553
J. Anat. (1979), 128, 3, pp. 553-562 With 8 figures Printed in Great Britain
Myotube formation in skeletal muscle regeneration M. ALLAM ALI* Department of Anatomy, University of Cambridge, Downing Street, Cambridge, CB2 3DY and Department of Anatomy, The Queen's University, Belfast
(Accepted 14 April 1978) INTRODUCTION
It is well known that mammalian skeletal muscle is capable of at least partial regeneration after injury (Field, 1960; Adam, Denny-Brown & Pearson, 1962), but the cellular mechanisms involved are still debated. Histological studies of (1) the early stages of repair after trauma (mechanical, Bintliff & Walker, 1960; Allbrook, 1962; thermal, liver, Shafiq & Gorycki, 1965; ischaemic, Clark, 1946; Reznik, 1969; chemical, Jirmanova & Thesleff, 1972), of (2) embryonic myogenesis (Copper & Konigsberg, 1961) and of (3) muscle development in tissue culture (Holtzer, Abbott & Lash, 1958; Allen & Pepe, 1965) have demonstrated a stage where sarcoplasmic ribbons or straps dominate the picture. Such straps are multinucleate and basiphilic. They have been called sarcoblasts (Godman, 1957; Betz, Firket & Reznik, 1966) and myotubes (Lash, Holtzer & Swift, 1957). The latter term will be used in the present text. Two explanations of the way myotubes are formed have been put forward. The first, the 'continuous' or 'budding' theory, postulates the outgrowth from the viable ends of the cut muscle fibres of nucleated sarcoplasmic buds (Miller, 1934; Gay & Hunt, 1954) which rapidly assume the form of myotubes. The second, the 'discontinuous' or 'embryonic' theory, states that in the early stages of regeneration mononucleated cells (myoblasts) appear in the wound area (Field, 1960; Price, Howes & Blumberg, 1964) where they proliferate and then fuse to form multinucleated myotubes (Sloper & Pegrum, 1967; Reznik, 1969) not at first connected with the cut ends of the original fibres. A few investigators believe that myotubes are formed by both these processes {Litver, 1962; Shafiq & Gorycki, 1965; Sloper, Bateson, Hindle & Warren, 1970). In general, the proponents of the 'continuous' theory have, in their experiments, inflicted more severe damage to muscle fibres than those who favour the 'discontinuous' theory: it seems possible therefore that the mode of repair is related to the severity of the injury. The present paper seeks to establish that either continuous or discontinuous modes of regeneration may occur, depending on the kind of damage inflicted on individual muscle fibres and their sarcolemmal sheaths. *
Present address: Anatomy Department, Faculty of Medicine, P.O. Box 2925, Riyadh, Saudi
Arabia.
554
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.
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Fig. 2. Electron micrograph of part of the damaged area 3 days after cut injury showing a
sarcoplasmic bud (Sb) extending from the viable stump of an injured muscle fibre (MF). Note the young mycfibrils (Mf) at the junction of stump and bud. x 9500.
ultramicrotome. They were stained with uranyl acetate and lead citrate and viewed in an AEI 801 or Siemens electron microscope. OBSERVATIONS
Light microscopy Cut muscle The wound area in the cut muscle during the first 2j days was heavily infiltrated with polymorphonuclear leucocytes and macrophages engaged in the removal of the damaged parts of the muscle fibres. Fibrin deposition, erythrocyte extravasation and marked oedema were further features of the wound gap created by contraction of the cut fibres (cf. Lash et al. 1957). After 3-4 days much of the wound debris had disappeared, and small sarcoplasmic buds were seen projecting from the ends of some of the cut muscle fibres. The lengths of these outgrowths varied considerably, sometimes extending as far as the middle of the wound gap. Figure 1 shows a group of such outgrowths 4 days after operation. Of particular interest was the location of the myonuclei in the injured fibres and their associated outgrowths. Near the site of injury the nuclei appeared to have moved into the interior of the muscle fibre and then into the central core of the new sarcoplasmic outgrowth. Such nuclei were larger and more granular than the normally peripherally situated nuclei of fibres further from the site of injury, and they had prominent nucleoli. The cytoplasm surrounding these enlarged nuclei was more basophilic than that around normal muscle nuclei. After the first week the wound gap was bridged by new fibrous tissue.
556
M. ALLAM ALI
Fig. 3. Cross section of a crushed area 3 days after injury showing several damaged muscle fibres surrounded by large numbers of inflammatory cells. Partially or completely separated cellular units (or nucleated fragments) with enlarged nuclei and dense nucleoli can be seen. These nucleated fragments are regarded as myoblastic cells. A mitotic figure is seen in one of them (top left). Resin-embedded. Methylene blue and van Gieson. x 1200.
Crushed muscle In general the muscle structure was much less disorganized after crushing than after cutting. In some areas, both muscle fibres and their sarcolemmal sheaths were clearly disrupted, but in others, though muscle fibres were broken, their sarcolemmal sheaths seemed to be intact (Fig. 3). In the areas where the sarcolemmal sheaths appeared intact sarcoplasmic buds were not seen; instead, the gaps in the muscle fibres were occupied by mononucleated cells inside the sarcolemmal tubes. Electron microscopy Cut muscle In some parts of the wound area sarcoplasmic outgrowths were seen with the electron microscope 3 days after injury (Fig. 2). The junction between the stump of an injured muscle fibre and such an outgrowth is illustrated in Figure 2, where some myofibrils and myofilaments are seen to extend in continuity between the stump and the beginning of the outgrowth. The more distal parts of the sarcoplasmic outgrowths showed no such formed elements, but were characterized by ribosome clusters.
Myotubes and muscle regeneration
557
Fig. 4. Cross section through a crushed area 3 days after injury, showing myoblasts within the folded basement membrane of a sarcolemmal tube. x 8000.
Crushed muscle In the wound area mononucleated cells were present, along with muscle debris, within intact sarcolemmal tubes (Fig. 4). Each cell possessed a clearly defined plasma membrane, and a large nucleus with one or more dense nucleoli. Collections of well defined myofibrils were seen in some cells, while in others there were only a very few rudimentary myofibrils (Fig. 5). The ribosome content also varied considerably from cell to cell. These cells were interpreted as myoblasts in various stages of differentiation. Figures 6 and 7 show three myoblasts lying close together within the same sarcolemmal tube. No cytoplasmic continuity between the cells was evident at this stage. Figure 8 shows a portion of the wound area after 4 days. Myoblasts seem to be fusing. Dense lines show where opposed plasma membranes are apparently losing their separate identities: such lines are not tangential profiles or tight junctions. DISCUSSION
The present study has shown certain aspects of the early stages of regeneration following trauma to rat skeletal muscle. It is concluded that both 'continuous' (i.e. from sarcoplasmic outgrowths) and 'discontinuous' (i.e. from new myoblasts) regeneration occurs, and that the 'continuous' mode of regeneration occurs when 36
ANA I28
558
M. ALLAM ALI
5
~
Fig. 5. Higher magnification of a portion of the preceding figure showing three adjacent myoblasts beneath a common folded basement membrane (large arrows). They exhibit varying stages of differentiation with varying collections of myofibrils (Mf) and ribosomes (small arrows). x 20500.
sarcolemmal integrity is destroyed, while the discontinuous mode characterizes sites where sarcolemmal tubes remain intact. This is in general agreement with Field (1960), who stressed that the mode of repair was related to the degree of preservation of muscle architecture, and in particular to the extent to which sarcolemmal sheaths remained intact. Likewise, Litver (1962) found in adult rabbit skeletal muscle that regeneration was of the 'continuous' type after a cutting or a heating injury and 'discontinuous' after a freezing or an electrical injury. Shafiq & Gorycki (1965), using the electron microscope, noted both types of regeneration after a thermal injury to the skeletal muscle of normal young mice. However, Sloper & Pegrum (1967) failed to find evidence of budding in crushed muscle in adult mice, and suggested that the illusion of budding resulted from secondary fusions between independently formed myotubes and surviving muscle fibres. They insisted that, to prove budding, cytoplasmic continuity must be demonstrated at the time when myotubes are being first formed some 3 or 4 days after injury. In the present study definite continuity between the sarcoplasmic straps and injured muscle fibres was apparent at both light and electron microscope levels on the third and fourth days. There is no doubt that direct budding from the old muscle fibres does occur, as many workers have affirmed, e.g. Clark (1946), Field (1960) and Shafiq (1970). However, there is little doubt that 'discontinuous' regeneration also occurs. This is shown by the appearance during the first 3 days of mononucleated cells inside
Myotubes and muscle regeneration
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Fig. 6. Longitudinal section of portion of a sarcolemmal tube in the crushed area 3 days after injury. Dense wavy basement membrane (Bm) outlines the tube, which is occupied by fusiform mononucleated cells (myoblasts). The cells are in close contact and oriented in the longitudinal axis of the tube. Several dense nucleoli are seen within the large nuclei. x 10000.
the basement membrane of some of the damaged muscle fibres, and their subsequent rapid differentiation into myoblasts, which then fuse. It is virtually impossible to crush a muscle in such a way that all fibres are damaged to a similar extent, so it is not surprising that budding may be seen at one place and myoblast formation at another (Sloper et al. 1970; Aloisi, 1970). The origin and mode of accumulation of the nuclei in the sarcoplasmic buds, and later in the myotubes derived from them, are undecided. However, the present study lends support to the view that nuclei migrate into the buds, without dividing, from the intact remnants of the muscle fibres (Capers, 1960; Shafiq, 1970). The origin of the myoblasts, whether from mononucleated fragments of damaged muscle fibres, or from satellite cells, or from mesenchymal connective tissue cells, is debated. This topic will be the subject of a further study. SUMMARY
Muscle fibre regeneration in the latissimus dorsi of adult rats has been studied by light and electron microscopy. Both 'continuous' and 'discontinuous' modes of regeneration occur. In 'continuous' regeneration sarcoplasmic buds grow from the damaged muscle fibres, their nuclei apparently originating from the healthy portion of the muscle fibre. 36-2
560
M. ALLAM ALI
Fig. 7. Higher magnification of portions of the myoblasts within the sarcolemmal tube seen in Fig. 6. The plasma membranes (Pm) are extremely close to one another, suggesting that fusion of the myoblasts may be about to take place. The cytoplasm of each myoblast contains clusters of ribosomes, several tubular elements, probably representing developing endoplasmic reticulum, mitochondria (M) and fat droplets (F). Focal areas of rudimentary myofibrillar structures can be seen towards the periphery of the cells (arrows). x 32000.
In 'discontinuous' regeneration mononucleated cells accumulate inside intact but vacated, sarcolemmal tubes. They differentiate into myofibril-containing myoblasts which later fuse. 'Continuous' regeneration occurs when the sarcolemmal sheaths are disrupted; 'discontinuous' regeneration when the sarcolemmal sheaths remain intact.
Myotubes and muscle regeneration
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J. Anat. (1979), 128, 3, pp. 553-562 With 8 figures Printed in Great Britain
Myotube formation in skeletal muscle regeneration M. ALLAM ALI* Department of Anatomy, University of Cambridge, Downing Street, Cambridge, CB2 3DY and Department of Anatomy, The Queen's University, Belfast
(Accepted 14 April 1978) INTRODUCTION
It is well known that mammalian skeletal muscle is capable of at least partial regeneration after injury (Field, 1960; Adam, Denny-Brown & Pearson, 1962), but the cellular mechanisms involved are still debated. Histological studies of (1) the early stages of repair after trauma (mechanical, Bintliff & Walker, 1960; Allbrook, 1962; thermal, liver, Shafiq & Gorycki, 1965; ischaemic, Clark, 1946; Reznik, 1969; chemical, Jirmanova & Thesleff, 1972), of (2) embryonic myogenesis (Copper & Konigsberg, 1961) and of (3) muscle development in tissue culture (Holtzer, Abbott & Lash, 1958; Allen & Pepe, 1965) have demonstrated a stage where sarcoplasmic ribbons or straps dominate the picture. Such straps are multinucleate and basiphilic. They have been called sarcoblasts (Godman, 1957; Betz, Firket & Reznik, 1966) and myotubes (Lash, Holtzer & Swift, 1957). The latter term will be used in the present text. Two explanations of the way myotubes are formed have been put forward. The first, the 'continuous' or 'budding' theory, postulates the outgrowth from the viable ends of the cut muscle fibres of nucleated sarcoplasmic buds (Miller, 1934; Gay & Hunt, 1954) which rapidly assume the form of myotubes. The second, the 'discontinuous' or 'embryonic' theory, states that in the early stages of regeneration mononucleated cells (myoblasts) appear in the wound area (Field, 1960; Price, Howes & Blumberg, 1964) where they proliferate and then fuse to form multinucleated myotubes (Sloper & Pegrum, 1967; Reznik, 1969) not at first connected with the cut ends of the original fibres. A few investigators believe that myotubes are formed by both these processes {Litver, 1962; Shafiq & Gorycki, 1965; Sloper, Bateson, Hindle & Warren, 1970). In general, the proponents of the 'continuous' theory have, in their experiments, inflicted more severe damage to muscle fibres than those who favour the 'discontinuous' theory: it seems possible therefore that the mode of repair is related to the severity of the injury. The present paper seeks to establish that either continuous or discontinuous modes of regeneration may occur, depending on the kind of damage inflicted on individual muscle fibres and their sarcolemmal sheaths. *
Present address: Anatomy Department, Faculty of Medicine, P.O. Box 2925, Riyadh, Saudi
Arabia.
554
M. ALLAM ALI ...: .
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.
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.......
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.
f
t
Sb.
.^~~~~~~~ *
S
t~Sb t _
*It.0 _
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...
4' .; :-
Fig. 2. Electron micrograph of part of the damaged area 3 days after cut injury showing a
sarcoplasmic bud (Sb) extending from the viable stump of an injured muscle fibre (MF). Note the young mycfibrils (Mf) at the junction of stump and bud. x 9500.
ultramicrotome. They were stained with uranyl acetate and lead citrate and viewed in an AEI 801 or Siemens electron microscope. OBSERVATIONS
Light microscopy Cut muscle The wound area in the cut muscle during the first 2j days was heavily infiltrated with polymorphonuclear leucocytes and macrophages engaged in the removal of the damaged parts of the muscle fibres. Fibrin deposition, erythrocyte extravasation and marked oedema were further features of the wound gap created by contraction of the cut fibres (cf. Lash et al. 1957). After 3-4 days much of the wound debris had disappeared, and small sarcoplasmic buds were seen projecting from the ends of some of the cut muscle fibres. The lengths of these outgrowths varied considerably, sometimes extending as far as the middle of the wound gap. Figure 1 shows a group of such outgrowths 4 days after operation. Of particular interest was the location of the myonuclei in the injured fibres and their associated outgrowths. Near the site of injury the nuclei appeared to have moved into the interior of the muscle fibre and then into the central core of the new sarcoplasmic outgrowth. Such nuclei were larger and more granular than the normally peripherally situated nuclei of fibres further from the site of injury, and they had prominent nucleoli. The cytoplasm surrounding these enlarged nuclei was more basophilic than that around normal muscle nuclei. After the first week the wound gap was bridged by new fibrous tissue.
556
M. ALLAM ALI
Fig. 3. Cross section of a crushed area 3 days after injury showing several damaged muscle fibres surrounded by large numbers of inflammatory cells. Partially or completely separated cellular units (or nucleated fragments) with enlarged nuclei and dense nucleoli can be seen. These nucleated fragments are regarded as myoblastic cells. A mitotic figure is seen in one of them (top left). Resin-embedded. Methylene blue and van Gieson. x 1200.
Crushed muscle In general the muscle structure was much less disorganized after crushing than after cutting. In some areas, both muscle fibres and their sarcolemmal sheaths were clearly disrupted, but in others, though muscle fibres were broken, their sarcolemmal sheaths seemed to be intact (Fig. 3). In the areas where the sarcolemmal sheaths appeared intact sarcoplasmic buds were not seen; instead, the gaps in the muscle fibres were occupied by mononucleated cells inside the sarcolemmal tubes. Electron microscopy Cut muscle In some parts of the wound area sarcoplasmic outgrowths were seen with the electron microscope 3 days after injury (Fig. 2). The junction between the stump of an injured muscle fibre and such an outgrowth is illustrated in Figure 2, where some myofibrils and myofilaments are seen to extend in continuity between the stump and the beginning of the outgrowth. The more distal parts of the sarcoplasmic outgrowths showed no such formed elements, but were characterized by ribosome clusters.
Myotubes and muscle regeneration
557
Fig. 4. Cross section through a crushed area 3 days after injury, showing myoblasts within the folded basement membrane of a sarcolemmal tube. x 8000.
Crushed muscle In the wound area mononucleated cells were present, along with muscle debris, within intact sarcolemmal tubes (Fig. 4). Each cell possessed a clearly defined plasma membrane, and a large nucleus with one or more dense nucleoli. Collections of well defined myofibrils were seen in some cells, while in others there were only a very few rudimentary myofibrils (Fig. 5). The ribosome content also varied considerably from cell to cell. These cells were interpreted as myoblasts in various stages of differentiation. Figures 6 and 7 show three myoblasts lying close together within the same sarcolemmal tube. No cytoplasmic continuity between the cells was evident at this stage. Figure 8 shows a portion of the wound area after 4 days. Myoblasts seem to be fusing. Dense lines show where opposed plasma membranes are apparently losing their separate identities: such lines are not tangential profiles or tight junctions. DISCUSSION
The present study has shown certain aspects of the early stages of regeneration following trauma to rat skeletal muscle. It is concluded that both 'continuous' (i.e. from sarcoplasmic outgrowths) and 'discontinuous' (i.e. from new myoblasts) regeneration occurs, and that the 'continuous' mode of regeneration occurs when 36
ANA I28
558
M. ALLAM ALI
5
~
Fig. 5. Higher magnification of a portion of the preceding figure showing three adjacent myoblasts beneath a common folded basement membrane (large arrows). They exhibit varying stages of differentiation with varying collections of myofibrils (Mf) and ribosomes (small arrows). x 20500.
sarcolemmal integrity is destroyed, while the discontinuous mode characterizes sites where sarcolemmal tubes remain intact. This is in general agreement with Field (1960), who stressed that the mode of repair was related to the degree of preservation of muscle architecture, and in particular to the extent to which sarcolemmal sheaths remained intact. Likewise, Litver (1962) found in adult rabbit skeletal muscle that regeneration was of the 'continuous' type after a cutting or a heating injury and 'discontinuous' after a freezing or an electrical injury. Shafiq & Gorycki (1965), using the electron microscope, noted both types of regeneration after a thermal injury to the skeletal muscle of normal young mice. However, Sloper & Pegrum (1967) failed to find evidence of budding in crushed muscle in adult mice, and suggested that the illusion of budding resulted from secondary fusions between independently formed myotubes and surviving muscle fibres. They insisted that, to prove budding, cytoplasmic continuity must be demonstrated at the time when myotubes are being first formed some 3 or 4 days after injury. In the present study definite continuity between the sarcoplasmic straps and injured muscle fibres was apparent at both light and electron microscope levels on the third and fourth days. There is no doubt that direct budding from the old muscle fibres does occur, as many workers have affirmed, e.g. Clark (1946), Field (1960) and Shafiq (1970). However, there is little doubt that 'discontinuous' regeneration also occurs. This is shown by the appearance during the first 3 days of mononucleated cells inside
Myotubes and muscle regeneration
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559
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;
Fig. 6. Longitudinal section of portion of a sarcolemmal tube in the crushed area 3 days after injury. Dense wavy basement membrane (Bm) outlines the tube, which is occupied by fusiform mononucleated cells (myoblasts). The cells are in close contact and oriented in the longitudinal axis of the tube. Several dense nucleoli are seen within the large nuclei. x 10000.
the basement membrane of some of the damaged muscle fibres, and their subsequent rapid differentiation into myoblasts, which then fuse. It is virtually impossible to crush a muscle in such a way that all fibres are damaged to a similar extent, so it is not surprising that budding may be seen at one place and myoblast formation at another (Sloper et al. 1970; Aloisi, 1970). The origin and mode of accumulation of the nuclei in the sarcoplasmic buds, and later in the myotubes derived from them, are undecided. However, the present study lends support to the view that nuclei migrate into the buds, without dividing, from the intact remnants of the muscle fibres (Capers, 1960; Shafiq, 1970). The origin of the myoblasts, whether from mononucleated fragments of damaged muscle fibres, or from satellite cells, or from mesenchymal connective tissue cells, is debated. This topic will be the subject of a further study. SUMMARY
Muscle fibre regeneration in the latissimus dorsi of adult rats has been studied by light and electron microscopy. Both 'continuous' and 'discontinuous' modes of regeneration occur. In 'continuous' regeneration sarcoplasmic buds grow from the damaged muscle fibres, their nuclei apparently originating from the healthy portion of the muscle fibre. 36-2
560
M. ALLAM ALI
Fig. 7. Higher magnification of portions of the myoblasts within the sarcolemmal tube seen in Fig. 6. The plasma membranes (Pm) are extremely close to one another, suggesting that fusion of the myoblasts may be about to take place. The cytoplasm of each myoblast contains clusters of ribosomes, several tubular elements, probably representing developing endoplasmic reticulum, mitochondria (M) and fat droplets (F). Focal areas of rudimentary myofibrillar structures can be seen towards the periphery of the cells (arrows). x 32000.
In 'discontinuous' regeneration mononucleated cells accumulate inside intact but vacated, sarcolemmal tubes. They differentiate into myofibril-containing myoblasts which later fuse. 'Continuous' regeneration occurs when the sarcolemmal sheaths are disrupted; 'discontinuous' regeneration when the sarcolemmal sheaths remain intact.
Myotubes and muscle regeneration
.1
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