Congenital myopathy associated with abnormal accumulation of desmin and dystrophin

Neuromusc. Disord., Vol. 2, No. 3, pp. 169-175, 1992 Printed in Great Britain

CONGENITAL

0960-8966/92 $5.00 + 0.00 Pergamon Press Ltd

MYOPATHY

ACCUMULATION

ASSOCIATED

OF DESMIN

WITH

AND

ABNORMAL

DYSTROPHIN

A. PRELLE,*M. MOGGIO,G. P. COM], A. GALLANTI,N. CHECCARELLI,N. BRESOLIN,P. CISCATO,F. FORTUNATO and G. SCARLATO Istituto di Clinica Neurologica, Centro Dino Ferrari, Universit/tdi Milano, Italy (Received6 February 1992; in revisedform 21 May 1992; accepted 11 June 1992)

studied a 5-yr-old boy clinically presenting congenital myopathy. Muscle biopsy showed sarcoplasmic accumulation of desmin filaments leading to diagnosis of desmin storage myopathy. An immunohistochemical study of other cytoskeletal proteins (actin, alpha-actinin, vimentin and dystrophin) was performed. Desmin positive areas reacted strongly with anti-midrod and C-terminus dystrophin antibodies. Probed with the same antibodies by Western blot, desmin and dystrophin showed normal molecular size but densitometric analysis demonstrated a parallel increase of both proteins. Our results indicate that intrasarcoplasmic desmin storage is associated with an abnormal accumulation of dystrophin. Since no other cytoskeletal proteins are accumulated this finding seems to be specific and suggests a possible structural and functional association between these two proteins in striated muscle. Abstract--We

Key words: Congenital myopathy, intermediate filament myopathies, desmin, dystrophin.

INTRODUCTION

We describe a 5-yr-old boy clinically resembling a case of a congenital neuromuscular disorder. Muscle biopsy revealed sarcoplasmic accumulation of desmin filaments leading to the diagnosis of desmin storage myopathy. This disease has been identified recently and described in both familiar and sporadic cases [1, 2]. Desmin, an intermediate filament, is a major component of the cytoskeleton, localized at the Z line [3, 4]. In order to clarify this pathology better we studied other cytoskeletal components by immunological analysis. Following recent data from literature we have also included dystrophin protein analysis [5]. CASE REPORT

A 5-yr-old boy came to our observation because of a delay in milestone development since birth. Neuropsychological development was also impaired. The family history was negative for neuromuscular disorders and no parental

* Author to whom correspondenceshould be addressedat: Istituto di Clinica Neurologica, Centro Dino Ferrari, Via F. Sforza 35, 20122 Milano, Italy.

consanguinity was reported. Labour and delivery were uncomplicated. Neurological examination showed proximal and distal muscular weakness with associated hypotonia. Cranial nerves and somatosensory examination were normal. Tendon reflexes were absent. An arched palate was present. The serum creatine kinase level was normal. Electromyography showed a myopathic pattern. Cardiological examination and electrocardiography were normal, as well as cerebral MRI. MATERIAL AND METHODS

Muscle biopsy was performed on the left quadriceps under general anaesthesia. Morphological studies

Muscle was frozen in isopentane cooled in liquid nitrogen; cryostat sections were prepared for histological and histochemical examination using standardized methods [6]. Acridine orange and fluorescein-alpha-bungarotoxin (from Molecular Probes) staining was performed as described previously [6, 7]. Electron microscopy studies were performed as described previously [8] and several ultra-thin sections were cut from ten different blocks. For immunohistochemical 169

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Fig. 1. HE, × 250, cross-section of patient muscle: subsarcolemmal eosinophilic areas are present in some fibres.

analysis weused different antibodies: monoclonal antibodies against desmin, vimentin, actin, and alpha-actinin from Amersham International; dystrophin monoclonal antibodies against the mid- rod (30 kDa) and COOH terminus domains from Novocastra. Immunofluorescent techniques were performed as described previously [9]; in particular, sections were collected on coverslips treated with polylysine 1% and incubated with the different primary antibodies for 2 h at RT: desmin and vimentin monoclonal antibodies were diluted 1 : 5 in 1% BSA/PBS, actin and alpha-actinin antibodies were diluted 1 : 100 in 1% BSA/PBS, dystrophin antibodies were used undiluted. The sections were rinsed in PBS three times for 5 min. As a second antibody we used a FITC-labelled sheep anti-mouse IgG (Amersham, Code No. 1031) diluted 1 : 25 in 1% BSA/PBS for desmin, vimentin, actin and alphaactinin antibodies. Sections with dystrophin antibodies were incubated for 1 h with a rhodamine-labelled anti-mouse IgG (from Dako) diluted 1 : 40 in P B S - I % B S A . Finally sections were rinsed in 0.1 M PBS pH 7.4, three times for 5 min, mounted in glycerol/PBS 1 : l and observed with a Leitz fluorescence microscope. Control sections were incubated with nonimmune serum and with PBS containing ! % BSA.

Biochemical studies

Immunoblot analyses of muscle samples were performed using a monoclonal anti-desmin antibody (Amersham) and two monoclonal antibodies against the dystrophin mid-rod (30 kDa) and C-terminus domains (Novocastra). Muscle samples were homogenized on a Potter homogenizer at 0*C in 10 volumes of sample buffer containing 75 mM Tris-HCl (pH 6.8 at 20°C), 15% (w/v) SDS, 20% (v/v) mercaptoethanol and 0.001% (w/v) bromophenol blue. The total protein concentration was determined by the Lowry method [10], before loading. A volume corresponding to 300 /tg protein was analysed on a 1.5 mm thick SDS-polyacrylamide gel using a linear resolving gel gradient of 412.5% and a 3.5% stacking gel. Proteins were separated in an overnight run at 0.6 mA cm ~at 15°C and subsequently blotted onto a nitrocellulose membrane, at a current of 1 A for 4 h, at ! 5°C. Molecular weight nitrocellulose strips were stained with amido black. The nitrocellulose was then cut into two parts at approximately 110 kDa molecular weight. The upper portion was probed with dystrophin primary antibodies (dilution 1 : 30), the lower part with the desmin antibody (dilution 1 : 5). The secondary antibody reaction (peroxidase-labelled anti-mouse IgG, dilution 1 : 100) was performed for 1 h at RT. Blots were

Desmin and Dystrophin Muscle Accumulation

171

Fig. 2. EM, thin section, uranyl acetate and lead citrate. (a) Original magnification x 12,000,a subsarcolemmalarea contains an abnormal electrondense material not surrounded by a membrane. (b) Original magnification x 20,000 enlargement of the electrondense material that appears granulo-filamentouswith a protein-like aspect. finally exposed to freshly prepared 0.05% diaminobenzidine in PBS with 0.025% hydrogen peroxide for 2 rain before rinsing with distilled water and drying. Blotted gels were fixed in 20% trichloroacetic acid, and stained with Coomassie brilliant blue, in order to quantitate post-transfer myosin (heavy chain). Quantitation was performed through densitometric analysis of the nitrocellulose signal and pre-transfer gel.

RESULTS

Morphology Muscle biopsy examination shows a variation in muscle fibre size with hypotrophic fibres among some hypertrophic ones. Twenty-three per cent of fibres reveal subsarcolemmal and intracytoplasmic areas, hyperchromic with Gomori

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al.

Fig. 3. Immunohistochemistrywith anti-desmin antibodies, × 100, cross-sectionof patient muscle: strong reaction is present in the subsarcolemmal area of many fibres.

Fig. 4. Immunohistochemistrywith anti-dystrophin C-terminus, x 400, cross-sectionof patient muscle:dystrophin is present at the sarcolemma of all muscle cells and it is overexpressed in the subsarcolemmalarea of some fibres. trichrome a n d eosinophilic with h a e m a t o x y l i n and eosin (Fig. I). These a b n o r m a l areas stain positively with oxidative m e t h o d s for N A D H a n d COX. A T P a s e reactions show negative staining in the same areas, which are present in both fibre types. Rare splittings a n d centrally

located nuclei are noted. Neither necrosis, n o r i n f l a m m a t o r y cells or connective tissue proliferation are present. Acridine orange staining does not show positive fibres for R N A . B u n g a r o t o x i n staining does n o t reveal neuromuscular junctions.

Des

Dys - - ~

2

3

II

I Dys - - ~

1

2

B 3

II

I

1

2

3

C 4

5

Fig. 5. Western blot analysis. (A) Lane 1: control; lane 2: patient; lane 3: DMD patient; (I) dystrophin (Dys) immunoreactivity(mid-rod domain); (II) desmin (Des) immunoreactivity. (B) Lane order as in (A); (I) dystrophin immunoreactivity(COOH-terminus); (ID this part of nitrocellulose was incubated only with the second antibody (mouse monoclonal IgG anti IgG). (C) Coomassie brilliant blue stained post-transfer gel. Lane 1: low molecular weights; 2: control; 3: patient; 4: DMD patient; 5: high molecular weights. See text for further details.

1

A

45

9"/.4

116.2

200

Kd

O

~

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At the ultrastructural level 32% of the examined fibres show abnormal areas containing an electrondense material with a filamentous, protein-like aspect, not surrounded by a membrane. Few mitochondrial aggregates are present between these filaments (Fig. 2). Immunohistochemical study of cytoskeletal proteins shows strong staining for desmin in these areas (Fig. 3). No positive staining is observed with anti-actin, alpha-actinin and vimentin antibodies. On the contrary dystrophin analysis shows a normal pattern at the cell surface of all myofibres but an abnormal accumulation of the protein in desmin positive areas with both antibodies (Fig. 4).

Biochemistry The patient's muscle homogenate was probed with the same antibodies by Western blot. Both desmin and dystrophin are the normal molecular size but densitometric analysis demonstrates a parallel increase of the two proteins (desmin: + 63.0%; dystrophin: + 67.5% of controls, n = 4), while the myosin heavy chain level is normal (Fig. 5). DISCUSSION We report a case of congenital neuromuscular disease resulting as an abnormal accumulation of desmin and dystrophin in muscle tissue. Diagnosis was made by muscle biopsy analysis which revealed focal intrasarcoplasmic accumulation of a granulo-filamentous material that reacted strongly with anti-desmin and anti-dystrophin antibodies. Biochemical studies confirmed these data. Since we cannot demonstrate an impairment of catabolism or a blockage in the metabolic pathway of these proteins, we do not consider this case a "storage disease" in the same sense as a glycogenosis or lipid myopathy. Myopathies with intermediate filament aggregates have been described previously by different authors, and few of them have been associated with desmin accumulation [1-3, 11]. Increased desmin has also been demonstrated in different infantile myopathies probably related to the presence of regenerating fibres or the persistence of immature fibres [12, 13]. However, in our case no regenerating fibres are present and the focal accumulation of desmin cannot be due to the expression of immature fibres. Our case seems to be different from the previously reported ones because of the clinical

appearance of congenital myopathy, the lack of cardiomyopathy and a negative family history. Moreover our results indicate that intrasarcoplasmic desmin accumulation is associated with an abnormal increase of dystrophin. Dystrophin accumulation was revealed using antibodies against different epitopes, the mid-rod and the C-terminus domains, both suggesting the presence of a normal protein in these areas. This aspect could obviously not be studied in the previous reports of intermediate filament myopathies made before the discovery of dystrophin. Desmin is a major component of intermediate filaments, localized along the Z line in striated muscle and stably associated with actin and alpha-actinin [3]. In particular it is present at the periphery of each disc, forming a honeycomblike net within the Z plane, and linking individual myofibrils laterally at their Z discs and to the sarcolemma [4]. This distribution suggests a role of a mechanical integrator for myofibrillar contraction [3]. Recent data suggest that desmin is also localized at the neuromuscular junction, highly concentrated in the post-synaptic domain [14]. Dystrophin is the protein product of the Duchenne muscular dystrophy (DMD) gene, recently characterized and localized at the sarcolemma [15, 16], and in very small amounts in the T tubules and in the triads [17]. The complete protein sequence indicates similarities with other cytoskeletal proteins such as alphaactinin and spectrin [5, 18, 19] and in particular the amino terminus of dystrophin displays similarity to the actin-binding site of alphaactinin [20]. EM studies of purified dystrophin have led to structural similarities with spectrin being hypothesized [21]. Moreover, like desmin, dystrophin is strongly expressed at the neuromuscular junction [22]. The functional role of dystrophin in normal striated muscle is not well defined but literature data suggest that it is a member of the cytoskeletal proteins, strictly associated with other intermediate filaments. Since desmin is bound to alpha-actinin [3] and dystrophin has structural homologies with alpha-actinin [20] we can hypothesize that desmin is also associated with dystrophin. In our case the parallel abnormal storage of desmin and dystrophin seems a specific finding, and actually no other cytoskeletal proteins are accumulated. This pathological finding supports the hypothesis of a structural and functional association between these two proteins in normal striated muscle. Therefore, we think dystrophin

Desmin and Dystrophin Muscle Accumulation

analysis should be performed in intermediate filament myopathies to extend our knowledge of the function of this protein and its pathological rote in these diseases.

REFERENCES

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