4) Expression of collagen VI α5 and α6 chains in human muscle and in Duchenne

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Expression of collagen VI

α5 and α6 chains in human muscle and in Duchenne

muscular dystrophy-related muscle

ﬁbrosis

Patrizia Sabatelli

a,

⁎

, Francesca Gualandi

b

, Sudheer Kumar Gara

c

, Paolo Grumati

d

, Alessandra Zamparelli

e

,

Elena Martoni

b

, Camilla Pellegrini

b

, Luciano Merlini

e

, Alessandra Ferlini

b

, Paolo Bonaldo

d

,

Nadir Mario Maraldi

e

, Mats Paulsson

c,f,g

, Stefano Squarzoni

a,

⁎

,1

, Raimund Wagener

c,f,1

a_{CNR-National Research Council of Italy, Institute of Molecular Genetics-IOR, Bologna, Italy} b_{Department of Experimental and Diagnostic Medicine, University of Ferrara, Italy} c

Center of Biochemistry, Medical Faculty, University of Cologne, D-50931, Germany

d

Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, Italy

e

Laboratory of Musculoskeletal Cell Biology, Rizzoli Orthopaedic Institute, Bologna, Italy

f

Center for Molecular Medicine Cologne (CMMC), University of Cologne, D-50931, Germany

g

Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, D-50931, Germany

a b s t r a c t

a r t i c l e i n f o

Article history: Received 8 August 2011

Received in revised form 13 December 2011 Accepted 19 December 2011 Keywords: Collagen VI Skeletal muscle Myotendinous junctions Fibrosis

Duchenne muscular dystrophy

Collagen VI is a major extracellular matrix (ECM) protein with a critical role in maintaining skeletal muscle functional integrity. Mutations in COL6A1, COL6A2 and COL6A3 genes cause Ullrich Congenital Muscular Dys-trophy (UCMD), Bethlem Myopathy, and Myosclerosis. Moreover, Col6a1−/−mice and collagen VI deficient zebrafish display a myopathic phenotype. Recently, two additional collagen VI chains were identified in humans, theα5 and α6 chains, however their distribution patterns and functions in human skeletal muscle have not been thoroughly investigated yet. By means of immunofluorescence analysis, the α6 chain was detected in the endomysium and perimysium, while theα5 chain labeling was restricted to the myotendi-nous junctions. In normal muscle cultures, theα6 chain was present in traces in the ECM, while the α5 chain was not detected. In the absence of ascorbic acid, theα6 chain was mainly accumulated into the cyto-plasm of a sub-set of desmin negative cells, likely of interstitial origin, which can be considered myo fibro-blasts as they expressedα-smooth muscle actin. TGF-β1 treatment, a pro-fibrotic factor which induces trans-differentiation offibroblasts into myofibroblasts, increased the α6 chain deposition in the extracellular matrix after addition of ascorbic acid. In order to define the involvement of the α6 chain in muscle fibrosis we studied biopsies of patients affected by Duchenne Muscular Dystrophy (DMD). We found that theα6 chain was dramatically up-regulated infibrotic areas where, in contrast, the α5 chain was undetectable. Our results show a restricted and differential distribution of the novelα6 and α5 chains in skeletal muscle when com-pared to the widely distributed, homologousα3 chain, suggesting that these new chains may play specific roles in specialized ECM structures. While theα5 chain may have a specialized function in tissue areas sub-jected to tensile stress, theα6 chain appears implicated in ECM remodeling during muscle fibrosis.

1. Introduction

Collagen VI is an extracellular matrix protein which forms a distinct microﬁbrillar network in most connective tissues. It was long consid-ered to consist of three genetically distinctα-chains (α1, α2 and α3), secreted into the extracellular matrix where they form an extended microﬁlamentous network (Chu et al., 1988; Knupp and Squire, 2001).

The collagen VI_{α1, α2 and α3 chains form heterotrimeric monomers} that are assembled intracellularly to dimers and tetramers. After secre-tion, ﬁlaments are formed by end to end interactions of the pre-assembled tetramers, forming characteristic beadedﬁlaments (Bruns, 1984) as visualized by the rotary shadowing electron microscopy tech-nique (von der Mark et al., 1984; Sabatelli et al., 2001).

Collagen VI has a critical role in maintaining skeletal muscle functional integrity; in fact, mutations in the COL6A1, COL6A2, and COL6A3 genes cause a group of inherited muscular dystrophies, namely Ullrich congen-ital muscular dystrophy (UCMD) (Camacho Vanegas et al., 2001), Bethlem myopathy (BM) (Jöbsis et al., 1996; Lampe and Bushby, 2005; Gualandi et al., 2009), and Myosclerosis myopathy (Merlini et al., 2008). Col6a1−/− mice show a complete absence of collagen VI chains (Bonaldo et al., 1998; Gara et al., 2008) and display a myopathic ⁎ Corresponding authors at: CNR - National Research Council of Italy, Institute of

Mo-lecular Genetics-IOR, via di Barbiano 1/10, 40136 Bologna, Italy. Tel.: + 39 051 6366779; fax: + 39 051 583593.

E-mail addresses:sabatelli@area.bo.cnr.it(P. Sabatelli),squarzoni@area.bo.cnr.it

(S. Squarzoni).

1_{These authors contributed equally.}

Contents lists available atSciVerse ScienceDirect

Matrix Biology

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phenotype associated with an impairment of the autophagyflux that de-termines the presence of dilated sarcoplasmic reticulum and abnormal mitochondria: these present a latent dysfunction, that causes an increased apoptosis of musclefibers (Irwin et al., 2003; Grumati et al., 2010). More-over, zebrafish models of the human collagen VI myopathies develop my-opathy and display mitochondrial ultrastructural changes along with an increased rate of spontaneous apoptosis (Telfer et al., 2010).

Collagen VI is also thought to be implicated in tissue remodelling and wound healing, as variations in collagen VI expression have been reported in many pathological conditions, like muscle and liverﬁbrosis (Freise et al., 2009). Fibrosis is the most conspicuous pathological change in dystrophic muscles; it is characterized by excessive accumu-lation of collagens and other ECM components, including collagen VI (Zanotti et al., 2007) and is regulated by mechanisms involving cell– cell and cell–matrix interactions, as well as by factors secreted into the ECM. TGF-_{β1 is one of the most potent regulators of tissue wound} healing andﬁbrosis (Cutroneo, 2007). It is highly expressed in regener-ating muscle after injury and in dystrophic muscles, including Duchenne muscular dystrophy patients and mdx mice (Zhou et al., 2006), carrying mutations in the dystrophin gene, and in MDC1A, caused by mutations in LAMA2 gene (Zanotti and Mora, 2006).

Recently, three novel collagen VI chains,α4, α5 and α6 were iden-tiﬁed (Fitzgerald et al., 2008; Gara et al., 2008). The new chains struc-turally resemble the collagen VIα3 chain, each of them consisting of seven VWA domains followed by a collagenous domain, two or three C-terminal VWA domains and chain speciﬁc domains. In mouse the novel chains show a differential and restricted expression (Gara et al., 2011). Since Col6a1 null mice do not deposit the new collagen VI chains in the extracellular matrix, it was proposed that their as-sembly and secretion requires the presence of theα1 chain and that the new chains may substitute for theα3 chain, probably forming (_{α1 α2 and α4), (α1 α2 and α5) or (α1 α2 and α6) heterotrimers} (Gara et al., 2008, 2011). Due to a large scale pericentric inversion, the human COL6A4 gene on chromosome 3 is broken into two pieces and has become a non-processed pseudogene (Wagener et al., 2009). In humans, COL6A5 mRNA expression is restricted to a few tissues, in-cluding lung, testis, and colon. In contrast, the COL6A6 mRNA was found in a wide range of fetal and adult tissues, including lung, kid-ney, liver, spleen, thymus, heart, skeletal muscle, pancreas, testis and uterus (Fitzgerald et al., 2008). Collagen VIα5 chain protein ex-pression in humans was so far studied only in skin, where it was found in a narrow zone just below the dermal epidermal basement membrane and around blood vessels (Sabatelli et al., 2011). It was earlier reported that the α5 chain is present in the epidermis (Söderhäll et al., 2007), but this observation has been challenged (Sabatelli et al., 2011). Collagen VIα6 protein expression in humans was found in kidney, skeletal and cardiac muscle, lung, blood vessels, pancreas, spleen and cartilage (Fitzgerald et al., 2008). However, col-lagen VI dependent disease is mainly manifested in muscle and the precise localization of the novel chains has not been comprehensively studied in this tissue yet. The discovery of two additional collagen VI chains in humans adds a layer of complexity to collagen VI function in the extracellular matrix (Fitzgerald et al., 2008; Gara et al., 2008).

To provide insight into the expression and function of theα5 and α6 collagen VI chains in human skeletal muscle, we studied these proteins and the corresponding mRNAs in normal muscle tissue and cell cultures, and in Duchenne Muscular Dystrophy (DMD) muscle with marked ﬁbro-sis, arisen independently from any mutation in the collagen VI genes. 2. Results

2.1. Immunoﬂuorescence analysis of the collagen VI α6 chain in normal skeletal muscle

To assess the tissue distribution of theα6 chain in human skeletal muscle, frozen sections of tibialis anterior and soleus, obtained from

normal subjects were stained with an antibody against the α6 chain. Theα6 chain localized both in the perimysium and endomy-sium, with a less homogeneous pattern in the endomysium as com-pared to labeling obtained for the homologousα3 chain (Fig. 1a). Double labeling with anti-α3 and α6 chains antibodies revealed a partial co-localization (Fig. 1b), thus to better characterize the differ-ential distribution ofα3 and α6 chain, we performed double labeling reactions with an antibody against the lamininγ1 chain, a marker of the basement membrane of myofibers and blood vessels, and with an antibody against collagen III, a component of thefibrillar matrix. The α6 chain appeared mainly interstitial, in fact it was absent from the basement membranes of both muscle and vessels, while it co-localized with collagen III (Fig. 1b). In contrast, theα3 chain was detected at the basement membrane of myofibers and blood vessels, as it co-localized with the lamininγ1 chain (Fig. 1b).

2.2. The collagen VIα5 chain is selectively localized at the basement membrane of myotendinous junctions

By immunofluorescence analysis, the α5 chain was not detected in the endoysium and perimysium of tibialis anterior (Fig. 2), however, when we analyzed a sample derived from the soleus muscle taken at the insertion to the Achille's tendon, we found a selective expres-sion of theα5 chain at the myotendinous junctions (MTJ) where it co-localized with the lamininα2 chain, a marker for the basement membrane of muscle fibers. The labeling pattern obtained with pFAK antibodies, which are selectively expressed at the MTJ, mirrored on the cytoplasmic side theα5 chain signal on the extracellular side at the MTJ level (Fig. 2). The _{α5 chain was absent in the} extra-junctional myofiber basement membrane and within the tendon (Fig. 2). Theα6 chain was also detected at the myotendinous junc-tions, though not as a speci_{fic localization but as a continuous labeling} of the endomysium. Similarly to theα5 chain, it was absent within the tendon (data not shown).

2.3. COL6A1 homozygous null mutations affect the deposition of the col-lagen VIα6 chain in the extracellular matrix of skeletal muscle of an UCMD patient

To assess if COL6A1 mutations affect the deposition of the novelα6 chain in the extracellular matrix of human muscle, in accordance with the reported Col6a1−/−mouse model (Gara et al., 2008), we studied the muscle biopsy of an UCMD patient carrying a homozygous dele-tion in exon 22 that causes a frameshift, resulting in a premature stop codon at residues 504–505 in the triple helical domain. At the protein level, this mutation causes a complete absence of theα1 chain both in patient's cells and medium of cultured skinﬁbroblasts (P3 inGiusti et al., 2005) and in muscle biopsy (Demir et al., 2004), similar to the Col6a1−/−mice phenotype (Bonaldo et al., 1998).

By immunoﬂuorescence analysis we found that both the α3 and theα6 chains were absent in the extracellular matrix (Supplementary Fig. 1); we could not determine the effect of the COL6A1 mutation on theα5 chain deposition since myotendinous junctions were not present in the patient's biopsy, as detected by lamininγ1 chain (Supplementary Fig. 1).

2.4. Collagen VI α5 and α6 chain expression in normal muscle cell cultures

We compared the expression and organization of theα5 and α6 chains with that of their homologue, theα3 chain, in a primary nor-mal muscle cell culture under different culture conditions, i.e. grown for 48 h (proliferating) or 7 days (long term), with and with-out 0.25 mM L-ascorbic acid, and after induction of myogenic differ-entiation. Theα3 chain was detected in the extracellular matrix of both proliferating and long-term samples, however the microﬁbrillar

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network appeared better organized and developed in long-term cul-tures (Fig. 3a). Theα6 and α5 chains were not detectable in prolifer-ating primary muscle cell cultures (not shown). In long-term cultures, slight ﬁlamentous, α6-containing deposits were detected in the

extracellular matrix (Fig. 3b), while theα5 chain was completely ab-sent (not shown). Theα6 antibody co-localized with α3-positive fil-amentous structures, however, an α3-based independent network could be also detected (Fig. 3b). COL6A5 transcripts were Fig. 1. Immunofluorescence analysis of collagen VI α6 and α3 chains in transversal muscle sections of tibialis anterior from a normal subject.a: The α6 chain is expressed both in the perimysium and endomysium, with a discontinuous pattern around the musclefibers. α3 chain immunolabeling is diffuse and homogeneous both in the perimysium and endomy-sium. Bar, 100μm. Nuclei were counterstained with DAPI (blue).b: α6 chain labeling appears discontinuous in the endomysium (arrows), and a partial co-localization with anti-α3 chain was shown with double labeling. Theα3 chain antibody strongly stains basement membranes, as demonstrated by double-labeling for the laminin γ1 chain (yellow fluores-cence in merge image); theα6 chain appears expressed in the interstitium between adjacent fibers and absent in basement membrane of both capillary vessels and myofibers. In agreement with an interstitial localization, theα6 chain co-localizes with collagen type III. Bar, 20 μm. Nuclei were counterstained with DAPI (blue).

Fig. 2. Immunoﬂuorescence analysis of collagen VI α6 in human muscle.a: Collagen VI α5 chain localization in transversal sections of tibialis anterior (extra-junctional, left panel) and of soleus at the insertion to the Achille's tendon (MTJ, middle and right panels). Theα5 chain (green) is absent in the interstitium of tibialis anterior, while it is selectively expressed at the myotendinous junction of soleus (arrows) where it co-localizes with the lamininα2 chain (red), a marker for basement membranes. Bar, 100 μm in left and middle panels, and 20μm in right upper and lower panels. Nuclei were counterstained with DAPI (blue).b: Double labeling with anti-α5 chain (green) and anti-pFAK (red) antibodies, of a section of soleus at the insertion to the Achille's tendon, reveals that theα5 chain is expressed at the extracellular side of myotendinous junctions, where pFAK labeling is concen-trated. The merged image shows a clear correspondence of the two labeling patterns. Bar, 20μm. Nuclei were counterstained with DAPI (blue).

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undetectable both in proliferating and in long-term cells. Both COL6A3 and COL6A6 transcript levels were increased in long-term cultures compared to proliferating cells (1.7:1 COL6A6 and 2.9:1 COL6A3) (Fig. 3c). Primary cultures derived from muscle biopsies may contain both interstitial and myogenic cells. To assess which cells produce theα6 chain, long-term muscle cultures were analyzed in the absence of ascorbic acid, to induce intracellular accumulation of under-hydroxylated collagen VI chains (Engvall et al., 1986). In agree-ment with a previous study which established that interstitial cells, and not myogenic cells, produce collagen VI (Zou et al., 2008), we found that theα3 chain was absent in myogenic (desmin-positive) cells, while it was accumulated in most of desmin-negative cells. The α6 chain was detected intracellularly only in a subset of desmin-negative cells, and, similarly to theα3 chain, it was absent in myogenic cells. Interestingly, most of the α6 chain-producing cells showed aﬂattened shape and were positive for α-smooth mus-cle actin (α-SMA), indicating that they could be myoﬁbroblasts, prob-ably of interstitial origin (Fig. 3d).

Under differentiating conditions, theα3 chain was present both in the extracellular matrix surrounding myotubes and inside the cyto-plasm of desmin-negative cells. The presence of this protein inside the cytoplasm can be ascribed to the absence of ascorbic acid in differ-entiating medium (Lattanzi et al., 2000). The α3 chain was not detected inside the cytoplasm of myotubes, conﬁrming that myogenic cells do not produce collagen VI (Zou et al., 2008). Theα6 chain was

faintly detected in the extracellular matrix as a poorly developed net-work. Some desmin-negative cells showed cytoplasmic labeling while the protein was absent within myotubes (Supplementary Fig. 2). The alpha5 chain was not detectable both in myotubes and desmin-negative cells, similarly to proliferating and long-term cultures (not shown). These data strongly indicate that regulation of bothα5 and α6 chains is not affected by myogenic differentiation.

2.5. Trans-differentiation of muscleﬁbroblasts into myoﬁbroblasts in-duced by TGFβ1 strongly increases collagen VI α6 chain deposition in the extracellular matrix of normal muscle cultures

TGFβ1 is a strong inducer of muscle fibroblasts activation, when applied to primary muscle cell cultures, and offibroblast differentia-tion into myofibroblasts, which acquire an α-SMA-positive pheno-type. On the contrary, TGFβ1 inhibits myogenic differentiation. To assess the effect of TGFβ1 on the expression of the novel collagen VI chains, normal primary muscle cells were treated for 10 days. As expected, we detected a strong increase in the number ofα-SMA pos-itive cells (Fig. 4a). In contrast, desmin expression was reduced, as well as the number of spontaneously differentiated myotubes (not shown). In these conditions, the amount ofα6 chain was increased and the secreted protein deposited in a widefilamentous network in the extracellular matrix (Fig. 4a). The α6 chain mainly co-localized with theα3 chain, however, a distinct α3 chain containing Fig. 3. Immunofluorescence analysis of collagen VI α3 and α 6 chains in normal muscle cultures.a: The collagen VI α3 chain (red fluorescence) is detected in the extracellular matrix of both proliferating and long-term (after ascorbic acid treatment for 7 days post-confluence) samples, however the arrangement of the collagen VI network appears better orga-nized in long term cultures. Bar, 100μm. Nuclei were counterstained with DAPI (blue).b: Collagen VI α6 chain (red fluorescence) deposits are detectable in the extracellular matrix of muscle cultures after ascorbic acid treatment for 7 days post-confluence. α6-containing structures (arrows, left upper panel) show a filamentous arrangement, as detected at higher magnification (right upper panel). Lower panels show double labeling with antibodies against the anti-α6 (red fluorescence) and α3 (green fluorescence) chains. Filamen-tousα6 chain-containing structures (red) co-localize with α3 chain-containing positive microfilaments, as revealed by the merged image (lower, right panel), however, an inde-pendentα3 chain-containing network (green fluorescence) can be also detected. Bar, 20 μm. Nuclei were counterstained with DAPI (blue).c: Real Time-PCR analysis of COL6A3 and COL6A6 transcripts. Reported values are the mean of two different experiments utilizing ACTB and GAPDH as reference transcripts. The relative ratios ofCOL6A3/Actin-GAPDH and COL6A6/Actin-GAPDH mRNAs in proliferating cells were normalized to 1. Both COL6A3 and COL6A6 transcript levels were increased in long-term cells (1.7:1 for COL6A6 and 2.9:1 for COL6A3).d: Immunofluorescence analysis of collagen VI α3 and α6 chains in long-term normal muscle cell cultures in the absence of ascorbic acid treatment. Samples were double-labeled for desmin (upper panels, greenfluorescence) and for α-smooth muscle actin (α-SMA; lower panels, green fluorescence). The collagen VI α3 chain (red fluores-cence, left, upper panel) appears to be absent in desmin-positive cells and accumulates in desmin-negative cells, indicating that thefibroblasts are the main source of α3 chain. Similarly, theα6 chain (red, right, upper panel) is absent in desmin-positive, but different from the α3 chain, it is detected intracellularly only in a subset of desmin-negative cells. Cell producing collagen VIα6 chain (red) are positive for α-smooth muscle actin (green, right, lower panel), indicating that they could be myofibroblasts. Bar, 20 μm. Nuclei were counterstained with DAPI (blue).

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network could also be detected (Fig. 4a). Both COL6A3 and COL6A6 transcript levels were markedly reduced in cells after long-term (10 days) TGFβ1 treatment (0.48:1 COL6A3 and 0.16:1 COL6A6). By analyzing mRNA levels at different time points, we detected constant-ly low transcriptional levels of COL6A6 mRNA starting from 24 hour of treatment. Differently, an initial up-regulation of COL6A3 mRNA at 24–48 hours was followed by a gradual decrease at prolonged TGFβ1 treatment (Fig. 4b). TGFβ1 treatment did not affect the expres-sion and synthesis of theα5 chain, both at the RNA and protein levels (not shown).

2.6. The collagen VIα6 chain, but not the α5 chain, is up-regulated in dystrophic muscles

As myofibroblasts play a major role upon fibrosis, we studied the expression of theα6 chain in muscle biopsies of five patients affected by Duchenne Muscular Dystrophy (DMD), a muscular dystrophy characterized by a markedfibrosis which is definitely independent from any mutation in collagen VI genes. Because of scarce availability of tissue, only the DMD815 biopsy was studied by means of immuno-fluorescence, Western blotting and mRNA analysis. The other DMD patients were studied either by immunofluorescence plus mRNA or by immunofluorescence plus Western blotting analysis.

By immunofluorescence analysis, the α6 chain antibody showed an increased amount of protein in the extracellular matrix of all DMD patients (Fig. 5a andSupplementary Fig. 3a). The localization of theα6 chain was interstitial, in fact no labeling was detected at the basement membrane of myofibers and blood vessels, as indicated by double labeling with anti-lamininγ1 antibody (Fig. 5a). By double staining, theα6 chain labeling correlated with collagen III positive areas, used as a marker offibrosis. Semiquantitative analysis revealed a strong correlation between the increase ofα6 chain and collagen III

protein amount (supplementary Fig. 3b,c). Also theα3 chain was in-creased in all DMD patients (Fig. 5b) and matched the degree of fibro-sis. Double labeling with antibodies against theα3 and α6 chains showed a partial co-localization, and confirmed the absence of the α6 chain from the basement membranes of vessels and myofibers. At high magnification, undulated/wavy bundles of fibrils of different size containing both theα6 and α3 chains were detected in fibrotic areas (Fig. 5c), suggesting that the α6 and α3 chains may co-assemble and cooperate in remodeling the extracellular matrix uponfibrosis. Western blot analysis confirmed the increase of α3 and _{α6 chains in the three DMD patients examined (DMD815,} DMD1112, DMD1179). The increased protein levels ofα3 and α6 chains matched with a general increase of collagen VI as shown by α1 immuno-blotting (Fig. 6a). The protein increase matched a rele-vant increase of COL6A6 transcript level, with a fold change that ran-ged from 10 to 14 in the three analyzed DMD muscles (DMD815, DMD1 and DMD23, Fig. 6b). Nevertheless, at the transcriptional level, the increase in COL6A3 mRNA infibrotic DMD muscles (2–4 fold changes) was significantly lower than the increase in COL6A6 mRNA. In contrast, theα5 chain and the corresponding transcript were not detected (not shown).

3. Discussion

The discovery of two additional collagen VI chains in humans (α5 andα6), which may substitute for the α3 chain, suggests the exis-tence of additional assembly forms of collagen VI (Fitzgerald et al., 2008; Gara et al., 2008, 2011). So far, the localization and the expres-sion pattern of the novel chains in human skeletal muscle, the tissue most affected by collagen VI deficiency, was mostly unknown, with the exception of their reported presence in commercially available samples such as fetal human skeletal muscle extract and non-Fig. 4. Immunofluorescence and Real Time-PCR analysis of α6 chain in normal muscle cultures after TGFβ1 treatment.a: After 10 days TGFβ1 treatment most of interstitial cells expressα-SMA (left upper panel, green fluorescence) indicating trans-differentiation into a myofibroblasts-like phenotype. In the absence of ascorbic acid (right upper panel) most ofα-SMA expressing cells accumulated the collagen VI α6 chain (red) intracellularly. After ascorbic acid treatment (left and right middle panels), the α6 chain (red) was abundantly secreted and arranged in a widefilamentous network in the extracellular matrix. Left and right lower panels show double labeling with antibodies against the α6 andα3 chains on samples treated with TGFβ1 for 10 days in presence of ascorbic acid. The α6 chain (red fluorescence) mainly co-localizes with the α3 chain (green) labeling. How-ever, a distinctα3−containing network can also be detected (green fluorescence). Bar, 20 μm . Nuclei were counterstained with DAPI (blue).b: Real Time-PCR analysis. Reported values are the mean of two different experiments utilizing ACTB and GAPDH as reference transcripts The relative ratios of COL6A3/Actin-GAPDH and COL6A6/Actin-GAPDH mRNAs in untreated cells were normalized to 1. COL6A3 and COL6A6 transcript levels are strongly reduced in cells after long-term (10 days) TGFβ1 treatment (0.48:1 for COL6A3 and 0.16:1 for COL6A6). Analysis of mRNA levels at different time points revealed constantly low transcriptional levels of COL6A6 mRNA starting from 24 hour of treatment. In contrast, an initial slight up-regulation of COL6A3 mRNA at 24–48 hours, was followed by a gradual decrease after prolonged TGFβ1 treatment.

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speciﬁed skeletal muscle tissue derived from a normal adult human tissue panel (Fitzgerald et al., 2008).

Our study aims to define more precisely the expression and distri-bution of collagen VIα5 and α6 chains in mature human skeletal muscle. In fact we show that in human skeletal muscle, the_{α5 and} α6 chains differ from the homologous α3 chain in a number of as-pects, e.g. localization pattern, cellular origin and involvement in ECM remodelling upon musclefibrosis. To distinguish between the different assembly forms (Gara et al., 2008), we used antibodies spe-cific for the individual α3, α5 and α6 chains (Sabatelli et al., 2011).

In normal skeletal muscle sections, theα5 chain was selectively detected at the basement membrane of myotendinous junctions. Our previous studies showed that theα5 chain localizes at junctional

sites also in human skin, in particular at the dermal-epidermal junc-tion (Sabatelli et al., 2011). These data indicate that α5 chain-containing collagen VI micro_{fibrils may have a specific function in} specialized tissue areas subjected to tensile stress. Myotendinous junctions are crucial elements in the transmission of mechanical force from the muscle via tendons to the skeletal elements. Several molecules have been identified at this site and mutations in their genes lead to pathologic changes of myotendinous junctions:α7β1 integrin, laminin 2, and the dystrophin glycoprotein complex are piv-otal at this site (Mayer et al., 1997; Pegoraro et al., 2002; Vainzof and Zatz, 2003). Tenascin, an oligomeric extracellular matrix protein, was one of the first myotendinous junction markers (Chiquet and

Fambrough, 1984). Notably, mutations in tenascin XB gene cause

Ehlers–Danlos syndrome (Zweers et al., 2003), with muscle weakness and contractures and secondary deﬁciency of collagen VI, overlapping with the collagen VI myopathies phenotype (Voermans et al., 2007), and pointing to a possible functional interaction between these myo-tendinous junction components. Thereforeα5 gene mutation screen-ing might result appealscreen-ing in those cases of Ehlers_{–Danlos disease} which are negative for known mutations.

Theα6 chain was less abundant than the homologous α3 chain. Its localization was mainly interstitial where it partially co-localized with theα3 chain. The most relevant difference between the α6 andα3 chain localization was the absence of the α6 chain in the base-ment membrane of both musclefibers and vessels, where in contrast theα3 chain was specifically detected. Considering that also the α5 chain is absent at the extrajunctional basement membrane, it is pos-sible that theα3-containing microfibrils represent the specific base-ment membrane collagen VI form. This hypothesis is supported by recent immunoelectron microscopy studies performed with anti-bodies specific for the collagen VI α1 α2 α3(VI) heterotrimer, which con_{firmed basement membrane localization of this collagen} VI form in skeletal musclefibers (Gara et al., 2011) and endothelial cells (Groulx et al., 2011). It is interesting to note that absence/reduc-tion of collagen VI at the basement membrane of muscle are features of UCMD/BM muscle biopsies obtained from patients carrying muta-tions in COL6A1, COL6A2 and COL6A3 genes (Ishikawa et al., 2004; Kawahara et al., 2007; Merlini et al., 2008; Gualandi et al., 2009). Basement membrane abnormalities have also been detected in mus-cle fibers of Ullrich congenital muscular dystrophy patients (Niiyama et al., 2002; Squarzoni et al., 2006) and in blood vessels of Myosclerosis myopathy patients (Merlini et al., 2008). These data point to a pivotal role ofα1 α2 α3 collagen VI form in maintaining basement membrane functional integrity.

As it has been shown for cartilage and skin, the expression of the novel chains in muscle also differs between man and mouse (Fitzgerald et al., 2008; Gara et al., 2011; Sabatelli et al., 2011). Whereas theα5 chain in man is selectively expressed at the myoten-dinous junction, in mouse theα5 chain is strongly expressed in peri-mysium and sparsely in epiperi-mysium and, in addition, in perineurium of intramuscular peripheral nerves and at the basement membrane of neuromuscular junctions (Gara et al., 2011). The differences in tis-sue distribution between man and mouse could be the result ofα4 chain gene loss in humans, which leads to a functional re-orientation of the remaining chains. In contrast to this, the expression of theα6 chain is similar in human and mouse skeletal muscle. In agreement with reported studies on the Col6a1 null mice model, the α6 collagen VI chain was not expressed in the skeletal muscle of a UCMD patient carrying an α1 null mutation. This result indicates that in humans, similarly to mice, theα6 chain co-assemble with theα1 chain (Gara et al., 2008).

To deﬁne the organization of novel collagen VI chains in the extra-cellular matrix we analyzed normal skeletal muscle cultures. Filamen-tousα6 chain deposits were detectable in the extracellular matrix only in long term cultures where α6 chain-containing networks were less abundant and organized than those containing the α3 Fig. 5. Immunoﬂuorescence analysis of α6 chain in DMD patients muscle biopsies.a:

Immunofluorescence analysis of the collagen VI α6 chain (red) and the laminin γ1 chain (green) in muscle sections of three patients affected by Duchenne muscular dys-trophy (DMD815, DMD1, DMD23).α6 chain labeling appears increased in the endomy-sium and perimyendomy-sium, with its increase correlating with the degree offibrosis. The α6 chain localization is strictly interstitial, as demonstrated by absence of co-localization with the lamininγ1 chain (right panels, green fluorescence),a marker for basal lamina. Bar, 100μm.b: Immunofluorescence analysis of the collagen VI α3 chain in DMD815 muscle sections revealed a marked increase infibrotic ares (red fluorescence, left panel). Double labeling with antibodies against–the laminin γ1 chain (green) reveals the presence ofα3 chain at the basal lamina of muscle fibers (yellow fluorescence in merge image, right panel).c: High magnification of a muscle section from patient DMD815 double labeled with antibodies against collagen VIα6 (red fluorescence) and -α3 (green fluorescence) chains showing the presence of undulated/wavy bundles offibrils of different size containing both the α6 and α3 chains. In the basement mem-brane of a musclefiber, only the α3 chain is found (arrows). Bar, 20 μm. Nuclei were counterstained with DAPI (blue).

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chain. A co-localization was observed, indicating that secretedα3 and α6 chains may co-assemble and form a common network, while the presence of a distinctα3-based network conﬁrmed the immunohis-tochemical pattern observed in tissue sections.

Theα5 chain was absent both at the protein and the mRNA level in proliferating and long term cultures. This is possibly due to the or-igin of the culture, in fact the muscle fragment we used to establish primary cultures did not include myotendinous junctions. On the other hand, the absence of theα5 chain demonstrates that it is dis-pensable for cell-substrate adhesion under normal conditions and might underscore a specificity of this chain for junctional structures. We determined which cells synthesizeα6 chain in a primary mus-cle culture, as this contains both myogenic (desmpositive) and in-terstitial (desmin-negative) cells. Collagen VI-producing cells could be identified by omitting ascorbic acid treatment (Prockop et al., 1976; Engvall et al., 1986), as they in this conditions display an intra-cellular staining with antibodies against collagen VI. Theα3 chain was accumulated in desmin-negative cells only, confirming that it is produced by interstitial but not by myogenic cells (Zou et al., 2008). The_{α6 chain was also absent in desmin-positive cells, but, in contrast} to theα3 chain, the α6 chain was detected intracellularly only in a subset of desmin-negative cells which resulted to beα-SMA positive, likely myofibroblasts of interstitial origin.

As TGFβ1 treatment induces transdifferentiation of interstitial muscle fibroblasts to a myofibroblast phenotype (Zanotti et al., 2010), we evaluated its effect onα5 and α6 chain expression and de-position in extracellular matrix. A striking increase in the number of α-SMA-positive cells was observed after 10 days treatment, indicat-ing that the treatment induced transdifferentiation. In addition, TGFβ1 treatment induced a marked increase of the α6 chain-containing filamentous network in the ECM, which only partially overlapped with labeling forα3 chains. In contrast, α5 chain expres-sion remained undetectable, even after TGFβ1 treatment. As expected, and in contrast with theα6 chain increase, desmin expres-sion was reduced, as well as the number of spontaneously differenti-ated myotubes.

Time course Real Time-PCR experiments showed down-regulation of COL6A6 mRNA levels, starting from 24 h of TGFβ1 treatment, indi-cating that in the cell culture model, despite a very early induction oc-curred, the increased protein amounts cannot be ascribed to a direct effect of TGFβ1 on COL6A6 transcription.

Previous studies reported that in vitro treatment with TGF-β1 in-duces the expression of several collagen VI interacting proteins, as collagens I and IV (Evans et al., 2003), biglycan (Todorova et al., 2011), as well asﬁbronectin and integrins (Reinboth et al., 2006; Honda et al., 2010). It is possible that TGF-β1 treatment improves the efﬁciency of the extracellular binding/assembly of the α6 chain by induction of collagen VI related-proteins, resulting in an increased deposition in the extracellular matrix.

Our results indicate that myofibroblasts are the main α6 chain producing cells and that TGF-β1 treatment enhances α6 chain secre-tion, pointing to a possible involvement of theα6 chain in fibrosis (Wipff and Hinz, 2008). Fibrosis is a conspicuous pathological change in dystrophic muscles. It is characterized by excessive accumulation of collagens and other ECM components including collagen VI. For this reason we evaluated the expression ofα5 and α6 chains in mus-cle biopsies of three DMD patients with different degrees offibrosis. We found that collagen VI was generally increased infibrotic muscle and, in particular, theα6 chain was strongly up-regulated in DMD muscle at protein level, as shown by Western blot, real time PCR and immuno-histochemical analysis. The increase of the_{α6 chain} was particularly evident infibrotic areas and maintained an intersti-tial localization at the endomysium, since no colocalization with base-ment membrane markers was detected.

On the basis of their high homology with theα3 chain, the colla-gen VIα5 and α6 chains are likely to interact with α1 and α2 chains, in agreement with previous studies on Col6a1 knockout mice (Gara et al., 2008, 2011) and on human skin (Sabatelli et al., 2011). This may give rise to homologous heterotrimers, which appear to function differently, most probably with regard to tissue adhesion. Specifically, in human skeletal muscle we hypothesize that theα3-containing col-lagen VI is involved in basement membranes adhesion to the intersti-tial extracellular matrix; theα5-containing collagen VI is involved in junctional adhesion at myotendinous junctions and dermal-epidermal junctions, and theα6-containing collagen VI has a role in adhesion to endomysium and in the development offibrosis, in asso-ciation withfibrillar collagen. Future studies will concern the role of the novel collagen VI chains in human disease. Moreover, since our data suggest specific roles in specialized ECM structures for α5 and α6 chains, being the α5 related to tensile stress and the α6 implicat-ed in ECM fibrotic remodelling, they might respectively represent new biomarkers for monitoring these stressing events.

Fig. 6. Western blot and RT-PCR analysis of collagen VI chains in muscle biopsies of DMD patients.a: Western blot analysis of skeletal muscles extracts from a healthy control (CTRL1,2) and from DMD815, DMD1112 and DMD1179 patients. Proteins were separated by SDS-PAGE under reducing conditions and detected with polyclonal antibodies speciﬁc for the collagen VIα6 and α3 and α1 chains; actin was used as loading control.b: Real Time-PCR analysis of COL6A6 transcripts in DMD skeletal muscle biopsies. Reported values are the mean of two different experiments utilizing ACTB and GAPDH as reference transcripts. The relative ratios of COL6A3/Actin-GAPDH and COL6A6/Actin-GAPDH mRNAs in control muscle were normalized to 1. The increased amount of collagen VIα3 and α6 chains in DMD muscles matched with an increase of the corresponding mRNAs. The COL6A6 transcript level showed a fold change that ranged from 10 to 14 in the three analyzed samples whereas the variation in the COL6A3 mRNA level was lower (2–4 fold change).

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4. Experimental procedures

4.1. Preparation of antibodies against the new collagen VI chains The purified recombinant collagen VI fragments were used to im-munize rabbits and guinea pigs. The antisera obtained were puri_fied by affinity chromatography on a column with antigen coupled to CNBr-activated Sepharose (GE Healthcare). The specific antibodies were eluted with 0.1 M glycine, pH 2.5, and the eluate was neutral-ized with 1 M Tris–HCl, pH 8.8 (Sabatelli et al., 2011).

4.2. Patients

All subjects studied granted informed consent. Skeletal muscle bi-opsies of one soleus at the insertion with Achille's tendon and two tibialis anterior from healthy subjects, tibialis anterior from _ﬁve DMD patients and gastrocnemius from one UCMD patient were fro-zen in isopentane pre-chilled in liquid nitrogen and stored in liquid nitrogen. All patients (Table 1) were previously diagnosed by genetic and immunohistochemical analysis.

4.3. Immunoﬂuorescence analysis

Frozen sections (7μm-thick) from muscles of healthy donors and offive DMD (DMD1, DMD815, DMD23, DMD1112 and DMD1179) and one UCMD patients (seeTable 1) were incubated with the affinity purified rabbit polyclonal antibody against the collagen VI α5 chain (diluted in PBS 1/40). For the immunofluorescence analysis of the col-lagen _{α6 chain, frozen sections from healthy donors and patients} werefixed with 2% paraformaldehyde in phosphate buffered saline (PBS), washed in PBS and permeabilized with 0.15% Triton X100, sat-urated with 5% normal goat serum (Sigma) and incubated with the rabbit or guinea pig antibody against theα6 chain diluted 1/40. All sections were developed with anti-rabbit or anti-guinea pig TRITC or FITC-conjugated IgG (DAKO) and double labeled with mouse monoclonal antibodies against the lamininγ1, laminin α2 chain, col-lagen type III (Chemicon), or with anti-pp125 FAK antibody (Santa Cruz) and revealed with anti-mouse or anti-rabbit FITC or TRITC-conjugated secondary antibodies (DAKO). An antibody against the collagen VIα1α2α3 chains (Fitzgerald) was used on sections of the UCMD patient to assess the expression of collagen VI. Samples were mounted with an anti-fading reagent (Molecular Probes) and ob-served with a Nikon epifluorescence microscope. Nuclei were coun-terstained with DAPI.

The proliferation of connective tissue in DMD muscle biopsies was evaluated by immunofluorescence analysis of collagen type III (Hantaï et al., 1985) and compared with the normal muscle biopsy. Double labeling with anti-α6 chain antibody was performed in order to evaluate whether collagen III-positive areas correlated with the alpha6 chain labeling. Five differentfields for each biopsy were taken with afixed exposure time by the NIS-Elements AR (Nikon) aging program using the multi-channel acquisition function. All im-ages, which included both signals for the collagen VIα6 chain and

for collagen III, were captured using an high-resolution CCD camera (Nikon). Semi-quantitative analysis was performed by a software de-veloped in cooperation with Nikon Italy (NIS-Elements 3.0 AR imag-ing program, Nikon). Theﬂuorescent areas were evaluated by the area fraction function, and the mean values were calculated. For graphic representation, area fraction mean values were expressed as percentage. Data were analyzed using the Mann–Whitney test and the signiﬁcance was calculated versus normal muscle.

4.4. Muscle cultures

Primary muscle cells derived from tibialis anterior of a normal subject were established as described (Cenni et al., 2005) and maintained in D-MEM plus 20% fetal calf serum (Rando and Blau, 1994). For immunofluorescence analysis of the collagen VI α5 and α6 chains muscle cells were grown onto coverslips for 48 hrs or 7 days, with and without 0.25 mML-ascorbic acid. When needed, cells were treated with 10 ng/ml TGF-β1 (Sigma). Differentiation to myotubes was induced by incubating subcon_{fluent cells for} 7 days with D− MEM plus 10% fetal calf serum (Lattanzi et al., 2000). Samples werefixed with cold methanol, washed with PBS and incubated with rabbit or guinea pig antibodies against the α5, α6 or α3 chains. Double labeling was performed with mouse monoclonal anti-desmin (Novocastra) or anti-alpha-SMA (SIGMA) antibodies. Nuclei were counterstained with DAPI

4.5. Quantiﬁcation of COL6A5 and COL6A6 transcripts by real-time PCR Total RNA was isolated from muscle sections of normal tibialis or tibialis from patients DMD815, DMD1 and DMD23 or from cell cul-tures by using RNeasy Kit (QIAGEN, Chatsworth, CA) and reverse transcribed by using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). In order to quantify the steady state level of COL6A5 and COL6A6 transcripts, commercially available TaqMan ex-pression assays (Applied Biosystems) were used for target genes (COL6A3: Hs00915102_m1 Ex 23–24; COL6A5 (COL29A1): Hs00542046_m1 exons 35–36; COL6A6: Hs01029204_m1 exons 12–13) and for β actin and GAPDH as housekeeping reference genes (ACTB Endogenous Control- GAPDH endogenous control). Real-time PCR was performed in triplicate on the Applied Biosystems Prism 7900HT system, using 10 ng of cDNA and default parameters. Evalua-tion of COL6A3 and COL6A6 transcripts levels was performed by the comparative CT method (ΔΔCT Method; Applied Biosystems User Bullettin #2). cDNAs from control muscle sections or untreated cells were utilized as calibrators.

4.6. Western blot analysis

Frozen sections (20μm) were prepared from skeletal muscles bi-opsies of two healthy donors and patients DMD815, DMD1112 and DMD1179. The sections were extracted by incubation for 10 min at 70 °C in a lysis solution containing 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 2% SDS, 1% Triton X-100, 0.5 mM dithiothreitol,

Table 1

Table of muscle biopsies.

Patient Age of biopsy (years_months) Sex Disease status Muscle type Mutation

DMD1 4.7 Male Slow in running Tibialis del 51

DMD815 10.2 Male Unable to climb stairs Tibialis splicing in 25c_3641-1G>A

DMD23 9.1 Male Difﬁculty with stairs Tibialis del45-52

DMD1112 0.9 Male Delayed motor milestones Tibialis c_5551C>T

DMD1179 11 Male Unable to climb stairs Tibialis del 1M-2

UCMD 1.5 Male Sitter, diffuse muscle wasting and weakness Gastrocnemius COL6A1 IG del 1456nt (Giusti et al., 2005)

CTRL2 10 Male Healthy donor Tibialis

CTRL1120 9 Female Healthy donor Tibialis

CTRL8 35 Male Healthy donor Tibialis

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1 mM ethylenediaminetetraacetic acid, 10% glycerol and protease in-hibitors. For each sample, the total protein content was determinate using_{“BCA Protein Assay Kit” (Pierce). Samples were reduced with} 5%β-mercaptoethanol and separated by SDS-PAGE on 3-8% (w/v) gradient polyacrylamide gels. Collagen VI α3 and α6 chains were detected using the af_{finity-purified antibodies developed in rabbit} (Sabatelli et al., 2011). Collagen VIα1 chain was detected using a spe-cific collagen VI α1 antibody (H-200, Santa Cruz Biotechnologies). As a loading control, actin was used (Sigma). Secondary antibodies con-jugated with horseradish peroxidase were employed (GE Healthcare) and bands detected by chemiluminescence (SuperSignal West Pico, Pierce).

Supplementary materials related to this article can be found on-line atdoi:10.1016/j.matbio.2011.12.003.

Acknowledgments

This work has been funded by the BIO-NMD Grant (EC, 7th FP, proposal #241665;“http://www.bio-nmd.eu” to AF as coordinator) and Italian Research Ministry“Prin 2008” grant no. 2008PB5S89 to PB, PS, SS, AF, by Fondazione CaRisBo and by the Deutsche Forschungsgemeinschaft (WA1338/2-6 and SFB 829) to RW and MP. SKG is a member of the International Graduate School in Genetics and Functional Genomics at the University of Cologne. The authors wish to thank Dr Rosa Curci for assistance in human muscle cultures establishment.

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