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A histochemical and electron microscopic study of a fast- and a slow-twitch muscle in genetically spastic mice
TISSUE & CELL 1981 13 (I) 61-69 ( 1981 Longman Group Ltd
MARTIN
0040-8166/8
1~00060061$02.00
A. LEVIN, PAUL DEGENNARO, ANTHONY ROSS, NORMA SERAFIN and JAMES A. STEWART*
A HISTOCHEMICAL AND ELECTRON MICROSCOPIC STUDY OF A FAST- AND SLOW-TWITCH MUSCLE IN GENETICALLY SPASTIC MICE
A
ABSTRACT. Fast and slow muscle fibers were studied in the flexor digitorum longus (FDL) and soleus (SOL) muscles, respectively. in control and spastic mice. Histochemical and electron microscopic studies indicated an increased number of mitochondria, a decreased deposition of glycogen and a vesiculation and distension of the sarcoplasmic reticulum in many fast-twitch fibers of the spastic FDL. Similar findings were not evident in the slow-twitch fibers of the spastic SOL. Since the spastic condition causes increased muscular activity as a result of more rapid and prolonged nerve impulse firing, these findings reinforce the idea that a muscle fiber’s oxidative capabilities are a function of its activity.
Introduction MICE, homozygous for the mutant spa gene, were discovered in 1960 in a hybrid population (Chai, 1961). The clinical manifestations of this spastic condition, which vary considerably from mouse to mouse, include: rapid tremors of the hind limbs and tail, reduced flexibility of trunk movements, and difficulty in righting when placed on its back. These conditions are most notable following any type of external stimulus. When stimulated, the animal appears hyperactive. Increased stimulation often results in the animal falling over on its side and being unable to right itself. Not all animals are this severely affected. This disorder can only be recognized in some animals as a tremor transmitted to the finger tips when the animal is lifted by its tail. Experimental evidence suggests that a metabolic defect in the brain, resulting in Department of Biology, Eastern Connecticut State College, Willimantic, Connecticut 06226. * Department of Biochemistry, University of New Hampshire, Durham. New Hampshire 03824. Received 22 January 1980. Revised 20 September 1980.
overproduction of an excitatory transmitter, underlies the spastic condition (Chai, 1961; Chai et al., 1962; Chatterjee and Hechtman, 1977; Stewart, 1979). The tremors, therefore, are a result of increased neural impulse activity to the muscles of the extremities and back. This, in effect, changes the patterns of activity of those muscles (and muscle fibers) involved. Physiologically, vertebrate fibers are classified as fast- and slow-twitch based upon their relative speeds of contraction. Slow-twitch fibers, which receive a constant level of nervous impulse activity, can maintain tension for long periods of time. This is in contrast to the sporadically stimulated fasttwitch fibers which are specialized for brief intermittent phasic activity (Brooke, 1970). During the past 20 years, the utilization of ultrastructural, histochemical, and biochemical techniques in mammalian muscle studies has led to much confusion and a variety of classifications. Generally, they correspond to three types based upon myofibrillar ATPase, oxidative enzymes, and the presence or absence of glycogen (see Close, 1972, for a review). These three types are best classified as fast-twitch glycolytic (FG), fast-twitch
62
LEVIN,
DEGENNARO,
oxidative-glycolytic (FOG), and slow-twitch oxidative (SO) (Peter et al., 1972). Other examples of nomenclature for FG, FOG, and SO fibers, respectively, include: (1) white, intermediate, and red (Gauthier, 1970; Dubowitz, 1970); (2) A, C, and B (Stein and Padykula, 1962); and (3) II(B), II(A), and I (Brooke and Kaiser, 1970; Dubowitz, 1970). These three fibers can also be demonstrated ultrastructurally (Gauthier, 1970; Tomanek, 1976). The FG fibers contain a sparse mitochondrial population with little if any subsarcolemmal accumulations of an extensive sarcotubular mitochondria, system and thin Z lines. The FOG fibers have fairly numerous intermyofibrillar mitochondria, a fair sarcotubular system and moderately wide Z lines. The SO fibers exhibit large numbers of intermyofibrillar and subsarcolemmal mitochondria, a fairly welldeveloped sarcotubular system and wide Z lines. This study was undertaken to determine whether histochemical and/or ultrastructural differences occur in the muscle fibers of spastic mice as a result of increased neural activity. The two muscles chosen for this study were the flexor digitorum longus (FDL), a fast-twitch muscle containing predominantly FG and FOG fibers, and the soleus (SOL), a slow-twitch oxidative muscle containing predominantly SO and FOG fibers with a small population of FG fibers. Materials and Methods Adult male and female spastic (spa/spa) and mice weighing benormal (spa/ + , +/+) tween 24 and 31 g were sacrificed by means of cervical dislocation. The SOL and the FDL muscles were prepared for histochemical and electron microscopic analysis as discussed below. Histochemistry The muscles were removed, placed on wooden tongue depressor blades and frozen by immersion for 5-10 min in a beaker containing methyl butane cooled to - 195°C with liquid nitrogen. The muscles were mounted on chucks, allowed to warm to - 20 to - 15°C and were sectioned at 10 pm on a Lab-Tek cryostat. Sections were picked up on coverslips and allowed to air dry at room temperature for 0.5-3.0 hr.
ROSS,
SERAFIN
AND
STEWART
Tissues were reacted for myofibrillar ATPase activity at pH 9.4 following pre-incubation at pH 4.35 (acid) or pH 10.4 (alkaline) (Padykula and Herman, 1955; Guth and Samaha, 1959, 1970). In addition, sections were stained for succinic dehydrogenase activity (Lillie, 1964) and glycogen using the periodic acid-Schiff (PAS) reaction (McManus, 1948). In aI1 cases, tissues were dehydrated in a graded series of ethanol and mounted with Histoclad. Electron microscopy Portions of the mid-belly region of the FDL and SOL muscles were removed, placed on wooden supports and fixed in buffered (pH 7.1) 2% glutaraldehyde. After 30-45 min initial fixation, all muscles were diced and allowed to fix for a total of 2+ hr. The tissues were rinsed in two changes of phosphate buffered sucrose (pH 7.1); the second rinse was of 12-14 hr duration at 4°C. The tissues were post-fixed in 1 % phosphatebuffered osmium (pH 7.1) for 1 hr, dehydrated in a graded ethanol series, infiltrated in a propylene oxide-Epon-Araldite mixture and embedded in an Epon-Araldite mixture. The resin was polymerized for 48 hr at 60°C. Thick and thin sections were made with glass knives on an LKB Ultratome II. Thick sections were stained with toluidine blue whereas thin sections were stained first with uranium, then lead. Thin sections were examined in a JEOL lOOB-II electron microscope at 80 kV. Ultrastructural observations were limited to FG and FOG fibers in the FDL and SO fibers in the SOL. Results Control muscles (a) Histochemical observations. All three fiber types (FT, FOG, and SO) were found in the soleus and flexor digitorum longus (FDL) muscles of control animals (Table 1). The FDL contained predominantly fast-twitch fibers of both types with the SO fibers making up between 2 and 5% of the total population. The total percentage of oxidative fibers (i.e., both SO and FOG fibers) was variable (between 40 and 65%) (Fig. 1). Periodic acid-Schiff activity (glycogen) was variable in both FG and FOG fibers with little or no activity associated with the SO fibers (Fig. 2).
FAST
AUD
SLOW
MUSCLES
IN
SPASTIC
hi
MICE
I.
Fig. Fibers from the mid-belly region of a control FDL muscle stained for succinic dehydrogenase activity. Note the moderate number of oxidativc fibers. Compsrc this with Fig. 3. x 150. Fig. 2 Flbera content
(PAS]
from
the mld.belly
showing
many
fibers
region with
ofa control moderate
FDL
musclestained
to heavy
glycogen
for glycogen depnzlts.
Fig. 3. Fibers from the mid-belly region of a spastic FDL muscle Fumed dehydrogenase activity. Note the large wmbcr of oxidative fibers. Compare 150.
x
Fig. 4. Fibers
from
content. These fibers, Jepoqition y 150.
the mid-belly taken
from
region a very
The soleus, a predominantly oxidativc muscle, possessed few FG fibers; all other fi hers were either SO or FOG The number of SO fibers varied between 45 and 60 y/i. In both muscles, SO fibers were clearly identifiable due to their low glycogen content
ofa
spastic
spastic
FDL
mouse,
x 150.
for wccinlc tiith Fig.
muscle stained
for glgcogen
show
no glycogen
llttle
or
I.
(PAS). high subsarcolemmal and intermyoiihrillar mitochondrial distribution (SDH) and intense ATPase activity following atid pre-incubation, which was reversed following alkaline pre-incubation. The FOG fibers exhibited moderate but variable glycogen
64
LEVIN,
DEGENNARO,
ROSS, SERAFIN
AND STEWART
Table 1. Histochemicaifiber types found in spastic and control muscles of the mouse Histochemical Myosin ATPase (alkaline preincubation) Control Expt.
Muscle Flexor digitorum longus Fiber types FG FOG SO Soleus Fiber types FG FOG SO
Myosin ATPase (acid preincubation) Control Expt.
reaction Periodic acidSchiff
Succinic dehydrogenase Control
Expt.
Control
Expt.
2-3 2-3 0
3 3 0
0 0 3
0 0 3
1 2 3
2-3*t 2-3*t 3
2-3 2-3 1
O-1
2-3 2-3 0
3 3 0
0 0 3
0 0 3
1
2 3
2 2-3 3
2-3 2
O-l O-l O-l
1
O-12 o-13
~~ 3, strong reaction; 2, moderate reaction; I, light reaction; 0, no reaction. * The greater the degree of spasticity, the higher the activity. t In many instances it was impossible to differentiate between FG and FOG fibers. $ The greater
the degree of spasticity,
the lower the activity.
deposits, large numbers of intermyofibrillar mitochondria and intense ATPase activity following alkaline pre-incubation, which was reversed following acid pre-incubation. The FG fibers exhibited characteristics similar to the FOG fibers except that few mitochondria were present (Table 1). (b) Ultrastructural observations. In both the FDL and SOL muscles, the appearance of the fibers was the same as that previously described for mammalian muscles (Gauthier, 1970; Tomanek, 1976). In the SO fibers of the soleus, large numbers of intermyofibrillar and subssrcolemmal mitochondria, a fairly well-developed sarcotubular system, and wide Z lines were found. Glycogen granules were rarely present (Fig. 5). In the FG fibers of the FDL, a sparse
mitochondrial population with mitochondria occasionally paired at the I bands, an extensive sarcotubular system, and thin Z lines were found (Fig. 7). The FOG fibers contained numerous mitochondria usually paired at the I bands, a fairly well-developed sarcotubular system and moderately wide Z lines (Fig. 8). In most of the fast fibers, glycogen granules were present. Spastic muscles (a) Histochemical observations. No discernible abnormalities were found in the spastic muscles following light microscopic observadistinct histochemical tions; however, changes were noted, especially in the FDL. In muscles isolated from animals showing severe mitochondrial activity was so spasticity, great in all fast fibers that it was impossible
Fig. 5. Electron micrograph of a SO fiber from a control SOL muscle demonstrating abundant
mitochondria
(M) wide Z lines (Z) and little or no glycogen.
x 24,000.
Fig. 6. Electron micrograph of a SO fiber from a spastic SOL muscle. This fiber is comparable to control muscle fibers. x 24,000. Fig. 7. Electron micrograph of a FG fiber from a control FDL muscle demonstrating the mitochondria (M) paired at the I bands (I) and relatively thin Z lines (Z). x 24,000.
FAST
AND
SLOW
MUSCLES
IN SPASTIC
to histochemically type the majority of the spastic muscle fibers based on their SDH activity (compare Figs. 1 and 3). In less severely affected mice, mitochondrial density was not as high. Glycogen deposits (as indicated by the PAS reaction) were severely depleted and virtually absent in the muscles of severely affected mice (Fig. 4). Increased mitochondrial density was not as striking in the soleus since this muscle is normally highly oxidative. In both muscles, the relative numbers of fast- and slow-twitch fibers remained unchanged as indicated by myosin ATPase activity. (b) Ultrastructural observations. All SO fibers studied in the soleus were comparable to control muscle fibers (Fig. 6). The most obvious changes found in the FDL muscles of severely spastic mice correlated well with the histochemical findings. It was not possible to readily differentiate between FG and FOG fibers. Most fibers studied exhibited increased intermyofibrillar mitochondria and very low (often absent) glycogen deposits (Fig. 9). Terminal cisternae of the sarcoplasmic reticulum were often distended; however, the sarcoplasmic reticulum membrane serations (SR feet) which are found in association with the membrane of the T-tubule always remained intact (Figs. 10, 11). In addition, many regions of extensive vesiculation of the sarcoplasmic reticulum were found (Fig. 10). Discussion Naturally and artificially induced changes in muscular activity have been shown to induce
MICE
histochemical, physiological, and ultrastructural changes in various muscle fibers. Intermittent long-term stimulation of the tibialis anterior and extensor digitorum muscles (fast muscles) of the rabbit with a frequency pattern simulating that of a slow muscle, increased mitochondrial density and enzymes of the citric acid cycle. Concomitant with this were decreases in enzymes of glycolytic metabolism (Pette et al., 1973) and increased fatigue resistance (Pette and Ramirez, 1975). Similar long-term stimulation of the slow rabbit soleus had no effect (Pette and Ramirez, 1975). Terjung (1976) found that intense exercise caused increased oxidative capacity in the muscles of rats. Prince and his colleagues (1976, 1977) demonstrated increased numbers of oxidative fibers in male long-distance runners and female collegiate field hockey players. Other more drastic experimental manipulations have produced changes that suggest a conversion of fast-twitch fibers to slowtwitch fibers. Tenotomy of synergists has been used. Schiafino and Bormioli (1973) found that following synergistic tenotomy, the extensor digitorum longus and flexor hallucis longus muscles of the rat showed increased succinic dehydrogenase activity, and fibers having low myosin ATPase activity (following alkaline pre-incubation) increased in numbers. Tomanek (1975) found similar results in the soleus and plantaris muscles of the kitten following synergistic tenotomy. Nerve cross-union studies also suggest the importance of the nerve in the physiological status of muscle fibers. Buller and his
Fig. 8. Electron micrograph of a FOG fiber from a control FDL muscle demonstrating abundant mitochondria and moderately wide Z lines. Also note the glycogen deposits. x 24.000. Fig. 9. Electron micrograph of a fast fiber from a spastic FDL muscle. Note the large number of mitochondria and virtual absence of glycogen. In severely affected mice, this was the predominant fiber type. x 24,000. Fig. 10. Electron micrograph of a fast fiber from a severely spastic FDL muscle. Note triad with distended terminal cisternae (C) and vesiculation of some regions of the sarcoplasmic reticulum (Sr). x 24,000. Fig. I I. Higher magnification (F) are intact. x 60,000.
67
of a triad similar to that shown in Fig. 10. The SR feet
68
LEVIN,
DEGENNARO,
colleagues (1960, 1965) found that following nerve cross-union of fast and slow muscles in adult cats, each muscle took on the twitch characteristics of the foreign nerve, Thus, the slow muscle became fast and the fast muscle became slow. In addition to physiological changes, biochemical and histochemical changes were observed following crossinervation of the cat FDL and soleus muscles. The myosin ATPase activity of the fast FDL approached that of the slow soleus whereas the activity of the soleus muscle increased only slightly (Buller and Lewis, 1965). Our histochemical and ultrastructural findings strongly suggest that increased muscular activity in the spastic condition, due to an increased rate of nerve firing, can induce adaptive changes in the oxidative and glycolytic capacity of muscle fibers. Just exactly how the nerve affects the muscle fibers has been a subject of much debate. There are two current hypotheses regarding the nerve’s effect upon the muscle: (1) a specific neurotropic substance is passed down the nerve causing the muscle to differentiate and be maintained in that state and, (2) the activity of a muscle fiber, as a consequence of nervous impulse propagation, is the main factor involved in the development and maintenance of muscle fiber types. The more or less constant nervous impulse activity to the fast fibers, resembling that encountered by a slow fiber, caused an increase in oxidative capacity. However, no fast fibers exhibited any other slow fiber characteristics, i.e., low myosin ATPase activity following alkaline pre-incubation and slow fiber ultrastructure. This, in conjunction with other studies involving increased use, suggests that the oxidative and glycolytic capacities, as may be indicative of fast or slow fibers, are depen-
dent upon the actual activity of the muscle fiber whereas differences in myosin ATPase
ROSS,
SERAFIN
AND STEWART
and ultrastructure between fast and slow fibers may be dependent upon some trophic substance found in the specific nerve. In this study, the only consistent ultrastructural difference (other than glycogen granule and mitochondrial content) in the fast fibers of spastic mice was the distension and vesiculation of the sarcoplasmic reticulum. Variations in the sarcoplasmic reticulum are the most prominent early ultrastructural abnormality described in many muscular diseases. Vesiculation and distension of the sarcoplasmic reticulum were found in embryonic and adult dystrophic muscles of chickens (Schotland, 1970; Shafiq et al., 1974; Allen and Murphy, 1978) and mice (Platzer and Powell, 1975). In many dystrophic disorders, other ultrastructural changes follow the appearance of sarcoplasmic reticulum abnormalities. This did not occur here. All muscle fibers maintained their integrity. Also, normal muscle function in the presence of increased activity is further suggested by the normal appearance of the ‘S.R. feet’ indicative of the maintenance of coupling between the T system and the sarcoplasmic reticulum (Franzini-Armstrong, 1972, 1973). These findings suggest that the sarcoplasmic reticulum changes in response to functional demand. Therefore, changes in the sarcoplasmic reticulum in many muscular disorders may be due to initial changes in the activity of the muscle fibers, this being followed by the pathological structural conditions associated with the particular disorder. Acknowledgements
The authors wish to thank Dr Grace Rovozzo and Mrs M. Grace Levin for kindly reading the manuscript and making valuable suggestions.
ALLEN, E. R. and MURPHY, B. J. 1978. Sarcotubular development in dystrophic skeletal muscle cells. Cell 7%~. Res., 194, 125-l 30. BROOKE, M. H. 1970. Some comments on neural influence on the two histochemical types of muscle fibers. In The Physiology and Biochemistry of Muscle ns a Food (eds. E. J. Brisky, R. G. Cassens and B. B. Marsh),
Vol. 2, pp. 131-153. University of Wisconsin Press, Madison. BROOKE. M. H. and KAISER, K. K. 1970. Three myosin ‘adenosine triphosphatase’ systems: the nature of their pH lability and sulfhydryl dependence. J. Hisrochem. Cytochem., 18, 670-672. BULLER, A. J., ECCLES, J. C. and ECCLES, R. M. 1960. Differentiation of fast and slow muscles in cat hind limb. .I. Physiol., 150, 399-416.
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AND
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MUSCLES
IN SPASTIC
MICE
69
BULLER. A. J. and LEWIS, D. M. 196.5. Further observations on mammalian cross-innervated skeletal muscle. J. Physiol., 178. 343-358. CHAI. C. K. 1961. Hereditary spasticity in mice. J. Heredity, 52, 241-243. &AI, C. K., ROBERTS, E. and SIDMAN. R. L. 1962. Influence of amino-oxyacetic acid, a y-aminobutyrate transaminase inhibitor, on hereditary spastic defect in the mouse. Proc. Sot. exp. Biol. Med., 109, 491.. 495. CHATTERIEE. S. and HECHTMAN, P. 1977. y-Aminobutyric acid metabolism in brain homogenates of the spastic mouse. Eiorhem. Genetics, 15, 147-151. CLOSE. R. 1972. Dynamic properties of mammalian skeletal muscle. Physiol. Rev., 52, 129-137. DUBOWITZ, V. 1970. Differentiation of fiber types in skeletal muscle. In The Physiology and Biochemirrn~ of Muscle as a Food (eds. E. J. Brisky, R. G. Cassens and B. B. Marsh). Vol. 2, pp. 103-130. University of Wisconsin Press, Madison. FRANZINI-ARMSTRONG, C. 1972. Studies of the triad. III. Structure of the junction in fast-twitch fibers. Tissr/e & Cell, 4, 469-478. FRANZINI-ARMSTRONG,C. 1973. Membrane systems in muscle fibers. The Structure and Function of Mosck, (ed. G. H. Bourne), Vol. 2, pp. 531-619. Academic Press, New York. GAIJTHIER, G. F. 1970. The ultrastructure of three fiber types in mammalian skeletal muscle. In The Physiology and Biochemistry of Muscle as a Food (eds. E. J. Brisky. R. C. Cassens and B. B. Marsh), Vol. 2. pp. 103S130. University of Wisconsin Press, Madison. GIITH. L. and SAMAHA, F. J. 1969. Qualitative differences between actomyosin ATPase of slow and I‘dSt mammalian muscle. Exp. Neural., 25, 138-l 52. G~TH, L. and SAMAHA, F. J. 1970. Procedure for the histochemical demonstration of actomyosin ATPase. Exp. Neural., 28, 365-367. LILLIE. R. D. 1964. Histopathologic Technic and Practical Histochemiatry. McGraw-Hill, New York. MCMANLIS,J. F. A. 1948. Histological and histochemical uses of periodic acid. Stain Tech., 23, 99-108. PADYKULA, H. A. and HERMAN, E. 1955. The specificity of the histochemical method for adenosine triphosphatase. J. Histochem. Cytochem., 3, 170-195. PETER. I., BARNARD, R., EDGERTON, V.. GILLESPIE,C. and STEMPEL,K. 1972. Metabolic profiles of three fiber types of skeletal muscle in guinea pigs and rabbits. Biochemistry, 11, 2627-2633. PE~TE. D., SMITH, M. E., STAUDTE.H. W. and VRBOVA, G. 1973. Effects of long-term electrical stimulation on some contractile and metabolic characteristics of fast rabbit muscles. fpiisers Arc/z., 338, 257-272. PETX, D. and RAMIREZ, B. V. 1975. Influence of intermittent long-term stimulation on contractile, histochemical and metabolic properties of fibre populations in fast and slow rabbit muscles. efliigers Arch., 361, l-7. PLATZER, A. C. and POWELL, J. A. 1975. Fine structure of prenatal and early postnatal dystrophic mouse muscle. J. Neural. Sci., 24, 109-126. PRINCE, F., HIKIDA, R. S. and HAGERMAN, F. 1976. Human muscle fiber types in power lifters. distance runners and untrained subjects. P’irgers Arch., 363, 19-26. PRINCE,F. P. and HIKIDA, R. S. 1977. Muscle fiber types in women athletes and non-athletes. Pfliiger.v Arch., 371, 161-165. SCHIAFFINO, S. and BORMIOLI, P. 1973. Adaptive changes in developing rat skeletal muscle in response to functional overload. Exp. Neural. 40, 126-137. SCHOTLAND,D. L. 1970. An electron microscopic investigation of myotonic dystrophy. J. Nruropath. esp Neural., 29, 241-252. SHAFIQ. S. A., ASIEDU, S., RYAN, D. and MILHORAT, A. T. 1974. Effect of denervation and hereditary muscular dystrophy on the differentiation of chicken fiber types with some observations on early changes in muscular dystrophy. Exploratory Concepts in Muscular Dystrophy, Control Mechanisms and Their re1ationship.s to Muscular Dystrophy and Related Neuromuscular Diseases (ed. A. T. Milherat), Excerpta Medica, Amsterdam. STEIN, J. M. and PADYKULA, H. A. 1962. Histochemical classification of individual skeletal muscle fibers of the rat. Am. J. Anat., 110, 103-124. STEWART,J. 1979. Personal communication. TERJI!NG, R. 1976. Muscle fiber involvement during training of different intensities and duration. Anr. J. Physiol., 230, 946-950. TOMANEK, R. 1975. A histochemical study of postnatal differentiation of skeletal muscle with reference to functional overload. Dev. Biol., 42, 305-3 14. TOMANEK.R. 1976. Ultrastructural differentiation of skeletal muscle fibers and their diversity. J. Ultrastruct., Rex.. 55. 2 12-227.
MARTIN
0040-8166/8
1~00060061$02.00
A. LEVIN, PAUL DEGENNARO, ANTHONY ROSS, NORMA SERAFIN and JAMES A. STEWART*
A HISTOCHEMICAL AND ELECTRON MICROSCOPIC STUDY OF A FAST- AND SLOW-TWITCH MUSCLE IN GENETICALLY SPASTIC MICE
A
ABSTRACT. Fast and slow muscle fibers were studied in the flexor digitorum longus (FDL) and soleus (SOL) muscles, respectively. in control and spastic mice. Histochemical and electron microscopic studies indicated an increased number of mitochondria, a decreased deposition of glycogen and a vesiculation and distension of the sarcoplasmic reticulum in many fast-twitch fibers of the spastic FDL. Similar findings were not evident in the slow-twitch fibers of the spastic SOL. Since the spastic condition causes increased muscular activity as a result of more rapid and prolonged nerve impulse firing, these findings reinforce the idea that a muscle fiber’s oxidative capabilities are a function of its activity.
Introduction MICE, homozygous for the mutant spa gene, were discovered in 1960 in a hybrid population (Chai, 1961). The clinical manifestations of this spastic condition, which vary considerably from mouse to mouse, include: rapid tremors of the hind limbs and tail, reduced flexibility of trunk movements, and difficulty in righting when placed on its back. These conditions are most notable following any type of external stimulus. When stimulated, the animal appears hyperactive. Increased stimulation often results in the animal falling over on its side and being unable to right itself. Not all animals are this severely affected. This disorder can only be recognized in some animals as a tremor transmitted to the finger tips when the animal is lifted by its tail. Experimental evidence suggests that a metabolic defect in the brain, resulting in Department of Biology, Eastern Connecticut State College, Willimantic, Connecticut 06226. * Department of Biochemistry, University of New Hampshire, Durham. New Hampshire 03824. Received 22 January 1980. Revised 20 September 1980.
overproduction of an excitatory transmitter, underlies the spastic condition (Chai, 1961; Chai et al., 1962; Chatterjee and Hechtman, 1977; Stewart, 1979). The tremors, therefore, are a result of increased neural impulse activity to the muscles of the extremities and back. This, in effect, changes the patterns of activity of those muscles (and muscle fibers) involved. Physiologically, vertebrate fibers are classified as fast- and slow-twitch based upon their relative speeds of contraction. Slow-twitch fibers, which receive a constant level of nervous impulse activity, can maintain tension for long periods of time. This is in contrast to the sporadically stimulated fasttwitch fibers which are specialized for brief intermittent phasic activity (Brooke, 1970). During the past 20 years, the utilization of ultrastructural, histochemical, and biochemical techniques in mammalian muscle studies has led to much confusion and a variety of classifications. Generally, they correspond to three types based upon myofibrillar ATPase, oxidative enzymes, and the presence or absence of glycogen (see Close, 1972, for a review). These three types are best classified as fast-twitch glycolytic (FG), fast-twitch
62
LEVIN,
DEGENNARO,
oxidative-glycolytic (FOG), and slow-twitch oxidative (SO) (Peter et al., 1972). Other examples of nomenclature for FG, FOG, and SO fibers, respectively, include: (1) white, intermediate, and red (Gauthier, 1970; Dubowitz, 1970); (2) A, C, and B (Stein and Padykula, 1962); and (3) II(B), II(A), and I (Brooke and Kaiser, 1970; Dubowitz, 1970). These three fibers can also be demonstrated ultrastructurally (Gauthier, 1970; Tomanek, 1976). The FG fibers contain a sparse mitochondrial population with little if any subsarcolemmal accumulations of an extensive sarcotubular mitochondria, system and thin Z lines. The FOG fibers have fairly numerous intermyofibrillar mitochondria, a fair sarcotubular system and moderately wide Z lines. The SO fibers exhibit large numbers of intermyofibrillar and subsarcolemmal mitochondria, a fairly welldeveloped sarcotubular system and wide Z lines. This study was undertaken to determine whether histochemical and/or ultrastructural differences occur in the muscle fibers of spastic mice as a result of increased neural activity. The two muscles chosen for this study were the flexor digitorum longus (FDL), a fast-twitch muscle containing predominantly FG and FOG fibers, and the soleus (SOL), a slow-twitch oxidative muscle containing predominantly SO and FOG fibers with a small population of FG fibers. Materials and Methods Adult male and female spastic (spa/spa) and mice weighing benormal (spa/ + , +/+) tween 24 and 31 g were sacrificed by means of cervical dislocation. The SOL and the FDL muscles were prepared for histochemical and electron microscopic analysis as discussed below. Histochemistry The muscles were removed, placed on wooden tongue depressor blades and frozen by immersion for 5-10 min in a beaker containing methyl butane cooled to - 195°C with liquid nitrogen. The muscles were mounted on chucks, allowed to warm to - 20 to - 15°C and were sectioned at 10 pm on a Lab-Tek cryostat. Sections were picked up on coverslips and allowed to air dry at room temperature for 0.5-3.0 hr.
ROSS,
SERAFIN
AND
STEWART
Tissues were reacted for myofibrillar ATPase activity at pH 9.4 following pre-incubation at pH 4.35 (acid) or pH 10.4 (alkaline) (Padykula and Herman, 1955; Guth and Samaha, 1959, 1970). In addition, sections were stained for succinic dehydrogenase activity (Lillie, 1964) and glycogen using the periodic acid-Schiff (PAS) reaction (McManus, 1948). In aI1 cases, tissues were dehydrated in a graded series of ethanol and mounted with Histoclad. Electron microscopy Portions of the mid-belly region of the FDL and SOL muscles were removed, placed on wooden supports and fixed in buffered (pH 7.1) 2% glutaraldehyde. After 30-45 min initial fixation, all muscles were diced and allowed to fix for a total of 2+ hr. The tissues were rinsed in two changes of phosphate buffered sucrose (pH 7.1); the second rinse was of 12-14 hr duration at 4°C. The tissues were post-fixed in 1 % phosphatebuffered osmium (pH 7.1) for 1 hr, dehydrated in a graded ethanol series, infiltrated in a propylene oxide-Epon-Araldite mixture and embedded in an Epon-Araldite mixture. The resin was polymerized for 48 hr at 60°C. Thick and thin sections were made with glass knives on an LKB Ultratome II. Thick sections were stained with toluidine blue whereas thin sections were stained first with uranium, then lead. Thin sections were examined in a JEOL lOOB-II electron microscope at 80 kV. Ultrastructural observations were limited to FG and FOG fibers in the FDL and SO fibers in the SOL. Results Control muscles (a) Histochemical observations. All three fiber types (FT, FOG, and SO) were found in the soleus and flexor digitorum longus (FDL) muscles of control animals (Table 1). The FDL contained predominantly fast-twitch fibers of both types with the SO fibers making up between 2 and 5% of the total population. The total percentage of oxidative fibers (i.e., both SO and FOG fibers) was variable (between 40 and 65%) (Fig. 1). Periodic acid-Schiff activity (glycogen) was variable in both FG and FOG fibers with little or no activity associated with the SO fibers (Fig. 2).
FAST
AUD
SLOW
MUSCLES
IN
SPASTIC
hi
MICE
I.
Fig. Fibers from the mid-belly region of a control FDL muscle stained for succinic dehydrogenase activity. Note the moderate number of oxidativc fibers. Compsrc this with Fig. 3. x 150. Fig. 2 Flbera content
(PAS]
from
the mld.belly
showing
many
fibers
region with
ofa control moderate
FDL
musclestained
to heavy
glycogen
for glycogen depnzlts.
Fig. 3. Fibers from the mid-belly region of a spastic FDL muscle Fumed dehydrogenase activity. Note the large wmbcr of oxidative fibers. Compare 150.
x
Fig. 4. Fibers
from
content. These fibers, Jepoqition y 150.
the mid-belly taken
from
region a very
The soleus, a predominantly oxidativc muscle, possessed few FG fibers; all other fi hers were either SO or FOG The number of SO fibers varied between 45 and 60 y/i. In both muscles, SO fibers were clearly identifiable due to their low glycogen content
ofa
spastic
spastic
FDL
mouse,
x 150.
for wccinlc tiith Fig.
muscle stained
for glgcogen
show
no glycogen
llttle
or
I.
(PAS). high subsarcolemmal and intermyoiihrillar mitochondrial distribution (SDH) and intense ATPase activity following atid pre-incubation, which was reversed following alkaline pre-incubation. The FOG fibers exhibited moderate but variable glycogen
64
LEVIN,
DEGENNARO,
ROSS, SERAFIN
AND STEWART
Table 1. Histochemicaifiber types found in spastic and control muscles of the mouse Histochemical Myosin ATPase (alkaline preincubation) Control Expt.
Muscle Flexor digitorum longus Fiber types FG FOG SO Soleus Fiber types FG FOG SO
Myosin ATPase (acid preincubation) Control Expt.
reaction Periodic acidSchiff
Succinic dehydrogenase Control
Expt.
Control
Expt.
2-3 2-3 0
3 3 0
0 0 3
0 0 3
1 2 3
2-3*t 2-3*t 3
2-3 2-3 1
O-1
2-3 2-3 0
3 3 0
0 0 3
0 0 3
1
2 3
2 2-3 3
2-3 2
O-l O-l O-l
1
O-12 o-13
~~ 3, strong reaction; 2, moderate reaction; I, light reaction; 0, no reaction. * The greater the degree of spasticity, the higher the activity. t In many instances it was impossible to differentiate between FG and FOG fibers. $ The greater
the degree of spasticity,
the lower the activity.
deposits, large numbers of intermyofibrillar mitochondria and intense ATPase activity following alkaline pre-incubation, which was reversed following acid pre-incubation. The FG fibers exhibited characteristics similar to the FOG fibers except that few mitochondria were present (Table 1). (b) Ultrastructural observations. In both the FDL and SOL muscles, the appearance of the fibers was the same as that previously described for mammalian muscles (Gauthier, 1970; Tomanek, 1976). In the SO fibers of the soleus, large numbers of intermyofibrillar and subssrcolemmal mitochondria, a fairly well-developed sarcotubular system, and wide Z lines were found. Glycogen granules were rarely present (Fig. 5). In the FG fibers of the FDL, a sparse
mitochondrial population with mitochondria occasionally paired at the I bands, an extensive sarcotubular system, and thin Z lines were found (Fig. 7). The FOG fibers contained numerous mitochondria usually paired at the I bands, a fairly well-developed sarcotubular system and moderately wide Z lines (Fig. 8). In most of the fast fibers, glycogen granules were present. Spastic muscles (a) Histochemical observations. No discernible abnormalities were found in the spastic muscles following light microscopic observadistinct histochemical tions; however, changes were noted, especially in the FDL. In muscles isolated from animals showing severe mitochondrial activity was so spasticity, great in all fast fibers that it was impossible
Fig. 5. Electron micrograph of a SO fiber from a control SOL muscle demonstrating abundant
mitochondria
(M) wide Z lines (Z) and little or no glycogen.
x 24,000.
Fig. 6. Electron micrograph of a SO fiber from a spastic SOL muscle. This fiber is comparable to control muscle fibers. x 24,000. Fig. 7. Electron micrograph of a FG fiber from a control FDL muscle demonstrating the mitochondria (M) paired at the I bands (I) and relatively thin Z lines (Z). x 24,000.
FAST
AND
SLOW
MUSCLES
IN SPASTIC
to histochemically type the majority of the spastic muscle fibers based on their SDH activity (compare Figs. 1 and 3). In less severely affected mice, mitochondrial density was not as high. Glycogen deposits (as indicated by the PAS reaction) were severely depleted and virtually absent in the muscles of severely affected mice (Fig. 4). Increased mitochondrial density was not as striking in the soleus since this muscle is normally highly oxidative. In both muscles, the relative numbers of fast- and slow-twitch fibers remained unchanged as indicated by myosin ATPase activity. (b) Ultrastructural observations. All SO fibers studied in the soleus were comparable to control muscle fibers (Fig. 6). The most obvious changes found in the FDL muscles of severely spastic mice correlated well with the histochemical findings. It was not possible to readily differentiate between FG and FOG fibers. Most fibers studied exhibited increased intermyofibrillar mitochondria and very low (often absent) glycogen deposits (Fig. 9). Terminal cisternae of the sarcoplasmic reticulum were often distended; however, the sarcoplasmic reticulum membrane serations (SR feet) which are found in association with the membrane of the T-tubule always remained intact (Figs. 10, 11). In addition, many regions of extensive vesiculation of the sarcoplasmic reticulum were found (Fig. 10). Discussion Naturally and artificially induced changes in muscular activity have been shown to induce
MICE
histochemical, physiological, and ultrastructural changes in various muscle fibers. Intermittent long-term stimulation of the tibialis anterior and extensor digitorum muscles (fast muscles) of the rabbit with a frequency pattern simulating that of a slow muscle, increased mitochondrial density and enzymes of the citric acid cycle. Concomitant with this were decreases in enzymes of glycolytic metabolism (Pette et al., 1973) and increased fatigue resistance (Pette and Ramirez, 1975). Similar long-term stimulation of the slow rabbit soleus had no effect (Pette and Ramirez, 1975). Terjung (1976) found that intense exercise caused increased oxidative capacity in the muscles of rats. Prince and his colleagues (1976, 1977) demonstrated increased numbers of oxidative fibers in male long-distance runners and female collegiate field hockey players. Other more drastic experimental manipulations have produced changes that suggest a conversion of fast-twitch fibers to slowtwitch fibers. Tenotomy of synergists has been used. Schiafino and Bormioli (1973) found that following synergistic tenotomy, the extensor digitorum longus and flexor hallucis longus muscles of the rat showed increased succinic dehydrogenase activity, and fibers having low myosin ATPase activity (following alkaline pre-incubation) increased in numbers. Tomanek (1975) found similar results in the soleus and plantaris muscles of the kitten following synergistic tenotomy. Nerve cross-union studies also suggest the importance of the nerve in the physiological status of muscle fibers. Buller and his
Fig. 8. Electron micrograph of a FOG fiber from a control FDL muscle demonstrating abundant mitochondria and moderately wide Z lines. Also note the glycogen deposits. x 24.000. Fig. 9. Electron micrograph of a fast fiber from a spastic FDL muscle. Note the large number of mitochondria and virtual absence of glycogen. In severely affected mice, this was the predominant fiber type. x 24,000. Fig. 10. Electron micrograph of a fast fiber from a severely spastic FDL muscle. Note triad with distended terminal cisternae (C) and vesiculation of some regions of the sarcoplasmic reticulum (Sr). x 24,000. Fig. I I. Higher magnification (F) are intact. x 60,000.
67
of a triad similar to that shown in Fig. 10. The SR feet
68
LEVIN,
DEGENNARO,
colleagues (1960, 1965) found that following nerve cross-union of fast and slow muscles in adult cats, each muscle took on the twitch characteristics of the foreign nerve, Thus, the slow muscle became fast and the fast muscle became slow. In addition to physiological changes, biochemical and histochemical changes were observed following crossinervation of the cat FDL and soleus muscles. The myosin ATPase activity of the fast FDL approached that of the slow soleus whereas the activity of the soleus muscle increased only slightly (Buller and Lewis, 1965). Our histochemical and ultrastructural findings strongly suggest that increased muscular activity in the spastic condition, due to an increased rate of nerve firing, can induce adaptive changes in the oxidative and glycolytic capacity of muscle fibers. Just exactly how the nerve affects the muscle fibers has been a subject of much debate. There are two current hypotheses regarding the nerve’s effect upon the muscle: (1) a specific neurotropic substance is passed down the nerve causing the muscle to differentiate and be maintained in that state and, (2) the activity of a muscle fiber, as a consequence of nervous impulse propagation, is the main factor involved in the development and maintenance of muscle fiber types. The more or less constant nervous impulse activity to the fast fibers, resembling that encountered by a slow fiber, caused an increase in oxidative capacity. However, no fast fibers exhibited any other slow fiber characteristics, i.e., low myosin ATPase activity following alkaline pre-incubation and slow fiber ultrastructure. This, in conjunction with other studies involving increased use, suggests that the oxidative and glycolytic capacities, as may be indicative of fast or slow fibers, are depen-
dent upon the actual activity of the muscle fiber whereas differences in myosin ATPase
ROSS,
SERAFIN
AND STEWART
and ultrastructure between fast and slow fibers may be dependent upon some trophic substance found in the specific nerve. In this study, the only consistent ultrastructural difference (other than glycogen granule and mitochondrial content) in the fast fibers of spastic mice was the distension and vesiculation of the sarcoplasmic reticulum. Variations in the sarcoplasmic reticulum are the most prominent early ultrastructural abnormality described in many muscular diseases. Vesiculation and distension of the sarcoplasmic reticulum were found in embryonic and adult dystrophic muscles of chickens (Schotland, 1970; Shafiq et al., 1974; Allen and Murphy, 1978) and mice (Platzer and Powell, 1975). In many dystrophic disorders, other ultrastructural changes follow the appearance of sarcoplasmic reticulum abnormalities. This did not occur here. All muscle fibers maintained their integrity. Also, normal muscle function in the presence of increased activity is further suggested by the normal appearance of the ‘S.R. feet’ indicative of the maintenance of coupling between the T system and the sarcoplasmic reticulum (Franzini-Armstrong, 1972, 1973). These findings suggest that the sarcoplasmic reticulum changes in response to functional demand. Therefore, changes in the sarcoplasmic reticulum in many muscular disorders may be due to initial changes in the activity of the muscle fibers, this being followed by the pathological structural conditions associated with the particular disorder. Acknowledgements
The authors wish to thank Dr Grace Rovozzo and Mrs M. Grace Levin for kindly reading the manuscript and making valuable suggestions.
ALLEN, E. R. and MURPHY, B. J. 1978. Sarcotubular development in dystrophic skeletal muscle cells. Cell 7%~. Res., 194, 125-l 30. BROOKE, M. H. 1970. Some comments on neural influence on the two histochemical types of muscle fibers. In The Physiology and Biochemistry of Muscle ns a Food (eds. E. J. Brisky, R. G. Cassens and B. B. Marsh),
Vol. 2, pp. 131-153. University of Wisconsin Press, Madison. BROOKE. M. H. and KAISER, K. K. 1970. Three myosin ‘adenosine triphosphatase’ systems: the nature of their pH lability and sulfhydryl dependence. J. Hisrochem. Cytochem., 18, 670-672. BULLER, A. J., ECCLES, J. C. and ECCLES, R. M. 1960. Differentiation of fast and slow muscles in cat hind limb. .I. Physiol., 150, 399-416.
FAST
AND
SLOW
MUSCLES
IN SPASTIC
MICE
69
BULLER. A. J. and LEWIS, D. M. 196.5. Further observations on mammalian cross-innervated skeletal muscle. J. Physiol., 178. 343-358. CHAI. C. K. 1961. Hereditary spasticity in mice. J. Heredity, 52, 241-243. &AI, C. K., ROBERTS, E. and SIDMAN. R. L. 1962. Influence of amino-oxyacetic acid, a y-aminobutyrate transaminase inhibitor, on hereditary spastic defect in the mouse. Proc. Sot. exp. Biol. Med., 109, 491.. 495. CHATTERIEE. S. and HECHTMAN, P. 1977. y-Aminobutyric acid metabolism in brain homogenates of the spastic mouse. Eiorhem. Genetics, 15, 147-151. CLOSE. R. 1972. Dynamic properties of mammalian skeletal muscle. Physiol. Rev., 52, 129-137. DUBOWITZ, V. 1970. Differentiation of fiber types in skeletal muscle. In The Physiology and Biochemirrn~ of Muscle as a Food (eds. E. J. Brisky, R. G. Cassens and B. B. Marsh). Vol. 2, pp. 103-130. University of Wisconsin Press, Madison. FRANZINI-ARMSTRONG, C. 1972. Studies of the triad. III. Structure of the junction in fast-twitch fibers. Tissr/e & Cell, 4, 469-478. FRANZINI-ARMSTRONG,C. 1973. Membrane systems in muscle fibers. The Structure and Function of Mosck, (ed. G. H. Bourne), Vol. 2, pp. 531-619. Academic Press, New York. GAIJTHIER, G. F. 1970. The ultrastructure of three fiber types in mammalian skeletal muscle. In The Physiology and Biochemistry of Muscle as a Food (eds. E. J. Brisky. R. C. Cassens and B. B. Marsh), Vol. 2. pp. 103S130. University of Wisconsin Press, Madison. GIITH. L. and SAMAHA, F. J. 1969. Qualitative differences between actomyosin ATPase of slow and I‘dSt mammalian muscle. Exp. Neural., 25, 138-l 52. G~TH, L. and SAMAHA, F. J. 1970. Procedure for the histochemical demonstration of actomyosin ATPase. Exp. Neural., 28, 365-367. LILLIE. R. D. 1964. Histopathologic Technic and Practical Histochemiatry. McGraw-Hill, New York. MCMANLIS,J. F. A. 1948. Histological and histochemical uses of periodic acid. Stain Tech., 23, 99-108. PADYKULA, H. A. and HERMAN, E. 1955. The specificity of the histochemical method for adenosine triphosphatase. J. Histochem. Cytochem., 3, 170-195. PETER. I., BARNARD, R., EDGERTON, V.. GILLESPIE,C. and STEMPEL,K. 1972. Metabolic profiles of three fiber types of skeletal muscle in guinea pigs and rabbits. Biochemistry, 11, 2627-2633. PE~TE. D., SMITH, M. E., STAUDTE.H. W. and VRBOVA, G. 1973. Effects of long-term electrical stimulation on some contractile and metabolic characteristics of fast rabbit muscles. fpiisers Arc/z., 338, 257-272. PETX, D. and RAMIREZ, B. V. 1975. Influence of intermittent long-term stimulation on contractile, histochemical and metabolic properties of fibre populations in fast and slow rabbit muscles. efliigers Arch., 361, l-7. PLATZER, A. C. and POWELL, J. A. 1975. Fine structure of prenatal and early postnatal dystrophic mouse muscle. J. Neural. Sci., 24, 109-126. PRINCE, F., HIKIDA, R. S. and HAGERMAN, F. 1976. Human muscle fiber types in power lifters. distance runners and untrained subjects. P’irgers Arch., 363, 19-26. PRINCE,F. P. and HIKIDA, R. S. 1977. Muscle fiber types in women athletes and non-athletes. Pfliiger.v Arch., 371, 161-165. SCHIAFFINO, S. and BORMIOLI, P. 1973. Adaptive changes in developing rat skeletal muscle in response to functional overload. Exp. Neural. 40, 126-137. SCHOTLAND,D. L. 1970. An electron microscopic investigation of myotonic dystrophy. J. Nruropath. esp Neural., 29, 241-252. SHAFIQ. S. A., ASIEDU, S., RYAN, D. and MILHORAT, A. T. 1974. Effect of denervation and hereditary muscular dystrophy on the differentiation of chicken fiber types with some observations on early changes in muscular dystrophy. Exploratory Concepts in Muscular Dystrophy, Control Mechanisms and Their re1ationship.s to Muscular Dystrophy and Related Neuromuscular Diseases (ed. A. T. Milherat), Excerpta Medica, Amsterdam. STEIN, J. M. and PADYKULA, H. A. 1962. Histochemical classification of individual skeletal muscle fibers of the rat. Am. J. Anat., 110, 103-124. STEWART,J. 1979. Personal communication. TERJI!NG, R. 1976. Muscle fiber involvement during training of different intensities and duration. Anr. J. Physiol., 230, 946-950. TOMANEK, R. 1975. A histochemical study of postnatal differentiation of skeletal muscle with reference to functional overload. Dev. Biol., 42, 305-3 14. TOMANEK.R. 1976. Ultrastructural differentiation of skeletal muscle fibers and their diversity. J. Ultrastruct., Rex.. 55. 2 12-227.