Loss of the sarcoglycan complex and sarcospan leads to muscular dystrophy in {beta}-sarcoglycan-deficient mice

doi:10.1093/hmg/8.9.1589

This Article

	Abstract
	FREE Full Text (PDF)
	Alert me when this article is cited
	Alert me if a correction is posted

Services

	Email this article to a friend
	Similar articles in this journal
	Similar articles in ISI Web of Science
	Similar articles in PubMed
	Alert me to new issues of the journal
	Add to My Personal Archive
	Download to citation manager
	Search for citing articles in: ISI Web of Science (77)
	Request Permissions

Google Scholar

	Articles by Araishi, K.
	Articles by Ozawa, E.
	Search for Related Content

PubMed

	PubMed Citation
	Articles by Araishi, K.
	Articles by Ozawa, E.

Social Bookmarking

	What's this?

Loss of the sarcoglycan complex and sarcospan leads to muscular dystrophy in [beta]-sarcoglycan-deficient mice

Human Molecular Genetics Pages 1589-1598 ©1999 Oxford University Press

Loss of the sarcoglycan complex and sarcospan leads to muscular dystrophy in [beta]-sarcoglycan-deficient mice
Introduction
Results
   Generation of BSG^-^/- mice
   BSG^-^/- mice exhibit a hypertrophic appearance and develop progressive muscular dystrophy
   The immunoreactivities of all sarcoglycans and sarcospan are lost in the sarcolemma of BSG^-^/- mice
   Sarcoglycans and sarcospan were practically undetectable in the membrane fractions of BSG^-^/- mice, although their mRNAs, except that for [beta]-sarcoglycan, were present
   The dystrophin-dystrophin-associated protein (DAP) complex in BSG^-^/- mice
Discussion
   BSG^-^/- mice develop muscular dystrophy
   [alpha]-, [beta]-, [gamma]- and [delta]-sarcoglycan as well as sarcospan were absent in the BSG^-^/- sarcolemma
   Dystrophin-DAP complex in the skeletal muscle of BSG^-^/- mice
Materials And Methods
   Targeting vector construction and transfection of the ES cell
   Generation of BSG^-^/- mice
   Northern blot analysis
   Antibodies
   Light microsome preparation
   Histology and immunohistochemistry
   Vital staining with EBD
   Serum CK assay
   Affinity chromatography
   Immunoprecipitation by anti-[alpha]-sarcoglycan, anti-[beta]-dystroglycan and anti-dystrophin antibodies
   Statistical analysis
Acknowledgements
References

Loss of the sarcoglycan complex and sarcospan leads to muscular dystrophy in [beta]-sarcoglycan-deficient mice

Kenji Araishi¹^{, 2}, Toshikuni Sasaoka¹, Michihiro Imamura¹, Satoru Noguchi¹^{, 3}, Hiroshi Hama¹, Eriko Wakabayashi¹, Mikiharu Yoshida¹, Tetsuro Hori², Eijiro Ozawa¹^{, +}

¹Division of Cell Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, 187-8502, Japan, ²Department of Physiology, Faculty of Medicine, Kyushu University, Fukuoka, 812-8582, Japan and ³Inheritance and Variation Group, PRESTO, Japan Science and Technology Corporation, Kawaguchi, Saitama, 332-0012, Japan

Received May 7, 1999; Revised and Accepted June 28, 1999

DDBJ/EMBL/GenBank accession nos AB024920-AB024923

[beta]-Sarcoglycan, one of the subunits of the sarcoglycan complex, is a transmembranous glycoprotein which associates with dystrophin and is the molecule responsible for [beta]-sarcoglycanopathy, a Duchenne-like autosomal recessive muscular dystrophy. To develop an animal model of [beta]-sarcoglycanopathy and to clarify the role of [beta]-sarcoglycan in the pathogenesis of the muscle degeneration in vivo, we developed [beta]-sarcoglycan-deficient mice using a gene targeting technique. [beta]-Sarcoglycan-deficient mice (BSG^-^/- mice) exhibited progressive muscular dystrophy with extensive degeneration and regeneration. The BSG^-^/- mice also exhibited muscular hypertrophy characteristic of [beta]-sarcoglycanopathy. Immunohistochemical and immunoblot analyses of BSG^-^/- mice demonstrated that deficiency of [beta]-sarcoglycan also caused loss of all of the other sarcoglycans as well as of sarcospan in the sarcolemma. On the other hand, laminin-[alpha]2, [alpha]- and [beta]-dystroglycan and dystrophin were still present in the sarcolemma. However, the dystrophin-dystroglycan complex in BSG^-^/- mice was unstable compared with that in the wild-type mice. Our data suggest that loss of the sarcoglycan complex and sarcospan alone is sufficient to cause muscular dystrophy, that [beta]-sarcoglycan is an important protein for formation of the sarcoglycan complex associated with sarcospan and that the role of the sarcoglycan complex and sarcospan may be to strengthen the dystrophin axis connecting the basement membrane with the cytoskeleton.

INTRODUCTION

Sarcoglycans, the proteins involved in sarcoglycanopathy (SGP), are transmembranous proteins. [alpha]-, [beta]-, [gamma]- and [delta]-sarcoglycan associate together to form a hetero-tetrameric complex, called the sarcoglycan complex (SGC), and are present in the sarcolemma (1). The SGC comprises a dystrophin-glycoprotein complex (DGC) together with dystrophin and the dystroglycan complex (1-6). The DGC has been considered as the mechanical link between the basement membrane and the intracellular cytoskeleton (5-10). However, the role of the SGC in preventing muscular dystrophy remains to be clarified.

On the basis of immunohistochemical data and other studies, Duchenne-like or severe childhood autosomal recessive muscular dystrophy (SCARMD) (11) was predicted to be caused by mutation of any one of the sarcoglycan genes, which gives rise to loss of or a great reduction in the entire SGC (7,12-13). This was realized from the finding that any of the sarcoglycan genes can be responsible for SCARMD (14-18). Thus, the diagnostic term SGP was introduced. However, problems such as why the entire SGC is lost or greatly reduced when one of the sarcoglycan genes is mutated and why muscular dystrophy ensues following loss of or a great reduction in the SGC have not been clarified. To resolve these problems, animal models of SGP are considered necessary.

There are three animal models of SGP. [alpha]-Sarcoglycan-deficient mice (19) and [gamma]-sarcoglycan-deficient mice (20,21) were developed by a gene targeting technique. A naturally occurring dystrophic hamster was found to have a mutation in the [delta]-sarcoglycan gene (22-24). However, there is no animal model of [beta]-SGP. Absence of an animal model of [beta]-sarcoglycan deficiency is the reason why the role of [beta]-sarcoglycan in the skeletal muscle has not been well characterized and the pathogenesis of [beta]-SGP remains unclarified.

In this study, to develop an animal model of [beta]-SGP and to investigate the pathogenesis of SGP by both immunohistochemical and biochemical methods in the case of [beta]-sarcoglycan deficiency, we generated [beta]-sarcoglycan-deficient (BSG^-^/-) mice. BSG^-^/- mice developed muscular dystrophy with hypertrophic appearance resembling the features in human SGP patients. Vital staining with Evans blue dye (EBD) of the muscles (25) in BSG^-^/- mice revealed the presence of EBD-containing muscle fibers in most skeletal muscles. Serum creatine kinase (CK) activity in BSG^-^/- mice was 100 times higher than that in their wild-type littermates. Immunohistochemical and biochemical analyses revealed that [beta]-sarcoglycan deficiency also led to the loss of all of four sarcoglycans as well as of sarcospan. Furthermore, the dystrophin-dystroglycan complex was unstable in the absence of the SGC and sarcospan. The SGC and sarcospan are thought to play an important role in stabilizing the dystrophin axis connecting the basement membrane and the cytoskeleton.

RESULTS

Generation of BSG^-^/- mice

We cloned and characterized the mouse [beta]-sarcoglycan gene. We constructed a targeting vector designed to disrupt exon 2 of this gene, which encodes the intracellular and membrane-spanning domains (Fig. 1a). Embryonic stem (ES) cell colonies that survived G418 and gancyclovir selection were analyzed by Southern blot analysis for the presence of homologous recombinants using 5[prime] and 3[prime] probes (Fig. 1a). Mutated ES cell clones were examined for integration of the single neomycin resistance (neo) gene sequence by hybridization to a neo probe. Homologous recombinants were injected into C57BL/6J blastocysts to generate chimeric mice. Chimeric males were mated with C57BL/6J females to generate the [beta]-sarcoglycan heterozygous mutant (BSG^+/-) mice through germline transmission. BSG^+/- mice were identified by Southern blotting and were interbred to obtain homozygous mutant (BSG^-^/-) mice (Fig. 1b). The genotype ratios of offspring from heterozygote matings were 23.8% (78/328) homozygous mutant, 51.8% (170/328) heterozygous mutant and 24.4% (80/328) wild-type genotype at 4 weeks of age. Since these ratios were close to a Mendelian distribution, there was no apparent survival disadvantage of BSG^-^/- mice.

Figure 1. Generation of BSG^-^/- mice by gene targeting. (a) Restriction map of the wild-type [beta]-sarcoglycan gene locus (top), the targeting vector (middle) and the targeted locus (bottom). A genomic region of 2.0 kb including exon 2 (E2) was deleted and replaced by a phosphoglycerate kinase promoter-driven neomycin phosphotransferase gene (NEO). The 5[prime]- and 3[prime]-flanking probes and the NEO probe used for Southern blot analysis are indicated. N, NcoI; HSV-TK, herpes simplex virus thymidine kinase gene. (b) Southern blot analysis of NcoI-digested genomic DNA from mouse tail biopsy samples obtained from littermates. Using the 5[prime]-flanking probe, the targeted locus gave an NcoI-digested fragment of 11.5 kb, whereas the wild-type allele gave a 21 kb band. +/+, wild-type; +/-, heterozygous; -/-, homozygous mutant. (c) Northern blot analysis. Exon 6 of [beta]-sarcoglycan cDNA hybridized with the correct sized transcript in 80 µg of total RNA extracted from the skeletal muscle of wild-type (+/+) and heterozygotes (+/-); homozygous (-/-) mutant tissue showed no [beta]-sarcoglycan transcript (top). As a control for equal loading, the same membrane was rehybridized with a G3PDH cDNA probe (bottom). (d) Western blot analysis. Using monoclonal antibody 5B1 against [beta]-sarcoglycan, [beta]-sarcoglycan was detected in the microsomal preparations of wild-type (+/+) and heterozygous (+/-) skeletal muscles, but not in the microsomal preparations of homozygous mutant (-/-) skeletal muscle.

To confirm that the mutation was a null mutation, we carried out northern blot analysis with exon 6 of [beta]-sarcoglycan cDNA as the probe and western blot analysis with anti-[beta]-sarcoglycan antibody. [beta]-Sarcoglycan mRNA was detected in wild-type and BSG^+/- mice but not in BSG^-^/- mutant mice on northern blotting (Fig. 1c). In western blot analysis, using monoclonal antibody 5B1 against [beta]-sarcoglycan, the 43 kDa [beta]-sarcoglycan protein was clearly detected in microsomal preparations from both wild-type and BSG^+/- mice, but not in those from BSG^-^/- mice (Fig. 1d).

BSG^-^/- mice exhibit a hypertrophic appearance and develop progressive muscular dystrophy

Intuitively, BSG^-^/- mice did not show any obvious difference from wild-type and BSG^+/- littermates up to 8 weeks of age. However, after 8 weeks of age BSG^-^/- mice showed a brawny appearance with hypertrophic shoulders and limbs (Fig. 2a). When the skin was removed, it was apparent that the limbs of 20-week-old BSG^-^/- female mice were hypertrophic and larger than those of their wild-type littermates (Fig. 2b and c) and the mass of their tibialis anterior (TA) muscle was about one and a half times higher than that of their wild-type littermates (Fig. 2d). The body weights of 20-week-old BSG^-^/- female mice were greater than those of their wild-type littermates (Fig. 2e). To evaluate the degree of muscular hypertrophy in BSG^-^/- mice, the masses of TA, gastrocnemius and quadriceps femoris muscles of 20-week-old BSG^-^/- mice and their wild-type littermates were measured and compared with the total body mass of the mice (Table 1). The muscles of the BSG^-^/- mice were significantly larger than those of the wild-type mice. In the skinned limbs of BSG^-^/- mice, whitish stripes were observed (arrow in Fig. 2c and d). These whitish stripes were never seen in their wild-type or BSG^+/- littermates. These stripes were demonstrated to be degenerating muscle fibers by histological analysis.

Figure 2. Characterization of BSG^-^/- mice. (a) (Left) Wild-type (+/+) female mouse; (right) BSG^-^/- (-/-) female littermate. Compared with the wild-type littermate, the BSG^-^/- female mouse showed limb hypertrophy at 20 weeks of age (arrowheads). Hindlimb of a wild-type mouse (b) and of a BSG^-^/- mouse (c). Animals were killed by anesthesia overdose and skinned. It was apparent that the TA and the gastrocnemius of the BSG^-^/- mouse were hypertrophic compared with those of its wild-type littermate (arrowheads). Whitish stripes were prominent in the muscles of BSG^-^/- mice (c, arrow). These stripes were demonstrated to represent degenerating muscle fibers. (d) Dissected TA muscles. The TA muscle of BSG^-^/- mice (right) was hypertrophic and bigger than that of its wild-type littermate (left). The TA muscle of BSG^-^/- mice contained whitish stripes demonstrated to represent muscle fiber degeneration (arrow). (e) Body weight of 20-week-old wild-type (+/+) and BSG^-^/- (-/-) mice. Female BSG^-^/-mice were bigger than female wild-type mice (29.7 3.6 versus 24.1 2.0 g). *Significant differences (P < 0.01) between female wild-type and BSG^-^/- mice. All data are presented as means SD.

Table 1. Muscle mass relative to total body mass (percentage of muscle weight relative to total body weight)

Muscle type Wild-type (n = 5) BSG^-^/- (n = 5) Ratio BSG^-^/-/wild-type

Quadriceps femoris 0.93 0.13 1.25 0.19^a 1.34

Tibialis anterior 0.26 0.03 0.43 0.06^b 1.65

Gastrocnemius 0.87 0.08 0.92 0.24^a 1.05

All data are presented as means SD.
^aSignificant difference between wild-type and BSG^-^/- (P < 0.05).
^bSignificant difference between wild-type and BSG^-^/- (P < 0.01).

To examine the histological characteristics of the muscles in these mutant mice, frozen sections of the quadriceps femoris, TA, soleus and gastrocnemius muscles were stained with hematoxylin and eosin (H&E) and evaluated at 2, 3, 4, 6, 8, 14 and 20 weeks of age. At 2 weeks of age, muscle fiber degeneration was clearly found in BSG^-^/- mice. Infiltration of mononuclear cells and degenerating fibers with phagocytosis were observed in the soleus of BSG^-^/- mice (Fig. 3a). Regenerating myofibers, with basophilic cytoplasm and centrally located nuclei, were also observed in both the soleus and TA muscles of 2-week-old BSG^-^/- mice (Fig. 3a and b). At 3 weeks of age, the areas exhibiting degeneration in the soleus muscle of BSG^-^/- mice were further extended compared with those observed in mice of 2 weeks of age, and mononuclear cell infiltration and an increase in the mass of connective tissue were prominent (Fig. 3c). At 8 weeks of age, degenerative changes, including necrotic fibers with phagocytosis, infiltration of mononuclear cells and increase in the mass of connective tissue, were prominent in BSG^-^/- mice. Regenerative changes were also observed. Muscle fibers with centrally located nuclei surrounded the degenerating areas and there were regenerating myocytes with basophilic cytoplasm in the areas showing cell proliferation of the quadriceps femoris muscle of BSG^-^/- mice (Fig. 3d and e). At 20 weeks of age, in the quadriceps femoris muscle of BSG^-^/- mice >95 % of myofibers (2653/2731 fibers) contained central nuclei (Fig. 3f). In their wild-type littermates, the number of cells with centrally located nuclei never exceeded 5% (37/1665 fibers). Variously sized muscle fibers with centrally located nuclei were observed in BSG^-^/- mice at 20 weeks of age (Fig. 3f).

Figure 3. Histological analysis of H&E stained skeletal muscle and cardiac muscle of BSG^-^/- mice and assessment of muscle fiber degeneration by EBD assay. Soleus (a) and TA (b) muscles from 2-week-old BSG^-^/- mice. At the center, there were necrotic muscle fibers with phagocytosis (asterisk), mononuclear cell infiltration (arrow) and muscle fiber regeneration with central nucleation (b, arrowhead). (c) Soleus muscle from 3-week-old BSG^-^/- mice shows necrosis, regeneration, connective tissue proliferation and mononuclear cell infiltration. (d) Quadriceps muscle from 8-week-old BSG^-^/- mice. There was muscle fiber degeneration in clusters and regeneration with central nuclei in the adjacent area (asterisk). (e) Quadriceps muscle from 8-week-old BSG^-^/- mice. A necrotic muscle fiber with phagocytosis was observed (asterisk). There were regenerating muscle fibers (arrow) and an increase in the mass of connective tissue. (f) Quadriceps muscle from 20-week-old BSG^-^/- mice. All of the muscle fibers shown were regenerated fibers with central nuclei, with prominent size variability. Fiber splitting was also observed in BSG^-^/- mice (asterisk). (g) Vital staining with EBD revealed degenerating muscle fibers in the quadriceps muscles of BSG^-^/- mice. Mice at 8 weeks of age were injected i.p. with EBD. Dye inclusion was extensively detected in most of the skeletal muscles of BSG^-^/- mice. Dye inclusion was not detected in the wild-type (+/+) mice (data not shown). (h) Cardiac muscles from 56-week-old BSG^-^/- mice. Scattered patches of fibrosis (arrow) were observed mainly in the left ventricular wall. Bars: (a, b, c and e) 10 µm; (d and h) 100 µm; (f and g) 50 µm.

In the diaphragm of BSG^-^/- mice, degenerative changes, including mononuclear cell infiltration, and regenerative changes, including muscle fibers with centrally located nuclei, were observed at 2 and 10 weeks of age and muscle fibers with centrally located nuclei and an increase in the mass of connective tissue were prominent in the diaphragm of 20-week-old BSG^-^/- mice (data not shown).

To assess the damage to muscle fibers in vivo, we injected EBD i.p. into BSG mutant mice of 6-8 weeks of age and examined EBD-containing muscle fibers at the microscopic level. In wild-type and BSG^+/- mice, EBD-containing fibers, which were considered to be damaged, were not seen (data not shown), whereas EBD was present in many muscle fibers of the quadriceps femoris, soleus, gastrocnemius and diaphragm muscles of BSG^-^/- mice and dye-containing fibers were clustered in all the muscles examined (Fig. 3g). In the TA muscles of BSG^-^/- mice, on the other hand, only a few small clustered EBD-containing muscle fibers were found.

At 8 weeks of age, the serum CK activity in BSG^-^/- mice (15 578 2432 IU/l, mean SD, n = 5) was ~100 times higher than that in their wild-type littermates (164.6 76 IU/l, n = 5).

BSG^-^/- mice lived for >1 year. At 6 weeks of age, we microscopically observed a few EBD-containing cardiac muscle fibers, but did not observe other obvious dystrophic change in the hearts of the BSG^-^/- mice. However, at 56 weeks of age we microscopically observed many scattered patches of fibrosis in the heart wall of the BSG^-^/-mice after H&E staining (Fig. 3h), whereas no obvious lesions were observed in the those of wild-type littermates.

The immunoreactivities of all sarcoglycans and sarcospan are lost in the sarcolemma of BSG^-^/- mice

The presence of the SGC, dystroglycan complex, sarcospan, dystrophin, utrophin and laminin-[alpha]2 in the muscle of BSG^-^/- mice was examined immunohistochemically (Fig. 4). All of the sarcoglycan subunits as well as sarcospan were absent in the muscle of BSG^-^/- mice, whereas they were present in the cell membrane in the muscle of wild-type mice. Antibodies to all other proteins (dystrophin, utrophin, the dystroglycans and laminin-[alpha]2) stained the cell membranes in the muscles of BSG^-^/- similarly to those of wild-type mice.

Figure 4. Immunofluorescence analysis of sarcolemmal proteins in the skeletal muscle of BSG^-^/- mice. Serial cryosections of skeletal muscle (quadriceps) of wild-type (+/+) and BSG^-^/- mice at 4 weeks of age were stained with antibodies against dystrophin (DYS), utrophin (Utr), [alpha]-dystroglycan ([alpha]-DG), [beta]-dystroglycan ([beta]-DG), laminin-[alpha]2 chain (Lam-[alpha]2), [alpha]-, [beta]-, [gamma]- and [delta]-sarcoglycan (SG) and sarcospan (SPN). The immunoreactivities of sarcoglycans and sarcospan were absent or extremely reduced in the salcolemma of BSG^-^/- mice. The immunoreactivities of dystrophin, utrophin and the dystroglycans in the sarcolemma of BSG^-^/- mice were almost equal to those in wild-type mice. Bar, 50 µm.

Sarcoglycans and sarcospan were practically undetectable in the membrane fractions of BSG^-^/- mice, although their mRNAs, except that for [beta]-sarcoglycan, were present

To examine the presence of sarcoglycans and sarcospan in the skeletal muscle of BSG^-^/- mice, we performed western blot analysis on isolated light microsome preparations of skeletal muscles from wild-type and BSG^-^/- mice (Figs 1d and 5a). Signals of [alpha]-, [gamma]- and [delta]-sarcoglycan and sarcospan were scarcely detected in, and [beta]-sarcoglycan was completely absent from, BSG^-^/- mice. Signals of dystrophin and [alpha]- and [beta]-dystroglycan were detected in the skeletal muscles of both wild-type and BSG^-^/- mice. These data were consistent with the results of immunohistochemical analyses in BSG^-^/- mice. Expression of the dihydropyridine receptor (DHPR) was examined as a positive control for light microsome preparations (26) and was detected equally in preparations of both BSG^-^/- and wild-type mice (Fig. 5a).

Figure 5. Western blot analyses of dystrophin-DAP components from light microsome fractions and northern blot analyses of dystrophin-DAP components in skeletal muscle total RNAs. (a) Western blot analysis of skeletal muscle light microsome preparations. Skeletal muscle light microsome fractions from wild-type (+/+) and BSG mutant (-/-) mice were analyzed by SDS-PAGE and western blotting using antibodies against individual DGC components. We used antibodies against the sarcoglycans ([alpha]-, [gamma]- and [delta]-SG; for [beta]-SG see Fig. 1d), the dystroglycans ([alpha]- and [beta]-DG), dystrophin (DYS) and sarcospan (SPN). To demonstrate equal loading of protein samples, we used the DHPR as a positive marker (26). (b) Eighty microgram samples of total skeletal muscle RNA from wild-type (+/+) and BSG^-^/- mice were electrophoresed on a 1% agarose gel containing formaldehyde. Blots were hybridized with [alpha]-, [gamma]- and [delta]-sarcoglycan, sarcospan and dystroglycan cDNA probes. [alpha]-, [gamma]- and [delta]-sarcoglycan, sarcospan and dystroglycan transcripts appeared in equal amounts in wild-type and BSG^-^/- mice (top). As a control for equal loading, the same membranes were rehybridized with a G3PDH cDNA probe (data not shown).

To determine whether the loss of all the sarcoglycan subunits as well as sarcospan reflected transcriptional alterations, we performed northern blot analyses of skeletal muscle-derived RNA. Expression of [alpha]-, [gamma]- and [delta]-sarcoglycan, sarcospan and dystroglycan mRNAs was the same in terms of their level and size in skeletal muscles of BSG^-^/- and wild-type mice (Fig. 5b). These data suggested that there were no transcriptional alterations in expression of the [alpha]-, [gamma]- and [delta]-sarcoglycan subunits and sarcospan.

The dystrophin-dystrophin-associated protein (DAP) complex in BSG^-^/- mice

To examine the dystrophin-DAP complex, we prepared a dystrophin-DAP complex from digitonin-solubilized wild-type and BSG^-^/- skeletal muscle homogenates followed by wheat germ agglutinin (WGA) affinity chromatography and immunoprecipitation. Immunoblot analysis revealed that [alpha]- and [beta]-dystroglycan, [alpha]-, [beta]-, [gamma]- and [delta]-sarcoglycan and sarcospan were detected in the WGA-bound fraction from the skeletal muscle of wild-type mice (Fig. 6a). In the skeletal muscle of BSG^-^/- mice, [alpha]-, [beta]-, [gamma]- and [delta]-sarcoglycan and sarcospan were not detectable in the WGA-bound fraction (Fig. 6a). To examine the possibility of the existence of the SGC, which was not associated with the dystroglycan complex in BSG^-^/- mice, we performed immunoprecipitation using anti-[alpha]-sarcoglycan antibodies (Fig. 6a). A polyclonal anti-[alpha]-sarcoglycan antibody immunoprecipitated [alpha]-, [beta]-, [gamma]- and [delta]-sarcoglycan and sarcospan together with [alpha]- and [beta]-dystroglycan and dystrophin from digitonin-solubilized muscle homogenates of wild-type mice (Fig. 6a). In contrast, none of the subunits of sarcoglycans, sarcospan, dystroglycans or dystrophin were detected in the immunoprecipitate of the skeletal muscle from BSG^-^/- mice (Fig. 6a).

Figure 6. Western blot analysis of WGA-bound fractions and immunoprecipitated fractions in digitonin-solubilized skeletal muscle homogenates from wild-type and BSG^-^/- mice. (a) (Top) Western blot analysis of WGA-bound fractions from skeletal muscles of wild-type (lane 1) and BSG^-^/- (lane 2) mice. Components of the dystroglycan complex, the SGC and sarcospan were resolved by SDS-PAGE. We used antibodies against [alpha]-, [beta]-, [gamma]- and [delta]-sarcoglycan ([alpha]-, [beta]-, [gamma]- and [delta]-SG), sarcospan (SPN) and [alpha]- and [beta]-dystroglycan ([alpha]- and [beta]-DG). (Bottom) Western blot analysis of fractions immunoprecipitated by anti-[alpha]-sarcoglycan antibody from skeletal muscles of wild-type (lane 1) and BSG^-^/- (lane 2) mice, as resolved by SDS-PAGE. We used antibodies against [alpha]-, [beta]-, [gamma]- and [delta]-sarcoglycan ([alpha]-, [beta]-, [gamma]- and [delta]-SG), sarcospan (SPN) and [alpha]- and [beta]-dystroglycan ([alpha]- and [beta]-DG). (b) Digitonin-solubilized skeletal muscle homogenates from wild-type and BSG^-^/- mice were equally divided into three groups. The first group was used for WGA affinity chromatography, the second for immunoprecipitation experiments using anti-dystrophin antibody and the third group for immunoprecipitation experiments using anti-[beta]-dystroglycan antibody. (Hmg) Western blot analyses of 50 µg of digitonin-solubilized skeletal muscle homogenates (starting material) from wild-type (lane 1) and BSG^-^/- mice (lane 2). We used antibodies against dystrophin (DYS) and [alpha]- and [beta]-dystroglycan ([alpha]- and [beta]-DG). (WGA) Western blot analysis of WGA-bound fractions from skeletal muscles of wild-type (lane 1) and BSG^-^/- (lane 2) mice. We used antibodies against dystrophin (DYS) and [alpha]- and [beta]-dystroglycan ([alpha]- and [beta]-DG). To demonstrate equal amounts of WGA-bound fractions, loaded samples were adjusted so that [alpha]-dystroglycan signals were equally detected in both wild-type and BSG^-^/- fractions. (WGA void) Western blot analysis of 50 µg of WGA void fractions from the wild-type (lane 1) and the BSG^-^/- (lane 2) mice. We used antibodies against dystrophin (DYS) and [alpha]- and [beta]-dystroglycan ([alpha]- and [beta]-DG). (DYSIP) Western blot analysis of fractions immunoprecipitated by anti-dystrophin antibody from wild-type (lane 1) and BSG^-^/- (lane 2) mice. We used antibodies against dystrophin (DYS) and [alpha]- and [beta]-dystroglycan ([alpha]- and [beta]-DG). To demonstrate equal amounts of fractions immunoprecipitated by the anti-dystrophin antibody, loaded samples were adjusted so that dystrophin signals were equally detected in both fractions from wild-type and BSG^-^/- mice. Arrowhead, intact [beta]-dystroglycan; double arrowheads, degraded [beta]-dystroglycan. ([beta]-DGIP) Western blot analysis of fractions immunoprecipitated by anti-[beta]-dystroglycan antibody from wild-type (lane 1) and BSG^-^/- (lane 2) mice, as resolved by SDS-PAGE. We used antibodies against dystrophin (DYS) and [alpha]- and [beta]-dystroglycan ([alpha]- and [beta]-DG). To demonstrate equal amounts of fractions immunoprecipitated by anti-[beta]-dystroglycan antibody, loaded samples were adjusted so that [beta]-dystroglycan signals were equally detected in both the wild-type and BSG^-^/- fractions. Arrowhead, intact [beta]-dystroglycan; double arrowheads, degraded [beta]-dystroglycan.

To examine the dystrophin-dystroglycan complex, either with or without the SGC and sarcospan, we prepared a dystrophin-dystroglycan complex from digitonin-solubilized wild-type and BSG^-^/- skeletal muscle homogenates followed by WGA affinity chromatography and immunoprecipitation.

Dystrophin and dystroglycans were detected similarly in the digitonin-solubilized skeletal muscle homogenates of both wild-type and BSG^-^/- mice (Fig. 6b, Hmg). In the BSG^-^/- WGA-bound fractions, signals of [beta]-dystroglycan and dystrophin were reduced compared with those in the WGA-bound fractions from wild-type mice, while signals of [alpha]-dystroglycan were similarly detected in BSG^-^/- and wild-type mice (Fig. 6b, WGA). On the other hand, [beta]-dystroglycan and dystrophin were clearly detected in the BSG^-^/- WGA void fractions, while they were not in the wild-type WGA void fractions (Fig. 6b, WGA void).

Monoclonal dystrophin antibody MANDRA1 immunoprecipitated dystrophin together with [alpha]- and [beta]-dystroglycan from the soluble skeletal muscle homogenates of wild-type mice (Fig. 6b, DYSIP). In the MANDRA1 immunoprecipitates of BSG^-^/- mice, signals of [alpha]- and intact [beta]-dystroglycan were reduced compared with those of the wild-type mice, whereas signals of dystrophin were equally detected in the case of BSG^-^/- and wild-type mice (Fig. 6b, DYSIP). The signal of degraded [beta]-dystroglycan was increased in the MANDRA1 immunoprecipitates of BSG^-^/- mice (Fig. 6b, DYSIP).

Polyclonal anti-[beta]-dystroglycan antibody PA3a immunoprecipitated [beta]-dystroglycan together with [alpha]-dystroglycan and dystrophin from the muscle homogenates of wild-type and BSG^-^/- mice (Fig. 6b, [beta]-DGIP). Signals of [alpha]-dystroglycan and dystrophin were greatly reduced in the muscle homogenates of BSG^-^/- mice compared with those of wild-type mice, whereas signals of [beta]-dystroglycan were equally detected in the case of BSG^-^/- and wild-type mice (Fig. 6b, [beta]-DGIP).

DISCUSSION

BSG^-^/- mice develop muscular dystrophy

In the present study we developed mice lacking the [beta]-sarcoglycan gene (BSG^-^/- mice). Histological analysis of the skeletal muscles by light microscopy revealed that BSG^-^/- mice had progressive muscular dystrophy. Pathological changes were found in the skeletal muscles of BSG^-^/- mice as early as 2 weeks of age and progressed as the mice got older. Degenerative changes in the muscle fibers of BSG^-^/- mice were most pronounced from 4 to 8 weeks of age. To assess the degenerative change in the skeletal muscles of BSG^-^/- mice, we performed vital staining with EBD and measured serum CK activity. In the skeletal muscles of BSG^-^/- mice, EBD-containing fibers were found, although no EBD was observed in the muscle fibers of wild-type mice. TA muscle is spared from EBD intrusion macroscopically in mdx mice (27). In the TA muscles of skinned carcasses of BSG^-^/- mice, EBD intrusion was hardly seen macroscopically, whereas a few clusters of EBD-containing muscle fibers were found upon microscopic observation. Serum CK activity in BSG^-^/- mice was ~100 times higher than that in wild-type mice. These two analyses indicate that the skeletal muscle fibers in BSG^-^/- mice are degenerative.

Around 14 weeks of age, regenerative changes were predominant in the skeletal muscle of BSG^-^/- mice and were associated with limb hypertrophy. These regenerative changes in BSG^-^/- skeletal muscle were also compatible with those in human sarcoglycanopathy, [alpha]- and [gamma]-sarcoglycan-deficient mice and [delta]-sarcoglycan-deficient dystrophic hamsters (11,19,20,22).

It has been reported that [delta]-sarcoglycan-deficient hamsters (22) and [gamma]-sarcoglycan deficient-mice (20) show cardiac involvement. BSG^-^/- mouse heart showed many fairly large patches of fibrosis and a small number of clusters of EBD-containing cells at 56 weeks of age; however, we did not observe natural death of mice during this period.

[alpha]-, [beta]-, [gamma]- and [delta]-sarcoglycan as well as sarcospan were absent in the BSG^-^/- sarcolemma

Immunohistochemical analysis of BSG^-^/- mice demonstrated that all of the four sarcoglycans, as well as sarcospan, were absent in the sarcolemma. However, laminin-[alpha]2, [alpha]- and [beta]-dystroglycan and dystrophin were present in the sarcolemma of BSG^-^/- mice. These results are similar to those reported in the skeletal muscle of patients with [beta]-SGP (15-16,28). In the case of the sarcoglycans, the results of immunohistochemical analyses of the skeletal muscles of BSG^-^/- mice were consistent with those of western blot analysis of the microsomal fractions of these mice, although [alpha]-, [gamma]- and [delta]-sarcoglycan mRNAs were normally transcribed. Our present study is also compatible with an in vitro study using CHO cells, which were transfected with the mutated [beta]-sarcoglycan gene and other wild-type sarcoglycan genes (29). These data prove the validity of the SGP hypothesis (7,12).

In addition to the marked reduction in expression of all three sarcoglycans, the amount of sarcospan protein was also significantly reduced in the membrane fractions of BSG^-^/- mice, although sarcospan mRNA was normally transcribed. Sarcospan is, similarly to the SGC, absent in BSG^-^/- mice. In [alpha]-sarcoglycan-deficient mice, sarcospan was also not detected in the sarcolemma (19). Immunohistochemical analysis of a [gamma]-SGP patient revealed that expression of sarcospan was markedly reduced in the sarcolemma (M. Imamura and E. Ozawa, unpublished data). These findings suggest a close relationship between sarcospan and the SGC. However, sarcospan may not be a component of the SGC, because it is not necessarily eluted together with sarcoglycans after treatment of the dystrophin-DAP complex with n-octyl [beta]-D-glucoside (1).

Furthermore, there were several differences in the immunohistochemical findings between BSG^-^/- mice and other sarcoglycan-deficient animals. In the [alpha]-sarcoglycan-deficient mice, expression of all of the four sarcoglycans in the sarcolemma was greatly reduced (19). In [gamma]-sarcoglycan-deficient mice, [alpha]-sarcoglycan was partially retained in the sarcolemma despite the great reduction in the other three sarcoglycans (20). In the [delta]-sarcoglycan deficient dystrophic hamster, all of the four sarcoglycans in the sarcolemma were lost (30-31). These data from animal models of SGP are consistent with those from human patients (28) and may reflect the molecular organization of the SGC (28,32).

Dystrophin-DAP complex in the skeletal muscle of BSG^-^/- mice

For further analysis of the dystrophin-DAP complex, we performed WGA affinity chromatography and immunoprecipitation using digitonin-solubilized muscle homogenates of BSG^-^/- mice. WGA affinity chromatography is a standard method to isolate the dystrophin-DAP complex from the skeletal muscle membrane lysate, because WGA binds the [alpha]-dystroglycan sugar chain. WGA-bound fractions of the skeletal muscles of wild-type mice contained [alpha]-, [beta]-, [gamma]- and [delta]-sarcoglycan, as well as sarcospan, in addition to the other components of the dystrophin-DAP complex; in contrast, we could detect none of the four sarcoglycans or sarcospan in the WGA-bound fractions of the skeletal muscles of BSG^-^/- mice. These observations suggest that [beta]-sarcoglycan plays a critical role in assembling the SGC with sarcospan and support the results of an in vitro study which revealed that [beta]-sarcoglycan is a core protein for assembling the SGC (32).

The dystrophin-dystroglycan complex in the skeletal muscle of wild-type mice was stably isolated by WGA affinity chromatography or by immunoprecipitation. In BSG^-^/- mice, however, only a small amount of the complete form of the dystrophin-dystroglycan complex was isolated by the same methods. In the dystrophic hamster lacking [delta]-sarcoglycan and also the other sarcoglycans, expression of [alpha]-dystroglycan in the sarcolemma is also reduced (33) and WGA affinity chromatography analyses reveal that dystrophin is apt to dissociate from [beta]-dystroglycan in the absence of the SGC (34). A protein-binding study using fusion proteins showed that [alpha]-dystroglycan binds not only to [beta]-dystroglycan but also to [alpha]-, [beta]- and [delta]-sarcoglycan (24). These results from BSG^-^/- mice, [delta]-sarcoglycan-deficient dystrophic hamsters and the protein-binding study could suggest that deficiency of the SGC and sarcospan causes instability of the dystrophin-dystroglycan complex. This may be one of the possible causes of human Duchenne-like muscular dystrophy in which the SGC is lost.

MATERIALS AND METHODS

Targeting vector construction and transfection of the ES cell

The mouse [beta]-sarcoglycan gene was isolated from a 129/SvJ mouse genomic library (Stratagene, La Jolla, CA) by hybridization with a human [beta]-sarcoglycan cDNA probe. A 15 kb genomic fragment containing exons 1-6 of the [beta]-sarcoglycan gene was subcloned and characterized by restriction mapping, nucleotide sequencing and Southern blot analysis. A 3.2 kb genomic fragment containing exon 1 as a 5[prime] homologous region and a 7.0 kb genomic fragment containing exons 3-5 as a 3[prime] homologous region were cloned into a pPNT vector (35). The targeting vector was designed to disrupt exon 2 encoding the intracellular and transmembrane domains of [beta]-sarcoglycan.

J1 ES cells were transfected with the linearized targeting vector DNA by electroporation (Gene Pulser set at 800 V and 3.0 µF; Bio-Rad, Hercules, CA). G418 (Gibco BRL Life Technologies, Rockville, MD) and gancyclovir (Hoffmann-La Roche, Basel, Switzerland) were added to the medium 24-48 h after the transfection for selection. ES cell lines with targeted disruption of the [beta]-sarcoglycan gene were identified by Southern blot analysis of NcoI-digested genomic DNA. The 5[prime]- and 3[prime]-flanking regions and the neomycin resistance gene were used as probes (Fig. 1a).

Generation of BSG^-^/- mice

Chimeric mice were generated by injecting the ES cells with a disrupted [beta]-sarcoglycan gene into C57BL/6J blastocysts and then implanting them into the uteri of pseudopregnant BDF1 recipients (Clea Japan, Tokyo, Japan). Chimeric male mice were obtained and germline transmission was examined by mating them with C57BL/6J females. Agouti coat color offspring were analyzed by Southern blot analysis. Heterozygous animals were interbred to generate homozygous mice. The genotypes were determined by Southern blot analysis or PCR on DNA obtained from a tail biopsy specimen (Fig. 1b). All animal handling procedures were in accordance with a protocol approved by the National Institute of Neuroscience (Tokyo, Japan).

Northern blot analysis

Total RNA from wild-type, heterozygous and homozygous skeletal muscle was extracted using TRIzol (Gibco BRL) according to the manufacturer's instructions. Eighty micrograms of total RNA was electrophoresed on a 1.0% agarose gel containing formaldehyde and transferred to a Hybond-N⁺ membrane (Amersham Pharmacia Biotech, Little Chalfont, UK). Membranes in the hybridization solution (ExpressHyb; Clontech, Palo Alto, CA) were hybridized with the probes under high stringency conditions according to the manufacturer's protocol. The mouse full-length [alpha], [beta], [gamma] and [delta]-sarcoglycan cDNAs (GenBank accession nos AB024920-AB024923), the dystroglycan cDNA, the sarcospan cDNA and the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) cDNA were used as hybridization probes.

Antibodies

Monoclonal antibodies against [alpha]-sarcoglycan (NCL-a-sarco), [beta]-sarcoglycan (NCL-b-sarco), [gamma]-sarcoglycan (NCL-g-sarco) and [beta]-dystroglycan (NCL-b-DG) were purchased from Novo Castra Laboratories (Newcastle-upon-Tyne, UK). Monoclonal antibodies against [alpha]-dystroglycan (clone VIA4-1) and the DHPR (clone D5-E1) were purchased from Upstate Biotechnology (Lake Placid, NY). A monoclonal antibody against dystrophin (MANDRA1) was purchased from Sigma (St Louis, MO). A monoclonal antibody against laminin-[alpha]2 (clone 4H8-2) was purchased from ALEXIS (Läufelfingen, Switzerland). Polyclonal antibody PA3a against [beta]-dystroglycan was raised as described previously (36). Rabbit polyclonal antibodies to [alpha]- and [beta]-sarcoglycan were raised against recombinant fragments of the cytoplasmic domain of human [alpha]-sarcoglycan (amino acids 316-387) and the extracellular domain of mouse [beta]-sarcoglycan (amino acids 96-154). [alpha]-Sarcoglycan cDNA fragments (0.4 kb) were amplified from a human skeletal muscle cDNA library (Clontech) by PCR using the following oligonucleotide primer set: 5[prime]-GCGGGAGGGAAGGCTGAAGAGAG-3[prime]; 5[prime]-AATTGGTGAGCAGAGCAGCAGAT-3[prime]. Amplification was carried out using LA-Taq (Takara, Kyoto, Japan) for 35 cycles, each cycle consisting of 94°C for 1 min, 55°C for 1 min and 72°C for 3 min. The amplified [alpha]-sarcoglycan DNA fragment was subcloned into the pCR2.1 vector (Invitrogen, NV Leek, The Netherlands) and the clone (pCR-aSG) was confirmed by sequencing to be human [alpha]-sarcoglycan cDNA. Then, the EcoRI fragment of pCR-aSG was ligated into the pGEX4T-1 vector (Amersham Pharmacia Biotech). Recombinant protein was purified from the soluble fraction of the cell lysates on a glutathione-Sepharose column and used as antigen. The amplification of [beta]-sarcoglycan cDNA fragments (176 bp) from a mouse full-length [beta]-sarcoglycan cDNA was carried out under the same conditions described above except that the primer set for PCR was: 5[prime]-CGGGATCCCCAAATGGGTGTGATAGCATGGAG-3[prime]; 5[prime]-GGAATTCTCACTTGGTCGTCCCTTGCTGGAAGAC-3[prime]. The amplified [beta]-sarcoglycan DNA fragment was digested with EcoRI and BamHI. Then, the EcoRI-BamHI fragment was ligated into the pGEX4T-1 vector. Purification of the GST-[beta]-sarcoglycan recombinant protein was carried out under the same conditions as those described above. The anti-[alpha]- and anti-[beta]-sarcoglycan antibodies were affinity purified on Sepharose 4B columns (Amersham Pharmacia Biotech) coupled with the relevant recombinant proteins. Antibody against a glutathione S-transferase moiety was removed by adsorption on Sepharose 4B columns coupled with glutathione S-transferase. An affinity-purified guinea pig polyclonal antibody against [delta]-sarcoglycan was raised against a recombinant fusion protein containing a human [delta]-sarcoglycan fragment (amino acids 84-290) with glutathione S-transferase under the same conditions as those described above. Rabbit polyclonal antibody to sarcospan was prepared against the synthetic peptide of KRAG, MGRKPSPRAQELPEEEARTC, with a terminal cysteine, coupled to bovine serum albumin. The anti-sarcospan antibody was affinity purified from rabbit antiserum with the peptide coupled to Sepharose 4B. Affinity-purified rabbit polyclonal antibodies against utrophin and dystrophin were prepared as described previously (37).

Light microsome preparation

The quadriceps femoris muscles (2 g) were dissected from wild-type, heterozygous and homozygous mice and light microsome fractions were prepared as described by Ohlendieck et al. (26). To minimize protein degradation, the light microsome fractions were prepared at 4°C. Briefly, muscles were homogenized twice for 30 s each in buffer A [20 mM sodium pyrophosphate, 20 mM sodium phosphate monohydrate, 1 mM magnesium chloride, 0.303 M sucrose, 0.5 mM EDTA, pH 7.0, 0.23 mM phenylmethanesulfonylfluoride (PMSF), 7 µg/ml Calpain inhibitor and 1× protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)] with a polytron homogenizer. The homogenate was centrifuged for 15 min at 14 000 g and the supernatant was centrifuged for 30 min at 30 000 g. The pellet, which contained mostly lysosomes and mitochondria, was discarded. Light microsomes were obtained from the supernatant by centrifugation for 40 min at 100 000 g. To remove the proteins that were non-specifically attached to the pellet, the light microsomes were washed with 0.6 M potassium chloride and then recentrifuged at 100 000 g for 40 min. The final pellets were completely dissolved in SDS sample buffer (10% glycerol, 2.3% SDS, 5% 2-mercaptoethanol, 62.5 mM Tris-HCl, pH 6.8) and boiled. The protein concentrations were determined by the Coomassie plus colorimetric assay (Pierce, Rockford, IL). A 50 µg protein fraction was electrophoresed on SDS-polyacrylamide gels and blotted on an immobilon-P membrane (Millipore, Bedford, MA). Primary antibodies were then added to the membrane, and peroxidase-labeled anti-IgG was subsequently used as the secondary antibody. Signal detection was performed according to the protocol of the ECL detection kit (Amersham Pharmacia Biotech).

Histology and immunohistochemistry

Thick cryosections (8 µm) were analyzed by immunofluorescence using specific antibodies as described previously (31). For H&E staining, the 8 µm thick sections were stained for 7 min each in hematoxylin and eosin and then dehydrated with ethanol and xylenes, mounted with Entellan neu (Merck, Darmstadt, Germany) and examined by light microscopy. All sections were photographed under an Olympus AX 70 microscope (Olympus Optical, Tokyo, Japan).

Vital staining with EBD

To detect damaged muscle fibers, EBD (10 mg/ml in phosphate-buffered saline) was injected i.p. into mice (0.1 ml/10 g body wt) as described by Matsuda et al. (25). The mice were killed 12 h after injection and their muscles were sectioned and examined under a fluorescence microscope (AX 70; Olympus Optical).

Serum CK assay

The serum CK activity was measured using the creatine kinase reagent (Merck) according to the manufacturer's instructions. Blood was obtained by intracardiac puncture of anesthetized mice at 8 weeks of age.

Affinity chromatography

For WGA affinity chromatagraphy (34), quadriceps femoris muscles (0.5 gram) from wild-type and BSG^-^/- mice were homogenized in 10 vol (w/v) of buffer B (1% digitonin, 500 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.25 mM PMSF, 7 µg/ml Calpain inhibitor and 1× protease inhibitor cocktail). After centrifugation at 60 000 g, the supernatant was incubated overnight at 4°C with WGA-Sepharose 6MB beads (Amersham Pharmacia Biotech). The beads were then washed extensively with buffer B and proteins were eluted with buffer B containing 0.6 M N-acetyl-D-glucosamine.

Immunoprecipitation by anti-[alpha]-sarcoglycan, anti-[beta]-dystroglycan and anti-dystrophin antibodies

Anti-[alpha]-sarcoglycan and anti-[beta]-dystroglycan polyclonal antibodies and anti-dystrophin monoclonal antibody were used for immunoprecipitation. All procedures were performed at 4°C. For immunoprecipitations, quadriceps femoris muscles (0.5 g) from wild-type and homozygous mice were homogenized and solubilized in 10 vol (w/v) of buffer IP (1% digitonin, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.25 mM PMSF, 7 µg/ml Calpain inhibitor and 1× protease inhibitor cocktail). After centrifugation at 60 000 g, the supernatant was incubated for 2 h at 4°C with protein G-Sepharose (Amersham Pharmacia Biotech). After removing the resin, the soluble fractions were incubated with anti-[alpha]-sarcoglycan, anti-[beta]-dystroglycan or anti-dystrophin antibodies overnight at 4°C and then reincubated for 2 h at 4°C with protein G-Sepharose (Amersham Pharmacia Biotech). Resins were washed extensively with buffer IP and eluted with 10% SDS solution.

Statistical analysis

All data are presented as means SD. Comparisons between groups were performed by the Mann-Whitney U-test. The null hypothesis was rejected at P < 0.01 or at P < 0.05.

ACKNOWLEDGEMENTS

We thank Dr T. Matsuzaki for managing our mouse facility, Dr Richard Mulligan for the pPNT vector, Dr En Li for the J1 ES cell line, and Dr A. Takaki, Dr S. Shioda, Ms Mizuguchi and our laboratory members for encouragement and discussions. This work was supported by Grants for Nervous and Mental Disorders (8A-1 and 9A-1), for Health Sciences Research and for the COE program from the Ministry of Health and Welfare, and grants from the Ministry of Education, Science, Sports and Culture and the Science and Technology Agency, Japan. E.W. was supported by the Japan Health Sciences Foundation.

REFERENCES

1. Yoshida, M., Suzuki, A., Yamamoto, H., Noguchi, S., Mizuno, Y. and Ozawa, E. (1994) Dissociation of the complex of dystrophin and its associated proteins into several unique groups by n-octyl [beta]-D-glucoside. Eur. J. Biochem., 222, 1055-1061. MEDLINE Abstract

2. Yoshida, M. and Ozawa, E. (1990) Glycoprotein complex anchoring dystrophin to sarcolemma. J. Biochem., 108, 748-752. MEDLINE Abstract

3. Ervasti, J.M., Ohlendieck, K., Kahl, S.D., Gaver, M.G. and Campbell, K.P. (1990) Deficiency of a glycoprotein component of the dystrophin complex in dystrophic muscle. Nature, 345, 315-319. MEDLINE Abstract

4. Ervasti, J.M. and Campbell, K.P. (1991) Membrane organization of the dystrophin-glycoprotein complex. Cell, 66, 1121-1131. MEDLINE Abstract

5. Ozawa, E., Yoshida, M., Suzuki, A., Mizuno, Y., Hagiwara, Y. and Noguchi, S. (1995) Dystrophin-associated proteins in muscular dystrophy. Hum. Mol. Genet., 4, 1711-1716. MEDLINE Abstract

6. Suzuki, A., Yoshida, M., Hayashi, K., Mizuno, Y., Hagiwara, Y. and Ozawa, E. (1994) Molecular organization at the glycoprotein-complex-binding site of dystrophin. Three dystrophin-associated proteins bind directly to the carboxy-terminal portion of dystrophin. Eur. J. Biochem., 220, 283-292. MEDLINE Abstract

7. Ozawa, E., Noguchi, S., Mizuno, Y., Hagiwara, Y. and Yoshida, M. (1998) From dystrophinopathy to sarcoglycanopathy: evolution of a concept of muscular dystrophy. Muscle Nerve, 21, 421-438. MEDLINE Abstract

8. Ozawa, E., Hagiwara, Y. and Yoshida, M. (1999) Creatine kinase, cell membrane and Duchenne muscular dystrophy. Mol. Cell. Biochem., 190, 143-151. MEDLINE Abstract

9. Campbell, K.P. (1995) Three muscular dystrophies: loss of cytoskeleton-extracellular matrix linkage. Cell, 80, 675-679. MEDLINE Abstract

10. Ohlendieck, K. (1996) Towards an understanding of the dystrophin-glycoprotein complex: linkage between the extracellular matrix and the membrane cytoskeleton in muscle fibers. Eur. J. Cell Biol., 69, 1-10. MEDLINE Abstract

11. Ben Hamida, M., Fardeau, M. and Attia, N. (1983) Severe childhood muscular dystrophy affecting both sexes and frequent in Tunisia. Muscle Nerve, 6, 469-480. MEDLINE Abstract

12. Mizuno, Y., Noguchi, S., Yamamoto, H., Yoshida, M., Suzuki, A., Hagiwara, Y., Hayashi, Y.K., Arahata, K., Nonaka, I., Hirai, S. and Ozawa, E. (1994) Selective defect of sarcoglycan complex in severe childhood autosomal recessive muscular dystrophy muscle. Biochem. Biophys. Res. Commun., 203, 979-983. MEDLINE Abstract

13. Matsumura, K., Tomè, F.M.S., Collin, H., Azibi, K., Chaouch, M., Kaplan, J.C., Fardeau, M. and Campbell, K.P. (1992) Deficiency of the 50K dystrophin-associated glycoprotein in severe childhood autosomal recessive muscular dystrophy. Nature, 359, 320-322. MEDLINE Abstract

14. Roberds, S.L., Leturcq, F., Allamand, V., Piccolo, F., Jeanpierr, M., Anderson, R.D., Lim, L.E., Lee, J.C., Tomè, F.M.S., Romero, N.B., Fardeau, M., Beckmann, J.S., Kaplan, J.-C. and Campbell, K.P. (1994) Missense mutations in the adhalin gene linked to autosomal recessive muscular dystrophy. Cell, 78, 625-633. MEDLINE Abstract

15. Bönnemann, C.G., Modi, R., Noguchi, S., Mizuno, Y., Yoshida, M., Gussoni, E., McNally, E.M., Duggan, D.J., Angelini, C., Hoffman, E.P., Ozawa, E. and Kunkel, L.M. (1995) [beta]-Sarcoglycan (A3b) mutations cause autosomal recessive muscular dystrophy with loss of the sarcoglycan complex. Nature Genet., 11, 266-273. MEDLINE Abstract

16. Lim, L.E., Duclos, F., Broux, O., Bourg, N., Sunada, Y., Allamand, V., Meyer, J., Richard, I., Moomaw, C., Slaughter, C., Tomè, F.M.S., Fardeau, M., Jackson, C.E., Beckmann, J.S. and Campbell, K.P. (1995) [beta]-Sarcoglycan: characterization and role in limb-girdle muscular dystrophy linked to 4q12. Nature Genet., 11, 257-265. MEDLINE Abstract

17. Noguchi, S., McNally, E.M., Ben Othmane, K., Hagiwara, Y., Mizuno, Y., Yoshida, M., Yamamoto, H., Bönnemann, C.G., Gussoni, E., Denton, P.H., Kyriakides, T., Middleton, L., Hentati, F., Ben Hamida, M., Nonaka, I., Vance, J.M., Kunkel, L.M. and Ozawa, E. (1995) Mutations in the dystrophin-associated protein [gamma]-sarcoglycan in chromosome 13 muscular dystrophy. Science, 270, 819-822. MEDLINE Abstract

18. Nigro, V., Moreira, E.S., Piluso, G., Vainzof, M., Belsito, A., Politano, L., Puca, A.A., Passos-Bueno, M.R. and Zats, M. (1996) The 5q autosomal recessive limb-girdle muscular dystrophy (LGMD2F) is caused by a mutation in the [delta]-sarcoglycan gene. Nature Genet., 14, 195-198. MEDLINE Abstract

19. Duclos, F., Straub, V., Moore, S.A., Venzke, D.P., Hrstka, R.F., Crosbie, R.H., Durbeej, M., Lebakken, C.S., Ettinger, A.J., Van der Meulen, J., Holt, K.H., Lim, L.E., Sanes, J.R., Davidson, B.L., Faulkner, J.A., Williamson, R. and Campbell, K.P. (1998) Progressive muscular dystrophy in [alpha]-sarcoglycan-deficient mice. J. Cell Biol., 142, 1461-1471. MEDLINE Abstract

20. Hack, A.A., Ly, C.T., Jiang, F., Clendenin, C.J., Sigrist, K.S., Wollmann, R.L. and McNally, E.M. (1998) [gamma]-Sarcoglycan deficiency leads to muscle membrane defects and apoptosis independent of dystrophin. J. Cell Biol., 142, 1279-1287. MEDLINE Abstract

21. Sasaoka , T., Mizumo, Y., Noguchi, S., Imamura, M., Wakabayahi, E., Yoshida, M. and Ozawa, E. (1998) Muscular dystrophic phenotype in the gamma-sarcoglycan-deficient mice. Am. J. Hum. Genet., 63 (suppl.), 2228-2228.

22. Homburger , F., Baker, J.R., Nixon, C.W. and Whitney, R. (1962) Primary, generalized polymyopathy and cardiac necrosis in an inbred line of Syrian hamsters. Med. Exp., 6, 339-345.

23. Nigro, V., Okazaki, Y., Belsito, A., Piluso, G., Matsuda, Y., Politano, L., Nigro, G., Ventura, C., Abbondanza, C., Molinari, A.M., Acampora, D., Nishimura, M., Hayashizaki, Y. and Puca, G.A. (1997) Identification of the Syrian hamster cardiomyopathy gene. Hum. Mol. Genet., 6, 601-607. MEDLINE Abstract

24. Sakamoto, A., Ono, K., Abe, M., Jasmin, G., Eki, T., Murakami, Y., Masaki, T., Toyo-oka, T. and Hanaoka, F. (1997) Both hypertrophic and dilated cardiomyopathies are caused by mutation of the same gene, [delta]-sarcoglycan, in hamster: an animal model of disrupted dystrophin-associated glycoprotein complex. Proc. Natl Acad. Sci. USA, 94, 13873-13878. MEDLINE Abstract

25. Matsuda, R., Nishikawa, A. and Tanaka, H. (1995) Visualization of dystrophic muscle fibers in mdx mouse by vital staining with Evans blue: evidence of apoptosis in dystrophin-deficient muscle. J. Biochem., 118, 959-964. MEDLINE Abstract

26. Ohlendieck, K., Ervasti, J.M., Snook, J.B. and Campbell, K.P. (1991) Dystrophin-glycoprotein complex is highly enriched in isolated skeletal muscle sarcolemma. J. Cell Biol., 112, 135-148. MEDLINE Abstract

27. Straub, V., Rafael, J.A., Chamberlain, J.S. and Campbell, K.P. (1997) Animal models for muscular dystrophy show different patterns of sarcolemmal disruption. J. Cell Biol., 139, 375-385. MEDLINE Abstract

28. Vainzof, M., Passos-Bueno, M.R., Canovas, M., Moreira, E.S., Pavanello, R.C.M., Marie, S.K., Anderson, L.V.B., Bönnemann, C.G., McNally, E.M., Nigro, V., Kunkel, L.M. and Zatz, M. (1996) The sarcoglycan complex in the six autosomal recessive limb-girdle muscular dystrophies. Hum. Mol. Genet., 5, 1963-1969. MEDLINE Abstract

29. Holt, K.H. and Campbell, K.P. (1998) Assembly of the sarcoglycan complex. J. Biol. Chem., 273, 34667-34670. MEDLINE Abstract

30. Roberds, S.L., Ervasti, J.M., Anderson, R.D., Ohlendieck, K., Kahl, S.D., Zoloto, D. and Campbell, K.P. (1993) Disruption of the dystrophin-glycoprotein complex in the cardiomyopathic hamster. J. Biol. Chem., 268, 11496-11499. MEDLINE Abstract

31. Mizuno, Y., Noguchi, S., Yamamoto, H., Yoshida, M., Nonaka, I., Hirai, S. and Ozawa, E. (1995) Sarcoglycan complex is selectively lost in dystrophic hamster muscle. Am. J. Pathol., 146, 530-536. MEDLINE Abstract

32. Chan, Y., Bönnemann, C.G., Lidov, H.G.W. and Kunkel, L.M. (1998) Molecular organization of sarcoglycan complex in mouse myotubes in culture. J. Cell Biol., 143, 2033-2044. MEDLINE Abstract

33. Straub, V., Duclos, F., Venzke, D.P., Lee, J.C., Cutshall, S., Leveille, C.J. and Campbell, K.P. (1998) Molecular pathogenesis of muscle degeneration in the [delta]-sarcoglycan-deficient hamster. Am. J. Pathol., 153, 1623-1630. MEDLINE Abstract

34. Iwata, Y., Nakamura, H., Mizuno, Y., Yoshida, M., Ozawa, E. and Shigekawa, M. (1993) Defective association of dystrophin with sarcolemmal glycoproteins in the cardiomyopathic hamster heart. FEBS Lett., 329, 227-231. MEDLINE Abstract

35. Tybulewicz, V., Crawford, C., Jackson, P., Bronson, R. and Mulligan, R. (1991) Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto oncogene. Cell, 65, 1153-1163. MEDLINE Abstract

36. Mizuno, Y., Yoshida, M., Yamamoto, H., Hirai, S. and Ozawa, E. (1993) Distribution of dystrophin isoforms and dystrophin-associated proteins 43DAG (A3a) and 50DAG (A2) in various monkey tissues. J. Biochem., 114, 936-941. MEDLINE Abstract

37. Imamura, M. and Ozawa, E. (1998) Differential expression of dystrophin isoforms and utrophin during dibutyryl-cAMP-induced morphological differentiation of rat brain astrocytes. Proc. Natl Acad. Sci. USA, 95, 6139-6144. MEDLINE Abstract

+To whom correspondence should be addressed. Tel: +81 42 346 1711; Fax: +81 42 346 1741; Email: ozawa{at}ncnaxp.ncnp.go.jp

This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: jnl.info{at}oup.co.uk
Last modification:
Copyright© Oxford University Press, 1999.

CiteULike

Connotea

Del.icio.us What's this?

This article has been cited by other articles:

R. Xu, S. DeVries, M. Camboni, and P. T. Martin
Overexpression of Galgt2 Reduces Dystrophic Pathology in the Skeletal Muscles of Alpha Sarcoglycan-Deficient Mice
Am. J. Pathol., July 1, 2009; 175(1): 235 - 247.
[Abstract] [Full Text] [PDF]

J. R. Guyon, J. Goswami, S. J. Jun, M. Thorne, M. Howell, T. Pusack, G. Kawahara, L. S. Steffen, M. Galdzicki, and L. M. Kunkel
Genetic isolation and characterization of a splicing mutant of zebrafish dystrophin
Hum. Mol. Genet., January 1, 2009; 18(1): 202 - 211.
[Abstract] [Full Text] [PDF]

J. Davis, M. V. Westfall, D. Townsend, M. Blankinship, T. J. Herron, G. Guerrero-Serna, W. Wang, E. Devaney, and J. M. Metzger
Designing Heart Performance by Gene Transfer
Physiol Rev, October 1, 2008; 88(4): 1567 - 1651.
[Abstract] [Full Text] [PDF]

N. Urasawa, M. R. Wada, N. Machida, K. Yuasa, Y. Shimatsu, Y. Wakao, S. Yuasa, T. Sano, I. Nonaka, A. Nakamura, et al.
Selective Vacuolar Degeneration in Dystrophin-Deficient Canine Purkinje Fibers Despite Preservation of Dystrophin-Associated Proteins With Overexpression of Dp71
Circulation, May 13, 2008; 117(19): 2437 - 2448.
[Abstract] [Full Text] [PDF]

K. Kobuke, F. Piccolo, K. W. Garringer, S. A. Moore, E. Sweezer, B. Yang, and K. P. Campbell
A common disease-associated missense mutation in alpha-sarcoglycan fails to cause muscular dystrophy in mice
Hum. Mol. Genet., May 1, 2008; 17(9): 1201 - 1213.
[Abstract] [Full Text] [PDF]

D. J. Milner and S. J. Kaufman
{alpha}7{beta}1 Integrin Does Not Alleviate Disease in a Mouse Model of Limb Girdle Muscular Dystrophy Type 2F
Am. J. Pathol., February 1, 2007; 170(2): 609 - 619.
[Abstract] [Full Text] [PDF]

L. M. Judge, M. Haraguchiln, and J. S. Chamberlain
Dissecting the signaling and mechanical functions of the dystrophin-glycoprotein complex
J. Cell Sci., April 15, 2006; 119(8): 1537 - 1546.
[Abstract] [Full Text] [PDF]

D. Danieli-Betto, A. Esposito, E. Germinario, D. Sandona, T. Martinello, A. Jakubiec-Puka, D. Biral, and R. Betto
Deficiency of {alpha}-sarcoglycan differently affects fast- and slow-twitch skeletal muscles
Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2005; 289(5): R1328 - R1337.
[Abstract] [Full Text] [PDF]

M. Imamura, Y. Mochizuki, E. Engvall, and S. Takeda
{varepsilon}-Sarcoglycan compensates for lack of {alpha}-sarcoglycan in a mouse model of limb-girdle muscular dystrophy
Hum. Mol. Genet., March 15, 2005; 14(6): 775 - 783.
[Abstract] [Full Text] [PDF]

S. C. Brown, S. Torelli, M. Brockington, Y. Yuva, C. Jimenez, L. Feng, L. Anderson, I. Ugo, S. Kroger, K. Bushby, et al.
Abnormalities in {alpha}-Dystroglycan Expression in MDC1C and LGMD2I Muscular Dystrophies
Am. J. Pathol., February 1, 2004; 164(2): 727 - 737.
[Abstract] [Full Text] [PDF]

S. Takeda, M. Kondo, J. Sasaki, H. Kurahashi, H. Kano, K. Arai, K. Misaki, T. Fukui, K. Kobayashi, M. Tachikawa, et al.
Fukutin is required for maintenance of muscle integrity, cortical histiogenesis and normal eye development
Hum. Mol. Genet., June 15, 2003; 12(12): 1449 - 1459.
[Abstract] [Full Text] [PDF]

R. H. CROSBIE, R. BARRESI, and K. P. CAMPBELL
Loss of sarcolemma nNOS in sarcoglycan-deficient muscle
FASEB J, November 1, 2002; 16(13): 1786 - 1791.
[Abstract] [Full Text] [PDF]

J. F. Watchko, T. L. O'Day, and E. P. Hoffman
Functional characteristics of dystrophic skeletal muscle: insights from animal models
J Appl Physiol, August 1, 2002; 93(2): 407 - 417.
[Abstract] [Full Text] [PDF]

D. J. Blake, A. Weir, S. E. Newey, and K. E. Davies
Function and Genetics of Dystrophin and Dystrophin-Related Proteins in Muscle
Physiol Rev, April 1, 2002; 82(2): 291 - 329.
[Abstract] [Full Text] [PDF]

P. D. Cote, H. Moukhles, and S. Carbonetto
Dystroglycan Is Not Required for Localization of Dystrophin, Syntrophin, and Neuronal Nitric-oxide Synthase at the Sarcolemma but Regulates Integrin alpha 7B Expression and Caveolin-3 Distribution
J. Biol. Chem., February 8, 2002; 277(7): 4672 - 4679.
[Abstract] [Full Text] [PDF]

T. Y. Nakamura, Y. Iwata, M. Sampaolesi, H. Hanada, N. Saito, M. Artman, W. A. Coetzee, and M. Shigekawa
Stretch-activated cation channels in skeletal muscle myotubes from sarcoglycan-deficient hamsters
Am J Physiol Cell Physiol, August 1, 2001; 281(2): C690 - C699.
[Abstract] [Full Text] [PDF]

M. Royuela, G. Hugon, F. Rivier, J. A. Fehrentz, J. Martinez, R. Paniagua, and D. Mornet
Variations in Dystrophin Complex in Red and White Caudal Muscles from Torpedo marmorata
J. Histochem. Cytochem., July 1, 2001; 49(7): 857 - 866.
[Abstract] [Full Text] [PDF]

H. Ueda, K. Ueda, T. Baba, and S. Ohno
{{delta}}- and {{gamma}}-Sarcoglycan Localization in the Sarcoplasmic Reticulum of Skeletal Muscle
J. Histochem. Cytochem., April 1, 2001; 49(4): 529 - 538.
[Abstract] [Full Text]

C. Cifuentes-Diaz, T. Frugier, F. D. Tiziano, E. Lacene, N. Roblot, V. Joshi, M. H. Moreau, and J. Melki
Deletion of Murine SMN Exon 7 Directed to Skeletal Muscle Leads to Severe Muscular Dystrophy
J. Cell Biol., March 5, 2001; 152(5): 1107 - 1114.
[Abstract] [Full Text] [PDF]

Y. Hagiwara, T. Sasaoka, K. Araishi, M. Imamura, H. Yorifuji, I. Nonaka, E. Ozawa, and T. Kikuchi
Caveolin-3 deficiency causes muscle degeneration in mice
Hum. Mol. Genet., December 1, 2000; 9(20): 3047 - 3054.
[Abstract] [Full Text] [PDF]

M. Imamura, K. Araishi, S. Noguchi, and E. Ozawa
A sarcoglycan-dystroglycan complex anchors Dp116 and utrophin in the peripheral nervous system
Hum. Mol. Genet., December 1, 2000; 9(20): 3091 - 3100.
[Abstract] [Full Text] [PDF]

V. Allamand and K. P. Campbell
Animal models for muscular dystrophy: valuable tools for the development of therapies
Hum. Mol. Genet., October 1, 2000; 9(16): 2459 - 2467.
[Abstract] [Full Text] [PDF]

G. E. Crawford, J. A. Faulkner, R. H. Crosbie, K. P. Campbell, S. C. Froehner, and J. S. Chamberlain
Assembly of the Dystrophin-Associated Protein Complex Does Not Require the Dystrophin Cooh-Terminal Domain
J. Cell Biol., September 18, 2000; 150(6): 1399 - 1410.
[Abstract] [Full Text] [PDF]

R. H. Crosbie, L. E. Lim, S. A. Moore, M. Hirano, A. P. Hays, S. W. Maybaum, H. Collin, S. A. Dovico, C. A. Stolle, M. Fardeau, et al.
Molecular and genetic characterization of sarcospan: insights into sarcoglycan-sarcospan interactions
Hum. Mol. Genet., August 12, 2000; 9(13): 2019 - 2027.
[Abstract] [Full Text] [PDF]

M. Yoshida, H. Hama, M. Ishikawa-Sakurai, M. Imamura, Y. Mizuno, K. Araishi, E. Wakabayashi-Takai, S. Noguchi, T. Sasaoka, and E. Ozawa
Biochemical evidence for association of dystrobrevin with the sarcoglycan-sarcospan complex as a basis for understanding sarcoglycanopathy
Hum. Mol. Genet., April 12, 2000; 9(7): 1033 - 1040.
[Abstract] [Full Text] [PDF]

C. S. Lebakken, D. P. Venzke, R. F. Hrstka, C. M. Consolino, J. A. Faulkner, R. A. Williamson, and K. P. Campbell
Sarcospan-Deficient Mice Maintain Normal Muscle Function
Mol. Cell. Biol., March 1, 2000; 20(5): 1669 - 1677.
[Abstract] [Full Text]

A. Hack, M. Lam, L Cordier, D. Shoturma, C. Ly, M. Hadhazy, M. Hadhazy, H. Sweeney, and E. McNally
Differential requirement for individual sarcoglycans and dystrophin in the assembly and function of the dystrophin-glycoprotein complex
J. Cell Sci., January 7, 2000; 113(14): 2535 - 2544.
[Abstract] [PDF]

L. A. Liu and E. Engvall
Sarcoglycan Isoforms in Skeletal Muscle
J. Biol. Chem., December 31, 1999; 274(53): 38171 - 38176.
[Abstract] [Full Text] [PDF]

G. Piluso, M. Mirabella, E. Ricci, A. Belsito, C. Abbondanza, S. Servidei, A. A. Puca, P. Tonali, G. A. Puca, and V. Nigro
gamma 1- and gamma 2-Syntrophins, Two Novel Dystrophin-binding Proteins Localized in Neuronal Cells
J. Biol. Chem., May 19, 2000; 275(21): 15851 - 15860.
[Abstract] [Full Text] [PDF]

R. Barresi, S. A. Moore, C. A. Stolle, J. R. Mendell, and K. P. Campbell
Expression of gamma -Sarcoglycan in Smooth Muscle and Its Interaction with the Smooth Muscle Sarcoglycan-Sarcospan Complex
J. Biol. Chem., December 1, 2000; 275(49): 38554 - 38560.
[Abstract] [Full Text] [PDF]

This Article

Abstract

FREE Full Text (PDF)

Alert me when this article is cited

Alert me if a correction is posted

Services

Email this article to a friend

Similar articles in this journal

Similar articles in ISI Web of Science

Similar articles in PubMed

Alert me to new issues of the journal

Add to My Personal Archive

Download to citation manager

Search for citing articles in:
ISI Web of Science (77)

Request Permissions

Google Scholar

Articles by Araishi, K.

Articles by Ozawa, E.

Search for Related Content

PubMed

PubMed Citation

Articles by Araishi, K.

Articles by Ozawa, E.

Social Bookmarking

What's this?

Muscle type	Wild-type (n = 5)	BSG^-^/- (n = 5)	Ratio BSG^-^/-/wild-type
Quadriceps femoris	0.93 0.13	1.25 0.19^a	1.34
Tibialis anterior	0.26 0.03	0.43 0.06^b	1.65
Gastrocnemius	0.87 0.08	0.92 0.24^a	1.05

				J. R. Guyon, J. Goswami, S. J. Jun, M. Thorne, M. Howell, T. Pusack, G. Kawahara, L. S. Steffen, M. Galdzicki, and L. M. Kunkel Genetic isolation and characterization of a splicing mutant of zebrafish dystrophin Hum. Mol. Genet., January 1, 2009; 18(1): 202 - 211. [Abstract] [Full Text] [PDF]

				J. Davis, M. V. Westfall, D. Townsend, M. Blankinship, T. J. Herron, G. Guerrero-Serna, W. Wang, E. Devaney, and J. M. Metzger Designing Heart Performance by Gene Transfer Physiol Rev, October 1, 2008; 88(4): 1567 - 1651. [Abstract] [Full Text] [PDF]

				N. Urasawa, M. R. Wada, N. Machida, K. Yuasa, Y. Shimatsu, Y. Wakao, S. Yuasa, T. Sano, I. Nonaka, A. Nakamura, et al. Selective Vacuolar Degeneration in Dystrophin-Deficient Canine Purkinje Fibers Despite Preservation of Dystrophin-Associated Proteins With Overexpression of Dp71 Circulation, May 13, 2008; 117(19): 2437 - 2448. [Abstract] [Full Text] [PDF]

				K. Kobuke, F. Piccolo, K. W. Garringer, S. A. Moore, E. Sweezer, B. Yang, and K. P. Campbell A common disease-associated missense mutation in alpha-sarcoglycan fails to cause muscular dystrophy in mice Hum. Mol. Genet., May 1, 2008; 17(9): 1201 - 1213. [Abstract] [Full Text] [PDF]

				D. J. Milner and S. J. Kaufman {alpha}7{beta}1 Integrin Does Not Alleviate Disease in a Mouse Model of Limb Girdle Muscular Dystrophy Type 2F Am. J. Pathol., February 1, 2007; 170(2): 609 - 619. [Abstract] [Full Text] [PDF]

				L. M. Judge, M. Haraguchiln, and J. S. Chamberlain Dissecting the signaling and mechanical functions of the dystrophin-glycoprotein complex J. Cell Sci., April 15, 2006; 119(8): 1537 - 1546. [Abstract] [Full Text] [PDF]

				D. Danieli-Betto, A. Esposito, E. Germinario, D. Sandona, T. Martinello, A. Jakubiec-Puka, D. Biral, and R. Betto Deficiency of {alpha}-sarcoglycan differently affects fast- and slow-twitch skeletal muscles Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2005; 289(5): R1328 - R1337. [Abstract] [Full Text] [PDF]

				M. Imamura, Y. Mochizuki, E. Engvall, and S. Takeda {varepsilon}-Sarcoglycan compensates for lack of {alpha}-sarcoglycan in a mouse model of limb-girdle muscular dystrophy Hum. Mol. Genet., March 15, 2005; 14(6): 775 - 783. [Abstract] [Full Text] [PDF]

				S. C. Brown, S. Torelli, M. Brockington, Y. Yuva, C. Jimenez, L. Feng, L. Anderson, I. Ugo, S. Kroger, K. Bushby, et al. Abnormalities in {alpha}-Dystroglycan Expression in MDC1C and LGMD2I Muscular Dystrophies Am. J. Pathol., February 1, 2004; 164(2): 727 - 737. [Abstract] [Full Text] [PDF]

				S. Takeda, M. Kondo, J. Sasaki, H. Kurahashi, H. Kano, K. Arai, K. Misaki, T. Fukui, K. Kobayashi, M. Tachikawa, et al. Fukutin is required for maintenance of muscle integrity, cortical histiogenesis and normal eye development Hum. Mol. Genet., June 15, 2003; 12(12): 1449 - 1459. [Abstract] [Full Text] [PDF]

				R. H. CROSBIE, R. BARRESI, and K. P. CAMPBELL Loss of sarcolemma nNOS in sarcoglycan-deficient muscle FASEB J, November 1, 2002; 16(13): 1786 - 1791. [Abstract] [Full Text] [PDF]

				J. F. Watchko, T. L. O'Day, and E. P. Hoffman Functional characteristics of dystrophic skeletal muscle: insights from animal models J Appl Physiol, August 1, 2002; 93(2): 407 - 417. [Abstract] [Full Text] [PDF]

				D. J. Blake, A. Weir, S. E. Newey, and K. E. Davies Function and Genetics of Dystrophin and Dystrophin-Related Proteins in Muscle Physiol Rev, April 1, 2002; 82(2): 291 - 329. [Abstract] [Full Text] [PDF]

				P. D. Cote, H. Moukhles, and S. Carbonetto Dystroglycan Is Not Required for Localization of Dystrophin, Syntrophin, and Neuronal Nitric-oxide Synthase at the Sarcolemma but Regulates Integrin alpha 7B Expression and Caveolin-3 Distribution J. Biol. Chem., February 8, 2002; 277(7): 4672 - 4679. [Abstract] [Full Text] [PDF]

				T. Y. Nakamura, Y. Iwata, M. Sampaolesi, H. Hanada, N. Saito, M. Artman, W. A. Coetzee, and M. Shigekawa Stretch-activated cation channels in skeletal muscle myotubes from sarcoglycan-deficient hamsters Am J Physiol Cell Physiol, August 1, 2001; 281(2): C690 - C699. [Abstract] [Full Text] [PDF]

				M. Royuela, G. Hugon, F. Rivier, J. A. Fehrentz, J. Martinez, R. Paniagua, and D. Mornet Variations in Dystrophin Complex in Red and White Caudal Muscles from Torpedo marmorata J. Histochem. Cytochem., July 1, 2001; 49(7): 857 - 866. [Abstract] [Full Text] [PDF]

				H. Ueda, K. Ueda, T. Baba, and S. Ohno {{delta}}- and {{gamma}}-Sarcoglycan Localization in the Sarcoplasmic Reticulum of Skeletal Muscle J. Histochem. Cytochem., April 1, 2001; 49(4): 529 - 538. [Abstract] [Full Text]

				C. Cifuentes-Diaz, T. Frugier, F. D. Tiziano, E. Lacene, N. Roblot, V. Joshi, M. H. Moreau, and J. Melki Deletion of Murine SMN Exon 7 Directed to Skeletal Muscle Leads to Severe Muscular Dystrophy J. Cell Biol., March 5, 2001; 152(5): 1107 - 1114. [Abstract] [Full Text] [PDF]

				Y. Hagiwara, T. Sasaoka, K. Araishi, M. Imamura, H. Yorifuji, I. Nonaka, E. Ozawa, and T. Kikuchi Caveolin-3 deficiency causes muscle degeneration in mice Hum. Mol. Genet., December 1, 2000; 9(20): 3047 - 3054. [Abstract] [Full Text] [PDF]

				M. Imamura, K. Araishi, S. Noguchi, and E. Ozawa A sarcoglycan-dystroglycan complex anchors Dp116 and utrophin in the peripheral nervous system Hum. Mol. Genet., December 1, 2000; 9(20): 3091 - 3100. [Abstract] [Full Text] [PDF]

				V. Allamand and K. P. Campbell Animal models for muscular dystrophy: valuable tools for the development of therapies Hum. Mol. Genet., October 1, 2000; 9(16): 2459 - 2467. [Abstract] [Full Text] [PDF]

				G. E. Crawford, J. A. Faulkner, R. H. Crosbie, K. P. Campbell, S. C. Froehner, and J. S. Chamberlain Assembly of the Dystrophin-Associated Protein Complex Does Not Require the Dystrophin Cooh-Terminal Domain J. Cell Biol., September 18, 2000; 150(6): 1399 - 1410. [Abstract] [Full Text] [PDF]

				R. H. Crosbie, L. E. Lim, S. A. Moore, M. Hirano, A. P. Hays, S. W. Maybaum, H. Collin, S. A. Dovico, C. A. Stolle, M. Fardeau, et al. Molecular and genetic characterization of sarcospan: insights into sarcoglycan-sarcospan interactions Hum. Mol. Genet., August 12, 2000; 9(13): 2019 - 2027. [Abstract] [Full Text] [PDF]

				M. Yoshida, H. Hama, M. Ishikawa-Sakurai, M. Imamura, Y. Mizuno, K. Araishi, E. Wakabayashi-Takai, S. Noguchi, T. Sasaoka, and E. Ozawa Biochemical evidence for association of dystrobrevin with the sarcoglycan-sarcospan complex as a basis for understanding sarcoglycanopathy Hum. Mol. Genet., April 12, 2000; 9(7): 1033 - 1040. [Abstract] [Full Text] [PDF]

				C. S. Lebakken, D. P. Venzke, R. F. Hrstka, C. M. Consolino, J. A. Faulkner, R. A. Williamson, and K. P. Campbell Sarcospan-Deficient Mice Maintain Normal Muscle Function Mol. Cell. Biol., March 1, 2000; 20(5): 1669 - 1677. [Abstract] [Full Text]

				A. Hack, M. Lam, L Cordier, D. Shoturma, C. Ly, M. Hadhazy, M. Hadhazy, H. Sweeney, and E. McNally Differential requirement for individual sarcoglycans and dystrophin in the assembly and function of the dystrophin-glycoprotein complex J. Cell Sci., January 7, 2000; 113(14): 2535 - 2544. [Abstract] [PDF]

				L. A. Liu and E. Engvall Sarcoglycan Isoforms in Skeletal Muscle J. Biol. Chem., December 31, 1999; 274(53): 38171 - 38176. [Abstract] [Full Text] [PDF]

				G. Piluso, M. Mirabella, E. Ricci, A. Belsito, C. Abbondanza, S. Servidei, A. A. Puca, P. Tonali, G. A. Puca, and V. Nigro gamma 1- and gamma 2-Syntrophins, Two Novel Dystrophin-binding Proteins Localized in Neuronal Cells J. Biol. Chem., May 19, 2000; 275(21): 15851 - 15860. [Abstract] [Full Text] [PDF]

				R. Barresi, S. A. Moore, C. A. Stolle, J. R. Mendell, and K. P. Campbell Expression of gamma -Sarcoglycan in Smooth Muscle and Its Interaction with the Smooth Muscle Sarcoglycan-Sarcospan Complex J. Biol. Chem., December 1, 2000; 275(49): 38554 - 38560. [Abstract] [Full Text] [PDF]