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* Department of Neuroscience, Brown University, Providence, Rhode Island, USA;
LifeCell Corporation, Branchburg, New Jersey, USA; and
Department of Physiology and Biophysics, University of Washington, Seattle, Washington, USA
1Correspondence: Department of Neuroscience, Brown Univrsity, 190 Thayer St., Box 1953, Providence, RI 02912, USA. E-mail: Justin_Fallon{at}brown.edu
SPECIFIC AIMS
Defects in the dystrophin-associated protein complex (DAPC) are the cause of most muscular dystrophies. The aims of this study were to 1) determine the function of the extracellular matrix proteoglycan biglycan in regulating the composition of the DAPC at the myofiber plasma membrane and 2) test the potential of biglycan as a therapeutic for muscular dystrophy.
PRINCIPAL FINDINGS
1. Biglycan null mice display a dystrophic phenotype
Mutations in any one of several DAPC components cause a wide range of muscular dystrophies. In previous work we have shown that biglycan binds to the DAPC component 
-dystroglycan. Therefore, we tested whether biglycan null mice display a dystrophic phenotype. First, we performed the Evans blue dye uptake assay to determine whether muscle membrane integrity is compromised in the biglycan null mice. We intravenously injected wild-type, biglycan null (bgn), and dystrophin-null (mdx) mice with Evans blue dye, and the extent of uptake into quadriceps femoris muscle fibers was assessed. Wild-type fibers exhibited very little dye uptake, mdx fibers exhibited almost complete uptake, and bgn fibers exhibited an intermediate perimembranous uptake. Next we examined the muscles of bgn mice histologically. A fraction of the myofibers in bgn null mice displayed centrally localized nuclei, a hallmark of regenerated fibers (5.08%±0.58 and 9.65%±1.34 in quadriceps and diaphragm, respectively). We did not observe extensive necrosis or mononuclear cell infiltration. Finally, we measured fiber diameters from quadriceps femoris, gastrocnemius, and diaphragm muscles. In the quadriceps and gastrocnemius, bgn null fibers were smaller than wild-type fibers. On the other hand, the diaphragm of biglycan null mice displayed a wider fiber size distribution. These results indicate that bgn muscle is mildly dystrophic, with increased membrane permeability and an increased number of fibers that have undergone degeneration and regeneration.
2. -Dystrobrevin, syntrophin, and nNOS expression is selectively reduced at the sarcolemma of bgn mice
We next compared the expression of individual DAPC proteins in the quadriceps femoris muscles of adult wild-type and bgn null mice. In all cases we compared biglycan null mice to either wild-type littermates or to congenic, age-matched wild-type animals. Sarcolemmal expression of the 2 chain of laminin, - and ßbeta;-dystroglycan, -, ßbeta;-, and 
-sarcoglycan and dystrophin was equivalent in wild-type and bgn null animals. However, expression levels of -dystrobrevin-1 and -2, - and ßbeta;1-syntrophin and nNOS were reduced at the sarcolemma of bgn mice (Fig. 1
A). In addition, we observed an increase in the intracellular staining of - and ßbeta;2-syntrophin in the larger diameter fibers and an increase in cytosolic ßbeta;1-syntrophin in every fiber of the bgn knockouts (Fig. 1A, B
).
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We also biochemically compared the expression levels of the -dystrobrevins and syntrophins in KCl-washed heavy microsomal membrane fractions (a mixture of plasma and intracellular membranes). The membrane-associated expression of -dystrobrevin-1 and -2 was decreased overall in bgn muscle. Conversely, levels of -, ßbeta;1-, and ßbeta;2-syntrophin were increased in the membrane fractions of bgn mice. The increase in syntrophins in these total membrane fractions is in accord with the increase in intracellular syntrophin expression seen via immunohistochemistry (Fig. 1C
). Finally, we compared nNOS expression between non-KCl-washed membrane fractions from wild-type and bgn muscle. nNOS expression was reduced in the bgn membrane fractions (Fig. 1D
).
3. Biglycan induces the redistribution of nNOS to the plasma membrane
We next developed a cell culture system to further examine the role of biglycan in targeting DAPC components to the cell surface. We cultured biglycan-deficient myotubes and incubated them with either purified recombinant biglycan core polypeptide (0.7 nM) or with vehicle alone for 4 h. Living myotubes were labeled with rhodamine--bungarotoxin to visualize AChRs and to demarcate the plasma membrane. We then fixed and permeabilized the cells and immunostained for nNOS. In untreated cells nNOS was distributed throughout the cytoplasm, with little labeling observed at the plasma membrane. Biglycan treatment increased nNOS levels at the myotube surface. We quantified this redistribution of nNOS by assigning each cell a score (1–4, 4 being the highest) representing the extent to which surface nNOS was present. In the absence of biglycan, the mean score was 2.0 ± 0.11, whereas in the presence of biglycan the mean score was 2.94 ± 0.12. These data demonstrate that treatment of biglycan null myotubes with biglycan core increases the amount of nNOS on the myotube plasma membrane (P<0.01; Kolmogorov-Smirnov test).
We also examined the localization of -dystrobrevin-1 and -2, and -, ßbeta;1-, and ßbeta;2-syntrophin in biglycan null myotubes in the absence or presence of biglycan. -Dystrobrevin-1 and -2 and ßbeta;2-syntrophin staining were widely expressed within the cell, but little cell surface proximal expression was detected. In contrast, -syntrophin and ßbeta;1-syntrophin were expressed throughout the cell including in a surface-proximal disposition. Biglycan treatment did not significantly alter the localization of these proteins.
4. The injection of purified biglycan protein restores the expression of -dystrobrevins and ßbeta;-syntrophins to the sarcolemma of bgn null muscle fibers in vivo
We asked whether intramuscular (i.m.) injection of bgn null mice with purified biglycan core polypeptide could directly restore the expression of the syntrophin-dystrobrevin-nNOS DAPC subcomplex to the sarcolemma. Purified recombinant biglycan core polypeptide (50 µg) was injected into the right quadriceps femoris muscles of 2-wk-old bgn null animals. Buffer alone was injected into the left quadriceps to provide intra-animal comparisons. The injection site was visualized by the inclusion of India ink in each solution. Quadriceps were dissected 4, 7, 11, and 14 days postinjection, sectioned, and immunostained. As shown in Fig. 2
A–D (upper left), i.m. injected biglycan becomes stably associated with the perimysium and sarcolemma. No biglycan immunoreactivity was observed on the vehicle-injected side at any time (Fig. 2A-D
, lower left). Eleven and 14 days after injection of biglycan we observed increased -dystrobrevin-1 and -2 and ßbeta;1- and ßbeta;2-syntrophin expression at the sarcolemma. Moreover, the increased expression of these intracellular DAPC proteins showed a tight spatial correlation with the exogenous biglycan (Fig. 2A-D
; compare upper left and upper right). No up-regulation was observed in the vehicle-injected muscle (Fig. 2A-D
; compare upper and lower right). We did not observe a change in -syntrophin or nNOS expression after biglycan injection. Thus, biglycan can be delivered to muscle in vivo and direct the localization of the -dystrobrevins and ßbeta;-syntrophins to the sarcolemma.
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CONCLUSIONS AND SIGNIFICANCE
In this study we demonstrate that biglycan regulates the targeting of a specific subset of DAPC components—the syntrophin-dystrobrevin-nNOS complex—to the sarcolemma. The mechanism by which biglycan signals to localize this dystrobrevin-syntrophin subcomplex is unknown (Fig. 3
). Intramuscular administration of purified biglycan core polypeptide can restore the sarcolemmal expression of -dystrobrevin-1 and -2, and ßbeta;1- and ßbeta;2-syntrophin in biglycan null mice. This result demonstrates that biglycan can directly induce an increase in protein expression and/or a relocalization of DAPC proteins from the cytoplasm to the plasma membrane. It also shows that i.m. injected biglycan protein appropriately localizes to the perimysium and sarcolemma and is stably associated with these sites for up to 2 wk after administration. These pharmacokinetic properties of biglycan, coupled with its ability to induce specific changes in the localization of some DAPC components in muscle, suggest it is a potential therapy for muscular dystrophy.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5124fje
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