Eur. J. Biochem. 193,623 - 628 (1 990) 0FEBS 1990
Myosin heavy chain isoform composition in striated muscle after denervation and self-reinnervation Anna JAKUBIEC-PUKA', Jolanta, KORDOWSKA ', Claudia CATANI' and Ugo CARRAROZ
Laboratory of Protein Metabolism, Nencki Institute of Experimental Biology, Warsaw, Poland
' Consiglio Nazionale delle Ricerche, Unit for Muscle Biology and Physiopathology, Institute of General Pathology, Padova, Italy (Received December 4, 1989/July 9, 1990) - EJB 89 1453
The total content of myosin heavy chains (MHC) and their isoform pattern were studied by biochemical methods in the slow-twitch (soleus) and fast-twitch (extensor digitorum longus) muscles of adult rat during atrophy after denervation and recovery after self-reinnervation. The pattern of fibre types, in terms of ultrastructure, was studied in parallel. After denervation, total MHC content decreased sooner in the slow-twitch muscle than in the fast-twitch. The ratio of MHC-1 and the MHC-2B isoforms to the MHC-2A isoform decreased in the slow and the fast denervated muscles, respectively. After reinnervation of the slow muscle, the normal pattern of MHC recovered within 10 days and the type 1 isoform increased above the normal. In the reinnervated fast muscle, the 2B/2A isoform ratio continued to decrease. Traces of the embryonic MHC isoform, identified by immunochemistry, were found in both denervated and reinnervated slow and fast muscles. A shift in fibre types was similar to that found in the MHC isoforms. Within 2 months of recovery a tendency to normalization was observed. The results show that (a) MHC-2B isoform and the morphological characteristics of the 2B-type muscle fibres are susceptible to lack of innervation, similar to those of type 1, (b) during muscle recovery induced by reinnervation the MHC isoforms and muscle fibres shift transiently to type 1 in the soleus and to type 2A in the extensor digitorum longus muscles, and (c) the embryonic isoform of MHC may appear in the adult skeletal muscles if innervation is disturbed.
During the last decade, plasticity of the striated muscle has been intensively studied (results are summarized in [l 31) and the general rules of denervation atrophy and of reinnervation recovery are well known. However, it is not fully understood how in the mature striated muscle a disturbed innervation influences remodelling of the contractile apparatus. We would like to stress that in the slow leg muscle (in adult rats) atrophying after denervation, the myosin filaments disappear before the actin filaments [4]; simultaneously the muscle content of myosin heavy chains (MHC) decreases considerably. After reinnervation, when the muscle begins to recover, its MHC content increases rapidly in parallel with recovery of the correct proportion and arrangement of thin and thick filaments [4, 51. Nevertheless, it is not known how the total content of MHC changes in the denervated and reinnervated fast muscle. Myosin polymorphism is characteristic of different types of skeletal muscle fibres. The isoform composition of myosin may become adapted to modifications of functional requirements (for review see [6, 71). In chronically denervated mixed muscles (2 - 6 months after the operation) expression of the fast isoform of myosin prevails, while after a long-term reinnervation (6 months of recovery) myosin composition is simiCorrespondence to A. Jakubiec-Puka, Nencki Institute of Experimental Biology, ul. Pasteura 3, PL-02-093 Warszawa, Poland Abbreviations. EDL, extensor digitorum longus muscle, MHC, myosin heavy chains; MLC, myosin light chains; SO, slow-twitch oxidative muscle fibres; FOG, fast-twitch oxidative-glycolytic muscle fibres; FG, fast-twitch glycolytic muscle fibres.
lar to that of the normal muscle [8, 91. On the other hand, it is not clear how the myosin isoforms change in the shortterm denervated muscle and whether any lack of innervation equally influences the expression of fast MHC isoforms. It is not known which isoforms of myosin are expressed in the adult muscle soon after reinnervation: whether it is (a) its own type, (b) another of the adult skeletal muscle fibres, or (c) the isoform(s) characteristic of the developing muscle. The latter is especially interesting, since the embryonic and neonatal myosins have been detected by immunohistochemistry in fibres of the denervated adult muscle [lo]. The present work was performed to describe how the MHC isoforms shift during short-term denervation and also immediately after reinnervation of the fast- and slow-contracting skeletal muscle. The study by biochemical methods were carried out in parallel with fibre-type characterization by ultrastructural criteria. Preliminary results have been presented [ll]. MATERIALS AND METHODS Experiments on rats
The soleus and extensor digitorum longus (EDL) muscles of female albino Wistar rats 2.5-3 months old (150-190 g) were studied. Denervation atrophy or recovery of the denervated muscle following reinnervation was produced under ether anaesthesia by, respectively, cutting or crushing the sciatic nerve, as previously described in detail [12, 131. After crushing the sciatic nerve, atrophy of the rat leg muscles
624 progresses for about 15 days at the same rate as that produced by cutting the nerve. After this time, recovery of the muscle begins owing to reinnervation [5, 131. Full recovery from denervation atrophy of most muscle characteristics (including normal histochemical pattern without type-grouping) takes about 4 months; the muscle then looks quite normal [12]. The muscles, after cutting or crushing the sciatic nerve, are thereafter called denervated and reinnervated, respectively. The experimental timing is expressed in number of days following the operation. Experimental soleus and EDL muscles were taken from the same leg. Muscles from the contralateral leg and from legs of nonoperated animals of the same group served as controls. In selected experiments leg muscles of 3-day-old rats were used as controls. Muscles from at least four animals were used in each experiment for biochemical analysis, and two animals for ultrastructural study. Preparation of myofihrils At the stated post-operative time, myofibrils were separated as described [4]. Either purified myofibrils or crude myofibrils were used. Crude myofibrils from both experimental and contralateral legs were obtained under exactly the same conditions, with a carefully controlled and monitored solvent volume during procedure, and were studied in parallel. Albumin was added to crude myofibrils as an internal quantitative standard. Purified myofibrils were obtained from crude myofibrils by washing several times with a low-ionic-strength buffer and filtering through layers of cheese cloth. The total content of protein in myofibrils was estimated by the method of Lowry et al. [14].
a
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Electrophoretic unalysis of M H C isqforms Purified myofibrils were used. Isoforms of MHC were separated by SDSIPAGE according to Carraro and Catani [16], as modified by Danieli Betto et al. [17]. Individual isoforms of MHC (1,2A and 2B) were distinguished according to electrophoretic mobility. Relative amounts of MHC isoforms were determined by comparing the degree of intensity of staining with Coomassie blue of the electrophoretic bands using a Shimadzu densitometer, model ‘3-930. Electrophoretic analysis qf M L C isoforms Purified myofibrils were used. Myosin light chains (MLC) were separated by two-dimensional gel electrophoresis as described [IS].
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” Estimation of the total M H C muscle content The total MHC content in the muscle was evaluated by estimating the total MHC in crude myofibrils, as described in detail [4, 51. Myofibrillar proteins were separated by SDSi PAGE according to Weber and Osborn [15], by subjecting samples containing 20-40 pg protein to 6-89’0 gels. After separation and staining with Coomassie blue, individual bands were cut out and eluted from the gels; the amount of protein was estimated colorimetrically. The intensity of protein staining was proportional up to 30 pg protein within one band. Content of the total MHC was referred to the albumin eluted from the same gel, then compared with the MHC content from other gels. Gels run in the same electrophoresis and stained and destained together were exclusively used for comparison. The data from each experiment was derived from analysis of at least four electrophoretic runs.
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201
Tlme after operation ldoys)
Fig. 1. Changes in mu.vcle muss (‘a,h ) , total M H C eontent (c, d j and M H C pattern (e, f ) in the soleus and EDL muscles. D, denervated (after cutting the nerve). R , reinnervated (after crushing the nerve). Electrophoregrams presented in Fig. 2 were scanned as described in Materials and Methods. The amounts of‘ the individual MHC isoforms are shown (e, f). Thc sum of all MHC in the sample was taken as 100%. Each point represents the result from at least four muscles. The bars indicate mean i SD in (a) and (b), and mean f SEM in (c) and (d)
Analysis of the embryonic M H C isoform Crude myofibrils were used. For identification of the embryonic MHC, myofibril proteins were separated by SDS/ PAGE and MHC were electrophoretically transferred to Hybond TM-C hybridization-transfer membrane (Amersham), as described [19].The blots were stained with the 0.2% solution of Ponceau red [20] and photographed. The blots were incubated with some purified monoclonal antibodies BFG6 for embryonic MHC [21], then with the affinity-purified anti-(mouse IgG) antibodies, conjugated with alkaline phosphatase (Kirkegard and Perry Laboratories Inc, USA). Blots
625
Fig. 2. SDSjPAGE of’MHC. (a) Soleus and (b) EDL. Lanes 1 - 5 , control myofibrils from muscles of non-operated animals from the same group as corresponding experimental myofibrils. Lanes 6 - 16, experimental myofibrils, denervated (after cutting the nerve) and reinnervated (after crushing the nerve). The experimental timing is expressed in number of days following the operation. Lane 1, control of lanes 6, 8, 10 and 12; lane 2, control of lanes 7 and 11 ; lane 3, control of lanes 14 and 15; lane 4. control of lanes 9 and 13; lane 5, control of lane 16. Lane 6,14-day denervated; lane 7,19-day denervated; lane 8,21 -day denervated; lane 9,24-day denervated; lane 10,19-day reinnervated, experiment 1; lane 11, 19-day reinnervated, experiment 2; lane 12, 21-day reinnervated; lane 13, 24-day reinnervated; lane 14, 26-day reinnervated; lane 15, 33-day reinnervated; lane 16, 61-day reinnervated
were developed as described [22], taking a photograph every 5 min during the phosphatase reaction.
Analysis of muscle fibre types To investigate the ultrastructure, samples were randomly selected from the central part of muscles and embedded in Epon. The type of muscle fibre was evaluated according to the ultrastructural characteristic of fibres [23, 241: (a) the Zline thickness; (b) the structure of the M-line and (c) that of sarcoplasmic reticulum; (d) the amount and shape of mitochondria, as described in [25]. Fibres were classified into three types [26]: a slow-twitch oxidative (SO), a fast-twitch oxidative-glycolytic (FOG), and a fast-twitch glycolytic (FG), which, as is well known, correspond in general to types 1, 2A and 2B [27], respectively. The types of all the fibres were evaluated under identical conditions.
RESULTS
Muscle mass and total MHC content The change in mass of the studied muscles was taken as a criterion of muscle atrophy and recovery. Fig. 1a and b shows that after one week the progress of atrophy is greater in the soleus than in the EDL denervated muscle. One week later the degree of atrophy became the same in both. After that time the mass of muscles of animals subjected to the sciatic nerve crushing began increasing due to reinnervation, while the mass of muscle of animals subjected to nerve cutting continued to decrease. After 2 months the mass of reinnervated muscle was comparable to that of the contralateral muscles. The total MHC content of experimental muscles, expressed as percentage of the contralateral muscle, is also shown (Fig. 1c and d). In the denervated EDL muscle (either by cutting or crushing the nerve) the total MHC content decreased to about 40% of the control value within 17 days. It continued to decrease as atrophy progressed in the muscle denervated by cutting the nerve (Fig. 1d). The rate of decrease of the total MHC content was similar to that of the muscle mass (compare Fig. 1 b and d). On the other hand, in the denervated soleus muscle the content of MHC decreased much
more relative to the muscle mass as early as in the second week after the operation of either cutting or crushing the nerve (compare Fig. 1a and c). The dates reported here are in full agreement with those previously published [4, 5, 131. During the third week after crushing the nerve, the total MHC muscle content stopped decreasing and began increasing. The total MHC content in the soleus increased much more rapidly than that in the EDL muscle (compare Fig. 1c and d), probably due to the fact that the degree of MHC loss was much higher in the soleus than in the EDL muscle just before reinnervation (17 days after operation the figures were 23 f 2% and 41 2% for the soleus and EDL, respectively; mean SEM of contralateral muscle). The results point to the lower dynamics of changes in the total MHC content in the EDL from those in the soleus muscle.
MH C isoforms SDSjPAGE with high glycerol concentration [17] allows for separation of MHC isoforins (Fig. 2). The MHC of the control soleus muscle contained 24% (19-27%) type 2A isoform and 76% (70-81%) type 1: traces of 2B isoform (0-3%) were also found. After denervation, changes in the proportion of the MHC isoforms appeared: the MHC-2A increased, while the MHC-1 decreased (Figs 1e and 2a). After reinnervation the MHC isoforins recovered to the control pattern within about 10 days and type 1 prevailed: it increased up to 94% of the total content of MHC. This higher-thannormal muscle proportion of the MHC-1 persisted for several weeks. N o sooner than 2 months after the operation, a tendency to normalization was observed (Figs 1e and 2a). 26 days after crushing the nerve, SDSjPAGE of MHC showed a band with the electrophoretic mobility of the 2B isoform, representing 8% of the total MHC in samples. The MHC of the control EDL muscle contained about 31% (24-36%) 2A isoform, about 67% (64 to 72%) type 2B and about 2% (0-3%) type 1. After denervation, the proportion of the 2A isoform increased to 41 - 52% of the total MHC content, whereas the proportion of the 2B isoform decreased to about 45% (Figs 1 f and 2b). During recovery of the EDL muscle after reinnervation, the normal pattern of MHC did not return, because the 2A isoform continued to
626
Fig. 3. Two-dimensional gel electrophoresis of MLC. (a) Soleus and (b) EDL on the 26 days after crushing the nerve. 1s and 2s: MLC characteristic for slow type myosin. IF, 2F and 3F: MLC characteristic for fast type myosin. The spots on the right of the marked MLC correspond to either those phosphorylated (2F) or those deaminated (IS, 1F). The spots on the lower-right of 1s and 2 s correspond to 1F and 2F, respectively. Thc assumed position of the embryonic MLC is indicated by an arrow
increase; the 2B isoform decreased to about 37% of the total MHC content. This pattern persisted for up to at least 2 months after the operation (Figs 1f and 2 b). The proportion of the MHC-1 isoform was maintained at the control level during the processes of both atrophy and recovery of the EDL muscle.
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Embryonic myosin A search was made for embryonic myosin in the experimental muscles by two-dimensional SDSjPAGE of the myosin light chain (MLC) and by immunoblotting of the MHC. Fig. 3 shows the MLC pattern in the muscles 27 days after crushing the nerve. The MLC pattern of the soleus was dominated by the slow types, while that of the EDL contained the three light chains peculiar to fast muscles. The embryonic isoform of MLC was not detected. However, when the MHC were transferred to nitrocellulose paper and reacted with a monoclonal antibody specific to the embryonic MHC, one band in the MHC patterns of the soleus and EDL muscles, after cutting and crushing the nerve, gave obvious positive results. The reaction, which was not detectable with the MHC of the control muscles of adult animals, was very intensive with MHC of muscle of 3-day-old rats (Fig. 4). These results demonstrated a presence of the embryonic isoform of MHC in the denervated and recovering slow and fast muscles of adult rats. Tjye of fibres by ultrastructural criteria
In parallel with studies of MHC, the fibre type was evaluated by ultrastructural criteria and the results are presented in Table 1. In the control soleus muscle, 90% of fibres showed characteristics of the SO type, 9 % of the FOG type and only a few fibres (about 1%) of both those types. In the control EDL muscle 61O h of fibres showed characteristics of the FG type, 33% FOG type and 2% SO type. Some fibres showed characteristics of two types simultaneously: i.e. about 3% of fibres of the F G and FOG types, and about 1% of SO and FOG types. In the denervated soleus muscle, a proportion of the SO fibres decreased, while that of the FOG type seemed to remain unchanged. Indeed, numerous fibres appeared to show characteristics of types SO and FOG. After reinnervation (4 6 weeks after crushing the nerve) the proportion of both SO and FOG fibres decreased compared with the denervated atrophying muscle, while the proportion of fibres simultaneously showing SO and FOG characteristics increased. Several fibres could not be defined by the ultrastructural criteria; they resembled those of the developing muscle since they
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Fig. 4. Immunohlots of MHCfrom experimental muscles with a monoclonal anti- (ernhryonic M H C ) antibody. (a) Blot stained with Ponceau red immediately after transfer; (b, c) blot after incubation with monoclonal antibody BFG6 for embryonic MHC, then with anti-(mouse IgG) antibodies conjugated with alkaline phosphatase. The photographs were taken after 10 min in (b) and after 35 min in (c) of development. MHC of myofibrils from (lane 1) leg muscles of the 3day-old rats, (lane 2) denervated soleus, 21 days after cutting the nerve, (lane 3) reinnervated soleus, 21 days after crushing the nerve, (lane 4) denervated EDL, 21 days after cutting the nerve, (lane 5 ) reinnervated EDL, 21 days after crushing the nerve, (lane 6 ) control EDL, from non-operated animals, (lane 7) control soleus, from nonoperated animals, and (lane 8) as lane 1
contained loosely arranged myofibrils surrounded by sarcoplasm rich in polysomes. In the denervated EDL muscle the proportion of FG fibres decreased and several fibres showed simultaneous characteristics of the types FOG/FG or FOG/SO. After reinnervation of the EDL muscle (4 - 6 weeks after crushing the nerve) the composition of fibres was, in general, similar to that in the denervated muscle, with one exception: fibres of the pure FG type were transiently absent, but all fibres showed FOG characteristics, alone or together with the FG or SO types. As late as 2 months after the operation the tendency to normalization of fibre types appeared in both the soleus and the EDL muscles. The results showed that in both fast and slow muscles the pattern of the fibre types was changed during the course of denervation atrophy and of recovery after reinnervation;
627 Table 1. Fibre composition in denervnted and reinnervated muscles The type of muscle fibres was evaluated according to the ultrastructural characteristic of fibres, as described in Materials and Methods. The fibres were classified into types SO, FOG and FG, and fibres showing characteristics of two types simultaneously, SO/FOG and FOG/FG. D) denervated (after cutting the sciatic nerve); (R) reinnervated (after crushing the sciatic nerve). The total number of fibres identified was taken as 100% Muscle
Time after operation
Total number of fibres identified
Proportion of total fibres
so
SO/FOG
83 70 56 69 87
90 57 27 61 82
1 21 39 26 16
9 9
64 58 66 84 63
2
1 15 13 21 2
33 43 45 29 30
____
several fibres appeared with two-type characteristics. At each stage, shifts in fibre types were in good agreement with those observed in the MHC isoforms.
DISCUSSION In the present study, we have shown that in denervated muscles there are clear changes in the proportion of the MHC isoforms which are peculiar to the soleus and EDL normal adult muscles. Indeed, the proportion of isoforms 1 and 2B decreased in the denervated soleus and EDL muscles, respectively, while in both muscles the proportion of the 2A isoform increased (Figs. l e , f, and 2). As the content of the slow myosin subunits decreased more than the fast myosin subunits, this confirmed a stricter dependence of slow myosin expression on innervation than that of fast myosin [28 - 301. To our knowledge this is the first paper to conclusively show that MHC-2B is much more susceptible to lack of innervation than is MHC-2A. This very interesting phenomenon has been confirmed by Schiaffino (personal communication). D'Albis and coworkers [31] found a decreased proportion of myosin specific for the 2B fibres, while that of the 2A fibres was increased in denervated muscle regenerating after cardiotoxin injury. Thus, it seems that MHC-2B is as dependent on innervation as type 1. One could assume that the relative increase of MHC-2A in both the denervated EDL and soleus muscles might come about as an effect of higher stability of the 2A isoform compared with the other isoforms in the denervated muscle, owing to its lower sensitivity to proteolytic degradation. On the other hand, the imbalance of isoforms could stem from their changed synthesis. The latter interpretation seems to be more probable, because the shift in the MHC pattern was correlated with the transformation of the ultrastructural characteristics of the fibre types. It is also possible that a relative increase in MHC-2A is in fact an increase in the MHC-2X subunit, comigrating with MHC-2A, as was found in the stimulated denervated muscle [32]. This paper indicates a particular feature of both the EDL and the soleus denervated muscles: in a single fibre, characteristics of two types can be detected simultaneously, and one of
FOG
__
FOG/FG
FG
Undefined
13 32 10
2 3 2 3 28 39 33 32
61 14 3 17 36
these is the 2A (FOG) type. The number of pure 2A fibres increased only slightly or even decreased (Table 1). Therefore, it seems that the relative increase in the MHC-2A isoform content in the myosin of denervated muscles, could result from fibre transformation rather than from preferential atrophy of type 1 (SO) and type 2B (FG) fibres. Transformation of fibres into the 2A type, observed in the denervated fast and slow muscles, suggests that innervation by the more specialised motor unit, fast or slow, suppresses an expression of proteins corresponding to the 2A type; lack of innervation allows for their expression. The same explanation may be given for expression of embryonic MHC in denervated muscle. After reinnervation of the slow soleus muscle, the type 1 isoform of MHC recovered rapidly then overproduction of this isoform took place (Figs 1 e and 2a). On the other hand, in the reinnervated EDL muscle, the 2A isoform of MHC still prevailed, while the 2B isoform continued to decrease (Figs 1f and 2b). Thus, in both recovering muscles, the proportion of MHC shifts to the slower isoforms. This is in agreement with the transformation of fibre types in a more oxidative pattern in reinnervated muscles (Table 1). We can interpret these phenomena by considering the recovering muscle as a muscle hypertrophying following increased activity. In such a case, muscle metabolism and myosin isoforms shift to a more oxidative pattern [6, 33, 341. Transformation of fibres following innervation by foreign motor neurons or following a transient double innervation seems least probable, taking into account the fact that in the reinnervated muscle, after appropriate nerve crushing, a type-grouping has not appeared (results not shown) [12]. The authors wish to thank Prof. S. Schiaffino for his kind gift of the monoclonal antibodies for embryonic MHC, and Mrs. H. Chomontowska and Mr. S. Belluco for their skilful technical assistance. We are also indebted to Prof. S. Schiaffino, Dr D. B i d , Dr E. Mussini and Dr L. Dalla Libera for discussing the results. This work was financed by the Polish Academy of Sciences (grant no. CPBP 04.01.3.07), by the Italian Consiglio Nazionale delle Ricerclze (grant to the Unit for Muscle Biology and Physiopathology) by the P r o p t t o Finulizzuto Tecnologie Biomediche c Sanitarie and by the Italian Ministero della Puhhlicu Istruzione 609'0 to Prof. U. Carraro.
628 REFERENCES 1. Pette, D., cd. (1980) Plasticity of muscle. Proceedings ? f a Syni-
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Myosin heavy chain isoform composition in striated muscle after denervation and self-reinnervation Anna JAKUBIEC-PUKA', Jolanta, KORDOWSKA ', Claudia CATANI' and Ugo CARRAROZ
Laboratory of Protein Metabolism, Nencki Institute of Experimental Biology, Warsaw, Poland
' Consiglio Nazionale delle Ricerche, Unit for Muscle Biology and Physiopathology, Institute of General Pathology, Padova, Italy (Received December 4, 1989/July 9, 1990) - EJB 89 1453
The total content of myosin heavy chains (MHC) and their isoform pattern were studied by biochemical methods in the slow-twitch (soleus) and fast-twitch (extensor digitorum longus) muscles of adult rat during atrophy after denervation and recovery after self-reinnervation. The pattern of fibre types, in terms of ultrastructure, was studied in parallel. After denervation, total MHC content decreased sooner in the slow-twitch muscle than in the fast-twitch. The ratio of MHC-1 and the MHC-2B isoforms to the MHC-2A isoform decreased in the slow and the fast denervated muscles, respectively. After reinnervation of the slow muscle, the normal pattern of MHC recovered within 10 days and the type 1 isoform increased above the normal. In the reinnervated fast muscle, the 2B/2A isoform ratio continued to decrease. Traces of the embryonic MHC isoform, identified by immunochemistry, were found in both denervated and reinnervated slow and fast muscles. A shift in fibre types was similar to that found in the MHC isoforms. Within 2 months of recovery a tendency to normalization was observed. The results show that (a) MHC-2B isoform and the morphological characteristics of the 2B-type muscle fibres are susceptible to lack of innervation, similar to those of type 1, (b) during muscle recovery induced by reinnervation the MHC isoforms and muscle fibres shift transiently to type 1 in the soleus and to type 2A in the extensor digitorum longus muscles, and (c) the embryonic isoform of MHC may appear in the adult skeletal muscles if innervation is disturbed.
During the last decade, plasticity of the striated muscle has been intensively studied (results are summarized in [l 31) and the general rules of denervation atrophy and of reinnervation recovery are well known. However, it is not fully understood how in the mature striated muscle a disturbed innervation influences remodelling of the contractile apparatus. We would like to stress that in the slow leg muscle (in adult rats) atrophying after denervation, the myosin filaments disappear before the actin filaments [4]; simultaneously the muscle content of myosin heavy chains (MHC) decreases considerably. After reinnervation, when the muscle begins to recover, its MHC content increases rapidly in parallel with recovery of the correct proportion and arrangement of thin and thick filaments [4, 51. Nevertheless, it is not known how the total content of MHC changes in the denervated and reinnervated fast muscle. Myosin polymorphism is characteristic of different types of skeletal muscle fibres. The isoform composition of myosin may become adapted to modifications of functional requirements (for review see [6, 71). In chronically denervated mixed muscles (2 - 6 months after the operation) expression of the fast isoform of myosin prevails, while after a long-term reinnervation (6 months of recovery) myosin composition is simiCorrespondence to A. Jakubiec-Puka, Nencki Institute of Experimental Biology, ul. Pasteura 3, PL-02-093 Warszawa, Poland Abbreviations. EDL, extensor digitorum longus muscle, MHC, myosin heavy chains; MLC, myosin light chains; SO, slow-twitch oxidative muscle fibres; FOG, fast-twitch oxidative-glycolytic muscle fibres; FG, fast-twitch glycolytic muscle fibres.
lar to that of the normal muscle [8, 91. On the other hand, it is not clear how the myosin isoforms change in the shortterm denervated muscle and whether any lack of innervation equally influences the expression of fast MHC isoforms. It is not known which isoforms of myosin are expressed in the adult muscle soon after reinnervation: whether it is (a) its own type, (b) another of the adult skeletal muscle fibres, or (c) the isoform(s) characteristic of the developing muscle. The latter is especially interesting, since the embryonic and neonatal myosins have been detected by immunohistochemistry in fibres of the denervated adult muscle [lo]. The present work was performed to describe how the MHC isoforms shift during short-term denervation and also immediately after reinnervation of the fast- and slow-contracting skeletal muscle. The study by biochemical methods were carried out in parallel with fibre-type characterization by ultrastructural criteria. Preliminary results have been presented [ll]. MATERIALS AND METHODS Experiments on rats
The soleus and extensor digitorum longus (EDL) muscles of female albino Wistar rats 2.5-3 months old (150-190 g) were studied. Denervation atrophy or recovery of the denervated muscle following reinnervation was produced under ether anaesthesia by, respectively, cutting or crushing the sciatic nerve, as previously described in detail [12, 131. After crushing the sciatic nerve, atrophy of the rat leg muscles
624 progresses for about 15 days at the same rate as that produced by cutting the nerve. After this time, recovery of the muscle begins owing to reinnervation [5, 131. Full recovery from denervation atrophy of most muscle characteristics (including normal histochemical pattern without type-grouping) takes about 4 months; the muscle then looks quite normal [12]. The muscles, after cutting or crushing the sciatic nerve, are thereafter called denervated and reinnervated, respectively. The experimental timing is expressed in number of days following the operation. Experimental soleus and EDL muscles were taken from the same leg. Muscles from the contralateral leg and from legs of nonoperated animals of the same group served as controls. In selected experiments leg muscles of 3-day-old rats were used as controls. Muscles from at least four animals were used in each experiment for biochemical analysis, and two animals for ultrastructural study. Preparation of myofihrils At the stated post-operative time, myofibrils were separated as described [4]. Either purified myofibrils or crude myofibrils were used. Crude myofibrils from both experimental and contralateral legs were obtained under exactly the same conditions, with a carefully controlled and monitored solvent volume during procedure, and were studied in parallel. Albumin was added to crude myofibrils as an internal quantitative standard. Purified myofibrils were obtained from crude myofibrils by washing several times with a low-ionic-strength buffer and filtering through layers of cheese cloth. The total content of protein in myofibrils was estimated by the method of Lowry et al. [14].
a
b
SOL
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weiaht of muscle
Electrophoretic unalysis of M H C isqforms Purified myofibrils were used. Isoforms of MHC were separated by SDSIPAGE according to Carraro and Catani [16], as modified by Danieli Betto et al. [17]. Individual isoforms of MHC (1,2A and 2B) were distinguished according to electrophoretic mobility. Relative amounts of MHC isoforms were determined by comparing the degree of intensity of staining with Coomassie blue of the electrophoretic bands using a Shimadzu densitometer, model ‘3-930. Electrophoretic analysis qf M L C isoforms Purified myofibrils were used. Myosin light chains (MLC) were separated by two-dimensional gel electrophoresis as described [IS].
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MHC type 1 M m - 0 MHC type 2A H * - - o MHC type 2 8 c .b - - 0
” Estimation of the total M H C muscle content The total MHC content in the muscle was evaluated by estimating the total MHC in crude myofibrils, as described in detail [4, 51. Myofibrillar proteins were separated by SDSi PAGE according to Weber and Osborn [15], by subjecting samples containing 20-40 pg protein to 6-89’0 gels. After separation and staining with Coomassie blue, individual bands were cut out and eluted from the gels; the amount of protein was estimated colorimetrically. The intensity of protein staining was proportional up to 30 pg protein within one band. Content of the total MHC was referred to the albumin eluted from the same gel, then compared with the MHC content from other gels. Gels run in the same electrophoresis and stained and destained together were exclusively used for comparison. The data from each experiment was derived from analysis of at least four electrophoretic runs.
D H
I
201
Tlme after operation ldoys)
Fig. 1. Changes in mu.vcle muss (‘a,h ) , total M H C eontent (c, d j and M H C pattern (e, f ) in the soleus and EDL muscles. D, denervated (after cutting the nerve). R , reinnervated (after crushing the nerve). Electrophoregrams presented in Fig. 2 were scanned as described in Materials and Methods. The amounts of‘ the individual MHC isoforms are shown (e, f). Thc sum of all MHC in the sample was taken as 100%. Each point represents the result from at least four muscles. The bars indicate mean i SD in (a) and (b), and mean f SEM in (c) and (d)
Analysis of the embryonic M H C isoform Crude myofibrils were used. For identification of the embryonic MHC, myofibril proteins were separated by SDS/ PAGE and MHC were electrophoretically transferred to Hybond TM-C hybridization-transfer membrane (Amersham), as described [19].The blots were stained with the 0.2% solution of Ponceau red [20] and photographed. The blots were incubated with some purified monoclonal antibodies BFG6 for embryonic MHC [21], then with the affinity-purified anti-(mouse IgG) antibodies, conjugated with alkaline phosphatase (Kirkegard and Perry Laboratories Inc, USA). Blots
625
Fig. 2. SDSjPAGE of’MHC. (a) Soleus and (b) EDL. Lanes 1 - 5 , control myofibrils from muscles of non-operated animals from the same group as corresponding experimental myofibrils. Lanes 6 - 16, experimental myofibrils, denervated (after cutting the nerve) and reinnervated (after crushing the nerve). The experimental timing is expressed in number of days following the operation. Lane 1, control of lanes 6, 8, 10 and 12; lane 2, control of lanes 7 and 11 ; lane 3, control of lanes 14 and 15; lane 4. control of lanes 9 and 13; lane 5, control of lane 16. Lane 6,14-day denervated; lane 7,19-day denervated; lane 8,21 -day denervated; lane 9,24-day denervated; lane 10,19-day reinnervated, experiment 1; lane 11, 19-day reinnervated, experiment 2; lane 12, 21-day reinnervated; lane 13, 24-day reinnervated; lane 14, 26-day reinnervated; lane 15, 33-day reinnervated; lane 16, 61-day reinnervated
were developed as described [22], taking a photograph every 5 min during the phosphatase reaction.
Analysis of muscle fibre types To investigate the ultrastructure, samples were randomly selected from the central part of muscles and embedded in Epon. The type of muscle fibre was evaluated according to the ultrastructural characteristic of fibres [23, 241: (a) the Zline thickness; (b) the structure of the M-line and (c) that of sarcoplasmic reticulum; (d) the amount and shape of mitochondria, as described in [25]. Fibres were classified into three types [26]: a slow-twitch oxidative (SO), a fast-twitch oxidative-glycolytic (FOG), and a fast-twitch glycolytic (FG), which, as is well known, correspond in general to types 1, 2A and 2B [27], respectively. The types of all the fibres were evaluated under identical conditions.
RESULTS
Muscle mass and total MHC content The change in mass of the studied muscles was taken as a criterion of muscle atrophy and recovery. Fig. 1a and b shows that after one week the progress of atrophy is greater in the soleus than in the EDL denervated muscle. One week later the degree of atrophy became the same in both. After that time the mass of muscles of animals subjected to the sciatic nerve crushing began increasing due to reinnervation, while the mass of muscle of animals subjected to nerve cutting continued to decrease. After 2 months the mass of reinnervated muscle was comparable to that of the contralateral muscles. The total MHC content of experimental muscles, expressed as percentage of the contralateral muscle, is also shown (Fig. 1c and d). In the denervated EDL muscle (either by cutting or crushing the nerve) the total MHC content decreased to about 40% of the control value within 17 days. It continued to decrease as atrophy progressed in the muscle denervated by cutting the nerve (Fig. 1d). The rate of decrease of the total MHC content was similar to that of the muscle mass (compare Fig. 1 b and d). On the other hand, in the denervated soleus muscle the content of MHC decreased much
more relative to the muscle mass as early as in the second week after the operation of either cutting or crushing the nerve (compare Fig. 1a and c). The dates reported here are in full agreement with those previously published [4, 5, 131. During the third week after crushing the nerve, the total MHC muscle content stopped decreasing and began increasing. The total MHC content in the soleus increased much more rapidly than that in the EDL muscle (compare Fig. 1c and d), probably due to the fact that the degree of MHC loss was much higher in the soleus than in the EDL muscle just before reinnervation (17 days after operation the figures were 23 f 2% and 41 2% for the soleus and EDL, respectively; mean SEM of contralateral muscle). The results point to the lower dynamics of changes in the total MHC content in the EDL from those in the soleus muscle.
MH C isoforms SDSjPAGE with high glycerol concentration [17] allows for separation of MHC isoforins (Fig. 2). The MHC of the control soleus muscle contained 24% (19-27%) type 2A isoform and 76% (70-81%) type 1: traces of 2B isoform (0-3%) were also found. After denervation, changes in the proportion of the MHC isoforms appeared: the MHC-2A increased, while the MHC-1 decreased (Figs 1e and 2a). After reinnervation the MHC isoforins recovered to the control pattern within about 10 days and type 1 prevailed: it increased up to 94% of the total content of MHC. This higher-thannormal muscle proportion of the MHC-1 persisted for several weeks. N o sooner than 2 months after the operation, a tendency to normalization was observed (Figs 1e and 2a). 26 days after crushing the nerve, SDSjPAGE of MHC showed a band with the electrophoretic mobility of the 2B isoform, representing 8% of the total MHC in samples. The MHC of the control EDL muscle contained about 31% (24-36%) 2A isoform, about 67% (64 to 72%) type 2B and about 2% (0-3%) type 1. After denervation, the proportion of the 2A isoform increased to 41 - 52% of the total MHC content, whereas the proportion of the 2B isoform decreased to about 45% (Figs 1 f and 2b). During recovery of the EDL muscle after reinnervation, the normal pattern of MHC did not return, because the 2A isoform continued to
626
Fig. 3. Two-dimensional gel electrophoresis of MLC. (a) Soleus and (b) EDL on the 26 days after crushing the nerve. 1s and 2s: MLC characteristic for slow type myosin. IF, 2F and 3F: MLC characteristic for fast type myosin. The spots on the right of the marked MLC correspond to either those phosphorylated (2F) or those deaminated (IS, 1F). The spots on the lower-right of 1s and 2 s correspond to 1F and 2F, respectively. Thc assumed position of the embryonic MLC is indicated by an arrow
increase; the 2B isoform decreased to about 37% of the total MHC content. This pattern persisted for up to at least 2 months after the operation (Figs 1f and 2 b). The proportion of the MHC-1 isoform was maintained at the control level during the processes of both atrophy and recovery of the EDL muscle.
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Embryonic myosin A search was made for embryonic myosin in the experimental muscles by two-dimensional SDSjPAGE of the myosin light chain (MLC) and by immunoblotting of the MHC. Fig. 3 shows the MLC pattern in the muscles 27 days after crushing the nerve. The MLC pattern of the soleus was dominated by the slow types, while that of the EDL contained the three light chains peculiar to fast muscles. The embryonic isoform of MLC was not detected. However, when the MHC were transferred to nitrocellulose paper and reacted with a monoclonal antibody specific to the embryonic MHC, one band in the MHC patterns of the soleus and EDL muscles, after cutting and crushing the nerve, gave obvious positive results. The reaction, which was not detectable with the MHC of the control muscles of adult animals, was very intensive with MHC of muscle of 3-day-old rats (Fig. 4). These results demonstrated a presence of the embryonic isoform of MHC in the denervated and recovering slow and fast muscles of adult rats. Tjye of fibres by ultrastructural criteria
In parallel with studies of MHC, the fibre type was evaluated by ultrastructural criteria and the results are presented in Table 1. In the control soleus muscle, 90% of fibres showed characteristics of the SO type, 9 % of the FOG type and only a few fibres (about 1%) of both those types. In the control EDL muscle 61O h of fibres showed characteristics of the FG type, 33% FOG type and 2% SO type. Some fibres showed characteristics of two types simultaneously: i.e. about 3% of fibres of the F G and FOG types, and about 1% of SO and FOG types. In the denervated soleus muscle, a proportion of the SO fibres decreased, while that of the FOG type seemed to remain unchanged. Indeed, numerous fibres appeared to show characteristics of types SO and FOG. After reinnervation (4 6 weeks after crushing the nerve) the proportion of both SO and FOG fibres decreased compared with the denervated atrophying muscle, while the proportion of fibres simultaneously showing SO and FOG characteristics increased. Several fibres could not be defined by the ultrastructural criteria; they resembled those of the developing muscle since they
b
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Fig. 4. Immunohlots of MHCfrom experimental muscles with a monoclonal anti- (ernhryonic M H C ) antibody. (a) Blot stained with Ponceau red immediately after transfer; (b, c) blot after incubation with monoclonal antibody BFG6 for embryonic MHC, then with anti-(mouse IgG) antibodies conjugated with alkaline phosphatase. The photographs were taken after 10 min in (b) and after 35 min in (c) of development. MHC of myofibrils from (lane 1) leg muscles of the 3day-old rats, (lane 2) denervated soleus, 21 days after cutting the nerve, (lane 3) reinnervated soleus, 21 days after crushing the nerve, (lane 4) denervated EDL, 21 days after cutting the nerve, (lane 5 ) reinnervated EDL, 21 days after crushing the nerve, (lane 6 ) control EDL, from non-operated animals, (lane 7) control soleus, from nonoperated animals, and (lane 8) as lane 1
contained loosely arranged myofibrils surrounded by sarcoplasm rich in polysomes. In the denervated EDL muscle the proportion of FG fibres decreased and several fibres showed simultaneous characteristics of the types FOG/FG or FOG/SO. After reinnervation of the EDL muscle (4 - 6 weeks after crushing the nerve) the composition of fibres was, in general, similar to that in the denervated muscle, with one exception: fibres of the pure FG type were transiently absent, but all fibres showed FOG characteristics, alone or together with the FG or SO types. As late as 2 months after the operation the tendency to normalization of fibre types appeared in both the soleus and the EDL muscles. The results showed that in both fast and slow muscles the pattern of the fibre types was changed during the course of denervation atrophy and of recovery after reinnervation;
627 Table 1. Fibre composition in denervnted and reinnervated muscles The type of muscle fibres was evaluated according to the ultrastructural characteristic of fibres, as described in Materials and Methods. The fibres were classified into types SO, FOG and FG, and fibres showing characteristics of two types simultaneously, SO/FOG and FOG/FG. D) denervated (after cutting the sciatic nerve); (R) reinnervated (after crushing the sciatic nerve). The total number of fibres identified was taken as 100% Muscle
Time after operation
Total number of fibres identified
Proportion of total fibres
so
SO/FOG
83 70 56 69 87
90 57 27 61 82
1 21 39 26 16
9 9
64 58 66 84 63
2
1 15 13 21 2
33 43 45 29 30
____
several fibres appeared with two-type characteristics. At each stage, shifts in fibre types were in good agreement with those observed in the MHC isoforms.
DISCUSSION In the present study, we have shown that in denervated muscles there are clear changes in the proportion of the MHC isoforms which are peculiar to the soleus and EDL normal adult muscles. Indeed, the proportion of isoforms 1 and 2B decreased in the denervated soleus and EDL muscles, respectively, while in both muscles the proportion of the 2A isoform increased (Figs. l e , f, and 2). As the content of the slow myosin subunits decreased more than the fast myosin subunits, this confirmed a stricter dependence of slow myosin expression on innervation than that of fast myosin [28 - 301. To our knowledge this is the first paper to conclusively show that MHC-2B is much more susceptible to lack of innervation than is MHC-2A. This very interesting phenomenon has been confirmed by Schiaffino (personal communication). D'Albis and coworkers [31] found a decreased proportion of myosin specific for the 2B fibres, while that of the 2A fibres was increased in denervated muscle regenerating after cardiotoxin injury. Thus, it seems that MHC-2B is as dependent on innervation as type 1. One could assume that the relative increase of MHC-2A in both the denervated EDL and soleus muscles might come about as an effect of higher stability of the 2A isoform compared with the other isoforms in the denervated muscle, owing to its lower sensitivity to proteolytic degradation. On the other hand, the imbalance of isoforms could stem from their changed synthesis. The latter interpretation seems to be more probable, because the shift in the MHC pattern was correlated with the transformation of the ultrastructural characteristics of the fibre types. It is also possible that a relative increase in MHC-2A is in fact an increase in the MHC-2X subunit, comigrating with MHC-2A, as was found in the stimulated denervated muscle [32]. This paper indicates a particular feature of both the EDL and the soleus denervated muscles: in a single fibre, characteristics of two types can be detected simultaneously, and one of
FOG
__
FOG/FG
FG
Undefined
13 32 10
2 3 2 3 28 39 33 32
61 14 3 17 36
these is the 2A (FOG) type. The number of pure 2A fibres increased only slightly or even decreased (Table 1). Therefore, it seems that the relative increase in the MHC-2A isoform content in the myosin of denervated muscles, could result from fibre transformation rather than from preferential atrophy of type 1 (SO) and type 2B (FG) fibres. Transformation of fibres into the 2A type, observed in the denervated fast and slow muscles, suggests that innervation by the more specialised motor unit, fast or slow, suppresses an expression of proteins corresponding to the 2A type; lack of innervation allows for their expression. The same explanation may be given for expression of embryonic MHC in denervated muscle. After reinnervation of the slow soleus muscle, the type 1 isoform of MHC recovered rapidly then overproduction of this isoform took place (Figs 1 e and 2a). On the other hand, in the reinnervated EDL muscle, the 2A isoform of MHC still prevailed, while the 2B isoform continued to decrease (Figs 1f and 2b). Thus, in both recovering muscles, the proportion of MHC shifts to the slower isoforms. This is in agreement with the transformation of fibre types in a more oxidative pattern in reinnervated muscles (Table 1). We can interpret these phenomena by considering the recovering muscle as a muscle hypertrophying following increased activity. In such a case, muscle metabolism and myosin isoforms shift to a more oxidative pattern [6, 33, 341. Transformation of fibres following innervation by foreign motor neurons or following a transient double innervation seems least probable, taking into account the fact that in the reinnervated muscle, after appropriate nerve crushing, a type-grouping has not appeared (results not shown) [12]. The authors wish to thank Prof. S. Schiaffino for his kind gift of the monoclonal antibodies for embryonic MHC, and Mrs. H. Chomontowska and Mr. S. Belluco for their skilful technical assistance. We are also indebted to Prof. S. Schiaffino, Dr D. B i d , Dr E. Mussini and Dr L. Dalla Libera for discussing the results. This work was financed by the Polish Academy of Sciences (grant no. CPBP 04.01.3.07), by the Italian Consiglio Nazionale delle Ricerclze (grant to the Unit for Muscle Biology and Physiopathology) by the P r o p t t o Finulizzuto Tecnologie Biomediche c Sanitarie and by the Italian Ministero della Puhhlicu Istruzione 609'0 to Prof. U. Carraro.
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