Introduction to Muscular DystrophyJOHN D. PORTER*Departments of Ophthalmology, Neurology, and Neuroscience, University Hospitals of Cleveland and Case Western Reserve University,Cleveland, Ohio 44106-5068

KEY WORDS muscular dystrophy; dystrophin; dystroglycan; sarcoglycan; laminin-2

This special issue of Microscopy Research and Tech-nique is devoted to the topic of muscular dystrophy. Themuscular dystrophies are heritable, primary muscledisorders, which involve axial and/or appendicularmusculature. The pattern of histological alterationsassociated with muscular dystrophy is diagnostic, aconsequence of cyclic degeneration and regeneration ofskeletal muscle (although cardiac and smooth musclealso may be involved). Muscle degeneration is typicallyprogressive, with an apparent increase in severity asconnective tissue accumulates and the intrinsic regen-erative capacity of muscle becomes exhausted. Thediagnosis of muscular dystrophy, at best, means a life ofovercoming diminished movement capacity, and, atworst, leads to more than a decade of progressivemuscle wasting and an all too early death.

This devastating class of neuromuscular disordershas attracted much attention from both basic scienceand clinical research communities. In the 13 yearssince the discovery that dystrophin represents the keyprotein missing in Duchenne muscular dystrophy (Hoff-man et al., 1987), studies have linked genetic diseaseloci to protein products to cellular phenotypes, therebygenerating testable models of the pathogenesis of thedystrophies. Many types of muscular dystrophy arenow known to result from the disruption of an oligo-meric protein complex in the muscle fiber membrane, orsarcolemma. But it still remains unclear precisely howthe absence of these specific sarcolemmal proteins leadsto muscle fiber death and there is, as yet, no adequatetherapy for any of the muscular dystrophies. Thisspecial journal issue assembles a knowledgeable set ofreviews that relate the current status of concepts andtechnologies in the field of muscular dystrophy. Thecommon themes are the determination of the location,interrelationships, and functional roles of the proteinsaffected in the various muscular dystrophies and studyof the means by which the absence of these proteinsleads to myofiber death. The series of articles concludeswith a hopeful look to the future, conveying insights intoprogress in the most promising therapeutic approaches.

YOU CAN’T TELL THE PLAYERSWITHOUT A SCORECARD

The story of muscular dystrophy is, in many ways, astory of the cellular localization of proteins—the ab-sence of any of the interlocking pieces of an intricatetransmembrane protein complex either yields a muscu-lar dystrophy or is embryo lethal. The dystrophin-glycoprotein complex, or DGC, lies at the center ofhypotheses regarding the pathogenesis of musculardystrophy (see Campbell, 1995). This complex consistsof dystrophin, the dystroglycans (a and b), the sarcogly-

cans (a, b, g, and d), the syntrophins (a, b1), thedystrobrevins (a, b), laminin-a2 (merosin), and sarco-span. Some components of this complex are relativelyrecent discoveries, so one cannot assume that all theplayers are yet known. Figure 1 depicts a schematicrepresentation of the currently accepted model of theskeletal muscle DGC. The C-terminus of dystrophinbinds F-actin, a component of the muscle cytoskeleton.Dystrophin domains near the N-terminus bind b-dystro-glycan and the cytosolic syntrophin complex. The inte-gral membrane protein, b-dystroglycan, in turn bindsa-dystroglycan and an intramembranous sarcoglycancomplex. a-Dystroglycan, in turn, is a receptor for themuscle-specific laminin-a2 in the extracellular matrix.The recently characterized protein, sarcospan, is associ-ated with the sarcoglycans and may be regarded as afunctional component of this DGC subcomplex (Crosbieet al., 1999). Finally, the dystrobrevins also are a recententry into the organization of the DGC (Blake et al.,1996; Sadoulet-Puccio et al., 1996), interacting withboth dystrophin and syntrophin. There are other sarco-lemmal-spanning proteins or protein complexes inmuscle, some of which can produce muscular dystrophy-like signs if disrupted (e.g., the a7 integrin to laminin-a2 linkage), but molecular genetic approaches haveshown that each component of the DGC is in some waycritical for myofiber development and survival, so func-tion and dysfunction of the DGC is the focus of thisissue. The first article, by Watkins, Cullen, Hoffman,and Billington goes beyond this brief introduction toprovide a contemporary treatment of the DGC by high-resolution immunocytochemistry.

LINKAGE BETWEEN THEDYSTROPHIN-GLYCOPROTEIN COMPLEX

AND MUSCULAR DYSTROPHYIn the decade since the identification of dystrophin,

molecular genetics has revolutionized the diagnosis ofmuscular dystrophy. Classification schemes that oncewere based upon fine, even subjective, differences inpatient motor behavior, pathology, and inheritancepatterns, now are organized around sarcolemmal pro-tein families and utilize easily delineated gene defects

Contract grant sponsor: National Institutes of Health; Contract grant num-bers: R01 EY09834, R01 EY12779, and P30 EY11373; Contract grant sponsor:Muscular Dystrophy Association; Contract grant sponsor: Research to PreventBlindness; Contract grant sponsor: Ohio Lions Eye Research Foundation;Contract grant sponsor: CWRU Visual Science Research Fund; Contract grantsponsor: Evenor Armington Fund.

*Correspondence to: John D. Porter, Ph.D., Department of Ophthalmology,University Hospitals of Cleveland and Case Western Reserve University, 11100Euclid Avenue, Cleveland, OH 44106-5068. E-mail: jdp7@po.cwru.edu

MICROSCOPY RESEARCH AND TECHNIQUE 48:127–130 (2000)

r 2000 WILEY-LISS, INC.

that can be employed in genetic counseling. We nowunderstand that mutations in any one component of theDGC produce primary loss of a specific protein, but alsoare often accompanied by secondary changes in theproper membrane targeting of other DGC components.The subsequent group of articles in this issue considersthe cellular localization and function of the DGC, asrevealed by naturally occurring and gene knockoutmodels of muscular dystrophy. First, the article bySewry provides a nice introduction to the topic ofinterdependence of DGC components by discussingprimary and secondary changes in the cellular localiza-

tion of the DGC in various human muscular dystro-phies. Studies such as those reviewed by Sewry gaverise to the first structural models of the DGC and to thenotion that this protein assembly might mechanicallystabilize the sarcolemma against damage from theconstant muscle fiber shortening and lengthening dur-ing contraction.

Progress in the dissection of the precise structure andfunction of the DGC would soon plateau if investigatorswere restricted to naturally occurring mutations inhumans and animals, but, as in many fields, theseefforts have been fostered by application of gene knock-

Fig. 1. Schematic of normal sarcolemmal organization of the DGCin health and disease, adapted from multiple published sources. Innormal skeletal muscle (top left), the muscle cytoskeleton (actin) isthought to be connected to the extracellular matrix by virtue of adystrophin–b-dystroglycan–a-dystroglycan–laminin-2 linkage. The rolethat other DGC components might play in a hypothesized mechanicalstabilization role is unclear. The other DGC components, however, areassociated with various cell signaling elements. Thus, it is likely thatcomprehensive models of DGC function will include both mechanical

and cell signaling roles, with both functions compromised and contrib-uting toward the pathology associated with muscular dystrophy.Primary and secondary alterations in the DGC are indicated fordystrophin deficiency (Duchenne muscular dystrophy), sarcoglycandeficiency (limb girdle muscular dystrophy), and laminin-2 deficiency(congenital muscular dystrophy). A red X through the affected proteinindicates primary gene mutations, while the associated secondarydisplacement of other DGC proteins from the sarcolemma is indicatedby a change in color to gray.

128 J.D. PORTER

out methodology. Strategic placement of mutationsallows determination of the key functional domains ofDGC proteins. This approach is reviewed in the seriesof three articles that follow that of Sewry. The genera-tion of transgenic animals with mutations that mimichuman muscular dystrophy provides considerable powerin addressing molecular disease mechanisms, includingtesting hypotheses of pathogenic cascades and genetherapy. Using this approach, progress has been madein the understanding of three muscular dystrophiesthat result from DGC mutations: Duchenne (dystro-phin), limb girdle (sarcoglycan, for most autosomalrecessive forms), and congenital muscular dystrophy(laminin-a2). Figure 1 details the DGC alterations inthese three types of muscular dystrophy. First, thearticle by Rafael and Brown reviews recent develop-ments in DGC protein localization and function inrelationship to Duchenne muscular dystrophy. The lossof dystrophin displaces other DGC proteins from thesarcolemma, leading to cyclic muscle degeneration andregeneration. Studies are described that use gene knock-outs and transgenic models to assess structure-functionrelationships of dystrophin and to demonstrate thepotential of a dystrophin homologue, utrophin, as arescue strategy. Next, Hack, Groh, and McNally focusupon the sarcoglycan complex, a set of four linkedproteins that lies off to the side of the main axis of themuscle cytoskeleton to extracellular matrix linkage.Disruption of the sarcoglycan complex, in limb girdlemuscular dystrophy or in gene knockout models, altersthe DGC and membrane function in ways that are verydifferent from dystrophinopathy, suggesting the exis-tence of alternative mechanisms for muscle fiber deathor rescue in muscular dystrophy. The article by Miyagoe-Suzuki, Nakagawa, and Takeda completes the series ofparallel articles on the DGC, opening a new molecularapproach to understanding laminin-a2-deficient con-genital muscular dystrophy. The dy/dy mouse, origi-nally a much studied model of muscular dystrophy, fellinto disfavor with the recognition that dystrophin waslinked to Duchenne dystrophy and that the mdx mouse,not dy/dy, was a model of dystrophinopathy. Subse-quent identification of the role of laminin-a2 in congeni-tal muscular dystrophy re-established the utility of thedy/dy mouse, with its similar laminin-a2 defect. Indiscussing the pathogenesis of laminin-a2 deficiency,this group reports findings from human congenitalmuscular dystrophy, the dy/dy mouse, and a newknockout mouse model of congenital muscular dystro-phy. Collectively, data from the transgenic and knock-out mouse strategies described in these reviews shouldnot only aid understanding of the functional roles of theDGC but may yield insight into therapies.

TRANSLATION OFDYSTROPHIN-GLYCOPROTEIN COMPLEXDEFECTS INTO MUSCLE FIBER DEATH

Until recently, it was thought that the DGC servedprincipally as a physical chain connecting muscle fibercytoskeleton to extracellular matrix, mechanically sta-bilizing the sarcolemma against contraction-associateddamage (see Campbell, 1995). The absence of a DGCcomponent then was hypothesized to break the mechani-cal linkage, destabilizing the sarcolemma, leading tocalcium influx through frank membrane defects or

leaky calcium channels, and subsequent necrotic myofi-ber degeneration via calcium-activated protease cas-cades. Yet, as is apparent in this journal issue, dystro-phin-, sarcoglycan-, and laminin-a2-deficiency each alterthe structural organization of the DGC but do notproduce the same consequences for the sarcolemma.The loss of sarcolemmal integrity observed in the mdxand sarcoglycan knockout mice contrasts with its pres-ervation in skeletal muscle of the dy/dy. This observa-tion supports the notion that the pathophysiology ofmuscular dystrophy may be more complex than simplystructural destabilization of the sarcolemma. Studies ofthe sarcoglycan complex, reviewed by Hack, Groh, andMcNally, have been particularly crucial in delineatingthe mechanisms behind DGC function and dysfunction.Sarcoglycan mutations may not destabilize the cytoskel-eton to extracellular matrix linkage, but instead appearto disrupt the scaffolding for cell signaling functions ofthe DGC, leading to myofiber apoptosis. Indeed, there isaccumulating evidence for a signaling role for variouscomponents of the DGC, including DGC-associatednitric oxide synthase (nNOS), signal transduction adap-tor (Grb2), calcium-dependent kinase regulator(calmodulin), and ecto-ATPase. As the articles in thisissue will show, it is now clear that the DGC as astructural scaffold for aggregation of signaling com-plexes is an emerging theme in muscular dystrophyand muscle biology.

In the pursuit of disease mechanisms, often theexceptional tissue response can provide vital mechanis-tic information. Andrade, Porter, and Kaminski arguethat the response of a specialized muscle group, extra-ocular muscle, might yield insights into the DGC inhealth and disease. Extraocular muscle is notablyspared in dystrophinopathy, sarcoglycanopathy, andlaminin-a2 deficiency, lacking the signs of muscle degen-eration or loss of sarcolemmal integrity that typifyother skeletal muscles. These authors ask: do muta-tions that disrupt the DGC in other muscles leave itintact in extraocular muscle or does extraocular musclesomehow neutralize the loss of DGC components. These,and other data, require a new look at the DGC, as loss ofan intact DGC may not mean, a priori, the degenerationof muscle. Elucidation of the mechanism(s) by which anintact DGC supports myofiber survival is not only vitalto understanding muscle biology, but also would signifi-cantly aid design of new treatment regimens for thisdevastating disease. The debate over the function of theDGC and the pathogenesis triggered by a disruptedDGC runs through several of the articles in this issue.

IF DYSTROPHIN-GLYCOPROTEIN COMPLEXORGANIZATION IS SO SIMPLE, WHY NO

CURE YET?The answer to this very legitimate question will be

obvious to the reader of this issue of Microscopy Re-search and Technique: the structure and function of theDGC are not as simple as the current structural biologymodels suggest. Designing treatment paradigms whenthe primary pathogenic mechanisms are not yet re-solved is problematic at best. However, the final threearticles in this series discuss the basic science behindtwo different approaches under development for theclinical management of muscular dystrophy.

129INTRODUCTION

The cyclic degeneration and regeneration of musclecharacterizes muscular dystrophy. There has long beenan assumption, still valid, that understanding themuscle regeneration process might lead to ameliorativetherapy for muscular dystrophy. Skeletal muscle is oneof those tissues that possess an inherent regenerativecapacity. A population of stem cells, satellite cells, formsnew muscle fibers via an asynchronous process requir-ing satellite cell activation, proliferation, and fusion toreplace those muscle fibers lost to injury or disease.Anderson, Moor, and Rector discuss factors that regu-late this regenerative response, including the loss ofproliferative capacity in muscular dystrophy. A naturaloutgrowth of an understanding of muscle regenerationis the idea that satellite cells from normal individualsmight be introduced into muscular dystrophy patientsand subsequently produce muscle fibers that are DGC-competent. Skuk and Tremblay review the controversyand progress that have been associated with myoblasttransfer. Progress in alleviating the central problemswith myoblast transfer, initial transplant rejection, andimmunologic interpretation of the re-introduced pro-tein as foreign, is detailed in this article. Finally, thearticle by Hartigan-O’Connor and Chamberlain consid-ers very recent progress in gene therapy for musculardystrophies. The re-introduction of DGC components inmice with primary dystrophin or sarcoglycan muta-tions has been shown to prevent the structural andfunctional deterioration of skeletal muscle. Chamber-lain reviews efforts designed to introduce gene con-structs into human muscular dystrophy patients.

Taken together, the reviews in this issue emphasizethe tremendous progress that has been made since thediscovery of the first protein link to muscular dystro-

phy, dystrophin. Much of this progress can be attrib-uted to the application of molecular genetic technologydesigned to ascertain the structure-function of theDGC. The next several years likely will see majoradvances in understanding the non-structural roles ofthe DGC in muscle development and homeostasis. Themajor challenge is to translate knowledge of the DGCinto effective therapy for the muscular dystrophy pa-tient.

ACKNOWLEDGMENTSThe efforts of Dr. Francisco Andrade in design and

construction of the figure, reviewing an earlier versionof this manuscript, and assisting with the topic andauthor selections for this special journal issue are muchappreciated. Beth Ann Benetz also provided assistancewith illustration preparation. The author is the recepi-ent of a Research to Prevent Blindness Senior ScientificInvestigator Award.

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Isoform diversity of dystrobrevin, the murine 87-kDa postsynapticprotein. J Biol Chem 271:7802–7810.

Campbell KP. 1995. Three muscular dystrophies: loss of cytoskeleton-extracellular matrix linkage. Cell 80:675–679.

Crosbie RH, Lebakken CS, Holt KH, Venzke DP, Straub V, Lee JC,Grady RM, Chamberlain JS, Sanes JR, Campbell KP. 1999. Mem-brane targeting and stabilization of sarcospan is mediated by thesarcoglycan subcomplex. J Cell Biol 145:153–165.

Hoffman EP, Brown RH, Jr, Kunkel LM. 1987. Dystrophin: the proteinproduct of the Duchenne muscular dystrophy locus. Cell 51:919–928.

Sadoulet-Puccio HM, Khurana TS, Cohen JB, Kunkel LM. 1996.Cloning and characterization of the human homologue of a dystro-phin related phosphoprotein found at the Torpedo electric organpost-synaptic membrane. Hum Mol Genet 5:489–496.

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