Ultrastructural abnormality of sarcolemmal nuclei in Emery-Dreifuss muscular dystrophy (EDMD)

Journal of the Neurological Sciences 159 (1998) 88–93

Ultrastructural abnormality of sarcolemmal nuclei in Emery-Dreifuss muscular dystrophy (EDMD) ´ a , *, D. Toniolo b , I. Hausmanowa-Petrusewicz a A. Fidzianska a

Department of Neurology, Medical School and Neuromuscular Unit, Polish Academy of Science, Warsaw, Poland b Institut of Biochemical Genetics, Pavia, Italy Received 11 November 1997; received in revised form 23 February 1998; accepted 25 March 1998

Abstract We performed ultrastructural studies on nuclear abnormalities in biopsied muscles from seven patients with EDMD, of three non-related families, and two sporadic cases. The diagnosis was based on clinical data and molecular findings. We detected different degrees of abnormalities in the sarcolemmal nuclei ranging from marked condensation of chromatin to complete damage of nuclear components. Other nuclei in the same muscle cell very often appeared normal. The extrusion of nuclear chromatin into sarcoplasm as a consequence of nuclear membrane disintegration was observed in numerous nuclei. All these nuclear changes are considered to be cytological indicators of nuclear dysfunction evoked by emerin deficiency.  1998 Elsevier Science B.V. All rights reserved. Keywords: EDMD; Emerin deficiency; Nuclear architecture changes

1. Introduction

2. Materials and methods

Emerin, the serine-rich 34 kDa protein encoded by Emery-Dreiffus muscular dystrophy (EDMD) gene, shows ubiquitous tissue distribution with the highest expression in skeletal and cardiac muscles [1,2]. Recently emerin was found localized at the nuclear membrane of normal skeletal, cardiac and smooth muscle [10,12], as well as in leukocytes and the skin [11]. In contrast, a deficiency of emerin was observed in immunofluorescent staining of skeletal, cardiac muscles, leukocytes and skin from EDMD patients [11,12]. The absence of emerin in the nuclear membrane of muscle cells in EDMD patients may lead to nuclear structural abnormalities. To elucidate the structural changes evoked by emerin deficiency, we examined muscle nuclei architecture in patients with EDMD.

We investigated seven affected EDMD males representing three non-related families and two sporadic cases. The individuals from two previously presented families [5] were reexamined, particularly those males who appeared unaffected during prior examination. One male, who was previously classified as normal at age ten, revaluated at age 26, showed clear signs of the disease such as contracture of elbows and low spine and atrioventricular heart block. In addition new individuals of one family (three males) and two sporadic cases were investigated. The diagnosis was based on clinical data, DNA analysis and lack of emerin in immunostaining procedure. The quadriceps, femoris and pectoralis muscles were investigated. Serial frozen sections for light microscopy were stained according to standard techniques. The original anti-emerin antibody (provided by Dr Toniolo) was used for immunohistochemical studies. For the specific identification of nuclear membrane, each section was

*Corresponding author. 0022-510X / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved. PII: S0022-510X( 98 )00130-0

´ et al. / Journal of the Neurological Sciences 159 (1998) 88 – 93 A. Fidzianska

89

double- stained for immunofluorescence with anti-emerin antibody and anti-human nuclear membrane antibody (MAB 1274 Chemicon) according to a previously described method [12]. For the identification of karyoskeletal abnormality we used anti-nuclear lamin antibodies (lamins A / C, B Novocastra). For electron microscopy, the muscle specimens were fixed in 3% gluteraldehyde in phosphate buffer and postfixed in 1% osmium tetroxide in the same buffer. Then they were dehydrated and embedded in spurrresin. Thin sections double-stained with uranyl acetate and lead citrate were examined with a JEM X / II electron microscope.

3. Results Light microscopical examination of the specimens of quadriceps muscles showed abnormal variation in muscle fibre size with the presence of many small muscle fibres, some of which occurred in small groups. Central location of nuclei, splitting and increased endomysial connective tissue were observed. The immunostaining of control muscle showed normal reactivity for nuclear membrane antibody (Fig. 1a). The antibody against emerin showed uniform, intensive labelling around each myonuclei (Fig. 2a). In contrast there was no labelling with emerin antibody in all nine EDMD patients (Fig. 2b). In addition, the reactivity of nuclear membrane antibody was weak and considerably less intensive then that observed in normal muscle (Fig. 1b). In electron microscopy, the complex of structural changes in the architecture of some nuclei was observed, while the other nuclei in the same muscle cell appeared normal (Fig. 3). Various degrees of chromatin aggregation and condensation was the most frequent finding. The nuclei, poorly endowed with euchromatin, were occupied by granular hypercondensed chromatin having the appearance of very dark nuclei (Fig. 4). The nucleoli, composed of dense closely packed granules, were observed in heterochromatic nuclei. Similar chromatin condensation

Fig. 1. Immunofluorescence staining of muscle specimens with antihuman nuclear membrane antibody. (a) Control muscle. Clear, positive staining of nuclear membrane, 31050. (b) Muscle of EDMD patient. Markedly diminished staining of nuclear membrane, 31050.

Fig. 2. Immunofluorescence staining of muscle specimens with antiemerin antibody. (a) Control muscle. Intensive staining of nuclear membrane, 31050. (b) Muscle of EDMD patient. Absence of immunostaining of nuclear membrane, 31050.

and aggregation were observed in endothelial cells, smooth muscles and in fibroblasts. Focal loss of nuclear membrane and karyoplasm extrusion into extranuclear space with chromatin bleb formation was the most characteristic alteration seen in the muscle of EDMD patients. The chromatin blebs, varying in size and shape devoid of membrane, indicated direct continuity with the main part of the nuclei (Fig. 5). The most intensive karyoplasm extrusion into sarcoplasm across disrupted nuclear mem-

Fig. 3. Nucleus with normal architecture, 324 000.

90

´ et al. / Journal of the Neurological Sciences 159 (1998) 88 – 93 A. Fidzianska

Fig. 4. Highly condensed, hyperchromatic nucleus, 324 000.

brane was manifested by large chromatin leaks (Fig. 6). Nuclear membrane disintegration and chromatin bleb formation was also observed in fibroblasts (Fig. 7). Another example of nuclear membrane permeability and chromatin extrusion was the appearance of ‘naked’ chromatin fragments located close to the nuclei (Fig. 8). For better documentation of nuclear membrane disruption and karyoplasm extrusion, we used anti-nuclear lamin anti-

Fig. 5. Karyoplasm extrusion (arrow) into extranuclear space, 324 000.

Fig. 6. A large chromatin leak (arrow head), 350 000.

bodies. In the control, normal muscle lamins were incorporated into the nuclear membrane as well as uniformly distributed throughout the karyoplasm (Fig. 9a). In the EDMD patients, the lamins reactivity was observed, not

Fig. 7. Nuclear membrane disintegration and chromatin bleb formation seen in fibroblasts, 324 000.

´ et al. / Journal of the Neurological Sciences 159 (1998) 88 – 93 A. Fidzianska

91

Fig. 8. ‘Naked’ chromatin fragment is located close to nucleus, 360 000.

only in karyoplasm, but also dispersed around nuclei as delicate clouds or tails (Fig. 9b). Besides the changes in the nuclear envelope presented above, other alterations of nuclear architecture were found in EDMD patients, namely a very peculiar and unique nuclear channel system embedded in dense chromatin. These single, membrane-bound tubules which presented circular profiles on transverse sections, measuring approximately 300–350 nm in diameter, were separated from adjacent heterochromatin by much less electron-dense narrow shells (Fig. 10). Several such profiles were occasionally present in the nuclei and sometimes tubules were interconnected forming a large channel system (Fig. 11). The continuity between channels and the inner membrane of the nuclear envelope suggests that the tubules arise by inner nuclear membrane invaginations. The ultrastructural studies also revealed the presence of intranuclear pseudoinclusions which were rounded, elongated or irregular in outline. They contained different cytoplasmic components

Fig. 9. Immunofluorescence staining of muscle specimens with antilamins. (a) Control muscle. Lamins are incorporated in the nuclear membrane as well as uniformly distributed through the karyoplasm, 31050. (b) Muscle of EDMD patient. Small chromatin fragments labelled by lamins close to nucleus, 31050.

Fig. 10. Single membrane-bound channels 300–350 nm in diameter arranged in a cluster (arrow), 340 000.

which were separated from the nuclear matrix by two membranes derived from invagination of the nuclear envelope (Fig. 12). More advanced degeneration of the nuclei was manifested by nuclear matrix fragmentation. The nuclear changes, as described, were observed in all studied cases. The number of degenerating nuclei increased with the age of the patients and there were more

Fig. 11. Unique channel system within the nuclear chromatin, 360 000.

92

´ et al. / Journal of the Neurological Sciences 159 (1998) 88 – 93 A. Fidzianska

Fig. 12. Deep channel indentations forming pseudoinclusions, 330 000.

numerous abnormal nuclei in older patients than in younger ones.

4. Discussion The present study revealed severe fine structural changes of nuclear architecture in skeletal muscle of EDMD patients in whom emerin deficiency was stated. The nuclei, devoid of emerin and with low anti-nuclear membrane reactivity, showed different degrees of structural abnormalities, ranging from marked condensation of chromatin to complete damage. It is interesting to note that abnormal nuclei were observed within muscle cells of normal pattern frequently next to normal nuclei. No abnormalities were found in the satellite cell nuclei. The most prominent changes in EDMD patients were condensation of nuclear chromatin, focal loss of nuclear membrane and chromatin extrusion into extranuclear space. The chromatin bleb formation, varying in size and shape, accompanied by nuclear membrane disruption, were frequently observed in skeletal and smooth muscle as well as in the fibroblasts. We used antibodies against lamin to demonstrate the karyoplasm extrusion into the extranuclear space. Lamin, part of the intermediate filament protein family, regulate nuclear chromatin organization [16]. In normal myonuclei they are closely associated with heterochromatin and uniformly distributed within nuclei [6]. The appearance of small chromatin fragments labelled by lamins situated close to nuclei provides strong evidence of karyoplasm flowing out, as seen at ultrastructural level. This phenomenon was more pronounced in older patients and tended to increase with duration of the disease. The highly electron dense nuclei observed in EDMD

patients usually occur by the impairment of nuclear DNA / RNA metabolism [4]. There is now increasing evidence supporting the idea that euchromatic nuclei are active in RNA and DNA synthesis, while the heterochromatic nuclei show little or no activity [8,15]. In agreement with this is the observation of regenerating muscle cells whereas while the synthesis of DNA and RNA reaches a high level, almost all nuclei are in the decondensed (euchromatic) form [3]. Conversely, various substances, which exhibit RNA synthesis, induce a condensation of nuclear chromatin [7]. The occurrence of numerous intranuclear tubules or cannalicula present as circular profiles is a unique finding never observed in neuromuscular disorders. These nuclear tubules may be clearly distinguished from other nuclear inclusions such as tubulofilementous structures [17], rodlet bodies [9], intranuclear hyaline inclusions [14], granular nuclear inclusion bodies [13] and replicated paramyxoviruses [4]. The origin and the significance of intranuclear channel systems seen in EDMD patients is not clear, but it is conceivable that these changes may represent the abnormality in nuclear membrane biosynthesis. Although the nucleus plays an important role in the muscle cell function such as replication, transcription and transport of both pre-ribosomal and pre-messenger RNAs, its alteration in muscle diseases are poorly understood. The abnormal nuclear morphology is considered to be not as important for the evaluation of human neuromuscular disorders as are the presence, absence or structural changes in the sarcoplasm constituents. Some nuclear changes and nuclear inclusions are of diagnostic significance. Structural changes that we present in myonuclei as well as in nuclei of fibroblasts of EDMD patients such as, intranuclear channel system formation, chromatin condensation, nuclear membrane disintegration and disruption, chromatin extrusion into extranuclear space, to our knowledge have not been noticed or reported in other neuromuscular disorders or in experimental in vivo conditions. Based on our observations it is tempting to speculate that the emerin loss and aberration in nuclear membrane biosynthesis could be responsible for nuclear structural abnormality. The nuclear function of emerin is at present not clear. It is only known that the protein is localized in the nuclear membrane and may form a complex with other membrane proteins. Further studies are necessary to elucidate the physiological and pathological role of emerin.

References [1] Bione S, Maestrini E, Rivella S, et al. Identification of a novel X-linked gene responsible for Emery-Dreiffus muscular dystrophy. Nat Genet 1994;8:323–7. [2] Bione S, Yates JRW, Warren ST, Merlini L, Morandi L, Toniolo D. Mutations in the Emery-Dreiffus muscular dystrophy gene and evidence for autosomal form. Neuromuscular Disord 1996;6:S15. [3] Engel WK. Muscle fibre regeneration in human neuromuscular

´ et al. / Journal of the Neurological Sciences 159 (1998) 88 – 93 A. Fidzianska

[4] [5] [6]

[7]

[8] [9]

[10]

diseases. In: Mauro M, editor. Muscle regeneration. New York: Raven Press 1979:285–96. Ghandially FN. Ultrastructural pathology of the cell and matrix. 3rd ed, vol. 1. London:Butterworths, 1988:1–158. Hausmanowa-Petrusewicz I. The Emery-Dreiffus disease. Neuropathol Pol 1988;26:265–81. Hozak P, Sasseville AMR, Raymond Y, Cook PR. Lamin proteins form an internal nucleoskeleton as well as peripheral lamina in human cells. J Cell Sci 1995;108:635–44. Kawabuchi M, Osame M, Kanaseki T. Morphogenesis of nuclear inclusions in the soleplate region of rat skeletal muscle fibres following daily administration of neostigmine. Tissue Cell Res 1989;256:137–44. Kornberg RD, Lorch Y. Chromatin structure and transcription. Annu Rev Cell Biol 1992;8:563–87. Lafarga M, Berciano MT, Andres MA. Protein synthesis inhibition induces perichromatin granulae accumulation and intranuclear rodlet formation in osmotically stimulated supraoptic neurons. Anat Embryol (Berlin) 1993;187:363–9. Manilal S, Man Nguyen thi, Sewry CA, Swry CA. The Emery-

[11]

[12]

[13]

[14] [15] [16] [17]

93

Dreiffus muscular dystrophy protein, emerin is a molecular membrane protein. Hum Mol Genet 1996;5:801–8. Manilal S, Sewry CA, Man Nguyen thi, Muntoni F, Morris GE. Diagnosis of X-linked Emery-Dreffus muscular dystrophy by protein analysis of leukocytes and skin with monoclonal antibodies. Neuromuscular Disord 1997;7:63–6. Nagano A, Koga, Ogawa M, et al. Emerin deficiency at the nuclear membrane in patients with Emery-Dreiffus muscular dystrophy. Nat Genet 1996;12;254–9. ¨ ¨ Schroder JM, Kramere KG, Hopf HC. Granular nuclear inclusion body disease: fine structure of tibial muscle and sural nerve. Muscle Nerve 1985:52–9. Soffer D. Neuronal intranuclear hyaline inclusion disease presenting as Friedrich’s ataxia. Acta Neuropathol (Berlin) 1985;65:322–9. Spector DL. Macromolecular domains within the cell nucleus. Annu Rev Cell Biol 1993;9:265–315. Steinert PM, Roop DR. Molecular and cellular biology of intermediate filaments. Annu Rev Biochem 1988;57:593–625. Tome FMS, Fardeau M. Nuclear changes in muscle disorders. Methods Achiev Exp Pathol 1986;12:261–96.