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Decorin and biglycan expression is differentially altered in several muscular dystrophies

Simona Zanotti, Tiziana Negri, Cristina Cappelletti, Pia Bernasconi, Eleonora Canioni, Claudia Di Blasi, Elena Pegoraro, Corrado Angelini, Patrizia Ciscato, Alessandro Prelle, Renato Mantegazza, Lucia Morandi, Marina Mora
DOI: http://dx.doi.org/10.1093/brain/awh635 2546-2555 First published online: 23 September 2005


Biglycan and decorin are small extracellular proteoglycans that interact with cytokines, whose activity they may modulate, and with matrix proteins, particularly collagens. To better understand their role in muscle fibrosis, we investigated expression of decorin and biglycan transcripts and protein in muscle of several forms of muscular dystrophy, and also expression of perlecan, an extracellular proteoglycan unrelated to collagen deposition. In Duchenne muscular dystrophy (DMD) and LAMA2-mutated congenital muscular dystrophy (MDC1A) we also quantitated transcript levels of the profibrotic cytokine TGF-β1. We examined muscle biopsies from nine DMD patients, aged 2–8 years; 14 BMD (Becker muscular dystrophy) patients (nine aged 1–5 years; five aged 30–37 years); four MDC1A patients (aged 2–7 years); six dysferlin-deficient patients (aged 19–53 years) with mutation ascertained in two, and normal expression of proteins related to limb girdle muscular dystrophies in the others; 10 sarcoglycan-deficient patients: seven with α-sarcoglycan mutation, two with β-sarcoglycan mutation and one with γ-sarcoglycan mutation (five aged 8–15 years; five aged 26–43 years); and nine children (aged 1–6 years) and 12 adults (aged 16–61 years) suspected of neuromuscular disease, but who had normal muscle on biopsy. Biglycan mRNA levels varied in DMD and MDC1A depending on the quantitation method, but were upregulated in BMD, sarcoglycanopathies and dysferlinopathy. Decorin mRNA was significantly downregulated in DMD and MDC1A, whereas TGF-β1 was significantly upregulated. Decorin mRNA was normal in paediatric BMD, but upregulated in adult BMD, sarcoglycanopathies and dysferlinopathy. Perlecan transcript levels were similar to those of age-matched controls in all disease groups. By immunohistochemistry, decorin and biglycan were mainly localized in muscle connective tissue; their presence increased in relation to increased fibrosis in all dystrophic muscle. By visual inspection, decorin bands on immunoblot did not differ from those of age-matched controls in all patient groups. However, when the intensity of the bands was quantitated against vimentin and normalized against sarcomeric actin, in DMD and MDC1A the ratio of band intensities was significantly lower than in age-matched controls. Variations in the transcript and protein levels of these proteoglycans in different muscular dystrophies probably reflect the variable disruption of extracellular matrix organization that occurs in these diseases. The significantly lowered decorin levels in DMD and MDC1A may be related to the increased TGF-β1 levels, suggesting a therapeutic role of decorin in these severe dystrophies.

  • proteoglycans
  • fibrosis
  • muscular dystrophy
  • decorin
  • biglycan
  • BMD = Becker muscular dystrophy
  • DAPs = dystrophin-associated proteins
  • DMD = Duchenne muscular dystrophy
  • TGF-β = transforming growth factor-β


Biglycan and decorin are closely similar, extracellularly located, small leucine-rich proteoglycans, distinguished from each other principally by the presence of one (decorin) or two (biglycan) chondroitin or dermatan sulphate side chains at their N-terminal ends (Krusius and Ruoslahti, 1986).

Numerous functions of biglycan and decorin have been documented including protein–protein interactions, cell adhesion, signal transduction, DNA repair and RNA processing (Vogel and Trotter, 1987; Winnemoller et al., 1991; Bidanset et al., 1992a; Kobe and Deisenhofer, 1994; De Luca et al., 1996; Kinsella et al., 1997; Santra et al., 2000). Functions of likely relevance in muscle diseases include binding to collagens, binding to transforming growth factor-β (TGF-β), and interaction of biglycan with α-dystroglycan (Bowe et al., 2000), a component of the dystrophin-associated protein (DAP) complex of the muscle cell membrane that is often disrupted in muscular dystrophies.

Decorin binds type I (Scott and Orford, 1981), II (Vogel et al., 1984), III (Thieszen and Rosenquist, 1995) and VI collagens (Bidanset et al., 1992b); biglycan binds type I (Schonherr et al., 1995) and VI collagens (Wiberg et al., 2002). The two proteoglycans also differ in tissue distribution with decorin found mainly in the body of the extracellular matrix and biglycan localized more closely around cells. Differences are also evident from gene disruption studies: The null biglycan mouse has an osteoporosis-like phenotype, with growth failure and reduced bone formation (Xu et al., 1998). The null decorin mouse has lax skin of markedly reduced tensile strength, with thin dermis, similar to that observed in the human Ehlers–Danlos syndrome (Corsi et al., 2002).

In vitro both decorin and biglycan form complexes with TGF-β (Yamaguchi et al., 1990; Noble et al., 1992; Hildebrand et al., 1994); decorin in particular sequesters TGF-β, forming complexes that modulate cytokine's biological activity (Hildebrand et al., 1994).

Fibrosis is a major characteristic of a wide spectrum of diseases affecting kidney, bladder, liver, lung, gut, heart and muscle. In muscle dystrophies such as congenital muscular dystrophies and Duchenne muscular dystrophy (DMD), the primary defect leads to continuous myofibre degeneration, while regeneration processes are unable to keep pace with the requirement for new fibres, and massive connective tissue deposition (fibrosis) occurs, with marked increases in levels of extracellular matrix proteins (Stephens et al., 1982). The mechanisms of these alterations are not completely understood, however increases in TGF-β production have been linked to fibrosis in muscle as well as kidney, liver and lung (Bernasconi et al., 1995; Sime et al., 1997; Coker and Laurent, 1998).

In the mdx mouse model of DMD, significantly increased synthesis of several proteoglycans, including decorin, has been reported (Càceres et al., 2000); biglycan expression was increased by immunohistochemical analysis (Bowe et al., 2000), but transcript levels were either unchanged or slightly increased, depending on the probe used by microarray analysis (Porter et al., 2004). In DMD muscle, a recent microarray and real-time PCR study demonstrated upregulation of biglycan transcript (Haslett et al., 2002).

To better understand the roles of decorin and biglycan in muscle fibrosis in muscular dystrophies, we analysed mRNA and protein expression of both, in the skeletal muscle of patients with DMD, Becker muscular dystrophy (BMD), LAMA2-mutated CMD (MDC1A), sarcoglycanopathies and dysferlinopathy, in comparison with normal age-matched controls. We also analysed the expression of the pro-fibrotic cytokine TGF-β1, and perlecan, another extracellular proteoglycan not involved in collagen organization.

Patients and methods

We examined muscle biopsies from nine DMD patients, aged 2–8 years; 14 BMD patients (nine aged 1–5 years; five aged 30–37 years); four MDC1A patients (age 2–7 years); six dysferlin-deficient patients (aged 19–53 years) with mutation ascertained in two, and normal expression of proteins related to limb girdle muscular dystrophies in the others; 10 sarcoglycan-deficient patients (SGD): seven with α-sarcoglycan mutation, two with β-sarcoglycan mutation, and one with γ-sarcoglycan mutation (five aged 8–15 years; five aged 26–43 years); and nine children (age 1–6 years) and 12 adults (age 16–61 years) suspected of neuromuscular disease, but who had normal muscle on biopsy.

Informed consent for biopsy and biopsy storage for research was obtained in all cases from patients or parents. Muscle samples were frozen in isopentane pre-cooled in liquid nitrogen and stored in liquid nitrogen pending use.

RNA extraction

Total RNA was isolated from 25 to 30 mg of muscle tissue using the SV Total RNA Isolation System (Promega Corporation, Madison, WI), according to the manufacturer's instructions, and stored at −80°C.

The quantity and purity of RNA were checked spectrophotometrically at 260 nm after diluting 5 μl 1 : 100 with nuclease-free water and assuming 1 absorbance unit equivalent to 40 μg of single-stranded RNA/ml. Purity was estimated as the ratio of absorbance at 260 nm to that at 280 nm.

First-strand cDNA synthesis

RNA (1 μg) was reverse transcribed in the presence of 1× first strand buffer (Invitrogen Life Technologies, Carlsbad, CA), 1 mM of each deoxynucleoside triphosphate (Promega), 8 pM random hexamers, 10 μM dithiothreitol, 1 IU/μl RNAase inhibitor (Roche Molecular Biochemicals, Basel, Switzerland) and 10 IU/μl M-MLV reverse transcriptase (Gibco, Life Technologies, Carlsbad, CA). The reaction mixture was incubated at room temperature for 10 min, at 37°C for 1 h and at 95°C for 5 min. The reaction product was stored at −20°C pending use.

cDNA integrity was assessed by amplification of human β-actin (GenBank accession no. BC004251) using specific primers (forward 5′-TCACCCACACTGTGCCCATCTACGA-3′ and reverse 5′-CAGCGGAACCGCTCATTGCCAATGG-3′). PCR conditions were: 94°C 1 min, 62°C 1 min, 72°C 1 min, for 35 cycles.

Synthesis of competitors

Homologous competitors were obtained by PCR reaction using specific primers designed to be amplifiable by the same primers as those amplifying the target (decorin, biglycan or perlecan) yet distinguishable from the target. The competitor for human decorin (GenBank accession no. NM_001920) was amplified using the primers: forward 5′-TGAAGGCCACTATCATCCTCC-3′ and reverse engineered primer (R-eng) 5′-TCACCAAAGGTGTAAATGCTCCACTTTGTCCAGACC-3′. The competitor for human biglycan (GenBank accession no. NM_001711) was amplified using the primers: 5′-TCACACCCACCTACAGCG-3′ and R-eng 5′-ATGTAGAGCTTCTGCAGCTTCCTGTTCTGCAGGTCC-3′. The competitor for human perlecan (GenBank accession no. NM_005529) was amplified using the primers: 5′-GGTTGCACCAAATGTCTGTG-3′ and R-eng 5′-TCTGATGGTAACTGGGGAGGACTCAATTCTGA-3′.

For the PCR reactions, an amount of template cDNA corresponding to 0.04 μg of total RNA was used.

For human decorin, use of the R-eng primer resulted in a fragment 142 bp shorter than the main PCR product of 245 bp. PCR conditions were: 94°C 1 min, 50°C 2 min, 72°C 1 min, for 5 cycles; then 94°C 1 min, 55°C 2 min, 72°C 1 min, for 25 cycles.

For biglycan, use of the R-eng primer produced a fragment 104 bp shorter than the main product of 271 bp. PCR conditions were: 94°C 1 min, 50°C 2 min, 72°C 1 min, for 5 cycles; then 94°C 1 min, 55°C 2 min, 72°C 1 min, for 25 cycles.

For perlecan, use of the R-eng primer produced a fragment 100 bp shorter than the main one of 273 bp. PCR conditions were: 94°C 1 min, 51°C 2 min, 72°C 1 min, for 5 cycles; then 94°C 1 min, 56°C 2 min, 72°C 1 min, for 25 cycles.

Purification of competitors

The total volume of PCR reaction (25 μl) was loaded onto a 2% agarose gel (agarose MP Roche) in 0.1 M Tris, 0.09 M boric acid, 1 mM EDTA buffer (Invitrogen), stained with 5 μg/ml ethidium bromide (Bio-Rad Laboratories, Hercules, CA) and electrophoresed at 90 mA for 45 min in a submarine gel unit (Bio-Rad). The competitor band was excised from the gel and purified using the NucleoSpin Extract 2-in-1 kit (Macherey-Nagel, Düren, Germany). The quality of the purified competitors was checked electrophoretically and quantified spectrophotometrically. The PCR competitor products were sequenced to check identity (3100 Genetic Analyzer, Applied Biosystems, CA).

Competitive PCR

PCR mixtures containing different known quantities of competitor were added to reverse-transcribed samples derived from 0.04 μg of total RNA in 25 μl reaction volume. The numbers of molecules of decorin competitor were: 6 × 104, 12.5 × 104, 2.5 × 104, 5 × 105, 7.5 × 105, 1.25 × 106, 2.5 × 106, and 5 × 106; the primers used were: forward 5′-TGAAGGCCACTATCATCCTCC-3′, reverse 5′-TCACCAAAGGTGTAAATGCTCC-3′ and the conditions were 94°C 1 min, 55°C 1 min, 72°C 1 min for 35 cycles. The numbers of copies of biglycan competitor were: 1.25 × 103, 2.5 × 103, 5 × 103, 104, 1.25 × 104, 2.5 × 104 and 5 × 104; the primers were: forward 5′-TCACACCCACCTACAGCG-3′ and reverse 5′-ATGTAGAGCTTCTGCAGCTTCC-3′ and the PCR conditions were: 94°C 1 min, 60°C 1 min and 72°C 1 min, for 35 cycles, The numbers of copies of perlecan competitor were: 6.5 × 104, 1.3 × 105, 2.6 × 105, 5.5 × 105, 8 × 105, 1.5 × 106 and 2.6 × 106; the primers were: forward 5′-GGTTGCACCAAATGTCTGTG-3′ and reverse 5′-TCTGATGGTAACTGGGGAGG-3′ and PCR conditions were: 94°C 1 min, 56°C 1 min and 72°C 1 min for 35 cycles. All primers were synthesized by Proligo Primers and Probes Boulder, CO.

Electrophoresis after competitive PCR was performed on 2% agarose gel. DNA bands were visualized with 5 μg/ml of ethidium bromide and photographed by Kodak EDAS290 digital camera (Eastman Kodak Company, Scientific Imaging Systems, New Haven, CO). Fluorescence quantification of competitors and targets employed a Fluor-S MultiImager densitometer (Bio-Rad) and the Quantity One software version 4.2.3 (Bio-Rad).

The logarithms of the fluorescence density of competitor and target were plotted against competitor copy numbers. The number of target RNA molecules was determined from the intersection of the regression curve on the x-axis (number of competitor copies) (Fig. 1). The results were expressed as number of copies of decorin, biglycan or perlecan mRNA/0.04 μg total RNA.

Fig. 1

Competitive PCR of decorin transcript. (A) PCR product of decorin target (upper band, 387 bp) and competitor (lower band, 245 bp) resolved on ethidium bromide stained 2% agarose gel. Lanes 1–8: correspond to 6 × 104, 12.5 × 104, 2.5 × 104, 5 × 105, 7.5 × 105, 1.25 × 106, 2.5 × 106 and 5 × 106 copies of decorin competitor, respectively. Lane 1: 100 bp ladder (DNA molecular weight marker XIV, Roche). (B) cDNA sample integrity confirmed by PCR of housekeeping gene β-actin (295 bp). (C) Representative plot used to determine the quantity of the target mRNA sequences in muscle sample.

Real-time PCR

The expression of decorin, biglycan and TGF-β1 mRNA was analysed by quantitative real-time PCR in DMD and MDC1A patients and age-matched controls. RNA was reverse transcribed using the High Capacity cDNA Archive kit (Applied Biosystems, CA), according to the manufacturer's instructions.

TaqMan Universal PCR MasterMix and Assays-on-Demand Gene Expression probes (Applied Biosystems) were used for the PCR step (Assay ID Decorin HS 00266491 m1; Assay ID Biglycan HS 00156076 m1; Assay ID TGF-β1 HS 00171257 m1). The primer sequences are not publicly available, but the manufacturer has established their validity. Reactions were performed in 96-well plates with 50 μl volumes. All samples were analysed in triplicate. Cycling parameters were 2 min at 50°C, 95°C for 10 min, and followed by 40 cycles of PCR (15 s at 95°C and 1 min at 60°C). Products were detected with Perkin-Elmer ABI Prism 7000 sequence detection system. The comparative Ct method was used for quantification. Values obtained were normalized against those of the human β-actin as endogenous control.


Immunohistochemical analyses were performed on 6 μm cryostat muscle sections on gelatinized slides. Polyclonal anti-decorin and anti-biglycan antibodies were a gift of Dr Larry Fisher (Matrix Biochemistry Unit, National Institute of Health, Bethesda, MD) and were characterized in his laboratory (Fisher et al, 1995). Monoclonal anti-decorin antibody was from R&D Systems Inc, Minneapolis, MN. Anti-heparan sulphate proteoglycan (HSPG or perlecan) and anti-collagen VI antibodies were rat and mouse monoclonals, respectively, from Chemicon International Temecula, CA.

Sections were incubated for 90 min in primary antibody (dilutions: anti-decorin polyclonal 1:200, anti-decorin monoclonal 1:25, anti-biglycan 1:50, anti-perlecan 1:500, anti-collagen VI 1:250), followed by incubation for 60 min in biotinylated anti-mouse, anti-rat or anti-rabbit IgG (Jackson ImmunoResearch Inc, West Grove, PA) 1:250, and finally rhodamine-avidin D (Vector Labs, Burlingame, CA) 1:250 for 60 min.

To obtain double immunolabelling, decorin or biglycan polyclonal antibodies were used in combination with collagen VI monoclonal mouse antibody (Chemicon). In all cases a mixture of the primary antibodies was used in the first incubation step, followed by biotinylated goat anti-rabbit IgG (Jackson), by rhodamine-avidin D (Vector Labs) and by Cy2-labelled goat anti-mouse IgG (Jackson).

All incubations were carried out at room temperature in a humid chamber. As controls, either sections were incubated with isotype-specific IgG (Dako, Copenhagen, Denmark) or primary antibody was omitted. Sections were examined either under a Zeiss Axioplan fluorescence microscope or a Bio-Rad–Nikon confocal microscope with krypton–argon laser.

Western blot

Western blot analysis of decorin was performed on muscle homogenates as described by Cooper et al. (2003). Briefly, two consecutive 8 μm thick frozen sections of muscle, ∼5 mm diameter, from each patient and control, were solubilized in 25 μl lysis buffer containing 4% SDS, 125 mM Tris–HCl pH 8.8, 40% glycerol, and 0.5 mM PMSF, sonicated, boiled, and centrifuged at 15 000 g for 5 min. The supernatants were electrophoresed on a 7.5% SDS–PAGE gel (0.75 mm thickness) and transferred to nitrocellulose membranes. Blocked membranes were probed with anti-decorin 1:250 (R&D), anti-vimentin 1:100 (Dako), and anti-sarcomeric actin (1:2000; Dako) monoclonal antibodies, followed by biotin-conjugated secondary antibody (1:2500; Jackson ImmunoResearch), by alkaline phosphatase-conjugated streptavidin (1:5000; Jackson ImmunoResearch) and by detection with BCIP/NBT substrate (Pierce, Rockford, IL).

Decorin was quantitated densitometrically using the Fluor-S-Max Multi-Imager (Bio-Rad) and the Quantity One software version 4.2.3 (Bio-Rad). Sarcomeric actin and vimentin were quantitated as indicators of how much muscle and connective tissue protein, respectively, was loaded onto each lane.

The outline of each band was defined by a software algorithm involving background measurements, to obtain a value for volume OD. The volume OD for each protein band was divided by the corresponding value of the vimentin band in that sample. The value obtained was divided by the value of the sarcomeric actin band. Densitometric estimates of decorin abundance were expressed as percentages of those in normal control samples.

Quantitation of fibrosis

The extent of connective tissue was measured on collagen VI-immunostained sections at ×20 magnification using the NIH Image software version 1.62 (http://rsb.info.nih.gov/nih-image/). At least three fields from each patient were analysed. Briefly, fields of equal size were photographed and digitalized; using Image, a threshold was applied to the photographs to obtain black and white images with areas positive for collagen in black and negative areas in white. Manual corrections were sometimes applied to eliminate non-muscle/non-fibrosis areas or to add areas not recognized by the software. The area positive for collagen VI was calculated as a percentage of the entire image. The mean ± standard deviation (SD) was then obtained for each disease group from the total of all analysed fields.

Statistical analysis

The Wilcoxon non-parametric test was used to determine whether differences between groups were significant at the P < 0.05 level.


Competitive PCR

Decorin mRNA (Fig. 2) was 2.10 × 106 ± 0.08 × 106 (copies per 0.04 μg RNA) in paediatric controls and 1.2 × 106 ± 0.12 × 106 in adult controls. In DMD and MDC1A samples, values for decorin mRNA were 0.70 × 106 ± 0.17 × 106 and 1 × 106 ± 0.06 × 106, respectively, both values being significantly lower (P < 0.05) than in paediatric controls.

Fig. 2

Expression of (A) decorin, (B) biglycan and (D) perlecan mRNA copies (by competitive PCR) and (C) extent of fibrosis in age-matched controls and DMD, paediatric BMD, paediatric SGD, MDC1A, adult BMD, adult SGD and adult dysferlin-deficient patients. Data are expressed as means ± SD. Asterisks indicate significantly different (P < 0.05) from age-matched control.

In all other forms of muscular dystrophy, except paediatric BMD, decorin mRNA levels were significantly higher (P < 0.05) than controls. In particular, decorin mRNA was 2.85 × 106 ± 0.08 × 106 in paediatric BMD; 2.7 × 106 ± 0.08 × 106 in adult BMD; 3.08 × 106 ± 0.07 × 106 in paediatric SGD; 2.7 × 106 ± 0.07 × 106 in adult SGD; and 3.3 × 106 ± 0.06 × 106 in dysferlin-deficient cases.

Biglycan mRNA (Fig. 2) was 2.20 × 104 ± 0.42 × 104 in paediatric controls and 1.38 × 104 ± 0.49 × 104 in adult controls. Biglycan transcript levels were significantly lower (P < 0.05) in DMD (1.8 × 104 ± 0.72 × 104) and MDC1A (1.8 × 104 ± 0.05 × 104) patients than paediatric controls, but significantly higher (P < 0.05) than age-matched controls in SGD (2.9 × 104 ± 0.12 × 104 children; 3.3 × 104 ± 0.32 × 104 adults), dysferlin-deficient myopathy (3.39 × 104 ± 0.21 × 104) and BMD (2.7 × 104 ± 0.4 × 104 children; 2.18 × 104 ± 0.58 × 104 adult).

mRNA levels of both decorin and biglycan increased with increasing fibrosis in all patient groups except DMD and MDC1A (Fig. 2). In controls, mRNA levels were higher in children than in adults in relation to the greater extent of connective tissue in children's muscle (Dubowitz and Brooke, 1973).

Perlecan mRNA levels (Fig. 2) did not differ significantly between patients and age-matched controls.

Real-time PCR

As biopsies from some patients analysed by competitive PCR were no longer available, muscle samples from four additional DMD patients aged 5–8 years were analysed. Decorin mRNA expression was a mean of 3.0 times lower in DMD than in aged-matched healthy controls (consistent with competitive PCR findings) and did not vary appreciably with age (Fig. 3).

Fig. 3

Changes in TGF-β1 and decorin transcript levels, relative to age-matched controls, in nine DMD and four MDC1A patients as determined by real-time PCR.

In MDC1A patients, decorin mRNA expression was a mean of 2.4 times lower than in age-matched controls (consistent with competitive PCR findings) (Fig. 3).

Biglycan transcript levels in DMD patients were a mean of 3.1 times higher than age-matched controls (cf. decrease by competitive PCR). Expression appeared to vary with age (3.5-fold increase in patients aged 6–12 years; 1.75-fold increase in those aged 2–5 years).

Biglycan transcript expression in MDC1A patients did not differ from that in age-matched controls (cf. decrease by competitive PCR).

TGF-β1 mRNA transcripts were a mean of 11.7 times higher in DMD and 29.3 times higher in MDC1A than age-matched controls (Fig. 3).


In normal controls decorin and biglycan were located prominently in the perimysium and endomysium; they were also expressed in association with fibre surfaces, and apparently in increasing quantities with increasing age (Figs 4 and 5).

Fig. 4

Representative photomicrographs showing immunolocalization of decorin, biglycan, perlecan and collagen VI in consecutive sections of muscle from paediatric controls and paediatric patients. Magnification ×200.

Fig. 5

Representative photomicrographs showing immunolocalization of decorin, biglycan, perlecan and collagen VI in consecutive sections of muscle from adult controls and adult patients. Magnification ×200.

In all patients' muscle both proteins were expressed similarly to controls except that the endomysial and perimysial compartments were more extensive in patients and hence positivity in sections was more extensive. In the endomysium, decorin positivity had a mesh-like appearance (Fig. 5). Everywhere decorin co-localized with collagen VI (Fig. 6). Biglycan, by contrast was generally less extensive in the endomysium and often prominently associated with fibre surfaces; furthermore the locations of biglycan and collagen VI rarely overlapped (Fig. 6).

Fig. 6

Confocal co-localization of decorin and collagen VI, and biglycan and collagen VI in muscle from a DMD patient. Magnification ×400.


By visual inspection, decorin bands on immunoblot did not differ from those of age-matched controls in all patient groups (Fig. 7). However, when the intensity of the bands was quantitated against vimentin (marker of connective tissue) normalized against sarcomeric actin (marker of muscle tissue), in DMD and MDC1A the ratio of band intensities was significantly lower (P < 0.05) than in age-matched controls (mean ratio of optical densities: 1.36 ± 0.13 in controls; 0.52 ± 0.19 in DMD; 0.47 ± 0.11 in MDC1A).

Fig. 7

Representative western blots of decorin in (A) paediatric control (lane 1), paediatric BMD (lane 2), DMD (lane 3), paediatric SGD (lane 4), and MDC1A (lane 5), (B) in adult control (lane 1), adult BMD (lane 2), and dysferlin deficient (lane 3). Decorin band intensities do not vary greatly with disease; sarcomeric actin bands also vary little; however, vimentin bands (marker of connective tissue) are more intense in DMD and MDC1A patients.

In BMD, SGD and dysferlin-deficient muscle, the ratios did not differ from those of age-matched control muscles. In our hands biglycan antibody did not work on immunoblot.


Muscular dystrophies share several clinical and pathological traits, but vary in severity, inheritance pattern and molecular defect. Several of them are due to mutations in genes encoding components of the DAP complex. We investigated interactions between DAP components and three extracellular matrix proteoglycans in fibrotic connective tissue of muscle from patients with DAP-related muscular disorders (DMD, BMD, sarcoglycanopathies and MDC1A) and also those with primary dysferlin deficiency as disease controls. We evaluated biglycan expression because of its ability to bind β-dystroglycan (Bowe et al., 2000), a DAP component, and indications that it interacts with β- and γ-sarcoglycan (Rafii et al., 2000) and also with the pro-fibrotic cytokine TGF-β1 (Hildebrand et al., 1994); decorin because of its similarities to biglycan; and perlecan as a matrix proteoglycan unrelated to collagen deposition.

We found significant alterations in the mRNA and protein levels of biglycan and decorin in the muscle diseases investigated. In particular we found marked reductions in decorin transcript levels in DMD and MDC1A patients compared with age-matched controls, whereas decorin transcript levels were significantly increased in all other diseases studied, except paediatric BMD.

By competitive PCR, we found that biglycan transcripts were consistently increased in all diseases except DMD and MDC1A where they were significantly lower than in controls (although SDs were high). However, by real-time PCR, biglycan transcripts were increased in DMD and unchanged in MDC1A.

For the real-time PCR study we included DMD patients aged 6–8 years, in addition to those aged 2–5.5 years included in the competitive PCR study. We found that biglycan transcript levels were highest in older DMD patients, in accord with the findings of Haslett et al. (2002) who examined DMD patients aged 5–7 years by microarray and real-time PCR.

The discrepancy between our competitive and real-time PCR results for biglycan is puzzling as there was no discrepancy for decorin. Apart from the fact that competitive PCR is less reliable as a quantitation method, it is also noteworthy that individual variation in DMD was high by competitive PCR, while the samples analysed by real-time PCR included those from older patients in whom variation was more contained. We suggest that biglycan expression may be more related to the extent of collagen deposition, which is quite variable in DMD, particularly during the early years of life. However, the measurement technique may also play a role, as suggested by a recent gene expression profiling study on the mdx mouse, which found that biglycan transcript levels were either unchanged or slightly increased, depending on the probe used (Porter et al., 2004).

Immunohistochemical analysis showed that decorin and biglycan proteins were prominently expressed in the fibrotic connective tissue of all muscle samples including those from DMD and MDC1A patients. This prompted us to quantitate decorin on western blot in relation to markers of connective tissue and muscle proteins. When decorin was compared with vimentin and normalized to sarcomeric actin it was greatly reduced in DMD and MDC1A, but not significantly different from control in the other diseases studied. We were unable to quantitate biglycan in this way.

Càceres et al. (2000) investigated protein synthesis in mdx mice finding increased synthesis of decorin and other proteoglycans, which they interpreted as related to the intense myoproliferation that characterizes this model. They suggested that increased decorin may play a role in allowing mdx muscle to escape the consequences of dystrophin absence, possibly by stabilizing the interaction between the DAP and the extracellular matrix.

An immunohistochemical study by Bowe et al. (2000) on mdx mice found that biglycan protein expression was increased in muscle, in accord with our immunological and transcript findings, and the transcript findings of Haslett et al. (2002). The latter authors suggested that the biglycan increase in muscular dystrophies might be related to collagen deposition and that, in DMD, increased biglycan expression compensates for the absence of dystrophin by stabilizing the interaction between DAP and the extracellular matrix (as also suggested for decorin). Biglycan may also stabilize this interaction in other DAP-related diseases. Unfortunately, elucidation of biglycan's role in muscle pathology is hindered by the non-availability of antibodies that work on immunoblot.

Both decorin and biglycan interact with TGF-β (Kolb et al., 2001a), a cytokine implicated in fibrosis (Sporn and Roberts, 1992). We found that TGF-β transcript levels were greatly increased in DMD and MDC1A patients by real-time PCR, as also reported in previous competitive PCR studies by our group (Bernasconi et al., 1995, 1999) and by Haslett et al. (2002) in DMD. Our findings of marked increase in TGF-β and clear decrease in decorin mRNA in DMD and MDC1A support decorin downregulation by TGF-β in these conditions, with consequent inability of decorin to modulate the biological activity of TGF-β leading to worsened fibrosis. TGF-β has been reported to downregulate decorin mRNA transcription in chondrocytes in vitro (Demoor-Fossard et al., 2001).

Whereas biglycan has been reported to have no effect on TGF-β-mediated fibrotic responses in the lung (Kolb et al., 2001a), decorin binding to TGF-β has been used to overcome TGF-β overproduction in different experimental models of fibrosis. Border et al. (1992) reported that administration of decorin inhibits increased extracellular matrix production and attenuates disease manifestations in the rat model of glomerulonephritis. Introduction of decorin, by direct injection in hamsters or by adenoviral gene transfer in mice reduces bleomycin-induced pulmonary fibrosis (Giri et al., 1997; Kolb et al., 2001b). Daily intraventricular injections of decorin have also been used successfully to attenuate gliosis and inflammation induced in rat cerebral hemisphere by penetrating incisional wounds (Logan et al., 1999). Similarly, in their reproducible muscle laceration injury model in mice, Fukushima et al. (2001) showed that decorin injection into muscle prevents fibrosis in a dose-dependent way and enhances muscle regeneration, resulting in near-complete functional recovery. Li et al. (2004) recently demonstrated that in vitro overproduction of extracellular matrix proteins by myoblasts genetically engineered to express TGF-β, was prevented by decorin treatment.

Myostatin, another member of the TGF-β family and negative regulator of skeletal muscle growth (McPherron et al., 1997) has been recently shown to bind decorin so that it becomes trapped in the extracellular matrix, resulting in downregulation of muscle cell growth (Miura et al., 2004).

Thus, several lines of evidence indicate that decorin may play fundamental roles in regulating extracellular matrix protein deposition via its interaction with TGF-β and collagens, and in regulating muscle cell growth by its interaction with myostatin.

The data of the present study, showing that decorin is reduced in muscle disease characterized by severe fibrosis provide further support to this scenario and suggest the use of this proteoglycan as a therapeutic agent in dystrophic muscle.


The authors thank Don Ward for help with the English. Telethon Italy is gratefully acknowledged for financial support to M.M. (Grant No. GTF02002), C.A. (Grant No. GTF02009), and A.P. (Grant No. GTF02008 to Dr M. Moggio).


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