64444ournal ofNeurology, Neurosurgery, and Psychiatry 1993;56:644-648

Familial desminopathy: myopathy with J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.56.6.644 on 1 June 1993. Downloaded from accumulation of -type intermediate filaments

Jiri Vajsar, Laurence E Becker, Robert M Freedom, E Gordon Murphy

Abstract lings who presented with cardiomyopathy and Two siblings developed cardiomyopathy clinically silent myopathy.7 During the follow- several years before slowly progressive ing years, however, they have both developed muscle weakness. Skeletal muscle biopsy slowly progressive muscle weakness. In this specimens showed subsarcolenumal cres- paper, we characterise histochemically, cents of dark eosinophilic material in immunohistochemically, and ultrastructurally both type I and type II fibres. Immuno- the increased number of deposits of subsar- histochemically the subsarcolemmal colemmal material found in repeated muscle material stained positively for the inter- biopsy specimens. mediate filament protein desmin and for the heat shock protein ubiquitin but for no other cytoskeletal proteins. Ultra- Patients and methods structurally the subsarcolemmal deposits PATENTS consisted of aggregates of granular and Patient I filamentous material arising from Z- A five year old girl whose parents are second bands. Follow up muscle biopsies six cousins presented with excessive tiredness, years later showed an increased number effort intolerance, and epigastric pain. of the muscle fibres that contained sub- Examination revealed gallop rhythm, sarcolemmal aggregates that stained enlarged left atrium, and deep T-wave inver- positively for desmin and ubiquitin. sion on ECG in the left precordial leads. These clinical and pathological features Cardiac catheterisation at six years showed characterise a rare familial myopathy elevated left ventricular end diastolic pressure associated with an unusual distribution and pulmonary artery wedge pressure consis- of desmin proteins tent with the reduced compliance of her left in skeletal and probably also cardiac ventricle. There was also evidence of thicken- muscle. ing of the left ventricular wall. Although she was not complaining of muscle weakness, an (7 Neurol Neurosurg Psychiatry 1993;56:644-648) EMG performed at the age of 11 showed myopathic changes in the gastrocnemius

muscle. Results of tests for nerve conduction, http://jnnp.bmj.com/ Intermediate filaments represent one of the creatine kinase, lactate dehydrogenase, and three main cytoskeletal protein filament sys- concentrations of total and free carnitine in tems of most vertebrate cells. The filaments, serum were all normal. She then had an open which are 10 to 15 nm wide, can be divided muscle biopsy from the triceps muscle. By the into four distinct sequence types, numbered I age of 17 she had developed muscle weak- to IV.' Acidic keratins are type I filaments; ness. Examination showed generally reduced neutral-basic keratins, type II; desmin, muscle bulk and diminished deep tendon vimentin, and glial fibrillary acidic protein, reflexes. Muscle strength was decreased to on September 23, 2021 by guest. Protected copyright. type III; and neurofilaments, type IV. Desmin four fifths in her arms and legs; proximal and vimentin represent the main components muscle groups were slightly weaker. She had The Hospital for Sick of intermediate filaments in skeletal and an incomplete Gowers' sign and tired easily Children, and the smooth muscle cells. Immunoelectron when running. The results of her mental University ofToronto, microscopy has shown that desmin surrounds examination were normal. Toronto, Ontario, Electrocardio- Canada the myofibrils at Z-disc levels and anchors the graphic examination showed left ventricular Division ofNeurology myofibrils in the transverse register as well as hypertrophy with strain. The results of J Vajsar to the sarcolemma." The network of inter- repeated measurements of creatine kinase in E G Murphy mediate filaments connects the plasma mem- serum and of forearm lactic ischaemic tests Division of brane skeleton with the nuclear lamina or were normal. A second biopsy of the triceps Neuropathology karyoskeleton. muscle was performed. L E Becker Abnormal expression of desmin has been Division of Cardiology R M Freedom seen in tumours derived from smooth and Patient 2 skeletal muscle,4 muscle Correspondence to: regenerating fibres,5 The first patient's brother developed transient Dr Vajsar, Division of and congenital and late onset myopathies congestive heart failure at two years. Neurology, 555 University with or Avenue, Toronto, Ontario, without cardiomyopathy.6 Cardio- Examination revealed palpable gallop and Canada M5G 1X8. myopathy may develop during the course of quadruple rhythm with third and fourth heart Received 25 June 1992 the disease but has not been reported as a sounds. ECG showed decreased contractility and in revised form 14 September 1992 presenting feature. of the left ventricle and diffuse S-T depres- Accepted 28 September 1992 We have previously reported on two sib- sion and T-wave inversion in the left precor- Familial desminopathy: myopathy with accumulation ofdesmin-type intermediatefilaments 645

Figure I Muscle biopsy obtained for electron microscopy. J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.56.6.644 on 1 June 1993. Downloaded from specimen shownng subsarcolemmal deposits of Limited muscle biochemistry showed nor- dark eosinophilic material A~~~~~~A mal activity of the following glycolytic in most musclefibres fi; ;~~~.K~~~~~~~~~~~;e ... enzymes: acid maltase, debranching enzyme, (HE, x 800). phosphorylase, phosphofructokinase, phos-

X...... phoglycerate kinase, and phosphoglycerate *; ~~~~~~~~~~~~~~~~~~~~~~~~~~.. mutase. ,~~~~~~~W, PATHOLOGICAL RESULTS Light microscopy, histochemistry, and immunohistochemistry Mild variation in fibre size and a few atrophic fibres were seen on cross and longitudinal sections in both patients. No fibre degenera- tion or regeneration, however, was present in any of the four specimens. There was dial leads. This boy, bor at termn, had devel- increased internalisation of nuclei and mild oped hypoxic-ischaemnic encephalopathy focal endomysial fibrosis. The most striking because of meconium aspiration at birth. finding was the presence of deposits of dark This was followed by a seizure disorder and eosinophilic subsarcolemmal material (fig 1). global developmental delay: he walked at the The subsarcolemmal deposits showed identi- age of 3 1/2 years and spoke only single words cal positive staining in all four specimens at the age of 6. Electromyography yielded (table). On ATPase, the material was seen in normal results and his serum creatine kinase both type I and II fibres. concentration was normal at the age of 5 The histogram showed that 75% (patient when he underwent a biopsy of the triceps 1) and 70% (patient 2) of fibres were type I. muscle. By the age of 11 he had difficulty ris- The average diameter of type I fibres in ing from the floor. Examination indicated he patient 1 increased over six years from 32 to was mentally retarded. Muscle bulk was 45 p and the average diameter of type II reduced and muscle strength was four fifthis fibres from 43 to 56,u. In patient 2 the size of diffusely but slightly better distally. Deep ten- type I fibres increased over six years from 21 don reflexes were brisk and a partial Gowers' to 36 ,u, whereas the size of type II fibres sign was present. Electrocardiographic exami- decreased from 25 to 21 p. nation showed left ventricular hypertrophy When the immunoperoxidase technique with strain. Serum creatine kinase concentra- with antisera to myosin, ubiquitin, vimentin, tion was again normnal and another biopsy of desmin, a actinin, actin, keratin, and glial fib- the triceps muscle was performed. rillary acidic protein was performed, crescents of subsarcolemmal material stained positive MUSCLE BIOPSIES only with antisera to desmin (fig 2a, 2b) and Two open biopsies from the right triceps ubiquitin (fig 3). The muscle fibres contain- muscle for each child were performed 6 years ing material positive for desmin were calculat- apart. Serial sections embedded in paraffi ed in three different microscopic fields (50 http://jnnp.bmj.com/ and frozen were prepared for conventional fibres/field). In patient 1 the deposits were histological, histochemical, and immunohis- initially present in an average of 22% of tochemical examinations (table). Dewaxed muscle fibres, which increased to 68% in the and rehydrated paraffin sections were stained second biopsy from the same muscle six years with commercially available antibodies later. In patient 2 the material was present (Dakopatts, Denmark) to localise desmin and initially in 14% and later in 66% of muscle

ubiquitin immunoreactivity. Semi-thin sec- fibres. on September 23, 2021 by guest. Protected copyright. tion fixed with osmium and stained with tolu- idine blue were examined and thin sections ELECTRON MICROSCOPY The findings on electron microscopy were similar for both children. There were focal Table Results of staining subsarcolemmal deposits in all subsarcolemmal areas of muscle fibres that four biopsy specimens in two siblings with cardiomyopathy andprogresssive muscle weakness showed aggregates of granular and filamen- tous material (fig 4, fig 5a, 5b). The aggre- Stain Staining or reactiviy gates varied in size and occasionally could be Gomori's modified trichrome Dark green discerned arising and streaming from Z- Hematoxylin and eosin Dark eosinophillic Pyronin Negative bands. Where the material was densely Periodic acid-Schiff Positive packed filaments could not be distinguished. Phosphorylase Negative Acid phosphatase Positive Areas of extensive aggregates showed mild Menadione nitroblue tetrazolumNeative accumulation of glycogen. Both the I-bands Succinic dehydrogen Negative and Z-bands were distorted in areas Lactic dehydrogenase Nepative adjacent Oil red 0 Negative to the electron-dense aggregates. Otherwise Congo red Negative Cytochrome oxidase Negative the myofibrillary alignment was normal. In Desmin Dark brown addition, there was extensive accumulation of Myosin Negative mitochondria in the beneath the a Actini Negative sarcoplasm Actin Negative electron dense aggregates. The mitochondria Vimentin Negative were mildly pleomorphic; some were elongat- Ubiquitin Positive ed or branched. Contiguous to the subsar- 646 Vajsar, Becker, Freedom, Murphy J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.56.6.644 on 1 June 1993. Downloaded from .k. s*%- ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.. AIL *...... ,

4W-S < ,; :.\ <.: .; \ . X .: .. -;.... A~~~~~~~~A i;N'F 4 ' ' t ts §t '' Figure 4 Electron micrograph oftriceps muscle showing / subsarcolemmal deposit in which aggregates seem to be arising and streamingfrom Z-bands (x 11 600).

.

Discussion We have described the unique clinical and pathological findings in two siblings who pre- IL sented with cardiomyopathy several years before they developed slowly progressive muscle weakness. Although initially there was no clinical evidence of skeletal myopathy, NINDwh biopsy specimens from the triceps muscles showed crescents of subsarcolemmal material in 22% of muscle fibres in patient 1 and in 14% in patient 2; six years later, when muscle weakness had developed, this material increased to 68% and 66%, respectively. The subsarcolemmal material in all four speci- mens stained positively for the intermediate filament protein desmin and the heat shock protein ubiquitin. Electron microscopy showed that the subsarcolemmal crescents contained aggregates of granular and filamen- Nk tous material. The aggregates streamed from Z-bands. Patients with non-progressive or slightly B progressive myopathies may have various http://jnnp.bmj.com/ Figure 2 Sections oftriceps muscle ofpatient 2 showing subsarcolemmal crescents of morphological changes in the muscle fibres. deposits ofdesmin-positive material with immunoperoxidase stain. (a) Transverse section In some myopathies the morphological x 320, (b) longitudinal x 400. abnormality may be characterised by an accu- mulation of abnormal material seen in various parts of the with both light and . electron microscopy.7 10 The material is often colemmal aggregates were sparse myelin fig electron dense at the ultrastructural level and ures ai and vacuolar structures with n consists of granules and intermediate fila- on September 23, 2021 by guest. Protected copyright. increased number of capillaries wit]h ments approximately 10 nm node. In the 15 increased pinocytic vesicles. case reports presented by Pellissier et al the electron dense material was immunoreactive for desmin and in one case the material was closely related to Z-bands.6 In our two patients the material positive for desmin was found in subsarcolemmal areas and also appeared related to Z-bands. Most of the cases reported by Pellissier et al, as well as ours, were familial, with age of onset between 2 and 48 years.6 All three of 111 their cases involving children and some of their adult patients developed cardiomyo- pathy after the onset of muscle weakness. Two children, aged 11 and 13 years, died of heart failure.10 On the other hand, our patients presented with cardiac symptoms early in life, long before their symptoms of Figure 3 Subsarcolemmal deposits showingpositive muscle weakness occurred. They predomi- immunoreactivity to ubiquitin with immunoperoxidase nantly experienced proximal muscle weakness stain, (x 1000). that was slowly progressive over the years. Familial desminopathy: myopathy with accumulation ofdesmin-type intermediatefilaments 647

Samat found very strong immunoreactivity J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.56.6.644 on 1 June 1993. Downloaded from for desmin and vimentin in the myofibres of neonates with myotubular myopathy."2 He suggested that the high level of expression of intermediate filament proteins suggests the arrest of morphogenesis in the developing muscle fibres. In some myopathies and in the strained muscle of elderly people, increased S.l At W; W~~~~~AIRW E s*4 ,3 w stainability for desmin in areas of Z-disc :S~~~~V ; :A', ...... streaming also occurs, suggesting that inter- mediate filaments are related to sarcomeroge- '.; t 1 9 b s % .4 S W :80, :. ik .. . .. nesis or repair of disrupted sarcomeres.3 During normal myogenesis in avian and mammalian muscle, desmin intermediate fila- ment proteins organise from a longitudinal to

A~~~~~~ a transverse circumferential network around Z-discs. When Tassin et al treated normal mice myotubes with tetrodotoxin, which pre- vents contractile activity of myotubes by blocking the voltage-dependent sodium ion .. . 4 channels, the treated myotubes displayed o .v aggregates of desmin intermediate filament

o -: e X .U 9 #. : .ff. .tB f#p^ S XN protein. 14 Also the normal organisation of * ...... i. S

.... desmin from a longitudinal to a transverse network was prevented. Both of these results *, £ .; ,. ;.h.vl ,c S ..0r-. were reversible: when the treatment with vg * B .'; ...... ai...... ,.;:S .. tetrodotoxin was stopped the desmin aggre- "ti., ::. gates disappeared and the normal organisa- ..... tion of the desmin network followed. These * y.0 ,. .2C0v,< , a } Y z., results suggested that aggregates may reflect a modification of binding desmin with the plas- :|R ma membrane in the presence of an abnormal

: ...... agent...... :. ....:.> : :...... : ' . :rzi;t Abnormal reorganisation of desmin inter- --! .9. :, 4 H::: mediate filaments caused by an unknown deleterious process is supported by another C'' s...... ;S...... d U* f. ..:.: A variety of cellular stresses including .. . .:.. - finding...... viral infection, toxicological injury, and ,. ....,... :P .: .. ...fM > experimental heat shock have been suggested :.e. '"';.. f:.... : < :e . t::.. B .. :iN:'.¢.: as the cause of collapse of intermediate fila- Figure 5 (a) Low magnification electron micrograph showing aggregates of subsarcolemmal granular andfilamentous material (x 18 870). (b) High magnification ments with ubiquitination in neurons, astro- *..:. ' .,.:: . http://jnnp.bmj.com/ of abnormal material showing mainlyfilaments andfew granules (x 68 000). cytes, and liver and muscle cells.'5 The . .. :. : ubiquitin and desmin-positive aggregates in our patients may thus represent a reorganised Most patients reported also have slowly pro- and collapsed network of desmin intermedi- gressive..muscle .. weakness,:.. mostly:.:proximal, ate filaments. but few have diffuse..weakness: ..:::with predomi- Thus increased immunoreactivity to nantly distal involvement." In our patients desmin seems to be a non-specific feature

the progressive natureNof Za.Zthe ; myopathy was indicating perturbation in both the develop- on September 23, 2021 by guest. Protected copyright. supported by the findings of repeat biopsies. ing and mature muscle fibres. The cause of In the second biopsy from the same muscle, the progressive accumulation of aggregates there were more fibres displaying accumula- positive for desmin, however, seen in muscle tion of material positive for desmin in a fibres of our patients and patients reported characteristic subsarcolemmal distribution. elsewhere, remains unclear. In addition, it is Desmin, a polypeptide consisting of non- not clear why the heart muscle in our patients helical N-terminal and C-terminal ends and was initially affected, a feature rather different an a helical middle domain, has a molecular from the other reported cases of myopathies weight of 52 000 d and consists of about 300 with desmin accumulation. Progressive disor- amino acids. Monoclonal or polyclonal anti- ganisation of desmin intermediate filaments bodies against desmin may have specificity in mature muscle fibres in response to delete- against any portion of the desmin molecule, rious stimuli with higher affinity for the car- which has about 30% to 70% of amino acids diac muscle is one possibility. Another may in common with other intermediate be the existence of a genetic predisposition filaments.4 During early development the pri- for excessive production or incorrect turnover mary generation of myotubes expresses both of desmin. A recent study localised the gene desmin and vimentin. Both are also expressed encoding the synthesis of desmin on chromo- in the early stages of activated satellite cells some 2q35,'6 but so far the isolated desmin seen in regenerating muscle.25i2 With matu- probe has not been used in clinical practice. ration in both myotubes and satellite cells, Finally, the fundamental issue of whether pri- vimentin is lost, whereas desmin persists. mary desmin accumulation may result in a 648 Vajsar, Becker, Freedom, Murphy

myopathy or whether it is a secondary result AT. Skeletal muscle in idiopathic cardiomyopathy. J J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.56.6.644 on 1 June 1993. Downloaded from Neurol Sci 1972;15:303-20. of progressive changes in the muscle caused 9 Fardeau M, Godet-Guillain J, Tome FMS, et al. Une by some other undetermined factor needs to nouvelle affection musculaire familiale, definie par l'accumulation intra-sarco-plasmique d'un materiel be resolved. granulo-filamentaire dense en microscopie electronique. Rev Neurol (Paris) 1978;134:411-25. 10 Goebel HH, Lenard HG, Langenbeck U, Mehl B. A form This manuscript was prepared with the assistance of medical of congenital muscular dystrophy. Brain Dev 1980;2: publications, The Hospital for Sick Children, Toronto, 387-400. Ontario. 11 Edstrom L, Thornell L-E, Eriksson A. A new type of hereditary distal myopathy with characteristic sarcoplas- mic bodies and intermediate (skeletin) filaments. Jf Neurol Sci 1980;47:171-90. 1 Steinert PM, Roop DR. Molecular and cellular biology of 12 Sarnat HB. Myotubular myopathy: arrest of morphogene- intermediate filaments. Annual Review Biochemistry sis of myofibres associated with persistence of fetal 1988;57:593-625. vimentin and desmin. Four cases compared with fetal 2 Thornell L-E. Immunohistochemistry of intermediate fila- and neonatal muscle. CanJ Neurol Sci 1990;17:109-23. ments [abstract]. JNeurol Sci 1990;98 (Suppl):58. 13 Jakobsson F, Borg K, Edstrom L. Fibre-type composition, 3 Granger BL, Lazarides E. The existence of an insoluble Z structure and cytoskeletal protein location of fibres in disc scaffold in chicken skeletal muscle. 1978; anterior tibial muscle. Comparison between young 15:1253-68. adults and physically active aged humans. Acta 4 Truong LD, Rangdaeng S, Cagle P, Ro JY, Hawkins H, Neuropathol (Berl) 1990;80:459-68. Font RL. The diagnostic utility of desmin. A study of 14 Tassin AM, Pinion-Raymond M, Paulin D, Rieger F. 584 cases and review of the literature. Am J Clin Pathol Unusual organization of desmin intermediate filaments 1990;93:305-14. in muscular dysgenesis and TTX-treated myotubes. Dev 5 Bornemann A, Schmalbruch H. The intracellular distribu- Biol 1988;129:37-47. tion of desmin and vimentin in regenerating rat muscles 15 Lowe J, Blanchard A, Morrell K, et al. Ubiquitin is a com- [abstract]. J Neurol Sci 1990;98:(Suppl):294. mon factor in intermediate filament inclusion bodies of 6 Pellissier JF, Pouget J, Charpin C, Figarella D. Myopathy diverse type in man, including those of Parkinson's dis- associated with desmin type intermediate filaments. An ease, Pick's disease, and Alzheimer's disease, as well as immunoelectron microscopic study. J Neurol Sci 1989; Rosenthal fibres in cerebellar astrocytoma, cytoplasmic 89:49-61. bodies in muscle, and Mallory bodies in alcoholic liver 7 Calderon A, Becker LE, Murphy GE. Subsarcolemmal disease. JPathol 1988;155:9-15. vermiform deposits in skeletal muscle, associated with 16 Viegas-Pequignot E, Li ZL, Dutrillaux B, Apiou F, Paulin familial cardiomyopathy: report of two cases of a new D. Assignment of human desmin gene to band 2q35 by entity. PediatrNeurosci 1987;13:108-12. nonradioactive in situ hybridization. Hum Genet 1989; 8 Shafiq SA, Sande MA, Carruthers RR, Killip T, Milhorat 83:33-6. http://jnnp.bmj.com/ on September 23, 2021 by guest. Protected copyright.