyperthermia in which there was no evidence of ongoing muscle fibre destruction [44, 45]. Furthermore, more recent studies have not confirmed the presence of elevated free intracellular calcium at rest [46-49]. Pressmar et al. reported transient elevations in calcium following osmotic shock [49]. The origin of the increased cytosolic free calcium where it is present remains unclear. Abnormal stretchinactivation calcium channels [50], and increased

310

O. HARDIMAN

activity of calcium leak channels [38] have been reported in cultures of Duchenne and mdx muscle. However, the density and significance of such channels in the pathogenesis of the disease remains unclear [29]. To summarize, accumulations of free intracellular calcium may occur in dystrophin-deficient disease, but this is not a consistent finding, and cannot directly account for the fibre degeneration. The evidence against a direct relationship between dystrophin deficiency, elevated levels of free intracytoplasmic calcium and fibre necrosis is further strengthened by the study of other diseases characterized by elevations in free cytosolic calcium including malignant hyperthermia [44, 45], and Brody's disease (Ca ++ adenosine triphosphatase deficiency [51]), in which ongoing fibre necrosis is not a feature despite consistent reports of elevated levels of free cytosolic calcium. The natural history of dystrophin-deficient diseases differs radically across species [15 19] and the human form can present with a number of different phenotypes [1-11]. Models that predict a primary role for calcium-induced muscle destruction must, therefore, permit the relative stability and subsequent hypertrophy of regenerated (dystrophin-deficient) muscle in the mdx mouse after 4-6 weeks [I 5, 16], the marked muscle hypertrophy with no weakness in cats [I 8], and the relatively benign clincial course that characterizes some of the atypical human phenotypes [1-11]. To date, these issues have not been convincingly addressed by the current hypotheses. Membrane signalling hypothesis

It is proposed that elevations in free cytosolic calcium in non-necrotic dystrophin-deficient fibres represent a complicated perturbation of membrane-associated signalling mechanisms. These signalling abnormalities could then produce an intracellular environment that predisposes to fibre destruction. The structure homologies of dystrophin to spectrin suggest that its primary function is cytoskeletal [21, 30]. Dystrophin is thought to aggregate as a homo-tetramer beneath the sarcolemma [21], and to bind with the extracellular matrix by interacting with transmembrane dystrophin-associated glycoproteins [21, 32]. However, since the discovery of the skeletal muscle form of dystrophin, other isoforms have been identified which lack the amino-terminal and rod-domains [21]. These isoforms are expressed in high levels in non-muscle tissue,

where they may not serve an obvious structural function [52]. Neural, cardiac and smooth muscle isoforms of dystrophin differ from skeletal muscle dystrophin in that they are not uniformly distributed along the membrane [21, 53]. In fact, in cardiac muscle, dystrophin staining with antibodies is absent in the fascia adherens of the intercalated discs, which are force transducing structures [21]. These considerations support the view that dystrophin may also subserve other functions in addition to the provision of mechanical stability. For example, it may function to stabilize membrane-bound proteins, and maintain local concentrations of interdependent membrane components [21, 53]. Skeletal muscle dystrophin contains a calcium-binding site which is thought to be nonfunctional [21]. However, it has been shown recently that dystrophin and two associated glycoproteins also have calmodulin-binding domains, suggesting that these proteins may be directly involved in signal transduction pathways [54]. The biological function(s) of these calmodulin-binding domains, and ramifications for their absence in dystrophin-deficient muscle remain to be elucidated further. A role for dystrophin in modulating signal transduction across the sarcolemmal membrane has not been examined in detail. The recent evidence that different isoforms of dystrophin may have diverse functions [21, 52, 53], may be consistent with previously described derangements in the activity of membrane-bound signalling proteins in DMD and BMD. It may be that dystrophin is required to facilitate transmembrane signalling by providing a subsarcolemmal structure that maintains local concentrations of essential coupling proteins. In 1976, Mawatari et al. reported an elevated basal activity of adenylyl cyclase in both whole muscle and mixed cultures from patients with DMD/ BMD [55]. This abnormality was specific to DMD/BMD. Activity was paradoxically reduced following stimulation with isoprenaline and epinephrine, which act primarily through beta2 and alpha-adrenergic receptors. Activation of the adenylyl cyclase complex by sodium fluroride (which is now known to bind and activate G~ proteins [56]) was much reduced when compared to normals. The nature and possible significance of these abnormalities of adenylyl cyclase activity in the pathophysiology of dystrophin-deficient muscle could not have been appreciated at the time of the study, as the details of receptor-effector coupling and trans-

Cell Signalling in DMD

membrane signalling had not been established [55-60]. In the context of a possible defect in cell signalling, and of a potential mechanism for hypertrophy, the activity of muscle adenylyl cyclase in DMD clearly requires re-investigation both in homogenates of whole muscle and in clonally derived myoblast cultures. This should prove informative in view of the advances in the understanding of the role of membrane-bound G-proteins (including the membrane-associated protein ras) [61] in receptor-mediated signal transduction. Recent findings that isoprenaline enhances myoblast fusion in normal but not in DMD cultures [62] also point to a defect in adenylyl cyclase activity in dystrophin-deficient muscle which requires further characterization. In addition to the putative abnormalities of adenylyl cyclase in DMD, it is proposed that dystrophin-deficient muscle may also exhibit hitherto unrecognized derangements in other signalling pathways as a consequence of defective receptor-effector coupling. Dystrophindeficient human cultures differ from agematched normals in their response to acetylcholine [43], glucocorticoids [63] and growth hormone (Hardiman and Sklar, unpublished observation). Mongini et al. have shown that stimulation of DMD muscle cultures with acetylcholine results in excessive elevations of free intracytoplasmic calcium when compared with normal values [43]. The mechanism by which this might occur remains unclear, but it has been suggested that it may relate to increased sensitivity of the sarcoplasmic reticulum [43]. Abnormal calcium release from the sarcoplasmic reticulum has been described in both DMD and m d x muscle following stimulation of chemically skinned fibres with caffeine [64-65]. It should perhaps be noted that skinning of intact fibres, which disrupt the sarcolemmal membrane, is likely to remove dystrophin and its associated glycoproteins. The process leaves intact the membrane of the sarcoplasmic reticulum, and the transverse tubule-sarcoplasmic reticulum junction [66]. In normal muscle, dystrophin is not present in the transverse tubular system, nor does it localize to the membrane of the sarcoplasmic reticulum [20, 67]. It is present, however, at the junctional t-system [68, 69]. Thus, the sarcoplasmic reticulum sensitivity to caffeine in dystrophin deficiency must be a consequence of a membrane or signalling abnormality located in the junctional t-system. Both the increased intracellular calcium release in response to ACh stimulation described by

311

Mongini et al. [43] in dystrophin-deficient fibres, and the increased sensitivity of the sarcoplasmic reticulum could relate to alterations in the synthesis of phosphoinositides by the membrane-bound enzyme phospholipase C, either as a consequence of abnormal coupling of the enzyme, or because of secondary abnormalities in the phospholipid content of the membrane [70]. IP3 is capable of releasing calcium from the sarcoplasmic reticulum [71], although the physiological significance of this action is unclear. The previously described excessive elevations of free intracellular calcium reported in dystrophin deficiency [37-39, 43, 72] could be interpreted as a generalized alteration in receptor signalling due to changes in G-protein activity and cAMP levels [73]. These changes would affect the activity of voltage-gated calcium channels which are modulated by both Gproteins and cAMP [74]. Further derangements may occur in membrane-bound phosphoinositide metabolism as a result of alterations in phospholipase C coupling [70]. If there is a generalized disruption of membrane signalling in dystrophin deficiency, it is not surprising that reports of elevated levels of intracellular free calcium are conflicting. Such elevations would be a function of the dynamics of signalling across the membrane, and would, thus, vary over time, across fibre types, in relation to receptor activation, and according to the particular metabolic profile of the fibre at the time of examination.

M U S C L E H Y P E R T R O P H Y IN D Y S T R O P H I N

DEFICIENCY

To investigate a possible link between dystrophin deficiency and muscle fibre hypertrophy, the signalling mechanisms associated with fibre hypertrophy must first be examined. Primary myogenesis, which occurs at 9 weeks gestation in humans is recapitulated to some extent in tissue culture. Myoblast differentiation in vitro is regulated by the MyoD family of helix-loophelix proteins [75, 76]. These include MyoD, myogenin, myf5 mrf4, and the inhibitory factor id [75]. These regulatory genes inhibit cell proliferation and have the capacity to activate the complete muscle differentiation programme. The multiplicity of muscle regulatory factors suggests either that there is a considerable redundancy in the system, or that each factor might regulate a distinct subset of genes [75-77].

312

O. HARDIMAN

Differential expression might, thus, underlie the diversity of myogenic cells during development. The relative importance of each member of the MyoD family in the different stages of human myogenesis and muscle regeneration has not been fully determined. Under normal circumstances, dystrophin expression occurs following myoblast fusion [78]. It is, therefore, unlikely that the absence of dystrophin in myoblasts should affect the signalling systems that promote myoblast fusion. If this is the case, the hypertrophy and degeneration associated with dystrophin deficiency is likely to occur after the onset ofmyoblast fusion, at a time when signals promoting skeletal muscle growth and hypertrophy prevail. Myotube formation is associated with a reduction in the expression of growth factor and hormone receptors [75]. However, alterations in circulating levels of hormones, growth factors and drugs (in addition to neural influences) can affect muscle mass in vivo, indicating that normal skeletal muscle fibres are dependent on ongoing trophic influences [77, 79]. The interplay between growth factors, oncogenes and myogenic regulators in postnatal muscle growth and regeneration is unclear. The degree of activation of satellite cells, which contribute to the growth of muscle fibres, varies with the type of stimulus: "normal" postnatal growth is associated with an increase in DNA content as a result of satellite cell recruitment [80, 81]. Treatment with the beta2 adrenergic agonist clenbuterol reportedly induces hypertrophy with minimal satellite cell recruitment [82]. It is not clear whether the fibre enlargement of dystrophin deficiency is due to satellite cell recruitment, or hypertrophy of existing fibres with no increase in the total number of nuclei. Following hypertrophic stimuli, Hesketh et al. have shown that expression of c-myc, fos, and possibly other oncogenes which are normally down-regulated in terminally differentiated muscle, is increased [83]. Stimulation with the betaadrenergic agonist clenbuterol produces a transient increase in the expression of c-myc. However, high levels ofc-myc, fos, jun and ras in vitro have been associated with marked downregulation of the activity of the muscle-specific helix-loop-helix proteins and consequent inhibition of differentiation [83]. The apparent correlation between elevated levels of myc and fibre hypertrophy remains to be elucidated: there is evidence that under particular circumstances,

myc-induced inhibition may be overcome, suggesting that muscle-specific gene expression and myc-induction are not necessarily mutually exclusive [83]. However, it is also possible that the hypertrophic response following stimulation with clenbuterol (and other agents such as the anabolic steroids) is associated with synthesis of autocrine growth factors (e.g. insulin-like growth factors 1 and II) in addition to c-myc. There is evidence that the IGFs may have an obligatory role in activation of the myogenic regulator proteins [84, 85]. In vitro, anti-sense IG-II oligonucleotides block differentiation in the rat cell line L6, and exogenous |GF up-regulates myogenin expression [86]. Clinical conditions characterized by decreased circulating levels of IGF are associated with muscle wasting [87, 88], and elevated levels of IGFs are associated with muscle anabolism [89]. Thus, control of the IGF secretion may be of pivotal importance in determining muscle bulk. It is clear that the cellular mechanisms by which physiological and pharmacological stimuli induce hypertrophy are poorly understood, but may involve similar signalling pathways which occur in primary myogenesis. To date, the effects on the MyoD family of myogenic regulators, and other factors that promote differentiation remains to be determined. A more complete understanding of the cellular processes that lead to physiological and pharmacological hypertrophy is likely to elucidate the mechanism of hypertrophy in dystrophin deficiency. At present, it is possible to draw parallels between the hypertrophic response of normal muscle to beta-adrenergic agonists [90], and the fibre hypertrophy of dystrophin deficiency in terms of alterations in membrane-bound signalling processes. In DMD, the overall up-regulation of basal cAMP (and calcium) could contribute to the muscle hypertrophy by a similar pathway to that induced by activation of the beta2 adrenergic receptor. Whether the activation of cellular oncogenes such as c-myc and c-fos is crucial to fibre hypertrophy remains to be examined in dystrophin-deficient muscle. The signalling pathways leading to increased synthesis of musclespecific protein may also involve autocrine expression of IGF I and II. Elevated levels of IGF I and myogenin mRNA have been identified in some dystrophin-deficient cultures (Sklar, personal communication), and up-regulation of IGF secretion has been proposed as a mechanism of clenbuterol-induced hypertrophy [82].

Cell Signalling in DMD THERAPEUTIC IMPLICATIONS

Glucocorticoid effects

Clinical improvement has been described in DMD patients following prednisone therapy [91]. Although steroids were initially used because of their immunosuppressive effects, the clinical improvement may also relate to direct effects of steroids on myogenesis. Steroids differentially affect the myogenic programme [78]; treatment of human muscle cultures with methylprednisolone can inhibit myoblast fusion and expression of myosin heavy chain, paradoxically, dystrophin expression can be simultaneously augmented. In addition, there is experimental data to indicate that adenylyl cyclase activity in some steroid treated cultures may be down-regulated [92]. Down-regulation may occur by up-regulation of inhibitory G proteins: this effect has also been demonstrated in vivo in cardiac muscle from deoxycorticosteronetreated rats [93], and/or by a direct effect on the catalytic subunit. If adenylyl cyclase activity is a priori abnormal in DMD cultures [55], the modulation of activity of the catalytic subunit by steroids may be contributing to the clinical improvement observed in Duchenne patients treated with steroids. It is also likely that steroids regulate, either directly or indirectly, the musclespecific transcriptional regulators from the MyoD family, as the programme of myogenesis is disrupted in some steroid-treated cultures [63]. Several pathways exist by which glucocorticoids might alter the transcription or action of these regulatory proteins, including the upregulation of id, altered expression of proto-oncogenes, regulation of the autocrine growth factors IGF I and II [94], or up-regulation of inhibitory G proteins (which enhance the expression of the myogenic regulator Myf4) [95]. Thus, in dystrophin deficiency, modulation of such signalling pathways for myogenesis by steroids could further attenuate the putitive pathways leading to fibre hypertrophy and subsequent necrosis. CONCLUSIONS

In summary, this review attempts to directly address the relationship between dystrophin deficiency, altered signalling mechanisms, and fibre hypertrophy and necrosis. It is clear that further experiments are required to confirm the hypothesis that dystrophin deficiency perturbs the normal signalling pathways in muscle. Such studies may clarify the mechanism(s) of fibre

313

hypertrophy and possibly the factors leading to fibre necrosis. If consistent signalling abnormalities can be demonstrated in dystrophin-deficient muscle, they will provide a functional assay of dystrophin deficiency. Furthermore, elucidation of the defects will provide opportunities for the development of new pharmacological strategies targeted at the defective pathways, and aimed at preventing the progressive hypertrophy and necrosis. Acknowledgements--The author is grateful to Professor Dr Reinhardt Rudel, Dr R. M. Sklar, Dr Kay Nolan and Professor R. P. Kernan for their comments on the manuscript, and to Mr Adam May for graphics. The work was supported by the Newman Scholar Fund University College Dublin, and the Health Research Board, Ireland. The author's Newman Scholarship is sponsored by Drug Research Corporation Ireland Ltd. REFERENCES

1. Hoffman E P, Brown R H Jr, Kunkel L M. Dystrophin, the protein product of the DMD locus. Cell 1987; 51: 919 18. 2. Engel A G. Duchenne dystrophy. In: Engel A G, Banker B Q eds. Myology: Basic and Clinical. New York: McGraw-Hill 1986; 1185 1240. 3. Gospe S M Jr, Lazaro R P, Lava N S, Grootscholten P M, Schott M O, Fischbeck K H. Familial X-linked myalgia and cramps: a non-progressive myopathy associated with a deletion in the dystrophin gene. Neurology 1989; 39: 1277-1280. 4. Gold R, Kress W, Meurers B, et al. Becker muscular dystrophy: detection of unusual disease courses by combined approach to dystrophin analysis. Muscle Nerve 1992; 15:241 218. 5. Thakker P B, Sharma A. Becker muscular dystrophy: an unusual presentation. Arch Dis Child 1993; 69:158 159. 6. Sunohara N, Arahata K, Hoffman E P, et al. Quadriceps myopathy: form fruste of BMD. Ann Neurol 1990; 28: 634~39. 7. Aridawa E, Hoffman E P, Kaido M, Nonaka I, Sugita H, Arahata K. The frequency of pateints with dystrophin abnormalities in a limb-girdle patient population. Neurology 1991; 41:1491 1496. 8. Hoffman E P, Fischbeck K H, Brown R H Jr, et al. Dystrophin characterization in muscle biopsies from Duchenne and Becket muscular dystrophy patients. New Engl J M e d 1988; 318:1363 1368. 9. Norman A, Coakely J, Thomas N, Harper P. Distinction of Becker from limb-girdle muscular dystrophy by means of dystrophin cDNA probes. Lancet 1989; 1: 466-468. 10. McDonald T D, Medori R, Younger D S, et al. Becker muscular dystrophy or spinal muscular atrophy?-Dystrophin studies resolve conflicting results of electromyography and muscle biopsy. Neuromuscular Disorders 1991; 1(3): 195 200. 11. Lunt P W, Cumming W J, Kingston H, et al. DNA probes in differential diagnosis of Becker muscular dystrophy and spinal muscular atrophy. Lancet 1989; I: 46-47. 12. Cooper B J. Animal models of Duchenne and Becker muscular dystrophy. Br M e d Bull 1989; 45(3): 703-718. 13. Hoffman E P, Gorospe R M. The animal models of Duchenne muscular dystrophy: windows on the pathophysiological consequences of dystrophin deficiency. Curr Top Membranes 199l, 38:113-155.

314 14. 15.

16.

17.

18.

19.

20.

21. 22.

23.

24.

25. 26.

27.

28.

29. 30.

31. 32. 33. 34.

35.

36.

0. HARDIMAN Kunkel E M, Hoffman E P. Duchenne/Becker muscular dystrophy. Br Med Bull 1989; 45(3): 630 643. Coulton G R, Morgan J E, Partridge T A, Sloper J C. The mdx mouse skeletal muscle myopathy 1. A histological morphometric and biochemical investigation. Neuropathol Appl Neurobiol 1988: 14:53 70. Coulton G R, Curtin M A, Morgan J E, Partridge T. The mdx mouse skeletal muscle myopathy. 2. Contractile properties. Neuropathol Appl Neurobio11988: 14(4): 299 314. Valentine B A, Cooper B J. Canine X-linked muscular dystrophy: selective involvement of muscle in neonatal dogs. Neuromuscular Disorders 1991: I(1): 31 38. Gaschen P F, Hoffman E P, Gorospe J F, et al. Dystrophin deficiency causes lethal muscle hypertrophy in cats. J Neurol Sci 1992; I10:149 159. McCully K, Giger U, Vet M, el al. Canine X-linked muscular dystrophy studied with in vivo phosphorous magnetic resonance spectroscopy. Musch, Nerve 1991: 14:1091 1098. Brook M. In: A Clinician's View of Neuromuscular Diseases. Baltimore: Williams & Wilkins, 1986: 46, 47, 133, 226 228. Ahn A H, Kunkel L M. The structural and functional diversity ofdystrophin. Nature Genet 1993; 3:283 291. Carpenter J L, Hoffman F C A, Romonul F C A, et al. Feline muscular dystrophy with dystrophin deficiency. Am J Pathol 1989; 135:909 919. Stedman H H, Sweeney H L, Shrager J B, et al. The mdx diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy. Nature 1991 ; 352:536 539. Duchenne de Boulonge. Dc la paralysie musculairc pseudohypertrophique ou paralysie myosclerosique. (Extrait des Archives generales de Medecinc.) Asselin, Paris, 1868. Emery A E H. Duehenne Muscular Dystraphy, 2nd Ed. Oxford: Oxford University Press, 1993. Karpati G, Carpenter S. Small caliber skeletal muscle fibres do not suffer deleterious consequences of dystrophin gene expression. Am J Med Genet 1986:25 653 658. Tinsley J M. Blake D J, Roche A, et al. Primary structure of dystrophin-related protein. Nature 1992: 360: 591--593. Matsumura K, Ervasti J M, Ohlendieck K, Kahl S K, Campbell K P. Association of dystrophin-related protein with dystrophin-associated proteins in mdx mouse muscle. Nature 1992; 360:588 590. Hurter O F. The membrane hypothesis of duchcnne Muscular Dystrophy: quest for functional evidence. J lnher Metab Dis 1992; I$: 565-577. Koenig M, Monaco A P, Kunkel L M. The complete sequence ofdystrophin predicts a rod-shaped cytoskeletal protein. Cell 1988:53 219 228. Bonilla E, Samitt C E, Miranda A F, et al. Duchenne Muscular Dystrophy: deficiency of dystrophin at the muscle cell surface. Cel 1988; 54: 447~,52. Ervasti J M, Campbell K P. Membrane organization of the dystrophin glycoprotein complex. Cell 1991; 66: 1121 1131. Bradley W G, Fulthorpe J J. Studies of sarcolemmal integrity in myopathic fibres. Neurology 1978:28 670 677. Morki B, Engel A G. Duchenne Dystrophy: electron microscopic findings pointing to a basic or early abnormality in the plasma membrane of the muscle fiber. Neurology 1975; 25:1111-1120. Delaporte C, Dautraux B, Rouche A, Fardeau M. Changes in surface morphology and basal laminae of cultured cells from Duchenne muscle dystrophy patients. J Neurol Sei 1990; 95:77 88. Menke A, Jockusch H. Decreased osmotic stability of

37. 38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50. 51.

52.

53. 54.

55.

dystrophin-less muscle cells from the mdx mouse. Nature 1991; 349:69 71. Turner P R, Fong P, Denetclaw W F, Steinhardt R A. Increased calcium influx in dystrophic muscle. J Cell Bio11991:115 1701 1712. Fong P, Turner P R, Denetclaw W F, Steinhardt R A. Increased activity of calcium leak channels in myotubes of Duchenne human and mdx mouse origin. Science 1990; 250:673 676. Bodensteiner J B, Engel A G. lntraccllular calcium accumulation in Duchenne dystrophy and other myopathies: a study of 567,000 muscle fibres in 114 biopsies. Neurology 1978: 28: 439M46. Dunn J F, Bannister M, Kemp G J, Publicover S J. Sodium is elevated in mdx muscles: ionic interactions in dystrophic cells. J Neurol Sei 1993; 114 76 80. Fong C N, Atwood H L, Charlton M P. Intracellular sodium-activity at rest and after tetanic stimulation in muscles of normal and dystrophic (dy/dy)C57Bl/6J mice. ILxp Neural 1986; 93:359 368. Rothman S M, Bischoff R. Electrophysiology of Duchenne dystrophy myotubes in tissue culutre. Ann Neurol 1983: 13:176 179. Mongini T, Ghigo D, Doriguzzi C, Bussolino F, et ol. Free cytoplasmic Ca ~' at rest and after cholinergic stimulus is increased m cultured muscle cells from Duchenne muscular dystrophy patients. Neurology 1988: 38: 476~-80. Condrescu M, Lopez J R, Medina P, Alamo L. Deticient function of the sarcoplasmic reticulum in patients susceptible to malignant hyperthermia. Muscle Nerve 1987; 10:238 241. Lopez J R, Sanchez V, Lopez 1, el al. Effects of extracellular magnesium on myoplasmic calcium in malignant hyperthermia-susceptible swine. Anaesthesioh)gy 1990; 73(1): 1(19 117. Gailly P H, Bofland B, Himpens B, Casteels R, Gillis J M. Critical evaluation of cytosolic calcium determination in resting muscle fibres from normal and dystrophic (mdx) mice. Cell Cah'ium 1993; 14:474 483. Rivet M, Cognard C, Rideau Y, Duport G, Raymond G. Calcium currents in normal and dystrophic human skeletal muscle cells in culture. Cell Cah'ium 1990: ! I: 507 514. Ribet-Bastide M, lmbert N, Cognard C, Duport G, Rideau Y, Raymond G. Changes in cytosolic resting ionized calcium and in calcium transients during in vitro development of normal and Duchenne muscular dystrophy cultured skeletal muscle measured by laser cytofluorimetry using indo-l. Cell Calcium (in press). Pressmar J, Brinkmeier H, Seewald M J, Jockusch H, Naumann T, Rudel R. Intracellular calcium concentrations are not elevated at rest in cultured muscle from DMD patients and in mdx mouse cultures. Pfluegers Arch (in press). Franco A Jr, Lansman J B. Calcium entry through stretch-inactivated ion channels in mdx myotubes. Nature 1990; 334: 670-673. Karpati G, Charuk J, Carpenter S, Jablecki C, Holland P. Myopathy caused by a deficiency of Ca ~ ~-adenosine triphosphatase in sarcoplasmic reticulum. ~lnn Neurol 1986: 20:38 49. Lederfein D, Levy Z, Augicr N, et al. A 71 kilodalton protein is a major product of the Duchennc muscular dystrophy gene in brain and other non-muscle tissues. Proe Natl Aead Sei USA 1992: 89: 5346- 5350. Ervasti J M, Campbell K P. Dystrophin and the membrane skeleton. Curr Opin Cell Bio11993: 5:82 87. Madhaven R, Massom L R, Jarrett J W. Calmodulin specifically binds three proteins of the dystrophin glycoprotein complex. Biochem Biaphys Res Commun 1992: 185:753 759. Mawatari S, Miranda A F. Rowland L P. Adenyl

Cell Signalling in DMD

56.

57.

58. 59. 60.

61.

62. 63.

64. 65.

66.

67. 68. 69~

70. 71. 72. 73. 74. 75.

cyclase abnormality in Duchenne muscular dystrophy: muscle cells in culture. Neurology 1976; 26:1021 -1026. Northup J K. Regulation of cyclic nucleotides in the nervous system. In: Siegel G, Agranoff B, Albers R W, Molinoff P, eds. Basic Neurochemistrv, 4th Edn New York: Raven Press, 1989; 349 372. Reithmann C, Gierschik P, Werden K, Jakobs K H. Role of inhibitory G protein alpha-subunits in adenylyl cyclase desensitization. Mol Cell Endocrin 1991; 82: C215 221. Hille B. G protein-coupled mechanisms and nervous signalling. Neuron 1992; 9:187 195. Ross E M. Signal sorting and amplification throught G protein-coupled receptors. Neurol 1989; 3:141 152. Homey C J. Vatner S F, Vatner D E. Beta-adrenergic receptor regualtion in the heart in pathophysiologic states, abnormal adrenergic responsiveness in cardiac disease. Ann Rev Physiol 1991; 53:137 159. Egan S E, Giddings B W, Brooks M W, Buday L, Sizeland A M , Weinberg R A. Association of Sos Ras exchange protein with Grb2 is implicated in tryosine kinase signal transduction and transformation. Nature 1993; 363:45 51. Hardiman O. Effects of isoprenaline on cultures of normal and dystrophic muscle. Irish J M e d S c i (in press) Hardiman O, Brown R H Jr, Beggs A, Specht L, Sklar R M. Differential effects on the fusion of Duchenne/ Becker and control muscle cultures; pharmacological detection of accelerated aging in dystrophic muscle. Neurology 1992; 42: 1085-1091. Takagi A, Sojima S, Masayosi I, Araki M. Increased leakage ofcalcium ion from the sarcoplasmic reticulum of the mdx mouse. J Neurol Sci 1992; II0: 160-164. Takagi A, Sunohara N, Ishihara T, Nonaka I, Sugita H. Malignant hyperthermia and related neuromuscular diseases: caffeine contracture of the skinned muscle fibres. Musele Nerve 1986; 6:510 514. Donaldson S K, Goldberg N D, Walseth T F, Huettemann D A. Voltage dependence of inositol 1,4,5,-triphosphate-induced calcium release in peeled skeletal muscle fibres. Proc Natl AcadSci USA 1988; 85: 5749 5753. Hoffman E P, Hudecki M S, Rosenberg P A, Pollina C M, Kunkel L M. Cell and fibre type distribution of dystrophin. Neuron 1988; 1:411-420. Zubrzycka-Gaarn E E, Bulman D E, Karpati G, et al. The DMD gene product is localized to the sarcolemma of human skeletal muscle. Nature 1988; 333(3): 46~469. Knudson C M, Joffman E P, Kahl S D, Kundel L M, Campbell K P. Characterization of dystrophin in skeletal muscle triads: evidence for the association of dystrophin with junctional t-system. J Biol Chem 1988; 263: 848~8484. Anderson J E. Myotube phospholipid synthesis and sarcolemmal ATPase activity in dystrophic (mdx) mouse muscle. Biochem Cell Biol 1991; 69:835 841. Jaimovich E. Chemical transmission at the triad: lnsP~? J Musc Res Cell MotiliO' 1991; 12: 316~320. Dunn J F, Radda G K. Total ion content of skeletal and cardiac muscle in the mdx mouse dystrophy: Ca' ' is elevated at all ages. J Neurol Sci 1991; 103: 226-231. Birmbaumer L, Abramowitz J, Brown A M. Receptoreffector coupling by G proteins. Biochim Biophys Act 1990; 1031:163 224. Tsien R W. Calcium channels, stores and oscillations. Ann Rev Cell Biol 1990; 6:715 760. Wright W E. Muscle basic helix-loop-helixproteins and the control ofmyogenesis. Curr Opin Genet Dev 1992; 2: 243 248.

76.

77.

78.

79. 80.

81.

82.

83.

84. 84.

85.

87.

88.

89.

90.

91.

92.

93.

94.

95.

315

Hughes S M, Taylor J M, Tapscott S J, Gurley C M, Carter W J, Peterson C A. Selective accumulation of MyoD and myogenin mRNAs in fast and slow adult skeletal muscle is controlled by innervation and hormones. Development 1993; 118:1137 1147. Miller J B. Myoblasts, myosins, myoDs and the diversification of muscle fibres. Neuromusc Disor 1991; I(1): 7-17. Hardiman O, Sklar R M, Brown R H. Methylprednisolone selectivity affects dystrophin expression in human muscle cultures. Neurology 1993; 43:342 345. Florini J R. Hormonal control of muscle growth. Muscle Nerve 1987; 10: 577-598. Allen R E, Boxhorn L K. Regulation of skeletal muscle satellite proliferation and differentiation by TGF-beta, IGF-I and FGF. J Cell Physiol 1989; 138:311-315. Moss F P, Liblond C P. Satellite cells as the source of nuclei in muscles of growing rats. Anat Rec 1971 ; 170: 421-436. Maltin C A, Delday M I. Satellite cells in innervated and denervated muscles treated with clenbuterol. Muscle Nerve 1992; 15:919 925. Hesketh J E, Whitelaw P A. The role of cellular oncogenes in myogenes and muscle cell hypertrophy. lnt J Biochem 1992; 24(2): 193 203. Olson E N. Proto-oncogenes in the regulatory circuit for myogenesis. Semin Cell Biol 1992; 3:127 136. Florini J R, Magri K A, Ewton D Z, James P I, Grindstaff K, Rotwein P S. "Spontaneous" differentiation of skeletal myoblasts is dependent upon autocrine secretion of insulin-like growth factor-ll. J Biol Chem 1991; 24:15917 15923. Florini J R, Ewton D Z, Magri K A. Hormones, growth factors and myogenic differentiation. Ann Rev Physiol 1991; 53: 201-216. Mehls O, Blum W F, Schaefer F, Tonshoff B, Scharer K. Growth failure in renal disease. Baillieres Clin Endocrhlol Met 1992; 6:665 685. Bernstein D, Jasper J R, Rosenfeld R G, Hintz R L. Decreased serum insulin-like growth factor-I associated with growth failure in newborn lambs with cyanotic heart disease. J Clin Invest 1992: 89:1128 1132. Rudman D, Feller A G, Hoskote M D, et al. Effects of human growth hormone in men over 60 years old. New Engl J M e d 1990; 323: 1-6. Yang Y T, McElligott M A. Multiple actions of betaadrenergic agonists on skeletal muscle and adipose tissue. Biochem J 1989; 261: 1-10. Mendell J, Moxley R T, Griggs R C, et al. Randomized double-blind six-month trial of prednisone in Duchenne's dystrophy. N E n g l J M e d 1988; 320: 15921597. Hardiman O. Methylprednisolone downregulates adenylyl cyclase activity in human muscle clones. Ann Neurol (in press). Bohm M, Gierschik P, Knoror A, Larisch K, Weismann K, Erdmann E. Desensitization of adenylate cyclase and increase of Gi alpha in cardiac hypertrophy due to acquired hypertension. H)pertension 1992; 20:103 112. Luo J, Murphy L J. Dexamethasone inhibits growth hormone induction of IGF-I mRNA in hypophysectomized rats and reduces IGF-I mRNA abundance in the intact rat. Endocrinology 1989; 125:165 171. Salminen A, Braun T, Buchberger A, Jurs S, Winter B, Arnold H H. Transcription of the muscle regulatory gene myf4 is regulated by serum components, peptide growth factors and signalling pathways involving G proteins. J Cell Biol 1991; 115(4): 905-917.