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P~t~r F. M. van DI~R VIgN,*'I"++ PAUL H. K. JAP,* RIa H. W. WeTZELs,§HE.nKJ. TER LAAK, FRANs C. s. RAMAEKERSflAD M. STADHOUDERS*and RoB c. A. SENGERS'I"

Departments of "Cell Biology and Histology. +Pediatrics, §Pathology and Neurology, University of Nijmegen, Nijmegen: and fDepartment of Molecular Cell Biology. Universityof Limburg, Maastricht. The Netherlands (Received 21 Jam,try/991: in final,/orm 13 June 1991)

Abstract--Postnatal centralization of muscle fibre nuclei, which were previously located subsarcolemmally, is described in a case of centronuclear myopathy (CNM) in a male patient with generalized ntuscle weakness since birth. A muscle biopsy was taken at the age of 11 months: no particular abnormalities were observed at this stage apart from an unusual variation in fibre size. A distinctly below average muscle fibre diameter, increased endomysial connective tissue, and features typical for CNM were found in a biopsy taken 9 yr later. Immunohistochemical studies using antibodies to desmin, vimentin, laminin and type IV collagen revealed altered staining patterns contpared with normal fibres. The abnormalities in the patterns of cytoskelctal proteins point to a defective regulation of the composition and org:mization of the cytoskeletal network during development, paralleled by ab|tormalities in the extraccllular matrix. K,'r words: Centronuclear myopathy, pathogencsis, cytoskeleton, basement membrane, extracellular matrix, immunohistochemistry.

INTROI)UCTION Tile first case of a muscular disease characterized by numerous centrally located nuclei in the muscle fibres was documented by Spiro et al. [I]. Since the morphological findings in the muscle biopsy suggested the presence of myotubcs, the disease was named "'myotubular myopathy", The general term "centronuclear myopathy'" (CNM) was suggested by Sher et al. [2]. Several authors described variations in clinical picture, age of onset, mode of inheritance and progression, which indicated that the term centronuclear myopathy may in fact cover more than one disease [3-7]. The pathogenesis of the disorder is not clear although some explanations have been put forward [8-1 I]: (I) an arrest of, or delay in, muscle fibre maturation: (2) an impairment of maturation preventing migration of nuclei to a peripheral location; and (3) degenerative processes causing secondary nuclear migration to the centre of the fibres. ,+Author to whom correspondence should be addressed: Department of Cell Biology and |listology. University of Nijmegen. P.O. Box9101,65001 i B Nijmegen.The Netherlands.

Disorganized cytoskeletal filaments have been observed in several myopathies [12,13] but few studies have investigated whether the presence of central nuclei is related to a disorganization of the cytoskeletal network. Because of the putative role of the cytoskeleton in the positioning of the nucleus, in its interaction with the cell membrane [14,15], it is tempting to speculate that some disorder in the cytoskeletal protein system might lead to centralization of nuclei in C N M . Using antibodies to intermediate filaments of the desmin (skeletin) type, no specific abnormalities in muscle biopsies of two patients presenting a slowly progressive form of C N M were observed [13], whereas in four cases of a severe, neonatal form of C N M desmin patterns comparable with foetal muscle were described [I 6]. Studies on the distribution pattern of the basement membrane constituents laminin and type IV collagen in C N M have not revealed differences in the localization of these components when compared with normal muscle [17.19]. Both laminin and type IV collagcn were always located in a sharply delineated ring representing the basement membrane surrounding m u s i c fibres, blood vessels and capillaries.

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in this report we describe a patient who suffered from a generalized muscle weakness since birth and from whom a muscle biopsy was taken at the age of II months. The biopsy showed no obvious abnormalities apart from too large a mean fibre diameter, and too large a vltriation in fibre size. A second biopsy was taken at the age of I0 yr. Tile biopsy lindings appeared to be typical for CNM. in addition to conventional ultrastructural and histochemical characterization of the muscle biopsies, we performed immunohistochemical studies with antibodies to the intermediate lilament proteins desmin and vimentin, and the basement membrane components laminin and type IV collagen, to investigate the organization and composition of the cytoskeletal network and the basement membrane of the pathologic myolibres. PATIENT AND MI'TI'IIOI)S

C a s e histor I'

The patient, a boy, was born as the first child of healthy, non-consanguineous parents after an uneventful pregnancy and delivery. From birth onwards he experienced hypotonia, feeding d i f ficulty and reached motor milestones with some delay. He showed facial weakness and ptosis, but no other signs of external ophthalmoplegia. He walked at 18 months but was never able to run. A biopsy of the quadriceps rot, sole was pcrlbrmcd elsewhere (by Dr M. tt. Brooke) at the age of I I months (biopsy I). The biopsy was delinitely abnormal, but a clear diagnosis could not be

made. He had a number of life threatening respiratory infections, during one of which he needed ventilatory support. He experienced increasing diltict, lty in walking and showed progressive lordosis, lixed hip Ilexion and equinovarus deformity of the feet. When we lirst saw him at theage ofahnost 10yr. he was a short statured boy. with a height of 126 cm (below third percentile}, a head circumference of 51 cm (between ItJlh and 50th percentile} and weighed 18.5 kg (below third percentile). General examination revealed no abnormalities and no cltrdiomyopathy was diagnosed. There was facial diplegia and bilateral ptosis withot, t limited extra-ocular muscle movement, and generalized weakness, more pronounced in tile distal than the proximal mt, scles. He was seriously handicapped by pes equinus left and right, with a slight varus and adductus deformity. The serum creatine kinase was not increased and the results of electromyographic investigations were equivocal. A needle biopsy of the quadriceps muscle (vastus lateralis) was perlbrmed and the tissue was embedded in epoxy-resin for ultrastructural examination (biopsy 2L A Z-lengthening procedure was performed on the right Achilles tendon and after uneventft, l recovery the left Achilles tendon was also treated. During the operation, a biopsy of tile soleus muscle was taken (biopsy 3). The wounds healed well. Subsequently an ankle orthosis was made on both sides, but the course was seriously complicated by delayed weaning from the respirator dt,e to tracheomalacia. This was eventually treated by

Centronuclear Myopathy

213

Table I. Summar~ (~f the histtx:hemical and morphometrical findings

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surgical removal of the eroded part of the trachea. Further recovery was uncomplicated.

tlistochemistrv and morphometry Sections of biopsy I stained with haematoxylin-eosin (H&E) and ATPase (high pH) were kindly put at our disposal by Dr Brooke. Frozen cryostat sections of biopsy 3 were used for staining with H&E and for ATPase (high pH) and other histochemical stainings [20]. Muscle fibre diameters were estimated using a MOP-Videoplan measuring instrument (Kontron, Munich, F.R.G.). [n cases of ellipsoid protilcs, the lesser diameter was considered to be the profile diameter [20].

lmmunohislochemistry Immunohistocheniistry could only be performed on material from biopsy 3, which was frozen immediately in liquid nitrogen and stored lit - 20"C. Air-dried, 6/tm eryostat sections were fixed in methanol ( - 2 0 " C , 5-10 rain) and ;lcetone ( - 20"C, I0 30 s). After air drying they were incubated with an antibody or antiserum for 30 rain at room temperature. The sections were rinsed in PBS (30 rain), and when double labelling was performed incttbated with a second antibody or antiserum, After rinsing in PBS (30 min) they were incubated with the appropriate anti-mouse and/or anti-rabbit immunoglobulins. labelled with fluorescein isothiocyanate (Nordic Immunology, Tilburg, The Netherlands), Texas Red (Southern Biotechnology Associates, Birmingham, AL, U.S.A.) or peroxidase (Dakopatts, Glostrup, Denmark). After washing in PBS for 30 rain, the sections were mounted in Gelvatol (Monsanto, St. Louis, MO, U.S.A.) or processed for the detection of peroxidase as described elsewhere [21]. Slides were viewed with a Leitz Dialux 20 EB microscope, equipped with epilluoreseent illumination. Pictures were taken using 400 ASA Tri-X fihn

(Kodak, Rochester, NY, U.S.A.) with an autonlatic camera. Tile following polyclonal and monoclonal antibodies were used in this study: (I) tile mot, se monoclonal antibody 4El0. specific for human laminin [22]; (2) a mouse monoclonal antibody to type IV collagen, raised to human kidney glomeruli [23]: (3) tile mouse monoclonal RV 202, specific for vimentin, the intermediate filament protein tk)und in most types of mesenchymal cells [24]; (4) an aflinity-puritled polyclonal rabbit antiserum (pVIM), raised against vimcntin isolated from calf lens by preparative gel electrophoresis [251; (5) the mouse monoclonal antibody RD 3111, specific for desmin, the muscle specific intermediate filament protein [24]; (6) a polyclonal rabbit antiserum (pDcs), raised against chicken gizzard muscle desmin as described bet\)re [25]; and (7) the mouse monocolonal antibodies 331)R5B4 and 330-R5D4, specific for embryonic myosin [26]. The monoclonal and polyclonal antibodies to vimentin and desmin are available from EuroDiagnostics B.V., Apeldoorn, The Netherlands. Soleus muscle from a 9-yr-old patient suffering from a clinical (possibly toxic) polyneuropathy (Figs 2 and 3), and a 6-yr-old patient suffering from Duchenne muscular dystrophy, in whose biopsy material large groups of basophilic regenerating fibres were present (Fig. 4), were used as controls.

Electron microscopy Material from biopsy 2 was prefixed in buffered 2% glutaraldehyde and postfixed in 2% OsO,. Following embedding in Epon 812, ultrathin sections were double contrasted (uranyl acetate, lead citrate), and examined in a Philips EM 300 or a Philips EM 301 electron microscope.

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Fig. 2. Indirect i m m u n o f l u o r e s c c n c e m i c r o g r a p h s of conlrt~l muscle (a} and of muscle tissue of the paliunl ~ i t h C N M (b o}, in~cubalcd 'a,ith the p o l y c h m a l a n t i s e r u m lu dcsmin tlt i1 or wilh the m o n o c h m a l desmin antibod,. (j o } l~a~rin/licatcs 25 llnl II I'],',;I i / f ,',;

Ilislochemical oh.~'ervations Histochemical and quantitative m o r p h o metrical tindings are depicted in Fig. I and sumn'tarized in Table 1. The first biopsy was characterized by a normal ratio o f type ! to type !! tibrcs. Only 0.7", of the fibres contained centrally located nuclei (Fig. la,c). I|ov~ever, there was a distinct variation in libre diameter and the average diameter ~,ts too large t\)r that age. Biopsy 2. taken 9 ~r later from the same muscle, shmscd numerous librcs with central nuclci and a mean muscle fibre diameter far betray normal. Fibrcs sho,,sed a somewhat

rounded appearance (Fig. 6a), endomysial connecti',e tissue was increased, and adipose tissue was spread throughout the whole biopsy, replacing about 25% o f the muscle tissue. Some librcs (espccially the librcs with the largest diameters) showed small or large central "'holes", a pcrinuclcar halo or a radial, spoke-like appearance of the cytoplasm. In longitudinal sections, intern,|l nuclei were located in chains t~,ith or ,aithout internuclear spaces. Nuclei were some,.~,hat rounded but not swollen and did not contain prominent nuclcoli. In biopsy 3, taken from anofl3er llltlscle, the same abnormalities ,acre f, rescnt tl:ig. Ib, d,L In H & E stained .~ctions no bh.ish, basophilic tibres ~ere observed.

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bnmunohistochemk'al ohservations (Figs 2 and 3) Monoclonal and polyclonal antibodies to desmin, vimentin, laminin and type IV collagen were used to characterize abnormalities of the cytoskeleton and the basement membrane in biopsy 3. In numerous fibres desmin organization was disturbed (Fig. 2b-o), rest, lting in either protein accumulations in the peripheral region of the fibres (Fig. 2i), or in irregularly organized aggregates surrounding the nuclci (Fig. 2 b g ) , irregular punctate reactivity in the myoplasm (Fig. 2h,k -m) or a circumscript reactivity bctwcen the nuclei (Fig. 2j). Vimentin, normally not present in mature muscle libres but only in the interstitial tissue (Fig. 3a), was detected as cytoplasmatic aggregates in numerous diseased muscle fibres (Fig. 3b). Staining by antibodies to laminin (Fig. 3c.d) and type IV collagen (Fig. 3e,f) was more intense

than in the control biopsy: each fibre was completely surrounded by a thickened layer of laminin (Fig. 3d) and type IV collagen (Fig. 3f). Blood vessel walls also showed increased rcactivity with the laminin and type IV collagen antibodies while the proliferated endomysial connective tissue showed no reactivity. These patterns were comparable with those obtained with peroxidase conjugated secondary antibodies, and wcre neither observed when incubation with the primary antibodics was omitted, nor in control mr, sole. In order to identif)' possible regenerating tibres we used two antibodies to embryonic myosin. No staining ~vas observed in any of the muscle tibres of our patient, including those with disturbed desmin distribution, with either antibody (Fig. 4a,b), while in a control patient a large group of rcgcncrating fibres showcd obvious staining with

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F i g 4. Indirect immunofluorescence unicrographs of muscle tissue of the patient with C N M (a,b, double labelling) and of control muscle (c,d. serial sections), incubated with the polychmal (a) or nmnoclonal (c) dcsmin antibodies or with 330-R5B4, ,t monochmal ;lntibody to embryonic myosin (b,d). Bars indicate 60/till la,b) or IIX)/:m (c,d),

both antibodies to embryonic myosin as wcll as the antibodies to dcsmin (Fig. 4c,d). Uhrastructural oh,wrvathms (Figs 5 and 6) Apart from the presence ofcentral nuclei, most muscle tibres showed a regular :trrangcment of the contractile apparatus, tlowevcr, spoke-like organization of the myofibrils, with widened intermyolilamentous spaces wits observed its well as structural aberrzttions such as replicated triads, target tibres, Z-disk streaming, and other myolilamentous disorganizations (Fig. 5c,d). In several tibres membrane-bound depositions of electron-grey or -opaque material were observed between the filaments (not ilhtstrated). Pcrinuclear spaces, present in most of the tibrcs, were occupied by mitochondria, lysosomal structures, small lipofuscin-like granules, glycogen, Golgi complexes, intermediate tilaments (Fig. 5a,b) as well as tilamentous bodies (Fig. 5c}. A replicated basal lamina surrounding muscle fibres and blood vessels was also seen (Fig. 6b,c). D I S ( ' [ NSI()N

Centronuclcar tnyopathy (CNM) consists of several subtypes with different characteristics

[3,8,27,28], the most commonly observed of which arc centrally located nuclei in a signiticant number of muscle tibres and a type I tibrc predominance. Several authors [3,27-29] have suggested that CNM can bc subdivided into the following categories: (I) a neonatal type with autosomal recessive inheritance, ("myotubular myopathy") [3]: (2) a severe neonatal type with X-chromosome linked inheritance ("X-linked myotubular myopathy", XLMTM) [3]; and (3) an autosomal dominant or sporadic type, with onset in childhood or adult life ("centronuclear myopathy") [3]. Considering the time of onset, the negative family history and the anamnestic data, the patient described in this report probably belongs to type 3. There has been much speculation about the pathogenesis of the disease. The original assumption ofSpiro et al. [I] that libres with central nuclei were non-maturated foetal myotubes was questioned by several authors [2,8,9]. In contrast to normal foetal myotubes, which have prominent nuclei and small amounts of contractile elements, most fibres in CNM muscle contain rckttivclv large areas with fully differentiated sarcomcrcs. The presence of myotibrillar disruptions, atrophy-associated abnormalities and

Centronuclear Myopathy

217

Fig. 5. Alterations in the organization of the cytoskeleton as ob~rved by electron microscopy. (a) Centrally located nuclei (n) with an internuclear space occupied by numerous organelles. At the right regular arranged myolilaments and the sarcolemma with a fibroblastic extension. (b) Disarrangement of intermediate-sized filaments (inset higher magnification ), mitochondria (arrows) and a Golgi area (asterisk) between bundles of myofilaments. (c) Perinuclear area containing glycogen, filamentous bodies (arrowheads), and replication of triads (arrow,); n indicates the nucleus. (d) Irregular electrondense accumulations of cross-sectioned Z-band material. Bars indicate I ,urn (a,b,c) or I0 pm (inset, d). s u b s a r c o l c m m a l l y located nuclei are m o r e suggestive o f o t h e r p a t h o l o g i c a l processes r a t h e r than an arrest in foetal muscle d e v e l o p m e n t . In X L M T M , however, evidence is a c c u m u l a t i n g that fibres with central nuclei m a y indeed be nond e v e l o p c d foetal muscle fibres [ 16,30,31 ]. Few follow-up studies have been p e r f o r m e d with C N M patients. T h e cases that have been

d o c u m e n t e d so far showed no o b v i o u s histologic changes in consecutive biopsies [4] o r decreased n u m b e r s o f central nuclei [32]. O n e case with p a t h o l o g i c a l features o f b o t h C N M a n d multicore disease has been described that in some aspects resembles o u r case [33]. Unlike a muscle biopsy taken d u r i n g the first year o f life, biopsies taken several years later revealed the presence o f

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"C Fig. 6. Micrographs showing the extracellular matrix in musctc tissue of the patient with CNM. (a) Proliferation of interstitial tissue as seen in a I/.tm tolluidine-blue stained section of Epon 812 embedded tissue (biopsy 2). Inset: a small libre enclosed by endomysial tissue. (b) Basal lamina reduplication of a myofibre with granular deposits (of cellular defecation); note increased interstitial tissue (asterisk). (c) Part of endothelial cells (venous capillary) with reduplication of the basal lamina; asterisk indicates lumen. Bars indicate 25 ,urn (a), I0 l t m (inset) or I/~m (b,c).

internal nuclei in a large number of fibres. It seems to indicate that in this patient muscutar weakness preceded the translocation of nuclei, it has been proposed by Hfilsmann et al. [9] that postnatal, secondary migration of nuclei to a more central position may be due to an autolytic or autodegenerative process. We present a case in which a boy suffering from generalized muscle weakness was biopsied 9 yr after the first examination. During this period, a number of changes had occurred, both within and between the muscle fibres. Apart from an unusual variation in fibre size, muscle fibre

maturation appeared normal. Subsequently, fibres did not grow to normal sizes and nuclei migrated to the centre of the fibres. These observations point to a secondary migration of nuclei to a central position in this case of CNM. Furthermore, the presence of hypertrophic Iibres in biopsy I and the presence of atrophic fibres in biopsies 2 and 3 indicate a delay or even an arrest in muscle fibre growth. This abnormal development is associated with pathological changes, such as the appearance of fibres with large wtcuoles, or radial or spoke-like structures, and a proliferation of endomysial tissue with

Centronuclear

excessive adipose tissue replacing muscle fibres. Several authors have proposed that proliferation of connective tissue may play a role in pathogenesis of various myopathies [18,19, 29]. Presumably, proliferated endomysial tissue encloses muscle fibres in such a way that growth in width, and possibly also in length, is hindered. In combination with the presence of a reduplicated sarcolemma, endomysial tissue proliferation may interfere with the contact of the fibres with anchoring structures to the supporting tissue [34], and consequently also with the co-ordinated contraction of muscle fibres and force-transmission. It has been suggested that in muscular dystrophic mice, the presence of increased connective tissue and an abnormal sarcolemma caused growth impairment and maturational defects in pathologic muscle fibres [35]. Our studies showed a distinct and even increase in laminin and type IV collagen immunoreactivity surrounding all muscle fibres (Fig. 3d,f). This is most likely to be related to the thickening and reduplication of the basal laminae around all muscle fibres and blood vessels (Fig. 6b,c). We are aware of one other immunohistochemical study of a CNM muscle biopsy [19], using anti-type IV collagen and laminin antibodies, in this case no significant differences were found when compared with control tissue. Irregular, focally increased immunoreactivity for laminin and type IV collagen is found in muscular dystrophies and around individual atrophic fibres in a variety of other neuromuscular disorders [17]. However, the regularity in the increase of endomysial type IV collagen and laminin throughout the muscular tissue, in the absence of extensive fibrosis of the perimysium, seems rather exceptional [17]. In this context it seems worthwhile to note that the desmin intermediate filament organization is severely disturbed within a high percentage of muscle fibres. We consider it highly unlikely that the aberrant desmin patterns are related to an immaturity of the fibres or are the result of regenerative processes. In a variety of myopathies, the desrain staining pattern is intense and diffusely distributed in immature and regenerating muscle fibres [36,37]. Also arguing against muscle fibre regeneration is the absence of basophilic fibres and large, vesicular nuclei with prominent nucleoli. To further exclude the possibility of fibre regeneration in this patient, we used antibodies specific for embryonic myosin. These

Myopathy

219

antibodies reveal distinctly positive fibres in normal foetal or neonatal human skeletal muscle tissue [26], stain regenerating fibres (Fig. 4d), but showed no positive reaction at all in our patient (Fig. 4b). Focal accumulation of intermediate-sized filaments in muscular disorders is often considered a rather non-specific abnormality [12,13,38]. In our case however, we observed a generalized disturbance of the desmin organization. It has been demonstrated in various cell types, including muscle cells, that desmin filaments contact both the nuclear surface and the plasma membrane [14,39--41]. It is tempting to speculate that in our CNM case, a disturbance of the regulatory mechanisms that govern the organization of the desmin intermediate filaments may result in an abnormal localization of the nuclei. In addition, the occurrence of intracellular cytoskeletal abnormalities and the extracellular thickening ofendomysial structures may also be related. Under normal conditions vimentin is only present in immature or regenerating muscle fibres [16,41]. Therefore the presence of a punctate localization of vimentin immunoreactivity in underdeveloped, but clearly not regenerating, muscle fibres may be due to an abnormal regulation of intermediate filament gene-expression. In this respect it is interesting to refer to Sarnat's suggestion [16] that persistence of"foetal" desmin and vimentin filaments hinders nuclear migration to a subsarcolemmal position during maturation of muscle fibres in XLMTM. From the above it will be clear that further studies on abnormalities in the development of the myofibre cytoskeletal system and of extracellular matrix components are needed for a better understanding of the pathogenesis of the various forms of CNM. The different staining patterns obtained with antibodies to desmin and vimentin might be of diagnostic significance in discerning between XLMTM and the childhood onset type of CNM. Acknowledgements--We thank M. de Bruyn,M. Florie,and

M. Klein Rot for technicalassistance, Dr M. Brooke for our disposal, Drs A. Wessels, U. Wewerand W. Hancockfor donationof antibodies,and Dr D. lies for criticallyreadingthe manuscript.

putting tissue sections at

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P . F . '~V~. VAN DER VEN et al.

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I I.

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23. 24. 25.

26.

27. 28.

29.

30. 31.

32. 33. 34. 35. 36.

37.

38. 39.

40.

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42.

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