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Congenital myotonic dystrophy
Journal of the Neurological Sciences, 1987, 77:59-68
59
Elsevier JNS 02749
Congenital myotonic dystrophy Changes in muscle pathology with ageing Yuzo T a n a b e and I k u y a N o n a k a Department of Pediatrics, Chiba University School of Medicine. Chiba 280 and Division of Ultrastructural Research, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187 (Japan)
(Received 25 February, 1986) (Revised, received 10 July, 1986) (Accepted 18 July, 1986)
SUMMARY Undifferentiated type 2C fibers and satellite cells were increased in number in younger patients with congenital myotonic dystrophy (CMD) indicating immaturity in muscle fiber growth. The changes found in a 38-year-old man with CMD were identical to those described in late onset myotonic dystrophy. Type 1 fibers were found to become predominant with age. This suggests that in this disorder fiber type transformation progresses with age, presumably due to abnormal neural influences or aberrant sarcolemmal responses.
Key words: Congenital myotonic dystrophy; Fiber type transformation; Maturational defect
INTRODUCTION The congenital form of myotonic dystrophy (CMD) is a disorder that has some characteristic clinical features in the neonatal period or early infancy, including muscle hypotonia, facial diplegia, respiratory and swallowing difficulties, and joint contractures (Vanier 1960; Dodge et al. 1965; Bell and Smith 1972; Harper 1979). Skeletal muscle pathology in infants with CMD has also been well described (Karpati et al. 1973;
Correspondence address: Dr. Y. Tanabe, Divisionof Ultrastructural Research, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187, Japan. 0022-510X/87/$03.50 © 1987Elsevier Science Publishers B.V. (BiomedicalDivision)
60 Farkas et al. 1974; Sarnat and Silbert 1976; Argov et al. 1980) and it was suggested that there was a delay in fetal muscle maturation possibly due to abnormal neural trophic influences (Sarnat and Silbert 1976). Thus, it can be assumed that undifferentiated type 2C fibers and satellite cells, mononuclear blastic cells located beneath the basal lamina of muscle fibers, are found in greater number in infantile CMD patients, as is seen also in fetal muscles (Ishikawa 1966; Allbrook et al. 1971 ; Brooke et al. 1971 ; Shultz 1976; Farkas-Bargeton et al. 1977; Colling-Saltin 1978). In this study, we determined the incidence of both type 2C fibers and satellite cells in CMD and the morphological alterations with age from neonatal period to adulthood, since it is unclear whether the skeletal muscle pathology in adults with CMD corresponds to that in the late (adult) onset myotonic dystrophy. MATERIALS AND M E T H O D S
We examined the 9 patients with CMD, 6 males and 3 females, ranging in age from 1 month to 39 years. Five patients had muscle biopsies in infancy (patients 1-5), 3 in late childhood (patients 6-8), and 1 (patient 9) in adulthood. All patients developed symptoms in infancy. The common clinical picture in most of these patients included extreme hypotonia, respiratory distress at birth, feeding difficulty and facial diplegia with a tent-shaped mouth. Talipes was present in 7 patients. In 3 infants, chest roentgenograms showed elevation of the right side of the diaphragm, as described previously (Chudley and Barmada 1979). Patient 9 was a 38-year-old man who had muscle hypotonia, weakness, facial diplegia and mental retardation since infancy. The diagnosis of myotonic dystrophy was confmned clinically in all the mothers of these patients. Biopsies of the biceps brachii or quadriceps femoris muscles were frozen in isopentane cooled in liquid nitrogen. Serial sections were stained with hematoxylin and eosin (HE), modified Gomori trichrome (mGT), and a battery ofhistochemical methods (Dubowitz and Brooke 1973). Histometric analyses of muscle fiber size and numbers were performed by measuring the lesser diameter of each of the 500 neighbouring muscle fibers in the ATPase preparations with a MOP 2 semi-automatic image analyzer (Kontron). Specimens from 4 infantile patients were examined electron-microscopically after initial fLxation in glutaraldehyde solution and post-fixation in 1 ~o OsO4 containing 1 ~ lanthanum nitrate. The tissue was then dehydrated in graded ethanol and propylene oxide, and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate. Five hundred cross-sectioned muscle fibers from each muscle were photographed at final magnifications of × 1000 or x 2000, and myonuclei, satellite cells, and satellite cell nuclei were counted. Cells enclosed by basement membrane, but not by plasma membrane, were denoted as satellite cells. Control muscles for histochemical morphometry were obtained from 9 infants aged 5-30 months who presented developmental delay, but whose biopsies did not show any diagnostic abnormal lrmding. Control specimens for the satellite cell populations were obtained from 8 infants aged 5-21 months without any apparent neuromuscular disorder.
61 RESULTS
Histopathological observations In 5 infants with CMD, the most outstanding histochemical feature was the presence of small round muscle fibers with large, occasionally internal, vesicular nuclei and basophilic cytoplasm (Fig. 1A). In almost all these infants, muscle fibers varied in size and did not demonstrate distinct fiber type differentiation on NADH-TR stain, but had a thin subsarcolemmal peripheral halo devoid of enzymatic activity (Karpati et al. 1973; Sarnat and Silbert 1976) (Fig. 1B). Another histochemical abnormality seen in all infantile biopsies except one was the presence of numerous acid phosphatase positive spots in the sarcoplasm of over 60-70~o of the fibers. Neither necrosis nor phagocytosis were present. Sarcoplasmic mass, ring fibers and small angular fibers were not observed. Oil red O and PAS stains showed no abnormality. The most striking pathological finding in older (childhood) patients was a markedly increased number of fibers with internal nuclei, ranging from 40 to 70 ~ of the fibers counted (Fig. 1C). There were also several angular fibers (2 patients) and a few necrotic fibers (1 patient). There were no fibers with peripheral halo on NADH-TR stain or with high acid phosphatase activity. In the adult patient, the overall pathological Findings were quite similar to those in late onset myotonic dystrophy (Carpenter and Karpati 1984): marked variation in fiber size, an abundance of internal nuclei, several pyknotic nuclear clumps, numerous basophilic sarcoplasmic masses, and scattered necrotic fibers undergoing phagocytosis. Moderate distorsions of the intermyofibrillar network pattern were demonstrated on NADH-TR reaction. Endo- and perimysiai fibrous tissue were increased (Fig. 1D). Intrafusal muscle fibers appeared to be normal and discrete fragmentation was not seen, even in the adult patient. The major pathological changes in the muscle biopsy at different ages in our patients is summarized in Table 1.
Morphometrical analyses Fiber size and fiber type distribution on histochemistry are summarized in Table 2. On histometrical analysis, type 1 fiber atrophy, defined as a difference in the mean diameter of type 1 and type 2 fibers of more than 12~o (Brooke and Engel 1969), was present in all infantile and 2 childhood patients. Seven patients showed type 2B fiber deficiency (less than 5~), and the incidence of type 2C fibers in 4 infantile (8.2-84.9~o) and 1 childhood patients was higher than that in controls, particularly in patient 2 in whom type 2C fibers constituted approximately 85~o of all muscle fibers. The mean diameter of fibers was less than 10 #m in patients 1, 2 and 4. Type 1 fiber predominance (more than 55~) was found in all but 3 infants. In the adult patient, almost all the fibers (96.5 ~ ) behaved as type 1 fibers on myofibrillar ATPase reactions. Muscle fiber type distribution occasionally differed from fascicle to fascicle in a number of patients, especially in patient 5, who had type 1 fiber predominance (63.0~o) without type t fiber atrophy in about half of the fascicles and type 1 fiber atrophy without predominance in the others (Fig. 2). The mean fiber diameter in the former
Fig. 1..4: Note the small round muscle fibers with large internal nuclei. Patient 2 (1 month old), HE, x 800. B: Fibers with peripheral halo devoid of enzyme ~ctivit) in subsarcolemmal region. Patient 2 (1 month old), N A D H - T R , x 960. C: An increase in number of muscle fibers with internal nuclei. Patient 7 (11 )'ears old), tiE. x 160. D: Many fibers with internal nuclei, numerous sarcoplasmic masses, small angular fibers, and several necrotic fibers. Patient 9 (39)'ears old), HE, x 160.
63 TABLE 1 SUMMARY OF SKELETAL MUSCLE PATHOLOGY AT DIFFERENT AGES IN PATIENTS WITH CMD Patient No. Sex Age at biopsy Variation in fiber size Type 1 fiber atrophy Type 2B fiber deficiency Increased number of type 2C fibers Peripheral halo High acid-p, activity Type 1 fiber predominance Internal nuclei Small angular fiber Necrosis Sarcoplasmic mass Fibrosis
1 M 1 mo
2 M 1 mo
3 F 2 mo
4 M 1 yr
5 M 1 yr
6 M 10 yr
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
7 F 11 yr +
8 F 12 yr +
9 M 39 yr +
+ +
+
+
+
+
+
+
+
+
+
+
+ +
+
+
+ +
+ + + + +
fascicles were significantly larger than those in the latter. The incidence of type 2C fibers was not different from fascicle to fascicle.
Satellite cell populations and electron microscopic observations The number o f myonuclei in 500 muscle fibers varied in the muscles of infantile patients. They were significantly increased in number in 2 patients, particularly in patient 5 who was 1 year old. In contrast, the ratio of satellite cell nuclei to subsarcolemmal nuclei decreased from 24.8% to 14.0% with age, although in all C M D infants it was higher than that in control infants (Table 3). Electron-microscopic examination (patients 1 and 2) showed similar and characteristic findings. There were a number of small caliber fibers with occasional central euchromatic and large convoluted nuclei. M o s t o f fibers contained loosely packed disorganized myofibrils. In the subsarcolemmal zone, the organelles including mitochondria and sarcoplasmic reticulum were almost absent, while small amounts of disorganized myofilaments and glycogen granules were seen. Multiple satellite cells within a c o m m o n basement m e m b r a n e were rarely seen in these two patients.
a h c o
23.6 8.7 42.0 65.0 a 63.0 a 34.1 64.7 a 98.6 a 74. I a 96.5 a 44.0 _+ 4.7
1 34.9 4.9 13.9 31.3 28.4 50.8 18.3 0 22.7 1.2 28.1 _+ 8.1
2A
Fiber type distribution (~o)
Type 1 fiber p r e d o m i n a n c e . Significantly increased n u m b e r of type 2C fibers. Type 1 fiber atrophy. Age of controls; 5 - 3 0 months, n = q. S D = s t a n d a r d deviation.
1 2 3 4 5 Fascicles A B 6 7 8 9 Controls e ( M e a n _+ SD)
Patient No.
FIBER SIZE AND FIBER TYPE DISTRIBUTION
TABLE 2
8.9 1.5 11.5 1.1 0.4 5.2 12.5 0.4 1.6 1.4 26.1 _+ 7.0
2B 32.6 b 84.9 b 32.6 b 2.6 8.2 b 9.9 b 4.5 1.0 1.6 0.9 1.8 _+ 1.2
2C
IN PATIENTS WITH CMD
7.3 8.5 12.8 9.2 18.9 11.4 16.2 30.9 34.4 47.0 20.7
1
i 1.6 c -+ 2.4 ¢ -+ 3.4 c _+ 2.4 c _+ 4.5 _+ 2.4 c _+ 3.3 c _+ 15.4 _+ 7.5 ~ _+ 17.2 _+ 3.6 _+ _+ _+ _+ _+ _+ _+
2.9 2.0 4.0 3.5 5.1 3.3 9.4 46.2 _+ 14.7 50.9 + 2.0 17.7 _+ 3.4
9.2 10,4 18.6 10.7 18.1 14.4 32.8
2A
Fiber d i a m e t e r ( m e a n _+ S D , / ~ m )
9.6_+ 2.1 11.1 + 3.9 17.2 -+ 4.5 12.5 + 2.2 14.9 -+ 3.3 16.9 _+ 4.6 41.9 -+ 9.6 25.7 _+ 4.1 46.8 _+ 13.8 39.4 _+ 15.5 18.8 _+ 2.7
2B
8.2 _+ 2.0 9.4 + 3.4 16.9 + 3.7 9.9 _+ 2.2 14.3 + 4.4 11.2 + 2.3 18.0 + 7.2 31.1 i 8.1 27.3 _+ 9.3 31,8 + 2.0 17.4 i 3.O
2C
4~
65
Fig. 2. A: Note the difference in fiber type distribution from fascicle to fascicle: type 1 fiber predominance in the left half of field, and normal distribution but type 1 fiber atrophy in the fight. The left and right halves of the field are illustrated in B and C respectively at high magnification. Patient 5 (1 year old). A - C : ATPase (pH4.3),A: x 250, B and C: x 500.
66 TABLE 3 SATELLITE CELL P O P U L A T I O N IN I N F A N T I L E PATIENTS W I T H C M D Patient No.
Age/Sex
Number of M F
Number of M N
Number of SC
Number of SN
Incidence of SC/MF
(°Jo) 1 2 3 5 Controls (Mean + SD) (n = 8)
lm/M lm/M 2m/F ly/M
500 500 500 500 500
90 104 183 281 126.6 + 15.1
84 63 41 61 14.6 + 4.2
30 31 32 45 11.6 _+3.0
16.8 12.8 8.2 12.3 2.9 _+0.9
Incidence of SN/(MN + SN)
(o~)
24.8 23.7 14.9 14.0 8.4 + 1.6
M F = muscle fibers, M N = myonuclei, SC = satellite cells, SN = satellite cell nuclei, SD = standard deviation.
DISCUSSION
Our morphometrical results in infants with CMD are further confn'mation of the concept that muscle fibers in CMD patients are immature (Karpati et al. 1973; Farkas etal. 1974; Sarnat and Silbert 1976; Argov etal. 1980). The incidence of undifferentiated type 2C fibers was increased from 8.2~o to 84.9~o in 4 patients, and the ratio of satelfite eell nuclei to subsarcolemmal nuclei was also significantly increased from 14.0~o to 24.8~ in 5 infantile patients. It has been known that undifferentiated type 2C fibers and the satellite cells increase in number during the process of muscle regeneration (Wakayama and Schotland 1979; Nonaka et al. 1981; Ishimoto et al, 1983) and during early muscle development (Ishikawa 1966; Allbrook et al, 1971; Brooke et al. 1971; Farkas-Bargeton et al. 1977). Since necrotic or regenerating fibers were rarely found in infantile CMD, we infer that these findings reflected muscle fiber immaturity. Another aim of this study was to determine changes in muscle pathology in CMD after the neonatal period, since previous studies had been limited mainly to infantile patients. Type 1 fiber predominance with many centronuclear fibers along with selective type 1 fiber atrophy were present in 3 childhood patients. The findings in 1 adult patient with CMD were consistent with those found in the late onset form of myotonic dystrophy. These results indicate that the nature of the muscle pathology in the early infantile stage of the congenital form of myotonic dystrophy is different from that in the late onset form, but that they come to resemble each other in the later stages as CMD patients survive into adulthood. The change of fiber type distribution with ~ may provide us with more important information on the pathogenesis of the disease. In our patients selective type 1 fiber atrophy persisted throughout infancy and childhood, and type 1 fiber predominance was found even in infancy and tended to be more marked in adulthood. The
67 pathogenesis of type 1 fiber predominance has not been established, but it is postulated that a single muscle fiber type has been steadily transformed during the evolution of this disease. This contention is supported by the findings in patient 5. There was type 1 fiber predominance in some fascicles, where the mean fiber diameter was larger than that in other fascicles with type 1 fiber atrophy but no type 1 fiber predominance. It may be that the fiber type distribution in the latter fascicles is transformed into that in the former. Since it has been shown that changing motor nerve innervation can lead to fiber type transformation (Dubowitz 1967; Karpati and Engel 1968), the fiber type changes in CMD may result from neural transformation, from type 2 to type 1, possibly induced by abnormal trophic factors. ACKNOWLEDGEMENTS
The authors wish to express their cordial thanks to Profs. Hironori Nakajima (Chiba University School of Medicine, Chiba, Japan), Ikuya Nonaka (National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan) and S. Mark Sumi (University of Washington, Seattle, U.S.A.) and for their kind suggestions and criticism on this work. REFERENCES Allbrook, D. B., M. F. Han and W.H. Kirkaldy-Willis (1971) Population of muscle satellite cells in relation to age and mitotic activity, Pathology, 3: 233-243. Argov, Z., D. Gardner-Medwin, M. A. Johnson and F.L. Mastaglia (1980) Congenital myotonic dystrophy: fiber type abnormalities in two cases, Arch. Neurol., 37: 693-696. Bell, D, B. and D.W. Smith (1972) Myotonic dystrophy in the neonate, J. Pedlar., 81: 83-86. Brooke, M.H. and W.K. Engel (1969) The histographic analysis of human muscle biopsies with regard to fiber types, Part 4 (Children's biopsies), Neurology (Minneap.), 19: 591-599. Brooke, M.H., E. Williamson and K.K. Kaiser (1971) The behavior of four fiber types in developing and reinnervated muscle, Arch. Neurol., 25: 360-366. Carpenter, S. and G. Karpati (1984) Pathology of Skeletal Muscle, Churchill Livingstone, New York, pp. 616-630. Chudley, A. E. and M. A. Barmada (1979) Diaphragmatic elevation in neonatal myotonic dystrophy, Amer. J. Dis. Child., 133: 1182-1185. Colling-Saltin, A. (1978) Enzyme histochemistry on skeletal muscle of human foetus, J. Neurol. Sci., 39: 169-185. Dodge, P. R., I. Gamstorp, R. K. Byers and P. Russell (1965) Myotonic dystrophy in infancy and childhood, Pediatrics, 35: 3-19. Dubowitz, V. (1967) Pathology of experimentally re-innervated skeletal muscle, ,/. Neurol. Neurosurg. Psychiat., 30:99-110. Dubowitz, V, and M.H. Brooke (1973) Muscle Biopsy ~ A Modem Approach, Saunders, London. Farkas, E., F.M.S. Tom6, M, Fardeau, M.L. Ars6nio-Nufies, P. Dreyfus and M.F. Diebler (1974) Histochemical and ultrastructural study of muscle biopsies in 3 cases of dystrophia myotonica in the newborn child, J. Neurol. Sci., 21 : 273-288. Farkas-Bargeton, E., M.F. Diebler, M.L. Ars6nio-Nufles, R. Wehl6 and B. Rosenberg (1977) Etude de la maturation histoch6mique, quantitative et ultrastructurale de muscle foetal humain, J. Neurol. Sci., 31: 245-259. Harper, P.S. (1979)Myotonic Dystrophy, S aunders, Philadelphia, pp. 170-206. Ishikawa, H. (1966) Electron microscopic observations of satellite cells with special reference to the development of mammalian skeletal muscles, Z. Anat. Entwickl-Gesch., 125: 43-63. Ishimoto, S., I. Goto, M. Ohta and Y. Kuroiwa (1983) A quantitative study of the muscle satellite cells in various neuromuscular disorders, J. Neurol. Sci., 62:303-314.
68 Karpati, G. and W.K. Engel (1968) 'Type grouping' in skeletal muscles after experimental reinnervation, Neurology' (Minneap.), 18: 447-455. Karpati, G., S. Carpenter, G. V. Watters, A. A. Eisen and F. Anderman (1973) Infantile myotonic dystrophy. Histochemical and electron microscopical features in skeletal muscle, Neurology (Minneap.L 23: 1066-1077. Nonaka, 1., A. Takagi and H. Sugita ( 1981) The significance of type 2C muscle fibers in Duchenne muscular dystrophy, Muscle & Nerve, 4: 326-333. Sarnat, H.B. and S.W. Silbert (1976) Maturational arrest of fetal muscle in neonatal myotonic dystrophy, Arch, Neurol., 33: 466-474. Shultz, E. (1976) A quantitative study of the satellite cell population in postnatal mouse lumbrical muscle, Anat. Rec., 185: 279-288. Vanier, T.M. (1960) Dystrophia myotonica in childhood, Brit. Med. J., II: 1284-1288. Wakayama, Y. and D.L. Schotland (1979) Muscle satellite cell populations in Duchenne dystrophy. In: A. Mauro (Ed.), Muscle Regeneration, Raven Press, New York, pp. 120-129.
59
Elsevier JNS 02749
Congenital myotonic dystrophy Changes in muscle pathology with ageing Yuzo T a n a b e and I k u y a N o n a k a Department of Pediatrics, Chiba University School of Medicine. Chiba 280 and Division of Ultrastructural Research, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187 (Japan)
(Received 25 February, 1986) (Revised, received 10 July, 1986) (Accepted 18 July, 1986)
SUMMARY Undifferentiated type 2C fibers and satellite cells were increased in number in younger patients with congenital myotonic dystrophy (CMD) indicating immaturity in muscle fiber growth. The changes found in a 38-year-old man with CMD were identical to those described in late onset myotonic dystrophy. Type 1 fibers were found to become predominant with age. This suggests that in this disorder fiber type transformation progresses with age, presumably due to abnormal neural influences or aberrant sarcolemmal responses.
Key words: Congenital myotonic dystrophy; Fiber type transformation; Maturational defect
INTRODUCTION The congenital form of myotonic dystrophy (CMD) is a disorder that has some characteristic clinical features in the neonatal period or early infancy, including muscle hypotonia, facial diplegia, respiratory and swallowing difficulties, and joint contractures (Vanier 1960; Dodge et al. 1965; Bell and Smith 1972; Harper 1979). Skeletal muscle pathology in infants with CMD has also been well described (Karpati et al. 1973;
Correspondence address: Dr. Y. Tanabe, Divisionof Ultrastructural Research, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187, Japan. 0022-510X/87/$03.50 © 1987Elsevier Science Publishers B.V. (BiomedicalDivision)
60 Farkas et al. 1974; Sarnat and Silbert 1976; Argov et al. 1980) and it was suggested that there was a delay in fetal muscle maturation possibly due to abnormal neural trophic influences (Sarnat and Silbert 1976). Thus, it can be assumed that undifferentiated type 2C fibers and satellite cells, mononuclear blastic cells located beneath the basal lamina of muscle fibers, are found in greater number in infantile CMD patients, as is seen also in fetal muscles (Ishikawa 1966; Allbrook et al. 1971 ; Brooke et al. 1971 ; Shultz 1976; Farkas-Bargeton et al. 1977; Colling-Saltin 1978). In this study, we determined the incidence of both type 2C fibers and satellite cells in CMD and the morphological alterations with age from neonatal period to adulthood, since it is unclear whether the skeletal muscle pathology in adults with CMD corresponds to that in the late (adult) onset myotonic dystrophy. MATERIALS AND M E T H O D S
We examined the 9 patients with CMD, 6 males and 3 females, ranging in age from 1 month to 39 years. Five patients had muscle biopsies in infancy (patients 1-5), 3 in late childhood (patients 6-8), and 1 (patient 9) in adulthood. All patients developed symptoms in infancy. The common clinical picture in most of these patients included extreme hypotonia, respiratory distress at birth, feeding difficulty and facial diplegia with a tent-shaped mouth. Talipes was present in 7 patients. In 3 infants, chest roentgenograms showed elevation of the right side of the diaphragm, as described previously (Chudley and Barmada 1979). Patient 9 was a 38-year-old man who had muscle hypotonia, weakness, facial diplegia and mental retardation since infancy. The diagnosis of myotonic dystrophy was confmned clinically in all the mothers of these patients. Biopsies of the biceps brachii or quadriceps femoris muscles were frozen in isopentane cooled in liquid nitrogen. Serial sections were stained with hematoxylin and eosin (HE), modified Gomori trichrome (mGT), and a battery ofhistochemical methods (Dubowitz and Brooke 1973). Histometric analyses of muscle fiber size and numbers were performed by measuring the lesser diameter of each of the 500 neighbouring muscle fibers in the ATPase preparations with a MOP 2 semi-automatic image analyzer (Kontron). Specimens from 4 infantile patients were examined electron-microscopically after initial fLxation in glutaraldehyde solution and post-fixation in 1 ~o OsO4 containing 1 ~ lanthanum nitrate. The tissue was then dehydrated in graded ethanol and propylene oxide, and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate. Five hundred cross-sectioned muscle fibers from each muscle were photographed at final magnifications of × 1000 or x 2000, and myonuclei, satellite cells, and satellite cell nuclei were counted. Cells enclosed by basement membrane, but not by plasma membrane, were denoted as satellite cells. Control muscles for histochemical morphometry were obtained from 9 infants aged 5-30 months who presented developmental delay, but whose biopsies did not show any diagnostic abnormal lrmding. Control specimens for the satellite cell populations were obtained from 8 infants aged 5-21 months without any apparent neuromuscular disorder.
61 RESULTS
Histopathological observations In 5 infants with CMD, the most outstanding histochemical feature was the presence of small round muscle fibers with large, occasionally internal, vesicular nuclei and basophilic cytoplasm (Fig. 1A). In almost all these infants, muscle fibers varied in size and did not demonstrate distinct fiber type differentiation on NADH-TR stain, but had a thin subsarcolemmal peripheral halo devoid of enzymatic activity (Karpati et al. 1973; Sarnat and Silbert 1976) (Fig. 1B). Another histochemical abnormality seen in all infantile biopsies except one was the presence of numerous acid phosphatase positive spots in the sarcoplasm of over 60-70~o of the fibers. Neither necrosis nor phagocytosis were present. Sarcoplasmic mass, ring fibers and small angular fibers were not observed. Oil red O and PAS stains showed no abnormality. The most striking pathological finding in older (childhood) patients was a markedly increased number of fibers with internal nuclei, ranging from 40 to 70 ~ of the fibers counted (Fig. 1C). There were also several angular fibers (2 patients) and a few necrotic fibers (1 patient). There were no fibers with peripheral halo on NADH-TR stain or with high acid phosphatase activity. In the adult patient, the overall pathological Findings were quite similar to those in late onset myotonic dystrophy (Carpenter and Karpati 1984): marked variation in fiber size, an abundance of internal nuclei, several pyknotic nuclear clumps, numerous basophilic sarcoplasmic masses, and scattered necrotic fibers undergoing phagocytosis. Moderate distorsions of the intermyofibrillar network pattern were demonstrated on NADH-TR reaction. Endo- and perimysiai fibrous tissue were increased (Fig. 1D). Intrafusal muscle fibers appeared to be normal and discrete fragmentation was not seen, even in the adult patient. The major pathological changes in the muscle biopsy at different ages in our patients is summarized in Table 1.
Morphometrical analyses Fiber size and fiber type distribution on histochemistry are summarized in Table 2. On histometrical analysis, type 1 fiber atrophy, defined as a difference in the mean diameter of type 1 and type 2 fibers of more than 12~o (Brooke and Engel 1969), was present in all infantile and 2 childhood patients. Seven patients showed type 2B fiber deficiency (less than 5~), and the incidence of type 2C fibers in 4 infantile (8.2-84.9~o) and 1 childhood patients was higher than that in controls, particularly in patient 2 in whom type 2C fibers constituted approximately 85~o of all muscle fibers. The mean diameter of fibers was less than 10 #m in patients 1, 2 and 4. Type 1 fiber predominance (more than 55~) was found in all but 3 infants. In the adult patient, almost all the fibers (96.5 ~ ) behaved as type 1 fibers on myofibrillar ATPase reactions. Muscle fiber type distribution occasionally differed from fascicle to fascicle in a number of patients, especially in patient 5, who had type 1 fiber predominance (63.0~o) without type t fiber atrophy in about half of the fascicles and type 1 fiber atrophy without predominance in the others (Fig. 2). The mean fiber diameter in the former
Fig. 1..4: Note the small round muscle fibers with large internal nuclei. Patient 2 (1 month old), HE, x 800. B: Fibers with peripheral halo devoid of enzyme ~ctivit) in subsarcolemmal region. Patient 2 (1 month old), N A D H - T R , x 960. C: An increase in number of muscle fibers with internal nuclei. Patient 7 (11 )'ears old), tiE. x 160. D: Many fibers with internal nuclei, numerous sarcoplasmic masses, small angular fibers, and several necrotic fibers. Patient 9 (39)'ears old), HE, x 160.
63 TABLE 1 SUMMARY OF SKELETAL MUSCLE PATHOLOGY AT DIFFERENT AGES IN PATIENTS WITH CMD Patient No. Sex Age at biopsy Variation in fiber size Type 1 fiber atrophy Type 2B fiber deficiency Increased number of type 2C fibers Peripheral halo High acid-p, activity Type 1 fiber predominance Internal nuclei Small angular fiber Necrosis Sarcoplasmic mass Fibrosis
1 M 1 mo
2 M 1 mo
3 F 2 mo
4 M 1 yr
5 M 1 yr
6 M 10 yr
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
7 F 11 yr +
8 F 12 yr +
9 M 39 yr +
+ +
+
+
+
+
+
+
+
+
+
+
+ +
+
+
+ +
+ + + + +
fascicles were significantly larger than those in the latter. The incidence of type 2C fibers was not different from fascicle to fascicle.
Satellite cell populations and electron microscopic observations The number o f myonuclei in 500 muscle fibers varied in the muscles of infantile patients. They were significantly increased in number in 2 patients, particularly in patient 5 who was 1 year old. In contrast, the ratio of satellite cell nuclei to subsarcolemmal nuclei decreased from 24.8% to 14.0% with age, although in all C M D infants it was higher than that in control infants (Table 3). Electron-microscopic examination (patients 1 and 2) showed similar and characteristic findings. There were a number of small caliber fibers with occasional central euchromatic and large convoluted nuclei. M o s t o f fibers contained loosely packed disorganized myofibrils. In the subsarcolemmal zone, the organelles including mitochondria and sarcoplasmic reticulum were almost absent, while small amounts of disorganized myofilaments and glycogen granules were seen. Multiple satellite cells within a c o m m o n basement m e m b r a n e were rarely seen in these two patients.
a h c o
23.6 8.7 42.0 65.0 a 63.0 a 34.1 64.7 a 98.6 a 74. I a 96.5 a 44.0 _+ 4.7
1 34.9 4.9 13.9 31.3 28.4 50.8 18.3 0 22.7 1.2 28.1 _+ 8.1
2A
Fiber type distribution (~o)
Type 1 fiber p r e d o m i n a n c e . Significantly increased n u m b e r of type 2C fibers. Type 1 fiber atrophy. Age of controls; 5 - 3 0 months, n = q. S D = s t a n d a r d deviation.
1 2 3 4 5 Fascicles A B 6 7 8 9 Controls e ( M e a n _+ SD)
Patient No.
FIBER SIZE AND FIBER TYPE DISTRIBUTION
TABLE 2
8.9 1.5 11.5 1.1 0.4 5.2 12.5 0.4 1.6 1.4 26.1 _+ 7.0
2B 32.6 b 84.9 b 32.6 b 2.6 8.2 b 9.9 b 4.5 1.0 1.6 0.9 1.8 _+ 1.2
2C
IN PATIENTS WITH CMD
7.3 8.5 12.8 9.2 18.9 11.4 16.2 30.9 34.4 47.0 20.7
1
i 1.6 c -+ 2.4 ¢ -+ 3.4 c _+ 2.4 c _+ 4.5 _+ 2.4 c _+ 3.3 c _+ 15.4 _+ 7.5 ~ _+ 17.2 _+ 3.6 _+ _+ _+ _+ _+ _+ _+
2.9 2.0 4.0 3.5 5.1 3.3 9.4 46.2 _+ 14.7 50.9 + 2.0 17.7 _+ 3.4
9.2 10,4 18.6 10.7 18.1 14.4 32.8
2A
Fiber d i a m e t e r ( m e a n _+ S D , / ~ m )
9.6_+ 2.1 11.1 + 3.9 17.2 -+ 4.5 12.5 + 2.2 14.9 -+ 3.3 16.9 _+ 4.6 41.9 -+ 9.6 25.7 _+ 4.1 46.8 _+ 13.8 39.4 _+ 15.5 18.8 _+ 2.7
2B
8.2 _+ 2.0 9.4 + 3.4 16.9 + 3.7 9.9 _+ 2.2 14.3 + 4.4 11.2 + 2.3 18.0 + 7.2 31.1 i 8.1 27.3 _+ 9.3 31,8 + 2.0 17.4 i 3.O
2C
4~
65
Fig. 2. A: Note the difference in fiber type distribution from fascicle to fascicle: type 1 fiber predominance in the left half of field, and normal distribution but type 1 fiber atrophy in the fight. The left and right halves of the field are illustrated in B and C respectively at high magnification. Patient 5 (1 year old). A - C : ATPase (pH4.3),A: x 250, B and C: x 500.
66 TABLE 3 SATELLITE CELL P O P U L A T I O N IN I N F A N T I L E PATIENTS W I T H C M D Patient No.
Age/Sex
Number of M F
Number of M N
Number of SC
Number of SN
Incidence of SC/MF
(°Jo) 1 2 3 5 Controls (Mean + SD) (n = 8)
lm/M lm/M 2m/F ly/M
500 500 500 500 500
90 104 183 281 126.6 + 15.1
84 63 41 61 14.6 + 4.2
30 31 32 45 11.6 _+3.0
16.8 12.8 8.2 12.3 2.9 _+0.9
Incidence of SN/(MN + SN)
(o~)
24.8 23.7 14.9 14.0 8.4 + 1.6
M F = muscle fibers, M N = myonuclei, SC = satellite cells, SN = satellite cell nuclei, SD = standard deviation.
DISCUSSION
Our morphometrical results in infants with CMD are further confn'mation of the concept that muscle fibers in CMD patients are immature (Karpati et al. 1973; Farkas etal. 1974; Sarnat and Silbert 1976; Argov etal. 1980). The incidence of undifferentiated type 2C fibers was increased from 8.2~o to 84.9~o in 4 patients, and the ratio of satelfite eell nuclei to subsarcolemmal nuclei was also significantly increased from 14.0~o to 24.8~ in 5 infantile patients. It has been known that undifferentiated type 2C fibers and the satellite cells increase in number during the process of muscle regeneration (Wakayama and Schotland 1979; Nonaka et al. 1981; Ishimoto et al, 1983) and during early muscle development (Ishikawa 1966; Allbrook et al, 1971; Brooke et al. 1971; Farkas-Bargeton et al. 1977). Since necrotic or regenerating fibers were rarely found in infantile CMD, we infer that these findings reflected muscle fiber immaturity. Another aim of this study was to determine changes in muscle pathology in CMD after the neonatal period, since previous studies had been limited mainly to infantile patients. Type 1 fiber predominance with many centronuclear fibers along with selective type 1 fiber atrophy were present in 3 childhood patients. The findings in 1 adult patient with CMD were consistent with those found in the late onset form of myotonic dystrophy. These results indicate that the nature of the muscle pathology in the early infantile stage of the congenital form of myotonic dystrophy is different from that in the late onset form, but that they come to resemble each other in the later stages as CMD patients survive into adulthood. The change of fiber type distribution with ~ may provide us with more important information on the pathogenesis of the disease. In our patients selective type 1 fiber atrophy persisted throughout infancy and childhood, and type 1 fiber predominance was found even in infancy and tended to be more marked in adulthood. The
67 pathogenesis of type 1 fiber predominance has not been established, but it is postulated that a single muscle fiber type has been steadily transformed during the evolution of this disease. This contention is supported by the findings in patient 5. There was type 1 fiber predominance in some fascicles, where the mean fiber diameter was larger than that in other fascicles with type 1 fiber atrophy but no type 1 fiber predominance. It may be that the fiber type distribution in the latter fascicles is transformed into that in the former. Since it has been shown that changing motor nerve innervation can lead to fiber type transformation (Dubowitz 1967; Karpati and Engel 1968), the fiber type changes in CMD may result from neural transformation, from type 2 to type 1, possibly induced by abnormal trophic factors. ACKNOWLEDGEMENTS
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