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Involvement of fast and slow twitch muscle fibres in avian muscular dystrophy
Journal of the Neurological Sciences, 1983, 61:217-233
217
Elsevier
I N V O L V E M E N T OF FAST A N D SLOW T W I T C H M U S C L E FIBRES IN AVIAN MUSCULAR DYSTROPHY
JOHN A. PIZZEY, ERIC A. BARNARD and PENELOPE J. BARNARD. Department of Biochemistry, Imperial College of Science and Technology, London (Great Britain)
(Received 28 March, 1983) (Accepted 28 April, 1983)
SUMMARY The extent of differential fibre type involvement in chicken muscular dystrophy can be assessed quantitatively by the statistical parameters of fibre area, nuclei content and nuclei distribution in the individual fibre types. Two muscles, the posterior latissimus dorsi (PLD) and the serratus metapatagialis (SMP), were found to have similar overall fibre type composition, although the latter contains two subtypes of type I fibres, one of which has not previously been recognised in avian muscle. In both muscles, type IIB fibres are most affected by the progressive pathology. Nuclear proliferation is one of the histopathological features which can be measured, and in the PLD, the mean number of total nuclei in type liB fibre cross-sections (N0 is increased from 2.23 in normal chickens to 3.70 in dystrophic chickens, by 60 days. The corresponding values for N~ in type liB muscle fibres o f the SMP at 50 days are 1.74 and 5.10. Likewise, statistical analyses of the distribution of the fibre areas and their variability demonstrate that the incidence of abnormality in chicken dystrophy is greatest in type liB fibres in both these muscles. Although type I fibres in the P L D are resistant to dystrophic change, it is noteworthy that in the SMP the type I fibres, also, are severely affected from an early stage, by these quantitative criteria. On the other hand, all fibres in a tonic muscle, the metapatagialis latissimus dorsi, are unaffected, as is true of all other tonic muscles previously studied. It is concluded that any twitch fibre type can, in principle, be affected by the actions of the gene concerned, and that this
This work was supported by the Muscular Dystrophy Group of Great Britain. Address reprint requests to Professor E.A. Barnard, Department of Biochemistry, Imperial College of Scienceand Technology,London, SW7 2AZ, Great Britain. 0022-510X/83/$03.00 © 1983 Elsevier SciencePublishers B.V.
218 expression can be greatly modified in individual muscles by various physiological features, for example their natural pattern of use or relative disuse.
Key words: C h i c k e n m u s c l e s - M u s c l e f i b r e s - M u s c u l a r d y s t r o p h y
INTRODUCTION
In the inherited muscular dystrophies of man or of experimental animals, the progress of the histopathological changes through the population of different muscle fibre types is a matter of considerable significance. If it were to be found that all fibre types are affected equally, this could, in the absence of a general neurological lesion, indicate a defect in a feature characteristic of the skeletal muscle cell in general. If, instead, selectivity is found for one fibre type, this could be due to a neurogenic defect in a particular class of motor neuron, since these are specific for the muscle fibre types (Burke et al. 1971), but in that case one might expect to see that same fibre type always affected wherever it occurs in muscles. The differential involvement of different muscles, well-known to occur in these myopathies, should be analysed to test the possibility that it arises from the variations in the proportions of fibre types in those muscles. In general, the pattern of selection of fibre types within one muscle is one of the basic parameters needed for the understanding of muscle pathology in such diseases. In the fibre type nomenclature employed for human muscles by Brooke and Kaiser (1970) and Dubowitz and Brooke (1973), adult human muscles were shown by those authors to contain generally three fibre types: I, IIA or liB, corresponding to slow-twitch oxidative, fast-twitch oxidative/glycolytic,or fast-twitch glycolytic, respectively, as described by others (Peter et al. 1972). Also, a very few human and mammalian muscles contain slow-tonic fibres (Hess 1970). It is well known that not all muscles are affected to the same extent in Duchenne muscular dystrophy (DMD). Thus, the rare tonic muscles in man such as the cremaster (Bonsett 1969) and some external ocular muscles (Gardner-Medwin 1980) are very resistant to the pathological change. In the affected muscles in DMD, some studies have suggested that type II fibres are primarily involved and type I are less affected (Baloh and Cancilla 1972; Johnson et al. 1973; Johnson and Kucukgalcin 1978; Ellis 1980). Further, an increasing predominance of the type I fibres and a deficiency of the type liB fibres in DMD or carrier muscle biopsies has generally been described (Brooke and Engel 1969; Dubowitz and Brooke 1973; Maunder-Sewry and Dubowitz 1981; Nonaka et al. 1981). However, Dubowitz and Brooke (1973) concluded that selective involvement of any one fibre type does not appear to be prominent in DMD, and others, also, have found no such selection there (Engel 1970; Buchthal et al. 1974). These comparisons are complicated in humans by the marked variability of fibre types, in both percentage and fibre size, within the normal population (Gollnick et al. 1972; Prince et al. 1976) and with exercise
219 and training (Andersen and Henriksson 1977; Jansson and Kaijser 1977). In inherited muscular dystrophy of the chicken, many of the histological, physical and biochemical features of D M D are reproduced (Asmundson and Julian 1956; Wilson et al. 1979; Barnard 1980) and, in particular, most of the histopathological changes are seen, both by electron (Libelius et al. 1979) and light microscopy (Barnard et al. 1982; Pizzey and Barnard 1983a). In such a model, the muscle at any stage can conveniently be analysed, in a population of genetically closely-related animals under standard conditions. It is already established that slow tonic muscles such as the anterior latissimus dorsi (Wilson et al. 1973), the plantaris or the adductor profundus (Barnard et al. 1982) are not significantly affected by the disease. A similar fibre type nomenclature to that noted above for mammals has recently been shown to account for the fibre types present in a wide range of chicken twitch muscles, although there are some secondary differences between a given fibre type in the two cases (Barnard et al. 1982). Using that nomenclature here, we can note that only type IIB fibres have previously been reported to be selected for pathological changes in the dystrophic chicken (Cosmos and Butler 1967; Ashmore and Doerr 1971; Beringer 1978). Type I fibres have been described as resistant to the disease (Shafiq et al. 1971; Ashmore et al. 1978). However, a detailed quantitative analysis of histopathological changes within the individual fibre types present in dystrophic muscles has not been reported, and it has seemed to us of interest to obtain quantitative expressions of the fibre type selectivity in the chicken muscles. We have included in this analysis two cutaneous muscles (a class hitherto unexamined in the dystrophic chicken), one of which contains an appreciable number of type I fibres. We show that these slowtwitch oxidative fibres can, indeed, be affected by the disease. Hence, it will be argued, factors additional to the neural control of muscle fibre type can regulate the pathology. MATERIALS A N D METHODS
Animals Normal New Hampshire chickens (London-line 412) and the corresponding line homozogous for muscular dystrophy (London-line 413) were bred and maintained in the Imperial College Dystrophic Chicken Research Facility. Both lines were derived from the corresponding University of California (Davis) lines (Wilson et al. 1979). Chickens were kept on a 12-h light cycle at 25°C and at 40-50~ relative humidity. All chickens had free access to food and water. Muscle specimens Chickens (35-90 days) were killed by cervical dislocation, and the muscle samples were at once removed. The serratus metapatagialis (SMP) is a white muscle, unlike the pigeon SMP (Khan 1979), and corresponds to the cutaneous muscle in another nomenclature (Saunders and Manton 1959). The metapatagialis latissimus dorsi (MLD) muscle was identified as described by Grim (1971). The
220
DLD
i
MLD SMP
A
Fig. 1. Schematic diagram of the dorsal view of the proximal wing muscles of the chicken showing the latissimus dorsi and associated dermal muscles. The DLD, MLD and SMP muscles all have dermal insertions. ALD = M. anterior latissimus dorsi; DLD = M. dorsocutaneous latissimus dorsi; MLD = M. metapatagialis latissirnus dorsi; SMP = M. serratus metapatagialis; ST = M. scapulotriceps.
locations of these muscles are illustrated in Fig. 1. Samples were taken from the mid-regions of the SMP, M L D and posterior latissimus dorsi (PLD) muscles from 5 normal and 5 dystrophic chickens. For the histological analysis and fibre-typing, the samples were immediately frozen in isopentane cooled by liquid nitrogen, and stored at - 8 5 °C prior to cutting 10-/~m sections at - 2 0 °C on a Bright cryostat, orientated for true transverse sections. End-plate localization was performed on samples which were frozen or fixed briefly in 2~o glutaraldehyde/0.1M sodium acetate (pH 5.9) at r o o m temperature.
Histological and histochemical techniques Frozen sections were stained for histological analysis with haematoxylin and eosin (HE). In addition, serial sections were stained (Dubowitz and Brooke 1973) for N A D H tetrazolium reductase ( N A D H - T R ) , or phosphorylase activity, or ATPase after pre-incubation at p H 4.3 or p H 4.6 or p H 9.4. Small samples of the fixed muscle were stained for acetylcholinesterase (ACHE) activity (Tsuji 1974) and teased in glycerin to separate fibres for end-plate counting. Longitudinal frozen sections were fixed for 30 min in 2~o glutaraldehyde and similarly stained for ACHE. All sections were photographed and individual fibres were typed as described elsewhere (Barnard et al. 1982) using 300-500 fibre cross-sections from each an!mal, i.e., a minimum of 1500 fibres of each genotype was analysed. All the type I fibres present in all of the sections of SMP and P L D muscle were analysed for the histological measurements, due to the relatively low frequency with which type I fibres occur in these muscles. Fibre areas were measured after projecting randomly-chosen negatives of ATPase-stained sections (pre-incubated at p H 4.6) onto the surface of the digit-
221 izing tablet of a Hewlett-Packard mini-computer. Recognition of fibre types was then performed by reference to micrographs of serial sections stained by the different methods described above. Diameters were randomly drawn across the negatives and, wherever possible, 100 fibres of each fibre type were measured for each bird. Fibres showing evidence of tangential sectioning (as indicated by an elliptical profile) were excluded. Normal and dystrophic fibre area histograms were compared using the Kolmogorov-Smirnov two-sample test (Sokal and Rohlf 1981). For each muscle sample the hypertrophy factor was calculated as described by Dubowitz and Brooke (1973), this giving a weighted score to abnormally large fibres. The number and distribution of nuclei in cross-sections of the muscle fibres were determined from HE sections, again with reference to serial sections for fibre typing as above. Again, wherever possible, for each bird, 100 fibres of each fibre type, falling on randomly-drawn diameters, were measured for nuclei number and distribution. Internal nuclei (Ni) were counted separately and the contents of normal and dystrophic fibres were compared using the t -test. RESULTS
Muscles and fibre types investigated The first muscle analysed was the PLD, a white, fast-contracting muscle, which is very commonly used for studies of avian dystrophic muscle. In the strain (413) of dystrophic chickens which we use, pathological change in this muscle becomes clearly established by about 4 weeks of age (being detectable even earlier), as assessed by histological changes and abnormal enzyme contents (Lyles et al. 1979; Barnard et al. 1982; Pizzey and Barnard 1983a). The second muscle, the SMP, is a small cutaneous muscle (Fig. 1) not previously analysed histologically in the dystrophic or normal chicken. The SMP muscle, like the PLD, is strongly affected by the disease (Fig. 2). Characteristic changes in the morphology of SMP muscle fibres can be detected by day 35 (see below). Between this stage and day 90, the progression of the pathology of the SMP muscle in dystrophic chickens is similar to that of the pure fasttwitch pectoral muscle, as has been described previously (Pizzey and Barnard 1983b). Fibre types in the SMP and PLD muscles were identified by the differential stainings and other criteria (Barnard et al. 1982) summarized in Table 1. The SMP was found thus to contain a mosaic structure of fibre types, mainly type liB but with some which are clearly recognisable as type I (Fig. 3). The third muscle examined, the MLD, is a small muscle lying dorsal to the PLD and inserting at one end into the skin (Fig. 1). Its fibres are multiplyinnervated (Fig. 4). They have been shown (Toutant et al. 1980) to be tonic in type, and consist of two sub-types which correspond to the sub-types IIIA and IIIB found (Barnard et al. 1982) in other slow-tonic multiply-innervated chicken muscles. These three muscles contain all the clearly-recognised subtypes of chicken skeletal muscle fibres.
Fig. 2. Frozen sections of the SMP muscle from normal (A) and dystrophic (B) chickens. In B, a hypertrophic, hypercontracted fibre is seen in the centre of the micrograph and many more myonuclei, especially in sarcolemmal positions, are seen. Haematoxylin and eosin ; bars = 60/tin. TABLE 1 SUMMARY OF FIBRE-TYPING CRITERIA ~ Criteria
Fibre type IA
IB
IIA
IIB
NADH-TR
•
•
•
C)
ATPase (4.3)
•
•
O
O
ATPase (4.6)
•
•
~
ATPase (9.4)
C)
~
•
•
Phosphorylase
O
C)
•
•
a Staining intensities are given on an arbitrary scale from 0 to 3 : C) = o, ~ = 1 +, •
= 2 +, •
= 3 +.
Fig. 3. Frozen SMP muscle sections, stained for myofibrillar ATPase after pre-incubation at p H 4.6 from normal (A) and dystrophic (B) chickens. In A, small type IB fibres (arrows) and one type IA fibre can be seen. In B, m a n y more type 1A fibres are present and several are hypertrophic and rounded in profile. Bars = 60 #m.
Fig. 4. Teased fibres from the M L D o f a 35-day dystrophic chicken. The muscle is stained for A C h E and several stained nerve endings can be seen along the length of the fibres. All fibres are multiplyinnervated. Bar = 100 gin.
224 Type I fibres Type I fibres represent a very small proportion ( < 3 ~ ) of the population in the PLD of normal chickens (Fig. 5). They are characteristically resistant to acid, but labile to alkali, pre-incubation for ATPase activity. They are also low in phosphorylase and high in N A D H . T R activity (Table 1). Thus, they show many similarities to mammalian type I fibres (Barnard et al. 1982). In both the P L D and the SMP muscles (and in all other chicken muscles we have examined), the mean cross-sectional fibre area (MFA) of type I fibres is smaller than that of the neighbouring type II fibres (Table 2) and the nuclei are virtually always found in sarcolemmal positions in type I fibres (Table 3). The SMP of normal chickens contains two sub-types of type I fibres (Table 1 ; Fig. 3). One of the sub-types (referred to here as type IA) is equivalent to the type I of the PLD. As in the PLD, type IA fibres of the SMP, of normal chickens represent only a very small proportion (<3~0) of the fibre population (Fig. 5). The normal SMP contains a larger class (411~0 of the total) of fibres which are also type I, on the basis of the histochemical criteria of Table 1, except that they show minor, but completely reproducible, differences in their resistances to acid or alkali pre-incubation for ATPase activity, and also in their staining reaction for N A D H . T R (Table 1 ; Fig. 6). The most striking feature of this fibre type is their extremely small cross-sectional area. In normal, 50-day chickens, this is 20-30 ~m 2 (Table 2), compared to 200/2m 2 in type IA fibres. As a consequence of this very small area of these fibres, extremely few nuclei are found in fibre cross-sections, and these are all in peripheral positions (Table 3). These fibres are classified here as type IB; we have not previously observed this fibre type in any of the other 12 muscles of the chicken examined. Both the IA and IB fibre types of the SMP muscle were shown by AChE staining of end-plates to be multiply-innervated. The end-plates in the IA fibres were closer to the en plaque morphology than the en grappe, but rather less condensed, as well as being smaller, than the en plaque endings of fast-twitch fibres (Fig. 7).
IA °~I00 ]
IB
IIA
lIB SMP
s04
)0 a_
E o
{--
I°° l
PLD
,o 50]
d~
0
Fig. 5. Fibre type proportions in the SMP and PLD muscles, measured on muscles of 5 normal (clear bars) and 5 dystrophic (black bars) chickens. Bars indicate SEM.
225 TABLE 2 A R E A A N A L Y S I S OF P L D A N D SMP M U S C L E FIBRES F R O M N O R M A L A N D D Y S T R O P H I C C H I C K E N S P L D values are from 3 normal and 3 dystrophic 70-day chickens, and SMP values are from 3 normal and 4 dystrophic 35-day chickens. Muscle
Fibre type a IA
IB
MFA
VC
SD b
HF
PLD N D
1346 1531
220 215
296 328
85 110
SMP N D
197 154
398 78 528* 83
MFA
105 23 170" 38*
IIA VC
630 516
SD b
15 20
HF
35 270*
lIB
MFA
VC
SD b
HF
1800 3270*
211 380 75 377* 1228"420"
MFA
VC
SD b
HF
2100 1774
421 632 80 759* 1358"450"
412 351
398 161 110 900* 312" 500*
Type IB fibres and type IIA fibres are absent in the P L D and SMP muscles, respectively. b SD: standard deviation. This reflects the variation in the pool of all fibres measured for any given type, and is not a reflection o f the variability between individual birds. * Values significantly different from normal chickens (P <0.01, by t-test). M F A = mean endomysial fibre area (/~m2); VC = variability coefficient; H F = hypertrophy factor. a
Fig. 6. Frozen section, stained for N A D H - T R , from the S M P muscle of a normal chicken. Nearly all fibres are type liB a n d produce a weak reaction product. Several, heavily-stained type IB (arrows) and one larger type IA fibre (double arrows) are also demonstrated. Bar = 60 #m.
226 TABLE 3 NUCLEI CONTENT AND DYSTROPHIC CHICKENS
DISTRIBUTION
IN PLD AND
SMP
MUSCLE
FIBRES OF NORMAL
AND
(A) Type l fibres Values are given as m e a n + SEM. P L D values were d e t e r m i n e d from the same chickens used for the values in Table 2. S M P values are f r o m 50-day chickens.
Muscle
Fibre type IB
1A Np
Ni
Nt
Np([',;)
0.82+_0.10 1.06+0.18
0.05_+0.01 0.07_+0.03
0.87_+0.13 1.13_+0.25
94.3_+3.6 93.8+4.7
0.83 +-0.17 1.76"-+0.17
0.09_+0.04 0.10_+0.02
0.92 _+0.20 1.86"_+0.09
89.9_+10.l 94.6-+3.8
Np
Ni
NI
Np('II;)
0.40_+0.12 0.71+-0.10
0.00+-0.00 0.11 _+ 0.11
I).40_+0.12 /).82_+0.25
100 _+0.00 89.0_+7.6
PLD N D
SMP N D
* Values significantly different f r o m n o r m a l ( P <0.02, by t-test). Np = p e r i p h e r a l nuclei; N i = internal nuclei; N~ = t o t a l nuclei.
TABLE 3 NUCLEI CONTENT AND DYSTROPHIC CHICKENS
DISTRIBUTION
1N P L D A N D
SMP
MUSCLE
FIBRES OF
NORMAL
AND
( B) Type l l fibres Values are given as m e a n _+ S E M a n d are t a k e n f r o m the same c h i c k e n s used for the values in T a b l e 3A.
Muscle
Fibre type I1B
IIA Np
Ni
NI
Np(%)
Np
Ni
Nt
Np(15~,)
1.80 + 0.42 2.25 _+0.32
0.37 +_0.09 /).61 _+0.16
2.17 _+0.64 2.86 +_0.86
82.95 -+ 3.7 78.67 _+2.6
1.23 +0.18 2.59*_+0.22
1.00+0.35 1.123-0.11
2.23 +0.53 3.70*+-0.33
62.5-+2.6 69.0_+1.4
0.94 _+0.06 3.14" _+0.51
0.80+_0.05 1.96+-1.12
1.74 +_0.36 5.10"+-1.63
55.7+4.1 63.3_+10.3
PLD
N D
SMP N
* Values significantly different from n o r m a l chickens ( P <0.02, by t-test).
In the dystrophic (D) animals the proportion of type I fibres of the PLD is increased above the normal (N) value, being about double at 7 weeks of age (Fig. 5). The histogram of type I fibre area distribution was unchanged in the dystrophic birds (not shown), so that the mean area and the standard deviation did not differ significantly between dystrophic and normal muscles (Table 2). The
227
Fig. 7. Frozen sections, stained for ACHE, from the SMP muscle of a normal chicken. In A, a type IA fibre can be seen with several stained nerve endings. The fibre diameter is too large to indicate a type IB fibre. In B, two large motor end-plates on adjacent type II fibres are shown. Bars = 60/~m. increases in the numbers of total myonuclei and peripheral myonuclei which characterize dystrophic chicken muscles fibres (McMurtry et al. 1972; Pizzey and Barnard 1983a) are absent in the type I fibres of the D-PLD, even up to 70 days of age (Table 3). Similarly, no hypertrophy, atrophy, fibre splitting, necrosis or phagocytosis was seen in the type I fibres in sections of the D - P L D from 30-70 days of age. As noted above, these fibres in the P L D muscle are, in fact, more precisely specified as type IA. In contrast to these, the type IA fibres in the D-SMP showed characteristic pathological changes. The number o f nuclei per fibre cross-section was doubled, and this increase occurred only at peripheral positions (Table 3). The distribution of type IA fibre areas is also markedly skewed compared to that of type IA fibres in the normal muscle (Fig. 8). Analyses o f fibre areas were made using also the variability coefficient (VC) and hypertrophy factor (HF), which are, respectively, estimations of variability within a muscle (VC = SD/mean x 1000) and of the contribution of abnormally large fibres (Dubowitz and Brooke 1973). These indices were both significantly higher in the D-SMP than in the N-SMP (Table 2). Hypertrophy and other pathological features such as the rounding of fibres were also seen in the type IA dystrophic fibres, as shown in Fig. 3, where the characteristic wide variation in fibre size is illustrated. These latter findings were seen consistently in a total of 5 dystrophic animals examined (involving about 300 type I fibres in these dystrophic SMP muscles ). The type IB fibres found in the SMP appear to be much more resistant to dystrophic change. Nuclei content is similar in type IB fibres o f both normal
228 IA
IB
0 1
22 3 (lO~m 2)
t~
S
. . . . . . . . . 2 ~ 6 8 (lO,um 2)
liB
."--:.. . . . . . . . . 10 12 2 /,
7 I .'-q, . , , Fq 6 28 210 12 l& 16 (10 ~Jm )
Fibre Area
Fig. 8. Area histograms of fibre types in the SMP muscle from normal (upper) and dystrophic (lower) 35-day chickens. The histograms are constructed from the pooled values of individual fibres in all of the muscle samples. Type IA and type liB fibres of the dystrophic SMP muscle show the greatest skewness in the histograms. Note the change of scale for type IB fibres. All histograms of dystrophic fibre types are different from normal (P <0.001).
and dystrophic birds, and the variability coefficient of the mean areas is also not significantly different between them. The high value for the latter parameter in both normal and dystrophic chickens in the present case is due to the exceptionally small mean area of this fibre type (Table 2). However, although dystrophic type IB fibres have not lost their polygonal profile by 70 days, hypertrophy is beginning before this stage, as can be seen from the mean areas and the hypertrophy factors listed in Table 2, and the area histograms shown in Fig. 8. Such hypertrophy was the only pathological change which we observed in any of the type IB fibres from dystrophic chickens up to 70 days. There was, however, an increase in the proportion of IB fibres in the D-SMP, as there was also of IA fibres (Fig. 5).
Type H fibres The majority of fibres in both the N-PLD and N-SMP are type IIB. The SMP has virtually no type IIA fibres (Fig. 5), while the PLD contains 15-20% in 8-week-old chickens (Beringer 1978). The normal type IIA fibres are slightly smaller in area than type IIB. The standard deviation of the mean areas is small for each fibre type, as shown in Table 2. This parameter is a measure of the range of fibre sizes within an individual muscle, and is not an expression of variability between different birds. Type IIA fibres of the D-PLD are often hypertrophic and frequently have a more rounded profile in cross-section, as found previously (Beringer 1978). Pathological features such as fibre splitting and necrosis were not seen here in any type IIA fibres of the D-PLD, up to 70 days of age. However, the mean area of the dystrophic type IIA fibres is much higher than in the type liB fibres in the D-PLD
229 (Table 2), because the latter population contains a large number of very small fibres, primarily due to longitudinal fibre splitting, atrophy or regeneration. However, since many type liB fibres are also severely hypertrophic, the standard deviation for that population is high. This causes the variability coefficient of dystrophic type liB fibres to be twice the value of the type IIA fibre population (Table 2). Thus, mean area values alone are not sufficient to reflect the relative involvement of different fibre types in dystrophic chicken muscles. Type liB fibres are again the most severely affected fibres in the SMP muscle in all dystrophic chickens examined up to 70 days, as determined by measurements of nuclei content and distribution (Table 3) and the statistical parameters of fibre area distribution (Table 2; Fig. 8). Phagocytosis and necrosis are associated only with type liB fibres in the P L D and SMP at this stage, although the incidence is only ~ 2~" of fibres in both the D-PLD and the D-SMP. The M L D muscle This muscle is almost purely tonic, as determined by histochemical criteria, in both normal and dystrophic chickens (Fig. 4). The proportions of the two tonic sub-types present, IliA and IIIB (Barnard et al. 1982), are unaltered by dystrophy. No changes in fibre size or shape, or in nuclei numbers and distribution are seen, nor any degenerative events (Fig. 9). This muscle is totally resistant to dystrophy up to the maximum period (90 days) studied here.
Fig. 9. Frozen section of the MLD muscle from a dystrophic chicken. The muscle is of normal appearance for chicken tonic muscle. There is little variation in fibre area and all myonuclei are in sarcolemmal positions. Haematoxylinand eosin; Bar = 60/~m.
230 DISCUSSION
The quantitative criteria of increased variability coefficient and skewed histograms of fibre area, increased total and peripheral nuclei and degenerative events, were sufficient to classify fibres as dystrophic or not, and these criteria behaved as a consistent set. We found thus that all twitch fibre types can be affected by muscular dystrophy in the chicken. A given twitch fibre may be either susceptible or resistant, depending on the muscle in which it is located. Thus, type IA fibres are unaffected in the D-PLD, as they are also on the red side of the sartorius and in the adductor superficialis (Barnard et al. 1982); they are, in contrast, strongly affected in the D-SMP. This is despite the fact that they are present as a similarly low percentage of total fibres in both the PLD and the SMP muscles, with many neighbouring dystrophic type liB fibres in each case. Since both the SMP and the P L D type I fibres are oxidative this, alone, cannot account for the resistance to dystrophy in type I fibres in the latter muscle. In some other muscles even type l i b fibres are resistant, as has recently been shown to be the case (at the ages studied here) in the red sartorius and the adductor superficialis muscles (Barnard et al. 1982), where these fibres are in a minority. This resistance has likewise been found in the type liB fibres (again a minority) of the complexus, a muscle of the neck used constantly and showing no pathologic changes in the dystrophic birds throughout life (Ashmore et al. 1973). Even within one muscle (the SMP), the two sub-types of type i fibre were found to differ in their expression of myopathy, type IB being much less affected. An increase in the proportion of type I fibres occurred whether they are of the group which showed myopathic change or not (Fig. 3). This is a more general phenomenon, in fact, since in other muscles which are affected, the resistant type I fibres increase in proportion, e.g. in the P L D (Barnard et al. 1982). It should be mentioned here that the two sub-types of type I fibres in the chicken SMP are not both found in the mammals, nor in the pigeon SMP. Khan (1979) found that the latter contained two type I sub-types, both of which were low in oxidative, but high in glycolytic enzymes. We found no evidence for a "slow-glycolytic" fibre type in the chicken SMP, nor in all the other chicken muscles which we have examined. However, one feature that is characteristic of type I avian fibres wherever they have been recognized is multiple innervation, as found here for the chicken SMP type IA and IB fibres, and previously for type I fibres in the pigeon SMP (Khan 1979), chicken sartorius (Ashmore et al. t978), P L D (Barnard et al. 1982) and biventer cervicis (Toutant et al. 1981 ; Barnard et al. 1982). The major difference between the two sub-types of type I fibres is the exceptionally small size of the type IB fibres in the SMP, which may be related to the specialised functions of subcutaneous muscles (see below). In conclusion, we can say that a given twitch fibre type does not necessarily respond in the same way to dystrophy in different muscles, so that a universal determination of the myopathy by the class of motor neurone can be ruled out.
231 It has long been recognised that chicken muscles vary in the extent to which they are affected by dystrophy. The most affected contain a majority of type IIB fibres, for example, the pectoral, biceps and PLD muscles. These are all involved in wing movement, but in a flightless bird such as the chicken, these muscles would get relatively little exercise, and this is, presumably, the reason for the high proportion of type IIB fibres in these muscles. In contrast, the pectoral muscle fibres in flying birds are generally oxidative (Talesara and Goldspink 1978). The amount of exercise a muscle gets may be related to the rate of expression of the disorder in both human and chicken dystrophy. It is thought that moderate exercise is beneficial for Duchenne patients (Vignos and Watkins 1966; Dubowitz and Heckmatt 1980) and there is an indication of this, also, in a limited application of exercise in the dystrophic chicken (Entrikin et al. 1978; Hudecki et al. 1978). The muscles which are completely or relatively spared in both Duchenne dystrophy (see Introduction) and chicken dystrophy are either tonic or tend to have a majority of type I fibres. This could be related to the frequent use of these muscles. The postural function of tonic muscles such as the ALD means that these muscles are virtually in constant use. We do not know yet if it is a constancy of stimulation in vivo, or the slower rate of stimulation, or some special neuronal factor, which confers the resistance of tonic muscles. In the oxidative twitch muscles with few type IIB fibres, we presume the latter are protected by the natural exercise of the muscle as a whole. The involvement of type I fibres of the SMP may be related to the specialised function of this muscle in maintaining tautness of the skin along the posterior edge of the wing during flight and as the wing is being folded (Bock 1973). It is probable that there is great variation in the use of this muscle between avian species. For example, the SMP of the chicken will rarely be used. Indeed, we suggest that the chicken SMP is used even less than the other muscles, mentioned above, which are involved in wing movement. This muscle in the chicken is very much smaller and thinner than in the pigeon, and appears to be vestigial in the former, flightless bird. Relative disuse may account for the involvement of type I fibres in the D-SMP, at stages when type I fibres in the D-PLD are unaffected. There are many other physiological features of muscle which are related, either directly or indirectly, to their fibre type composition. For example, type IIB-rich muscles are more readily fatigued than those with a majority of oxidative fibres and the latter are also generally more vascular (Vrbov~i et al. 1978). Whatever the nature of the primary lesion in chicken dystrophy, the expression of the disease in different muscles may be modified by such phYsiological factors. Thtts 'it is seen that both quantitative fibre type composition and the degree of use and nature of stimulation must be considered in interpreting the differential involvement of muscles in the muscular dystrophies.
232 REFERENCES Andersen, P. and J. Henriksson (1977) Training-induced changes in the subgroups of human type II skeletal muscle fibers, Aeta Physiol. Stand., 99: 123-125. Ashmore, C. R. and L. Doerr (197l) Postnatal development of fiber types in normal and dystrophic skeletal muscle of the chick, Exp. Neurol., 30: 431~t46. Ashmore, C.R., P.B. Addis, L. Doerr and H. Stokes (1973) Development of muscle fibers in the complexus muscle of normal and dystrophic chicks, J. Histoehem. Cytoehem., 21: 266278. Ashmore, C. R., P. Vigneron, L. Marger and L. Doerr (1978) Simultaneous cytochemical demonstration of muscle fiber types and acetylcholinesterase in muscle fibers of dystrophic chickens, Exp. Neurol., 60:68 82. Asmundson, V. S. and L. M. Julian (1956) Inherited muscle abnormality in the domestic fowl, J. Hered., 47 : 248-252. Baloh, R. and P.A. Cancilla (1972) An appraisal of histochemical fiber types in Duchenne muscular dystrophy, Neurology (Minneap.), 22 : 1243 1252. Barnard, E.A. (1980) Characterisation of inherited muscular dystrophy in the chicken. In: F.C. Rose and P.O. Behan (Eds.), Animal Models, o[' Neurological Disease, Pitman Medical Ltd., Tunbridge Wells, U.K., pp. 17 33. Barnard, E.A., J. M. Lyles and J, A. Pizzey (1982) Fibre types in chicken skeletal muscles and their changes in muscular dystrophy, J. Physiol. (Lond.), 331: 333-354. Beringer, T. (1978) Stereologic analysis of normal and dystrophic ~W myofibers, Exp. Neurol., 61 : 380-394. Bock, W.M. (1973) Structure and function of the pigeon M. serratus metapatagialis, Amer. Zool., 13: 1344. Bonsett, C.A. (1969) Studies of Pseudohypertrophic Muscular Dystrophy, Thomas, Springfield, IL. Brooke, M.H. and W.K. Engel (1969) The histographic analysis of human muscle biopsies with regard to fiber types, Part 1 (Adult male and female), Neurology (Minneap.), 19: 221~33. Brooke, M.H. and K.K. Kaiser (1970) Muscle fiber types How many and what kind'! Arch. Neurol. (Chic.), 23: 369-379. Buchthal, F., Z. Kamieniecka and H. Schmalbruch (1974) Fibre types in normal and diseased human muscles and their physiological correlates. In: A.T. Milhorat (Ed.), Exploratory Concepts' in Muscular Dystrophy and Related Disorders, 1I, Excerpta Medica, Amsterdam, pp. 526-551. Burke, R. E., D.M. Levine, F.G. Zajac, P. Tsairis and W. K. Engel (1971) Mammalian motor units Physiological-histochemical correlation in three types of cat gastrocnemius, Science, 174:709 712. Cosmos, E. and J. Butler (1967) Differentiation of fiber types in muscles of normal and dystrophic chickens. In: A.T. Milhorat (Ed.), Exploratory Coneepts in Muscular Dystrophy and Related Disorders, I, Excerpta Medica, Amsterdam, pp. 197 204. Dubowitz, V. and M.H. Brooke (1973) Muscle biopsy A Modern Approach, Saunders, London. Dubowitz, V. and J. Heckmatt (1980) Management of muscular dystrophy, Brit. Med. Bull., 36: 139-144. Ellis, D.A. (1980) Intermediary metabolism of muscle in Duchenne muscular dystrophy, Brit. Med. Bull., 36:165 171. Engel, W. K. (1970) Selective and nonselective susceptibility of muscle fiber types A new approach to human neuromuscular diseases, Arch. Neurol. (Chic.), 22:97 117. Entrikin, R.K., G.T. Patterson, P.M. Weidoff and B.W. Wilson (1978) Righting ability and skeletal muscle properties of phenytoin-treated dystrophic chickens, Exp. Neurol., 61: 650-663. Gardner-Medwin, D. (1980) Clinical features and classification of muscular dystrophies, Brit. Med. Bull., 36:109 115. Gollnick, P. D., R. B. Armstrong, C.W. Saubert IV, K. Piehl and B. Saltin (1972) Enzyme activity and fiber composition in skeletal muscle of untrained and trained men, J. Appl. Physiol., 33:312 319. Grim, M. (1971) Development of the primordia of the latissimus dorsi muscle of the chicken, Folia Morph., 19:252 258. Hess, A. (1970) Vertebrate slow muscle fibres, Physiol. Rev., 50: 40-62. Hudecki, M.S., C. Pollina, A.K. Bhargava, J.E. Fitzpatrick, C.A. Privitera and D. Schmidt (1978) Effect of exercise on chickens with hereditary muscular dystrophy, Exp. Neurol., 61 : 65-73. Jansson, E. and L. Kaijser (1977) Muscle adaptation to extreme endurance training in man, Acta Physiol. Scand., 100:315 324. Johnson, M. A. and D. K. K ucukgalcin (1978) Patterns of abnormal histochemical fibre type differentiation in human muscle biopsies, J. Neurol. Sci.. 37:159 178.
233 Johnson, M.A., J. Polgar, D. Weightman and D. Appleton (1973) Data on the distribution of fibre types in thirty-six human muscles - - An autopsy study, J. Neurol. Sci., 18:111-129. Khan, M.A. (1979) Histochemical and ultrastructural characteristics of a new fibre type in avian striated muscle, Histochem. J., 11 : 321-335. Libelius, R., I. Jirmanov~i, I. Lundquist, S. Thesleff and E.A. Barnard (1979) T-tubule endocytosis in dystrophic chicken muscle and its relation to muscle fiber degeneration, Acta Neuropath. (Berl.), 48 : 31-38. Lyles, J.M., I. Silman and E.A. Barnard (1979) Developmental changes in levels and forms of cholinesterases in muscles of normal and dystrophic chickens, J. Neurochem., 33: 727-738. McMurtry, S. L., L. M. Julian and V. S. Asmundson (1972) Hereditary muscular dystrophy of the chicken Quantitative histopathological findings of the pectoralis at 6 weeks of age, Arch. Path., 94: 217-224. Maunder-Sewry, C.A. and V. Dubowitz (1981) Needle muscle biopsy for carrier detection in Duchenne muscular dystrophy, J. Neurol. Sci., 49: 305-324. Nonaka, I., A. Takagi and H. Sugita (1981) The significance of type 2C muscle fibers in Duchenne muscular dystrophy, Muscle & Nerve, 4: 326-333. Peter, J. B., R.J. Barnard, V.R. Edgerton, C.A. Gillespie and K.E. Stempel (1972) Metabolic profiles of three fiber types of skeletal muscle in guinea pigs and rabbits, Biochemistry, 11 : 2627-2633. Pizzey, J.A. and E.A. Barnard (1983a) Structural change in muscles of the dystrophic chicken, Part 1 (Quantitative indices), Neuropath. Appl. Neurobiol., 9: 21-38. Pizzey, J.A. and E.A. Barnard (1983b) Structural change in muscles of the dystrophic chicken, Part 2 (Progression of the histopathology in the pectoralis muscle), Neuropath. Appl. Neurobiol., 9: 149-164. Prince, F.P., R.S. Hikida and F.C. Hagerman (1976) Human muscle fibre types in powerlifters, distance runners and untrained subjects, Pfliigers Arch. ges. Physiol., 363: 19-26. Saunders, J. T. and S. M. Manton (1959) Manual of Practical Vertebrate Morphology, Oxford University Press, London. Shafiq, S.A., V. Askanas and A.T. Milhorat (1971) Fiber types and preclinical changes in chicken muscular dystrophy, Arch. Neurol. (Chic.), 25: 560-571. Sokal, R. R. and F.J. Rohlf (1981) Biometry, 2nd edition, W. H. Freeman, San Francisco, CA. Talesara, G. L. and G. Goldspink (1978) A combined histochemical and biochemical study of myofibrillar ATPase in pectoral, leg and cardiac muscle of several species of bird, Histochem. J., 10: 695-710. Toutant, J.P., M. Toutant, D. Renaud and G. Le Douarin (1980) Fast histochemical fibre type in chicken tonic muscles. I.R.C.S. Med. Sci., 8: 139. Toutant, J.P., T. Rouad and G.H. Le Douarin (1981) Histochemical properties of the biventer cervicis muscle of the chick - - A relationship between multiple innervation and slow-tonic fibre types, Histochem. J., 13: 481-493. Tsuji, S. (1974) On the chemical basis of thiocholine methods for demonstration of acetylcholinesterase activities, Histochem., 42: 99-110. Vignos, P.J. and M.P. Watkins (1966) The effect of exercise in muscular dystrophy, J. Amer. Med. Ass., 197: 121-126. Vrbov~., G., T. Gordon and R. Jones (1978) Nerve -Muscle Interaction, Chapman and Hall, London. Wilson, B.W., S.G. Linkhart and P.A. Neiberg (1973) Acetylcholinesterase in singly and multiply innervated muscles of normal and dystrophic chickens, J. Exp. Zool., 186: 187-192. Wilson, B.W., W.R. Randall, G.T. Patterson and R.K. Entrikin (1979) Major physiologic and histochemical characteristics of inherited dystrophy of the chicken, Ann. N.Y. Acad. Sci., 317: 224~246. -
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217
Elsevier
I N V O L V E M E N T OF FAST A N D SLOW T W I T C H M U S C L E FIBRES IN AVIAN MUSCULAR DYSTROPHY
JOHN A. PIZZEY, ERIC A. BARNARD and PENELOPE J. BARNARD. Department of Biochemistry, Imperial College of Science and Technology, London (Great Britain)
(Received 28 March, 1983) (Accepted 28 April, 1983)
SUMMARY The extent of differential fibre type involvement in chicken muscular dystrophy can be assessed quantitatively by the statistical parameters of fibre area, nuclei content and nuclei distribution in the individual fibre types. Two muscles, the posterior latissimus dorsi (PLD) and the serratus metapatagialis (SMP), were found to have similar overall fibre type composition, although the latter contains two subtypes of type I fibres, one of which has not previously been recognised in avian muscle. In both muscles, type IIB fibres are most affected by the progressive pathology. Nuclear proliferation is one of the histopathological features which can be measured, and in the PLD, the mean number of total nuclei in type liB fibre cross-sections (N0 is increased from 2.23 in normal chickens to 3.70 in dystrophic chickens, by 60 days. The corresponding values for N~ in type liB muscle fibres o f the SMP at 50 days are 1.74 and 5.10. Likewise, statistical analyses of the distribution of the fibre areas and their variability demonstrate that the incidence of abnormality in chicken dystrophy is greatest in type liB fibres in both these muscles. Although type I fibres in the P L D are resistant to dystrophic change, it is noteworthy that in the SMP the type I fibres, also, are severely affected from an early stage, by these quantitative criteria. On the other hand, all fibres in a tonic muscle, the metapatagialis latissimus dorsi, are unaffected, as is true of all other tonic muscles previously studied. It is concluded that any twitch fibre type can, in principle, be affected by the actions of the gene concerned, and that this
This work was supported by the Muscular Dystrophy Group of Great Britain. Address reprint requests to Professor E.A. Barnard, Department of Biochemistry, Imperial College of Scienceand Technology,London, SW7 2AZ, Great Britain. 0022-510X/83/$03.00 © 1983 Elsevier SciencePublishers B.V.
218 expression can be greatly modified in individual muscles by various physiological features, for example their natural pattern of use or relative disuse.
Key words: C h i c k e n m u s c l e s - M u s c l e f i b r e s - M u s c u l a r d y s t r o p h y
INTRODUCTION
In the inherited muscular dystrophies of man or of experimental animals, the progress of the histopathological changes through the population of different muscle fibre types is a matter of considerable significance. If it were to be found that all fibre types are affected equally, this could, in the absence of a general neurological lesion, indicate a defect in a feature characteristic of the skeletal muscle cell in general. If, instead, selectivity is found for one fibre type, this could be due to a neurogenic defect in a particular class of motor neuron, since these are specific for the muscle fibre types (Burke et al. 1971), but in that case one might expect to see that same fibre type always affected wherever it occurs in muscles. The differential involvement of different muscles, well-known to occur in these myopathies, should be analysed to test the possibility that it arises from the variations in the proportions of fibre types in those muscles. In general, the pattern of selection of fibre types within one muscle is one of the basic parameters needed for the understanding of muscle pathology in such diseases. In the fibre type nomenclature employed for human muscles by Brooke and Kaiser (1970) and Dubowitz and Brooke (1973), adult human muscles were shown by those authors to contain generally three fibre types: I, IIA or liB, corresponding to slow-twitch oxidative, fast-twitch oxidative/glycolytic,or fast-twitch glycolytic, respectively, as described by others (Peter et al. 1972). Also, a very few human and mammalian muscles contain slow-tonic fibres (Hess 1970). It is well known that not all muscles are affected to the same extent in Duchenne muscular dystrophy (DMD). Thus, the rare tonic muscles in man such as the cremaster (Bonsett 1969) and some external ocular muscles (Gardner-Medwin 1980) are very resistant to the pathological change. In the affected muscles in DMD, some studies have suggested that type II fibres are primarily involved and type I are less affected (Baloh and Cancilla 1972; Johnson et al. 1973; Johnson and Kucukgalcin 1978; Ellis 1980). Further, an increasing predominance of the type I fibres and a deficiency of the type liB fibres in DMD or carrier muscle biopsies has generally been described (Brooke and Engel 1969; Dubowitz and Brooke 1973; Maunder-Sewry and Dubowitz 1981; Nonaka et al. 1981). However, Dubowitz and Brooke (1973) concluded that selective involvement of any one fibre type does not appear to be prominent in DMD, and others, also, have found no such selection there (Engel 1970; Buchthal et al. 1974). These comparisons are complicated in humans by the marked variability of fibre types, in both percentage and fibre size, within the normal population (Gollnick et al. 1972; Prince et al. 1976) and with exercise
219 and training (Andersen and Henriksson 1977; Jansson and Kaijser 1977). In inherited muscular dystrophy of the chicken, many of the histological, physical and biochemical features of D M D are reproduced (Asmundson and Julian 1956; Wilson et al. 1979; Barnard 1980) and, in particular, most of the histopathological changes are seen, both by electron (Libelius et al. 1979) and light microscopy (Barnard et al. 1982; Pizzey and Barnard 1983a). In such a model, the muscle at any stage can conveniently be analysed, in a population of genetically closely-related animals under standard conditions. It is already established that slow tonic muscles such as the anterior latissimus dorsi (Wilson et al. 1973), the plantaris or the adductor profundus (Barnard et al. 1982) are not significantly affected by the disease. A similar fibre type nomenclature to that noted above for mammals has recently been shown to account for the fibre types present in a wide range of chicken twitch muscles, although there are some secondary differences between a given fibre type in the two cases (Barnard et al. 1982). Using that nomenclature here, we can note that only type IIB fibres have previously been reported to be selected for pathological changes in the dystrophic chicken (Cosmos and Butler 1967; Ashmore and Doerr 1971; Beringer 1978). Type I fibres have been described as resistant to the disease (Shafiq et al. 1971; Ashmore et al. 1978). However, a detailed quantitative analysis of histopathological changes within the individual fibre types present in dystrophic muscles has not been reported, and it has seemed to us of interest to obtain quantitative expressions of the fibre type selectivity in the chicken muscles. We have included in this analysis two cutaneous muscles (a class hitherto unexamined in the dystrophic chicken), one of which contains an appreciable number of type I fibres. We show that these slowtwitch oxidative fibres can, indeed, be affected by the disease. Hence, it will be argued, factors additional to the neural control of muscle fibre type can regulate the pathology. MATERIALS A N D METHODS
Animals Normal New Hampshire chickens (London-line 412) and the corresponding line homozogous for muscular dystrophy (London-line 413) were bred and maintained in the Imperial College Dystrophic Chicken Research Facility. Both lines were derived from the corresponding University of California (Davis) lines (Wilson et al. 1979). Chickens were kept on a 12-h light cycle at 25°C and at 40-50~ relative humidity. All chickens had free access to food and water. Muscle specimens Chickens (35-90 days) were killed by cervical dislocation, and the muscle samples were at once removed. The serratus metapatagialis (SMP) is a white muscle, unlike the pigeon SMP (Khan 1979), and corresponds to the cutaneous muscle in another nomenclature (Saunders and Manton 1959). The metapatagialis latissimus dorsi (MLD) muscle was identified as described by Grim (1971). The
220
DLD
i
MLD SMP
A
Fig. 1. Schematic diagram of the dorsal view of the proximal wing muscles of the chicken showing the latissimus dorsi and associated dermal muscles. The DLD, MLD and SMP muscles all have dermal insertions. ALD = M. anterior latissimus dorsi; DLD = M. dorsocutaneous latissimus dorsi; MLD = M. metapatagialis latissirnus dorsi; SMP = M. serratus metapatagialis; ST = M. scapulotriceps.
locations of these muscles are illustrated in Fig. 1. Samples were taken from the mid-regions of the SMP, M L D and posterior latissimus dorsi (PLD) muscles from 5 normal and 5 dystrophic chickens. For the histological analysis and fibre-typing, the samples were immediately frozen in isopentane cooled by liquid nitrogen, and stored at - 8 5 °C prior to cutting 10-/~m sections at - 2 0 °C on a Bright cryostat, orientated for true transverse sections. End-plate localization was performed on samples which were frozen or fixed briefly in 2~o glutaraldehyde/0.1M sodium acetate (pH 5.9) at r o o m temperature.
Histological and histochemical techniques Frozen sections were stained for histological analysis with haematoxylin and eosin (HE). In addition, serial sections were stained (Dubowitz and Brooke 1973) for N A D H tetrazolium reductase ( N A D H - T R ) , or phosphorylase activity, or ATPase after pre-incubation at p H 4.3 or p H 4.6 or p H 9.4. Small samples of the fixed muscle were stained for acetylcholinesterase (ACHE) activity (Tsuji 1974) and teased in glycerin to separate fibres for end-plate counting. Longitudinal frozen sections were fixed for 30 min in 2~o glutaraldehyde and similarly stained for ACHE. All sections were photographed and individual fibres were typed as described elsewhere (Barnard et al. 1982) using 300-500 fibre cross-sections from each an!mal, i.e., a minimum of 1500 fibres of each genotype was analysed. All the type I fibres present in all of the sections of SMP and P L D muscle were analysed for the histological measurements, due to the relatively low frequency with which type I fibres occur in these muscles. Fibre areas were measured after projecting randomly-chosen negatives of ATPase-stained sections (pre-incubated at p H 4.6) onto the surface of the digit-
221 izing tablet of a Hewlett-Packard mini-computer. Recognition of fibre types was then performed by reference to micrographs of serial sections stained by the different methods described above. Diameters were randomly drawn across the negatives and, wherever possible, 100 fibres of each fibre type were measured for each bird. Fibres showing evidence of tangential sectioning (as indicated by an elliptical profile) were excluded. Normal and dystrophic fibre area histograms were compared using the Kolmogorov-Smirnov two-sample test (Sokal and Rohlf 1981). For each muscle sample the hypertrophy factor was calculated as described by Dubowitz and Brooke (1973), this giving a weighted score to abnormally large fibres. The number and distribution of nuclei in cross-sections of the muscle fibres were determined from HE sections, again with reference to serial sections for fibre typing as above. Again, wherever possible, for each bird, 100 fibres of each fibre type, falling on randomly-drawn diameters, were measured for nuclei number and distribution. Internal nuclei (Ni) were counted separately and the contents of normal and dystrophic fibres were compared using the t -test. RESULTS
Muscles and fibre types investigated The first muscle analysed was the PLD, a white, fast-contracting muscle, which is very commonly used for studies of avian dystrophic muscle. In the strain (413) of dystrophic chickens which we use, pathological change in this muscle becomes clearly established by about 4 weeks of age (being detectable even earlier), as assessed by histological changes and abnormal enzyme contents (Lyles et al. 1979; Barnard et al. 1982; Pizzey and Barnard 1983a). The second muscle, the SMP, is a small cutaneous muscle (Fig. 1) not previously analysed histologically in the dystrophic or normal chicken. The SMP muscle, like the PLD, is strongly affected by the disease (Fig. 2). Characteristic changes in the morphology of SMP muscle fibres can be detected by day 35 (see below). Between this stage and day 90, the progression of the pathology of the SMP muscle in dystrophic chickens is similar to that of the pure fasttwitch pectoral muscle, as has been described previously (Pizzey and Barnard 1983b). Fibre types in the SMP and PLD muscles were identified by the differential stainings and other criteria (Barnard et al. 1982) summarized in Table 1. The SMP was found thus to contain a mosaic structure of fibre types, mainly type liB but with some which are clearly recognisable as type I (Fig. 3). The third muscle examined, the MLD, is a small muscle lying dorsal to the PLD and inserting at one end into the skin (Fig. 1). Its fibres are multiplyinnervated (Fig. 4). They have been shown (Toutant et al. 1980) to be tonic in type, and consist of two sub-types which correspond to the sub-types IIIA and IIIB found (Barnard et al. 1982) in other slow-tonic multiply-innervated chicken muscles. These three muscles contain all the clearly-recognised subtypes of chicken skeletal muscle fibres.
Fig. 2. Frozen sections of the SMP muscle from normal (A) and dystrophic (B) chickens. In B, a hypertrophic, hypercontracted fibre is seen in the centre of the micrograph and many more myonuclei, especially in sarcolemmal positions, are seen. Haematoxylin and eosin ; bars = 60/tin. TABLE 1 SUMMARY OF FIBRE-TYPING CRITERIA ~ Criteria
Fibre type IA
IB
IIA
IIB
NADH-TR
•
•
•
C)
ATPase (4.3)
•
•
O
O
ATPase (4.6)
•
•
~
ATPase (9.4)
C)
~
•
•
Phosphorylase
O
C)
•
•
a Staining intensities are given on an arbitrary scale from 0 to 3 : C) = o, ~ = 1 +, •
= 2 +, •
= 3 +.
Fig. 3. Frozen SMP muscle sections, stained for myofibrillar ATPase after pre-incubation at p H 4.6 from normal (A) and dystrophic (B) chickens. In A, small type IB fibres (arrows) and one type IA fibre can be seen. In B, m a n y more type 1A fibres are present and several are hypertrophic and rounded in profile. Bars = 60 #m.
Fig. 4. Teased fibres from the M L D o f a 35-day dystrophic chicken. The muscle is stained for A C h E and several stained nerve endings can be seen along the length of the fibres. All fibres are multiplyinnervated. Bar = 100 gin.
224 Type I fibres Type I fibres represent a very small proportion ( < 3 ~ ) of the population in the PLD of normal chickens (Fig. 5). They are characteristically resistant to acid, but labile to alkali, pre-incubation for ATPase activity. They are also low in phosphorylase and high in N A D H . T R activity (Table 1). Thus, they show many similarities to mammalian type I fibres (Barnard et al. 1982). In both the P L D and the SMP muscles (and in all other chicken muscles we have examined), the mean cross-sectional fibre area (MFA) of type I fibres is smaller than that of the neighbouring type II fibres (Table 2) and the nuclei are virtually always found in sarcolemmal positions in type I fibres (Table 3). The SMP of normal chickens contains two sub-types of type I fibres (Table 1 ; Fig. 3). One of the sub-types (referred to here as type IA) is equivalent to the type I of the PLD. As in the PLD, type IA fibres of the SMP, of normal chickens represent only a very small proportion (<3~0) of the fibre population (Fig. 5). The normal SMP contains a larger class (411~0 of the total) of fibres which are also type I, on the basis of the histochemical criteria of Table 1, except that they show minor, but completely reproducible, differences in their resistances to acid or alkali pre-incubation for ATPase activity, and also in their staining reaction for N A D H . T R (Table 1 ; Fig. 6). The most striking feature of this fibre type is their extremely small cross-sectional area. In normal, 50-day chickens, this is 20-30 ~m 2 (Table 2), compared to 200/2m 2 in type IA fibres. As a consequence of this very small area of these fibres, extremely few nuclei are found in fibre cross-sections, and these are all in peripheral positions (Table 3). These fibres are classified here as type IB; we have not previously observed this fibre type in any of the other 12 muscles of the chicken examined. Both the IA and IB fibre types of the SMP muscle were shown by AChE staining of end-plates to be multiply-innervated. The end-plates in the IA fibres were closer to the en plaque morphology than the en grappe, but rather less condensed, as well as being smaller, than the en plaque endings of fast-twitch fibres (Fig. 7).
IA °~I00 ]
IB
IIA
lIB SMP
s04
)0 a_
E o
{--
I°° l
PLD
,o 50]
d~
0
Fig. 5. Fibre type proportions in the SMP and PLD muscles, measured on muscles of 5 normal (clear bars) and 5 dystrophic (black bars) chickens. Bars indicate SEM.
225 TABLE 2 A R E A A N A L Y S I S OF P L D A N D SMP M U S C L E FIBRES F R O M N O R M A L A N D D Y S T R O P H I C C H I C K E N S P L D values are from 3 normal and 3 dystrophic 70-day chickens, and SMP values are from 3 normal and 4 dystrophic 35-day chickens. Muscle
Fibre type a IA
IB
MFA
VC
SD b
HF
PLD N D
1346 1531
220 215
296 328
85 110
SMP N D
197 154
398 78 528* 83
MFA
105 23 170" 38*
IIA VC
630 516
SD b
15 20
HF
35 270*
lIB
MFA
VC
SD b
HF
1800 3270*
211 380 75 377* 1228"420"
MFA
VC
SD b
HF
2100 1774
421 632 80 759* 1358"450"
412 351
398 161 110 900* 312" 500*
Type IB fibres and type IIA fibres are absent in the P L D and SMP muscles, respectively. b SD: standard deviation. This reflects the variation in the pool of all fibres measured for any given type, and is not a reflection o f the variability between individual birds. * Values significantly different from normal chickens (P <0.01, by t-test). M F A = mean endomysial fibre area (/~m2); VC = variability coefficient; H F = hypertrophy factor. a
Fig. 6. Frozen section, stained for N A D H - T R , from the S M P muscle of a normal chicken. Nearly all fibres are type liB a n d produce a weak reaction product. Several, heavily-stained type IB (arrows) and one larger type IA fibre (double arrows) are also demonstrated. Bar = 60 #m.
226 TABLE 3 NUCLEI CONTENT AND DYSTROPHIC CHICKENS
DISTRIBUTION
IN PLD AND
SMP
MUSCLE
FIBRES OF NORMAL
AND
(A) Type l fibres Values are given as m e a n + SEM. P L D values were d e t e r m i n e d from the same chickens used for the values in Table 2. S M P values are f r o m 50-day chickens.
Muscle
Fibre type IB
1A Np
Ni
Nt
Np([',;)
0.82+_0.10 1.06+0.18
0.05_+0.01 0.07_+0.03
0.87_+0.13 1.13_+0.25
94.3_+3.6 93.8+4.7
0.83 +-0.17 1.76"-+0.17
0.09_+0.04 0.10_+0.02
0.92 _+0.20 1.86"_+0.09
89.9_+10.l 94.6-+3.8
Np
Ni
NI
Np('II;)
0.40_+0.12 0.71+-0.10
0.00+-0.00 0.11 _+ 0.11
I).40_+0.12 /).82_+0.25
100 _+0.00 89.0_+7.6
PLD N D
SMP N D
* Values significantly different f r o m n o r m a l ( P <0.02, by t-test). Np = p e r i p h e r a l nuclei; N i = internal nuclei; N~ = t o t a l nuclei.
TABLE 3 NUCLEI CONTENT AND DYSTROPHIC CHICKENS
DISTRIBUTION
1N P L D A N D
SMP
MUSCLE
FIBRES OF
NORMAL
AND
( B) Type l l fibres Values are given as m e a n _+ S E M a n d are t a k e n f r o m the same c h i c k e n s used for the values in T a b l e 3A.
Muscle
Fibre type I1B
IIA Np
Ni
NI
Np(%)
Np
Ni
Nt
Np(15~,)
1.80 + 0.42 2.25 _+0.32
0.37 +_0.09 /).61 _+0.16
2.17 _+0.64 2.86 +_0.86
82.95 -+ 3.7 78.67 _+2.6
1.23 +0.18 2.59*_+0.22
1.00+0.35 1.123-0.11
2.23 +0.53 3.70*+-0.33
62.5-+2.6 69.0_+1.4
0.94 _+0.06 3.14" _+0.51
0.80+_0.05 1.96+-1.12
1.74 +_0.36 5.10"+-1.63
55.7+4.1 63.3_+10.3
PLD
N D
SMP N
* Values significantly different from n o r m a l chickens ( P <0.02, by t-test).
In the dystrophic (D) animals the proportion of type I fibres of the PLD is increased above the normal (N) value, being about double at 7 weeks of age (Fig. 5). The histogram of type I fibre area distribution was unchanged in the dystrophic birds (not shown), so that the mean area and the standard deviation did not differ significantly between dystrophic and normal muscles (Table 2). The
227
Fig. 7. Frozen sections, stained for ACHE, from the SMP muscle of a normal chicken. In A, a type IA fibre can be seen with several stained nerve endings. The fibre diameter is too large to indicate a type IB fibre. In B, two large motor end-plates on adjacent type II fibres are shown. Bars = 60/~m. increases in the numbers of total myonuclei and peripheral myonuclei which characterize dystrophic chicken muscles fibres (McMurtry et al. 1972; Pizzey and Barnard 1983a) are absent in the type I fibres of the D-PLD, even up to 70 days of age (Table 3). Similarly, no hypertrophy, atrophy, fibre splitting, necrosis or phagocytosis was seen in the type I fibres in sections of the D - P L D from 30-70 days of age. As noted above, these fibres in the P L D muscle are, in fact, more precisely specified as type IA. In contrast to these, the type IA fibres in the D-SMP showed characteristic pathological changes. The number o f nuclei per fibre cross-section was doubled, and this increase occurred only at peripheral positions (Table 3). The distribution of type IA fibre areas is also markedly skewed compared to that of type IA fibres in the normal muscle (Fig. 8). Analyses o f fibre areas were made using also the variability coefficient (VC) and hypertrophy factor (HF), which are, respectively, estimations of variability within a muscle (VC = SD/mean x 1000) and of the contribution of abnormally large fibres (Dubowitz and Brooke 1973). These indices were both significantly higher in the D-SMP than in the N-SMP (Table 2). Hypertrophy and other pathological features such as the rounding of fibres were also seen in the type IA dystrophic fibres, as shown in Fig. 3, where the characteristic wide variation in fibre size is illustrated. These latter findings were seen consistently in a total of 5 dystrophic animals examined (involving about 300 type I fibres in these dystrophic SMP muscles ). The type IB fibres found in the SMP appear to be much more resistant to dystrophic change. Nuclei content is similar in type IB fibres o f both normal
228 IA
IB
0 1
22 3 (lO~m 2)
t~
S
. . . . . . . . . 2 ~ 6 8 (lO,um 2)
liB
."--:.. . . . . . . . . 10 12 2 /,
7 I .'-q, . , , Fq 6 28 210 12 l& 16 (10 ~Jm )
Fibre Area
Fig. 8. Area histograms of fibre types in the SMP muscle from normal (upper) and dystrophic (lower) 35-day chickens. The histograms are constructed from the pooled values of individual fibres in all of the muscle samples. Type IA and type liB fibres of the dystrophic SMP muscle show the greatest skewness in the histograms. Note the change of scale for type IB fibres. All histograms of dystrophic fibre types are different from normal (P <0.001).
and dystrophic birds, and the variability coefficient of the mean areas is also not significantly different between them. The high value for the latter parameter in both normal and dystrophic chickens in the present case is due to the exceptionally small mean area of this fibre type (Table 2). However, although dystrophic type IB fibres have not lost their polygonal profile by 70 days, hypertrophy is beginning before this stage, as can be seen from the mean areas and the hypertrophy factors listed in Table 2, and the area histograms shown in Fig. 8. Such hypertrophy was the only pathological change which we observed in any of the type IB fibres from dystrophic chickens up to 70 days. There was, however, an increase in the proportion of IB fibres in the D-SMP, as there was also of IA fibres (Fig. 5).
Type H fibres The majority of fibres in both the N-PLD and N-SMP are type IIB. The SMP has virtually no type IIA fibres (Fig. 5), while the PLD contains 15-20% in 8-week-old chickens (Beringer 1978). The normal type IIA fibres are slightly smaller in area than type IIB. The standard deviation of the mean areas is small for each fibre type, as shown in Table 2. This parameter is a measure of the range of fibre sizes within an individual muscle, and is not an expression of variability between different birds. Type IIA fibres of the D-PLD are often hypertrophic and frequently have a more rounded profile in cross-section, as found previously (Beringer 1978). Pathological features such as fibre splitting and necrosis were not seen here in any type IIA fibres of the D-PLD, up to 70 days of age. However, the mean area of the dystrophic type IIA fibres is much higher than in the type liB fibres in the D-PLD
229 (Table 2), because the latter population contains a large number of very small fibres, primarily due to longitudinal fibre splitting, atrophy or regeneration. However, since many type liB fibres are also severely hypertrophic, the standard deviation for that population is high. This causes the variability coefficient of dystrophic type liB fibres to be twice the value of the type IIA fibre population (Table 2). Thus, mean area values alone are not sufficient to reflect the relative involvement of different fibre types in dystrophic chicken muscles. Type liB fibres are again the most severely affected fibres in the SMP muscle in all dystrophic chickens examined up to 70 days, as determined by measurements of nuclei content and distribution (Table 3) and the statistical parameters of fibre area distribution (Table 2; Fig. 8). Phagocytosis and necrosis are associated only with type liB fibres in the P L D and SMP at this stage, although the incidence is only ~ 2~" of fibres in both the D-PLD and the D-SMP. The M L D muscle This muscle is almost purely tonic, as determined by histochemical criteria, in both normal and dystrophic chickens (Fig. 4). The proportions of the two tonic sub-types present, IliA and IIIB (Barnard et al. 1982), are unaltered by dystrophy. No changes in fibre size or shape, or in nuclei numbers and distribution are seen, nor any degenerative events (Fig. 9). This muscle is totally resistant to dystrophy up to the maximum period (90 days) studied here.
Fig. 9. Frozen section of the MLD muscle from a dystrophic chicken. The muscle is of normal appearance for chicken tonic muscle. There is little variation in fibre area and all myonuclei are in sarcolemmal positions. Haematoxylinand eosin; Bar = 60/~m.
230 DISCUSSION
The quantitative criteria of increased variability coefficient and skewed histograms of fibre area, increased total and peripheral nuclei and degenerative events, were sufficient to classify fibres as dystrophic or not, and these criteria behaved as a consistent set. We found thus that all twitch fibre types can be affected by muscular dystrophy in the chicken. A given twitch fibre may be either susceptible or resistant, depending on the muscle in which it is located. Thus, type IA fibres are unaffected in the D-PLD, as they are also on the red side of the sartorius and in the adductor superficialis (Barnard et al. 1982); they are, in contrast, strongly affected in the D-SMP. This is despite the fact that they are present as a similarly low percentage of total fibres in both the PLD and the SMP muscles, with many neighbouring dystrophic type liB fibres in each case. Since both the SMP and the P L D type I fibres are oxidative this, alone, cannot account for the resistance to dystrophy in type I fibres in the latter muscle. In some other muscles even type l i b fibres are resistant, as has recently been shown to be the case (at the ages studied here) in the red sartorius and the adductor superficialis muscles (Barnard et al. 1982), where these fibres are in a minority. This resistance has likewise been found in the type liB fibres (again a minority) of the complexus, a muscle of the neck used constantly and showing no pathologic changes in the dystrophic birds throughout life (Ashmore et al. 1973). Even within one muscle (the SMP), the two sub-types of type i fibre were found to differ in their expression of myopathy, type IB being much less affected. An increase in the proportion of type I fibres occurred whether they are of the group which showed myopathic change or not (Fig. 3). This is a more general phenomenon, in fact, since in other muscles which are affected, the resistant type I fibres increase in proportion, e.g. in the P L D (Barnard et al. 1982). It should be mentioned here that the two sub-types of type I fibres in the chicken SMP are not both found in the mammals, nor in the pigeon SMP. Khan (1979) found that the latter contained two type I sub-types, both of which were low in oxidative, but high in glycolytic enzymes. We found no evidence for a "slow-glycolytic" fibre type in the chicken SMP, nor in all the other chicken muscles which we have examined. However, one feature that is characteristic of type I avian fibres wherever they have been recognized is multiple innervation, as found here for the chicken SMP type IA and IB fibres, and previously for type I fibres in the pigeon SMP (Khan 1979), chicken sartorius (Ashmore et al. t978), P L D (Barnard et al. 1982) and biventer cervicis (Toutant et al. 1981 ; Barnard et al. 1982). The major difference between the two sub-types of type I fibres is the exceptionally small size of the type IB fibres in the SMP, which may be related to the specialised functions of subcutaneous muscles (see below). In conclusion, we can say that a given twitch fibre type does not necessarily respond in the same way to dystrophy in different muscles, so that a universal determination of the myopathy by the class of motor neurone can be ruled out.
231 It has long been recognised that chicken muscles vary in the extent to which they are affected by dystrophy. The most affected contain a majority of type IIB fibres, for example, the pectoral, biceps and PLD muscles. These are all involved in wing movement, but in a flightless bird such as the chicken, these muscles would get relatively little exercise, and this is, presumably, the reason for the high proportion of type IIB fibres in these muscles. In contrast, the pectoral muscle fibres in flying birds are generally oxidative (Talesara and Goldspink 1978). The amount of exercise a muscle gets may be related to the rate of expression of the disorder in both human and chicken dystrophy. It is thought that moderate exercise is beneficial for Duchenne patients (Vignos and Watkins 1966; Dubowitz and Heckmatt 1980) and there is an indication of this, also, in a limited application of exercise in the dystrophic chicken (Entrikin et al. 1978; Hudecki et al. 1978). The muscles which are completely or relatively spared in both Duchenne dystrophy (see Introduction) and chicken dystrophy are either tonic or tend to have a majority of type I fibres. This could be related to the frequent use of these muscles. The postural function of tonic muscles such as the ALD means that these muscles are virtually in constant use. We do not know yet if it is a constancy of stimulation in vivo, or the slower rate of stimulation, or some special neuronal factor, which confers the resistance of tonic muscles. In the oxidative twitch muscles with few type IIB fibres, we presume the latter are protected by the natural exercise of the muscle as a whole. The involvement of type I fibres of the SMP may be related to the specialised function of this muscle in maintaining tautness of the skin along the posterior edge of the wing during flight and as the wing is being folded (Bock 1973). It is probable that there is great variation in the use of this muscle between avian species. For example, the SMP of the chicken will rarely be used. Indeed, we suggest that the chicken SMP is used even less than the other muscles, mentioned above, which are involved in wing movement. This muscle in the chicken is very much smaller and thinner than in the pigeon, and appears to be vestigial in the former, flightless bird. Relative disuse may account for the involvement of type I fibres in the D-SMP, at stages when type I fibres in the D-PLD are unaffected. There are many other physiological features of muscle which are related, either directly or indirectly, to their fibre type composition. For example, type IIB-rich muscles are more readily fatigued than those with a majority of oxidative fibres and the latter are also generally more vascular (Vrbov~i et al. 1978). Whatever the nature of the primary lesion in chicken dystrophy, the expression of the disease in different muscles may be modified by such phYsiological factors. Thtts 'it is seen that both quantitative fibre type composition and the degree of use and nature of stimulation must be considered in interpreting the differential involvement of muscles in the muscular dystrophies.
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