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Myopathy with respiratory failure and typical myofibrillar lesions
Journal of the Neurological Sciences, 1990, 96:211-228
211
Elsevier
JNS 03318
Myopathy with respiratory failure and typical myofibrillar lesions Lars EdstrOm, Lars-Eric Thornell, Jaan Albo, Sven Landin and Margareta Samuelsson Departments of Neurology, Karolinska Hospital and Danderyd Hospital, Stockholm (Sweden), Department of Anatomy, Umed University and Department of Neurology, Orebro Hospital, Une~ (Sweden) (Received 11 October, 1989) (Revised, received 28 December, 1989) (Accepted 28 December, 1989)
SUMMARY
16 patients representing 7 different pedigrees exhibited an unusual, adult onset limb-girdle myopathy with typical clinical hallmarks. In a majority of cases there was evidence of an autosomal dominant inheritance. A prominent early finding in all cases was respiratory muscle weakness, and in many of these an acute respiratory incapacity was the reason for the In'st neurological examination. Neck flexor and sometimes foot extensor weakness were other early symptoms. The clinical picture seems to be at variance with that of the more well known hereditary myopathies. Electrophysiological analysis confirmed a myopathy and serum muscle enzyme concentrations were normal or slightly elevated. Muscle biopsy findings revealed myofibrillar changes which, at the light microscopy level, included plaques that stained strongly with rhodamineconjugated phalloidin, a specific marker for F-actin. At the ultrastructural level, these plaques were observed to be composed of moderately dense, thin filaments and were related to splitting of Z-discs or formed extensions from Z-discs. We believe that the muscle biopsy changes revealed by cytochemical and ultrastructural observations indicate defective myofibrillogenesis, and the possibility of defective actin polymerization is discussed. A conclusive answer requires further immunocytochemical and immunoelectrophoretic studies and possibly the application of molecular genetics.
Correspondence to: Dr. Lars Edstr6m, Department of Neurology, Karolinska Hospital, S-104 01 Stockholm, Sweden. 0022-510X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
212 Key words: Myopathy; Muscle dystrophy; Respiratory failure; Respiratory muscles; Sarcomere; Z-disc; Actin; Rhodamine-phalloidin; Desmin
INTRODUCTION Serious respiratory failure as a prominent early symptom of a hereditary myopathy is uncommon, although many respiratory problems commonly accompany more advanced stages of muscle dystrophies, such as Duchennes disease and myotonic dystrophy. Disorders with early respiratory failure include late onset Pompes disease (acid maltase deficiency), in which one-third of the cases involve early respiratory failure (Rosenow and Engel 1978) and respiratory involvement may occur in all cases and is often a cause of death. In nemaline myopathies (Kuitonnen et al. 1972; Dubowitz 1978), fatal progressive respiratory failure is also frequently encountered in the first and second decade of life. Among the group of disorders exhibiting muscle fiber inclusions similar to the cytoplasmic bodies of Engel (1962), occasional cases with early respiratory failure are reported (Jerusalem et al. 1979; Patel et al. 1983; Banker 1986; Winter et al. 1986). The present paper reports 16 cases representing 7 families who exhibit a limbgirdle syndrome with neck flexor weakness and a prominent involvement of respiratory muscles. The disease may typically be inherited as an autosomal dominant trait. The muscle biopsy findings included various myopathic changes, and characteristic myofibrillar changes were found in all biopsies. At the light microscopy level, there were plaques of material in many fibres that exhibited strong fluorescence of rhodamine-conjugated phaUoidin, which selectively binds to F-actin. We conclude that all these cases represent one and the same disorder. A preliminary report has been published elsewhere (EdstrOm and Thornell 1986).
MATERIALSAND METHODS Most biopsies were taken from the vastus lateralis of the quadriceps (VL) or the anterior tibial muscle (TA), but occasionally from the deltoid muscle (cf. Table 1). Most biopsies were obtained by means of a percutaneous modified Radner technique (Henriksson 1979), but some using open surgery.
Methodsfor specimenpreparation Specimens intended for ordinary light and fluorescence microscopy were frozen in Freon 13 cooled by liquid nitrogen (at - 190 °C) and kept in a refrigerator (at 75 °C) until 4-10 #m thick sections were cut in a cryostat at - 25 °C. Specimens intended for transmission electron microscopy (TEM) were pinned between needles on a piece of cork and fixed in 2.5~o gluthar aldehyde in phosphate buffer. Proceduresfor light microscopy. Stainings for hematoxylin-eosin and trichrome modified according to Engel and Cunningham (1963) were employed as well as histo-
213 chemical stainings for myosin-adenosine triphosphatase (myosin-ATPase) with alkaline and acid preincubations, NADH-TR, succinic dehydrogenase, fat, glycogen and acid phosphatase (cf. Dubowitz 1985). For visualization of F-actin in fluorescence microscopy, we used rhodamineconjugated phalloidin, which binds selectively to polymerized actin filaments (Molecular Probes Inc., Junction City, Oregon, U.S.A.). For visualization of the intermediate filament subunit desmin, we used a well characterized monoclonal antibody whose specificity has been described previously (Virtanen et al. 1986). The antibody was identified under fluorescence microscopy after labelling with a secondary FITC-conjugated rabbit anti-mouse serum. Procedures for transmission electron microscopy. After fLxation,the samples were washed in buffer and cut into blocks approximately 2 x 0.5 ram, postfixed in OsO 4 in PBS, dehydrated in acetone and embedded in Vestopal or Epon. 1.0-0.5-#m thick sections were cut on an LKB ultratome IV and stained with toluidine blue for light microscopy. After identification of areas of interest, ultrathin sections were produced and stained with uranyl acetate and lead citrate and examined in a Philips 300 or a JEOL 1200EX electron microscope.
RESULTS
Clinical hallmarks A summary of clinical and laboratory findings is presented in Table 1. Pedigree maps of three families presenting several members with typical symptoms are displayed in Fig. la-c. All cases were characterized by a proximal muscle weakness of the upper and lower extremities, with early affection of neck flexors and respiratory muscles, especially the diaphragm. There was sometimes an early involvement of dorsal foot extensors. Other distal muscles were spared for the most part but fmger extensors were involved in 4 cases. Cranial nerve innervated muscles were mostly spared, except for the sternocleidomastoid. In one patient there was, however, a bilateral weakness of the eye bulb abductors since early childhood and in one patient a slight myopathic face was observed. Muscle reflexes were normal or decreased but never totally inhibited. Time of onset varied from between the second and fifth decade. It must, however, be emphasized that the time of onset of some minor neuromuscular deficits is very difficult to establish. A majority of the patients admitted weakness of neck flexor muscles since early childhood on specific inquiry. The course of the disease seemed to be slowly progressive. No affection of the central nervous system or the sensory peripheral pathways has been observed, and no clear-cut clinical signs of cardiomyopathy were found.
Laboratory findings Electromyography (EMG) revealed a myopathy in all cases, but in one patient occasional muscles exhibited a mixture of neurogenic and myogenic changes. Peripheral
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IV Fig. 1. a-c. Preliminary pedigree maps of family 1-3, indicated PI-P3 in Table 1. In (b) there are 5 more siblings in generation II who are said to be unaffected which we have not been allowed to establish. Two affected sons of a first cousin of members of generation II are included in the study (case 8 and 9 in Table 1) but as we know too little about other individuals in this branch of the family they are not indicated in the map.
216
Fig. 2. a-c. Cryostat serial cross sections of muscle fibres exhibiting myopathic changes typical of this disorder. In (a), stained with rhodamine-phalloidin for detection of F-actin, there are numerous plaques of high fluorescence. In (b), trichrome stained, the same areas are visible as dark spots. In (c) stained for acid phosphatase it can be seen that the large fibres, filled with rhodamine fluorescent plaques, also have an elevated content of acid phosphatase activity, indicating increased autophagy. A cross in a fibre observed in all three sections facilitates comparison. 200 x.
217 nerve conduction velocity (NCV) was normal in all cases investigated. In a few cases reliable values were not obtained. There was little or no increase in serum muscle enzymes.
Muscle biopsy observations All the muscle biopsies exhibited typical plaques as described below and the frequency of fibres with such typical changes varied between 5 and 30~o in individual biopsies. Both type I and II fibres were involved. Light microscopy findings. The differentiation of muscle fibres into type I and type II with subtypes (Brooke and Kaiser 1970) was well preserved. There were no signs of type-grouping or abnormal fiber type dominance, but muscle fiber splitting and small groups of atrophic fibres were commonly found. In stainings for oxidative enzymes, targetoid and moth-eaten fibres were seen. Thus there were commonly irregularities of stainability for succinic dehydrogenase and NADH-TR but no typical ragged-red fibres or large areas of focal loss of enzyme activity. Typically, the number of muscle fibres exhibiting pathological changes varied considerably between different fascicles within one and the same biopsy. Several fascicles with almost normal muscle fibres were found to alternate with fascicles with many changes in large groups of fibres. Typical findings in affected fascicles revealed at the light microscopy level are illustrated in Fig. 2. Muscle fibres were of pronounced size difference, exhibiting slightly irregular fiber structure, and often contained smooth, opaque plaques of reddish or dark green color when stained for trichrome as seen in Fig. 2b. They were highly eosinophilic in hematoxyline-eosin (not illustrated). In crosssections produced in series with those stained for trichrome and labelled with rhodamine phalloidin (Fig. 2a), the plaques were highly fluorescent. In the fibres exhibiting fluorescence, there was an increased stainability for acid phosphatase as well in most cases (Fig. 2c). In longitudinal sections we observed that the plaques with high rhodamine fluorescence were focal (Fig. 3). Patches and streaks of high fluorescence with longitudinal and/or transverse orientation were seen (Fig. 3). By comparing sections cut in series and stained for rhodamine-phaUoidin and desmin, respectively, we observed that desmin was absent in the lesions of high rhodamine fluorescence (Fig. 3a, b). In longitudinal sections, however, we observed that the normal transverse striated pattern of desmin staining seen in unaffected areas of muscle fibres was often broken up in the areas of high phalloidin fluorescence (Fig. 3c, d, e, f). In addition, minor focal longitudinal strands of high desmin staining were observed in otherwise normal looking transverse striated pattern (Fig. 4). The fact that even slightly myopathic muscles with a high percentage of normally structured muscle fibres always displayed rhodamine phalloidin fluorescent plaques in scattered fibres pointed to these structures as representing an early and possibly pathognomonic phenomenon. Muscle fibre ultrastructure. In all muscle biopsies investigated, the most prominent pathological features of the muscle fibres were changes related to the myofibrils. In slightly affected fibres, some moderately electron dense material was seen within the myofibrils (Fig. 5a). Under higher magnification it was apparent that the material was
218
Fig. 3. a-f. Serial cross sections stained with rhodamine phalloidin in a and anti-desmin in b. Note complete lack of staining for desmin (b) in the plaques, which are highly fluorescent when stained with rhodaminephalloidin (a). Serial longitudinal sections are also stained with rhodamine-phalloidin (c) and anti-desmin (d). Note the focal appearance of high fluorescence in (c) and the longitudinal highly fluorescent streaks in (d). Parts of muscle fibres seen at high magnifications are stained with rhodamine phalloidin (e and f). Note the focal plaques and streaks of high fluorescence, a + b, 630x ; c + d, 300x ; e + f, 1500 x.
p r e s e n t b e t w e e n split Z - d i s c s a n d also f o r m e d s t r a n d s c o n n e c t e d to the Z - d i s c s (Fig. 5b). I n s o m e fibres there were areas with p r o n o u n c e d myofibrillar alterations a n d m o d e r a t e l y d e n s e f d a m e n t o u s m a s s e s (Figs. 6a, 7a). I n other fibres there were extensive dissolved m y o f i l a m e n t s a n d the fibres c o n t a i n e d long s t r a n d s or irregular collections o f
219
Fig. 4. a and b. Longitudinal sections stained with anti
moderately dense material (Fig. 6b). This material was never as dense as the Z-discs of normal myofibrils or as dense as enlarged Z-discs, Z-disc bodies or Z-rods, which were occasionally seen (Fig. 7). Irrespective of whether the moderately dense material occurred between Z-discs, formed short patches, strands of several sarcomeres in length or filamentous bodies, regular periodicities as in Z-rods were never seen. In thin sections allowing adequate resolution, thin filaments (4-6 nm) and amorphous material were resolved to be a main constituent of the aberrant myofibriUar material (Fig. 8). In between the aberrant myofibrils and the filamentous bodies, loosely arranged filaments, ribosomes and sarcotubules were seen. The filaments were of three classes: thin filaments (4-6 nm), intermediate filaments (7-11 nm), and thick filaments (12-16 nm). Mitochondria were sparse in the areas of dissoluted myofilaments. Other myopathic features included minicores and Z-disc streaming, autophagic vacuoles and myelin figures.
220
Fig. 5. a and b. Electron micrographs of a longitudinal ultrathin section. In (a) there is an area with an irregularity in the sarcomeric pattern, which is further magnified in (h). Note the streaks of medium-dense filamentous material related to the Z-discs and sometimes trapped between two Z-discs. An extraordinarily long streak is indicated by two arrows in both sections, a, 4400 x ; b, 17000 x .
221
Fig. 6. a and b. Electron micrographs of longitudinal ultrathin sections. Remnants of intact myofilaments (MF) in otherwise dissolved muscle fibres can be seen in (a). A filamentous m a s s is indicated by an arrow. Long strands of filamentous material (F) close to a nucleus (N) and an autolytic vacuole (V) can b¢ seen in (b). Two arrows point at dense Z-line material related to strands of medium dense filamentous material. 4000 x .
222
Fig. 7. a and b. Electron micrographs of longitudinal ultrathin sections. A magnification of the filamentous mass visible in Fig. 6a can be seen in (a). Note the content of thin filaments, irregularly arranged. The filamentous mass has an electron density that is lower than that of the Z-disc material indicated by arrows in (a) and (b). Medium-dense filamentous material encircling the dense Z-disc material can be seen in (b). a, 20 000 × ; b, 15 000 ×.
223
Fig. 8. a and b. Electron micrographs of longitudinal ultrathin sections. Streaks and patches of moderately dense material extend from Z-discs to Z-discs. Under higher magnification (b), the streaks seem to be composed of thin filaments (4-6 nm) and additional dense material. However, no apparent periodicity is seen. Note the difference in density between Z-discs, the aberrant lesions and the rest of the myofibrils. Note also that the thin filaments enter the myofibriUar lesions, a, 20000 x ; b, 75000 x.
224 DISCUSSION The similarities in clinical picture and muscle biopsy findings between these patients representing different pedigrees of Swedish origin strongly suggest that this is one and the same disorder. It is possible but still unproven that they might be descended from one and the same family. Does this disorder conform to any previously reported limb-girdle syndromes with characteristic muscle biopsy findings? A case reported by Jerusalem et al. (1979) and a case by Winter et al. (1986) had some similarities with ours, including early respiratory failure, but in the latter case the authors reported polydactylyand stiffness of the vertebral column resembling rigid spine syndrome. A mother and two of her teenaged children were reported by Patel et al. (1983) to be suffering from a limb-girdle syndrome with neck flexor weakness but also facial muscle involvement, which we observed only in one case. The children developed respiratory incapacity. In these three reports, the hematoxylin-eosin stainings revealed eosinophilic inclusions at the border of some muscle fibres, but the ultrastructural findings in all three reports indicated the presence of cytoplasmic bodies with the criteria given by Macdonald and Engel (1969), i.e. a spheroid structure with a dense core surrounded by a fdamentous radiating corona. This differs from our present observations of myofibrillar alterations including aggregates of thin filaments but not with an organization corresponding to the typical cytoplasmic bodies mentioned above. We have observed typical cytoplasmic bodies, e.g., in cases of polymyositis, but they did not stain specifically with rhodamine-phalloidin (unpublished observations). In nemaline myopathy there is a regular lattice pattern that characterizes the rod bodies and that is not found in the inclusions presented here (for review, see Fardeau 1982). The spheroid body myopathy of Goebel et al. (1978) had no clinical or histopathological similarity with the cases here. When the specificity of our present muscle biopsy observations is considered, the alterations of the myofibrils in relation to the Z-discs seem to be an early and typical change at the ultrastructural level. In more advanced stages of muscle fibre degeneration there were dissolved myofdaments, and strands of thin f'daments and of cytoplasmic bodies of moderate density were observed. These changes were observed at the light microscopy level as structures with strong binding of rhodamine phalloidin, and corresponding structural changes could be discerned already in routine stainings. There was undoubtedly an increased amount of actin in the fdamentous plaques related to the altered myoflbrils, as phalloidin selectively binds to F-actin (Haugland 1985). This agrees with the ultrastructural observations that thin filaments were resolved in both the filamentous masses and in the altered myofibrils. These f'daments were in continuity with Z-discs. However, they were of a lower density than the filaments of the Z-discs, but higher than those of the I-bands. Furthermore, although some amorphous material was seen to be attached to the filaments they did not show the typical structural pattern of the Z-discs or Z-rods (Goldstein et al. 1979, 1980, 1982; Yamaguchi et al. 1983, 1985). The main longitudinal component of the Z-disc is thought to be composed of actin
225 filaments, whereas the bridging structures are likely to be alpha-actinin (Isobe et al. 1988; Yamaguchi et al. 1983). However, recently a number of new proteins have been identified in the Z-disc, such as Z-protein (Muguruma et al. 1981), Zeugmatin (Maher et al. 1985), and Cap Z 36/32 (Casella et al. 1987). Their exact location is so far unknown. Aberrant I-bands have previously been observed in so-called double Z-discs in myofibrils of heart Purkinje fibres and suggested to be indicative of sarcomerogenesis (Legato 1970; ThorneU 1973). The strong rhodamine-phalloidin fluorescence, and the higher electron density, compared to the I-band, that characterizes the filamentous masses described here, might be explained by a higher density of actin fdaments than in the I-band or by an abnormal polymerization of actin. Clearly, the normal lengthdetermining mechanism for polymerization of actin to actin filaments seems to be out of order in the areas of abnormal myofilamental structure, as the filaments extending from one Z-disc to the next were sometimes several sarcorneres in length. This lengthdetermining mechanism is not yet fully understood, but several of the above mentioned proteins may be involved, e.g. alpha-actinin as well as beta-actinin (Furatsu et al. 1988). Other proteins related to the Z-disc and the sarcomere are nebulin and titin (connectin), two high molecular weight proteins which are thought to make up an elastic component of the myofibrils (Wang 1985; Maruyama 1986; Furst et al. 1988). There have been reports of a decrease of these proteins in muscles from patients with X-linked muscle dystrophies (Wood et al. 1987). In some case reports of cytoplasmic body myopathies (Nakashima et al. 1970; Kinoshita et al. 1975; Clark et al. 1978; Wolburg et al. 1982), it was suggested that this morphological phenomenon might be a specific marker of a basic defect of myof'dament biosynthesis. It is obvious from the present study that desmin, the subunit of muscle intermediate fdaments, was lacking within the plaques of high rhodamine fluorescence. This is in contrast to observations of disorders with cytoplasmic inclusions containing desmin (Edstr0m et al. 1980; Porte et al. 1980; Stoeckel et al. 1981; Osborn and Goebel 1983; Rappaport et al. 1988). Osborn and Goebel (1983) identified intermediate (desmin) filaments in the corona around the electron-dense core of cytoplasmic bodies in a case of congenital myopathy. We described a Swedish dominantly inherited myopathy that started in hand flexors and had a rapid progress, although respiratory muscles were never affected (Edstr0m et al. 1980). The muscle biopsy findings were characteristic (Edstr0m et al. 1980; EdstrOm and Wroblewski 1981). There were numerous sarcoplasmic dense bodies that varied in appearance. Some were composed of filamentous material and others were more electron-dense and completely or partly surrounded by a membrane. As we found an increased content of intermediate filaments we suggested that these bodies represented sequential changes in a degradation of desmin filaments. Thus, clinically and histopathologically, the disorder described earlier differs profoundly from that described in our present report. Here we observed, however, areas of myofibrils that contained streaks of desmin spanning over a few sarcomeres, similar to the areas of Z-streaming reported previously (Thornell et al. 1983) and regarded as an unspecific alteration.
226 When considering the possible basic defect responsible for the disorder, it must be born in mind that it should not only explain the occurrence of aggregates of actin filaments but also the inclination of the muscle cells to degenerate progressively. In recent years, changes in the sarcomere-associated cytoskeleton have attracted increasing interest in relation to the pathogenesis of muscle fibre degeneration. Defects of such organelles may be fatal to muscle fibres during contraction, and especially against high resistance. This concept is valid for, e.g., the intermediate filaments (EdstrOm et al. 1980; Thornell et al. 1983; Rappaport et al. 1988), the intrasarcomeric cytoskeletal filaments (Wood et al. 1987) and the plasma membrane related cytoskeletal protein dystrophin, which is lacking in Duchenne muscular dystrophy (Arahata et al. 1988; Watkins et al. 1988). It must however be emphasized that the connection between reduced amount of cytoskeletal proteins and the developement of muscle fibre degeneration is far from understood. The development of new immunohistochemical and immunoelectrophoretic techniques to analyse components of the sarcomere should further elucidate factors responsible for the etiology of the present hereditary myopathy and related disorders.
ACKNOWLEDGEMENTS This study was supported by grants from the Swedish Medical Research Council (projects 12X-3875 and 12X-3934), the Karolinska Institute and the University of Umeli. Ms. Birgitta Hedberg and Birgitta Lindegren have provided excellent technical assistance with the muscle biopsies. We are grateful to all those colleagues who have helped us with patient records and muscle biopsy material, including Drs. B. Andersson, H. Fahlgren, K . G . Henriksson, B. Hindfeldt, R. Libelius, B. Nilsson and P.O. Osterman.
REFERENCES Arahata, K., Shoichi, I., Ishiguro,T., Tsukahara, T., Suhara, Y., Eguchi,C., Ishihara, T., Nonaka, I., Ozawa, E. and Sugita, H. (1988) Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide. Nature, 333: 861-863. Banker, B.Q. (1986) The congenital myopathies. In: A.G. Engel and B.Q. Banker (Eds.), Myology, McGraw-Hill Book Company, New York, vol. 2, pp. 1525-1581. Brooke, M.H. and Kaiser, K.K. (1970) Muscle fibre types. How many and what kind? Arch. NeuroL, 23: 369-379. Casella, J.F., Craig, S.W.,Maack, D.J. and Brown, A. E. (1978). Cap Z (36/32), a barbed and actin-capping protein, is a component of the Z-line of skeletal muscle. J. Cell Biol., 105: 371-379. Clark, T.R., D'Agostino, A.N., Wilson, J., Brooks, R.R. and Cole G.C. (1978) Autosomal dominant myofibrillarinclusion body myopathy:clinical, histologic, histochemical, and ultrastructural characteristics. Neurology, 28: 399. Dubowitz, V. (1978) Muscle Disorders in Childhood, Saanders, London. Dubowitz, V. (1985) Muscle Biopsy: A Practical Approach, Balliere TindaU, London, Philadelphia, Toronto. Edstr6m, L. and Thornell, L-E. (1986) A dominantly inherited late onset myopathy with an early involvement of respiratory muscles and an abnormal accumulation of actin around the Z-discs.Muscle Nerve, $9, 203.
227 Edstrtm, L. and Wroblewski, R. (1981) Sarcoplasmic bodies in distal myopathy compared with nemalin¢ rods. J. Neurol. Sci., 49: 341-352. Edstrtm, L., Thornell, L-E. and Eriksson, A. (1980) A new type of hereditary distal myopathy with characteristic sarcoplasmic bodies and intermediate (skeletin) filaments. J. Neurol. Sci., 47: 171-190. Engel, W. K. (1962) The essentiality ofhisto- and cytochemical studies of skeletal muscle in the investigation of neuromuscular disease. Neurology (Minn.), 12: 778-794. Engel, W.K. and Cunningham, G.C. (1963) Rapid examination of muscle tissue. An improved trichrome method for fresh frozen biopsy sections. Neurology (Minn.), 13: 219-226. Fardeau, M. (1982) Congenital myopathies. In: F.L. Mastaglia and J. Walton (Eds.), Skeletal Muscle Pathology, Churchill-Livingstone, Edinburgh, London, Melbourne, New York, pp. 161-203. Furatsu, T., Asami, Y. and Ishiwata, S. (1988) Beta-actinin: a capping protein at the end of thin filaments in skeletal muscle. J. Biochem., 103: 61-71. Furst, D.O., Osborn, M., Nave, R. and Weber, K. (1988) The organization of titin filaments in the half-sarcomere revealed by monoclonal antibodies in immunoelectron microscopy: a map of ten nonrepetitive epitopes starting at the Z-line extends close to the M-line. J. Cell Biol., 106: 1563-1572. Goebd, H.H., Muller, J., Gillen, H.W. and Merritt, A.D. (1978) Antosomal dominant "spheroid body myopathy'. Muscle Nerve, 1: 14. Goebel, H.H., Schloon, H. and Lenard, H.G. (1981) Congenital myopathy with cytoplasmic bodies. Neuropediatrics, 12: 166-180. Goldstein, M.A., Schroeter, J. P. and Sass, R.L. (1979) The Z-lattice in canine cardiac muscle. J. Cell Biol., 83: 187-204. Goldstein, M. A., Stromer, J. P. and Sass, R. L. (1980) Optical reconstruction of nemaline rods. Exp. Neurol., 70: 83-97. Goldstein, M.A., Schroeter, J.P. and Sass, R.L. (1982) The Z-band lattice in a slow skeletal muscle. J. Muscle Res. Cell Motil., 3: 333-348. Haugland, R.P. (1985) Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes Inc., Junction City, U.S.A. Henriksson, K.G. (1979) "Semi-open" muscle biopsy technique- A simple outpatient procedure. Acta Neurol. Scand., 59: 317-332. Isobe, Y., Warner, F. and Lemanski, L. (1988) Three dimensional immunogold localization of alpha-actinin within the cytoskeletal networks of cultured cardiac muscle and nonmuscle cells. Proc. Natl. Acad. Sci. U.S.A., 85: 6758-6762. Jerusalem, F., Ludin, H., Bischoff, A. and Hartman, N. (1979) Cytoplasmic body neuromyopathy presenting as respiratory failure and weight loss. J. Neurol. Sci., 41: 1-9. Kinoshita, M., Satoyoshi, E. and Suzuki, Y. (1975) Atypical myopathy with myofibrillar aggregates. Arch. Neurol., 32: 417-420. Kuitonnen, P., Rapola, J., Noponen, A.L. and Donner, M. (1972) Nemaline myopathy. Report of 4 cases and review of the literature. Acta Pediatr. Scand., 61: 353-361. Legato, M.J. (1970) Sarcomerogenesis in human myocardium. J. Mol. Cell Cardiol., 1: 425-437. MacDonald, R. D. and Engel, A.G. (1969) The cytoplasmic body: another structural anomaly of the Z-disc. Acta Neuropathol., 14: 99-107. Maher, P.A., Cox, G.F. and Singer, J. (1985) Zeugrnatin: a new high molecular weight protein associated with Z-lines in adult and early embryonic striated muscle. J. Cell Biol., 101: 1871-1885. Maruyama, K. (1986) Connectin, an elastic filamentous protein of striated muscle. Int. Rev. Cytol., 104: 81-114. Muguruma, M., Kobayashi, K., Fukazawa, T., Ohashi, K. and Muruyama, K. (1981) A new 320.000 dalton protein located in the Z lines of vertebrate skeletal muscle. J. Biochem. (Tokyo), 89: 1981-1984. Nakashima, N., Tamura, Z., Okamoto, S. and Goto, H. (1970) Inclusion bodies in human neuromuscular disorder. Arch. Neurol., 22: 270-278. Ohashi, K., Mikawa, T., Muruyama, K. (1982) Localization of Z-protein in isolated Z-disc sheets of chicken leg muscle. J. Cell Biol., 95: 85-90. Osborn, M. and Goebel, H. H. (1983) The cytoplasmic bodies in a congenital myopathy can be stained with antibodies to desmin, the muscle specific intermediate filament protein. Acta Neuropathol., 62: 149-152. Patel, H., Berry, K., MacLeod, P. and Dunn, H.G. (1983) Cytoplasmic body myopathy. J. Neurol. Sci., 60: 281-292. Porte, A., Stoeekel, M. E., Sacrez, A. and Batzenschlager, A. (1980) Unusual familial cardiomyopathy with storage of intermediate filaments in the cardiac muscular cells. Virchows Arch. A, 386: 43-58. Rappaport, L., Contard, F., Samuel, J.L., Delcayre, C., Marotte, F., Tome, F. and Fardeau, M. (1988) Storage of phosphorylated desmin in a familial myopathy. FEBS Lett., 231: 421-425.
228 Rosenow, E.C. and Engel, A.G. (1978) Acid maitase deficiency in adults presenting as respiratory failure. Am. J. Med., 64: 485-493. Stoeckel, M.E., Osborn, M., Porte, A., Sacrez, A., Batzenschlager, A. and Weber, K. (1981) An unusual familial cardiomyopathy characterized by aberrant accumulations of desmin-type intermediate filaments. Virchows Arch. A, 393: 53-60. Thornell, L-E. (1973) Ultrastructural variations of Z-band in cow Purkinje fibres. J. Mol. Cell Cardiol., 5: 409-417. Thornell, L-E., Eriksson, A. and Edstr6m, L. (1983) Intermediate filaments in human myopathies. In: R. M. Dowben and J.W. Shay (Eds.), Cell andMuscle Motility, Plenum Press, New York and London, vol. 4 pp. 85-136. Virtanen, J., Kallajock, M., N~iv~lnen,O., Paranko, J., Thornell, L-E., Miettinen, M. and Letho, V. (1986) Pertubular myoid cells of human and rat testis are smooth muscle cells that contain desmin type intermediate filaments. Anat. Rec., 215: 10-20. Wang, K. (1985) Sarcomere-associated cytoskeletal lattice in striated muscle. In: J.W. Shay (Ed.), Cell and Muscle Motility, Plenum Press, New York and London, vol. 6 pp. 315-369. Watkins, S.C., Hoffman, E.P., Slayter, K.S. and Kunkel, L.M. (1988) Immunoelectron microscopic localization of dystrophin in myofibres. Nature, 333: 863-866. Winter, J.H., Neilly, J.B., Henderson, A.F., Stevenson, R.D., Doyle, D., Wiles, C.M. and Spencer, G.T. (1986) Life-threatening respiratory failure due to a previous undescribed myopathy. Quart. J. Med., 61: 1171-1178. Wolburg, H., Schlote, W., Langohr, H.D., Pfeiffer, J., Reiher, K.H. and Heckl, R.W. (1982) Slowly progressive congenital myopathy with cytoplasmic bodies. Clin. NeuropathoL, 1: 55. Wood, D., Zeviani, M., Prelle, A., Bonilla, E., Salviati, G., Miranda, A., DiMauro, S. and Rowland, U (1987) Is nebulin the defect gene product in Duchenne muscular dystrophy? N. Engl. J. Med., 316: 107-108. Yamaguchi, M., Robson, R. M. and Stromer, M. H. (1983) Evidence for actin involvement in cardiac Z-lines and Z-line analogues. J. Cell Biol., 96: 435-442. Yamaguchi, M., Izumimoto, M., Robson, R.M. and Stromer, M.H. (1985) Fine structure of wide and narrow vertebrate muscle Z-lines. A proposed model and computer simulation of Z-line architecture. J. Mol. BioL, 184: 621-644.
211
Elsevier
JNS 03318
Myopathy with respiratory failure and typical myofibrillar lesions Lars EdstrOm, Lars-Eric Thornell, Jaan Albo, Sven Landin and Margareta Samuelsson Departments of Neurology, Karolinska Hospital and Danderyd Hospital, Stockholm (Sweden), Department of Anatomy, Umed University and Department of Neurology, Orebro Hospital, Une~ (Sweden) (Received 11 October, 1989) (Revised, received 28 December, 1989) (Accepted 28 December, 1989)
SUMMARY
16 patients representing 7 different pedigrees exhibited an unusual, adult onset limb-girdle myopathy with typical clinical hallmarks. In a majority of cases there was evidence of an autosomal dominant inheritance. A prominent early finding in all cases was respiratory muscle weakness, and in many of these an acute respiratory incapacity was the reason for the In'st neurological examination. Neck flexor and sometimes foot extensor weakness were other early symptoms. The clinical picture seems to be at variance with that of the more well known hereditary myopathies. Electrophysiological analysis confirmed a myopathy and serum muscle enzyme concentrations were normal or slightly elevated. Muscle biopsy findings revealed myofibrillar changes which, at the light microscopy level, included plaques that stained strongly with rhodamineconjugated phalloidin, a specific marker for F-actin. At the ultrastructural level, these plaques were observed to be composed of moderately dense, thin filaments and were related to splitting of Z-discs or formed extensions from Z-discs. We believe that the muscle biopsy changes revealed by cytochemical and ultrastructural observations indicate defective myofibrillogenesis, and the possibility of defective actin polymerization is discussed. A conclusive answer requires further immunocytochemical and immunoelectrophoretic studies and possibly the application of molecular genetics.
Correspondence to: Dr. Lars Edstr6m, Department of Neurology, Karolinska Hospital, S-104 01 Stockholm, Sweden. 0022-510X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
212 Key words: Myopathy; Muscle dystrophy; Respiratory failure; Respiratory muscles; Sarcomere; Z-disc; Actin; Rhodamine-phalloidin; Desmin
INTRODUCTION Serious respiratory failure as a prominent early symptom of a hereditary myopathy is uncommon, although many respiratory problems commonly accompany more advanced stages of muscle dystrophies, such as Duchennes disease and myotonic dystrophy. Disorders with early respiratory failure include late onset Pompes disease (acid maltase deficiency), in which one-third of the cases involve early respiratory failure (Rosenow and Engel 1978) and respiratory involvement may occur in all cases and is often a cause of death. In nemaline myopathies (Kuitonnen et al. 1972; Dubowitz 1978), fatal progressive respiratory failure is also frequently encountered in the first and second decade of life. Among the group of disorders exhibiting muscle fiber inclusions similar to the cytoplasmic bodies of Engel (1962), occasional cases with early respiratory failure are reported (Jerusalem et al. 1979; Patel et al. 1983; Banker 1986; Winter et al. 1986). The present paper reports 16 cases representing 7 families who exhibit a limbgirdle syndrome with neck flexor weakness and a prominent involvement of respiratory muscles. The disease may typically be inherited as an autosomal dominant trait. The muscle biopsy findings included various myopathic changes, and characteristic myofibrillar changes were found in all biopsies. At the light microscopy level, there were plaques of material in many fibres that exhibited strong fluorescence of rhodamine-conjugated phaUoidin, which selectively binds to F-actin. We conclude that all these cases represent one and the same disorder. A preliminary report has been published elsewhere (EdstrOm and Thornell 1986).
MATERIALSAND METHODS Most biopsies were taken from the vastus lateralis of the quadriceps (VL) or the anterior tibial muscle (TA), but occasionally from the deltoid muscle (cf. Table 1). Most biopsies were obtained by means of a percutaneous modified Radner technique (Henriksson 1979), but some using open surgery.
Methodsfor specimenpreparation Specimens intended for ordinary light and fluorescence microscopy were frozen in Freon 13 cooled by liquid nitrogen (at - 190 °C) and kept in a refrigerator (at 75 °C) until 4-10 #m thick sections were cut in a cryostat at - 25 °C. Specimens intended for transmission electron microscopy (TEM) were pinned between needles on a piece of cork and fixed in 2.5~o gluthar aldehyde in phosphate buffer. Proceduresfor light microscopy. Stainings for hematoxylin-eosin and trichrome modified according to Engel and Cunningham (1963) were employed as well as histo-
213 chemical stainings for myosin-adenosine triphosphatase (myosin-ATPase) with alkaline and acid preincubations, NADH-TR, succinic dehydrogenase, fat, glycogen and acid phosphatase (cf. Dubowitz 1985). For visualization of F-actin in fluorescence microscopy, we used rhodamineconjugated phalloidin, which binds selectively to polymerized actin filaments (Molecular Probes Inc., Junction City, Oregon, U.S.A.). For visualization of the intermediate filament subunit desmin, we used a well characterized monoclonal antibody whose specificity has been described previously (Virtanen et al. 1986). The antibody was identified under fluorescence microscopy after labelling with a secondary FITC-conjugated rabbit anti-mouse serum. Procedures for transmission electron microscopy. After fLxation,the samples were washed in buffer and cut into blocks approximately 2 x 0.5 ram, postfixed in OsO 4 in PBS, dehydrated in acetone and embedded in Vestopal or Epon. 1.0-0.5-#m thick sections were cut on an LKB ultratome IV and stained with toluidine blue for light microscopy. After identification of areas of interest, ultrathin sections were produced and stained with uranyl acetate and lead citrate and examined in a Philips 300 or a JEOL 1200EX electron microscope.
RESULTS
Clinical hallmarks A summary of clinical and laboratory findings is presented in Table 1. Pedigree maps of three families presenting several members with typical symptoms are displayed in Fig. la-c. All cases were characterized by a proximal muscle weakness of the upper and lower extremities, with early affection of neck flexors and respiratory muscles, especially the diaphragm. There was sometimes an early involvement of dorsal foot extensors. Other distal muscles were spared for the most part but fmger extensors were involved in 4 cases. Cranial nerve innervated muscles were mostly spared, except for the sternocleidomastoid. In one patient there was, however, a bilateral weakness of the eye bulb abductors since early childhood and in one patient a slight myopathic face was observed. Muscle reflexes were normal or decreased but never totally inhibited. Time of onset varied from between the second and fifth decade. It must, however, be emphasized that the time of onset of some minor neuromuscular deficits is very difficult to establish. A majority of the patients admitted weakness of neck flexor muscles since early childhood on specific inquiry. The course of the disease seemed to be slowly progressive. No affection of the central nervous system or the sensory peripheral pathways has been observed, and no clear-cut clinical signs of cardiomyopathy were found.
Laboratory findings Electromyography (EMG) revealed a myopathy in all cases, but in one patient occasional muscles exhibited a mixture of neurogenic and myogenic changes. Peripheral
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IV Fig. 1. a-c. Preliminary pedigree maps of family 1-3, indicated PI-P3 in Table 1. In (b) there are 5 more siblings in generation II who are said to be unaffected which we have not been allowed to establish. Two affected sons of a first cousin of members of generation II are included in the study (case 8 and 9 in Table 1) but as we know too little about other individuals in this branch of the family they are not indicated in the map.
216
Fig. 2. a-c. Cryostat serial cross sections of muscle fibres exhibiting myopathic changes typical of this disorder. In (a), stained with rhodamine-phalloidin for detection of F-actin, there are numerous plaques of high fluorescence. In (b), trichrome stained, the same areas are visible as dark spots. In (c) stained for acid phosphatase it can be seen that the large fibres, filled with rhodamine fluorescent plaques, also have an elevated content of acid phosphatase activity, indicating increased autophagy. A cross in a fibre observed in all three sections facilitates comparison. 200 x.
217 nerve conduction velocity (NCV) was normal in all cases investigated. In a few cases reliable values were not obtained. There was little or no increase in serum muscle enzymes.
Muscle biopsy observations All the muscle biopsies exhibited typical plaques as described below and the frequency of fibres with such typical changes varied between 5 and 30~o in individual biopsies. Both type I and II fibres were involved. Light microscopy findings. The differentiation of muscle fibres into type I and type II with subtypes (Brooke and Kaiser 1970) was well preserved. There were no signs of type-grouping or abnormal fiber type dominance, but muscle fiber splitting and small groups of atrophic fibres were commonly found. In stainings for oxidative enzymes, targetoid and moth-eaten fibres were seen. Thus there were commonly irregularities of stainability for succinic dehydrogenase and NADH-TR but no typical ragged-red fibres or large areas of focal loss of enzyme activity. Typically, the number of muscle fibres exhibiting pathological changes varied considerably between different fascicles within one and the same biopsy. Several fascicles with almost normal muscle fibres were found to alternate with fascicles with many changes in large groups of fibres. Typical findings in affected fascicles revealed at the light microscopy level are illustrated in Fig. 2. Muscle fibres were of pronounced size difference, exhibiting slightly irregular fiber structure, and often contained smooth, opaque plaques of reddish or dark green color when stained for trichrome as seen in Fig. 2b. They were highly eosinophilic in hematoxyline-eosin (not illustrated). In crosssections produced in series with those stained for trichrome and labelled with rhodamine phalloidin (Fig. 2a), the plaques were highly fluorescent. In the fibres exhibiting fluorescence, there was an increased stainability for acid phosphatase as well in most cases (Fig. 2c). In longitudinal sections we observed that the plaques with high rhodamine fluorescence were focal (Fig. 3). Patches and streaks of high fluorescence with longitudinal and/or transverse orientation were seen (Fig. 3). By comparing sections cut in series and stained for rhodamine-phaUoidin and desmin, respectively, we observed that desmin was absent in the lesions of high rhodamine fluorescence (Fig. 3a, b). In longitudinal sections, however, we observed that the normal transverse striated pattern of desmin staining seen in unaffected areas of muscle fibres was often broken up in the areas of high phalloidin fluorescence (Fig. 3c, d, e, f). In addition, minor focal longitudinal strands of high desmin staining were observed in otherwise normal looking transverse striated pattern (Fig. 4). The fact that even slightly myopathic muscles with a high percentage of normally structured muscle fibres always displayed rhodamine phalloidin fluorescent plaques in scattered fibres pointed to these structures as representing an early and possibly pathognomonic phenomenon. Muscle fibre ultrastructure. In all muscle biopsies investigated, the most prominent pathological features of the muscle fibres were changes related to the myofibrils. In slightly affected fibres, some moderately electron dense material was seen within the myofibrils (Fig. 5a). Under higher magnification it was apparent that the material was
218
Fig. 3. a-f. Serial cross sections stained with rhodamine phalloidin in a and anti-desmin in b. Note complete lack of staining for desmin (b) in the plaques, which are highly fluorescent when stained with rhodaminephalloidin (a). Serial longitudinal sections are also stained with rhodamine-phalloidin (c) and anti-desmin (d). Note the focal appearance of high fluorescence in (c) and the longitudinal highly fluorescent streaks in (d). Parts of muscle fibres seen at high magnifications are stained with rhodamine phalloidin (e and f). Note the focal plaques and streaks of high fluorescence, a + b, 630x ; c + d, 300x ; e + f, 1500 x.
p r e s e n t b e t w e e n split Z - d i s c s a n d also f o r m e d s t r a n d s c o n n e c t e d to the Z - d i s c s (Fig. 5b). I n s o m e fibres there were areas with p r o n o u n c e d myofibrillar alterations a n d m o d e r a t e l y d e n s e f d a m e n t o u s m a s s e s (Figs. 6a, 7a). I n other fibres there were extensive dissolved m y o f i l a m e n t s a n d the fibres c o n t a i n e d long s t r a n d s or irregular collections o f
219
Fig. 4. a and b. Longitudinal sections stained with anti
moderately dense material (Fig. 6b). This material was never as dense as the Z-discs of normal myofibrils or as dense as enlarged Z-discs, Z-disc bodies or Z-rods, which were occasionally seen (Fig. 7). Irrespective of whether the moderately dense material occurred between Z-discs, formed short patches, strands of several sarcomeres in length or filamentous bodies, regular periodicities as in Z-rods were never seen. In thin sections allowing adequate resolution, thin filaments (4-6 nm) and amorphous material were resolved to be a main constituent of the aberrant myofibriUar material (Fig. 8). In between the aberrant myofibrils and the filamentous bodies, loosely arranged filaments, ribosomes and sarcotubules were seen. The filaments were of three classes: thin filaments (4-6 nm), intermediate filaments (7-11 nm), and thick filaments (12-16 nm). Mitochondria were sparse in the areas of dissoluted myofilaments. Other myopathic features included minicores and Z-disc streaming, autophagic vacuoles and myelin figures.
220
Fig. 5. a and b. Electron micrographs of a longitudinal ultrathin section. In (a) there is an area with an irregularity in the sarcomeric pattern, which is further magnified in (h). Note the streaks of medium-dense filamentous material related to the Z-discs and sometimes trapped between two Z-discs. An extraordinarily long streak is indicated by two arrows in both sections, a, 4400 x ; b, 17000 x .
221
Fig. 6. a and b. Electron micrographs of longitudinal ultrathin sections. Remnants of intact myofilaments (MF) in otherwise dissolved muscle fibres can be seen in (a). A filamentous m a s s is indicated by an arrow. Long strands of filamentous material (F) close to a nucleus (N) and an autolytic vacuole (V) can b¢ seen in (b). Two arrows point at dense Z-line material related to strands of medium dense filamentous material. 4000 x .
222
Fig. 7. a and b. Electron micrographs of longitudinal ultrathin sections. A magnification of the filamentous mass visible in Fig. 6a can be seen in (a). Note the content of thin filaments, irregularly arranged. The filamentous mass has an electron density that is lower than that of the Z-disc material indicated by arrows in (a) and (b). Medium-dense filamentous material encircling the dense Z-disc material can be seen in (b). a, 20 000 × ; b, 15 000 ×.
223
Fig. 8. a and b. Electron micrographs of longitudinal ultrathin sections. Streaks and patches of moderately dense material extend from Z-discs to Z-discs. Under higher magnification (b), the streaks seem to be composed of thin filaments (4-6 nm) and additional dense material. However, no apparent periodicity is seen. Note the difference in density between Z-discs, the aberrant lesions and the rest of the myofibrils. Note also that the thin filaments enter the myofibriUar lesions, a, 20000 x ; b, 75000 x.
224 DISCUSSION The similarities in clinical picture and muscle biopsy findings between these patients representing different pedigrees of Swedish origin strongly suggest that this is one and the same disorder. It is possible but still unproven that they might be descended from one and the same family. Does this disorder conform to any previously reported limb-girdle syndromes with characteristic muscle biopsy findings? A case reported by Jerusalem et al. (1979) and a case by Winter et al. (1986) had some similarities with ours, including early respiratory failure, but in the latter case the authors reported polydactylyand stiffness of the vertebral column resembling rigid spine syndrome. A mother and two of her teenaged children were reported by Patel et al. (1983) to be suffering from a limb-girdle syndrome with neck flexor weakness but also facial muscle involvement, which we observed only in one case. The children developed respiratory incapacity. In these three reports, the hematoxylin-eosin stainings revealed eosinophilic inclusions at the border of some muscle fibres, but the ultrastructural findings in all three reports indicated the presence of cytoplasmic bodies with the criteria given by Macdonald and Engel (1969), i.e. a spheroid structure with a dense core surrounded by a fdamentous radiating corona. This differs from our present observations of myofibrillar alterations including aggregates of thin filaments but not with an organization corresponding to the typical cytoplasmic bodies mentioned above. We have observed typical cytoplasmic bodies, e.g., in cases of polymyositis, but they did not stain specifically with rhodamine-phalloidin (unpublished observations). In nemaline myopathy there is a regular lattice pattern that characterizes the rod bodies and that is not found in the inclusions presented here (for review, see Fardeau 1982). The spheroid body myopathy of Goebel et al. (1978) had no clinical or histopathological similarity with the cases here. When the specificity of our present muscle biopsy observations is considered, the alterations of the myofibrils in relation to the Z-discs seem to be an early and typical change at the ultrastructural level. In more advanced stages of muscle fibre degeneration there were dissolved myofdaments, and strands of thin f'daments and of cytoplasmic bodies of moderate density were observed. These changes were observed at the light microscopy level as structures with strong binding of rhodamine phalloidin, and corresponding structural changes could be discerned already in routine stainings. There was undoubtedly an increased amount of actin in the fdamentous plaques related to the altered myoflbrils, as phalloidin selectively binds to F-actin (Haugland 1985). This agrees with the ultrastructural observations that thin filaments were resolved in both the filamentous masses and in the altered myofibrils. These f'daments were in continuity with Z-discs. However, they were of a lower density than the filaments of the Z-discs, but higher than those of the I-bands. Furthermore, although some amorphous material was seen to be attached to the filaments they did not show the typical structural pattern of the Z-discs or Z-rods (Goldstein et al. 1979, 1980, 1982; Yamaguchi et al. 1983, 1985). The main longitudinal component of the Z-disc is thought to be composed of actin
225 filaments, whereas the bridging structures are likely to be alpha-actinin (Isobe et al. 1988; Yamaguchi et al. 1983). However, recently a number of new proteins have been identified in the Z-disc, such as Z-protein (Muguruma et al. 1981), Zeugmatin (Maher et al. 1985), and Cap Z 36/32 (Casella et al. 1987). Their exact location is so far unknown. Aberrant I-bands have previously been observed in so-called double Z-discs in myofibrils of heart Purkinje fibres and suggested to be indicative of sarcomerogenesis (Legato 1970; ThorneU 1973). The strong rhodamine-phalloidin fluorescence, and the higher electron density, compared to the I-band, that characterizes the filamentous masses described here, might be explained by a higher density of actin fdaments than in the I-band or by an abnormal polymerization of actin. Clearly, the normal lengthdetermining mechanism for polymerization of actin to actin filaments seems to be out of order in the areas of abnormal myofilamental structure, as the filaments extending from one Z-disc to the next were sometimes several sarcorneres in length. This lengthdetermining mechanism is not yet fully understood, but several of the above mentioned proteins may be involved, e.g. alpha-actinin as well as beta-actinin (Furatsu et al. 1988). Other proteins related to the Z-disc and the sarcomere are nebulin and titin (connectin), two high molecular weight proteins which are thought to make up an elastic component of the myofibrils (Wang 1985; Maruyama 1986; Furst et al. 1988). There have been reports of a decrease of these proteins in muscles from patients with X-linked muscle dystrophies (Wood et al. 1987). In some case reports of cytoplasmic body myopathies (Nakashima et al. 1970; Kinoshita et al. 1975; Clark et al. 1978; Wolburg et al. 1982), it was suggested that this morphological phenomenon might be a specific marker of a basic defect of myof'dament biosynthesis. It is obvious from the present study that desmin, the subunit of muscle intermediate fdaments, was lacking within the plaques of high rhodamine fluorescence. This is in contrast to observations of disorders with cytoplasmic inclusions containing desmin (Edstr0m et al. 1980; Porte et al. 1980; Stoeckel et al. 1981; Osborn and Goebel 1983; Rappaport et al. 1988). Osborn and Goebel (1983) identified intermediate (desmin) filaments in the corona around the electron-dense core of cytoplasmic bodies in a case of congenital myopathy. We described a Swedish dominantly inherited myopathy that started in hand flexors and had a rapid progress, although respiratory muscles were never affected (Edstr0m et al. 1980). The muscle biopsy findings were characteristic (Edstr0m et al. 1980; EdstrOm and Wroblewski 1981). There were numerous sarcoplasmic dense bodies that varied in appearance. Some were composed of filamentous material and others were more electron-dense and completely or partly surrounded by a membrane. As we found an increased content of intermediate filaments we suggested that these bodies represented sequential changes in a degradation of desmin filaments. Thus, clinically and histopathologically, the disorder described earlier differs profoundly from that described in our present report. Here we observed, however, areas of myofibrils that contained streaks of desmin spanning over a few sarcomeres, similar to the areas of Z-streaming reported previously (Thornell et al. 1983) and regarded as an unspecific alteration.
226 When considering the possible basic defect responsible for the disorder, it must be born in mind that it should not only explain the occurrence of aggregates of actin filaments but also the inclination of the muscle cells to degenerate progressively. In recent years, changes in the sarcomere-associated cytoskeleton have attracted increasing interest in relation to the pathogenesis of muscle fibre degeneration. Defects of such organelles may be fatal to muscle fibres during contraction, and especially against high resistance. This concept is valid for, e.g., the intermediate filaments (EdstrOm et al. 1980; Thornell et al. 1983; Rappaport et al. 1988), the intrasarcomeric cytoskeletal filaments (Wood et al. 1987) and the plasma membrane related cytoskeletal protein dystrophin, which is lacking in Duchenne muscular dystrophy (Arahata et al. 1988; Watkins et al. 1988). It must however be emphasized that the connection between reduced amount of cytoskeletal proteins and the developement of muscle fibre degeneration is far from understood. The development of new immunohistochemical and immunoelectrophoretic techniques to analyse components of the sarcomere should further elucidate factors responsible for the etiology of the present hereditary myopathy and related disorders.
ACKNOWLEDGEMENTS This study was supported by grants from the Swedish Medical Research Council (projects 12X-3875 and 12X-3934), the Karolinska Institute and the University of Umeli. Ms. Birgitta Hedberg and Birgitta Lindegren have provided excellent technical assistance with the muscle biopsies. We are grateful to all those colleagues who have helped us with patient records and muscle biopsy material, including Drs. B. Andersson, H. Fahlgren, K . G . Henriksson, B. Hindfeldt, R. Libelius, B. Nilsson and P.O. Osterman.
REFERENCES Arahata, K., Shoichi, I., Ishiguro,T., Tsukahara, T., Suhara, Y., Eguchi,C., Ishihara, T., Nonaka, I., Ozawa, E. and Sugita, H. (1988) Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide. Nature, 333: 861-863. Banker, B.Q. (1986) The congenital myopathies. In: A.G. Engel and B.Q. Banker (Eds.), Myology, McGraw-Hill Book Company, New York, vol. 2, pp. 1525-1581. Brooke, M.H. and Kaiser, K.K. (1970) Muscle fibre types. How many and what kind? Arch. NeuroL, 23: 369-379. Casella, J.F., Craig, S.W.,Maack, D.J. and Brown, A. E. (1978). Cap Z (36/32), a barbed and actin-capping protein, is a component of the Z-line of skeletal muscle. J. Cell Biol., 105: 371-379. Clark, T.R., D'Agostino, A.N., Wilson, J., Brooks, R.R. and Cole G.C. (1978) Autosomal dominant myofibrillarinclusion body myopathy:clinical, histologic, histochemical, and ultrastructural characteristics. Neurology, 28: 399. Dubowitz, V. (1978) Muscle Disorders in Childhood, Saanders, London. Dubowitz, V. (1985) Muscle Biopsy: A Practical Approach, Balliere TindaU, London, Philadelphia, Toronto. Edstr6m, L. and Thornell, L-E. (1986) A dominantly inherited late onset myopathy with an early involvement of respiratory muscles and an abnormal accumulation of actin around the Z-discs.Muscle Nerve, $9, 203.
227 Edstrtm, L. and Wroblewski, R. (1981) Sarcoplasmic bodies in distal myopathy compared with nemalin¢ rods. J. Neurol. Sci., 49: 341-352. Edstrtm, L., Thornell, L-E. and Eriksson, A. (1980) A new type of hereditary distal myopathy with characteristic sarcoplasmic bodies and intermediate (skeletin) filaments. J. Neurol. Sci., 47: 171-190. Engel, W. K. (1962) The essentiality ofhisto- and cytochemical studies of skeletal muscle in the investigation of neuromuscular disease. Neurology (Minn.), 12: 778-794. Engel, W.K. and Cunningham, G.C. (1963) Rapid examination of muscle tissue. An improved trichrome method for fresh frozen biopsy sections. Neurology (Minn.), 13: 219-226. Fardeau, M. (1982) Congenital myopathies. In: F.L. Mastaglia and J. Walton (Eds.), Skeletal Muscle Pathology, Churchill-Livingstone, Edinburgh, London, Melbourne, New York, pp. 161-203. Furatsu, T., Asami, Y. and Ishiwata, S. (1988) Beta-actinin: a capping protein at the end of thin filaments in skeletal muscle. J. Biochem., 103: 61-71. Furst, D.O., Osborn, M., Nave, R. and Weber, K. (1988) The organization of titin filaments in the half-sarcomere revealed by monoclonal antibodies in immunoelectron microscopy: a map of ten nonrepetitive epitopes starting at the Z-line extends close to the M-line. J. Cell Biol., 106: 1563-1572. Goebd, H.H., Muller, J., Gillen, H.W. and Merritt, A.D. (1978) Antosomal dominant "spheroid body myopathy'. Muscle Nerve, 1: 14. Goebel, H.H., Schloon, H. and Lenard, H.G. (1981) Congenital myopathy with cytoplasmic bodies. Neuropediatrics, 12: 166-180. Goldstein, M.A., Schroeter, J. P. and Sass, R.L. (1979) The Z-lattice in canine cardiac muscle. J. Cell Biol., 83: 187-204. Goldstein, M. A., Stromer, J. P. and Sass, R. L. (1980) Optical reconstruction of nemaline rods. Exp. Neurol., 70: 83-97. Goldstein, M.A., Schroeter, J.P. and Sass, R.L. (1982) The Z-band lattice in a slow skeletal muscle. J. Muscle Res. Cell Motil., 3: 333-348. Haugland, R.P. (1985) Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes Inc., Junction City, U.S.A. Henriksson, K.G. (1979) "Semi-open" muscle biopsy technique- A simple outpatient procedure. Acta Neurol. Scand., 59: 317-332. Isobe, Y., Warner, F. and Lemanski, L. (1988) Three dimensional immunogold localization of alpha-actinin within the cytoskeletal networks of cultured cardiac muscle and nonmuscle cells. Proc. Natl. Acad. Sci. U.S.A., 85: 6758-6762. Jerusalem, F., Ludin, H., Bischoff, A. and Hartman, N. (1979) Cytoplasmic body neuromyopathy presenting as respiratory failure and weight loss. J. Neurol. Sci., 41: 1-9. Kinoshita, M., Satoyoshi, E. and Suzuki, Y. (1975) Atypical myopathy with myofibrillar aggregates. Arch. Neurol., 32: 417-420. Kuitonnen, P., Rapola, J., Noponen, A.L. and Donner, M. (1972) Nemaline myopathy. Report of 4 cases and review of the literature. Acta Pediatr. Scand., 61: 353-361. Legato, M.J. (1970) Sarcomerogenesis in human myocardium. J. Mol. Cell Cardiol., 1: 425-437. MacDonald, R. D. and Engel, A.G. (1969) The cytoplasmic body: another structural anomaly of the Z-disc. Acta Neuropathol., 14: 99-107. Maher, P.A., Cox, G.F. and Singer, J. (1985) Zeugrnatin: a new high molecular weight protein associated with Z-lines in adult and early embryonic striated muscle. J. Cell Biol., 101: 1871-1885. Maruyama, K. (1986) Connectin, an elastic filamentous protein of striated muscle. Int. Rev. Cytol., 104: 81-114. Muguruma, M., Kobayashi, K., Fukazawa, T., Ohashi, K. and Muruyama, K. (1981) A new 320.000 dalton protein located in the Z lines of vertebrate skeletal muscle. J. Biochem. (Tokyo), 89: 1981-1984. Nakashima, N., Tamura, Z., Okamoto, S. and Goto, H. (1970) Inclusion bodies in human neuromuscular disorder. Arch. Neurol., 22: 270-278. Ohashi, K., Mikawa, T., Muruyama, K. (1982) Localization of Z-protein in isolated Z-disc sheets of chicken leg muscle. J. Cell Biol., 95: 85-90. Osborn, M. and Goebel, H. H. (1983) The cytoplasmic bodies in a congenital myopathy can be stained with antibodies to desmin, the muscle specific intermediate filament protein. Acta Neuropathol., 62: 149-152. Patel, H., Berry, K., MacLeod, P. and Dunn, H.G. (1983) Cytoplasmic body myopathy. J. Neurol. Sci., 60: 281-292. Porte, A., Stoeekel, M. E., Sacrez, A. and Batzenschlager, A. (1980) Unusual familial cardiomyopathy with storage of intermediate filaments in the cardiac muscular cells. Virchows Arch. A, 386: 43-58. Rappaport, L., Contard, F., Samuel, J.L., Delcayre, C., Marotte, F., Tome, F. and Fardeau, M. (1988) Storage of phosphorylated desmin in a familial myopathy. FEBS Lett., 231: 421-425.
228 Rosenow, E.C. and Engel, A.G. (1978) Acid maitase deficiency in adults presenting as respiratory failure. Am. J. Med., 64: 485-493. Stoeckel, M.E., Osborn, M., Porte, A., Sacrez, A., Batzenschlager, A. and Weber, K. (1981) An unusual familial cardiomyopathy characterized by aberrant accumulations of desmin-type intermediate filaments. Virchows Arch. A, 393: 53-60. Thornell, L-E. (1973) Ultrastructural variations of Z-band in cow Purkinje fibres. J. Mol. Cell Cardiol., 5: 409-417. Thornell, L-E., Eriksson, A. and Edstr6m, L. (1983) Intermediate filaments in human myopathies. In: R. M. Dowben and J.W. Shay (Eds.), Cell andMuscle Motility, Plenum Press, New York and London, vol. 4 pp. 85-136. Virtanen, J., Kallajock, M., N~iv~lnen,O., Paranko, J., Thornell, L-E., Miettinen, M. and Letho, V. (1986) Pertubular myoid cells of human and rat testis are smooth muscle cells that contain desmin type intermediate filaments. Anat. Rec., 215: 10-20. Wang, K. (1985) Sarcomere-associated cytoskeletal lattice in striated muscle. In: J.W. Shay (Ed.), Cell and Muscle Motility, Plenum Press, New York and London, vol. 6 pp. 315-369. Watkins, S.C., Hoffman, E.P., Slayter, K.S. and Kunkel, L.M. (1988) Immunoelectron microscopic localization of dystrophin in myofibres. Nature, 333: 863-866. Winter, J.H., Neilly, J.B., Henderson, A.F., Stevenson, R.D., Doyle, D., Wiles, C.M. and Spencer, G.T. (1986) Life-threatening respiratory failure due to a previous undescribed myopathy. Quart. J. Med., 61: 1171-1178. Wolburg, H., Schlote, W., Langohr, H.D., Pfeiffer, J., Reiher, K.H. and Heckl, R.W. (1982) Slowly progressive congenital myopathy with cytoplasmic bodies. Clin. NeuropathoL, 1: 55. Wood, D., Zeviani, M., Prelle, A., Bonilla, E., Salviati, G., Miranda, A., DiMauro, S. and Rowland, U (1987) Is nebulin the defect gene product in Duchenne muscular dystrophy? N. Engl. J. Med., 316: 107-108. Yamaguchi, M., Robson, R. M. and Stromer, M. H. (1983) Evidence for actin involvement in cardiac Z-lines and Z-line analogues. J. Cell Biol., 96: 435-442. Yamaguchi, M., Izumimoto, M., Robson, R.M. and Stromer, M.H. (1985) Fine structure of wide and narrow vertebrate muscle Z-lines. A proposed model and computer simulation of Z-line architecture. J. Mol. BioL, 184: 621-644.