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Adaptive changes in developing rat skeletal muscle in response to functional overload
EXPERIMENTAL
NEUROLOGY
Adaptive S. National Institute
40, 126-137 (1973)
Changes in Developing Rat Skeletal Muscle in Response to Functional Overload SCHIAFFINO
AND
Research Council Unit of General Pathology, Received
SANDRA
PIEROBON
BORMIOLI
1
for Muscle Biology and Physiopathology, Uwiversity of Padova, 35100 Padova, Italy
November
27,1972
The histochemical profile of rat skeletal muscles can be modified in response to the increased functional overload induced by incapacitation of synergistic muscles. We have investigated) the effect of synergist elimination in rats of different age, in order to compare the relative plasticity of immature versus mature muscle fibers and the role of peripheral factors on fiber type differentiation. The chronic effects of increased functional overload were studied in the rat extensor digitorum longus (EDL) and extensor hallucis longus (EHL) muscles after extirpation of the synergistic tibialis anterior muscle. After tibialectomy at birth there was a marked increase in succinate dehydrogenase activity in overloaded muscles : the typical “white” or mitochondriapoor fibers, which constitute about 40% of the fiber population in the control muscles, were altogether absent in the hypertrophic EDL and< EHL. However, the activity of another mitochondrial enzyme, a-glycerophosphate dehydrogenase, was unchanged or, in some cases, even slightly decreased in most fibers of the overloaded muscles. The proportion of fibers with low myosin ATPase activity increased from 4% of the total fiber population in the control EDL to 10-1.5s in the hypertrophic muscle, and comparable changes were seen in EHL. Tibialectomy performed 1 or 2 months after birth, by contrast, induced rather limited or, in the case of EDL, almost negligible changes in the histochemical profile both with respect to mitochondrial oxidative enzymes and myosin ATPase. The findings show that the adaptive response of skeletal muscle to synergist elimination is markedly influenced by the degree of maturity of the muscles at the time the overload is imposed.
INTRODUCTION Mammalian skeletal muscle fibers are functionally and structurally heterogeneous. A variety of fiber types can be distinguished on the basis 1 This investigation was supported in part by a grant from Muscular Associations of America, Inc. to Prof. M. Aloisi. 126 Copyright All rights
1973 by Academic Press, IX. reproduction in any form reserved.
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of histochemical and ultrastructural criteria, whose functional correlates have not yet been completely defined. The distinctive features of the different fiber types become manifest at relatively late stages of development, apparently through a process of diversification of an initially undifferentiated fiber population. Thus, in the rat, histochemical and ultrastructural differentiation of skeletal muscle fibers takes place during the first weeks of postnatal development (7, 11, 33)) in the same period when physiological differences between fast and slow muscles become established (4). The diversification of mammalian skeletal muscle fibers is subsequent to and possibly causally dependent upon the formation of stable neuromuscular comlections. The importance of nerve influence in inducing and maintaining structural and functional differences between skeletal muscle fibers has been clearly demonstrated by cross-innervation experiments, but it is still a moot point whether the effect of nerve is mediated by the impulsedirected pattern of activity imposed on the muscle or by other impulseindependent mechanisms (see 13). The role of peripheral factors in the differentiation of muscle fibers and motor units remains likewise to be elucidated. In the attempt to clarify the mechanisms controlling fiber type differentiation in skeletal muscles we have investigated the effect of excessive functional overload induced by elimination of a synergist at early stages of muscle development. RIATERIALS
AND
METHODS
The extensor digitorum longus (EDL) and extensor hallucis longus (EHL) muscles of the rat were studied at different time intervals after extirpation of the synergistic tibialis anterior muscle. This procedure results in compensatory hypertrophy and hyperplasia of the overloaded muscles (Schiaffino, Pierobon Bormioli, and Aloisi, in preparation). The operation was performed upon l-day-old, 7-day-old, l-month-old, and 2-month-old Wistar rats. From 1 to 18 months after the operation the animals were killed, and the hypertrophic muscles were processed for histochemistry together with the corresponding muscles of the unoperated contralateral limb which were used as control. Experimental and control muscles were pinned side by side onto a plastic support under similar conditions of stretching and frozen in liquid nitrogen. Transverse sections were cut in a cryostat through the mid-belly of the muscles and incubated for the demonstration of succinate dehydrogenase (SDH) (23). mitochondrial a-glycerophosphate dehydrogenase (wGPDH) (IS), ,&hyclroxybutyrate dehydrogenase (/+HBDH) (241, and myosin ATPase (25 ) after preincubation in acid or alkali (14). The sections were fixed in formaldehyde before preincubation in alkali (14). The histochemical differentiation of EDL and EHL was also investigated in unoperated rats at 1, 7, 15, 21, and 30 days after birth.
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RESULTS The time course of histochemical maturation of EDL and EHL muscles during postnatal development seems to be essentially similar to that of other rat muscles (7, 11). In the newborn rat there was no recognizable histochemical difference between the various muscle cells, except for faint variations with the ATPase reaction. By 7 days dark (type II) and pale (type I) fibers could be clearly distinguished with the regular ATPase reaction and reversal of the staining pattern occurred after preincubation in acid. In the subsequent weeks type II fibers underwent further diversification into two subtypes displaying different sensitivity to formaldehyde fixation (14, 35). The relative proportions of type I and type II fibers did not change significantly in the course of postnatal development. Type II fibers represent about 96% of the total fiber population in EDL and about 91% in EHL. The two muscles appeared homogeneous in the newborn and 7-day-old rat when stained for the histochemical demonstration of mitochondrial oxidative enzymes. Differentiation into fibers with higher and lower SDH activity was first apparent during the second week, but “white” fibers with very low SDH activity were not seen until 3-4 weeks after birth. At this age both EDL and EHL exhibited the typical mosaic pattern of fibers with varying SDH activity, inversely correlated with fiber size, characteristic of the adult muscles (32). The a-GPDH activity was very low in muscles of the newborn and 7-day-old rat and increased markedly during the subsequent weeks, whereas the /?-HBDH reaction remained at low levels in most EDL and EHL fibers at all ages. It is of interest that an inverse distribution of these two mitochondrial enzyme activities is established during the same periods of postnatal development in the slow-twitch soleus muscle (see also 28, 30). The histochemical differentiation of EDL and EHL muscles was markedly affected by tibialectomy at birth, as shown in Figs. 1 and 2. The major differences concerned mitochondrial oxidative enzymes. “White” fibers with very low SDH activity, which constitute about 40% of the fiber population in the normal mature EDL and EHL, were completely absent in hypertrophic muscles, which were composed exclusively of fibers with moderate to high SDH activity. The resulting uniform staining pattern contrasted with the highly diversified histochemical profile of control muscles (Fig. 1 A-C). At variance with control, subsarsolemmal accumulations of SDH reaction product were seen in practically all fibers of the hypertrophic muscles (Fig. 1 B). The intensity of a-GPDH activity was unchanged or slightly decreased in most fibers of EDL and EHL after tibialectomy at birth (Fig. 1 D) ; there was also a significant proportion of fibers with almost negative a-GPDH reaction, which were rare in control muscles and were identified as fibers with low myosin ATPase activity
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in serial sections. On the contrary, the ,&HBDH reaction appeared somewhat increased with respect to control, especially in fibers from the deep regions of the EDL. The histochemical profile with the ATPase reaction was also modified by tibialectomy at birth. Hypertrophic muscles were still predominantly composed of type II fibers, but the proportion of type I fibers was variably increased up to 10-15s of the total fiber population in EDL and even more in EHL (Fig. 2). Dimensional hypertrophy of the single fibers was also generally more marked for type I than for type II fibers in overloaded muscles (Fig. 2 D). Furthermore, at variance with control, there was no variation in reaction intensity between type II fibers after prefixation in formaldehyde and preincubation in alkali (Fig. 2 A, B). Formaldehyde-sensitive fibers, which represent about 40% of the total fiber population in control muscles and correspond with white fibers in succinate dehydrogenase preparations, were in fact extremely rare in hypertrophic muscles. Similar though less marked histochemical changes were found in EDL and EHL muscles after tibialectomy performed at 7 days of age; by contrast, only slight modifications were seen in animals operated upon at 1 or 2 months of age. In the latter case, as shown in Fig. 3 A, the histochemical profile with the ATPase reaction was generally unchanged in the hypertrophic EDL. Also, the normal mosaic pattern with the SDH reaction was maintained (Fig. 3 B), although there was an occasional slight increase in SDH activity especially in the deep areas of the muscle. No detectable difference between hypertrophic and control muscles was apparent in sections incubated for the demonstration of LU-GPDH and ,&HBDH activities. Comparable, albeit more variable results were obtained with EHL muscle after tibialectomy at adult age. In this case we occasionally observed a significant increase in the proportion of type I fibers with the ATPase reaction and a more uniform staining pattern after incubation for SDH with a shift toward a higher oxidative activity. These changes, when present, were, however, less pronounced than those observed in animals operated upon at birth. DISCUSSION In keeping with a previous report (15), the present study shows that the histochemical profile of rat skeletal muscles can be altered by longlasting functional overload imposed by elimination of synergistic muscles ; in addition, our study shows that this adaptive response is markedly influenced by the degree of maturity of the muscles at the time of operation. The greater plasticity of the undifferentiated muscles fibers of the newborn rat was especially evident in the transformation of the histochemical profile with SDH and myosin ATPase. The response of mature muscles, particu-
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larly EDL, to the elimination of synergists, was by contrast rather limited in our system. This does not imply that the capacity of transformation of skeletal muscle fibers in response to peripheral stimuli becomes irreversibly restricted during muscle development, since in different systems, presumably in relation with heavier functional loads imposed on the muscles, important histochemical modifications have been shown to occur also at later postnatal stages. Thus, the proportion of fibers with low myosin ATPase activity is increased in the plantaris muscle after extirpation of the synergistic gastrocnemius and soleus muscles in adult rats (15). The finding that the total number of muscle fibers is markedly increased during long-term compensatory hypertrophy (Schiaffino, Pierobon Bormioli, and Aloisi, in preparation) complicates the interpretation of the histochemical changes. The increased proportion of type I fibers might in fact result from uneven hyperplasia of the two fiber populations rather than from a transformation of type II into type I fibers. The extensive changes in the histochemical profile with the SDH reaction, on the other hand, are best interpreted as the result of an altered diversification of developing skeletal muscle fibers, such that fibers destined to become “white” or mitochondria poor did mature into “red” or mitochondria rich. The capacity for this conversion is apparently maintained, though perhaps to a lesser degree, also in adult muscles (9, 12, 15, 21). The functional significance of the histochemical changes in overloaded muscles remains to be determined. JVe have previously shown that the contraction time of the fast-twitch EDL muscle does not change after tibialectomy at birth (17). The contraction time of the plantaris muscle is likewise apparently unaltered by long-term incapacitation of gastrocnemius and soleus in adult rats (2). Since a close correlation seems to exist between myosin ATPase activity and intrinsic speed of shortening in different muscles (l), one might have expected that an increase in the proportion of fibers with low histochemically detectable myosin ATPase should have been accompanied by a lengthening of the contraction time in the overloaded muscles. However, the time course of the isometric twitch may be determined by other factors, in addition to myosin ATPase activity such as the kinetics of calcium release and uptake by the sarcoplasmic reticulum or the affinity of troponin for calcium (see 6). Furthermore although the evidence seems convincing that the ATPase activity demouFIG. 1. Rat EDL muscle, 4 months after extirpation of the synergistic tibialis anterior at birth. A, X 40 ; B,C. X 225 ; D, X 50. SDH activity is markedly increased in the hypertrophic EDL (A, left side, and B) in comparison with the contralateral muscle (A, right side, and C). Note the more homogeneous appearance of the hypertrophic muscle due to absence of “white” fibers. Mitochondrial or-GPDH activity is slightly decreased in most muscle fibers of the hypertrophic EDL (D, lower side) in comparison with the control (upper side).
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--
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.11.
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strated histochemically in skeletal muscle fibers by the calcium-precipitation method is really and exclusively due to myosin ATPase (see 14, 34), the intensity of histochemical staining does not always parallel the level of biochemical activity (16) ; difficulties can, therefore, be encountered in attempting to correlate histochemical and physiological data (29). On the other hand, at least in our system, the increase in the proportion of fibers with low myosin ATPase activity during compensatory hypertrophy was very slight, their number never exceeding 10-15s of the total fiber population. This minor proportion of fibers cannot be expected to affect in a detectable way the performance of the whole muscle in vitro, though it may have an important role in the normal muscle function in V&O, when the different motor units act independently from each other. The changes in mitochondrial oxidative activities observed at late stages of compensatory muscle hypertrophy contrast with the apparently transient modifications described in the acute phases of the hypertrophic process (17) and are presumably related to the higher energy demand that the vicarious function entails. The marked increase in SDH activity in fibers of muscles subjected to excessive functional overload since birth suggests that mitochondrial capacity for oxidative phosphorylation is much enhanced in these muscles. This seems to represent a common adaptive response of most skeletal muscles to excessive use, irrespective of the type of overwork to which the muscle is subjected (21).2 However, the histochemical transformation in our system appears quantitatively much more pronounced than that previously reported in rat or guinea pig muscles after different exercise programs (9, 12, 21). The observation that SDH activity is positively correlated with resistance to fatigue in normal fasttwitch muscle fibers (3, 10) suggests that there should be a significant 2 An exception is represented are presumably already adapted
by slow-twitch muscles, to continuous activity (9,
such 15).
as the
soleus
which
FIG. 2. Rat EDL (A-C) and EHL (D) muscles, 3 months after tibialectomy at birth. A, X 16; B, X 64; C, X 16 ; D, X 35. A, section incubated for myosin ATPase activity, after fixation in formaldehyde and preincubation in alkali: there is an increase in the number of type I fibers in the hypertrophic EDL (lower side) in comparison with the contralateral muscle (upper side). Furthermore, type II fibers display a uniformly high intensity of reaction in the hypertrophic muscle, whereas in the control there is a large proportion of fibers with intermediate degree of activity. B, higher magnification of a central area in A, comprising portion of hypertrophic (lower side) and contralateral (upper side) muscles. C, serial section of the one shown in A, incubated for myosin ATPase after pretreatment in acid: numerous type I fibers, standing out against the background of the unstained type II fibers, are predominantly concentrated in the deep areas of the hypertrophic EDL. D, EHL, myosin ATPase after pretreatment in acid: note the striking increase in number and size of type I fibers in the hypertrophic muscle (at right) in comparison with the contralateral (at left).
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FIG. 3. Rat EDL muscle, 5 months after tibialectomy performed upon Z-month-old animals. X 13. Serial sections incubated for SDH (A) and) for myosin ATPase after pretreatment in acid (B) There is no obvious difference in the histochemical profile between hypertrophic (at right) and contralateral (at left) muscles.
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increase in the resistance to fatigue of EDL and EHL muscles after tibialectomy at birth, It is of interest, in this respect, that the ultrastructural profile of most fibers in the hypertrophic EDL is characterized by abundant mitochondrial complement, relatively thick Z band, and rich development of the sarcoplasmic reticulum (our unpublished observations), which seems to be typical of fast, but fatigue-resistant, muscle fibers (32). The increase in SDH activity during long-lasting compensatory hypertrophy of EDL and EHL was not accompanied by a parallel increase in mitochondrial wGPDH activity. This indicates that mitochondria from overloaded muscles differ in composition from those of normal muscles. A similar dissociation between mitochondrial oxidative activities has been previously observed in intensively trained muscles (19, 20). As the activity level of mitochondrial wGPDH in skeletal muscles is strictly correlated with the activity level of glycolytic enzymes (26, 27 j these findings suggest that the glycolytic capacity is unchanged or slightly decreased in hypertrophy muscles. The factors responsible for the histochemical and ultrastructural changes accompanying compensatory hypertrophy in developing muscles are not fully understood. It has been shown that the acute hypertrophic response of rat skeletal muscles to incapacitation of their synergists can take place independently of intact neural circuits and appears predominantly due to a direct effect of increased mechanical stretching (17, 22. 31). The transition from this acute stretch hypertrophy to the subsequent stages of the hypertrophic process, which gradually evolves toward a really compensatory state, seems to require, on the other hand, the active participation of the neural components. The finding that long-term functional overload of rat muscles due to incapacitation of their synergists is followed by hypertrophy of the corresponding nerve fibers (8) supports, indeed, the view that adaptive changes of the whole neuromuscular system occur under these conditions. One important point which remains to be settled, in this respect, concerns the motor unit composition of the hypertrophic muscles. In normal EDL there is probably only one motor unit which is relatively slow in comparison with the others, its contraction time being actually intermediate between that of most EDL units and that of most soleus units (5) : it is possible though still unproved, that this unit is composed of the rare fibers with low myosin ATPase activity. The increased number of these fibers during compensatory lypertrophy in rats operated upon at birth may either reflect an increased size of this preexisting motor unit or the presence of additional units with similar properties. The latter might in turn arise from transformation of fast motor units or, alternatively, from de novo formation of intermediate or slow motor units, which would imply an increase in the number of motor nerve fibers to hypertrophic muscles (see 8).
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REFERENCES 1. BARANY, M. 1967. ATPase activity of myosin correlated with speed of muscle shortening. 1. Gen. Physiol. SO: 197-218. 2. BINE;HORST, R. A. 1969. The effect of training on some isometric contraction characteristics of a fast muscle. Pfliigers Arch. 309: 193-202. 3. BURKE, R. E., D. N. LEMNE, F. E. ZAJAC, P. TSAIRIS, and W. K. ENGEL. 1971. Mammalian motor units : physiological-histochemical correlation in three types in cat gastrocnemius. Science 174: 709712. 4. CLOSE, R. 1964. Dynamic properties of fast and slow skeletal muscles of the rat during development. J. Physiol. 173: 74-95. 5. CLOSE, R. 1967. Properties of motor units in fast and slow skeletal muscles of the rat. J. Physiol. 193: 45-55. 6. CLOSE, R. I. 1972. Dynamic properties of mammalian skeletal muscles. Physiol. Rev. SZ: 129-197. 7. DUBOWITZ, V. 1963. Enzymatic maturation of skeletal muscle. Natwe (London) 197: 1215. 8. EDDS, M. V., JR. 1950. Hypertrophy of nerve fibers to functionally overloaded muscles. J. Camp. Newel. 93: 259-275. 9. EDGERTON, V. R., L. GERCHMAN, and R. CARROW. 1969. Histochemical changes in rat skeletal muscle after exercise. Exp. Neural. 24 : 110-123. 10. EDSTRGM, L., and E. KUGELBERG. 1968. Histochemical composition, distribution of fibers and fatiguability of single motor units. J. Newol. Neurosurg. Psychiat. 31: 424-433. 11. ENGEL, W. K., and G. KARPATI. 1968. Impaired skeletal muscle maturation following neonatal neurectomy. Dezlelop. Biol. 17 : 713-723. 12. FAULKNER, J, A., L. C. MAXWELL,, D. A. BROOK, and D. A. LIEBERMAN. 1971. Adaptation of guinea pig plantaris muscle fibers to endurance training. Amer. J. Physiol. 221: 291-297. 13. GUTH, L. 1968. “Trophic” influences of 14. GUTH, L., and F. J. SAMAHA. 1969.
15. 16. 17. 18. 19.
nerve on muscle. Physiol. Rev. 48: 645-687. Qualitative differences between actomyosin ATPase of slow and fast mammalian muscle. Exp. Neural. 25: 138-152. GUTH, L., and H. YELLIN. 1971. The dynamic nature of the so-called “fiber types” of mammalian skeletal muscle. Exp. Neural. 31 : 277-300. GUTH, L., and F. J. SAMAHA. 1972. Erroneous interpretations which may result from application of the “myofibrillar ATPase” histochemical procedure to developing muscle. Exp. Neural. 34: 465-475. GUTMANN, E., S. SCHIAFFINO, and V. HANZLIKOVA. 1971. Mechanism of compensatory hypertrophy in skeletal muscle of the rat. Exp. Neztrol. 31: 451-464. HESS, R., and A. G. E. PEXRSE. 1961. Histochemical and homogenization studies of mitochondrial a-glycerophosphate dehydrogenase in the nervous system. Nature (London) 191: 718-719. HOLLOSZY, J. 0. 1967. Biochemical adaptations in muscle. Effects of exercise on mitochondrial oxygen uptake and respiratory enzyme activity in skeletal muscle. J. Biol.
Chem.
242:
2278-2282.
20. HOLLOSZY, J. O., and L. B. OSCAI. 1969. Effect of exercise on, or-glycerophosphate dehydrogenase activity in skeletal muscle. Arch. Biochem. Bioghys. 130: 653-656. 21. KOWALSKI, K., E. E. GORDON, A. MARTINEZ, and J. ADAMEK. 1969. Changes in enzyme activities of various muscle fiber types in rat induced by different exercises. J. Histochem. Cytochem. 17: 60-607.
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22. MACKOVA, E., and P. HNIK. 1971. “Compensatory” muscle hypertrophy in the rat induced by tenotomy of synergistic muscles. Exjerientia 27: 1039-1040. 23. NACHLAS, M. M., K. C. Tsou, E. DE SOUZA, C. S. CHENG, and A. M. SELIGMAN. 1957. Cytochemical demonstration of succinic dehydrogenase by the use of a new fi-nitrophenyl substituted ditetrazole. J. Hi-stochem. Cytochem. 5 : 42@436. 24. NACHLAS, M. M., D. G. WALKER, and A. M. SELIGMAN. 1958. A histochemical method for the demonstration of diphosphopiridine nucleotide diaphorase. 25.
26. 27. 28.
29. 30. 31. 32. 33. 34. 35.
J. Biophys. PADYKULA,
Biochem.
Cytol.
4: 29-46.
H. A., and E. HERMAN. 1955. Factors affecting the activity of adenosine triphosphatase and other phosphatases as measured by histochemical techniques. J. Histochem. C$ochem. 3 : 161-169. PETTE, D., ancI T. BUTCHER. 1963. Proportionskostante Gruppen in Beziehung zur Differenzierung der Enzymaktivititsmuster von Skelett-muskeln des Kaninchens. 2. Physiol. Chem. 331: 180-195. PETTE, D. 1966. Mitochondrial enzyme activities, pp. 28-49. In “Regulation of Metabolic Processes in Mitochondria.” J. M. Tager, S. Papa, E. Quagliariello, and E. C. Slater (Eds.). Elsevier, Amsterdam. PIETER KARK, R. A., J. P. BLASS, J. AVIGAN, and W. K. ENGEL. 1971. The oxidation of p-hydroxybutyric acid by small quantities of type-pure red and white skeletal muscle. Z. Viol. Chem. 246: 45604566. ROBBINS, N., G. KARPATI, and W. K. ENGEL. 1969. Histochemical and contractile properties in the cross-innervated guinea pig soleus muscle. Arch. Neural. 20 : 318-329. ROMAXLTL, I. C. A. 1964. Enzymes in muscle. I. Histochemical studies of enzymes in individual muscle fibers. Arch. Newel. 11: 355-368. SCHIAFFINO, S., and V. HANZLIKOVA. 1970. On the mechanism of compensatory hypertrophy in skeletal muscles. E.rperientia 26 : 152-153. SCHIAFFINO, S., V. HANZLIKOVA, and S. PIEROBON. 1970. Relations between structure and function in rat skeletal muscle fibers. J. Cell. Biol. 47: 107-119. SCHIAFFINO. S. 1972. Differentiation of the contractile apparatus in vertebrate skeletal muscles. Boll. Zool. 39 : 139-158. SCHIAFFINO, S., and S. PIEROBON BORMIOLI. 1973. Histochemical characterization of adenosine triphosphatases in skeletal muscle fibers by selective extraction procedures. J. Histochrm. Cytochem. 21 : 142-145. STEIN, J. M., and H. A. PADYULA. 1962. Histochemical classification of individual skeletal muscle fibers of the rat. Amer. J. Anat. 110: 103-123.
NEUROLOGY
Adaptive S. National Institute
40, 126-137 (1973)
Changes in Developing Rat Skeletal Muscle in Response to Functional Overload SCHIAFFINO
AND
Research Council Unit of General Pathology, Received
SANDRA
PIEROBON
BORMIOLI
1
for Muscle Biology and Physiopathology, Uwiversity of Padova, 35100 Padova, Italy
November
27,1972
The histochemical profile of rat skeletal muscles can be modified in response to the increased functional overload induced by incapacitation of synergistic muscles. We have investigated) the effect of synergist elimination in rats of different age, in order to compare the relative plasticity of immature versus mature muscle fibers and the role of peripheral factors on fiber type differentiation. The chronic effects of increased functional overload were studied in the rat extensor digitorum longus (EDL) and extensor hallucis longus (EHL) muscles after extirpation of the synergistic tibialis anterior muscle. After tibialectomy at birth there was a marked increase in succinate dehydrogenase activity in overloaded muscles : the typical “white” or mitochondriapoor fibers, which constitute about 40% of the fiber population in the control muscles, were altogether absent in the hypertrophic EDL and< EHL. However, the activity of another mitochondrial enzyme, a-glycerophosphate dehydrogenase, was unchanged or, in some cases, even slightly decreased in most fibers of the overloaded muscles. The proportion of fibers with low myosin ATPase activity increased from 4% of the total fiber population in the control EDL to 10-1.5s in the hypertrophic muscle, and comparable changes were seen in EHL. Tibialectomy performed 1 or 2 months after birth, by contrast, induced rather limited or, in the case of EDL, almost negligible changes in the histochemical profile both with respect to mitochondrial oxidative enzymes and myosin ATPase. The findings show that the adaptive response of skeletal muscle to synergist elimination is markedly influenced by the degree of maturity of the muscles at the time the overload is imposed.
INTRODUCTION Mammalian skeletal muscle fibers are functionally and structurally heterogeneous. A variety of fiber types can be distinguished on the basis 1 This investigation was supported in part by a grant from Muscular Associations of America, Inc. to Prof. M. Aloisi. 126 Copyright All rights
1973 by Academic Press, IX. reproduction in any form reserved.
Dystrophy
SKELETAL
MUSCLE
127
of histochemical and ultrastructural criteria, whose functional correlates have not yet been completely defined. The distinctive features of the different fiber types become manifest at relatively late stages of development, apparently through a process of diversification of an initially undifferentiated fiber population. Thus, in the rat, histochemical and ultrastructural differentiation of skeletal muscle fibers takes place during the first weeks of postnatal development (7, 11, 33)) in the same period when physiological differences between fast and slow muscles become established (4). The diversification of mammalian skeletal muscle fibers is subsequent to and possibly causally dependent upon the formation of stable neuromuscular comlections. The importance of nerve influence in inducing and maintaining structural and functional differences between skeletal muscle fibers has been clearly demonstrated by cross-innervation experiments, but it is still a moot point whether the effect of nerve is mediated by the impulsedirected pattern of activity imposed on the muscle or by other impulseindependent mechanisms (see 13). The role of peripheral factors in the differentiation of muscle fibers and motor units remains likewise to be elucidated. In the attempt to clarify the mechanisms controlling fiber type differentiation in skeletal muscles we have investigated the effect of excessive functional overload induced by elimination of a synergist at early stages of muscle development. RIATERIALS
AND
METHODS
The extensor digitorum longus (EDL) and extensor hallucis longus (EHL) muscles of the rat were studied at different time intervals after extirpation of the synergistic tibialis anterior muscle. This procedure results in compensatory hypertrophy and hyperplasia of the overloaded muscles (Schiaffino, Pierobon Bormioli, and Aloisi, in preparation). The operation was performed upon l-day-old, 7-day-old, l-month-old, and 2-month-old Wistar rats. From 1 to 18 months after the operation the animals were killed, and the hypertrophic muscles were processed for histochemistry together with the corresponding muscles of the unoperated contralateral limb which were used as control. Experimental and control muscles were pinned side by side onto a plastic support under similar conditions of stretching and frozen in liquid nitrogen. Transverse sections were cut in a cryostat through the mid-belly of the muscles and incubated for the demonstration of succinate dehydrogenase (SDH) (23). mitochondrial a-glycerophosphate dehydrogenase (wGPDH) (IS), ,&hyclroxybutyrate dehydrogenase (/+HBDH) (241, and myosin ATPase (25 ) after preincubation in acid or alkali (14). The sections were fixed in formaldehyde before preincubation in alkali (14). The histochemical differentiation of EDL and EHL was also investigated in unoperated rats at 1, 7, 15, 21, and 30 days after birth.
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RESULTS The time course of histochemical maturation of EDL and EHL muscles during postnatal development seems to be essentially similar to that of other rat muscles (7, 11). In the newborn rat there was no recognizable histochemical difference between the various muscle cells, except for faint variations with the ATPase reaction. By 7 days dark (type II) and pale (type I) fibers could be clearly distinguished with the regular ATPase reaction and reversal of the staining pattern occurred after preincubation in acid. In the subsequent weeks type II fibers underwent further diversification into two subtypes displaying different sensitivity to formaldehyde fixation (14, 35). The relative proportions of type I and type II fibers did not change significantly in the course of postnatal development. Type II fibers represent about 96% of the total fiber population in EDL and about 91% in EHL. The two muscles appeared homogeneous in the newborn and 7-day-old rat when stained for the histochemical demonstration of mitochondrial oxidative enzymes. Differentiation into fibers with higher and lower SDH activity was first apparent during the second week, but “white” fibers with very low SDH activity were not seen until 3-4 weeks after birth. At this age both EDL and EHL exhibited the typical mosaic pattern of fibers with varying SDH activity, inversely correlated with fiber size, characteristic of the adult muscles (32). The a-GPDH activity was very low in muscles of the newborn and 7-day-old rat and increased markedly during the subsequent weeks, whereas the /?-HBDH reaction remained at low levels in most EDL and EHL fibers at all ages. It is of interest that an inverse distribution of these two mitochondrial enzyme activities is established during the same periods of postnatal development in the slow-twitch soleus muscle (see also 28, 30). The histochemical differentiation of EDL and EHL muscles was markedly affected by tibialectomy at birth, as shown in Figs. 1 and 2. The major differences concerned mitochondrial oxidative enzymes. “White” fibers with very low SDH activity, which constitute about 40% of the fiber population in the normal mature EDL and EHL, were completely absent in hypertrophic muscles, which were composed exclusively of fibers with moderate to high SDH activity. The resulting uniform staining pattern contrasted with the highly diversified histochemical profile of control muscles (Fig. 1 A-C). At variance with control, subsarsolemmal accumulations of SDH reaction product were seen in practically all fibers of the hypertrophic muscles (Fig. 1 B). The intensity of a-GPDH activity was unchanged or slightly decreased in most fibers of EDL and EHL after tibialectomy at birth (Fig. 1 D) ; there was also a significant proportion of fibers with almost negative a-GPDH reaction, which were rare in control muscles and were identified as fibers with low myosin ATPase activity
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in serial sections. On the contrary, the ,&HBDH reaction appeared somewhat increased with respect to control, especially in fibers from the deep regions of the EDL. The histochemical profile with the ATPase reaction was also modified by tibialectomy at birth. Hypertrophic muscles were still predominantly composed of type II fibers, but the proportion of type I fibers was variably increased up to 10-15s of the total fiber population in EDL and even more in EHL (Fig. 2). Dimensional hypertrophy of the single fibers was also generally more marked for type I than for type II fibers in overloaded muscles (Fig. 2 D). Furthermore, at variance with control, there was no variation in reaction intensity between type II fibers after prefixation in formaldehyde and preincubation in alkali (Fig. 2 A, B). Formaldehyde-sensitive fibers, which represent about 40% of the total fiber population in control muscles and correspond with white fibers in succinate dehydrogenase preparations, were in fact extremely rare in hypertrophic muscles. Similar though less marked histochemical changes were found in EDL and EHL muscles after tibialectomy performed at 7 days of age; by contrast, only slight modifications were seen in animals operated upon at 1 or 2 months of age. In the latter case, as shown in Fig. 3 A, the histochemical profile with the ATPase reaction was generally unchanged in the hypertrophic EDL. Also, the normal mosaic pattern with the SDH reaction was maintained (Fig. 3 B), although there was an occasional slight increase in SDH activity especially in the deep areas of the muscle. No detectable difference between hypertrophic and control muscles was apparent in sections incubated for the demonstration of LU-GPDH and ,&HBDH activities. Comparable, albeit more variable results were obtained with EHL muscle after tibialectomy at adult age. In this case we occasionally observed a significant increase in the proportion of type I fibers with the ATPase reaction and a more uniform staining pattern after incubation for SDH with a shift toward a higher oxidative activity. These changes, when present, were, however, less pronounced than those observed in animals operated upon at birth. DISCUSSION In keeping with a previous report (15), the present study shows that the histochemical profile of rat skeletal muscles can be altered by longlasting functional overload imposed by elimination of synergistic muscles ; in addition, our study shows that this adaptive response is markedly influenced by the degree of maturity of the muscles at the time of operation. The greater plasticity of the undifferentiated muscles fibers of the newborn rat was especially evident in the transformation of the histochemical profile with SDH and myosin ATPase. The response of mature muscles, particu-
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larly EDL, to the elimination of synergists, was by contrast rather limited in our system. This does not imply that the capacity of transformation of skeletal muscle fibers in response to peripheral stimuli becomes irreversibly restricted during muscle development, since in different systems, presumably in relation with heavier functional loads imposed on the muscles, important histochemical modifications have been shown to occur also at later postnatal stages. Thus, the proportion of fibers with low myosin ATPase activity is increased in the plantaris muscle after extirpation of the synergistic gastrocnemius and soleus muscles in adult rats (15). The finding that the total number of muscle fibers is markedly increased during long-term compensatory hypertrophy (Schiaffino, Pierobon Bormioli, and Aloisi, in preparation) complicates the interpretation of the histochemical changes. The increased proportion of type I fibers might in fact result from uneven hyperplasia of the two fiber populations rather than from a transformation of type II into type I fibers. The extensive changes in the histochemical profile with the SDH reaction, on the other hand, are best interpreted as the result of an altered diversification of developing skeletal muscle fibers, such that fibers destined to become “white” or mitochondria poor did mature into “red” or mitochondria rich. The capacity for this conversion is apparently maintained, though perhaps to a lesser degree, also in adult muscles (9, 12, 15, 21). The functional significance of the histochemical changes in overloaded muscles remains to be determined. JVe have previously shown that the contraction time of the fast-twitch EDL muscle does not change after tibialectomy at birth (17). The contraction time of the plantaris muscle is likewise apparently unaltered by long-term incapacitation of gastrocnemius and soleus in adult rats (2). Since a close correlation seems to exist between myosin ATPase activity and intrinsic speed of shortening in different muscles (l), one might have expected that an increase in the proportion of fibers with low histochemically detectable myosin ATPase should have been accompanied by a lengthening of the contraction time in the overloaded muscles. However, the time course of the isometric twitch may be determined by other factors, in addition to myosin ATPase activity such as the kinetics of calcium release and uptake by the sarcoplasmic reticulum or the affinity of troponin for calcium (see 6). Furthermore although the evidence seems convincing that the ATPase activity demouFIG. 1. Rat EDL muscle, 4 months after extirpation of the synergistic tibialis anterior at birth. A, X 40 ; B,C. X 225 ; D, X 50. SDH activity is markedly increased in the hypertrophic EDL (A, left side, and B) in comparison with the contralateral muscle (A, right side, and C). Note the more homogeneous appearance of the hypertrophic muscle due to absence of “white” fibers. Mitochondrial or-GPDH activity is slightly decreased in most muscle fibers of the hypertrophic EDL (D, lower side) in comparison with the control (upper side).
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--
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.11.
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strated histochemically in skeletal muscle fibers by the calcium-precipitation method is really and exclusively due to myosin ATPase (see 14, 34), the intensity of histochemical staining does not always parallel the level of biochemical activity (16) ; difficulties can, therefore, be encountered in attempting to correlate histochemical and physiological data (29). On the other hand, at least in our system, the increase in the proportion of fibers with low myosin ATPase activity during compensatory hypertrophy was very slight, their number never exceeding 10-15s of the total fiber population. This minor proportion of fibers cannot be expected to affect in a detectable way the performance of the whole muscle in vitro, though it may have an important role in the normal muscle function in V&O, when the different motor units act independently from each other. The changes in mitochondrial oxidative activities observed at late stages of compensatory muscle hypertrophy contrast with the apparently transient modifications described in the acute phases of the hypertrophic process (17) and are presumably related to the higher energy demand that the vicarious function entails. The marked increase in SDH activity in fibers of muscles subjected to excessive functional overload since birth suggests that mitochondrial capacity for oxidative phosphorylation is much enhanced in these muscles. This seems to represent a common adaptive response of most skeletal muscles to excessive use, irrespective of the type of overwork to which the muscle is subjected (21).2 However, the histochemical transformation in our system appears quantitatively much more pronounced than that previously reported in rat or guinea pig muscles after different exercise programs (9, 12, 21). The observation that SDH activity is positively correlated with resistance to fatigue in normal fasttwitch muscle fibers (3, 10) suggests that there should be a significant 2 An exception is represented are presumably already adapted
by slow-twitch muscles, to continuous activity (9,
such 15).
as the
soleus
which
FIG. 2. Rat EDL (A-C) and EHL (D) muscles, 3 months after tibialectomy at birth. A, X 16; B, X 64; C, X 16 ; D, X 35. A, section incubated for myosin ATPase activity, after fixation in formaldehyde and preincubation in alkali: there is an increase in the number of type I fibers in the hypertrophic EDL (lower side) in comparison with the contralateral muscle (upper side). Furthermore, type II fibers display a uniformly high intensity of reaction in the hypertrophic muscle, whereas in the control there is a large proportion of fibers with intermediate degree of activity. B, higher magnification of a central area in A, comprising portion of hypertrophic (lower side) and contralateral (upper side) muscles. C, serial section of the one shown in A, incubated for myosin ATPase after pretreatment in acid: numerous type I fibers, standing out against the background of the unstained type II fibers, are predominantly concentrated in the deep areas of the hypertrophic EDL. D, EHL, myosin ATPase after pretreatment in acid: note the striking increase in number and size of type I fibers in the hypertrophic muscle (at right) in comparison with the contralateral (at left).
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FIG. 3. Rat EDL muscle, 5 months after tibialectomy performed upon Z-month-old animals. X 13. Serial sections incubated for SDH (A) and) for myosin ATPase after pretreatment in acid (B) There is no obvious difference in the histochemical profile between hypertrophic (at right) and contralateral (at left) muscles.
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increase in the resistance to fatigue of EDL and EHL muscles after tibialectomy at birth, It is of interest, in this respect, that the ultrastructural profile of most fibers in the hypertrophic EDL is characterized by abundant mitochondrial complement, relatively thick Z band, and rich development of the sarcoplasmic reticulum (our unpublished observations), which seems to be typical of fast, but fatigue-resistant, muscle fibers (32). The increase in SDH activity during long-lasting compensatory hypertrophy of EDL and EHL was not accompanied by a parallel increase in mitochondrial wGPDH activity. This indicates that mitochondria from overloaded muscles differ in composition from those of normal muscles. A similar dissociation between mitochondrial oxidative activities has been previously observed in intensively trained muscles (19, 20). As the activity level of mitochondrial wGPDH in skeletal muscles is strictly correlated with the activity level of glycolytic enzymes (26, 27 j these findings suggest that the glycolytic capacity is unchanged or slightly decreased in hypertrophy muscles. The factors responsible for the histochemical and ultrastructural changes accompanying compensatory hypertrophy in developing muscles are not fully understood. It has been shown that the acute hypertrophic response of rat skeletal muscles to incapacitation of their synergists can take place independently of intact neural circuits and appears predominantly due to a direct effect of increased mechanical stretching (17, 22. 31). The transition from this acute stretch hypertrophy to the subsequent stages of the hypertrophic process, which gradually evolves toward a really compensatory state, seems to require, on the other hand, the active participation of the neural components. The finding that long-term functional overload of rat muscles due to incapacitation of their synergists is followed by hypertrophy of the corresponding nerve fibers (8) supports, indeed, the view that adaptive changes of the whole neuromuscular system occur under these conditions. One important point which remains to be settled, in this respect, concerns the motor unit composition of the hypertrophic muscles. In normal EDL there is probably only one motor unit which is relatively slow in comparison with the others, its contraction time being actually intermediate between that of most EDL units and that of most soleus units (5) : it is possible though still unproved, that this unit is composed of the rare fibers with low myosin ATPase activity. The increased number of these fibers during compensatory lypertrophy in rats operated upon at birth may either reflect an increased size of this preexisting motor unit or the presence of additional units with similar properties. The latter might in turn arise from transformation of fast motor units or, alternatively, from de novo formation of intermediate or slow motor units, which would imply an increase in the number of motor nerve fibers to hypertrophic muscles (see 8).
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