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Effects of denervation on the distribution of myosin isozymes in skeletal muscle fibers
EXPERIMENTAL
NEUROLOGY
76, 331-346 (1982)
Effects of Denervation on the Distribution of Myosin lsozymes in Skeletal Muscle Fibers GERALDINE Department
of Anatomy,
F. GAUTHIER
AND ANN
University of Massachusetts Massachusetts 01605 Received
November
W. HOBBS’
Medical
School,
Worcester,
IO, 1981
The pattern of distribution of myosin isozymes was examined with respect to the fiber population in the denervated rat diaphragm. Myosin was demonstrated by indirect immunofluorescence using antibodies against chicken myosin and by localization of ATPase activity. In the normal diaphragm, a heterogeneous fast-twitch muscle, the component fibers react with antibodies against fast or slow myosin, but usually not with both. By 8 weeks after removal of the nerve supply, all fibers reacted with antibodies specific for fast myosin, and many reacted with anti-slow myosin as well. This indicates either that multiple forms of myosin coexist within individual muscle fibers or that a unique myosin(s) is present which has determinants in common with both fast and slow isozymes. This pattern was observed for at least 24 weeks after denervation. At all stages, the localization of alkali-stable and acidstable ATPase activity correlated well with the response to anti-fast and anti-slow myosin, respectively. We conclude that the normal adult neuromuscular relationship is required for the preferential distribution of fast and slow myosins into different populations of fibers. New myosin is nevertheless synthesized in the absence of the nervous system, and characteristics of both fast and slow isozymes are evident even after prolonged denervation. Abbreviations: anti-AL&antibodies against myosin from the slow anterior latissimus dorsi, anti-Al-antibody specific for the N-terminal sequence of the alkali 1 light chain of “fast” myosin. ’ We are indebted to Dr. Susan Lowey for her generous gift of antibodies used in this study and for many helpful discussions. We are grateful to Dr. John V. Walsh for making the facilities available for recording the compound action potentials and for help in obtaining and interpreting the physiologic data. We thank Christopher D. Hebert for his assistance in preparing the photographic illustrations and Vachik Hacopian for his help in some of the cytochemical preparations used for the preliminary survey of denervated muscles. This study was supported by grants from the U.S. Public Health Service (AM-26727) and from the Muscular Dystrophy Association. Please send correspondence to Dr. Geraldine F. Gauthier, Department of Anatomy, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester. MA 01605. 331 0014-4886/82/050331-16$02.00/O Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved
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INTRODUCTION The protein composition of a skeletal muscle is clearly influenced by the nervous system. The effects of altering the pattern of innervation vary, depending on whether the muscle is fast or slow, and changes in fast muscles tend to be more pronounced. When a fast muscle is experimentally crossreinnervated by a nerve which formerly supplied a slow muscle, the speed of contraction is decreased and the myosin acquries characteristics of “slow” myosin (2, 29, 50, 58). Chronic stimulation at low frequency likewise slows the contraction time of a fast muscle and induces synthesis of slow myosin (41, 46, 47, 5 1). Surgical removal of the nerve supply also leads to a decrease in the speed of a fast muscle (23, 32, 36, 38) but the myosin composition varies, depending on the age and species of animal and the time after denervation (23). Within the fast category of skeletal muscles, it was reported that myosin ATPase activity and the proportion of “fast” light chains are either reduced (23, 55) or unchanged (27) after denervation. It was also reported, on the basis of light chain and heavy chain composition, that “slow” myosin was decreased, leaving “fast” myosin as the predominant isozyme in the denervated muscle (6, 7). The latter findings as well as observations on muscles denervated during postnatal development (30, 43) were interpreted to indicate that synthesis of slow myosin is dependent on the nervous system, but that fast myosin is maintained even after its removal. There is, in addition, a change in the cytochemical and ultrastructural composition of heterogeneous fast muscles which suggests a preferential atrophy of “fast” fibers ( 11, 27, 3 1, 56). The possibility exists, however, that the shift from a heterogeneous to a more homogeneous fiber population also reflects a transformation of fiber type accompanied by synthesis of a new type of myosin. By using antibodies against fast and slow myosin as markers, we attempted to determine whether or not removal of the nerve supply affects the distribution of myosin isozymes with respect to the microscopic pattern of fibers in a fast-twitch muscle. The immunocytochemical approach permits identification of specific isozymes in individual fibers ( 12, 14) and, in addition, the presence of more than one type of myosin in a particular fiber can be demonstrated in serial sections after exposure to antibodies specific for each of the isozymes. Our observations indicate that the normal adult pattern of segregation of fast and slow myosins into different populations of fibers is lost soon after denervation, but that immunochemical properties associated with slow as well as fast myosin can be demonstrated even after several months.
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MATERIALS Surgical
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AND METHODS Procedures
Young adult male albino rats (approximately 90 days old) were anesthetized with ether. The left hemidiaphragm was denervated by a lateral approach between the 9th and 10th ribs (14) and a 5-mm segment of the phrenic nerve was removed to prevent reinnervation. The intercostal approach eliminates the possibility of persistent innervation from the “accessory” phrenic nerve, which joins the phrenic nerve rostra1 to the point of excision (22). Normal adult male animals of comparable age at the time of killing were used as controls. All denervated animals were examined to ensure that the proximal phrenic nerve had not reestablished contact with the diaphragm. Effectiveness of the surgical procedure was also verified by the uniformly thin (nearly transparent) appearance of the,muscle relative to the intact diaphragm. Preparation
of Skeletal
Muscle
The abdominal surface of the left hemidiaphragm was exposed by a midventral incision in the body wall, and thin strips of muscle (3 mm) in the ventral third of the costal region were isolated by blunt dissection, tied to a splint and then excised. Each tied strip of denervated muscle was sutured, together with a strip of normal diaphragm, and frozen in isopentane cooled to -160°C with liquid nitrogen. Zmmunocytochemistry
Antibodies against slow myosin (anti-ALD) and against the N-terminal sequence of the alkali 1 light chain (anti-Al) of fast myosin were provided by Dr. Susan Lowey, and their specificities are described elsewhere (13). Antibody specific for the heavy chain of ALD myosin was prepared by absorbing anti-ALD myosin with an excess of purified ALD light chains (16). All specimens of denervated diaphragm were mounted with a specimen of normal diaphragm and sectioned simultaneously so that the experimental and normal muscles could be compared under identical conditions. Transverse cryostat sections (4 pm) were cut at -20°C and incubated as described by Gauthier and Lowey (13). The sections were exposed, without fixation, to unlabeled rabbit anti-Al or anti-ALD antibody, and subsequently treated with fluorescein-labeled goat anti-rabbit immunoglobulin. They were examined with a Zeiss fluorescence microscope equipped with
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an epi-illumination system. A xenon XBO 75 W/DC lamp, a narrow-band FITC excitation filter (485/20 nm), and a band-pass barrier filter (520 to 560 nm) were used with a Zeiss Neofluar 16/0.4 objective. The images were recorded on Kodak type 103 a-G spectroscopic plates. Enzyme Cytochemistry Transverse cryostat sections (10 pm) serial to those used for immunocytochemistry were used to localize ATPase activity according to the procedure described by Guth and Samaha (26). Sections were fixed 5 min at 4°C in 2% Formalin containing 0.068 M CaC12 and 0.33 M sucrose buffered with 0.2 M sodium cacodylate, at pH 7.6. This was followed by preincubation 15 min at room temperature in 0.018 M CaC12, and 0.1 M 2-amino2 methyl-1-propanol buffer (Sigma, No. 221), at pH 10.4. Serial sections were preincubated without fixation for 15 min at room temperature in 0.05 M potassium acetate and 0.018 M CaCl,, at pH 4.35. All sections then were incubated 45 min at 37°C in a medium containing 0.0027 M ATP, 0.018 M CaCl*, and 0.05 M KCl, at pH 9.4 (Sigma, No. 221). RESULTS Antibodies against myosin from the slow anterior latissimus dorsi (ALD) of the chicken were used to localize “slow” myosin with respect to individual muscle fibers in the rat diaphragm. Antibody specific for the difference peptide (Al) which is unique to the alkali 1 light chain (LClr) of chicken pectoralis myosin was used to identify “fast” myosin. The presence of fast and slow types of myosin is verified by the localization of ATPase activity. The ATPase activity of adult skeletal muscle fibers is stable after either acid or alkali preincubation, but usually not both (25). Fibers which react with antibodies specific for fast myosin have alkali-stable ATPase activity, whereas those which react with anti-slow myosin have acid-stable activity. This enzymic activity is not associated necessarily with the myofibrillar proteins exclusively, particularly after acid preincubation (10, 24). Nevertheless, the procedure serves as a useful preliminary indicator when surveying large numbers of experimental animals. At all stages of denervation, the localization of alkali-stable and acid-stable ATPase activity correlated well with the response to anti-fast (anti-Al) and anti-slow myosin (antiALD), respectively. In the normal adult rat diaphragm, fibers reacted with either anti-fast or anti-slow myosin, and only occasionally with both antibodies. This indicates that fast and slow isozymes were largely segregated into different populations of fiber types (see Figs. 2a, b and 3a, b, lower sections). After denervation of the diaphragm, fast and slow types of myosin were frequently observed within single fibers. Within the 1st week, there was an increase in the cross-sectional area occupied by fibers with acid-
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FIG. 1. Rat diaphragm, denervated 4 weeks. a-Alkali-stable, b-acid-stable ATPase. In this and all subsequent illustrations a specimen of normal diaphragm was sectioned simultaneously with the denervated muscle and is situated below it in each micrograph. In the normal control (lower sections), fibers had either acid- or alkali-stable ATPase activity, hence the pattern in (a) is reciprocal to that observed in (b). In the denervated muscle (upper sections), many fibers had activity after either acid or alkali preincubation. Those fibers which had acid-stable ATPase activity (b) also had moderate alkali-stable activity (a) and all other fibers had high alkali-stable activity. X226.
stable ATPase activity and a positive response to anti-slow myosin. This is consistent with the well known phenomenon of hypertrophy, a transient feature of the denervated diaphragm which affects the small (slow) fibers in particular (9, 62). There was also a preferential atrophy of the large (fast) fibers, which became even more pronounced at 4 weeks (14). At 4 weeks, many fibers reacted with antibody against both fast and slow myosin. They also had ATPase activity after either acid or alkali preincubation. Fibers which had acid-stable activity (Fig.lb), also had at least low or moderate alkali-stable activity compared with unreactive fibers in the control muscle (Fig. 1a). This most likely represents an increase in the synthesis of a fast type of myosin in fibers which otherwise would have only a slow type. By 8 weeks, all fibers reacted with anti-fast myosin (Figure 2a and 3a).
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FIG. 3. Rat diaphragm, denervated 8 weeks. a-Anti-fast, h-anti-slow myosin. All denervated fibers reacted with anti-fast myosin (a), and many reacted with anti-slow myosin as well (b). The control muscle (lower section) contained an example (left) of the occasional normal fiber which reacted with both antibodies. X226.
Hence those fibers which reacted with anti-slow myosin (Figs. 2b and 3b) had a positive response to both antibodies, and this indicates that fast and slow types of myosin coexisted in many individual fibers or that there was a myosin(s) with immunologic properties of both isozymes. All fibers likewise had high alkali-stable ATPase activity (Fig. 2c), and the fibers which reacted with anti-slow myosin also had high acid-stable ATPase activity (Fig. 2d). At 12 weeks after denervation the distribution of myosin was similar to that observed at 8 weeks. Between 15 and 24 weeks the alkali-stable FIG. 2. Rat diaphragm, denervated 8 weeks. a-Anti-fast, b-anti-slow myosin, c-alkalistable, d-acid-stable ATPase. Response to the two antibodies was reciprocal in the normal muscle (compare lower sections in a and b). All denervated fibers reacted with anti-fast myosin, although some stained more intensely than others. They also had alkali-stable ATPase activity (c). Many fibers reacted with anti-slow myosin, and the same fibers had acid-stable ATPase activity (d). Hence a substantial population of fibers had properties of both fast and slow isozymes of myosin. X226.
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FIG. 6. Rat diaphragm, denervated 70 weeks. a-Alkali-stable, bacid-stable ATPase. All fibers had alkali-stable ATPase activity. Many fibers had acid-stable activity as well, although the level was somewhat lower than in the control muscle. X226.
ATPase activity remained high in all fibers, but acid-stable activity was often diminished. In some animals there was little or no activity (Fig. 4a, b), whereas in others activity was as intense as that seen at 8 weeks (Fig. 5a, b), and this could be demonstrated for at least 24 weeks. Acid-stable ATPase activity as well as a positive response to anti-slow myosin were evident, though at a lower level, in muscles that had been denervated for 60 or 70 weeks (Fig. 6a, b). To determine whether or not the positive response to anti-slow myosin in the denervated muscle reflected a property of the light chain or heavy chain, anti-ALD myosin was absorbed with total ALD light chains. In FIG. 4. Rat diaphragm, denervated 20 weeks. a-Alkali-stable, &acid-stable ATPase. All fibers had alkali-stable ATPase activity (a), but, in this experimental animal, there was almost no acid-stable activity (b). X226. FIG. 5. Rat diaphragm, denervated 20 weeks. a-Alkali-stable, b-acid-stable ATPase. All fibers had alkali-stable ATPase activity, and many fibers had substantial acid-stable activity as well. X226.
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serial sections of &week denervated muscle, this antibody continued to react with the same fibers that had reacted with unabsorbed antibody, but the level of fluorescence was somewhat diminished relative to the control muscle. This indicates that the heavy chain was involved in the response to anti-slow myosin, although the light chain may also contribute to the staining pattern. Examination of electrically stimulated muscles provided strong evidence that denervation was complete and that reinnervation had not occurred. At 8 weeks after denervation, electrical stimulation of the proximal phrenic nerve in the left cervical region or of the surviving distal stump elicited no visible twitch response in the denervated hemidiaphragm at 0.3, 3.0 or 30 V for 2 ms. In an isolated diaphragm preparation, no compound action potential was recorded when the distal stump of the phrenic nerve was stimulated 4.5 ms at 3.8 V. In addition, no action potential was detected when the contralateral phrenic was stimulated, which indicates that there was no obvious crossing of phrenic motoneurons to muscle fibers of the opposite side. In summary, our observations indicate that there was an increase in the proportion of muscle fibers having a fast type of myosin within 4 weeks after denervation. By 8 weeks, the myosin in many fibers had characteristics of both fast and slow isozymes. Then, beginning by about 15 weeks, there was a trend toward a loss of properties associated with slow myosin, but a slow type of myosin continued to be present until at least 24 weeks, and in lesser amounts for as long as 70 weeks. DISCUSSION Significance
of the Immunocytochemical
Observations
The preferential distribution of fast and slow myosin into different fiber types characteristic of the rat diaphragm is no longer evident following denervation. By 8 weeks, a substantial number of fibers react with antibodies against fast as well as slow myosin, and the same fibers have both alkali- and acid-stable ATPase activity. This can be interpreted to mean that fast and slow isozymes coexist within individual muscle fibers or that a distinctive myosin(s) is present which has properties of both isozymes. The present results do not exclude either of these possibilities, but there is a precedent for the second interpretation. We showed previously that in developing skeletal muscles there was a uniform response by all fibers to anti-fast and anti-slow myosin (15, 16). In developing muscles, there are light chains which are equivalent to those of adult fast myosin and also
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small amounts of slow light chain (16,33,53), and, in the rat, an embryonic light chain which is not present in adult muscles (59). The heavy chain also appears to be different from that of adult myosin in both the rat (60, 61) and the chicken (3, 45). It was recently reported that there is a normal light chain pattern consisting of fast and slow forms in the denervated rat diaphragm at 2 months, but that at 6 months, there is, as in embryonic muscle, very little light chain or heavy chain characteristic of slow myosin (6, 7). Unlike the developing muscle, however, there is apparently no embryonic light chain (8). The possibility exists that there is, in denervated muscle, yet another distinctive myosin, but this cannot be resolved without chemical analysis. Nevertheless, the immunocytochemical observations provide evidence for a slow type of isozyme. It is unlikely that this is the original myosin that was present in the “slow fibers” at the time of denervation. A substantial amount of myosin observed in the denervated diaphragm probably represents newly synthesized protein. Information on the turnover rate of skeletal muscle myosin is limited, and published data vary greatly (35, 54). Some of the discrepancy may reflect technical problems in measuring protein turnover. For example, isotope data often include values for reutilization of labeled precursor, with the result that the half-life may be overestimated. In addition, the balance between synthesis and degradation may vary, depending on the physiologic state (18, 40). Recently published data based on turnover rates for mixed muscle proteins suggest that the half-life of myosin is 8 days for the diaphragm and 15 days for the gastrocnemius and plantaris of the rat (39), and this is consistent with a half-life value of 15 days obtained for the myosin heavy chain in the rat longissimus dorsi (Dr. Radovan Zak, Department of Medicine, University of Chicago, personal communication). The breakdown of muscle protein is greatly accelerated by denervation ( 17, 19, 20, 57). Using the rat diaphragm, it was calculated that the rate constant for protein degradation is more than doubled by 3 days after denervation (57). Hence, it is likely that the myosin present in this muscle at the time of denervation would no longer be observed by 8 weeks or at the even longer intervals examined in this study. There is also a measurable increase in protein synthesis after denervation (17, 19-21). In addition, our observations indicate that denervated fibers have synthesized a new type of myosin. Because fast and slow myosins are usually associated with separate fiber types in the diaphragm, the dual staining pattern implies that these fibers are capable of synthesizing an isozyme other than that which is normally present, and that may be unique to the altered physiologic state.
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Functional
HOBBS
Implications
As indicated earlier, there is general agreement that the speed of muscle contraction is slowed after denervation. Despite this, some investigators reported that a shift in myosin composition to a pattern in which a fast type of isozyme predominates can be induced by denervation of adult, developing, or regenerating skeletal muscles (6, 7, 34, 43). It is possible, however, as discussed above, that the myosin in the experimentally altered or undifferentiated muscles consists of molecules which are not equivalent to either the fast or slow adult forms. It is also possible that speed of contraction is unrelated to the type of myosin. Isometric twitch contraction time correlates well with molecular properties of the membrane systems (28). Moreover, the sarcoplasmic membranes of the now slowly contracting muscles become disorganized after denervation of either adult (11) or developing muscles (48). In addition, calcium transport activity of the sarcoplasmic reticulum is decreased in denervated muscle (37, 49). In the absence of a precisely ordered membrane system, it is likely that excitationcontraction coupling would be impaired and that speed of contraction might be slow regardless of the type of myofibillar protein present. Relationship
to the Nerve Supply
The extent to which the nervous system influences myosin synthesis is not certain. Data on ATPase activity and gel electrophoresis patterns and also on immunochemical properties of developing or denervated muscles often have been interpreted to indicate that fast but not slow myosin can be synthesized in the absence of the nervous system (6, 30, 42, 43). Slow myosin is said to be present in developing muscles only in those cells which have been contacted by a motoneuron (44). Nevertheless, slow myosin light chains are evident in muscle cell cultures, where the nerve supply is absent (33, 52) and also in muscles which have been surgically denervated (6). Cultured cells, moreover, react strongly with antibody specific for slow myosin (5). Evidence that the myosin of embryonic muscle fibers may not be equivalent to either fast or slow adult myosin makes it likely that both adult isozymes require an adult nervous system. The presence of a distinctive “embryonic” myosin in developing skeletal muscles of both the rat and chicken (16, 59, 60) and also of another distinctive “neonatal” isozyme which precedes synthesis of adult myosin [(61); P. A. Benfield, G. W a11er, and S. Lowey, in preparation]., may be related to the state of differentiation of the nerve supply. Even though neuromuscular contacts exist at stages where embryonic or neonatal myosin predominate, the structural configuration differs significantly from that of the adult neuromuscular junction. Muscle fibers receive a “polyneuronal”
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innervation pattern at these stages ( 1, 4) and, in addition, many ultrastructural features of the adult neuromuscular junction are not yet apparent (Gauthier, unpublished observations). We observed previously that, coincident with polyneuronal innervation, properties of fast and slow myosin are apparent in all muscle fibers (15, 16). In the present paper, we have shown that when the nerve supply to the rat diaphragm is removed, myosin(s) having immunological cross reactivity with adult fast and slow myosin continues to be synthesized by the muscle fibers. We conclude, regardless of whether the newly synthesized myosin is a fast or a slow, or a unique isozyme, that a fully differentiated neuromuscular relationship is required for the selective pattern of distribution of fast and slow myosin isozymes characteristic of normal adult skeletal muscles. REFERENCES I. ATSUMI,S. 1977. Development of neuromuscular junctions of fast and slow muscles in the chick embryo: a light and electron microscopic study. J. Neurocyrol. 6, 691-709. 2. BARANY, M., AND R. I. CLOSE. 1971. The transformation of myosin in cross-innervated rat muscles. J. Physiol. (London) 213: 455-474. 3. BENFIELD, P. A., S. LOWEY, AND D. D. LEBLANC. 1981. Fractionation and characterization of myosins from embryonic chicken pectoralis muscle. Biophys. J. 33: 243a. 4. BENNETT, M. R., AND A. G. PE~IGREW. 1974. The formation of synapses in striated muscle during development. J. Physiol. (London) 241: 51 S-545. 5. CANTINI, M., S. SARTORE, AND W. S. SCHIAFFINO. 1980. Myosin types in cultured muscle cells. J. Cell Biol. 85: 903-909. 6. CARRARO, U., C. CATANI, AND D. BIRAL. 1979. Selective maintenance of neurotrophitally regulated proteins in denervated rat diaphragm. Exp. Neurol. 63: 468-475. 7. CARRARO, U., C. CATANI, AND L. DALL.A LIBERA. 1981. Myosin light and heavy chains in rat gastrocnemius and diaphragm muscles after chronic denervation or reinnervation. Exp.
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consequences of denervation in fast and slow skeletal muscles and their relationship to neural control over muscle differentiation. Biochem. J. 126: 1099-l 110. MILEDI, R., AND C. R. SLATER. 1969. Electron-microscopic structure of denervated skeletal muscle. Proc. R. Sot. Lond. B. 174: 253-269. MILLWARD, D. J., P. C. BATES, G. J. LAURENT, AND C. C. Lo. 1978. Factors affecting protein breakdown in skeletal muscle. Pages 619-644 in H. L. SEGALAND D. J. DOYLE, Eds., Protein Turnover and Lysosome Function. Academic Press, New York. MILLWARD, D. J., P. J. GARLICK, D. 0. NNANYELUGO, AND J. C. WATERLOW. 1976. The relative importance of muscle protein synthesis and breakdown in the regulation of muscle mass. Biochem. J. 156: 185-188. PETTE, D., W. MULLER, E. LEISNER, AND G. VRBOV~. 1976. Time dependent effects on contractile properties, fibre populations, myosin light chains and enzymes of energy metabolism in intermittently and continuously stimulated fast twitch muscles of the rabbit. Pjiigers Arch. 364: 103- 112. RUBINSTEIN, N. A., AND H. HOLTZER. 1979. Fast and slow muscles in tissue culture synthesize only fast myosin. Nature (London) 280: 323-325. RUBINSTEIN, N. A., AND A. M. KELLY. 1978. Myogenic and neurogenic contributions to the development of fast and slow twitch muscles in rat. Devel. Biol. 62: 473-485. RUBINSTEIN, N. A., AND A. M. KELLY. 198 1. Development of muscle fiber specialization in the rat hindlimb. J. Cell Biol. 90: 128-144. RUSHBROOK, J. I., AND A. STRACHER. 1979. Comparison of adult, embryonic, and dystrophic myosin heavy chains from chicken muscle by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and peptide mapping. Proc. Natl. Acad. Sci. USA 76: 4331-4334. SALMONS, S., AND F. A. SR~TER. 1976. Significance of impluse activity in the transformation of skeletal muscle type. Nature (London) 263: 30-34. SALMONS, S., AND G. VRBOV~. 1969. The influence of activity on some contractile characteristics of mammalian fast and slow musc1es.J. Physiol. (London) 201: 535-549. SCHIAFFINO, S., AND P. SEITEMBRINI. 1970. Studies on the effect of denervation in developing muscle. I. Differentiation of the sarcotubular system. Virchows Arch. B. 4: 345-356.
49. SR~TER, F. A. 1970. Effect of denervation on fragmented sarcoplasmic reticulum of white and red muscle. Exp. Neural. 29: 52-64. 50. SR~TER, F. A., J. GERGELY, AND A. L. LUFF. 1974. The effect of cross-reinnervation of the synthesis of myosin light chains. Biochem. Biophys. Rex Commun. 56: 84-89. 51. SR~TER, F. A., J. GERGELY, S. SALMONS, AND F. ROMANUL. 1973. Synthesis by fast muscle of myosin light chains characteristic of slow muscle in response to long-term stimulation. Nature New Biol. 241: 17-19. 52. SRIHARI, T., AND D. PETTE. 1981. Myosin light chains in normal and electrostimulated cultures of embryonic chicken breast muscle. FEBS Lett. 123: 312-314. 53. STOCKDALE, F. E., N. RAMAN, AND H. BADEN. 1981. Myosin light chains and the developmental origin of fast muscle. Proc. Natl. Acad. Sci. USA 78: 931-935. 54. SWICK, R. W.,, AND H. SONG. 1974. Turnover rates of various muscle proteins. J. Anim. Sci. 38: 1150-1157. 55. SYROV~, I. 1976. The relation between ATPase activity and light chains of myosin in developing, adult and denervated muscles of several animal species. Physiol. Bohemoslov.
25: 295-300.
TOMANEK, R. J., AND D. D. LUND. 1973. Degeneration of different types of skeletal muscle fibres. I. Denervation. J. Anat. 116: 395-407. 57. TURNER, L. V., AND P. J. GARLICK. 1974. The effect of unilateral phrenicectomy on the 56.
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rate of protein synthesis in rat diaphragm in vivo. Biochim. Biophys. Acfa 349: 109113. WEEDS, A. G., D. R. TRENTHAM, C. J. C. KEAN, AND A. J. BULLER. 1974. Myosin from cross-reinnervated cat muscles. Nature (London) 247: 135-139. WHALEN, R. G., G. S. BUTLER-BROWNE, AND F. GROS. 1978. Identification of a novel form of myosin light chain present in embryonic muscle tissue and cultured muscle cells. J. Mol. Eiol. 126: 415-431. WHALEN, R. G., K. SCHWARTZ, P. BOUVERET, S. M. SELL, AND F. GROS. 1979. Contractile protein isozymes in muscle development: identification of an embryonic form of myosin heavy chain. Proc. Natl. Acad. Sci. USA 16: 5197-5201. WHALEN, R. G., S. M. SELL, G. S. BUTLER-BROWNE, K. SCHWARTZ, P. BOUVERET, AND I. PINSET-HARSTROM. 1981. Three myosin heavy-chain isozymes appear sequentially in rat muscle development. Nature (London) 292: 805-809. YELLIN, H. 1974. Changes in fiber types of the hypertrophying denervated hemidiaphragm. Exp. Neural. 42: 412-428.
NEUROLOGY
76, 331-346 (1982)
Effects of Denervation on the Distribution of Myosin lsozymes in Skeletal Muscle Fibers GERALDINE Department
of Anatomy,
F. GAUTHIER
AND ANN
University of Massachusetts Massachusetts 01605 Received
November
W. HOBBS’
Medical
School,
Worcester,
IO, 1981
The pattern of distribution of myosin isozymes was examined with respect to the fiber population in the denervated rat diaphragm. Myosin was demonstrated by indirect immunofluorescence using antibodies against chicken myosin and by localization of ATPase activity. In the normal diaphragm, a heterogeneous fast-twitch muscle, the component fibers react with antibodies against fast or slow myosin, but usually not with both. By 8 weeks after removal of the nerve supply, all fibers reacted with antibodies specific for fast myosin, and many reacted with anti-slow myosin as well. This indicates either that multiple forms of myosin coexist within individual muscle fibers or that a unique myosin(s) is present which has determinants in common with both fast and slow isozymes. This pattern was observed for at least 24 weeks after denervation. At all stages, the localization of alkali-stable and acidstable ATPase activity correlated well with the response to anti-fast and anti-slow myosin, respectively. We conclude that the normal adult neuromuscular relationship is required for the preferential distribution of fast and slow myosins into different populations of fibers. New myosin is nevertheless synthesized in the absence of the nervous system, and characteristics of both fast and slow isozymes are evident even after prolonged denervation. Abbreviations: anti-AL&antibodies against myosin from the slow anterior latissimus dorsi, anti-Al-antibody specific for the N-terminal sequence of the alkali 1 light chain of “fast” myosin. ’ We are indebted to Dr. Susan Lowey for her generous gift of antibodies used in this study and for many helpful discussions. We are grateful to Dr. John V. Walsh for making the facilities available for recording the compound action potentials and for help in obtaining and interpreting the physiologic data. We thank Christopher D. Hebert for his assistance in preparing the photographic illustrations and Vachik Hacopian for his help in some of the cytochemical preparations used for the preliminary survey of denervated muscles. This study was supported by grants from the U.S. Public Health Service (AM-26727) and from the Muscular Dystrophy Association. Please send correspondence to Dr. Geraldine F. Gauthier, Department of Anatomy, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester. MA 01605. 331 0014-4886/82/050331-16$02.00/O Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved
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INTRODUCTION The protein composition of a skeletal muscle is clearly influenced by the nervous system. The effects of altering the pattern of innervation vary, depending on whether the muscle is fast or slow, and changes in fast muscles tend to be more pronounced. When a fast muscle is experimentally crossreinnervated by a nerve which formerly supplied a slow muscle, the speed of contraction is decreased and the myosin acquries characteristics of “slow” myosin (2, 29, 50, 58). Chronic stimulation at low frequency likewise slows the contraction time of a fast muscle and induces synthesis of slow myosin (41, 46, 47, 5 1). Surgical removal of the nerve supply also leads to a decrease in the speed of a fast muscle (23, 32, 36, 38) but the myosin composition varies, depending on the age and species of animal and the time after denervation (23). Within the fast category of skeletal muscles, it was reported that myosin ATPase activity and the proportion of “fast” light chains are either reduced (23, 55) or unchanged (27) after denervation. It was also reported, on the basis of light chain and heavy chain composition, that “slow” myosin was decreased, leaving “fast” myosin as the predominant isozyme in the denervated muscle (6, 7). The latter findings as well as observations on muscles denervated during postnatal development (30, 43) were interpreted to indicate that synthesis of slow myosin is dependent on the nervous system, but that fast myosin is maintained even after its removal. There is, in addition, a change in the cytochemical and ultrastructural composition of heterogeneous fast muscles which suggests a preferential atrophy of “fast” fibers ( 11, 27, 3 1, 56). The possibility exists, however, that the shift from a heterogeneous to a more homogeneous fiber population also reflects a transformation of fiber type accompanied by synthesis of a new type of myosin. By using antibodies against fast and slow myosin as markers, we attempted to determine whether or not removal of the nerve supply affects the distribution of myosin isozymes with respect to the microscopic pattern of fibers in a fast-twitch muscle. The immunocytochemical approach permits identification of specific isozymes in individual fibers ( 12, 14) and, in addition, the presence of more than one type of myosin in a particular fiber can be demonstrated in serial sections after exposure to antibodies specific for each of the isozymes. Our observations indicate that the normal adult pattern of segregation of fast and slow myosins into different populations of fibers is lost soon after denervation, but that immunochemical properties associated with slow as well as fast myosin can be demonstrated even after several months.
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ISOZYMES
MATERIALS Surgical
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AND METHODS Procedures
Young adult male albino rats (approximately 90 days old) were anesthetized with ether. The left hemidiaphragm was denervated by a lateral approach between the 9th and 10th ribs (14) and a 5-mm segment of the phrenic nerve was removed to prevent reinnervation. The intercostal approach eliminates the possibility of persistent innervation from the “accessory” phrenic nerve, which joins the phrenic nerve rostra1 to the point of excision (22). Normal adult male animals of comparable age at the time of killing were used as controls. All denervated animals were examined to ensure that the proximal phrenic nerve had not reestablished contact with the diaphragm. Effectiveness of the surgical procedure was also verified by the uniformly thin (nearly transparent) appearance of the,muscle relative to the intact diaphragm. Preparation
of Skeletal
Muscle
The abdominal surface of the left hemidiaphragm was exposed by a midventral incision in the body wall, and thin strips of muscle (3 mm) in the ventral third of the costal region were isolated by blunt dissection, tied to a splint and then excised. Each tied strip of denervated muscle was sutured, together with a strip of normal diaphragm, and frozen in isopentane cooled to -160°C with liquid nitrogen. Zmmunocytochemistry
Antibodies against slow myosin (anti-ALD) and against the N-terminal sequence of the alkali 1 light chain (anti-Al) of fast myosin were provided by Dr. Susan Lowey, and their specificities are described elsewhere (13). Antibody specific for the heavy chain of ALD myosin was prepared by absorbing anti-ALD myosin with an excess of purified ALD light chains (16). All specimens of denervated diaphragm were mounted with a specimen of normal diaphragm and sectioned simultaneously so that the experimental and normal muscles could be compared under identical conditions. Transverse cryostat sections (4 pm) were cut at -20°C and incubated as described by Gauthier and Lowey (13). The sections were exposed, without fixation, to unlabeled rabbit anti-Al or anti-ALD antibody, and subsequently treated with fluorescein-labeled goat anti-rabbit immunoglobulin. They were examined with a Zeiss fluorescence microscope equipped with
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an epi-illumination system. A xenon XBO 75 W/DC lamp, a narrow-band FITC excitation filter (485/20 nm), and a band-pass barrier filter (520 to 560 nm) were used with a Zeiss Neofluar 16/0.4 objective. The images were recorded on Kodak type 103 a-G spectroscopic plates. Enzyme Cytochemistry Transverse cryostat sections (10 pm) serial to those used for immunocytochemistry were used to localize ATPase activity according to the procedure described by Guth and Samaha (26). Sections were fixed 5 min at 4°C in 2% Formalin containing 0.068 M CaC12 and 0.33 M sucrose buffered with 0.2 M sodium cacodylate, at pH 7.6. This was followed by preincubation 15 min at room temperature in 0.018 M CaC12, and 0.1 M 2-amino2 methyl-1-propanol buffer (Sigma, No. 221), at pH 10.4. Serial sections were preincubated without fixation for 15 min at room temperature in 0.05 M potassium acetate and 0.018 M CaCl,, at pH 4.35. All sections then were incubated 45 min at 37°C in a medium containing 0.0027 M ATP, 0.018 M CaCl*, and 0.05 M KCl, at pH 9.4 (Sigma, No. 221). RESULTS Antibodies against myosin from the slow anterior latissimus dorsi (ALD) of the chicken were used to localize “slow” myosin with respect to individual muscle fibers in the rat diaphragm. Antibody specific for the difference peptide (Al) which is unique to the alkali 1 light chain (LClr) of chicken pectoralis myosin was used to identify “fast” myosin. The presence of fast and slow types of myosin is verified by the localization of ATPase activity. The ATPase activity of adult skeletal muscle fibers is stable after either acid or alkali preincubation, but usually not both (25). Fibers which react with antibodies specific for fast myosin have alkali-stable ATPase activity, whereas those which react with anti-slow myosin have acid-stable activity. This enzymic activity is not associated necessarily with the myofibrillar proteins exclusively, particularly after acid preincubation (10, 24). Nevertheless, the procedure serves as a useful preliminary indicator when surveying large numbers of experimental animals. At all stages of denervation, the localization of alkali-stable and acid-stable ATPase activity correlated well with the response to anti-fast (anti-Al) and anti-slow myosin (antiALD), respectively. In the normal adult rat diaphragm, fibers reacted with either anti-fast or anti-slow myosin, and only occasionally with both antibodies. This indicates that fast and slow isozymes were largely segregated into different populations of fiber types (see Figs. 2a, b and 3a, b, lower sections). After denervation of the diaphragm, fast and slow types of myosin were frequently observed within single fibers. Within the 1st week, there was an increase in the cross-sectional area occupied by fibers with acid-
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FIG. 1. Rat diaphragm, denervated 4 weeks. a-Alkali-stable, b-acid-stable ATPase. In this and all subsequent illustrations a specimen of normal diaphragm was sectioned simultaneously with the denervated muscle and is situated below it in each micrograph. In the normal control (lower sections), fibers had either acid- or alkali-stable ATPase activity, hence the pattern in (a) is reciprocal to that observed in (b). In the denervated muscle (upper sections), many fibers had activity after either acid or alkali preincubation. Those fibers which had acid-stable ATPase activity (b) also had moderate alkali-stable activity (a) and all other fibers had high alkali-stable activity. X226.
stable ATPase activity and a positive response to anti-slow myosin. This is consistent with the well known phenomenon of hypertrophy, a transient feature of the denervated diaphragm which affects the small (slow) fibers in particular (9, 62). There was also a preferential atrophy of the large (fast) fibers, which became even more pronounced at 4 weeks (14). At 4 weeks, many fibers reacted with antibody against both fast and slow myosin. They also had ATPase activity after either acid or alkali preincubation. Fibers which had acid-stable activity (Fig.lb), also had at least low or moderate alkali-stable activity compared with unreactive fibers in the control muscle (Fig. 1a). This most likely represents an increase in the synthesis of a fast type of myosin in fibers which otherwise would have only a slow type. By 8 weeks, all fibers reacted with anti-fast myosin (Figure 2a and 3a).
336
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FIG. 3. Rat diaphragm, denervated 8 weeks. a-Anti-fast, h-anti-slow myosin. All denervated fibers reacted with anti-fast myosin (a), and many reacted with anti-slow myosin as well (b). The control muscle (lower section) contained an example (left) of the occasional normal fiber which reacted with both antibodies. X226.
Hence those fibers which reacted with anti-slow myosin (Figs. 2b and 3b) had a positive response to both antibodies, and this indicates that fast and slow types of myosin coexisted in many individual fibers or that there was a myosin(s) with immunologic properties of both isozymes. All fibers likewise had high alkali-stable ATPase activity (Fig. 2c), and the fibers which reacted with anti-slow myosin also had high acid-stable ATPase activity (Fig. 2d). At 12 weeks after denervation the distribution of myosin was similar to that observed at 8 weeks. Between 15 and 24 weeks the alkali-stable FIG. 2. Rat diaphragm, denervated 8 weeks. a-Anti-fast, b-anti-slow myosin, c-alkalistable, d-acid-stable ATPase. Response to the two antibodies was reciprocal in the normal muscle (compare lower sections in a and b). All denervated fibers reacted with anti-fast myosin, although some stained more intensely than others. They also had alkali-stable ATPase activity (c). Many fibers reacted with anti-slow myosin, and the same fibers had acid-stable ATPase activity (d). Hence a substantial population of fibers had properties of both fast and slow isozymes of myosin. X226.
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MYOSIN
ISOZYMES
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FIG. 6. Rat diaphragm, denervated 70 weeks. a-Alkali-stable, bacid-stable ATPase. All fibers had alkali-stable ATPase activity. Many fibers had acid-stable activity as well, although the level was somewhat lower than in the control muscle. X226.
ATPase activity remained high in all fibers, but acid-stable activity was often diminished. In some animals there was little or no activity (Fig. 4a, b), whereas in others activity was as intense as that seen at 8 weeks (Fig. 5a, b), and this could be demonstrated for at least 24 weeks. Acid-stable ATPase activity as well as a positive response to anti-slow myosin were evident, though at a lower level, in muscles that had been denervated for 60 or 70 weeks (Fig. 6a, b). To determine whether or not the positive response to anti-slow myosin in the denervated muscle reflected a property of the light chain or heavy chain, anti-ALD myosin was absorbed with total ALD light chains. In FIG. 4. Rat diaphragm, denervated 20 weeks. a-Alkali-stable, &acid-stable ATPase. All fibers had alkali-stable ATPase activity (a), but, in this experimental animal, there was almost no acid-stable activity (b). X226. FIG. 5. Rat diaphragm, denervated 20 weeks. a-Alkali-stable, b-acid-stable ATPase. All fibers had alkali-stable ATPase activity, and many fibers had substantial acid-stable activity as well. X226.
340
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serial sections of &week denervated muscle, this antibody continued to react with the same fibers that had reacted with unabsorbed antibody, but the level of fluorescence was somewhat diminished relative to the control muscle. This indicates that the heavy chain was involved in the response to anti-slow myosin, although the light chain may also contribute to the staining pattern. Examination of electrically stimulated muscles provided strong evidence that denervation was complete and that reinnervation had not occurred. At 8 weeks after denervation, electrical stimulation of the proximal phrenic nerve in the left cervical region or of the surviving distal stump elicited no visible twitch response in the denervated hemidiaphragm at 0.3, 3.0 or 30 V for 2 ms. In an isolated diaphragm preparation, no compound action potential was recorded when the distal stump of the phrenic nerve was stimulated 4.5 ms at 3.8 V. In addition, no action potential was detected when the contralateral phrenic was stimulated, which indicates that there was no obvious crossing of phrenic motoneurons to muscle fibers of the opposite side. In summary, our observations indicate that there was an increase in the proportion of muscle fibers having a fast type of myosin within 4 weeks after denervation. By 8 weeks, the myosin in many fibers had characteristics of both fast and slow isozymes. Then, beginning by about 15 weeks, there was a trend toward a loss of properties associated with slow myosin, but a slow type of myosin continued to be present until at least 24 weeks, and in lesser amounts for as long as 70 weeks. DISCUSSION Significance
of the Immunocytochemical
Observations
The preferential distribution of fast and slow myosin into different fiber types characteristic of the rat diaphragm is no longer evident following denervation. By 8 weeks, a substantial number of fibers react with antibodies against fast as well as slow myosin, and the same fibers have both alkali- and acid-stable ATPase activity. This can be interpreted to mean that fast and slow isozymes coexist within individual muscle fibers or that a distinctive myosin(s) is present which has properties of both isozymes. The present results do not exclude either of these possibilities, but there is a precedent for the second interpretation. We showed previously that in developing skeletal muscles there was a uniform response by all fibers to anti-fast and anti-slow myosin (15, 16). In developing muscles, there are light chains which are equivalent to those of adult fast myosin and also
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ISOZYMES
IN DENERVATED
MUSCLE
341
small amounts of slow light chain (16,33,53), and, in the rat, an embryonic light chain which is not present in adult muscles (59). The heavy chain also appears to be different from that of adult myosin in both the rat (60, 61) and the chicken (3, 45). It was recently reported that there is a normal light chain pattern consisting of fast and slow forms in the denervated rat diaphragm at 2 months, but that at 6 months, there is, as in embryonic muscle, very little light chain or heavy chain characteristic of slow myosin (6, 7). Unlike the developing muscle, however, there is apparently no embryonic light chain (8). The possibility exists that there is, in denervated muscle, yet another distinctive myosin, but this cannot be resolved without chemical analysis. Nevertheless, the immunocytochemical observations provide evidence for a slow type of isozyme. It is unlikely that this is the original myosin that was present in the “slow fibers” at the time of denervation. A substantial amount of myosin observed in the denervated diaphragm probably represents newly synthesized protein. Information on the turnover rate of skeletal muscle myosin is limited, and published data vary greatly (35, 54). Some of the discrepancy may reflect technical problems in measuring protein turnover. For example, isotope data often include values for reutilization of labeled precursor, with the result that the half-life may be overestimated. In addition, the balance between synthesis and degradation may vary, depending on the physiologic state (18, 40). Recently published data based on turnover rates for mixed muscle proteins suggest that the half-life of myosin is 8 days for the diaphragm and 15 days for the gastrocnemius and plantaris of the rat (39), and this is consistent with a half-life value of 15 days obtained for the myosin heavy chain in the rat longissimus dorsi (Dr. Radovan Zak, Department of Medicine, University of Chicago, personal communication). The breakdown of muscle protein is greatly accelerated by denervation ( 17, 19, 20, 57). Using the rat diaphragm, it was calculated that the rate constant for protein degradation is more than doubled by 3 days after denervation (57). Hence, it is likely that the myosin present in this muscle at the time of denervation would no longer be observed by 8 weeks or at the even longer intervals examined in this study. There is also a measurable increase in protein synthesis after denervation (17, 19-21). In addition, our observations indicate that denervated fibers have synthesized a new type of myosin. Because fast and slow myosins are usually associated with separate fiber types in the diaphragm, the dual staining pattern implies that these fibers are capable of synthesizing an isozyme other than that which is normally present, and that may be unique to the altered physiologic state.
342
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Functional
HOBBS
Implications
As indicated earlier, there is general agreement that the speed of muscle contraction is slowed after denervation. Despite this, some investigators reported that a shift in myosin composition to a pattern in which a fast type of isozyme predominates can be induced by denervation of adult, developing, or regenerating skeletal muscles (6, 7, 34, 43). It is possible, however, as discussed above, that the myosin in the experimentally altered or undifferentiated muscles consists of molecules which are not equivalent to either the fast or slow adult forms. It is also possible that speed of contraction is unrelated to the type of myosin. Isometric twitch contraction time correlates well with molecular properties of the membrane systems (28). Moreover, the sarcoplasmic membranes of the now slowly contracting muscles become disorganized after denervation of either adult (11) or developing muscles (48). In addition, calcium transport activity of the sarcoplasmic reticulum is decreased in denervated muscle (37, 49). In the absence of a precisely ordered membrane system, it is likely that excitationcontraction coupling would be impaired and that speed of contraction might be slow regardless of the type of myofibillar protein present. Relationship
to the Nerve Supply
The extent to which the nervous system influences myosin synthesis is not certain. Data on ATPase activity and gel electrophoresis patterns and also on immunochemical properties of developing or denervated muscles often have been interpreted to indicate that fast but not slow myosin can be synthesized in the absence of the nervous system (6, 30, 42, 43). Slow myosin is said to be present in developing muscles only in those cells which have been contacted by a motoneuron (44). Nevertheless, slow myosin light chains are evident in muscle cell cultures, where the nerve supply is absent (33, 52) and also in muscles which have been surgically denervated (6). Cultured cells, moreover, react strongly with antibody specific for slow myosin (5). Evidence that the myosin of embryonic muscle fibers may not be equivalent to either fast or slow adult myosin makes it likely that both adult isozymes require an adult nervous system. The presence of a distinctive “embryonic” myosin in developing skeletal muscles of both the rat and chicken (16, 59, 60) and also of another distinctive “neonatal” isozyme which precedes synthesis of adult myosin [(61); P. A. Benfield, G. W a11er, and S. Lowey, in preparation]., may be related to the state of differentiation of the nerve supply. Even though neuromuscular contacts exist at stages where embryonic or neonatal myosin predominate, the structural configuration differs significantly from that of the adult neuromuscular junction. Muscle fibers receive a “polyneuronal”
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ISOZYMES
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innervation pattern at these stages ( 1, 4) and, in addition, many ultrastructural features of the adult neuromuscular junction are not yet apparent (Gauthier, unpublished observations). We observed previously that, coincident with polyneuronal innervation, properties of fast and slow myosin are apparent in all muscle fibers (15, 16). In the present paper, we have shown that when the nerve supply to the rat diaphragm is removed, myosin(s) having immunological cross reactivity with adult fast and slow myosin continues to be synthesized by the muscle fibers. We conclude, regardless of whether the newly synthesized myosin is a fast or a slow, or a unique isozyme, that a fully differentiated neuromuscular relationship is required for the selective pattern of distribution of fast and slow myosin isozymes characteristic of normal adult skeletal muscles. REFERENCES I. ATSUMI,S. 1977. Development of neuromuscular junctions of fast and slow muscles in the chick embryo: a light and electron microscopic study. J. Neurocyrol. 6, 691-709. 2. BARANY, M., AND R. I. CLOSE. 1971. The transformation of myosin in cross-innervated rat muscles. J. Physiol. (London) 213: 455-474. 3. BENFIELD, P. A., S. LOWEY, AND D. D. LEBLANC. 1981. Fractionation and characterization of myosins from embryonic chicken pectoralis muscle. Biophys. J. 33: 243a. 4. BENNETT, M. R., AND A. G. PE~IGREW. 1974. The formation of synapses in striated muscle during development. J. Physiol. (London) 241: 51 S-545. 5. CANTINI, M., S. SARTORE, AND W. S. SCHIAFFINO. 1980. Myosin types in cultured muscle cells. J. Cell Biol. 85: 903-909. 6. CARRARO, U., C. CATANI, AND D. BIRAL. 1979. Selective maintenance of neurotrophitally regulated proteins in denervated rat diaphragm. Exp. Neurol. 63: 468-475. 7. CARRARO, U., C. CATANI, AND L. DALL.A LIBERA. 1981. Myosin light and heavy chains in rat gastrocnemius and diaphragm muscles after chronic denervation or reinnervation. Exp.
Neurol.
72: 40 1-4 12.
8. CARRARO, U., C. CATANI, L. DALLA LIBERA, M. VASCON, AND G. ZANELLA. 1981. Differential distribution of tropomyosin subunits in fast and slow rat muscles and its changes in long-term denervated hemidiaphragm. FEBS Let?. 128: 233-236. 9. FENG, T. P., AND D. X. Lu. 1965. New lights on the phenomenon of transient hypertrophy in the denervated hemidiaphragm of the rat. Sci. Sin. 19: 1772-1784. 10. GAUTHIER, G. F. 1967. On the localization of sarcotubular ATPase activity in mammalian skeletal muscle. Histochemie 11: 97- 111. 11. GAUTHIER, G. F., AND R. A. DUNN. 1973. Ultrastructural and cytochemical features of mammalian skeletal muscle fibres following denervation. J. Cell Sci. 12: 525-547. 12. GAUTHIER, G. F., AND S. LOWEY. 1977. Polymorphism of myosin among skeletal muscle fiber types. J. Cell Biol. 74: 760-779. 13. GAUTHIER, G. F., AND S. LOWEY. 1979. Distribution of myosin isoenzymes among skeletal muscle fiber types. J. Cell Biol. 81: 10-25. 14. GAUTHIER, G. F., AND S. F. SCHAEFFER. 1974. Ultrastructural and cytochemical manifestations of protein synthesis in the peripheral sarcoplasm of denervated and newborn skeletal muscle fibres. J. Cell Sci. 14: 113- 137. 15. GAUTHIER, G. F., S. LOWEY, AND A. W. HOBBS. 1978. Fast and slow myosin in developing muscle fibres. Nature (London) 274: 25-29.
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16. GAUTHIER, G. F., S. LOWEY, P. A. BENFIELD, AND A. W. HOBBS. 1982. Distribution and properties of myosin isozymes in developing avian and mammalian skeletal muscle fibers. J. Cell Biol. 92. 17. GOLDBERG, A. L. 1969. Protein turnover in skeletal muscle. II. Effects of denervation and cortisone on protein catabolism in skeletal muscle. J. Biol. Chem. 244: 3223-3229. 18. GOLDBERG, A. L., AND J. F. DICE. 1974. Intracellular protein degradation in mammalian and bacterial cells. Annu. Rev. Biochem. 43: 835-869. 19. GOLDBERG, A. L., C. JABLECKI, AND J. B. LI. 1974. Effects of use and disuse on amino acid transport and protein turnover in muscle. Ann. N. Y. Acud. Sci. 228: 190-201. 20. GOLDSPINK, D. F. 1976. The effects of denervation on protein turnover of rat skeletal muscle. Biochem. J. 156: 71-80. 21. GOLDSPINK, D. F. 1980. The influence of contractile activity and the nerve supply on muscle size and protein turnover. Pages 525-539, in D. PETTE, Ed., Plasticity of Muscle. W. de Gruyter, New York/Berlin. 22. GOTTSCHALL, J., AND H. GRUBER. 1977. The accessory phrenic nerve in the rat. Anat. Embryol.
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23. GUTMANN, E., J. MELICHNA, AND I. SYROV~. 1972. Contraction properties and ATPase activity in fast and slow muscle of the rat during denervation. Exp. Neural. 36: 488497. 24. GUTH, L. 1973. Fact and artifact in the histochemical procedure for myofibrillar ATPase. Exp.
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