Journal of

J Neurol (1987) 234:137-145

Neurology © Springer-Verlag1987

Rimmed vacuolar distal myopathy An ultrastructurai study H. Mizusawa 1, H. Kurisaki 2, M. Takatsu 3, K. Inoue 4, T. Mannen 4, Y. Toyokura 5, and T. Nakanishi 1 1 Department of Neurology, Institute of Clinical Medicine, University of Tsukuba, Ibaraki 305, Japan 2 Department of Neurology, National Tokyo Hospital, Kiyose 204, Japan 3 Department of Neurology, Teikyo Medical School, Tokyo 173, Japan 4 Department of Neurology, Institute of Brain Research, Faculty of Medicine, University of Tokyo, Tokyo 113, Japan 5 Tokyo Metropolitan Geriatric Hospital, Tokyo 173, Japan

Summary. A n ultrastructural study of biopsied muscles was performed in seven patients with rimmed vacuolar distal myopathy, which was characterized by prominent rimmed vacuoles in the muscle fibers. The earliest changes noted were focal proliferation of the Golgi's apparatus and mitochondrial degeneration with myofibrillar loss. A proliferation of the T-system appeared later. Secondary lysosomes (autophagosomes) could be noted much later and gradually increased in number. Autophagosomes tended to coalesce and became larger autophagic vacuoles, which were surrounded in part by relatively preserved myofibrils and partly by a single membrane. Gently curved laminated structures (tubulomembranous structures) were seen in the degenerating muscle fibers and also in relatively intact fibers, satellite cells, and interstitial cells in all cases. They were closely associated with lipofuscin-like material. These findings suggest that an abnormality of the lysosomal system might be essential in the pathogenesis of rimmed vacuolar distal myopathy. Key words: Rimmed vacuolar distal myopathy - Autophagic vacuole - Golgi's apparatus - Lysosome - Tubulomembranous structure

tinct clinical entity characterized by: (1) transmission as an autosomal recessive trait with many sporadic cases; (2) onset in young adulthood; (3) early and preferential involvement of the tibialis anterior and extensor digitorum et hallucis muscles of the lower leg, the hamstrings and adductors of the thigh; (4) normal or mild elevation of serum CK activity; (5) mainly myopathic EMG with certain "neuropathic" features; (6) peculiar histopathological findings of rimmed vacuoles in muscle fibers associated with certain "neuropathic" features, such as angular fibers, clustering of atrophic fibers, pyknotic nuclear clumps and fiber type predominance [14-16]. These clinical features were quite different from those in Welander's late distal myopathy [20], infantile distal myopathy of Magee and De Jong [9] and Miyoshi's distal muscular dystrophy [12]. The disease has tentatively been named rimmed vacuolar distal myopathy (RVDM). Electron microscopically, RVs correspond to vacuoles containing membranous whorls, dense bodies, glycogen granules and other amorphous, granular and fibrillar materials, which have been considered to be autophagic in nature [3]. Although RVs or autophagic vacuoles (AVs) in the muscle fiber are not particularly rare findings [3], little is known about the pathogenesis of this change. The purpose of this report is to show ultrastructurally the degenerating process of muscle fibers with AVs in RVDM.

Introduction Materials and methods Recently, a form of distal myopathy has attracted wide attention owing to the presence of prominent rimmed vacuoles (RVs) in the muscle fibers [13-16]. This variety forms a dis-

Muscle biopsy was performed in 7 patients with RVDM (1 male, 6 females; aged 26-48 years); see Table 1. Diagnoses

Table 1. A brief summary of the clinical and light-microscopic features of seven cases with rimmed vacuolar distal myopathy Case, sex

1F

2F

3F

4F

5F

6M

7F

Age at biopsy (years) Age at onset (years) Gait disturbance Biopsied muscle Rimmed vacuole

27 25 + Gc, Qf +++, +

48 38 ++ Ta, Qf* +, -

30 18 ++ Qf, Bc* + + + , ++

27 26 ++ Gc ++

31 26 +++ Ta, Qf ++, +

26 25 ++ Gc +++

29 26 + Gc, Qf ++, +

F = female; M = male; Gc = gastrocnemius muscle; Ta = tibialis anterior; Qf = quadriceps femoris; Bc = biceps brachii; + + + = marked; ++ = moderate; + = mild; - = absent; * = not examined by electron microscopy Offprint requests to: H. Mizusawa, Division of Neuropathology, Department of Pathology, Montefiore Medical Center, 111 East 210th Street, Bronx, NY 10467, USA

138

Fig. 1. Gastrocnemius muscle of case 6. A focal proliferation of Golgi's apparatus is noted between relatively intact myofibrils. ( × 54,300)

Fig. 2. Quadriceps femoris of case 3. Golgi's apparatus is markedly proliferated with myofibrillar loss and several autophagosomes. T-tubules, stained dark with lanthanum, show a certain proliferation with honeycomb structures. I and Z bands of the myofibrils seem to be more apt to degenerate than A and M bands. (× 21,300)

139

Fig. 3. Quadriceps femoris of case 7. T-tubules are markedly proliferated with mitochondrial degeneration and autophagosomes. Adjacent myofibrils are relatively well preserved. (× 8,200)

Fig. 4. Quadrieeps femoris of case 3. There is a subsarcolemmal proliferation of T-tubules stained dark with lanthanum. (× 16,400)

Fig, 5. Qastrocnemius muscle of case 6. There are numerous autophagosomes of various sizes and contents, such as membranous whorls and granular substances, among which are disorganized myofibrils, degenerated mitochondria, and proliferated T-tubules. Golgi's apparatus is no longer noticeable in this advanced lesion. (× 8,200)

Fig. 6. Gastrocnemius muscle of case 6. The upper part of the lower muscle fiber is compactedly occupied by numerous autophagic vacuoles and membranous whorls, among which few organellae remain. Some vacuoles are coalescing and forming larger vacuoles. The right upper and lower corners represent parts of two such large autophagic vacuoles that are partly bound by a single membrane. These huge vacuoles probably correspond to rimmed vacuoles seen through a light microscope. (× 3,300)

141

Fig. 7. Quadriceps femoris of case 5. Two tubulomembranous structures associated with lipofuscin-like granules and lipid droplets are located near the nucleus beneath the sarcolemma. (x 22,000)

Fig. 8. Quadriceps femoris of case 3. There are a few large tubulomembranous structures with many membranous whorls in a lesion of vacuolar degeneration. (× 8,600)

142 lead citrate were examined using a Joel 100 CX electron microscope. The specimens from case 3 were also stained with 2% lanthanum nitrate during osmium fixation.

Results

Fig. 9. A Quadriceps femoris of case 7. A tubulomembranous structure in a membrane-bound vacuole contains a few lipofuscin-like granules (x 13,000). B Quadriceps femoris of case 1. The periodicity of the lamination is approximately 15mm in this tubulomembranous structure (x 26,000). C Quadriceps femoris of case 7. A tubulomembranous structure consists of a little laminated structure and an abundant electron-dense materiai. (x 20,000) were made after full examinations exhibited the characteristic features of R V D M mentioned above. The clinical and lightmicroscopic features of all cases, except case 3, have been previously reported [15]. Specimens were biopsied from the quadriceps femoris, gastrocnemius, or tibialis anterior muscle and frozen in isopentane chilled in liquid nitrogen. Serial frozen sections were stained with H & E, Gomori trichrome, N A D H - T R , oil red O, PAS, phosphorylase, nonspecific esterase, routine ATPase, and ATPase with preincubation at p H 4.6 and 4.2. Light microscopy revealed prominent rimmed vacuoles in the muscle fibers in each of the cases (Table 1). A part of the specimen was fixed with cacodylate-buffered 2% glutaraldehyde and postfixed with 1% osmium tetroxide. After dehydration in an ascending series of ethanol and embedding in Epoxy resin, ultrathin sections stained with uranyl acetate and

The outstanding ultrastructural feature was marked vacuole formation. The vacuoles were quite varied in size and content. Indeed, the sizes ranged from a small endocytotic vesicle to almost the entire cross-sectional area of a muscle fiber. They were so-called AVs and contained numerous membranous whorls, various cytoplasmic debris, and glycogen granules. Fuller investigation of relatively intact muscle fibers disclosed scattered focal areas of vesiculotubular proliferation and mitochondrial changes where myofibrils did not exist (Fig. 1). Some tubular profiles were arranged as parallel stacks of cisternae, and others were extended in bizarre shapes. These tubulosaccular structures morphologically seemed to be Golgi's apparatus rather than T-tubules. Moreover, the structure was neither stained with lanthanum nor connected directly with evident T-tubules. In those lesions, mitochondria were markedly enlarged and proliferated without any specific inclusion, Although myofibrils surrounding the area were relatively intact, some of them showed disorganization such as Z-band streaming. In these areas, Z and I bands seemed to be liable to degenerate rather than M and A bands. In the areas of advanced myofibrillar loss, the proliferation of Golgi's apparatus became more prominent (Fig. 2). A proliferation of T-tubules and small vacuoles also appeared. The vacuoles were surrounded by a single or double membrane and seemed to be autophagosomes. T-tubule proliferation was observed in various patterns, such as honeycomb structures and bizarrely elongated profiles that were easily recognized by lanthanum stain (Figs. 3, 4). In the more advanced lesions, autophagosomes that were empty, or contained various membranous whorls or granular substances, increased both in number and size (Fig. 5). Among these autophagosomes, there were also free membranous whorls, degenerated mitochondria, glycogen particles, and randomly oriented filaments that appeared to be degenerated thin and thick myofilaments. A proliferation of Ttubules was still noticeable. Occasionally, there were isolated terminal cisternae of sarcoplasmic reticulum, which had an electron-dense granular appearance Autophagosomes tended to coalesce and form larger vacuoles. Very large autophagic vacuoles were also common findings (Fig. 6). They were surrounded partly by a single membrane and partly by relatively preserved myofibrils. Gently curved laminated structures were noted near myonuclei and in the area of vacuolar degeneration mentioned above in every case (Figs. 7, 8). They were usually associated with lipid droplets and electron-dense materials similar to lipofuscin. The ratio between the laminated structure and electron dense material varied from one inclusion body to another (Fig. 9). Some portions of the structures were rubbed out as if they were painted with a rough brush; therefore, the lamination was frequently so vague that its periodicity could not always be easily determined. However, it measured approximately 15 nm in some inclusions. This cytoplasmic inclusion seemed to be identical to the so-called tubulomembranous structure

Fig. 10A, !i. Quadriceps femoris of case 3. A A tubulomembranous structure in a satellite cell (lanthanum stain, x 11,500). B A tubulomembranous structure with electron-dense laminated bundles and a large lipid droplet in an interstitial cell. (x 23,000)

Fig. 11. Quadriceps femoris of case 5. Filamentous inclusions, approximately 20 nm in diameter, are noted in the nucleus of a degenerated muscle fiber. (× 18,600)

144 (TMS) described first in tubulomembranous myopathy [4]. TMS was also encountered in satellite cells, interstitial cells, and muscle fibers, some of which showed only minimal degeneration (Fig. 10). Intranuclear filamentous inclusion was noted in only one myonucleus in the samples examined from case 5 (Fig. 11). The filament measured approximately 20 nm in diameter. No similar inclusion was found in the cytoplasm. In contrast, cytoplasmic fibrous bodies consisting of 6- to 8-nm filaments were occasionally encountered in all cases. Cytoplasmic bodies were also a common finding.

Discussion Numerous AVs and membranous whorls are characteristic features in R V D M [6, 7, 10, 15, 16], although AVs per se are not disease-specific [2, 3]. R V D M also has other important features, such as angular fibers, pyknotic nuclear clumps, and fiber-type predominance. However, AVs probably play a very important role in the degeneration of muscle fibers since there are scattered atrophic fibers with RVs in the very early stages of muscle involvement [15]. It has also been stressed that there is no prominent necrosis of muscle fibers, such as that found in Duchenne's muscular dystrophy [15, 17]. AVs, autophagosomes, or secondary lysosomes have been considered to be derived from the fusion of primary lysosomes and phagosomes [1]. Therefore, AVs per se are closely related to the activation of the lysosomal system. The elevation of lysosomal enzymes such as cathepsins, acid phosphatase and [3glucuronidase has also been reported in biopsy muscle specimens from cases with R V D M [19]. As mentioned in Results, the tubulosaccular structure was regarded to be Golgi's apparatus because of the morphological similarity and the absence of staining with lanthanum. Usually, Golgi's apparatus is hardly seen outside the vicinity of the nucleus in the normal muscle fiber [1]; therefore, proliferation of this structure deep in the muscle fiber is clearly pathological. Our observation of a prominent Golgi's apparatus as an early change is thought to be important morphological evidence that activation of the lysosomal system might be essential in the pathogenesis of RVDM. These lesions of vacuolar degeneration were also accompanied by mitochondrial degeneration. Although it is a good marker of the lesion, mitochondrial degeneration is likely to be secondary rather than primary, since it lacks more specific changes such as paracrystalline inclusion. In these lesions, myofibrils were also lacking and relatively intact myofibrils surrounded them. The mechanism of myofibrillar loss has not yet been disclosed; furthermore, what causes proliferation of Golgi's apparatus or activates the lysosomal system remains unclear. McDonald and Engel have described how phagosomes were formed from T-tubulus in experimental chloroquine myopathy [11]. We also recognized marked proliferation of Ttubules. However, the proliferation was not apparent at a very early stage. Furthermore, no direct relation between Ttubules and phagosomes was confirmed in our study with lanthanum stain or in the literature [16]. Therefore, changes in T-system might contribute to the development of vacuolar degeneration but be secondary to the degeneration of various organellae in RVDM.

TMS in RVDM have previously been described by Kuzuhara et al. [8]. This report has further confirmed the close relationship between TMS and RVDM. TMS were very closely associated with a lipofuscin-like material, which was thought to be a residual body or a form of secondary lysosome. Another important finding was that TMS existed not only in degenerated muscle fibers and interstitial cells but also in satellite cells and relatively intact muscle fibers. These facts suggested that TMS are related to the lysosomal system and that the abnormality of the system that produces TMS may not be simply secondary to severe degeneration of muscle fibers. Therefore, we consider TMS to be also an important feature in RVDM, although it is not specific to the disease. Intranuclear inclusions consisting of 20-nm filaments are so similar to those described for inclusion body myositis that R V D M might not be distinguishable from a histopathological viewpoint alone [15]. The combination of peculiar clinical features and histopathological findings is necessary to make the diagnosis of R V D M [15]. However, the intranuclear inclusions are not as common in R V D M as in inclusion body myositis [15]. Furthermore, similar inclusions have also been encountered in Duchenne's muscular dystrophy and hypokalemic myopathy [5, 18]. Therefore, the significance of these filamentous inclusions in R V D M is still not apparent.

Acknowledgements. The authors thank Professor Ikuya Nonaka and Professor Hideo Sugita, National Center for Nervous, Mental and Muscular Disorders (NCNMMD), for their constant encouragement. The authors also thank Ms. Akiko Isawa for the technical assistance. This work was supported partly by a grant from the Project Research Department of the University of Tsukuba and partly by Grant no. 8408 from NCNMMD from the Ministry of Health and Welfare, Japan.

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