Adhesion molecules were studied in regenerating skeletal muscle immunohistochemically and ultrastructurally after a standardized trauma. In normal muscle, extracellular matrix (ECM) protein tenascin was restricted to myotendinous junctions (MTJ), while the integrin &-subunit was present also on the sarcolemma. After injury, tenascin increased on the outer surface of regenerating myofibers, where cellular fibronectin also accumulated. Later, tenascin concentrated at the tips of regenerating myofibers, where new MTJs were formed. The p,-subunit disappeared on necrotized myofibers and reappeared on regenerating fibers in a thicker layer. The regenerating myofibers were invested by a basal lamina, except for the growth cones at the distal ends, which were laminin-negative until the formation of MTJs occurred. These results indicate that regenerating muscle cells are attached to the ECM in a way that allows both growth of the muscle cells across the scar and their use before the regeneration is completed. Key words: muscle regeneration tenascin integrin laminin fibronectin MUSCLE & NERVE 15~482-489 1992
TIM0 HURME, MD, and HANNU KALIMO, MD
Transformation oi' the contraction force created by muscles into movement requires firm attachment of the rnyofibers to the structures to be moved. 'lliis at.tachment is mediated by tendons, compact bundles of connective tissue mainly composed of type I ~ o l l a g e nT. ~h e fusion of myofibers with the teridoris is realized by fbrmation of specialized myotendinous .junctions (MTJ), where the cytoskeletal, transmembrane (matrix receptor), a n d extracellular matrix (ECM) proteins interact. In muscle rupture, the functional continuity of the muscle-connective tissue complex is broken, a n d the muscle contract.ion only pulls the stumps of the breached ~nyofibersapart instead of approaching their tendinous cnds. '1'0 restore the functional continuity of tendon- niyofibertendon, the myofibers must regenerate, a n d the stumps of the ruptured fibers rejoined firmly
From the Department of Surgery (Dr Hurme). the Department of Pathology (Dr. Kalimo) and Paavo Nurmi Center (Dr. Hurme), University of Turku and University Central Hospital of Turku. Turku, Finland. Acknowledgments: The skilled technical assistance of Ms.Liisa Lempiainen and Paula Merilahti. and the excellent photography by Mr. Jaakko Liippo is gratefully acknowledged. Dr. lsmo Virtanen is thanked for the antiserum kindly provided to us. We also thank Drs. Markku Jalkane, Eero Vuorio. and Jvriki Heino for their exDert advice and criticism. Address reprint .requests to Tim0 Hurme. MD, Department of Surgery, University of Turku, Kiinamyllynkatu 4-8, SF-20520 Turku, Finland. Accepted for publication Aprili 8, 1991 CCC 0148-639X/92/040482-08 $04.00 0 1992 John Wiley & Sons, Inc
482
Adhesion in Muscle Regeneration
enough to withstand the tension created by the muscle contraction. Thus, optimal healing of muscle rupture necessitates balanced regeneration of both the muscle and connective tissue components.':' In the well-documented experimental model of muscle injury, a strike with a blunt hammer o n rat gastrocnemius causes rupture of myofibers to the depth of about half of the thickness o f the muscle.'5 Disrupted muscle fibers contract, and a gap is formed between the stumps, which is first filled with a hematoma to be gradually replaced by proliferating granulation tissue. T h e regeneration of the necrotized parts of the disrupted muscle fibers occurs completely inside the preserved part of basal lamina cylinder^.'^ When reaching the edge of the ruptured basal lamina, the regenerating muscle cells begin to penetrate through the granulation tissue. Occasional primitive M T j s a r e found at the tips of growing muscle cells, which provide the attachment between the ECM and muscle cell. This growth o f t h e regenerating myofibers through the connective tissue must be directed by intricate interactions between the sarcolemma and components of ECM about which knowledge is still very scant. Because of its abundance in the M T J s , tenascin,.5," is obviously o n e of' the key ECM protein candidates for providing firm anchorage of the myofibers to the connect.ive tissue. T h e attachment of cells to extracellular tenascin has been
'"
'
MUSCLE & NERVE
April 1992
suggested to be mediated by a receptor molecule of the integrin superfamily, which are heterodimers of two transmembrane subunits (Y and p, the latter (PI) being common to several adesion receptors (pl subfamily), including the receptor for fibroectin, l 7 laminin, lYand fibrillar collagen.22 A few analyses of tenascin and integrin P,-subunit have been done on developing chicken skeletal muscle,3v7but we are not aware of studies on their role in muscle regeneration. Fibronectin and laminin are both components of the basal lamina which invests each myofiber, including the MTJ area. Thus, these two basal lamina molecules must also be integral in the attachment of myofibers to the ECM. In this study we have analyzed the distribution of tenascin, cellular fibronectin, integrin p subunit, and laminin immunoreactivities during the healing of skeletal muscle after contusion injury, and correlated these findings with the ultrastructural features. The main focus is to clarify the role of these proteins in the re-establishment of the connection between the stumps of the ruptured myofibers to allow the breached muscle to contract again as a unit. MATERIALS AND METHODS
Fifty adult male Wistar rats were used in this study. A standard partial transverse contusion injury was induced to the left calf of each animal under light ether anesthesia using a spring-loaded hammer." T h e time of traumatization was designated as day 0. During the posttraumatization time, the animals were allowed to move freely. The animals were sacrificed by cervical dislocation under ether anesthesia on days 2, 5, 7, 14, and 21, nine rats at each time-point. Five injured animals served as controls. For immunohistochemical studies, the gastrocnemius muscle was dissected free immediately after death. The muscle was divided in the sagittal midline, and the medial half was snap frozen in freon 22 cooled with liquid nitrogen. Serial 5-pm sections were cut for morphological investigations. Routine sections were stained with modified Herovici."' In the immunohistochemical studies, the following antibodies were used: polyclonal rabbit antihuman tenascin, antihuman integrin P I subunit (both antisera are cross-reactive with corresponding rat antigen, manufacturer's information) and antirat laminin (Telios Pharmaceuticals, San Diego, CA), and monoclonal mouse antibody to human ED fragment of fibronectin (the sequence present only in cellular fibronectin; the an-
Adhesion in Muscle Regeneration
tibody, which also crossreacts with the corresponding rat antigen is a kind gift from Dr. I. Virtanen, see ref. 23). T h e bound primary antibodies were visualized using the appropriate avidin- biotinperoxidase method for either mouse or rabbit antibodies (Vector Laboratories, Burlingame, CA) with diaminobenzidine as the chromogen. Light hematoxylin counterstaining was used to discern cellular structures. For ultrastructural analysis, the gastrocnemius muscle was fixed in freshly prepared 4% phosphate-buffered paraformaldehyde at a standard length, cut sagittally at the midline, refixed with glutaraldehyde, postosmicated, dehydrated, and embedded in Epon. Semithin sections were stained with toluidine blue on the basis of which informative areas were selected. Thin sections were double stained with uranyl acetate and lead citrate, and examined in a Jeol JEM 100 C electron microscope. RESULTS
The light and electron microscopic features of the regenerating myofibers have been described in detail in our previous arti~1e.I~ When the regenerating muscle cells extended out of the old basal lamina cylinders, they began to penetrate the central zone (CZ) on days 5 to 7 as thin branches with the
FIGURE 1. Regenerated myofibers (nos. 1-3) in the RZ on day 7 are bound by a strongly laminin-positive basal lamina up to the growth cone at the tip, where the immunoreactivity is weak or absent (arrows). Antilaminin hematoxylin counterstain (bar-50
+
P-4.
MUSCLE & NERVE
April 1992
483
FIGURE 2. The distal end of the thin regenerating myofiber branch 21 days after the trauma. Myotendinous-junction-like structures have developed at the tips of 3 branches (nos. 1 -3),whereas the fourth (no. 4) has a cytoplasmic tip, which may still be advancing. Tip no. 1 also has cytoplasm with abundant vesicles, which are a sign of growth, even though a focal accumulation of subsarcolemmal material has occurred at the most distal end. Bundles of collagen (C) and thinner filaments, (F, possibly fibrillin) are present in the extracellular space. Myofibrils extend all the way to the tip nos. 2 and 3, and the thin filaments fuse with the dark subsarcolemmal material (bar-1 pm).
structure of myotube. 'These w e r e surrounded by a basal larnina as shown by die strong irrirriuriopositivity with antiserum to rat laminin (Fig. 1) u p to the growth cone (cf. ref. 14, Fig. 7); i.e., the advancing tip of the regenerating muscle cell. From day 7 onward, increasing numbers of the growth cones began to change their appearance. Basal lamina appeared on the outer surface of the sarcolemma, as did a layer of dark material subsarcolemmally, i.e., the tip assumed the appearance of a primitive MTJ (Fig. 2 ) . Both the myofibers and the endomysial structures were clearly negative with aiititenascin antibody both in the controls and in the surviving zone (SZ) of the injured rnuscle. Tenascin immunoreactivity was found only at myotendirious junctions as a thick, irregular band, which fused with the positively stained tendon o r intramuscular fascia (Fig. 3 ) . On day 2 , strong imniuriopositivity fbr tenascin was observed, as diffuse staining of the matrix in the granulation tissue in the CZ. In the regeneration zone (KZ) o n &y 5, antitenas& iInmLlnol-eactivity decorated the basal lamiria cylinclers filled l)y macrophages, which had phagocytosetl the necrotized part of the rnyofibers (Fig. 4). Definite inimunoreactivity also extended along the outer sur-
face of ruptured niyofibers proximally to their presei-ved parts in the SZ, where the endorn ysial space was widened a n d usually contained reactive cells (macoptiages and fibroblasts) (Fig. 5). O n day 5, much of the phagocytosis had been conipleted and inyoblast proliferation within the basal lamina cylinders had begun. When the regeneration progressed and myo-
Tenascin Immunoreactivity.
484
Adhesion in Muscle Regeneration
FIGURE 3. Myotendinous junctions at the attachment site of the myofibers to the intramuscular fascia (F) in the surviving zone of a rat 14 days after the trauma. Note the presence of tenascin immunoreactivity only at the MTJs (two marked with arrow), AntitenaScin + whereas the SarcOlemmais otherwise negative, hematoxylin counterstain (bar-100 pm)
MUSCLE & NERVE
April 1992
FIGURE 4. The old preserved basal lamia cylinders of the necrotized parts of myofibers in the RZ are delineated by tenascin immunoreaction product (arrows), which is also present on the preserved part of a myofiber (arrowhead). The extracellular matrix also stains weakly, but as a definite positive (asterisk). Antitenascin + hematoxylin counterstain (bar-100 km).
FIGURE 6. The regenerated myofibers on day 21 end in the connective tissue, which stains strongly tenascin-positive, Tenascin is also seen along the myofibers (arrows). The scar tissue on the right not associated with myofibers is tenascin-negative (asterisk). Antitenascin + hematoxylin counterstain (bar-100
wm).
tubes extended out of the preserved old basal lamina cylinders on days 7 to 14, these frequently formed ramified muscle cells, which pierced through the ECM of the CZ. Antitenascin positivity persisted on the sarcolemma of these myofiber branches, with accentuation at their growing tips as well as in the thickened endomysium of the regenerated parts of myofibers, until the end of the observation period on day 2 1. T h e surface of the intact myofibers in the SZ remained antiteascin negative (cf. Fig. 5 ) . In the connective tissue component of the healing scar, the septum between the stumps approaching each other retained its
FIGURE 5. A transverse section through the SZ close to the trauma on day 14. The endomysial space is widened. Tenascin irnmunoreactivity is present on the surviving parts of the myofibers, often on one side only (arrows), as well as a reticular network around the cells in the endomysial space. Note that fibers obviously farther from the lesion on the right are negative. Antitenascin + hematoxylin counterstain (bar-100 km).
Adhesion in Muscle Regeneration
FIGURE 7. (A) The preserved parts of the myofibers in the SZ are bound by a thin delicate line of integrin p,-subunit immunoreactivity. (B)In the MTJs, the irnmunoreactivity is accentuated, whereas in the underlying fascia to which the myofibers attach only thin streaks around fibroblasts are visible. The wall of a small artery (asterisk) is also strongly integrin &-subunit positive. Anti-integrin &-subunit + hematoxylin counterstain (bar100 pn).
MUSCLE & NERVE
April 1992
485
immunoreactivity, whereas the tenascin staining seemed to disappear from that connective tissue which did not associate with the regenerating myofibers (Fig. 6). Immunoreactivity for integrin P,-subunit in control animals, and in the SZ of injured rats, was identified around the intact myofibers as a thin delicate line (Fig. 7A). At the myotendinous junctions, the reactivity was stronger and formed broad bands, but in the adjoining tendon it was present only as streaks surrounding the fibroblasts within the dense connective tissue (Fig. 7B). Two days after the injury, integrin p,-subunit immunoreactivity in the RZ on the necrotized lntegrin p,-Subunit.
parts of the myofibers had more or less disappeared (Fig. 8). I t was observed on the proliferating fibroblasts between the basal lamina cylinders and on cells (endothelial and pericytic) of thc reconstructing capillary network, whereas the ECM between the cells did not stain. This basic staining pattern persisted throughout the regeneration period. O n day 14, the immunoreactivity on the regenerated parts of the myofibers was still stronger than that in the SZ, and a broad irnmunopositive band was often seen surrounding the tips of the myofiber stumps, most probably reflecting the folding of sarcolemma when M'rJs were formed (Fig. 9). In the intact muscle of the control rats and in the SZ of the injured gastronemius muscle, positive reaction with antibody to the cellular fibronectin was see in the epi- and perimysium arid around the individual muscle fibers with accentuation in the MTJ, whereas the sarcoplasm of all myofibers was negative (Fig. 10). During the healing process, the connective tissue formed in the CZ and that extending between the regenerating myofibers in the RZ, was clearly positive throughout the exaniination period (Fig. 1 l), though some decease in the staining seemed to occur at the later stages.
Cellular Fibronectin.
Laminin. In the controls and SZ of injured rats, expected positive reaction with antirat laminin antiserum was seen around individual muscle cell. At the in.jury site, the necrotiLed parts were surrounded with a thickened positive line corresponding to the corrugation of the ruptured basal
FIGURE 8. (A) On day 2, the necrotized parts of the myofibers have been phagocytosed by macrophages, which fill the preserved basal lamina cylinders (one marked with an arrow). Because the basal lamina of the necrotized parts becomes corrugated it appears thicker than that of the preserved fibers (arrowhead). (B)A nearby section demonstrates the loss of immunoreactivity for integin p,-subunit around the necrotic myofibers, which appear here as clusters of macrophages (two marked with arrows). The preserved fibers are demarcated by a thin line of integrin @,-subunitirnrnunoreactivity.(A) Antilarninin + hernatoxylincounterstain, (B) Anti-integrin p,-subunit + hematoxylin counterstai (bar-100 prn).
486
Adhesion in Muscle Regeneration
FIGURE 9. The sarcolernma of tne regenerating myofibers stains strongly positive with anti-integrin p,-subunit serum, with accentuation at their tips (arrows) on day 14. Anti-integrin p,subunit + hematoxylin counterstain (bar-50 km).
MUSCLE 8, NERVE
April 1992
FIGURE 10. MTJ region from the SZ on day 2. lmmunoreactivity for cellular fibronectin delineates all muscle fibers and is accentuated at the MTJs (arrows). The blood vessels (arrowheads) are also strongly positive, whereas the ECM stains more weakly. Anticellular fibronectin hematoxylin counterstain (bar-50 pm).
+
lamina cylinder (cf. Fig. 8A). T h e regenerating muscle cells extending into the connective tissue of the C Z were surrounded by a delicate thin line, which seemed to be lacking at the very tip (growth cone) of most regenerating fibers (cf. Fig. 1). When the regnerating ends formed the M'lJs, a broad band of laminin immunoreactivity appeared in concordance with the EM results, which showed a thick, well-developed basal lamina at the MTJ (cf. Fig. 2). DISCUSSION
Tenascin was discovered, from different tissues under various names (e.g., myotendinous antigen,
FIGURE 11. The newly formed patchy MTJs at the tips of the regenerating myofibers, on day 14, stain as strongly as their normal counterparts (arrows). The blood vessels and the surrounding ECM stain similarly as in the normal MTJ region. Anticellular fibronectin + hernatoxylin counterstain (bar-50 pm).
Adhesion in Muscle Regeneration
cytotactin, hexarachion, glioma-mesechymal extracellular matrix antigen) at about the same time by several workers (see ref. 8). Among the first researchers were Chiquet and Fambrough,"." who isolated "myotendinous antigen" from developig chicken muscle-tendon complex. Later studies revealed that "myotedinous antigen" is a major ECM glycoprotein, a disulfide-linked hexamer with molecular weight larger than 1000 kD. Tenascin is abundantly expessed during morphogenesis in many different tissues. This expression ascribes an important role to tenascin in the differentiation, especially in the epithelial-mesenchymal interaction. On the contrary, its distribution in adult animals is very resticted, myotendinous junctions being the most conspicuous location. Our results conform, in general, to those in previous reports on chicken muscle,"."32' which all underline the functional specificity of tenascin normally present only at the MTJs. The absence in the endomysium shows that, in intact muscle, tenascin does not contribute to the lateral binding of myofibers to their neighbors," which suggests that tenascin is used only when very strog attachment is required.
Production of Tenascin in Regenerating Muscle.
Abundant accumulation of synthesized tenascin in the trauma zone around the proliferating fibroblasts was already observed on day 2, when the phagocytosis of the necrotized parts of myofibers was still occurring and satellite cells had not yet been activated (on the basis of desmin negativity, data not shown). This indicates that it is the fibroblasts that are responsible for the early synthesis of tenascin. This concords well with the results of Sanes et a].'* and Gatchalian et who demonstrated that fibroblasts, which accumulated around the motor end-plates in muscle after denervation, actively synthesized tenascin already 2 days after the nerve section. Interestingly, tenascin was also abundantly accumulated around the basal lamina of the necrotized muscle fibers, which may well reflect the affinitiy of tenascin to bind with the proteoglycans in the basal lamina. On the basis of the immunohistochemical data, it appears likely that the tenascin around the viable, regenerating muscle cells is also synthesized by the fibroblasts, whose number increases in the endomysium between the stumps. T h e definite localization of the tenascin synthesis must await the results of in situ hybidization (in progress).
MUSCLE 8, NERVE
April 1992
487
Function of Tenascin in Traumatized Muscle. Each of tenascin's 6 arms has structural homology with three functionally different molecules: close to the central knob there is a domain with 13 repeats of epidermal growth factor (EGF)-like sequences. This is followed by 8 to 15 fibronectin type I11 domains and the arm ends, with a terminal knob of fibrinogen-like domain.8 On the basis of this molecular structure and cell-binding studies in vitro, tenascin has been suggested to have two contradictory functions." In healing muscle, the EGF domains of the tenascin molecule may, on one hand, stimulate cell proliferation, possibly that of satellite cells as well, and, on the other hand, the type I11 fibronectin-like sequences can provide firm attachment for the growing myofibers. The two above-mentioned contrary functions are inevitably needed, when the muscle is used before it has completely healed (as a freely moving rat does). At the same time that the myofibers are growing, the regenerating myofibers must be attached to the surrounding connective tissue firmly enough to withstand the contraction force. Our results on the distribution of tenascin immunoreactivity agrees well with this dual function. Newly synthesized tenascin accumulated mainly along the sides of- the regenerating myofibers, i.e., the healing myofibers; on the contrary, the intact fibers exploit tenascin to attach to their surrounding ECM, also laterally. At the same time, such attachment could allow the tip of the regenerating fiber to advance freely through the ECM. T h e ultrastructural findings corroborate this interpretation: the regenerating myofibers had a growth cone at their advancing ends, whereas, at later stages, a MTJ developed at their tips, which most likely indicates stagnation of the growth and the formation of a stronger attachment. Concordantly, the scar tissue between the stumps remained antitenascin-positive until the end of' the observation period, even though it disappeared from the other parts of the scar. T h e localization of tenascin molecules in relation to the basal lamina cannot be discerned at the light microscopic level of resolution. Tenascin's receptor for cell-matrix adhesion has been suggested to belong to the integrin superfamily, with the p-subunit identical to that of receptors for fibronectin (FNR),I7 laminin,I9 and collagen," but with a yet unknown a-subunit dif-
488
Adhesion in Muscle Regeneration
ferent from that of the FNR.' On the other hand, a transmembrane proteoglycan, syndecan, has been proposed to be the receptor molecule for ten a ~ c i n . ' Interestingly, ~ Chiquet-Ehrismann et al.' identified a strong cell-binding site near the tips of the 6 arms of tenascin, which binding could not be blocked by the RGD-sequence peptides. Specific studies on possible tenascin receptors on muscle cells have not been reported. T h e interaction of tenascin with other molecules in the ECM is equally controversial: Erickson and Bourdon' clearly state that tenascin does not bind to collagen, laminin, or fibronectin. The only ECM protei with avidity toward tenascin being chondroitin sulfate proteoglycan.6.1 On the other hand, interactions of tenascin with fibronectin7.' and have been described. The presence of abundant tenascin immunoreactivity along the sides of regenerating myofibers would agree with avidity to fibronectin of the basal lamina.
'
p,-Subunit
Distribution in Regenerating Muscle.
The P-subunit is known to be the same in several inte rin molecules, including fibronectin, l 2 lamininJ9 and tenascin receptors.' The P I-subunit immunoreactivity, discernible as a thin line along the intact myofibers, most likely reflects the binding of sarcolemma to either fibronectin or laminin present in the basal lamina investing each myofiher.' In the MT-J region, the sarcolemma forms multiple folds covered by similarly folded thick basal lamina. In accordance, a band-like thicker PI-subunit ininiunoreactivity was observed at the M'rJs. In this location, and during the regeneration over the lateral aspects of myofibers, some p,-subunits most likely belong to tenascin receptors. Fibronectin is also abundant in the MTJ region. Application of antibodies to the specific a-subunits (so far unknown in the tenascin receptor) will provide the answers to the relative importance of tenascin and fibronectin for myofiber adhesion to ECM. In conclusion, our results show that there are intricate interactions between the regenerating muscle fibers and the surrounding ECM. At the same time that growth of regenerating myofibers through the scar tissue occurs, these are attached to the ECM firmly enough to allow effective contraction of the muscle before the regeneration is com pleted.
MUSCLE & NERVE
April 1992
REFERENCES
1. Alhelda SM, Buck CA: Integrins and other cell adhesion molecules. FASEB J 1990;4:2868-2880. 2. Bourdon MA, Ruoslahti E: Tenascin mediates cell attachmerit through an KGD-dependent receptor. J Cell Biol 1989;108: 1149- 1155. 3. Bozyczko D, Decker C, Muschler J, Horwitz AF: Integrinon developing arid adult skeletal muscle. Exp Cell Re.< l989;183;?2-31. 4. Burgeson RE: New collagens, new concepts. Ann Re71 Crll Rzol 1988;4:551-577. 5. Chiquet M, Famhrough DM: Chick myotendinous antigen. I. A monoclonal antibody as a marker for tendon and muscle morphogenesis. J Cell Biol 1984;98: 1926- 1936. 6. Chiquet M, Fambrough DM: Chick myotendinous antigen. 11. A novel extracellular glycoprotein complex consisting of large disulfide-linked subunits. J Cell Rid 1984;98: 19371946. 7. Chiquet-Ehrismann R, Kalla P, Pearson
TIM0 HURME, MD, and HANNU KALIMO, MD
Transformation oi' the contraction force created by muscles into movement requires firm attachment of the rnyofibers to the structures to be moved. 'lliis at.tachment is mediated by tendons, compact bundles of connective tissue mainly composed of type I ~ o l l a g e nT. ~h e fusion of myofibers with the teridoris is realized by fbrmation of specialized myotendinous .junctions (MTJ), where the cytoskeletal, transmembrane (matrix receptor), a n d extracellular matrix (ECM) proteins interact. In muscle rupture, the functional continuity of the muscle-connective tissue complex is broken, a n d the muscle contract.ion only pulls the stumps of the breached ~nyofibersapart instead of approaching their tendinous cnds. '1'0 restore the functional continuity of tendon- niyofibertendon, the myofibers must regenerate, a n d the stumps of the ruptured fibers rejoined firmly
From the Department of Surgery (Dr Hurme). the Department of Pathology (Dr. Kalimo) and Paavo Nurmi Center (Dr. Hurme), University of Turku and University Central Hospital of Turku. Turku, Finland. Acknowledgments: The skilled technical assistance of Ms.Liisa Lempiainen and Paula Merilahti. and the excellent photography by Mr. Jaakko Liippo is gratefully acknowledged. Dr. lsmo Virtanen is thanked for the antiserum kindly provided to us. We also thank Drs. Markku Jalkane, Eero Vuorio. and Jvriki Heino for their exDert advice and criticism. Address reprint .requests to Tim0 Hurme. MD, Department of Surgery, University of Turku, Kiinamyllynkatu 4-8, SF-20520 Turku, Finland. Accepted for publication Aprili 8, 1991 CCC 0148-639X/92/040482-08 $04.00 0 1992 John Wiley & Sons, Inc
482
Adhesion in Muscle Regeneration
enough to withstand the tension created by the muscle contraction. Thus, optimal healing of muscle rupture necessitates balanced regeneration of both the muscle and connective tissue components.':' In the well-documented experimental model of muscle injury, a strike with a blunt hammer o n rat gastrocnemius causes rupture of myofibers to the depth of about half of the thickness o f the muscle.'5 Disrupted muscle fibers contract, and a gap is formed between the stumps, which is first filled with a hematoma to be gradually replaced by proliferating granulation tissue. T h e regeneration of the necrotized parts of the disrupted muscle fibers occurs completely inside the preserved part of basal lamina cylinder^.'^ When reaching the edge of the ruptured basal lamina, the regenerating muscle cells begin to penetrate through the granulation tissue. Occasional primitive M T j s a r e found at the tips of growing muscle cells, which provide the attachment between the ECM and muscle cell. This growth o f t h e regenerating myofibers through the connective tissue must be directed by intricate interactions between the sarcolemma and components of ECM about which knowledge is still very scant. Because of its abundance in the M T J s , tenascin,.5," is obviously o n e of' the key ECM protein candidates for providing firm anchorage of the myofibers to the connect.ive tissue. T h e attachment of cells to extracellular tenascin has been
'"
'
MUSCLE & NERVE
April 1992
suggested to be mediated by a receptor molecule of the integrin superfamily, which are heterodimers of two transmembrane subunits (Y and p, the latter (PI) being common to several adesion receptors (pl subfamily), including the receptor for fibroectin, l 7 laminin, lYand fibrillar collagen.22 A few analyses of tenascin and integrin P,-subunit have been done on developing chicken skeletal muscle,3v7but we are not aware of studies on their role in muscle regeneration. Fibronectin and laminin are both components of the basal lamina which invests each myofiber, including the MTJ area. Thus, these two basal lamina molecules must also be integral in the attachment of myofibers to the ECM. In this study we have analyzed the distribution of tenascin, cellular fibronectin, integrin p subunit, and laminin immunoreactivities during the healing of skeletal muscle after contusion injury, and correlated these findings with the ultrastructural features. The main focus is to clarify the role of these proteins in the re-establishment of the connection between the stumps of the ruptured myofibers to allow the breached muscle to contract again as a unit. MATERIALS AND METHODS
Fifty adult male Wistar rats were used in this study. A standard partial transverse contusion injury was induced to the left calf of each animal under light ether anesthesia using a spring-loaded hammer." T h e time of traumatization was designated as day 0. During the posttraumatization time, the animals were allowed to move freely. The animals were sacrificed by cervical dislocation under ether anesthesia on days 2, 5, 7, 14, and 21, nine rats at each time-point. Five injured animals served as controls. For immunohistochemical studies, the gastrocnemius muscle was dissected free immediately after death. The muscle was divided in the sagittal midline, and the medial half was snap frozen in freon 22 cooled with liquid nitrogen. Serial 5-pm sections were cut for morphological investigations. Routine sections were stained with modified Herovici."' In the immunohistochemical studies, the following antibodies were used: polyclonal rabbit antihuman tenascin, antihuman integrin P I subunit (both antisera are cross-reactive with corresponding rat antigen, manufacturer's information) and antirat laminin (Telios Pharmaceuticals, San Diego, CA), and monoclonal mouse antibody to human ED fragment of fibronectin (the sequence present only in cellular fibronectin; the an-
Adhesion in Muscle Regeneration
tibody, which also crossreacts with the corresponding rat antigen is a kind gift from Dr. I. Virtanen, see ref. 23). T h e bound primary antibodies were visualized using the appropriate avidin- biotinperoxidase method for either mouse or rabbit antibodies (Vector Laboratories, Burlingame, CA) with diaminobenzidine as the chromogen. Light hematoxylin counterstaining was used to discern cellular structures. For ultrastructural analysis, the gastrocnemius muscle was fixed in freshly prepared 4% phosphate-buffered paraformaldehyde at a standard length, cut sagittally at the midline, refixed with glutaraldehyde, postosmicated, dehydrated, and embedded in Epon. Semithin sections were stained with toluidine blue on the basis of which informative areas were selected. Thin sections were double stained with uranyl acetate and lead citrate, and examined in a Jeol JEM 100 C electron microscope. RESULTS
The light and electron microscopic features of the regenerating myofibers have been described in detail in our previous arti~1e.I~ When the regenerating muscle cells extended out of the old basal lamina cylinders, they began to penetrate the central zone (CZ) on days 5 to 7 as thin branches with the
FIGURE 1. Regenerated myofibers (nos. 1-3) in the RZ on day 7 are bound by a strongly laminin-positive basal lamina up to the growth cone at the tip, where the immunoreactivity is weak or absent (arrows). Antilaminin hematoxylin counterstain (bar-50
+
P-4.
MUSCLE & NERVE
April 1992
483
FIGURE 2. The distal end of the thin regenerating myofiber branch 21 days after the trauma. Myotendinous-junction-like structures have developed at the tips of 3 branches (nos. 1 -3),whereas the fourth (no. 4) has a cytoplasmic tip, which may still be advancing. Tip no. 1 also has cytoplasm with abundant vesicles, which are a sign of growth, even though a focal accumulation of subsarcolemmal material has occurred at the most distal end. Bundles of collagen (C) and thinner filaments, (F, possibly fibrillin) are present in the extracellular space. Myofibrils extend all the way to the tip nos. 2 and 3, and the thin filaments fuse with the dark subsarcolemmal material (bar-1 pm).
structure of myotube. 'These w e r e surrounded by a basal larnina as shown by die strong irrirriuriopositivity with antiserum to rat laminin (Fig. 1) u p to the growth cone (cf. ref. 14, Fig. 7); i.e., the advancing tip of the regenerating muscle cell. From day 7 onward, increasing numbers of the growth cones began to change their appearance. Basal lamina appeared on the outer surface of the sarcolemma, as did a layer of dark material subsarcolemmally, i.e., the tip assumed the appearance of a primitive MTJ (Fig. 2 ) . Both the myofibers and the endomysial structures were clearly negative with aiititenascin antibody both in the controls and in the surviving zone (SZ) of the injured rnuscle. Tenascin immunoreactivity was found only at myotendirious junctions as a thick, irregular band, which fused with the positively stained tendon o r intramuscular fascia (Fig. 3 ) . On day 2 , strong imniuriopositivity fbr tenascin was observed, as diffuse staining of the matrix in the granulation tissue in the CZ. In the regeneration zone (KZ) o n &y 5, antitenas& iInmLlnol-eactivity decorated the basal lamiria cylinclers filled l)y macrophages, which had phagocytosetl the necrotized part of the rnyofibers (Fig. 4). Definite inimunoreactivity also extended along the outer sur-
face of ruptured niyofibers proximally to their presei-ved parts in the SZ, where the endorn ysial space was widened a n d usually contained reactive cells (macoptiages and fibroblasts) (Fig. 5). O n day 5, much of the phagocytosis had been conipleted and inyoblast proliferation within the basal lamina cylinders had begun. When the regeneration progressed and myo-
Tenascin Immunoreactivity.
484
Adhesion in Muscle Regeneration
FIGURE 3. Myotendinous junctions at the attachment site of the myofibers to the intramuscular fascia (F) in the surviving zone of a rat 14 days after the trauma. Note the presence of tenascin immunoreactivity only at the MTJs (two marked with arrow), AntitenaScin + whereas the SarcOlemmais otherwise negative, hematoxylin counterstain (bar-100 pm)
MUSCLE & NERVE
April 1992
FIGURE 4. The old preserved basal lamia cylinders of the necrotized parts of myofibers in the RZ are delineated by tenascin immunoreaction product (arrows), which is also present on the preserved part of a myofiber (arrowhead). The extracellular matrix also stains weakly, but as a definite positive (asterisk). Antitenascin + hematoxylin counterstain (bar-100 km).
FIGURE 6. The regenerated myofibers on day 21 end in the connective tissue, which stains strongly tenascin-positive, Tenascin is also seen along the myofibers (arrows). The scar tissue on the right not associated with myofibers is tenascin-negative (asterisk). Antitenascin + hematoxylin counterstain (bar-100
wm).
tubes extended out of the preserved old basal lamina cylinders on days 7 to 14, these frequently formed ramified muscle cells, which pierced through the ECM of the CZ. Antitenascin positivity persisted on the sarcolemma of these myofiber branches, with accentuation at their growing tips as well as in the thickened endomysium of the regenerated parts of myofibers, until the end of the observation period on day 2 1. T h e surface of the intact myofibers in the SZ remained antiteascin negative (cf. Fig. 5 ) . In the connective tissue component of the healing scar, the septum between the stumps approaching each other retained its
FIGURE 5. A transverse section through the SZ close to the trauma on day 14. The endomysial space is widened. Tenascin irnmunoreactivity is present on the surviving parts of the myofibers, often on one side only (arrows), as well as a reticular network around the cells in the endomysial space. Note that fibers obviously farther from the lesion on the right are negative. Antitenascin + hematoxylin counterstain (bar-100 km).
Adhesion in Muscle Regeneration
FIGURE 7. (A) The preserved parts of the myofibers in the SZ are bound by a thin delicate line of integrin p,-subunit immunoreactivity. (B)In the MTJs, the irnmunoreactivity is accentuated, whereas in the underlying fascia to which the myofibers attach only thin streaks around fibroblasts are visible. The wall of a small artery (asterisk) is also strongly integrin &-subunit positive. Anti-integrin &-subunit + hematoxylin counterstain (bar100 pn).
MUSCLE & NERVE
April 1992
485
immunoreactivity, whereas the tenascin staining seemed to disappear from that connective tissue which did not associate with the regenerating myofibers (Fig. 6). Immunoreactivity for integrin P,-subunit in control animals, and in the SZ of injured rats, was identified around the intact myofibers as a thin delicate line (Fig. 7A). At the myotendinous junctions, the reactivity was stronger and formed broad bands, but in the adjoining tendon it was present only as streaks surrounding the fibroblasts within the dense connective tissue (Fig. 7B). Two days after the injury, integrin p,-subunit immunoreactivity in the RZ on the necrotized lntegrin p,-Subunit.
parts of the myofibers had more or less disappeared (Fig. 8). I t was observed on the proliferating fibroblasts between the basal lamina cylinders and on cells (endothelial and pericytic) of thc reconstructing capillary network, whereas the ECM between the cells did not stain. This basic staining pattern persisted throughout the regeneration period. O n day 14, the immunoreactivity on the regenerated parts of the myofibers was still stronger than that in the SZ, and a broad irnmunopositive band was often seen surrounding the tips of the myofiber stumps, most probably reflecting the folding of sarcolemma when M'rJs were formed (Fig. 9). In the intact muscle of the control rats and in the SZ of the injured gastronemius muscle, positive reaction with antibody to the cellular fibronectin was see in the epi- and perimysium arid around the individual muscle fibers with accentuation in the MTJ, whereas the sarcoplasm of all myofibers was negative (Fig. 10). During the healing process, the connective tissue formed in the CZ and that extending between the regenerating myofibers in the RZ, was clearly positive throughout the exaniination period (Fig. 1 l), though some decease in the staining seemed to occur at the later stages.
Cellular Fibronectin.
Laminin. In the controls and SZ of injured rats, expected positive reaction with antirat laminin antiserum was seen around individual muscle cell. At the in.jury site, the necrotiLed parts were surrounded with a thickened positive line corresponding to the corrugation of the ruptured basal
FIGURE 8. (A) On day 2, the necrotized parts of the myofibers have been phagocytosed by macrophages, which fill the preserved basal lamina cylinders (one marked with an arrow). Because the basal lamina of the necrotized parts becomes corrugated it appears thicker than that of the preserved fibers (arrowhead). (B)A nearby section demonstrates the loss of immunoreactivity for integin p,-subunit around the necrotic myofibers, which appear here as clusters of macrophages (two marked with arrows). The preserved fibers are demarcated by a thin line of integrin @,-subunitirnrnunoreactivity.(A) Antilarninin + hernatoxylincounterstain, (B) Anti-integrin p,-subunit + hematoxylin counterstai (bar-100 prn).
486
Adhesion in Muscle Regeneration
FIGURE 9. The sarcolernma of tne regenerating myofibers stains strongly positive with anti-integrin p,-subunit serum, with accentuation at their tips (arrows) on day 14. Anti-integrin p,subunit + hematoxylin counterstain (bar-50 km).
MUSCLE 8, NERVE
April 1992
FIGURE 10. MTJ region from the SZ on day 2. lmmunoreactivity for cellular fibronectin delineates all muscle fibers and is accentuated at the MTJs (arrows). The blood vessels (arrowheads) are also strongly positive, whereas the ECM stains more weakly. Anticellular fibronectin hematoxylin counterstain (bar-50 pm).
+
lamina cylinder (cf. Fig. 8A). T h e regenerating muscle cells extending into the connective tissue of the C Z were surrounded by a delicate thin line, which seemed to be lacking at the very tip (growth cone) of most regenerating fibers (cf. Fig. 1). When the regnerating ends formed the M'lJs, a broad band of laminin immunoreactivity appeared in concordance with the EM results, which showed a thick, well-developed basal lamina at the MTJ (cf. Fig. 2). DISCUSSION
Tenascin was discovered, from different tissues under various names (e.g., myotendinous antigen,
FIGURE 11. The newly formed patchy MTJs at the tips of the regenerating myofibers, on day 14, stain as strongly as their normal counterparts (arrows). The blood vessels and the surrounding ECM stain similarly as in the normal MTJ region. Anticellular fibronectin + hernatoxylin counterstain (bar-50 pm).
Adhesion in Muscle Regeneration
cytotactin, hexarachion, glioma-mesechymal extracellular matrix antigen) at about the same time by several workers (see ref. 8). Among the first researchers were Chiquet and Fambrough,"." who isolated "myotendinous antigen" from developig chicken muscle-tendon complex. Later studies revealed that "myotedinous antigen" is a major ECM glycoprotein, a disulfide-linked hexamer with molecular weight larger than 1000 kD. Tenascin is abundantly expessed during morphogenesis in many different tissues. This expression ascribes an important role to tenascin in the differentiation, especially in the epithelial-mesenchymal interaction. On the contrary, its distribution in adult animals is very resticted, myotendinous junctions being the most conspicuous location. Our results conform, in general, to those in previous reports on chicken muscle,"."32' which all underline the functional specificity of tenascin normally present only at the MTJs. The absence in the endomysium shows that, in intact muscle, tenascin does not contribute to the lateral binding of myofibers to their neighbors," which suggests that tenascin is used only when very strog attachment is required.
Production of Tenascin in Regenerating Muscle.
Abundant accumulation of synthesized tenascin in the trauma zone around the proliferating fibroblasts was already observed on day 2, when the phagocytosis of the necrotized parts of myofibers was still occurring and satellite cells had not yet been activated (on the basis of desmin negativity, data not shown). This indicates that it is the fibroblasts that are responsible for the early synthesis of tenascin. This concords well with the results of Sanes et a].'* and Gatchalian et who demonstrated that fibroblasts, which accumulated around the motor end-plates in muscle after denervation, actively synthesized tenascin already 2 days after the nerve section. Interestingly, tenascin was also abundantly accumulated around the basal lamina of the necrotized muscle fibers, which may well reflect the affinitiy of tenascin to bind with the proteoglycans in the basal lamina. On the basis of the immunohistochemical data, it appears likely that the tenascin around the viable, regenerating muscle cells is also synthesized by the fibroblasts, whose number increases in the endomysium between the stumps. T h e definite localization of the tenascin synthesis must await the results of in situ hybidization (in progress).
MUSCLE 8, NERVE
April 1992
487
Function of Tenascin in Traumatized Muscle. Each of tenascin's 6 arms has structural homology with three functionally different molecules: close to the central knob there is a domain with 13 repeats of epidermal growth factor (EGF)-like sequences. This is followed by 8 to 15 fibronectin type I11 domains and the arm ends, with a terminal knob of fibrinogen-like domain.8 On the basis of this molecular structure and cell-binding studies in vitro, tenascin has been suggested to have two contradictory functions." In healing muscle, the EGF domains of the tenascin molecule may, on one hand, stimulate cell proliferation, possibly that of satellite cells as well, and, on the other hand, the type I11 fibronectin-like sequences can provide firm attachment for the growing myofibers. The two above-mentioned contrary functions are inevitably needed, when the muscle is used before it has completely healed (as a freely moving rat does). At the same time that the myofibers are growing, the regenerating myofibers must be attached to the surrounding connective tissue firmly enough to withstand the contraction force. Our results on the distribution of tenascin immunoreactivity agrees well with this dual function. Newly synthesized tenascin accumulated mainly along the sides of- the regenerating myofibers, i.e., the healing myofibers; on the contrary, the intact fibers exploit tenascin to attach to their surrounding ECM, also laterally. At the same time, such attachment could allow the tip of the regenerating fiber to advance freely through the ECM. T h e ultrastructural findings corroborate this interpretation: the regenerating myofibers had a growth cone at their advancing ends, whereas, at later stages, a MTJ developed at their tips, which most likely indicates stagnation of the growth and the formation of a stronger attachment. Concordantly, the scar tissue between the stumps remained antitenascin-positive until the end of' the observation period, even though it disappeared from the other parts of the scar. T h e localization of tenascin molecules in relation to the basal lamina cannot be discerned at the light microscopic level of resolution. Tenascin's receptor for cell-matrix adhesion has been suggested to belong to the integrin superfamily, with the p-subunit identical to that of receptors for fibronectin (FNR),I7 laminin,I9 and collagen," but with a yet unknown a-subunit dif-
488
Adhesion in Muscle Regeneration
ferent from that of the FNR.' On the other hand, a transmembrane proteoglycan, syndecan, has been proposed to be the receptor molecule for ten a ~ c i n . ' Interestingly, ~ Chiquet-Ehrismann et al.' identified a strong cell-binding site near the tips of the 6 arms of tenascin, which binding could not be blocked by the RGD-sequence peptides. Specific studies on possible tenascin receptors on muscle cells have not been reported. T h e interaction of tenascin with other molecules in the ECM is equally controversial: Erickson and Bourdon' clearly state that tenascin does not bind to collagen, laminin, or fibronectin. The only ECM protei with avidity toward tenascin being chondroitin sulfate proteoglycan.6.1 On the other hand, interactions of tenascin with fibronectin7.' and have been described. The presence of abundant tenascin immunoreactivity along the sides of regenerating myofibers would agree with avidity to fibronectin of the basal lamina.
'
p,-Subunit
Distribution in Regenerating Muscle.
The P-subunit is known to be the same in several inte rin molecules, including fibronectin, l 2 lamininJ9 and tenascin receptors.' The P I-subunit immunoreactivity, discernible as a thin line along the intact myofibers, most likely reflects the binding of sarcolemma to either fibronectin or laminin present in the basal lamina investing each myofiher.' In the MT-J region, the sarcolemma forms multiple folds covered by similarly folded thick basal lamina. In accordance, a band-like thicker PI-subunit ininiunoreactivity was observed at the M'rJs. In this location, and during the regeneration over the lateral aspects of myofibers, some p,-subunits most likely belong to tenascin receptors. Fibronectin is also abundant in the MTJ region. Application of antibodies to the specific a-subunits (so far unknown in the tenascin receptor) will provide the answers to the relative importance of tenascin and fibronectin for myofiber adhesion to ECM. In conclusion, our results show that there are intricate interactions between the regenerating muscle fibers and the surrounding ECM. At the same time that growth of regenerating myofibers through the scar tissue occurs, these are attached to the ECM firmly enough to allow effective contraction of the muscle before the regeneration is com pleted.
MUSCLE & NERVE
April 1992
REFERENCES
1. Alhelda SM, Buck CA: Integrins and other cell adhesion molecules. FASEB J 1990;4:2868-2880. 2. Bourdon MA, Ruoslahti E: Tenascin mediates cell attachmerit through an KGD-dependent receptor. J Cell Biol 1989;108: 1149- 1155. 3. Bozyczko D, Decker C, Muschler J, Horwitz AF: Integrinon developing arid adult skeletal muscle. Exp Cell Re.< l989;183;?2-31. 4. Burgeson RE: New collagens, new concepts. Ann Re71 Crll Rzol 1988;4:551-577. 5. Chiquet M, Famhrough DM: Chick myotendinous antigen. I. A monoclonal antibody as a marker for tendon and muscle morphogenesis. J Cell Biol 1984;98: 1926- 1936. 6. Chiquet M, Fambrough DM: Chick myotendinous antigen. 11. A novel extracellular glycoprotein complex consisting of large disulfide-linked subunits. J Cell Rid 1984;98: 19371946. 7. Chiquet-Ehrismann R, Kalla P, Pearson
