sarcoglycans in muscular dystrophy

Sarcoglycans in Muscular DystrophyANDREW A. HACK,1 MARGARET E. GROH,2 ELIZABETH M. MCNALLY2,3*1Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 606372Department of Medicine, University of Chicago, Chicago, Illinois 606373Department of Human Genetics, University of Chicago, Chicago, Illinois 60637

KEY WORDS dystrophin-glycoprotein complex; muscle degeneration; animal models; pathogen-esis

ABSTRACT Muscular dystrophy is a heterogeneous genetic disease that affects skeletal andcardiac muscle. The genetic defects associated with muscular dystrophy include mutations indystrophin and its associated glycoproteins, the sarcoglycans. Furthermore, defects in dystrophinhave been shown to cause a disruption of the normal expression and localization of the sarcoglycancomplex. Thus, abnormalities of sarcoglycan are a common molecular feature in a number ofdystrophies. By combining biochemistry, molecular cell biology, and human and mouse genetics, agrowing understanding of the sarcoglycan complex is emerging. Sarcoglycan appears to be animportant, independent mediator of dystrophic pathology in both skeletal muscle and heart. Theabsence of sarcoglycan leads to alterations of membrane permeability and apoptosis, two sharedfeatures of a number of dystrophies. b-sarcoglycan and d-sarcoglycan may form the core of thesarcoglycan subcomplex with a- and g-sarcoglycan less tightly associated to this core. Therelationship of e-sarcoglycan to the dystrophin-glycoprotein complex remains unclear. Animalslacking a-, g- and d-sarcoglycan have been described and provide excellent opportunities for furtherinvestigation of the function of sarcoglycan. Dystrophin with dystroglycan and laminin may be amechanical link between the actin cytoskeleton and the extracellular matrix. By positioning itself inclose proximity to dystrophin and dystroglycan, sarcoglycan may function to couple mechanical andchemical signals in striated muscle. Sarcoglycan may be an independent signaling or regulatorymodule whose position in the membrane is determined by dystrophin but whose function is carriedout independent of the dystrophin-dystroglycan-laminin axis. Microsc. Res. Tech. 48:167–180,2000. r 2000 Wiley-Liss, Inc.

DYSTROPHIN-GLYCOPROTEINCOMPLEX (DGC)

Dystrophin, the gene product of the Duchenne Muscu-lar Dystrophy locus, is a large cytoskeletal proteinassociated with the plasma membrane of cardiac andskeletal muscle (Burghes et al., 1987; Koenig et al.,1987; Monaco et al., 1986). The absence of a transmem-brane domain within the primary sequence of dystro-phin lead to the hypothesis that dystrophin interactsdirectly or indirectly with integral membrane proteinsin its role at the sarcolemma (Fig. 1). Biochemicalisolation of dystrophin confirmed that dystrophin co-purifies as part of a macromolecular complex thatincludes of a number of transmembrane glycoproteins(Campbell and Kahl, 1989; Ervasti and Campbell,1991; Suzuki et al., 1992; Yoshida and Ozawa, 1990).Within the dystrophin-glycoprotein complex (DGC) is asubcomplex, sarcoglycan, which consists of a numbercomponents that are expressed exclusively in cardiacand skeletal muscle. Initial biochemical characteriza-tion of the sarcoglycan complex focused on the 50- and35-kD dystrophin-associated glycoproteins (DAGs).These studies revealed that at least three glycosylatedsubunits were present in the sarcoglycan subcomplex, a50-kD (A2 or 50-kD DAG), a 43-kD (A3b), and a 35-kD(A4 or 35-kD DAG) (Yoshida et al., 1994). Furthermore,crosslinking data revealed a close association betweenthe 50- and 35-kD subunits and suggested that all the

sarcoglycans but the 50-kD subunit could be crosslinkedto dystrophin (Yoshida and Ozawa, 1990).

Sarcoglycan Is a Subunit Within the DGCEarly observations using antibodies raised to the

entire DGC indicated that the 50- and 35-kD compo-nents of the DGC, now known as a- and g-sarcoglycan,were specifically absent from children with an autoso-mally inherited form of muscular dystrophy (Mat-sumura et al., 1992; Mizuno et al., 1994; Passos-Buenoet al., 1993). This form of muscular dystrophy, SevereChildhood Autosomal Recessive Muscular Dystrophy(SCARMD), was genetically linked to chromosome 13q12(Azibi et al., 1993; Ben Othmane et al., 1992; El Kerchet al., 1994). SCARMD is a common cause of musculardystrophy in Northern Africa and Mediterranean coun-tries (Ben Hamida et al., 1983). The clinical picture inSCARMD is quite similar to Duchenne Muscular Dys-trophy save for the absence of cognitive impairment(Ben Hamida et al., 1996; Miladi et al., 1999). Malesand females are affected equally, and cardiac involve-

Contract grant sponsor: NIH; Contract grant sponsor: American Heart Associa-tion; Contract grant sponsor: Heart Research Foundation; Contract grantsponsor: Muscular Dystrophy Association.

*Correspondence to: E. M. McNally, M.D., Ph.D., Department of Medicine,Section of Cardiology, University of Chicago, 5841 S. Maryland Ave. MC 6088,Chicago, IL 60637. E-mail: [email protected]

Received 10 August 1999; accepted in revised form 14 September 1999

MICROSCOPY RESEARCH AND TECHNIQUE 48:167–180 (2000)

r 2000 WILEY-LISS, INC.

ment may occur (Ben Hamida et al., 1983). Age of onsetis in the first decade with demise in the late second toearly third decade. It was thought that the 50-kDdystrophin-associated protein (50 DAG) may play a rolein SCARMD since it was shown to be specifically absentfrom SCARMD muscle biopsies (Matsumura et al.,1992).

a-Sarcoglycan (Adhalin, A2, 50-kD DAG)The 50-kD DAG (A2) was cloned initially from rabbit

skeletal muscle (Roberds et al., 1993a). Its primarysequence revealed a type I transmembrane proteinwith an amino-terminal signal sequence, a single trans-membrane domain, two predicted N-linked glycosyla-tion sites, and a cytoplasmic carboxyl-terminus (Fig. 2).The gene product was initially termed adhalin (‘‘adhal,’’

the arabic for muscle) because it was specifically absentin children with SCARMD. The human adhalin genewas cloned and mapped to chromosome 17q21, thusexcluding it as the cause of chromosome 13-linkedSCARMD (McNally et al., 1994). While adhalin was notfound to be the genetic cause of chromosome 13-linkedSCARMD, missense mutations in the human adhalingene were identified in patients with limb girdle muscu-lar dystrophy (LGMD) (Roberds et al., 1994). To date, alarge number of different mutations have been de-scribed in the human adhalin gene (Carrie et al., 1997;Duggan et al., 1997; Kawai et al., 1995; Piccolo et al.,1995). The most common mutation, R77C, rests in aCpG island. Haplotype analysis suggested that thismutation has arisen on more than one genetic back-ground, arguing that this site is a ‘‘hot spot’’ for

Fig. 1. The dystrophin-glycoprotein complex (DGC) and associ-ated proteins. The integral components of the DGC are shownschematically. Arrows indicate diseases caused by the absence of oneor more protein associated with the muscle plasma membrane. Defectsin dystrophin result in Duchenne/Becker muscular dystrophy. Defectsin sarcoglycan result in multiple forms of Limb Girdle Muscular

Dystrophy. Defects in Laminin-a2 result in Congenital musculardystrophy. CMD, congenital muscular dystrophy; LGMD, Limb GirdleMuscular Dystrophy; DMD/BMD, Duchenne muscular dystrophy/Becker muscular dystrophy; SPN, sarcospan; ABD, actin bindingdomain; CYS, cysteine-rich region; COOH, carboxy-terminal region;NOS, nitric oxide synthase.

168 A.A. HACK ET AL.

mutation (Bueno et al., 1995; Carrie et al., 1997). Thereare a number of conserved cysteines within the extracel-lular domain of adhalin. Thus, the introduction of anadditional cysteine residue may perturb secondarystructure. With the identification of the other sarcogly-can subunits came an appreciation for sarcoglycan as acomplex. In light of this, adhalin was renamed a-sarco-glycan.

Many mutations in a-sarcoglycan produce instabilityof a-sarcoglycan accompanied by secondary instabilityof the other sarcoglycan subunits (Carrie et al., 1997;Duggan et al., 1997). Some missense mutations ina-sarcoglycan may be associated with a slightly lesssevere phenotype. The presence of a milder phenotypemay correlate with residual a-sarcoglycan protein(Eymard et al., 1997). Reports of cardiomyopathy ina-sarcoglycan patients are also relatively rare suggest-ing that its role in cardiac and skeletal muscle maydiffer (Eymard et al., 1997). A splice form of humana-sarcoglycan was described that is predicted to gener-ate a nontransmembrane form of a-sarcoglycan (Mc-Nally et al., 1994). This splice form does not appear tobe present in other organisms, and the protein productcorresponding to this splice form has not been identified(McNally, unpublished results).

b-Sarcoglycan (A3b)There are two 43-kD elements in the DGC. The first

of these is the b-subunit of dystroglycan and is widelyexpressed in many different tissue types (Ibraghimov-Beskrovnaya et al., 1992). The second 43-kD DAG ispart of the sarcoglycan complex (Yoshida et al., 1994).This subunit, named b-sarcoglycan, was found to har-bor mutations in patients with severe and mild forms ofmuscular dystrophy (Bonnemann et al., 1995; Lim etal., 1995). Unlike a-sarcoglycan, b-sarcoglycan is a typeII transmembrane protein (Fig. 2). The primary se-quence revealed that b-sarcoglycan has a single trans-membrane domain and three predicted N-linked glyco-sylation sites. Characteristic of type II transmembraneproteins, there are conserved charged residues immedi-ately before the transmembrane domain. The amino-terminal cytoplasmic domain contains a large hydropho-bic region. b-sarcoglycan is expressed in cardiac andskeletal muscle. b-sarcoglycan mRNA is expressed inother tissues including brain, but expression of theprotein has not been detected in tissues outside ofstriated muscle (Fougerousse et al., 1998). Two mis-sense mutations were reported in the Southern IndianaAmish associated with a milder, adult onset limb girdlemuscular dystrophy (Duclos et al., 1998a; Lim et al.,

Fig. 2. Schematic representation of the five sarcoglycan proteins.a-sarcoglycan and e-sarcoglycan share 62% similarity at the aminoacid level. g-sarcoglycan and d-sarcoglycan share 69% similarity at theamino acid level. b-sarcoglycan shares low homology to g- andd-sarcoglycans, the highest homology is in the conserved cluster ofcysteines homologous to EGF. a- and e-sarcoglycan are type I trans-membrane proteins while b-, g- and d-sarcoglycan are type II trans-

membrane proteins. Cysteines are represented with filled circles in allfive sarcoglycans. The position of these cysteine residues is perfectlyconserved in a- and e-sarcoglycan. The lengths of the extracellulardomains and the positions of the cysteine residues are also wellconserved between b-, g- and d-sarcoglycan. a-sg, a-sarcoglycan; e-sg,e-sarcoglycan; b-sg, b-sarcoglycan; g-sg, g-sarcoglycan; d-sg, d-sarcogly-can.

169SARCOGLYCAN IN MUSCULAR DYSTROPHY

1995). However, other missense mutations have beenfound in patients with a more severe, childhood onsetcourse (Bonnemann et al., 1996, 1998). Generally,mutations in b-sarcoglycan appear to result in aninstability of the entire sarcoglycan complex (Bon-nemann et al., 1995; Lim et al., 1995). Missense muta-tions that affect the membrane-proximal portion ofb-sarcoglycan seem to be associated with a severedystrophic course (Bonnemann et al., 1998). It shouldbe emphasized that there is great variability in thephenotype associated with mutations in the sarcogly-can genes. Importantly, not all variability can be suffi-ciently explained by the type of mutation nor the genethat is mutated.

g-Sarcoglycan (35-kD DAG, A4)The 35-kD DAG (A4) was cloned from rabbit and

human skeletal muscle and found to encode a type IItransmembrane protein (Fig. 2) (Noguchi et al., 1995).The gene for g-sarcoglycan is expressed exclusively inheart and skeletal muscle. The gene product wastermed g-sarcoglycan, and the human gene was mappedto chromosome 13q12, the location implicated by ge-netic linkage analysis in SCARMD patients. A singlecommon mutation was found in patients of North Africandescent (Noguchi et al., 1995). This mutation, D521-T,destabilizes the protein and results in secondary insta-bility of the other sarcoglycan subunits. The carboxyl-terminus of g-sarcoglycan has homology to b- andd-sarcoglycan with a number of conserved cysteinesubunits in a fixed position similar to cysteine-containingEGF-like proteins (Fig. 3) (McNally et al., 1996b). Frame-shifting mutations at the extreme carboxyl-terminusthat affect these conserved cysteine residues alsoproduce instability of the entire g-sarcoglycan protein.Few missense mutations have been reported in g-sarco-glycan. One missense mutation (C283Y) alters oneof the conserved cysteine residues, again highlightingthe importance of these residues for proper folding andassembly of the sarcoglycan complex (Piccolo et al.,1996).

d-SarcoglycanA fourth sarcoglycan protein was identified by elec-

tronic database homology (Nigro et al., 1996b). d-sarco-glycan encodes a 35-kD type II transmembrane proteinthat is related to g-sarcoglycan (Fig. 2). d-sarcoglycan is58% homologous to g-sarcoglycan at the nucleotidelevel and 69% similar at the amino acid level. Nothernblot analysis shows the predominant d-sarcoglycanmRNA is expressed in heart and skeletal muscle. Asplice form, utilizing an alternative exon 8, appears tobe expressed outside of skeletal and cardiac muscle andlacks the conserved cysteine residues (Jung et al.,1996). The muscle form of g-sarcoglycan includes, likeb- and g-sarcoglycan, a cluster of conserved cysteineresidues at its extracellular carboxyl-terminus (Fig. 3).Mutations in d-sarcoglycan are found in patients withmuscular dystrophy, although mutations in d-sarcogly-can are a relatively rare form of muscular dystrophy(Fanin et al., 1997; Nigro, 1996a). Like g-sarcoglycan,mutations in the cysteine residues are critical andassociate with a severe phenotype (Moreira et al.,1998). Because of the homology between g- and d-sarco-glycan, initial biochemical characterization of sarcogly-can did not distinguish the two proteins (Ervasti andCampbell, 1991; Yoshida and Ozawa, 1990; Yoshida etal., 1994). Both genes are composed of eight exonswidely separated and spanning at least 100 kB in thegenome. The intron-exon borders are conserved be-tween d- and g-sarcoglycan. Thus, these two genes mayhave arisen from a gene duplication event (McNally etal., 1996a; Nigro et al., 1996). d-sarcoglycan is onlyweakly homologous to b-sarcoglycan, with most of thishomology in the extracellular domain (Figs. 2, 3). Theextracellular domains of b-, g-, and d-sarcoglycan arenearly identical in length (225 amino acids, 224 aminoacids, and 224 amino acids, respectively) and sharestrong homology in the EGF-like domain found in theirextreme carboxyl-termini (Fig. 3).

e-SarcoglycanA fifth sarcoglycan has been identified by electronic

database homology (Ettinger et al., 1997; McNally et

Fig. 3. Homology between b-, g- and d-sarcoglycan epidermalgrowth factor-like regions. b-, g- and d-sarcoglycan share conservedcysteine residues that are homologous to those seen in epidermalgrowth factor (EGF). The corresponding amino acids from eachsarcoglycan are shown above the consensus for an EGF-like repeat.

Cysteines are underlined and identical amino acids are noted with avertical bar. Note the high degree of homology in extreme carboxyl-termini of all three sarcoglycans. The stop codon is marked by a (*) andthe final amino acid is numbered.


al., 1998). This sarcoglycan, termed e-sarcoglycan ishomologous to a-sarcoglycan. Like a-sarcoglycan, e-sar-coglycan is a type I transmembrane protein with anamino-terminal signal sequence and a transmembranedomain (Fig. 2). There are a number of shared cysteineresidues and their positioning is perfectly conservedbetween a- and e-sarcoglycan. The carboxyl-terminus ispredicted to reside in the cytoplasm, and an alternativesplice form suggests that there may be alternativecytoplasmic tails present on e-sarcoglycan. e-sarcogly-can maps to chromosome 7q21–22 (McNally et al.,1998), and the mouse e-sarcoglycan gene maps tomouse chromosome 6, in a region syntenic to human7q21 (McNally et al., 1998). e-sarcoglycan’s gene struc-ture is highly conserved to that of a-sarcoglycan; bothgenes share an identical intron-exon structure and arecomposed of at least nine exons (McNally et al., 1998). Aprocessed pseudogene for e-sarcoglycan is present inthe human genome at 2q22 (McNally et al., 1998). It isunlikely that this pseudogene produces any proteinsince it does not maintain an open reading frame.Unlike a-sarcoglycan, e-sarcoglycan is widely ex-pressed in many different tissue types (Ettinger et al.,1997; McNally et al., 1998). e-sarcoglycan is also ex-pressed early in development with an expression pat-tern that is similar to dystroglycan (Ly and McNally,unpublished results; Williamson et al., 1997). Antibod-ies to e-sarcoglycan indicate that it is found at themuscle membrane although its exact relationship to theother sarcoglycans and the DGC is unclear (Ettinger etal., 1997). e-sarcoglycan appears to be decreased in themdx mouse, which lacks dystrophin (Sicinski et al.,1989), but appears to be maintained in primary a- org-sarcoglycan deficiency (Duclos et al., 1998b; Hack etal., 1998).

Mutations in the SarcoglycansNomenclature reflecting the sarcoglycans subcom-

plex was proposed, and adhalin was renamed a-sarco-glycan. The 42-kD DAG (A3b) was termed b-sarcogly-can, and the gene encoding the 35-kD DAG was namedg-sarcoglycan. The propensity for mutations in these

genes to affect the musculature of the limbs andproximal trunk lead to the nomenclature ‘‘limb-girdlemuscular dystrophy’’ (LGMD). The LGMDs were fur-ther classified to reflect the genetic heterogeneity thatunderlies these disorders (Bushby and Beckmann,1995). LGMDs type 1 are autosomal dominant disor-ders. LGMDs type 2 are recessive disorders and includemutations in a-sarcoglycan (LGMD type 2D), b-sarcogly-can (LGMD type 2E), g-sarcoglycan (LGMD type 2C),and d-sarcoglycan (LGMD 2F). A summary of thesarcoglycans is shown in Table 1.

There is considerable variability in the phenotypeassociated with sarcoglycan mutations. Nonsense andframeshifting mutations tend to cause more disruptionof the sarcoglycan complex and, therefore, a moresevere clinical outcome (Bonnemann et al., 1995;Eymard et al., 1997; Lim et al., 1995; Noguchi et al.,1995). However, it must be noted that exceptions to thisgeneralization do exist. Particularly, missense muta-tions in b-sarcoglycan can be found in patients with asevere phenotype and nearly complete absence of alldetectable sarcoglycan staining at the membrane (Bon-nemann et al., 1996, 1998). The type of mutation doesnot account for all the variability associated with thephenotype in LGMD patients. A single common muta-tion is present in g-sarcoglycan (D521-T), and thismutation is associated with a variable clinical outcomeeven within a single family (McNally et al., 1996a).Therefore, other genetic factors and/or environmentalfactors may influence the phenotype of these disorders.Overall, the spectrum associated with sarcoglycan genemutations overlaps the spectrum associated with dystro-phin mutations in Duchenne and Becker MuscularDystrophy. One notable exception is the lack of cogni-tive impairment seen with sarcoglycan mutations (BenHamida et al., 1983; Ben Hamida and Hentati, 1989;Miladi et al., 1999). Normally, dystrophin is expressedin the central and peripheral nervous system. In con-trast, a-, b-, g-, and d-sarcoglycan are expressed incardiac and skeletal muscle. Thus, the sarcoglycancomplex is muscle specific, and its defects appearlimited to cardiac and skeletal muscle.

TABLE 1. The sarcoglycans*

Genelocation Disease

Type oftransmembrane

proteinExpression

pattern

SizeUnglycosylated size(glycosylation sites) Homology Other names

a 17q21 LGMD 2D Type I Skeletal 50 KD e-sarcoglycan AdhalinCardiac 41 KD 50 kD DAG

(2 N-linked) A2b 4q21 LGMD 2E Type II Skeletal 43 KD Weak homology to 43 kD DAG

Cardiac 31 KD g- and d-sarcoglycan A3bBrain (3 N-linked)Peripheral nerve

g 13q12 LGMD 2C Type II Skeletal 35 KD d-sarcoglycan 35 kD DAGCardiac 31 KD A4

(1 N-linked)d 5q33 LGMD 2F Type II Skeletal 35 KD g-sarcoglycan

Cardiac 31 KDSmoothPlacenta

(2 N-linked)

e 7q21 None described Type I All tissues 50 KD a-sarcoglycan45 KD(1 N-linked)

*Shown above are features of each of the sarcoglycan genes. Mutations in the sarcoglycan genes cause Limb Girdle Muscular Dystrophies (LGMDs). a ande-sarcoglycan are homologous type I transmembrane proteins. b-, g- and d-sarcoglycans are related type II transmembrane proteins with homology in theirextracellular carboxyl-termini. Shown in italics are patterns of mRNA expression that have not been confirmed for protein expression. Alternative mRNA splice formsare present for a-, d-, and e-sarcoglycan (see text).


Membrane-Cytoskeletal-Extracellular MatrixInteractions and the DGC

In addition to sarcoglycan, the DGC includes dystro-phin, the syntrophins, dystroglycan, and sarcospan.The role of the DGC is not completely understood, butclues are emerging as to its potential role as a mechano-sensory transducer. Its location at the plasma mem-brane of heart and skeletal muscle suggests its role as acytoskeleton-membrane-extracellular matrix (ECM)crosslinker. Furthermore, the absence of the DGC inDMD is thought to result in a combination of mechani-cal and, potentially, nonmechanical defects that alterplasma membrane permeability (Brooks, 1998; Dupont-Versteegden et al., 1994; Fowler et al., 1990; Hack et al.,1999; Hayes et al., 1993; Hayes and Williams, 1996;Hayes and Williams, 1997; Hutter, 1992; Hutter et al.,1991; Law et al., 1995; Lynch et al., 1993; McArdle etal., 1991; Moens et al., 1993; Mokhtarian et al., 1999;Pasternak et al., 1995; Petrof et al., 1993; Sacco et al.,1992; Weller et al., 1990). In DMD, the DGC compo-nents including dystroglycan, the syntrophins, and thesarcoglycans are reduced at the plasma membrane as aresult of a primary defect in dystrophin (Ervasti et al.,1990; Ohlendieck and Campbell, 1991; Ohlendieck etal., 1993). Therefore, the mechanical and nonmechani-cal defects that arise in muscular dystrophy may occurin part from the loss of these proteins. Dystrophin andthe syntrophins, the cytoplasmic components of theDGC, can directly interact (Adams et al., 1993; Ahn andKunkel, 1995; Ahn et al., 1994; Castello et al., 1996;Peters et al., 1997; Suzuki et al., 1994, 1995; Yang et al.,1995). Syntrophin interacts with a number of otherelements including nitric oxide synthase (Hashida-Okumura et al., 1999; Venema et al., 1997), voltagegated sodium channels (Gee et al., 1998; Schultz et al.,1998), stress activated protein kinase-3 (Hasegawa etal., 1999), caveolin via NOS (Venema et al., 1997),calmodulin (Iwata et al., 1998), and phosphatidylinosi-tol 4,5-bisphosphate (Venema et al., 1997). Thus, thesyntrophins appear to form a subcomplex of the DGCthat interacts with the DGC by way of dystrophin andthe dystrophin-related molecules, the dystrobrevins(Froehner et al., 1997).

Dystroglycan is composed of two subunits, a and b. a-and b-dystroglycan are produced from a single gene andthe cleavage of a single protein precursor (Durbeej etal., 1998; Hemler, 1999; Henry and Campbell, 1996).The gene for human dystroglycan maps to chromosome3p25 (Ibraghimov-Beskrovnaya et al., 1993). Dystrogly-can is important in the early development of theembryo since mice homozygously lacking dystroglycandie at day 6 of development due to a defect in Reichert’smembrane, one of the earliest basement membranes inthe embryo (Williamson et al., 1997). Recent investiga-tion suggests that dystroglycan is important for base-ment membrane formation more generally as well(Henry and Campbell, 1998; Montanaro et al., 1999).The sarcoglycans appear to form another subcomplexwithin the DGC. The sarcoglycans can be isolated fromthe remainder of the DGC in the presence of b-octyl-Dglucoside (Yoshida et al., 1994). Most recently sarco-span, a 25-kD component of the DGC, was identified(Crosbie et al., 1997). Sarcospan is a member of thetetraspan family, a group of proteins thought to bemolecular facilitators. Other tetraspan proteins medi-

ate interactions among transmembrane proteins includ-ing the integrins (Maecker et al., 1997). Recent datasuggests that sarcoglycan is required for proper target-ing of sarcospan to the muscle plasma membrane(Crosbie et al., 1999).

Organization of SarcoglycanThe a-, b-, g-, and d-sarcoglycans together form a

tetrameric complex with a 1:1:1:1 stoichiometry (Junget al., 1996). Early studies of muscle biopsies fromLGMD patients suggested that a mutation in any onesarcoglycan produced instability of the remaining sarco-glycans (Bonnemann et al., 1995; Duggan et al., 1997;Lim et al., 1995; Noguchi et al., 1995). It is now clearthat this is an oversimplification, and that the excep-tions to this rule may provide clues as to the interac-tions within the sarcoglycan complex. Further studiesof additional patients, aided by studies of animalmodels of sarcoglycan deficiency, revealed that not allsarcoglycans are equally destabilized by a mutation inany one sarcoglycan. For example, LGMD-2C patientswith mutations in g-sarcoglycan frequently retain a-sar-coglycan present at the membrane (Vainzof et al.,1996). Thus, a-sarcoglycan may be less tightly associ-ated with g-sarcoglycan. Further supporting this idea,crosslinking studies from C2C12 cells indicate thata-sarcoglycan can be more easily separated from theremainder of the sarcoglycan complex (Chan et al.,1998). C2C12 cells express sarcoglycans upon differen-tiation from myoblasts to myotubes. Immunoprecipita-tion studies performed under increasing amounts ofdetergent were able to separate a-sarcoglycan from b,g, and d-sarcoglycan (Chan et al., 1998). Gel overlayexperiments also indicate that a-sarcoglycan and g-sar-coglycan do not associate as readily as the other sarco-glycan subunits (Sakamoto et al., 1997). Further sup-porting this notion is the observation that mutations ina-sarcoglycan (LGMD type 2D) may also be associatedwith residual g-sarcoglycan immunostaining (Vainzofet al., 1996). Whether this residual protein is functionalis unknown. A small study of patients with LGMDmutations suggests no clear correlate of residual sarco-glycan staining and clinical outcome (Vainzof et al.,1996).

Although such indirect immunofluorescent stainingstudies may be difficult to quantify, a secondary de-crease in dystrophin has been observed in g-sarcogly-can patients (Vainzof et al., 1996). This may imply adirect association between g-sarcoglycan and dystro-phin, although no such direct interaction has beendemonstrated. Crosslinking studies from C2C12 myo-tubes indicated that d-sarcoglycan may interact di-rectly with b-dystroglycan. Furthermore, these studiessupport the idea that b- and d-sarcoglycan are tightlyassociated (Chan et al., 1998). Immunostaining ofpatients with b- and d-sarcoglycan mutations alsosupport the idea that these two subunits of the sarcogly-can complex are more tightly associated (Vainzof et al.,1996). Finally, using a heterologous expression systemwith tagged proteins, Holt and Campbell (1998) showedthat in order for efficient and proper targeting to theplasma membrane the four sarcoglycans, a, b, g, and d,need to be expressed. To summarize these data, d-sarco-glycan may be closely linked to dystroglycan, whileg-sarcoglycan may be in proximity to dystrophin. b-, g-,and d-sarcoglycan, and, in particular, b- and d-sarcogly-


can are tightly associated while a-sarcoglycan may beless tightly linked to the complex (Fig. 1). The relation-ship of e-sarcoglycan to the other sarcoglycans is un-known.

ANIMAL MODELS OF SARCOGLYCANDEFICIENCY

While human mutation studies have been instrumen-tal in the identification of the sarcoglycans, such stud-ies are limited by adequate material for study. Animalmodels, lacking each of the sarcoglycans, provide aunique opportunity to investigate the complex physiol-ogy and molecular biology of sarcoglycan-deficiency inways not possible in vitro or using patient biopsysamples. Furthermore, animal models are the idealsubstrate in which to initiate testing of new therapeuticstrategies including gene therapy.

Naturally occurring as well as induced mutations inthe murine dystrophin gene result in a myopathicphenotype similar to Duchenne/Becker muscular dystro-phy (Chapman et al., 1989; Cox et al., 1993; Sicinski etal., 1989). The study of these mice, as well as otheranimal models of DMD/BMD (Carpenter et al., 1989;Cooper et al., 1988), has been extremely valuable to ourunderstanding of how perturbations in dystrophin andthe DGC result in muscle degeneration. At present,there are animal models of three of these diseases,LGMD-2D, LGMD-2C, and LGMD-2F, which are theresult of mutations in the a-, g- and d-sarcoglycangenes, respectively. In the case of a- and g-sarcoglycan,gene targeting in the mouse was used to produce thesemutations (Duclos et al., 1998b; Hack et al., 1998). Anaturally occurring deletion in the Syrian hamsterd-sarcoglycan gene provides a model of d-sarcoglycandeficiency (Homburger et al., 1962; Nigro et al., 1997).

Cardiomyopathic Hamster as a Model ford-Sarcoglycan Deficiency and LGMD-2F

Prior to the discovery of the dystrophin-glycoproteincomplex, the cardiomyopathic hamster (CMH) wascharacterized as an animal model of both cardiac andskeletal myopathy (Homburger et al., 1962). It wasafter the identification of dystrophin and the DGC thatthe relationship between the CMH and sarcoglycanbegan to evolve. Biochemical analyses demonstrated adeficit of the 50-kD dystrophin-associated protein (a-sarcoglycan) as well as a defect in the association of theDGC with dystrophin (Iwata et al., 1993ab; Roberds etal., 1993b). Further investigation revealed that a 43-and 35-kD dystrophin-associated protein (b- and g/d-sarcoglycan) were also deficient in the CMH (Mizuno etal., 1995). A deletion in the region of the promoter andfirst exon of d-sarcoglycan was found in the CMH andestablished it as the first animal model of sarcoglycandeficiency (Nigro et al., 1997).

Recent work has delineated the CMH mutation moreprecisely. A 30-kb deletion encompasses two of the threealternative first exons of d-sarcoglycan (Sakamoto etal., 1999). Although these exons do not contain codingsequences, they are each expressed in a tissue-re-stricted fashion (Fig. 4). Specifically, exon 1A is ex-pressed in heart and weakly in stomach (smoothmuscle); exon 1B is expressed in heart, skeletal muscleand stomach; and exon 1C is expressed in heart,skeletal muscle, and most strongly in stomach. Exons1B and 1C are deleted in the CMH and as a result little

d-sarcoglycan message is detected in heart and skeletalmuscle (Nigro et al., 1997; Sakamoto et al., 1999). Theremaining message seen in the CMH heart can beascribed to exon 1A. Furthermore, d-sarcoglycan tran-scripts with exon 1A and exon 2 (the first coding exon)can still be detected in stomach from the CMH, suggest-ing that d-sarcoglycan protein may still be produced atdetectable levels in stomach smooth muscle (Sakamotoet al., 1999).

Since the initial identification of the cardiomyopathichamster (BIO1.50), a number of sub-strains have beendeveloped that display either a dilated or hypertrophiccardiomyopathy (Jasmin and Eu, 1979; Sole, 1986). Allof these strains possess the identical d-sarcoglycanmutation (Sakamoto et al., 1997). Thus, the alterationin phenotype must arise from differences in the geneticbackground of each substrain. In the future, the identi-fication of genetic modifier loci may reveal new genescritical to the development and severity of cardiomyopa-thy.

Phenotypically, the CMH is characterized by myocar-dial and skeletal muscle necrosis beginning at 1 to 2months of age (Homburger et al., 1962). Successiverounds of degeneration and regeneration result inmuscular dystrophy. The cardiomyopathy in these ani-mals is striking and often lethal. Altered calciumhandling in the CMH heart has been implicated in thedevelopment of disease.Administration of calcium chan-nel blockers has been shown to reduce the appearanceof cardiomyopathy (Bhattacharya et al., 1982, 1987;Johnson and Bhattacharya, 1993; Watanabe et al.,1998). The mechanism leading to this alteration incalcium levels is unclear.

Vital staining with Evans blue dye reveals mem-brane permeability defects in the CMH that are similarto those seen in the dystrophin-deficient mdx mouse(Matsuda et al., 1995; Straub et al., 1997, 1998). Thesemembrane disruptions may be the source of calciumelevation in CMH muscle and thus may be causative inits dramatic cardiac disease. However, specific defectsin the electrophysiologic or biochemical properties of anumber of ion channels in CMH muscle have also beendescribed. These defects include alterations in theactivity or expression of sarcoplasmic reticulum (SR)Ca21-release channels (Finkel et al., 1992; Ueyama etal., 1998), SR and sarcolemmal calcium ATPases (Kuoet al., 1987, 1992; Panagia et al., 1984), Na1/Ca1

exchanger (Panagia et al., 1986), L-type calcium chan-nels (Thuringer et al., 1996b), and potassium channelsIto1, IKr, and IK1 (Lodge and Normandin, 1997; Thur-inger et al., 1996a).

The primary defect in d-sarcoglycan in the CMHhamster results in a secondary decrease in or absence ofthe other sarcoglycans, a, b, and g (Iwata et al., 1993b;Mizuno et al., 1995; Roberds et al., 1993b). It has beenreported that the expression of a- and b-dystroglycanare compromised in the CMH (Holt et al., 1998; Iwataet al., 1993b; Roberds et al., 1993b; Sakamoto et al.,1997; Straub et al., 1998). Yet others have also reportedthat one or both of the dystroglycans remain normallyexpressed (Mizuno et al., 1995; Straub et al., 1998).Nevertheless, it has been suggested that the absence ofsarcoglycan results in a defective association of dystro-phin with the remaining glycoproteins of the DGC(Iwata et al., 1993a,b), and that the association be-tween a-dystroglycan and b-dystroglycan may be weak-


ened by the reduction in sarcoglycan (Holt et al., 1998;Straub et al., 1998).

Three groups have recently reported stable restora-tion of the sarcoglycan complex using a d-sarcoglycan-containing adenovirus or adeno-associated virus (Greel-ish et al., 1999; Holt et al., 1998; Li et al., 1999). In thissystem, the restoration of d-sarcoglycan induces thestable formation of the sarcoglycan heterotetramer(Greelish et al., 1999; Holt et al., 1998; Li et al., 1999)and reverses the susceptibility of CMH muscle to Evansblue uptake, a marker of increased damage permeabil-ity (Greelish et al., 1999; Holt et al., 1998). Interest-ingly, overexpression of d-sarcoglycan in the cytoplasmof individual transduced muscle cells results in thereturn of the other sarcoglycans at the membrane (Li etal., 1999). Furthermore, effective local delivery of virusthrough the circulatory system can be achieved withthe addition of histamine to increase vascular perme-ability (Greelish et al., 1999). In all three cases, im-

mune response to the infection and expression of d-sar-coglycan appeared minimal (Greelish et al., 1999; Holtet al., 1998; Li et al., 1999). In one study where it wasstudied longer term, expression was sustained for atleast 6 months (Holt et al., 1998). The sustainedexpression and apparent minimal immune response isstrikingly different than similar experiments replacingdystrophin in the mdx mouse (Karpati et al., 1997).This difference may reflect altered immune propertiesin the hamster or tolerance from low level residuald-sarcoglycan expression in the CMH.

gsg2/2 Mice as a Model for g-SarcoglycanDeficiency and LGMD-2C

Mice lacking g-sarcoglycan (gsg-/-) have been gener-ated by homologous recombination in embryonic stemcells and are the only animal model described to datewith a primary deficiency of g-sarcoglycan (Hack et al.,1998). These animals show dystrophic changes in both

Fig. 4. Alternative splicing of d-sarcoglycan first exons in thecardiomyopathic hamster (CMH, Syrian BIO 14.6). d-sarcoglycan usesthree alternatively spliced first exons, labeled E1A, E1B and E1C.Each first exon is spliced to exon 2 (E2) in a tissue specific pattern(Sakamoto et al., 1999). Both the larger (.100 kb) locus and anenlargement of the deleted region are depicted. A deletion of 28.9 kb

affects the first exons as shown. Tissue expression for each alternativeexon 1 is depicted below that exon for heart, skeletal muscle, andstomach smooth muscle. -, no expression detected; 1 low expressionlevel; 11 moderate expression level; 111, high expression level; skmuscle, skeletal muscle; sm muscle, smooth muscle.


skeletal muscle and heart that are representative of thepathology seen in LGMD-2C and similar to the cardio-myopathic hamster. Because they show pathology inboth skeletal and cardiac muscle, gsg-/- mice are anexcellent model of the proximal muscle disease seen inLGMD-2C and the cardiac disease that is increasinglybeing recognized as an integral component of DGC-mediated muscular dystrophy (Fadic et al., 1996; Mel-acini et al., 1999; van der Kooi et al., 1998). gsg-/-miceon a mixed genetic background show increased mortal-ity that is likely due to cardiomyopathy. Similar tocardiomyopathy in CMH, cardiomyopathy in gsg-/- micemay be dilated or hypertrophic (Fig. 5) (Hack et al.,1998; Sakamoto et al., 1997). The availability of mul-tiple strains in the mouse as well as genetic maps andmarkers may permit the mapping of genetic modifierloci using gsg-/- mice.

Mice lacking g-sarcoglycan have been valuable forexpanding our understanding of the cellular conse-quences of sarcoglycan deficiency. gsg-/- muscle shows anotable increase in the rate of myonuclear apoptosis,suggesting that programmed cell death may play a rolein the pathogenesis of LGMD. Like mice lacking dystro-phin (mdx) and the cardiomyopathic hamster (BIO14.6), membrane disruptions are a feature of gsg-/-

muscle as assessed by Evans blue uptake and therelease of muscle creatine kinase into the blood stream(Hack et al., 1998). Significantly, the expression ofdystrophin, dystroglycan, and laminin appears unaf-fected by the loss of g-sarcoglycan when assessed byimmunostaining of skeletal and cardiac muscle (A.A.Hack and E.M. McNally, unpublished observations).Therefore the absence of g-sarcoglycan is likely suffi-cient to cause muscle degeneration independent of thedystrophin-dystroglycan-laminin mechanical link. Al-though the absence of dystrophin may confer furtherdeficits on DMD muscle, it should be noted that thesecondary absence of sarcoglycan appears sufficient toaccount for all of the pathology seen in the mdx mouseand possibly, in human disease as well.

Sgca-Null Mice as a Model fora-Sarcoglycan-Deficiency and LGMD-2D

Recently, mice lacking a-sarcoglycan (Sgca-null) weregenerated (Duclos et al., 1998b). Like mice lackingg-sarcoglycan, Sgca-null mice show progressive muscu-lar dystrophy similar to human LGMD. Like LGMD-2Dpatients, cardiomyopathy does not seem to be a promi-

nent feature of Sgca-null mice (Duclos et al., 1998b;Melacini et al., 1999). These animals are a valuableresource, which are interesting not only as a model ofthe human disease LGMD-2D but also in comparison togsg-/- mice with which they share both similarities andnotable differences (Hack et al., 1998).

Sgca-null mice show membrane permeability defects,increased Evans blue uptake, and serum pyruvatekinase elevation, similar to the CMH and gsg-/- mice.Thus, membrane permeability changes appear to be ageneral feature of sarcoglycan and dystrophin defi-ciency. This having been said, it is not clear from any ofthese models whether or not changes in plasma mem-brane integrity are causative for muscle degenerationor simply a result of it.

Not surprisingly, the loss of a-sarcoglycan has conse-quences for the stability of the other sarcoglycans. Infact, it appears that when a-sarcoglycan is absent, b-,g-, and d-sarcoglycan fail to be targeted to the plasmamembrane (Duclos et al., 1998b). This is also true forsarcospan, a recently identified tetraspan moleculethat is an integral component of the DGC and appearsto require the sarcoglycans for its stabilization andmembrane localization (Crosbie et al., 1999, 1997;Duclos et al., 1998b). e-sarcoglycan is unaffected by theabsence of the other sarcoglycans in both gsg-/- andSgca-null mice (Duclos et al., 1998b; Hack et al., 1998).Taken together, these findings are similar but notidentical to those reported for gsg-/- mice. However,both point to a specific disruption of the integrity of thesarcoglycan complex as a result of either a- or g-sarco-glycan deficiency.

One interesting difference between these two mousemodels is that Sgca-null mice appear to express lessdystrophin and a-dystroglycan at the sarcolemma thannormal animals. This decrease in dystrophin is accom-panied by an increase in utrophin expression, presum-ably as a compensatory mechanism (Duclos et al.,1998b). Although the molecular basis for these differ-ences between gsg-/- and Sgca-null mice is unclear, theylikely reflect heterogeneity in the interactions betweenmembers of the sarcoglycan complex as well as in theirrelationship to dystrophin and dystroglycan. For ex-ample, a-sarcoglycan may interact with dystrophinand/or dystroglycan and be critical for their localizationor function. The residual a-sarcoglycan seen gsg-/-

muscle may be sufficient to perform this function and

Fig. 5. Cardiomyopathy in mice lacking g-sarcoglycan. gsg-/- miceon a mixed genetic background show cardiomyopathy that can beeither dilated (A) or hypertrophic (B) compared to normal controls (C).The presence of both left and right ventricular disease suggests an

intrinsic role for sarcoglycan in the heart. Like the cardiomyopathichamster, this variation in disease expression may be influenced bygenetic modifier loci. Cardiomyopathy is a feature of human LGMD-2Cas well as gsg-/- mice.


thus dystrophin and dystroglycan are normal in gsg-/-

mice.

THEORIES OF PATHOGENESISMechanical-Weakness and

Contraction-Induced DamageIt has long been suspected that muscular dystrophy

is a disease of enhanced susceptibility to mechanically-induced plasma membrane disruption (Carpenter andKarpati, 1979; Mokri and Engel, 1975; Roses et al.,1975; Schmalbruch, 1975; Schotland et al., 1977). Inthis model, muscle lacking one or more proteins isrendered more vulnerable to mechanical injury. Sarco-lemmal disruption would then result in the influx ofcalcium (as well and the influx and outflow of a numberof other critical ions and proteins), the activation ofnumerous proteases and subsequent segmental necro-sis (Petrof, 1998). The identification of dystrophin asthe protein product of the Duchenne muscular dystro-phy locus and a component of the cortical actin cytoskel-eton of muscle added strength to this hypothesis(Burghes et al., 1987; Monaco et al., 1986). Studies ofisolated muscle and primary muscle cultures from mdxmice that lack dystrophin identified mechanical defectsat the single muscle and cellular levels, respectively(Brooks, 1998; Moens et al., 1993; Mokhtarian et al.,1999; Pasternak et al., 1995; Petrof et al., 1993; Welleret al., 1990). Furthermore, these findings have alsobeen corroborated in vivo by exercise and immobiliza-tion protocols designed to assess the role of mechanicalstress the degeneration of dystrophin-deficient muscle(Brooks, 1998; Brussee et al., 1997, 1998; McCully etal., 1991; Mokhtarian et al., 1999; Vilquin et al., 1998).

Sarcoglycan deficiency is a feature of both DMD andLGMD 2C-F. Therefore, the loss of sarcoglycan may be amajor pathologic mediator in both groups of severemuscular dystrophies. Curiously, mechanical defectsand elevated calcium levels do not appear to be afeature of the loss of g-sarcoglycan (Hack et al., 1999;Hassoni and Cullen, 1999). These findings support anonmechanical role of g-sarcoglycan. Defects in thisnonmechanical function are likely present in both DMDand LGMD 2C-F.

Specific Defect in the Regulationof Calcium Channels

At the same time that membrane damage was firstobserved in DMD patient muscle biopsy samples, it wasalso noted that intramyocyte calcium was elevated inthese tissues (Bodensteiner and Engel, 1978; Finger-man et al., 1984; Mongini et al., 1988). This elevation in[Ca]i was suspected to be a secondary consequence ofmembrane tearing. However, there remains some de-bate about the accuracy of these observations as well asthe presence of elevated [Ca]I (Gailly et al., 1993; Head,1993; Pressmar et al., 1994; Rivet-Bastide et al., 1993).With progress in patch-clamp recording, and the identi-fication of the mdx mouse as a model of dystrophin-deficiency (Sicinski et al., 1989), electrophysiologic inter-rogation of ion homeostasis in the absence of dystrophinshowed striking, specific defects in both calcium leakchannels (Hopf et al., 1996; Turner et al., 1991, 1993)and in stretch-inactivated calcium channels (Francoand Lansman, 1990; Franco-Obregon and Lansman,1994) in mdx myotubes in culture.

These findings suggested that the mechanism of celldeath in muscular dystrophy might be more compli-cated than previously suspected. Although no directinteraction between dystrophin or the DGC and eitherchannel has been demonstrated, it remains plausiblethat they are either directly or indirectly regulated bysome component of the DGC, possibly sarcoglycan.Furthermore, the interaction between syntrophin andvoltage gated sodium channels further emphasizes thepotential for DGC-ion channel interactions. Sarcogly-can is well positioned at the plasma membrane to be adirect or indirect modulator of calcium flux.

Sarcoglycan as an IndependentSignaling Modulator/Regulator

Sarcoglycan may be an independent signaling mod-ule that is simply localized in the membrane by dystro-phin but functions separately from the other compo-nents of the DGC. This signaling activity may regulatecalcium channels, as suggested above, or perform otherunknown functions critical to muscle survival. Theprimary structure of sarcoglycan is reminiscent of a cellsurface receptor although its potential ligands, bothintra- and extracellular are not known. Each of thesarcoglycan subunits appears to be serine phosphory-lated (Yamamoto et al., 1993). g-, b- and d-sarcoglycaneach contain an epidermal growth factor-like domain attheir extreme carboxyl-terminus that may be a bindingsight for a yet unidentified ligand (Fig. 3) (McNally etal., 1996b).

A potential interaction between the sarcoglycans andthe integrins has been described (Yoshida et al., 1998).Immunoprecipitation studies from the rat cell line,L6E9, showed an association with integrin a5b1, aprominent integrin in muscle, and the sarcoglycans.Moreover, in culture conditions that favor integrinsignaling (RGD peptide), an increase in tyrosine phos-phorylation was noted for a and g-sarcoglycan. Anti-sense treatment of the cells with anti-a and anti-gsarcoglycan oligonucleotides reduced focal adhesionproteins. In this way, sarcoglycan may be involved inbidirectional signaling and regulation of the integrina5b1 in striated muscle (Yoshida et al., 1998). It isintriguing to speculate that sarcospan may play a rolein the integrin-DGC interaction, as tetraspan proteinshave previously been shown to be involved in couplingintegrins (as well as other molecules) to other signalingcomplexes (Maecker et al., 1997; Wright and Tomlin-son, 1994). Sarcoglycan and sarcospan appear to beclosely associated and both are absent when sarcogly-can is disrupted (Crosbie et al., 1999). Thus, the ab-sence of sarcoglycan/sarcospan could be sufficient touncouple the dystrophin-dystroglycan axis from theintegrins in skeletal and cardiac muscle. In this way,muscle degeneration induced by either dystrophin orsarcoglycan mutations would result from a disruptionof the proper coupling between these two independent,integrated mechanical and signaling complexes.

Recent work has identified an ecto-ATPase domainwithin a-sarcoglycan which may represent its functionat the sarcolemma (Betto et al., 1999). In this way,a-sarcoglycan may serve to modulate periplasmalem-mal extracellular ATP concentration which in turnregulate the activity of purinergic receptors on skeletaland cardiac muscle. This possibility is particularly


intriguing since the P2X7 receptor has been shown tobe present in skeletal and cardiac muscle and to becapable of conducting calcium as well as sufficient toinduce apoptosis in non-muscle cell types (Rassendrenet al., 1997; Schulze-Lohoff et al., 1998; Surprenant etal., 1996). Thus, the secondary reduction in a-sarcogly-can-mediated ecto-ATPase activity in gsg-/- musclemight be sufficient to induce pore formation by P2X7and explain the apoptosis seen in this model of LGMD.

Integrated Mechanosensory SystemA coupled system of mechanical resistance and signal

induction is widely believed to exist in muscle as well asother tissues (Chicurel et al., 1998). The sarcoglycancomplex may serve the role of integrating mechanicalinputs from the dystrophin-dystroglycan complex withother sensors and transducers of similar signals inmuscle. In this way, the DGC would be both a mechani-cally important linkage between the actin cytoskeletonand the basement membrane as well as a sensor ofmechanical stress generated along this axis. Thus, theabsence of sarcoglycan and possibly, sarcospan, couldbe sufficient to uncouple the dystrophin-dystroglycanaxis from the integrins in skeletal and cardiac muscle.In this way, muscle degeneration induced by eitherdystrophin or sarcoglycan mutations would result froma disruption of the proper coupling between these twoindependent, integrated mechanical and signaling com-plexes.

CONCLUDING REMARKSDramatic advances have been made in the last 5

years since the first discovery of a-sarcoglycan. Fourdifferent genes involved in human disease have beenidentified, and additionally, a new concept of the DGChas evolved (Fig. 1). Future efforts at therapy for the allmuscular dystrophies will focus on restoring the DGCin all its roles, mechanical and sensory. The existence ofnew animal models will greatly advance the testing andimplementation of these therapies as well as advanceour knowledge of membrane, cytoskeleton, and extracel-lular matrix interactions.

ACKNOWLEDGMENTSWe acknowledgeAmy Murphy for her help in prepara-

tion of this manuscript. A. A. Hack is supported by theMedical Scientist Training Program and by a predoc-toral fellowship from the American Heart Association,Midwest Affiliate, Inc. E. M. McNally is a CulpeperMedical Scholar.

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