I. INTRODUCTION

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Duchenne muscular dystrophy (DMD) is a severe X-linked recessive, progressive muscle-wasting disease affecting ~1 in 3,500 boys (146). Patients are usually confined to a wheelchair before the age of 12 and die in their late teens or early twenties usually of respiratory failure. A milder form of the disease, Becker muscular dystrophy (BMD), has a later onset and a much longer survival. Both disorders are caused by mutations in the DMD gene that encodes a 427-kDa cytoskeletal protein called dystrophin. The vast majority of DMD mutations result in the complete absence of dystrophin, whereas the presence of low levels of a truncated protein is seen in BMD patients. In addition to these diseases, mutations in the genes encoding many components of the dystrophin-associated protein complex (see below) cause other forms of muscular dystrophy such as the limb-girdle muscular dystrophies and congenital muscular dystrophy.

There is currently no effective therapy for DMD, although various strategies are being developed driven by the increasing understanding of the molecular processes involved in the progression of the muscle weakness. This review summarizes the current knowledge of the gene and protein as well as the disease process and also illustrates how these studies have led to a broader understanding of muscle function.

	II. DUCHENNE MUSCULAR DYSTROPHY

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Typically, DMD patients are clinically normal at birth, although serum levels of the muscle isoform of creatine kinase are elevated. The first symptoms of DMD are generally observed between the ages of 2 and 5 years (135, 259), with the child presenting with a waddling gait or difficulty in climbing stairs. There is often a delay in the achievement of motor milestones, including a delay in walking, unsteadiness, and difficulty in running. Subsequently, the onset of pseudohypertrophy of the calf muscles, proximal limb muscle weakness, and Gowers' sign (the use of the child's arms to climb up his body when going from a lying to standing position) suggest DMD (188). Eventually, decreased lower-limb muscle strength and joint contractures result in wheelchair dependence, usually by the age of 12 (146). Weakness of the arms occurs later along with progressive kyphoscoliosis. Most patients die in their early twenties as a result of respiratory complications due to intercostal muscle weakness and respiratory infection. Death can also be the result of cardiac dysfunction with cardiomyopathy and/or cardiac conduction abnormalities observed in some patients (146).

In individuals affected by BMD (24), the clinical course is similar to that of DMD, although the onset of symptoms and the rate of progression are delayed. More than 90% of patients are still alive in their twenties, with some patients remaining mobile until old age (146). There is a continuous clinical spectrum between a mildly affected BMD patient and a severely affected DMD patient. BMD and DMD patients also present with mild cognitive impairment, indicating that brain function is also abnormal in these disorders (reviewed in Refs. 42, 335).

Normal skeletal muscle consists of muscle fibers that are evenly spaced, angular, and of a relatively uniform size. Muscle, being a syncytium, is multinucleated with nuclei located at the periphery of the fiber. Fetal DMD muscle is histologically normal except for occasional eosinophilic hypercontracted fibers (34, 145, 304). Necrotic or degenerating muscle fibers are characteristically seen in all postnatal DMD muscle biopsies even before muscle weakness is clinically observed. Degenerating fibers are often seen in clusters (grouped necrosis), and studies of longitudinal and serial transverse muscle sections show this process is often confined to segments of the muscle fiber (186, 438). These necrotic fibers are subject to phagocytosis, and muscle biopsies from DMD patients reveal the presence of inflammatory cells at perimysial and endomysial sites (12, 13). These cells are predominantly macrophages and CD4+ lymphocytes (330). A secondary sign of muscle fiber necrosis, at least in the early stages of the dystrophinopathies, is the active regeneration of muscle to replace or repair lost or damaged fibers (438). Early regenerating fibers are recognized by virtue of their small diameter, basophilic RNA-rich cytoplasm, and large, centrally placed myonuclei (29, 56, 438). Eventually, the regenerative capacity of the muscles is lost and muscle fibers are gradually replaced by adipose and fibrous connective tissue, giving rise to the clinical appearance of pseudohypertrophy followed by atrophy (reviewed in Ref. 146). The combination of progressive fibrosis and muscle fiber loss results in muscle wasting and ultimately muscle weakness.

	III. DYSTROPHIN: GENE AND PROTEIN

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The identification of the DMD gene on the X chromosome was the first triumph of positional cloning and opened up a new era in DMD research (280, 354). The gene was localized to Xp21 by studies of rare female DMD patients with balanced X;autosome translocations with the translocation breakpoint in Xp21 (54). This localization was confirmed using DNA markers (123), and the disease was shown to be allelic with a milder disease of similar clinical course, BMD (273). The gene was eventually identified by taking advantage of a patient with a large deletion who suffered from four X-linked phenotypes including DMD (162). The DMD gene is the largest described, spanning ~2.5 Mb of genomic sequence (Fig. 1) (98, 355) and is composed of 79 exons (98, 355, 417). The full-length 14-kb mRNA transcribed from the DMD locus was found to be predominantly expressed in skeletal and cardiac muscle with smaller amounts in brain and covered a large genomic region (280, 351, 354). The protein product encoded by this transcript was named dystrophin since the lack of it causes dystrophy (280).

View larger version (32K): [in this window] [in a new window]   Fig. 1. Schematic showing the organization of the human Duchenne muscular dystrophy (DMD) gene and the dystrophin-related protein family. The DMD gene is 2.5 Mb and encodes 7 different protein isoforms. The "full-length" dystrophin transcripts are transcribed from promoters (depicted by arrows) in the 5'-end of the gene. Each mRNA encodes a 427-kDa protein that only differs in its NH2-terminal sequences. The three products are designated Dp427 (B), Dp427 (M), and Dp427 (P) to reflect their tissue-specific expression pattern; B is in the brain, M in muscle, and P in cerebellar Purkinje cells. The smaller isoforms are produced from distally located promoters expressed in the retina (R: Dp260), brain (B3: Dp140), Schwann cells (S: Dp116) or are general (G: Dp71) ubiquitously expressed. The identifiable domains in the cysteine-rich (CR) region and COOH terminus (CYS) of dystrophin are identified. These are the WW domain, the EF hands, the ZZ domain, and the paired coiled-coil (CC). The four proline-rich hinge regions are designated 1-4. The binding sites for -dystroglycan (DG), syntrophin (SYN), and the dystrophin family binding site (DFB) are shown for each protein (dotted lines). The organization of the utrophin protein shows that it is very similar to dystrophin, whereas the DRP2 and the dystrobrevins proteins only have sequence similarity to the COOH-terminal regions of dystrophin as shown. Three -dystrobrevin isoforms are expressed in muscle representing successive COOH-terminal truncations. The -dystrobrevin-1 isoform has additional COOH-terminal sequence that contains the sites for tyrosine phosphorylation (Y). -Dystrobrevin is not expressed in muscle and is most similar to -dystrobrevin-1 but lacks the sites for tyrosine phosphorylation.

Expression of the full-length dystrophin transcript is controlled by three independently regulated promoters. The brain (B), muscle (M), and Purkinje (P) promoters consist of unique first exons spliced to a common set of 78 exons (Fig. 1) (53, 93, 185, 274, 315, 374). The names of these promoters reflect the major site of dystrophin expression. The B promoter drives expression primarily in cortical neurons and the hippocampus of the brain (19, 93, 185), while the P promoter is expressed in the cerebellar Purkinje cells and also skeletal muscle (185, 231). The M promoter results in high levels of expression in skeletal muscles and cardiomyocytes and also at low levels in some glial cells in the brain (19, 93). These three promoters are situated within a large genomic interval of ~400 kb (Fig. 1) (53).

The DMD gene also has at least four internal promoters that give rise to shorter dystrophin transcripts that encode truncated COOH-terminal isoforms. These internal promoters can be referred to as retinal (R), brain-3 (B3), Schwann cell (S), and general (G). Each of these promoters utilizes a unique first exon that splices in to exons 30, 45, 56, and 63, respectively, to generate protein products of 260 kDa (Dp260) (134a), 140 kDa (Dp140) (295), 116 kDa (Dp116) (72), and 71 kDa (Dp71) (43, 241, 291). Dp71 is detected in most nonmuscle tissues including brain, kidney, liver, and lung (43, 237, 238, 241, 291, 436, 439) while the remaining short isoforms are primarily expressed in the central and peripheral nervous system (72, 134a, 295, 439). Dp140 has also been implicated in the development of the kidney (142). These COOH-terminal isoforms contain the necessary binding sites for a number of dystrophin-associated proteins (see sect. VI, E and F), and although the molecular and cellular function of these isoforms has not been elucidated, they are thought to be involved in the stabilization and function of nonmuscle dystrophin-like protein complexes.

Alternative splicing at the 3'-end of the dystrophin gene generates an even greater number of isoforms (40, 152). These splice variants not only affect full-length dystrophin but are also found in the shorter isoforms such as Dp71. This differential splicing may regulate the binding of dystrophin to dystrophin-associated proteins at the membrane (114).

Dystrophin is 427-kDa cytoskeletal protein that is a member of the -spectrin/-actinin protein family (282). This family is characterized by an NH₂-terminal actin-binding domain followed by a variable number of repeating units known as spectrin-like repeats. Dystrophin can be organized into four separate regions based on sequence homologies and protein-binding capabilities (Fig. 1). These are the actin-binding domain at the NH₂ terminus, the central rod domain, the cysteine-rich domain, and the COOH-terminal domain. The NH₂ terminus and a region in the rod domain of dystrophin bind directly to but do not cross-link cytoskeletal actin (reviewed in Refs. 425, 512). The rod domain is composed of 24 repeating units that are similar to the triple helical repeats of spectrin. This repeating unit accounts for the majority of the dystrophin protein and is thought to give the molecule a flexible rodlike structure similar to -spectrin. These -helical coiled-coil repeats are interrupted by four proline-rich hinge regions (281).

At the end of the 24th repeat is the fourth hinge region that is immediately followed by the WW domain. The WW domain is a recently described protein-binding module found in several signaling and regulatory molecules (50). The WW domain binds to proline-rich substrates in an analogous manner to the src homology-3 (SH3) domain (313). Although a specific ligand for the WW domain of dystrophin has not been determined, this region mediates the interaction between -dystroglycan and dystrophin, since the cytoplasmic domain of -dystroglycan is proline rich (see below). However, the entire WW domain of dystrophin does not appear to be required for the interaction with dystroglycan because Dp71, a dystrophin isoform that contains only part of the WW domain, is reported to bind to -dystroglycan (421). Interestingly, transgenic mice overexpressing Dp71 in dystrophin-deficient muscle restore -dystroglycan and the DPC at the membrane but do not prevent muscle degeneration (113, 202).

The WW domain separates the rod domain from the cysteine-rich and COOH-terminal domains. The cysteine-rich domain contains two EF-hand motifs that are similar to those in -actinin and that could bind intracellular Ca²⁺ (282). The ZZ domain is also part of the cysteine-rich domain and contains a number of conserved cysteine residues that are predicted to form the coordination sites for divalent metal cations such as Zn²⁺ (395). The ZZ domain is similar to many types of zinc finger and is found both in nuclear and cytoplasmic proteins. The ZZ domain of dystrophin binds to calmodulin in a Ca²⁺-dependent manner (11). Thus the ZZ domain may represent a functional calmodulin-binding site and may have implications for calmodulin binding to other dystrophin-related proteins. The ZZ domain does not appear to be required for the interaction between dystrophin and -dystroglycan (412).

The COOH terminus of dystrophin contains two polypeptide stretches that are predicted to form -helical coiled coils similar to those in the rod domain (47). Each coiled coil has a conserved repeating heptad (a,b,c, d,e,f,g)_n similar to those found in leucine zippers where leucine predominates at the "d" position (reviewed in Refs. 68, 310). This domain has been named the CC (coiled coil) domain. Approximately 3-5% of proteins have coiled-coil regions. Coiled coils are well-characterized protein interaction domains. The CC region of dystrophin forms the binding site for dystrobrevin and may modulate the interaction between syntrophin and other dystrophin-associated proteins (see sect. VI) (47, 430).

The frequency of DMD coupled with a high new mutation rate (1 × 10⁴ genes/generation) has led to the characterization of hundreds of independent mutations. Mutations that cause DMD generally result in the absence, or much reduced levels, of dystrophin protein while BMD patients generally make some partially functional protein. There is some correlation between mutations in the DMD gene and the resulting phenotype. The study of such mutations has revealed the importance of a number of the structural domains of dystrophin and facilitated the design of dystrophin "mini-genes" for gene therapy approaches (reviewed in Ref. 9).

Approximately 65% of DMD and BMD patients have gross deletions of the DMD gene (279, 353). After the characterization of many such mutations, it became apparent that the size and position of the deletion within the DMD gene often did not correlate with the clinical phenotype observed. This observation can be largely explained by the reading frame theory of Monaco et al. (352). This argues that if a deletion leads to the expression of an internally truncated transcript without shifting the normal open reading frame, then a smaller, but functional version of dystrophin could be produced. This scenario would be consistent with a BMD phenotype. If, on the other hand, the deletion creates a translational frameshift, then premature termination of translation will result in the synthesis of a truncated protein. This latter scenario is often associated with extremely low levels of dystrophin expression due to mRNA or protein instability and results in a DMD phenotype. With the use of this reading frame theory and the knowledge of exon structure of the DMD gene, it has been possible in many cases to predict whether a young male is likely to develop BMD or DMD (279). However, there are exceptions to this reading frame rule (22, 316, 514), and there are cases in which complete dystrophin deficiency may be associated with a relatively benign phenotype (216).

The vast majority of large deletions detected in BMD and DMD cluster around two mutation "hot spots" (279, 281), although the reasons for this are unclear. It is possible, however, that the chromatin structure in Xp21 influences the occurrence of deletion or recombinant hotspots. Deletion cluster region I spans exons 45-53 (25) and removes part of the rod domain, while deletion cluster region II spans exons 2-20 and removes some or all of the actin-binding sites together with part of the rod domain (296). Most of the breakpoints occurring in cluster region II occur in the large introns 1 and 7. Most of these large deletions can be detected using a simple multiplex PCR test that screens the exons most commonly deleted and allows accurate genetic counseling in the majority of affected families via DNA-based diagnostics (26, 85).

One-third of DMD cases are caused by very small deletions and point mutations, most of which introduce premature stop codons (293, 419). Unlike the large deletions that cluster in two regions of the DMD gene, small deletions and point mutations appear to be evenly distributed throughout the gene (169, 398, 419). Although it might be predicted that such mutations would give rise to normal amounts of truncated protein, usually very little or no protein is detected, indicating that the corresponding transcripts or the truncated proteins are unstable (228). This has disappointing implications for the functional dissection of the dystrophin protein, since many mutations do not generate any information regarding the importance of a particular domain. Despite this setback, a small number of useful mutations have been identified that generate a mutated or truncated protein and convey information regarding the functional importance of the different dystrophin domains.

At the NH₂ terminus of dystrophin, the importance of the actin-binding domain was demonstrated by the identification of missense mutation (Arg for Leu-54) that resulted in a DMD phenotype associated with reduced amounts of protein (398). Furthermore, DMD patients have been described with in-frame deletions of exons 3-25 and produce normal amounts of truncated protein (488).

The rod domain of dystrophin has been found to accommodate large in-frame deletions without serious clinical consequences. The most notable example was the discovery of a patient with an in-frame deletion of 46% of the dystrophin coding sequence which resulted in only a mild case of BMD (deletions of exons 17-48) (147). This observation suggests that the rod domain acts as a spacer between the actin binding domain and the cysteine-rich and COOH-terminal domains of dystrophin, and truncation of this region merely shortens the bridge between these two functional regions without adversely affecting the function of the protein. Indeed, this deletion has been the basis of a dystrophin mini-gene that was incorporated into expression plasmids as well as retroviral and adenoviral vectors for transfer to muscle fibers in vivo (1, 139, 407). Furthermore, this mini-dystrophin was able to restore the normal muscle phenotype in transgenic mdx mice (391, 504). Other large deletions of the rod domain have also been observed in BMD patients (305, 514).

Although few missense mutations have been described in DMD patients, two informative substitutions have been identified in the cysteine-rich domain. The substitution of a conserved cysteine residue with a tyrosine at position 3340 results in reduced but detectable levels of dystrophin. This mutation alters one of the coordinating residues in the ZZ domain (Fig. 1 and sect. IIID) that is thought to interfere with the binding of the dystrophin-associated protein -dystroglycan (294). Another reported substitution of an aspartate residue to a histidine residue at position 3335 is also thought to affect the -dystroglycan binding site, and although there was normal localization and amounts of dystrophin detected, a severe phenotype resulted (184). Interestingly, the cysteine-rich domain is never deleted in BMD patients, suggesting that this domain is critical for dystrophin function (402).

A small number of cases have been reported in which an abnormally truncated protein that is deleted for the COOH terminus is synthesized and localized at the sarcolemma. A DMD patient was found to have a deletion that removed almost the entire cysteine-rich and COOH-terminal domain (39, 229) (Fig. 1 and sect. IIID). The abnormal protein was normally localized but resulted in a severe clinical phenotype. Another DMD patient has been reported to be deleted for everything 3' of exon 50 but again generates a truncated protein that is localized to the sarcolemma (222). These examples illustrate the functional importance of the cysteine-rich and COOH-terminal domains of dystrophin that presumably reflects their interactions with other dystrophin-associated proteins (see sect. VI, E and F).

Finally, cases of X-linked cardiomyopathy are caused by mutations in the DMD gene that abolish the cardiac gene expression of dystrophin, while retaining expression in skeletal muscle. This condition involves ventricular wall dysfunction, dilated cardiomyopathy, and cardiac failure in the absence of skeletal myopathy (153). Mutations in the muscle-specific M-promoter selectively abolish expression in the heart.

	IV. THE MDX MOUSE AND OTHER DYSTROPHIN-DEFICIENT ANIMALS

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The discovery of dystrophin allowed the recognition of other animals with lesions in their orthologous genes. Dystrophin-deficient mice, dogs, and cats (which arose by spontaneous mutation) and more recently nematodes [in which the DMD gene has undergone targeted disruption (35)] play a number of important roles in research into the functions of dystrophin. To a greater or lesser extent they provide models of DMD and allow study of the pathophysiological processes at work. The ease with which the murine genome can be manipulated has made the mdx mouse particularly useful in testing functional hypotheses. These animals also allow initial testing of putative treatments for DMD and indeed have been used in screening strategies for such treatments (8, 200).

This section aims to describe the phenotypes of the known dystrophin-deficient vertebrates.

The mdx mouse was initially identified because of raised serum creatine kinase levels (an enzyme released from damaged muscle) and was then found to have muscle pathology (67). It lacks full-length dystrophin (228) because of a point mutation in exon 23 of the DMD gene, which forms a premature stop codon (443). The mdx mouse retains expression of some COOH-terminal dystrophin isoforms, but mice lacking these too have been generated by ethyl-nitroso-urea induced and insertional mutagenesis (90, 112, 246, 505). These animals are phenotypically similar to the mdx mouse, arguing that full-length dystrophin is the functionally significant isoform in muscle.

Obvious weakness is not a feature, and the life span of mdx mice is not grossly reduced (311, 383). It has therefore been suggested that this mutant is not a helpful model of DMD (122). However, it is clear that simple in vivo tests can demonstrate muscle dysfunction (79, 403). True muscle hypertrophy is an important feature of mdx muscle (unlike DMD), but normalized force production and power output are significantly reduced (311). Muscle fiber necrosis occurs and is particularly frequent during a crisis period at 3-4 wk (469). There is a vigorous regenerative response as evidenced by frequent expression by fibers of the fetal myosin heavy chain isoform, and the majority of fibers become centrally nucleated, as occurs in muscle regeneration after nonspecific insults (109, 132, 211). After the crisis period, central nucleation remains frequent, although expression of fetal myosin heavy chain declines. Degeneration and regeneration continue; however, mdx muscle in which regeneration has been blocked by -irradiation shows a decline in total fiber numbers and does so as fast at 15-21 wk as at 2-8 wk (378). Further satellite cells (the undifferentiated muscle precursor cell which proliferates in regeneration) continue to express markers of activation (260). In the diaphragm (in which pathology appears most marked), muscle fiber loss and collagen deposition are significant (456). Atrophy and fibrosis are also features in limb muscles of older mdx mice (382). It is clear then that the mdx mice show many features of DMD but at later times relative to life span than patients. Why this should be is not clear but may relate to differences in the murine biology of muscle regeneration (186). Despite this, the mdx mouse has been a key resource in the exploration of dystrophic pathophysiology.

Several dystrophin-deficient dogs have been identified and the causative genetic lesion defined in at least three (186, 437, 441, 509). The best-characterized phenotype is the golden retriever (the GRMD dog) (104). Muscle weakness becomes apparent at 2 mo and progresses; life span is significantly reduced (491). Histologically muscle shows necrosis, fibrosis, and regeneration (489). The GRMD dog shows perhaps the closest similarity to DMD and has been used to test potential treatments (21).

Hypertrophic feline muscular dystrophy (HFMD) occurs in cats harboring a deletion of the dystrophin muscle and Purkinje promoters; muscle levels of dystrophin are therefore much reduced though nonzero (171, 510). Animals have an abnormal gait and histologically necrosis is present but fibrosis is not seen and hypertrophy is very marked. This later feature causes death in some individuals. Although this odd phenotype could be due to the particular mutation, a previous less well-characterized dystrophin-deficient cat also showed prominent hypertrophy, suggesting that this may be a feature of feline pathophysiology (81). Clinically, therefore, the HFMD cat seems a poor model of DMD.

	V. PATHOPHYSIOLOGY OF DYSTROPHIN-DEFICIENT MUSCLE

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This section describes the pathophysiological features of dystrophin-deficient muscle and the possible relationships between them. For the purposes of this review, we have divided data about dystrophin deficiency into two sets. One set of results flowed very directly from the discovery of dystrophin; biochemical and genetic techniques have then allowed the identification of binding partners and homologs. Investigation of the changes that occur in the expression of these molecules in dystrophin-deficient muscle has been a fruitful task, and this set of results is discussed in sections VI and VII. The second set of data in contrast have come from lines of investigation that could at least in principle have been carried out without detailed knowledge of dystrophin. These results are discussed in this section.

A.  Abnormalities of the Muscle Cell

1.  Membrane structure and function

In 1975 Mokri and Engel used electron microscopy to describe the ultrastructural features of DMD muscle (349). They noted absent or disrupted sections of sarcolemma overlying wedge-shaped areas of abnormal cytoplasm, the so-called delta lesions. This observation, subsequently confirmed, together with the high levels of several cytosolic proteins in the blood of patients with DMD, gave rise to the theory that the primary pathology of DMD muscle might be an abnormal fragility and leakiness of the cell membrane (349, 422). Although no equivalent to the delta lesion has been found in the mdx mouse (120, 481) or GRMD dog (489), there is good evidence that dystrophin-deficient muscle is characterized by increased permeability to macromolecules flowing in and out of the cell and that this abnormal permeability is made worse by mechanical stress.

DMD and mdx muscle contain an increased number of fibers that stain positively for endogenous extracellular proteins (albumin, IgG, IgM) (34, 95, 460). For example, Clarke et al. (95) examined the triceps of 12-wk-old mice and found that 25% of fibers stained for albumin in the mdx muscle and only 4% in normal muscle. A similar pattern can be seen using exogenous vital dyes that are normally excluded from muscle cells. mdx mice to whom Procion orange or Evans blue (which binds tightly to albumin) has been administered show an increased number of fibers containing the dye (55, 327, 460). Recently, an albumin targeted contrast agent has been developed that allows visualization of these changes in vivo by magnetic resonance imaging (457). To demonstrate that these differences reflect an increased permeability of some dystrophin-deficient muscle cells and not just an increased number of necrotic cells (which do take up these dyes), it is important that the dyes can be shown to accumulate in nonnecrotic cells. Several studies of, for example, Evans blue do demonstrate this (107, 460), but some others have not (319, 457).

These dyes are not taken up uniformly between or within muscles; typically groups of dye-positive fibers are seen and at widely different frequencies in different muscles (460). When animals are exercised on a treadmill, the number of dye-positive fibers increases in both normal and mdx but remains much higher in mdx muscle (65, 95).

An increased number of permeable fibers which increases further with mechanical stress can also be demonstrated in isolated muscle preparations. These also allow more precise control of applied stress than in live animal studies (255, 347, 390, 503). For example, Petrof et al. (390) applied a variety of mechanical stress/electrical stimulation protocols to isolated normal and mdx muscles and then counted fibers that had taken up Procion orange for the fluid in which the muscle was bathed. There were about fivefold more dye-positive fibers in mdx muscle under all the stress/stimulation protocols (including when no stress/stimulation had been applied). The most dye-positive fibers were seen after the application of "eccentric" contractions when a stimulated muscle is lengthened. The number of dye-positive fibers after different protocols was correlated with the peak mechanical stress but not the number of electrical stimulations (390).

Does an equivalent phenomenon occur in single fibers or cultured myotubes? Menke and Jockusch (339, 340) have subjected myotubes to hyposmolar stress before assessing their uptake of horseradish peroxidase and their release of several endogenous proteins. They concluded that mdx myotubes leak more (340).

There is thus good evidence that dystrophin-deficient muscle contains fibers that allow ingress of molecules normally excluded from the cytoplasm and that this tendency is enhanced after muscle has been put under mechanical stress. Why should this be? There is evidence that cells and especially muscle cells experience frequent transient cell membrane disruptions that are repaired by active resealing mechanisms (333). These disruptions are more frequent after mechanical stress (332). Does the absence of dystrophin render muscle cell membranes more susceptible to these disruptions? The costameres (a rectilinear array of proteins including vinculin and -spectrin which lies just under the sarcolemma in register with the sarcomeres) are deranged in mdx muscle (380, 506). Cytoskeletal -actin is normally tightly bound to the sarcolemma but is not in mdx muscle (427). There are therefore structural and functional deficits within the sarcolemmal cytoskeleton that could plausibly leave the membrane vulnerable to mechanical damage. Several attempts have been made to define biophysical abnormalities of the dystrophin-deficient sarcolemma and supporting cytoskeleton. Results are not conclusive. Several studies have measured the pressure which, when applied via a patch clamp, ruptures the membrane of myotubes. mdx and control myotubes do not differ (163, 164, 243), nor could a difference be found in the stress, strain, or energy required to rupture isolated muscles (289). In contrast, the stiffness of the subsarcolemmal cytoskeleton is decreased fourfold in mdx myotubes (381). The biophysical correlate of the enhanced permeability of dystrophin-deficient muscle cells therefore remains rather obscure. A clearer view may come from a more sophisticated theory of how membrane and sarcolemmal cytoskeleton behave under stress (242). Another possibility is that dystrophin plays a role in the resealing mechanisms mentioned above (333).

Two observations should be mentioned that may perhaps be linked to the above phenomena. First, there is evidence that the rate of progression of the pathological process (assessed histologically) may be altered by manipulating the levels of activity of mdx mice. Immobilizing a limb by splinting or neurotomy reduces pathology (265, 345, 348). Second, the tension that mdx muscle can develop drops faster than in normal muscle as it is subjected to repeated eccentric contractions (64, 347, 428). It may be that this is due to accumulating membrane "damage." An alternative explanation might invoke changes in fiber type composition and therefore in the isoforms of sarcomeric proteins expressed (which are known to occur in mdx muscle; Refs. 101, 390). However, single fibers isolated from normal and mdx muscle and subjected to chemical membrane disruption do not differ in the rate at which a force deficit develops during eccentric contractions (312). The contrast between this finding and the results in whole muscle may imply a causative role for the membrane.

Calcium homeostasis is critical to many aspects of muscle function (31), and early suggestions that it might be perturbed in dystrophin-deficient muscle stemmed from several observations. Hypercontracted fibers are the earliest morphological abnormality of DMD and were ascribed to persistently raised intracellular [Ca²⁺] ([Ca²⁺]_i) (119). DMD muscle biopsies showed an increase in the number of fibers positive for a histochemical calcium stain (49). It was hypothesized therefore that [Ca²⁺]_i is raised in dystrophin-deficient muscle and that this is an important cause of the pathophysiological processes leading to cell death (138). This speculation has spawned much investigation.

Spectroscopic studies demonstrate that the total calcium content of DMD muscle is raised even at an early stage (33, 34, 324). Examination of mdx and GRMD muscle broadly agrees (140, 409, 490). However, these studies could not distinguish the intracellular component of the total; this had to await a methodological advance.

A) [CA²⁺]_i. Some fluorescent calcium chelators (e.g., fura 2) have different excitation/emission spectra in their bound and unbound states. When introduced into cells, therefore, and after appropriate calibration, they allow determination of [Ca²⁺]_i (466). These techniques can be applied to muscle fibers or myotubes (but not intact animals). In 1988, two groups reported the use of this technique to show that the [Ca²⁺]_i of DMD myotubes and mdx myofibers was about double that of controls (356, 484). The technique has been widely taken up and applied using seemingly similar protocols, but reported data are in conflict. Steinhardt and co-workers (160, 236, 482, 483) confirmed and extended their original observations in dystrophin-deficient myotubes and fibers, and an independent group confirmed a doubling of [Ca²⁺]_i over controls in mdx myotubes (17). However, others have found no change (102, 166, 220, 292, 397, 413). One of these groups in the course of a further study found a small (20%) but statistically significant increase in [Ca²⁺]_i from mdx fibers over controls (485).

How can this conflict be explained? Part of the difficulty may be methodological, and the issues of calibration, altered handling of the dye by dystrophin-deficient cells, variable subcellular compartmentalization of different dyes, and techniques for introducing the dye into cells have been raised (182, 183, 236). Another variable may be the history of the cells used. Some studies prepared myofibers using an enzymatic disassociation step; others used purely mechanical steps. In addition, after fusion of myoblasts has been induced, myotubes show spontaneous contractions only after some days have elapsed. Given the role that mechanical stresses have been postulated to play in dystrophin-deficient cells, this may be an important factor. One of the above groups found no differences from controls in [Ca²⁺]_i in noncontracting mdx myotubes but large increases when tubes were cultured using conditions that promote spontaneous contractions. Stopping the contractions with tetrodotoxin reduced mdx [Ca²⁺]_i back to control values (248, 413). Steinhardt and co-workers (236) too have reported that chronic but not acute treatment with tetrodotoxin reduces [Ca²⁺]_i in mdx myotubes back to control values.

Investigation of the changes in [Ca²⁺]_i after electrical or K⁺-induced depolarization have also not achieved unanimity. Several found a normal peak value but a slower return to baseline in dystrophin-deficient preparations (102, 250, 356, 484, 485), but some found no change at all (220) and some a higher peak and slower decline (248). The considerations set out above may explain some of this variation.

Some of these investigators have used these techniques to examine how the absence of dystrophin alters changes in [Ca²⁺]_i when myotubes or fibers are challenged by increased external calcium concentrations and/or hyposmotic shock. Here there is agreement that larger rises in [Ca²⁺]_i occur in dystrophin-deficient cells (128, 249, 292, 397, 399, 482, 484).

The data so far apply to values for [Ca²⁺]_i averaged over the whole of the cytoplasm of the cell. Are there differences in regional [Ca²⁺] between cells with and without dystrophin that could be missed because of this? In mdx myofibers challenged by raised external [Ca²⁺], Turner et al. (482) saw regional [Ca²⁺]_i rise more close to the sarcolemma than deep within the fiber. However, they could not confirm this finding in myotubes, and further characterization of subcellular variation was beyond achievable resolution. Two more recent studies have however addressed the issue using different techniques. Allard and colleagues (317) (who found no difference from controls in whole cell [Ca²⁺]_i in mdx fibers) used patch-clamp measurements in estimate subsarcolemmal [Ca²⁺]_i in fibers. By measuring characteristics of calcium-activated K⁺ channels with the patch clamp in both the cell-attached and inside-out configurations, they estimated that [Ca²⁺]_i at the sarcolemma was threefold greater in mdx than wild-type fibers (102, 317). In the other study, myotubes were transfected with various DNA constructs that express a calcium-sensitive photoprotein tagged with different signal proteins that target to different subcellular regions (415). [Ca²⁺]_i at the sarcoplasmic reticulum (SR) was almost 50% greater in mdx than control myotubes. No differences could be demonstrated in cytoplasmic [Ca²⁺]_i, although the authors caution that the photoprotein signal is insensitive in the relevant range. The peak of the depolarization-induced transient was raised above control in mitochondria but not in bulk cytoplasm or subsarcolemma (at least in younger cultures; in 11-day myotubes the peak was greater in all three regions). The authors interpret their findings as consistent with an increase in cytoplasmic [Ca²⁺]_i, which is amplified in the SR.

In summary, data exist showing an increase in [Ca²⁺]_i in dystrophin-deficient myofibers and myotubes (especially after a challenge to calcium homeostasis) and also higher levels of calcium in the SR. It appears that consensus has been reached that conflicting data can largely be understood on the basis of methodological considerations (423). It should be remembered that all these are in vitro data; we are ignorant of [Ca²⁺]_i changes in intact animals.

B) CALCIUM FLUXES. An increase in [Ca²⁺]_i in dystrophin-deficient cells might arise from abnormal fluxes of calcium into the cytoplasm from outside the cell or from within the SR. What evidence is there for such calcium flows?

C) FLOWS OF CALCIUM INTO THE CELL. Different approaches to recording the rate of calcium entry into a cell are available. One uses the phenomenon of manganese quenching of the fluorescence of calcium-sensitive dyes like fura 2. If it is assumed that the divalent ions Mn²⁺ and Ca²⁺ enter a cell in the same way, then the rate of signal quenching after Mn²⁺ are introduced extracellularly gives a measure of calcium influx. Using this technique, two groups have demonstrated that the calcium entry in mdx myotubes and fibers is about double that in normal controls (236, 485). However, there was disagreement about the pharmacological features of the flow. Hopf et al. (236) found that nifedipine doubled the quenching rate, whereas Tutdibi et al. (485) found no change.

Another approach is to use patch-clamp techniques to study calcium channels. Franco and Lansman (163) have described abnormalities in mechanosensitive calcium channels. They found a calcium channel activity in normal myotubes that had a low opening probability and was activated by stretching. In mdx myotubes they also found a calcium channel activity that had a high opening probability and was inactivated by stretch. This activity was not found in control myotubes, and it was suggested that it might be responsible for extra calcium influx into mdx myotubes. Although this second channel activity could not be shown to occur in mdx myofibers, the authors showed that in this situation the open probability of the first kind of mechanosensitive channel was greater in mdx than control fibers (164, 217).

However, Steinhardt and co-workers (160) have described abnormalities in a different calcium channel activity in myotubes. They demonstrated a leak channel (i.e., voltage independent) activity in normal myotubes which in mdx myotubes had a threefold greater open probability. Nifedipine (an antagonist of L-type voltage-dependant calcium channels) increased the activity ascribed to this channel. The channel was also shown to be calcium selective (482).

These two groups agree that they are describing different phenomena (160, 164, 482). Franco-Obregon and Lansman (164) speculate that the leak type activity is an artifact of degenerating cultures. However, a leak channel activity increase in mdx myotubes has been confirmed independently (80). Moreover, Steinhardt and colleagues (236) managed to extend their original observations from myotubes to myofibers where again increased activity of calcium leak channels in dystrophin-deficient cells was seen (although the quantitative electrophysiological features of the channel were different in myofibers and tubes). In a separate study by this group in normal muscle, it was demonstrated that this channel had the properties of a capacitance current (i.e., was responsive to the state of intracellular calcium stores). Pharmacological antagonists of the activity were also described (235). However, the molecular correlate of this activity is unknown. That this activity is causally related to the rise in [Ca²⁺]_i in dystrophin-deficient muscle cells is evidenced by the ability of a leak channel antagonist to return [Ca²⁺]_i to normal (484). Data relevant to the cause of the increased calcium leak channel activity is considered in the section considering the role of proteolysis in dystrophin deficiency.

Carlson and Officer (76, 78, 80) have offered an alternative explanation for calcium leak channel activity and its increase in dystrophin deficiency. Using patch-clamp recordings from myotubes, they distinguished two types of channel activity: one a calcium leak channel and one attributed to acetylcholine receptor activity. These activities did not occur in single patches as independent events, and in mdx patches studied over long periods their relative frequencies changed. This prompted the speculation that calcium leak channel activity might be associated with acetylcholine receptors that had altered in some way and that this alteration was occurring more frequently in the context of a dystrophin-deficient membrane. The nature of this change in acetylcholine receptors has not yet been further defined.

D) FLUXES INTO THE SR. As mentioned above, some groups have found that the transient rise in [Ca²⁺]_i after depolarization is exaggerated or abnormally prolonged in dystrophin-deficient muscle preparations. This slowing of sequestration could be due to dysfunction of the SR Ca²⁺-ATPase or secondary to increased calcium levels with in the SR. Attempts have been made to directly examine SR Ca²⁺-ATPase activity, but the results are in conflict. Two studies of the tensions developed in mdx myofiber after manipulations that cause the SR to empty and refill concluded that calcium uptake by the SR was normal (269, 465). However, a study of the Ca²⁺-ATPase activity of SR vesicle preparations demonstrated almost a halving of the maximum uptake rate in mdx muscle. Turner et al. (482) have presented data that do not suggest an intrinsic problem of the SR calcium pump (482). Lowering the calcium concentration external to a mdx myotube brings its [Ca²⁺]_i back down to normal levels. Under these circumstances, the kinetics of the [Ca²⁺]_i transient also become normal.

Abnormal levels of several proteases are a feature of a wide variety of muscle diseases (224, 287, 385, 493). Changes in protease expression or activity in DMD or mdx muscle may therefore be nonspecific features, causally far removed from the primary pathological process (264, 286). However, there are data indicating that proteases and in particular calpains may have an important role in the pathophysiology of dystrophin deficiency. Protein degradation rates in isolated normal muscle (as assessed by tyrosine release) can be raised or lowered by manipulations that raise or lower [Ca²⁺]_i (165, 523). Turner et al. (484) having found a raised [Ca²⁺]_i in mdx myofibers therefore studied tyrosine release rates in isolated mdx muscle. Proteinolysis occurred 80% faster than in normal muscle, but this difference could be abolished by lower extracellular calcium concentrations (and perhaps therefore normalizing [Ca²⁺]_i). This result was subsequently confirmed, and the effect was shown to be blocked by leupeptin (a thiol protease inhibitor) (314). Steinhardt's group (482) went on to show that leupeptin not only blocked the extra proteolysis of mdx myotubes but also normalized their [Ca²⁺]_i and the open probability of their calcium leak channels (483). The exaggerated increase in [Ca²⁺]_i seen in mdx myotubes after hyposmolar shock is also abolished by a protease inhibitor (292). These are not the results that would have been expected if the abnormalities of calcium influx were a direct result of dystrophin deficiency. An alternative hypothesis was therefore put forward in which transient membrane ruptures allow an influx of calcium. This then causes local activation of proteases which modify calcium leak channels to cause further calcium ingress. Thus a vicious circle might be established in which calcium homeostasis becomes deranged. Two further studies in support of this notion have been performed (7, 328). McCarter and Steinhardt (328) simulated the initial steps in this process by using a patch clamp to rupture the membrane of a normal myotube. The patch clamp was then reattached either close to (<5 µm) or far from (50 µm) the rupture, and the calcium leak channel activity was measured. Channels close to the lesion had fourfold increased open probability. Incubating with leupeptin abolished this effect (328). Alderton and Steinhardt (7) used a more direct technique than tyrosine release to assess proteolysis in myotubes: hydrolysis of a fluorogenic calpain substrate. They confirmed that proteolysis occurs faster in mdx myotubes than controls and that this can be stopped by lowering external calcium concentration and by an antagonist of calcium leak channel activity. A variety of proteolysis inhibitors showed that most of the extra proteolysis was not due to lysosomal or proteosomal pathways (7). Candidates for this proteolytic activity include m- and µ-calpain (75). Evidence to specifically implicate calpains in the pathology of dystrophin-deficient muscle has also been presented (452). A difficulty here is that the regulation of calpain activity is complex and controversial. In particular, equating active calpain with the product of its autolytic lysis may not be justified (75). Direct evidence for the role of calpain activity in the pathophysiology of DMD is therefore lacking.

The hypothesis that the primary abnormality of dystrophin-deficient muscle is vulnerability to oxidative damage arose initially from two sorts of observation. First, DMD and mdx muscle show biochemical hallmarks of oxidative damage (363). Of course, this could be a nonspecific secondary feature (161). Second (but no more specifically), muscle diseases in which oxidative damage may play a primary role show features in common with DMD (338). However, there is now stronger evidence implicating oxidative damage early in the dystrophic process.

In the mdx mouse, there is very little necrosis before the wave of degeneration that occurs at around 3 wk, and during this time serum creatinine kinase levels are normal (327). Disatnik et al. (134) assayed the muscles of such very young mice for a marker of lipid peroxidation and for expression of several genes encoding antioxidants. They found that these were increased in mdx muscle at 2 wk. The same investigators studied the resistance of mdx and control myoutubes in culture to damage from a range of toxins; some were classed as pro-oxidants (e.g., hydrogen peroxide) and some nonoxidants (e.g., staurosporine which promotes apoptosis by inhibiting a range of protein kinases) (408). The mdx myotubes were more vulnerable to the pro-oxidants than controls. There was no difference with nonoxidants. The mdx and control myoblasts (before the expression of dystrophin) did not show differential toxicity with the pro-oxidants.

Necrotic myofibers are a feature of dystrophic muscle. Several investigators have looked for the features of myofibers undergoing apoptosis or programmed cell death in dystrophin-deficient muscle (4, 433). In mdx muscle, myonuclei showing the internucleosomal DNA fragmentation characteristic of apoptosis can be found (110, 319, 434, 473). They are present at 2 wk when necrosis is not a feature (473), and their numbers decline thereafter (433). The search for apoptotic myonuclei in DMD has been less clear cut. Some studies have found none (27, 251, 342), another found that that 10% of intact myofibers showed signs of DNA fragmentation and another that apoptotic nuclei were present but most were in satellite cells and macrophages. The reason for these differences is not clear.

What significance does the occurrence of apoptosis in (at least) mdx muscle have? The distinction between necrosis and apoptosis may not be rigid, and different intensities of a cellular insult may cause apoptosis and necrosis (372). It would be unsafe then to infer from the occurrence of apoptosis and necrosis in dystrophin-deficient muscle that different pathological processes must be at work.

Finally, Sandri et al. (435) compared the effect of cis-platinum (an inducer of apoptosis) on cultured mdx and control myotubes. They demonstrated more apoptotic myotubes in the mdx cells. Cis-platinum may achieve some of its effect by the generation of free radicals, so this result is consistent with the increased vulnerability of mdx myotubes to oxidative damage (318, 408).

B.  Abnormalities of the Muscle Tissue

1.  Vascular problems

In DMD muscle (and its animal models), necrotic fibers often occur in clusters. An explanation put forward to explain this "grouped necrosis" highlights a role for vascular dysfunction (leading to focal areas of ischemia). To support this, microembolization was found to produce pathology in rabbit muscle reminiscent of DMD (338). However, this model was subsequently criticized (57, 205), and structural studies revealed no striking vascular abnormality (155, 278, 343).

More recent work has focused on nitric oxide (NO) and its roles in muscle. NO is a vasodilator and a key modulator of vascular tone (157). In skeletal muscle, NO is produced by endothelial cells and by muscle fibers themselves which express neuronal-type nitric oxide synthase (nNOS) (277, 365). In DMD and mdx muscle however, nNOS disappears from its normal position at the sarcolemma, becoming cytoplasmic (60, 88). Could it be that loss of nNOS causes disregulation of vascular tone, ischemia, and the pathology of DMD? This seems not to be the case because mice in whom the nNOS gene has been disrupted do not have muscle disease (89, 239). Nor does it seem that the relocalization of nNOS from sarcolemma to cytoplasm (where it could conceivably have deleterious effects) contributes to the pathology; mdx mice crossed with nNOS-deficient mice have a phenotype indistinguishable from mdx (118).

However, evidence is available that the lack of nNOS in dystrophin-deficient muscle may still play a part in the pathological process. Sympathetic nervous input to muscle vasculature causes vasoconstriction. However, the relationship between vascular tone and sympathetic input differs in resting and exercising muscle. For a given increase in sympathetic input, vascular tone increases more in resting than in exercising muscle. The mechanisms responsible for this metabolic modulation of sympathetic vasoconstriction seem to depend on NO and nNOS because the effect is abolished by NOS inhibitors or in nNOS-deficient mice. It has now been demonstrated that this metabolic modulation is also much reduced in children with DMD and in mdx mice (432, 471). It is possible therefore that this deficit could cause functional ischemia of areas of muscle during exercise; although not in itself sufficient to cause disease, this might exacerbate some other pathological process (115).

Once necrosis starts, DMD and mdx muscle contain an increased number of a variety of inflammatory cells (14, 330, 364, 481). In the mdx mouse, the time course of the increase in CD4 and CD8 T lymphocytes mirrors that of the necrosis, peaking at 4-8 wk before declining. Are these cells reactive, and do they themselves contribute to cell death or some other pathological feature? This question has been addressed by a number of investigators using genetic or other manipulations to remove specific sets of inflammatory cells or mediators (454). Preliminary reports of mdx mice deficient in either mast cells or macrophages saw no change in histology at 4 wk (186), while mdx mice unable to produce tumor necrosis factor (TNF; a T cell-derived cytokine) developed in some muscles rather worse pathology (453) than mdx. The mdx mice missing perforin (a cytotoxic molecule secreted by T lymphocytes) have also been analyzed (455). In these some reduction in apoptotic and necrotic fibers was seen at the time point examined. In another model, antibody-mediated depletion of either CD4 or CD8 T cells was found to reduce pathology as assessed by a "histopathological index" (454). The contribution of T lymphocytes to the progressive fibrosis seen particularly in the mdx diaphragm has also been studied. Crosses of mdx with nude mice (that lack T cells) show some reduction in fibrosis at 12 and 24 wk (361). Transforming growth factor-1 (TGF-1) has been muted as a mediator of fibrosis in DMD (32, 516), but this has not yet been directly tested.

Muscle from normal mice and humans is capable of regeneration after extensive damage. That this process is occurring too in mdx mice is clear from experiments in which regeneration has been inhibited. The effect of -irradiation to make plain the importance of ongoing regeneration in the mdx phenotype has been referred to above (378). Similarly, mdx mice that also carry mutations in genes important in muscle regeneration (for example, fibroblast growth factor-6, Mnf, and MyoD) develop very severe muscle disease (158, 170, 334). However, both in patients with DMD and mdx mice regeneration eventually fails to keep up with ongoing necrosis so that atrophy occurs. Studies that have compared regeneration of normal and mdx muscle after damage by toxins or the like seem to confirm that mdx muscle especially in older animals regenerates less well than normal (252, 410, 522). Why should this be?

Myofibers themselves are postmitotic, but skeletal muscle contains a population of mononuclear muscle precursor cells within the basement membrane of the fibers (325). These satellite cells proliferate and fuse during regeneration (362). Although it has been demonstrated recently that populations of cells exist in other tissues that can gain access to muscle via the circulation and contribute to muscle regeneration, satellite cells are responsible for the predominant part of muscle regeneration (154). Is there some defect in satellite cells in dystrophin-deficient muscle? Studies from patients with DMD largely show an increase in satellite cell numbers (497, 500). However, muscle precursor cells isolated from DMD muscle are capable of fewer replications in vitro than age-matched controls (411, 502). This may reflect the larger number of replications they have already undergone in vivo (at least as assessed by decreasing telomere length) (124). These data seem to support the notion that because of a greater rate of turnover the satellite cell population becomes exhausted in dystrophin-deficient muscle. However, this work has been criticized on the grounds that the extraction of precursor cells from muscle is highly inefficient, retaining only a small fraction of the in vivo population (375, 411). This small fraction may not be representative. Indeed, it has become apparent that satellite cells are heterogeneous and contain functionally distinct subpopulations (23, 400). Renault et al. (411) were able to demonstrate a population of radiation-resistant precursor cells that was absent in mdx muscle. However, analysis of whole fiber cultures (which may allow study of a more representative pool of satellite cells) provided no evidence for a progressive general exhaustion of myogenic potential (48). The extent to which sustained high satellite cell turnover is responsible for impaired muscle regeneration is therefore uncertain. An alternative possibility is that some feature of the muscle environment (for example, factors secreted by fibroblasts or muscle fibers themselves) becomes inimical to regeneration. There is some evidence that TGF-1 and insulin-like growth factor binding protein-5 may play such a role, but this hypothesis is yet to be tested in vivo (336, 337).

Thus abnormalities of NO modulation of vascular tone may well contribute to pathology but cannot alone explain it. The same may be true of the inflammatory, fibrotic, and regenerative processes in dystrophin-deficient muscle. Further insights into these processes may come from microarray and other technologies that allow examination of changes in many mRNA levels in dystrophin-deficient tissues and thus reveal groups of up- or downregulated genes (86, 94, 151, 478). For example, Hoffman and colleagues (94) used high-density oligonucleotide arrays to compare the abundance of 6,000 mRNA species between normal, dystrophin-deficient, and -sarcoglycan-deficient muscle.

Important abnormalities of dystrophin-deficient muscle cells have been demonstrated in three areas: calcium homeostasis, an increased susceptibility to oxidative toxins, and increased (and stress enhancable) membrane permeability. Confirmation that the absence of dystrophin is indeed responsible for these abnormalities comes from experiments in which dystrophin has been restored. This has been achieved by making mdx mice transgenic for a construct consisting of a muscle and heart-specific promoter and a full-length dystrophin cDNA (111). This mouse makes dystrophin at supraphysiological levels. Comparison of mdx with normal mice has shown that myotube calcium homeostasis and susceptibility to oxidative stress (111, 130, 133) become normal. How are these various abnormalities related? One possible scheme (7) is outlined in Figure 2. It highlights the abnormal permeability of mechanically stressed muscle cells as the primary problem and links this through changes in protease and calcium channel activity to explain how a cell with badly deranged calcium homeostasis could result. This could in turn trigger necrosis or apoptosis. It is the case, however, that details of several of these steps are missing, for example, the molecular identity of the abnormal calcium channel and the biophysical nature of the membrane deficit. Other schemes have been suggested (77), and it should be recognized that the hierarchy of physiological derangements at play in dystrophin-deficient muscle remains uncertain.

View larger version (14K): [in this window] [in a new window]   Fig. 2. The pathophysiology of dystrophin deficiency. This diagram illustrates the scheme described by Steinhardt and others. For references, see text.

	VI. DYSTROPHIN-ASSOCIATED PROTEIN COMPLEX

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The dystrophin-associated protein complex (DPC) was identified because dystrophin was found to be enriched in muscle membrane fractions eluted from a wheat germ agglutinin (WGA) column (74, 149, 519). WGA is a plant lectin that has high affinity for N-acetylglucosamine, a common constituent found in the glycans of some glycoproteins. WGA-affinity chromatography was subsequently used to purify a complex of dystrophin-associated proteins and glycoproteins from rabbit skeletal (149, 519).

The consensus view of the DPC stoichiometry is that dystrophin is linked to the sarcolemma of normal muscle by a protein complex composed of at least 10 different proteins (Fig. 3 and Table 1). In contrast to spectrin that appears to be a functional heterodimer, the dystrophin complex is monomeric (426). This complex spans the membrane and links the actin-based cytoskeleton to the muscle basal lamina. Thus the DPC can be thought of as a scaffold connecting the inside of a muscle fiber to the outside.

View larger version (30K): [in this window] [in a new window]   Fig. 3. The dystrophin-associated protein complex (DPC) in skeletal muscle. Dystrophin binds to cytoskeletal actin at its NH2 terminus. At its COOH terminus, dystrophin is associated with a number of integral and peripheral membrane proteins that can be classified as the dystroglycan subcomplex, the sarcoglycan-sarcospan subcomplex, and the cytoplasmic subcomplex. The cytoplasmic subcomplex includes the syntrophins (syn) and -dystrobrevin (DB). The sarcoglycan-sarcopsan subcomplex comprises the sarcoglycans (, , , ) and sarcospan. The extracellular component of the dystroglycan complex, -dystroglycan (DG), binds to laminin-2 in the extracellular matrix and -dystroglycan (DG) in the sarcolemma. In turn, -dystroglycan binds to the dystrophin, thus completing the link between the actin-based cytoskeleton and the extracellular matrix. Additional DPC binding partners are omitted for clarity, but a full list of the proteins can be found in Table 1.

The DPC can be divided into several separate subcomplexes based on their location within the cell and their physical association with each other. Using detergent extraction and two-dimensional gel electrophoresis, Yoshida et al. (520) showed that the DPC could be dissociated into three distinct complexes. These complexes are the 1) the dystroglycan complex, 2) the sarcoglycan:sarcospan complex, and 3) the cytoplasmic, dystrophin-containing complex. Each of these subcomplexes is considered in detail below.

Dystroglycan was the first component of the DPC to be cloned (244). The single dystroglycan gene produces a precursor protein that is processed by an unidentified protease to produce - and -dystroglycan. The dystroglycan gene is composed of only two exons, and there is no evidence of alternative splicing, although several glycoforms are produced (245). The relative molecular weights of -dystroglycan differ in different tissues as a result of the aforementioned differential glycosylation (see below). In muscle, -dystroglycan has a molecular mass of 156 kDa, whereas -dystroglycan is 43 kDa. In brain, -dystroglycan has a molecular mass of 120 kDa and was independently identified as a protein called cranin (447, 448).

-Dystroglycan has a single transmembrane domain and is inserted into the muscle plasma membrane with the COOH terminus on the cytoplasmic side. In contrast, -dystroglycan is located in the extracellular matrix where it is thought to be directly associated with -dystroglycan through multiple covalent interactions. The extreme COOH terminus of -dystroglycan contains several proline residues that are required for dystroglycan binding to dystrophin (261, 412, 463, 464). The last 15 amino acids of -dystroglycan appear to bind directly to the cysteine-rich region of dystrophin. This region of -dystroglycan is proline rich and contains a site for tyrosine phosphorylation (258). Recently, the crystal structure of -dystroglycan bound to dystrophin has been determined (240). The structure of this region of dystrophin shows that dystroglycan forms contacts with both the WW domain and EF hands of dystrophin, emphasizing the functional importance of both of these domains to the dystrophin family of related proteins.

The COOH terminus of -dystroglycan also binds to the adaptor protein Grb2 (517) (Table 2). This interaction is mediated by the SH3 domain of Grb2 that binds to proline-rich sequences in the cyoplasmic tail of -dystroglycan. This interaction raises the possibility that -dystroglycan may participate in the transduction of extracellular-mediated signals to the muscle cytoskeleton (517). Interestingly, in vitro studies show that dystrophin inhibits the interaction between Grb2 and