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. 2008 Jun 5;213(3):284–295. doi: 10.1111/j.1469-7580.2008.00921.x

Costameric proteins in human skeletal muscle during muscular inactivity

Giuseppe Anastasi 1, Giuseppina Cutroneo 1, Giuseppe Santoro 1, Alba Arco 1, Giuseppina Rizzo 1, Placido Bramanti 2, Carmen Rinaldi 1, Antonina Sidoti 1, Aldo Amato 1, Angelo Favaloro 1
PMCID: PMC2732038  PMID: 18537849

Abstract

Costameres are regions that are associated with the sarcolemma of skeletal muscle fibres and comprise proteins of the dystrophin–glycoprotein complex and vinculin–talin–integrin system. Costameres play both a mechanical and a signalling role, transmitting force from the contractile apparatus to the extracellular matrix in order to stabilize skeletal muscle fibres during contraction and relaxation. Recently, it was shown that bidirectional signalling occurs between sarcoglycans and integrins, with muscle agrin potentially interacting with both types of protein to enable signal transmission. Although numerous studies have been carried out on skeletal muscle diseases, such as Duchenne muscular dystrophy, recessive autosomal muscular dystrophies and other skeletal myopathies, insufficient data exist on the relationship between costameres and the pathology of the second motor nerve and between costameric proteins and muscle agrin in other conditions in which skeletal muscle atrophy occurs. Previously, we carried out a preliminary study on skeletal muscle from patients with sensitive-motor polyneuropathy, in which we analysed the distribution of sarcoglycans, integrins and agrin by immunostaining only. In the present study, we have examined the skeletal muscle fibres of ten patients with sensitive-motor polyneuropathy. We used immunofluorescence and reverse transcriptase PCR to examine the distribution of vinculin, talin and dystrophin, in addition to that of those proteins previously studied. Our aim was to characterize in greater detail the distribution and expression of costameric proteins and muscle agrin during this disease. In addition, we used transmission electron microscopy to evaluate the structural damage of the muscle fibres. The results showed that immunostaining of α7B-integrin, β1D-integrin and muscle agrin appeared to be severely reduced, or almost absent, in the muscle fibres of the diseased patients, whereas staining of α7A-integrin appeared normal, or slightly increased, compared with that in normal skeletal muscle fibres. We also observed a lower level of α7B- and β1D-integrin mRNA and a normal, or slightly higher than normal, level of α7A-integrin mRNA in the skeletal muscle fibres of the patients with sensitive-motor polyneuropathy, compared with those in the skeletal muscle of normal patients. Additionally, transmission electron microscopy of transverse sections of skeletal muscle fibres indicated that the normal muscle fibre architecture was disrupted, with no myosin present inside the actin hexagons. Based on our results, we hypothesize that skeletal muscle inactivity, such as that found after denervation, could result in a reorganization of the costameres, with α7B-integrin being replaced by α7A-integrin. In this way, the viability of the skeletal muscle fibre is maintained. It will be interesting to clarify, by future experimentation, the mechanisms that lead to the down-regulation of integrins and agrin in muscular dystrophies.

Keywords: agrin, costameres, integrins, muscular diseases, skeletal muscle

Introduction

Costameres were first identified in skeletal and cardiac muscle as densely clustered patches of vinculin that are associated with the sarcolemma (Pardo et al. 1983a). The patches are positioned over the I-bands of each underlying sarcomere, and form two rows that flank each Z-line. Based on the location of costameres and the presence of vinculin, Pardo and others proposed that costameres anchor Z-lines to the sarcolemma, and thus help to maintain the spatial organization of the myofibrils during muscle contraction and relaxation (Pardo et al. 1983b; Craig & Pardo, 1983). This hypothesis was supported by earlier electron microscopy data that showed fibrous or plaque-like connections exist between the sarcolemma and those Z-lines of the myofibrils which lie closest to the membrane (Granger & Lazarides, 1978; Street, 1983) and festooning of the sarcolemma in contracted muscle fibres (Myklebust et al. 1980; Chiesi et al. 1981; Shear & Bloch, 1985). In addition, other proteins that are also involved in cytoskeletal–membrane interactions have been identified at sites coincident with costameres (Lazarides, 1978; Repasky et al. 1982; Craig & Pardo, 1983; Belkin et al. 1986). The results of all these studies have shown that costameres consist of cytoskeletal proteins that are associated with the sarcolemma (vinculin, talin, dystrophin), signalling proteins [nitric oxide synthase (NOS), syntrophins and dystrobrevin], transmembrane proteins (integrins, β-dystroglycan and sarcoglycans) and extracellular proteins (α-dystroglycan and muscle agrin). Because of their constituents, costameres are considered as ‘proteic machinery’ and they appear to comprise two protein complexes that provide important connections between the extracellular matrix and the muscle fibres: the dystrophin–glycoprotein complex (DGC) and the vinculin–talin–integrin system (Danowski et al. 1992; Ervasti & Campbell, 1993; Mondello et al. 1996).

The results of our recent studies on normal skeletal and cardiac muscle (Anastasi et al. 2003a) showed, for the first time, that costameres are localized to different regions in different fibres. In some fibres, they are located in the regions of the sarcolemma above the I-bands whereas in others they are positioned over the A-bands. We hypothesized that the region of the sarcolemma that is occupied by the costameres depends on the metabolic type of the fibre (fast or slow). In particular, we proposed that costameres are localized to I-bands in slow fibres, whereas they are localized to A-bands in fast fibres. Moreover, the co-localization of sarcoglycans and integrins suggested that bidirectional signalling occurs between the two, perhaps via an unknown protein that interacts with both groups of proteins (Anastasi et al. 2003a,b, 2004a).

A potential candidate for this hypothetical interacting protein is agrin, which plays a key role in signalling between the cell and the extracellular matrix (Martin & Sanes, 1997) and regulates the organization of cytoskeletal proteins (Bezakova & Lomo, 2001). Agrin is a heparin sulphate proteoglycan that was originally purified from the basal lamina and has been shown to induce clustering of acetylcholine receptors (AChRs) on cultured myotubes (Hagiwara & Fallon, 2001). Muscle agrin is involved in the organization of costameres in both junctional and extrajunctional regions of myofibres (Lisman & Fallon, 1999). In addition, neural agrin, which is released by motor nerve terminals into the synaptic cleft (McMahan, 1990), is essential also for the formation of neuromuscular junctions (Gautum et al. 1996) due to its ability to cluster acetylcholine receptors in vitro (Gesemann et al. 1995; Hagiwara & Fallon, 2001).

Integrins are a large family of transmembrane heterodimeric receptors that are vital for the maintenance of muscle fibre viability and cell adhesion to the extracellular matrix (Hynes, 1992). They serve a dual purpose: they link the extracellular matrix with the actin cytoskeleton and they allow the bidirectional transmission of signals between the extracellular matrix and the cytoplasm (Schwartz et al. 1995; Burridge & Chrzanowska-Wodnicka, 1996). The predominant integrin in adult skeletal and cardiac muscle is the β1D isoform (Belkin et al. 1996). The results of our recent investigations, carried out on human skeletal muscle affected by sarcoglycanopathy, showed that staining for α7B- and β1D-integrin is reduced and is lacking for β-, γ- and δ-sarcoglycan (Anastasi et al. 2004b).

Although numerous studies have been carried out on muscle diseases such as Duchenne muscular dystrophy, recessive autosomal muscular dystrophies and other myopathies (Calvo et al. 2000; Ginjaar et al. 2000; Dalkilic & Kunkel, 2003), insufficient data exist on the relationship between costameres, muscle agrin and the pathology of the second motor nerve. In a previous report (Anastasi et al. 2006), we presented our preliminary results of immunofluorescence studies of skeletal muscle fibres from patients with sensitive-motor polyneuropathy and demonstrated that α7B-integrin is replaced by α7A-integrin, its myogenic precursor (Belkin et al. 1996).

In thie present report, we have expanded these data on the skeletal muscle of patients with sensitive-motor polyneuropathy. We have added the analysis of other costameric proteins, such as vinculin, talin and dystrophin, and have also performed transmission electron microscopy and reverse transcriptase PCR (RT-PCR) analysis. These investigations were done to characterize in more detail the distribution and expression of costameric proteins and muscle agrin in this disease, and to confirm our previous data by other methods.

Materials and methods

Muscle biopsy samples

Initially, we examined skeletal muscle biopsies from two patients who did not have any coexisting neuromuscular pathology, but had undergone orthopaedic surgery for other reasons. Subsequently, biopsies were taken from the gastrocnemius muscle of ten patients who were affected by sensitive-motor polyneuropathy during orthopaedic surgery. The biopsy specimens were analysed using immunohistochemistry, transmission electron microscopy and RT-PCR. The age of the patients was between 30 and 60 years and all gave their informed consent. The investigation conformed with the principles outlined in the Declaration of Helsinki.

Immunohistochemistry

The biopsies were fixed in 3% paraformaldehyde in 0.2m phosphate buffer, pH 7.4, for 2h at room temperature. They were then washed extensively with 0.2m phosphate buffer, pH 7.4, and then with phosphate-buffered saline (PBS), containing 12 and 18% sucrose. The samples were snap-frozen in liquid nitrogen and 20-µm sections were prepared in a cryostat for use in a protocol to perform immunofluorescence. The sections were placed on glass slides that were coated with 0.5% gelatin and 0.005% chromium potassium sulphate.

To block non-specific binding sites and to permeabilize the membranes, the sections were preincubated with 1% bovine serum albumin (BSA), 0.3% Triton X-100 in PBS at room temperature for 15min. Finally, the sections were incubated with primary antibodies. The following primary antibodies were used: anti-α-sarcoglycan diluted 1:100, anti-β-sarcoglycan diluted 1:200, anti-γ-sarcoglycan diluted 1:100, anti-δ-sarcoglycan diluted 1:50, and anti-dystrophin diluted 1:20 (all from Novocastra Laboratories, Newcastle Upon Tyne, Uk); anti-agrin diluted 1:100 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); anti-vinculin diluted 1:100, and anti-talin diluted 1:100 (both from Sigma Chemicals, St. Louis, MO, USA); anti-α7B-integrin diluted 1:50, anti-β1D-integrin diluted 1:50, and anti-α7A-integrin diluted 1:100 (synthetic peptides from the COOH terminal region; kindly provided by the laboratory of Professor Tarone, University of Turin). Primary antibodies were detected using Texas Red-conjugated IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Slides were finally washed in PBS and sealed with mounting medium.

The sections were then analysed and images acquired using a Zeiss LSM 5 DUO (Carl Zeiss, Jena, Germany) confocal laser scanning microscope. All images were digitized at a resolution of 8 bits into an array of 2048×2048 pixels. Optical sections of fluorescent specimens were obtained using an HeNe laser (wavelength=543nm) and an Ar laser (wavelength=458nm) at a 62-s scanning speed with up to eight averages; 1.50-µm-thick sections were obtained using a pinhole of 250. For each reaction, at least 100 individual fibres were examined. Contrast and brightness were established by examining the most brightly labelled pixels and choosing the settings that allowed clear visualization of the structural details while keeping the pixel intensity at its highest (∼200). Each image was acquired within 62s, in order to minimize photodegradation. The ‘display profile’ function of the laser scanning microscope was used to show the intensity profile across an image along a freely selectable line. The intensity curves are shown in graphs below the scanned images.

Digital images were cropped and figure montages were prepared using Adobe Photoshop 7.0 (Adobe System, Palo Alto, CA).

Transmission electron microscopy

For electron microscopy, the muscle samples were dissected from surrounding connective tissue and prefixed in 4% glutaraldehyde fixative buffered by phosphate at pH 7.2–7.4 for 3h at room temperature. The samples were then fastened to an applicator stick, and placed in the identical fixative at 4°C for a further 2h. When attaching the samples, we were careful not to damage the muscular architecture.

After prefixation, the samples were fixed in 1% osmium oxide in 0.1m phosphate buffer, pH 7.2, containing 0.54% glucose. The specimens were then dehydrated in ethanol and embedded in Durcupan™ (Sigma-Aldrich). Semithin sections were cut and evaluated by light microscopy in order to define more accurately the field of observation. Ultrathin sections, obtained using an LKB 2088 Ultratome V (LKB Bromma, Stockholm, Sweden) and mounted on 200- to 300-mesh copper grids were stained with uranyl acetate in 70% ethanol and lead citrate solutions, according to the protocol of Reynolds (1963), and then examined under a Philips CM-10 transmission electron microscope (Philips Scientific, Eindhoven, The Netherlands).

Digital images were cropped and figure montages were prepared using Adobe Photoshop 7.0.

RT-PCR

Expression of α7A-, α7B- and β1D-integrin isoforms in the muscle biopsies of the ten patients with sensitive-motor polyneuropathy was evaluated by RT-PCR.

Total RNA isolation

Samples containing 50–100mg of tissue were homogenized using a power homogenizer (Ultra Turrax, IKA-Werke GmbH, Staufen, Germany). Total RNA was isolated by a single-step RNA isolation procedure (TRIzol® Reagents, Invitrogen) (Chomczynski & Sacchi, 1987) that uses a monophasic solution of phenol and guanidine isothiocyanate.

RT-PCR analysis

RT-PCR was carried out using the GeneAmp Gold RNA PCR Reagent kit (Applied Biosystems, Foster City, CA, USA) in a GeneAmp PCR System 9600 thermal cycler (Applied Biosystems,). In the first step, an initial RT reaction was carried out in a volume of 20µL containing 3µg of total RNA, 10U RNase inhibitor, 10mm DTT, 15U Multiscribe reverse transcriptase and 1.25µm oligo d(T)16 using the following thermal cycler conditions: 10min at 25°C, followed by 12min at 42°C. In the second step, a PCR was performed in a volume of 50µL containing 5µL of cDNA from the first step (RT) as a template, 2.5U AmpliTaq Gold DNA polymerase and each primer at a concentration of 0.2µm. The following primer pairs were used: for α7A-integrin, 5′-CGGGCCAACATCACAGTGAA-3’ (forward) and 5′-TGCCCCAGTTGTTCCTCAG-3’ (reverse); for α7B-integrin, 5′-CGGGCCAACATCACAGTGAA-3’ (forward) and 5′-TGAAGAATCCCATCTTCCAC-3’ (reverse); for β1D-integrin, 5′-CCAGAGTGTCCCACTGGTC-3’ (forward) and 5′-AATAGGACTCTTGTAAATCGG-3’ (reverse). PCR conditions were as follows: an initial 10-min denaturation step at 95°C, followed by 35 cycles of denaturation at 94°C for 40s, annealing at 62°C for 40s and extension at 72°C for 45s with a final extension at 72°C for 10min.

To ensure that equal quantities of input RNA were used for the RT-PCR, β-actin mRNA was amplified by an independent RT-PCR that was performed using the following primers: 5′-TCACCCACACTGTGCCCATCTACGA-3’ (forward) and 5′-CAGCGGAACCGCTCATTGCCAATGG-3’ (reverse), and reaction conditions of denaturation at 95°C for 5min, 35 cycles of 94°C for 30s, 54°C for 30s and 72°C for 1min, and a final extension at 72°C for 7min (Li & Yeung, 2002).

The amplified products were resolved on a 1.5% agarose gel and visualized by ethidium bromide staining. The product sizes were as follows: 322bp for α7A-integrin (containing exon 26), 208bp for α7B-integrin (without exon 26), 200bp for β1D-integrin and 290bp for β-actin. The amplified β-actin products were loaded in each lane 15min after the start of electrophoresis.

Digital images were cropped and figure montages were prepared using Adobe Photoshop 7.0.

Results

Immunohistochemistry

In order to analyse the distribution of components of the DGC and the vinculin–talin–integrin system, which comprise the costameres, we performed immunofluorescence analysis of the skeletal muscle samples taken from patients with sensitive-motor polyneuropathy. The 20-µm-thick cryosections, which had been obtained from the samples, were analysed by acquiring a stack of 16 sections, with a scan step size of 0.8µm.

First, we analysed the biopsy samples of the two patients who did not have any neuromuscular disease. These control reactions, carried out using a single antibody each time, showed normal intermittent, or periodic, staining patterns for α-sarcoglycan (Fig. 1A), β-sarcoglycan (Fig. 1B), γ-sarcoglycan (Fig. 1C), δ-sarcoglycan (Fig. 1D) and dystrophin (Fig. 1E). The same normal immunostaining pattern was seen for vinculin (Fig. 2A), talin (Fig. 2B), α7A-integrin (Fig. 2C), α7B-integrin (Fig. 2D), β1D-integrin (Fig. 2E) and, finally, muscle agrin (Fig. 2F).

Fig. 1.

Fig. 1

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Compound panel showing immunohistochemical findings in normal human skeletal muscle. Skeletal muscle fibres were immunolabelled with antibodies against α-sarcoglycan (A), β-sarcoglycan (B), γ-sarcoglycan (C), δ-sarcoglycan (D) and dystrophin (E). All tested proteins showed a costameric distribution and a normal immunostaining pattern.

Fig. 2.

Fig. 2

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Compound panel showing immunohistochemical findings in normal human skeletal muscle. Skeletal muscle fibres were immunolabelled with antibodies against vinculin (A), talin (B), α7A-integrin (C), α7B-integrin (D), β1D-integrin (E) and agrin (F). All tested proteins showed a costameric distribution and a normal immunostaining pattern.

Secondly, we performed immunostaining on longitudinal sections of skeletal muscle from the ten patients with sensitive-motor polyneuropathy using a single antibody each time. All of the sarcoglycan isoforms were detected to varying degrees along the sarcolemma (Fig. 3). In particular, immunostaining of α-sarcoglycan (Fig. 3A), β-sarcoglycan (Fig. 3B) and δ-sarcoglycan (Fig. 3D) was clearly evident and the immunostaining of γ-sarcoglycan was slightly reduced compared with that of the control (Fig. 3C). Dystrophin immunofluorescence appeared to be normal (Fig. 3E). However, when observed at higher resolution, it became apparent that the fibres were not uniformly stained for these proteins, but showed intermittent or periodic staining along the sarcolemma, which confirms that these proteins are also distributed in costameres in pathological conditions.

Fig. 3.

Fig. 3

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Compound panel showing immunostaining of longitudinal sections of skeletal muscle fibres of patients with sensitive-motor polyneuropathy. The sections were immunolabelled with antibodies against α-sarcoglycan (A), β-sarcoglycan (B), γ-sarcoglycan (C), δ-sarcoglycan (D) and dystrophin (E). Sarcoglycans were detectable along the sarcolemma; dystrophin showed a normal immunostaining pattern.

Immunostaining of components of the vinculin–talin–integrin system showed that vinculin (Fig. 4A) and talin (Fig. 4B) immunofluorescence was reduced, but still clearly detectable. By contrast, the immunostaining of α7A-integrin appeared to be normal, and even slightly increased (Fig. 4C), when compared with that of the control specimens. The immunofluorescence of α7B-integrin (Fig. 4D) and β1D-integrin (Fig. 4E) was severely reduced. Finally, virtually no immunostaining of muscle agrin could be seen (Fig. 4F).

Fig. 4.

Fig. 4

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Compound panel showing immunostaining of longitudinal sections of skeletal muscle fibres of patients with sensitive-motor polyneuropathy. The sections were immunolabeled with antibodies against proteins of the vinculin–talin–integrin system and muscle agrin: (A) vinculin; (B) talin; (C) α7A-integrin; (D) α7B-integrin; (E) β1D-integrin; (F) muscle agrin. α7A immunostaining appeared to be normal, or slightly increased; immunofluorescence for α7B-integrin, β1D-integrin and muscle agrin was severely reduced and nearly absent.

To confirm the protein staining patterns, we used the ‘display profile’ software function of the laser scanning microscope for selected samples. This additional analysis, which reveals the fluorescence intensity profile across an image along a freely selectable line, converted the immunofluorescence signal into a graph. The display profiles of the control specimens showed clear peaks of fluorescence for α7A-integrin (Fig. 5A). The profiles for α7B-integrin (Fig. 5B), β1D-integrin (Fig. 5C) and agrin (Fig. 5D) also showed obvious peaks of fluorescence. By applying this analysis to the samples of muscle fibres taken from the patients with sensitive-motor polyneuropathy, it was possible to show that the peaks of α7A-integrin fluorescence were increased in magnitude in these samples (Fig. 6A). By contrast, the fluorescence intensities of α7B-integrin (Fig. 6B), β1D-integrin (Fig. 6C) and muscle agrin (Fig. 6D) were severely reduced compared with those of the controls.

Fig. 5.

Fig. 5

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Display profiles of longitudinal sections of normal human skeletal muscle fibres: (A) α7A-integrin; (B) α7B-integrin; (C) β1D-integrin; (D) muscle agrin. All analysed samples showed fluorescence peaks with normal values.

Fig. 6.

Fig. 6

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Display profiles of longitudinal sections of human skeletal muscle fibres of patients with sensitive-motor polyneuropathy. (A) α7A-integrin; (B) α7B-integrin; (C) β1D-integrin; (D) muscle agrin. The α7A-integrin fluorescence peaks were of normal magnitude, while α7B-integrin, β1D-integrin and muscle agrin fluorescence intensities were absent.

Transmission electron microscopy

We used transmission electron microscopy to evaluate skeletal muscle damage and to assess particular ultrastructural features of the muscle taken from patients with sensitive-motor polyneuropathy. For this purpose, we analysed cross-sections of the skeletal muscle biopsies taken from the patients with sensitive-motor polyneuropathy, and compared them with cross-sections of normal skeletal muscle (Fig. 7A). The images obtained showed that the normal geometry of the thick and thin filaments was lost which we suggest was due to the absence of components of the DGC and vinculin-talin-integrin system. In fact, myosin thick filaments were not present inside the actin hexagon, and as a consequence, the muscle cytoarchitecture was disordered (Fig. 7B).

Fig. 7.

Fig. 7

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Transverse sections of normal human skeletal muscle (A) and skeletal muscle from patients with sensitive-motor polyneuropathy (B) observed by transmission electron microscopy. In B, the normal geometry of the thick and thin filaments is absent and the normal muscular architecture is disordered.

RT-PCR

In order to determine whether the increased levels of α7A-integrin protein are due to increased amounts of α7A-integrin mRNA, we performed RT-PCR with primers specific for the integrins. Using RNA samples isolated from control and diseased skeletal muscle fibres, we confirmed that, in comparison with those of healthy individuals (Fig. 8A), levels of α7A-integrin mRNA were increased in the skeletal muscle of patients with sensitive-motor polyneuropathy. mRNA levels of the α7B- and β1D-integrin isomers were lower than normal (Fig. 8B).

Fig. 8.

Fig. 8

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RT-PCR. (A) Control sample. Representative 1.5% agarose gel electrophoresis showing RT-PCR products amplified from one of the unaffected control individuals. In each lane an aliquot of the corresponding β-actin amplification reaction was included as a control to ensure equal quantities of input RNA. Lane 1: α7A-integrin and β-actin; lane 2: α7B-integrin and β-actin; lane 3: β1D-integrin and β-actin; lane 4: 100-bp ladder. (B) Representative 1.5% agarose gel electrophoresis showing RT-PCR products amplified from a sample from one of the affected patients. Lane 1: α7A-integrin and β-actin; lane 2: α7B-integrin and β-actin; lane 3: β1D-integrin and β-actin; lane 4: 100-bp ladder.

Discussion

Costameres are composed of large complexes of integral and peripheral membrane proteins that are linked to the contractile apparatus by intermediate filaments and to the extracellular matrix by laminin (Pardo et al. 1983a). These structures have three major functions: (1) to keep the plasma membrane, or sarcolemma, in register with nearby contractile structures; (2) to protect the sarcolemma against contraction-induced damage; and (3) to transmit some of the forces of contraction to the extracellular matrix.

The primary function of skeletal muscle is to generate mechanical forces for movement and stabilization. These forces are generated in sarcomeres and are transmitted longitudinally to myotendinous junctions and laterally to the extracellular matrix, and therefore to tendons (Street, 1983; Monti et al. 1999). Lateral forces are transmitted by protein complexes, one of which consists of F-actin and dystrophin underneath the sarcolemma, another which consists of β-dystroglycan and associated proteins in the sarcolemma, and a third which consists of α-dystroglycan and laminin-2 outside the sarcolemma (Ervasti & Campbell, 1993). In general, cytoskeletal proteins form dynamic structures that respond to mechanical and other signals by undergoing short- or long-term changes in shape (Ingber, 1997).

Here, we studied costameric proteins of the DGC and the vinculin–talin–integrin system, together with muscle agrin, in skeletal muscle from adult patients with sensitive-motor polyneuropathy. Our results using immunofluorescence showed that: (1) immunofluorescence was detectable for all the sarcoglycans tested, although to varying degrees; (2) staining for dystrophin, vinculin and talin was normal or slightly reduced; (3) staining for α7B- and β1D-integrin and muscle agrin appeared to be severely reduced or almost absent; and (4) staining of α7A-integrin appeared to be normal or slightly increased. Our results from RT-PCR analysis confirmed that the mRNA levels of integrins reflected the protein levels. Finally, transmission electron microscopy of transverse muscle sections revealed a disordering of the normal structure of muscle fibres, with the absence of myosin inside the actin hexagon, which showed that ultrastructural damage had occurred due to muscular inactivity.

The normal, or slightly decreased, immunostaining for dystrophin, sarcoglycans, vinculin and talin, under conditions of muscular inactivity, demonstrated and confirmed that, although these proteins are involved in muscular dystrophies, they are not involved in pathologies that affect the motor innervation of skeletal muscle (Campbell, 1995; Wewer & Engvall, 1996). However, our most important results are those that show an increased amount of α7A-integrin and decreased amounts of α7B- and β1D-integrins in the skeletal muscle of patients with sensitive-motor polyneuropathy. Previous reports demonstrated the key role of integrins in the formation of the postsynaptic membrane, and the results suggested that the increased expression of integrins may enhance the development and stability of neuromuscular junctions and restore muscle viability in dystrophic mice (Burkin et al. 1998, 2000). Therefore, enhanced expression of α7A-integrin, the myogenic precursor of α7B-integrin, may compensate for the decreased expression of other proteins, such as α7B- and β1D-integrin, and thus maintain both the mechanical and the signal transduction capacities of skeletal muscle.

The results of previous studies showed that muscle agrin is regulated by neural agrin (Bezakova et al. 2001; Bezakova & Lomo, 2001; Hagiwara & Fallon, 2001). Neural agrin, which is released by motor nerve terminals into the synaptic cleft (McMahan, 1990), is essential for the formation of neuromuscular junctions and acts by activating muscle-specific kinase (MuSK) (Gautum et al. 1996). Moreover, Glass and others have reported that (1) MuSK-deficient myotubes are unresponsive to agrin, (2) a dominant negative MuSK mutant inhibits agrin-induced AChR clustering and (3) chemical cross-linkers can attach agrin to MuSK on the myotube surface (Glass et al. 1996, 1997). However, agrin does not bind to soluble forms of MuSK or to MuSK expressed in non-muscle cells (Glass et al. 1996). Consequently, it is therefore likely that there are additional receptors in skeletal muscle that link agrin to MuSK or act in parallel to MuSK (Martin & Sanes, 1997). In our opinion, the integrins are logical candidates for the role of co-receptor. In fact, the integrins transmit many important signals that originate from components of the extracellular matrix and therefore they might be directly involved in agrin signalling (Hynes, 1992, 1996). In fact, integrins have previously been implicated as components or modulators of the signal transduction pathway of agrin (Martin & Sanes, 1997).

Based on our results, we hypothesize that muscle inactivity, for example as a result of denervation, could alter and modify the organization of costameres in two ways. First, muscular atrophy followed by inactivity could result in a lack of neural agrin, which would affect the gene expression of muscle agrin, sarcoglycans and integrins. The reduction in muscle agrin would, in turn, result in low levels of expression of the muscle-specific integrins, α7B- and β1D-integrin. In this way, the loss of α7B-integrin could be compensated for by the increased expression of α7A-integrin, which would enable muscle fibre viability to be maintained. Secondly, the mechanical stresses on cell surface receptors such as the integrins, which physically attach the cytoskeleton to the extracellular matrix, decrease in muscle atrophy because of reduced muscle contraction and relaxation. It has been suggested that mechanical signals can be transduced into a biochemical response through force-dependent changes in the geometry of the cytoskeletal scaffold (Ingber, 1997). This process relies on ‘tensegrity’ in the cell, i.e. the maintenance of cell shape by continuous tension. Therefore, the mechanical changes, which result from muscular inactivity, could produce changes in chemical signals that affect the structural organization of the costameres, and the loss of α7B-integrin could be replaced and reinforced by the increase in α7A-integrin.

These data, which confirm the results of our previous studies on costameric proteins, reveal a new avenue of research, which will have the aim of understanding the variations in the protein composition and structural arrangement of costameres during conditions of muscular inactivity. Our results could also be useful in the study of other muscle diseases because they highlight the need to verify the importance of, and the changes in, tensegrity in the muscle fibres. One of the challenges in this area of research will be to understand how these chemical and physical signals, which are responsible for the control of cell behaviour, correlate with muscle damage. Thus, it will be important to perform additional experiments in order to clarify the mechanism that leads to the down-regulation of α7B- and β1D-integrin and agrin.

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