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Regional injury and the terminal differentiation of satellite cells in stretched avian slow tonic muscle
DEVELOPMENTAL
BIOLOGY
151,
459-472
(19%)
Regional Injury and the Terminal Differentiation of Satellite Cells in Stretched Avian Slow Tonic Muscle P.K. WINCHESTERAND Department
of Cell Biology and Neuroscience,
The University
W.J. GONYEA~ of Texas Southwestern
Accepted February
Medical
Center, Dallas, Texas 75235
25, 1992
In the avian stretch model, the application of a weight overload to the humerus induces enlargement of the anterior latissimus dorsi (ALD) muscle and an increase in muscle fiber number which is accompanied by satellite cell activation. Myofiber injury may be an important stimulus to muscle fiber hyperplasia; therefore, light and electron microscopic evaluation was undertaken to determine if myofiber injury occurs in the stretch-enlarged ALD muscle of the adult quail. Autoradiographic studies were used to determine the terminal differentiation of labeled myogenic cells. A weight equal to 10% of body mass was attached to one wing of 2’7adult quail and 3 birds were euthanized at 9 intervals of stretch, from 1 to 30 days. Birds were injected with tritiated thymidine at intervals ranging from 1 hr to 3 days prior to euthanization. Labeled nuclei were detected by light microscopic examination and identified by electron microscopy of a serial section. Three regions of the muscle wore examined for disorganization of contractile elements, presence of cytoplasmic vacuoles, and/or phagocytic cell infiltration. The percentage of fibers exhibiting one or more of these criterion was significantly greater in the stretched ALD by Days 5 and 7 and declined at Day 10, reaching near control values by Day 14. Myofiber necrosis and phagocytic cell infiltration were only observed in the middle and distal regions of the stretched ALD muscle. Traditional signs of regeneration and repair were observed, including clusters of labeled myoblast-like cells and myotube formation within an existing basal lamina. New myotube formation with labeled central nuclei was also noted in the interstitial space, outside of basal lamina of persisting fibers. Labeled myonuclei were observed in the stretched fibers. These results demonstrate that chronic stretch produces regional injury and fiber degeneration and resultant regeneration in the ALD muscle of the adult quail. This may be an important stimulus for new fiber formation in this model. 0 15x12 Academic Press, Inc.
In the avian stretch model, the application of a weight overload to the humerus induces new fiber formation and fiber hypertrophy of the anterior latissimus dorsi (ALD) muscle (Sola et al, 1973; Kennedy et ok, 1988; Alway et ah, 1989,199O). Satellite cells have been shown to be activated in the stretched-enlarged ALD muscle from young chicken (Kennedy et ah, 1988) and adult quail (Winchester et al, 1991a), It has been proposed that satellite cells proliferate and differentiate to form new fibers in the stretched avian muscle (Kennedy et ah, 1988; Alway et ak, 1989, 1990; Winchester et ak, 1991a). Satellite cells play an important role during postnatal muscle growth by fusing with growing myofibers to increase the number of true myonuclei (Moss and Leblond, 1971). This mechanism may also occur in the hypertrophic fibers of the stretched adult muscle as suggested in the compensatory hypertrophy model (Salleo et al, 1983).
1To whom reprint requests should be sent at Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical School, 5323 Harry Hines Blvd., Dallas, TX 75235.
Activation and proliferation of satellite cells are well known to be associated with injury to the myofibers. The progenies of these activated satellite cells are assumed to give rise to a population of myogenic precursor cells that replaces necrotic myofibers in the regenerating muscle (reviewed in Campion, 1984). Although certain growth factors and hormones have been shown to have a mitogenic effect in vitro (reviewed in Grounds, 1991), little is known about the factors that directly or indirectly control the activation and proliferation of satellite cells in viva. Bischoff (1990a) has recently demonstrated that satellite cells can be stimulated to proliferate on rat myofibers killed with Marcaine, but not on viable myofibers. This suggests that the proliferative response of satellite cells to mitogens is depressed in the presence of an intact plasmalemma. In view of this, we hypothesized that stretch would result in muscle fiber injury and necrosis in the enlarged ALD muscle of the adult quail and this may be an important stimulus for muscle fiber hyperplasia observed in this model. Other investigators have examined structural changes seen in the stretched avian muscle (Khan, 1986; Ashmore et al, 1988; Kennedy et al, 1988). Kennedy and
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(D 1892 by Academic Press, Inc. of reproduction in any form reserved.
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FIG. 1. Percentage of change in muscle mass in the stretched ALD muscle relative to the intra-animal control muscle at nine different intervals of stretch. Bars represent means f SE. Stretched muscle mass was significantly greater than the contralateral muscle mass after 5, 7, 10, 14, 21, and 30 days of stretch. *P < 0.05.
colleagues (1988) reported myofibril disruption and Z band alterations in the stretched ALD muscle of young chicken. They suggested that this type of injury did not lead to muscle fiber death but could play a role in the activation and proliferation of myogenic precursor cells and resultant formation of nascent fibers. In contrast to this, Ashmore and co-workers (1988) reported the necrosis, vacuolation, and infiltration of phagocytic cells in the stretched patagialis (PAT) muscle of adult chicken. If muscle fiber death is observed in the stretched avian muscle, then the progeny of activated satellite cells would play an important role in the regeneration of the necrotic fibers. Before the role of satellite cells can be determined in the stretched avian muscle, autoradiographic studies following the terminal differentiation of labeled myogenic precursor cells would need to be done before conclusions could be drawn concerning their contribution to muscle enlargement in this model. The present study was undertaken to quantify pathological changes in the stretch-enlarged ALD muscles of adult quail. Ultrastructural studies using autoradiographic techniques of thymidine-labeled cells were conducted to trace the terminal differentiation of satellite cells in the stretched ALD muscle. Part of this work has appeared in abstract form (Winchester et al., 1991b).
50 * 1 II i 0123
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DAYS OF STRETCH FIG. 2. Percentage of total fibers counted that were injured in the proximal, middle, and distal regions of the control and stretched ALD muscles at nine different intervals of stretch. The control data from the nine intervals of stretch has been pooled and plotted at Day 0 of stretch. Bars represent means + SE. Percentage of injured myofibers in the stretched muscle is significantly greater than control values in the middle region at Days 5 and 7 and in the distal region at Days 5,7, and 10 days of stretch. *P < 0.05.
MATERIALS
Animal
AND METHODS
Procedures
Six-week-old adult male Corturnix quail (Truslow Farms, MA) were obtained for this study. The quail were housed one per cage with 12-hr cycles of light. Quail were fed Purina 18% protein turkey starter and water ad l&turn. The birds were weighed daily for a minimum of 2 weeks to ensure stable body weights prior to the experimental period. Hypertrophy was induced in the ALD muscles by the application of a cuff weight
FIG. 3. Light micrographs of a group of fibers in cross section followed at 200-pm intervals through the middle region of an ALD muscle that had been stretched for 5 days. Several of the fibers show degenerative changes. Toluidine blue stain was used. One fiber (asterisks) has been followed and shows a spectrum of degenerative changes. (A) The fiber appears relatively normal. (B) The fiber shows vaeuolation. (C) Contractile protein is disrupted and disorganized. (D) Only a basal lamina tube remains. Magnification, ~588; bar, 50 pm.
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at the same lengths. The apex of the ventricle was cannulated and the bird was perfused with 50 ml minimal essential medium (MEM, GIBCO Industries, NY), followed by 50 ml 2% glutaraldehyde (Ladd Research Industries, VT) in 0.1 M cacodylate buffer (pH 7.2-7.4). The ALD muscles were removed, cleaned of excessive connective tissue, weighed, and then stored in fresh fixative at 4°C. Autoradiography
and Electron Microscopy
Bundles of fibers (200-500 fibers) were gently teased from each muscle, rinsed in cacodylate buffer, postfixed in 1% osmium tetroxide, and stained overnight in 0.5% uranyl acetate. Muscle bundles were dehydrated in graded alcohols and embedded in Epon (Ladd Research Industries). The embedded muscle fiber bundles were cut transversely into 1 mm segments and mounted in cross section on Epon blocks. In some muscles, additional bundles were mounted longitudinally for sectioning. Two thick sections (0.5 and 1.0 pm) were cut from
FIG. 4. A degenerating fiber from the distal region of a muscle that had been stretched for 5 days. Contractile protein is foamy; membrane bound vacuoles (v) and a pyknotic nuclei (pn) can be seen. MagniAcation, x 3920; bar, 4 am.
filled with lead pellets secured proximal to the humeroulnar joint of the right wing of each bird. The mass of the cuff weight corresponded to IO% of the bird’s body mass. The wing weight was maintained for 1, 2, 3, 5, 7, 10,14,21, or 30 days, The unweighted left wing served as an intra-animal control. The animals tolerated the wing weight well, without change in their daily activity. Body weight did not change over the experimental period (pre, 162.9 + 14.9 g vs post, 153.4 f 15.3 g). Birds received either a single injection of tritiated thymidine ([*HlTdR) (sp act 5 Ci/mmole, ICN Radiochemicals, Irvine, CA) at a dosage of 2 &i/g body weight or two injections each at a dosage of 1.0 &i/g body weight. Birds survived up to 3 days postinjection before being euthanized. By limiting the time between injection and euthanization to 3 days, problems with label dilution was minimized (McGeachie and Grounds, 198’7). Three birds from each of the nine intervals of stretch were anesthetized by an intramuscular injection of Nembutal (0.15 mg/g body weight), Each bird was injected with heparin (1.0 IU/g body weight) and wings were pinned to ensure that the ALD muscles were fixed
FIG. 5. Two degenerating fibers from the middle region of a muscle that had been stretched for 3 days. One fiber has been infiltrated by red blood cells. Magnification, x2000; bar, 4 Grn.
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the muscle. A minimum of 500 fibers were counted in three regions of the control and stretched ALD muscles. Samples from three control and experimental muscles at nine different intervals of stretch were analyzed under an oil immersion lens (x600). Criteria for myofiber injury included the following: loss of organization of the contractile elements; foamy, vacuolated cytoplasm; and/or presence of phagocytic cell infiltration. The percentage of injured myofibers was expressed as a percentage of total fibers counted in each region of the control and stretched ALD muscles. Electron microscopy was also used for ultrastructural examination of fibers identified as injured with light microscopy. In one muscle, serial sections were taken every 8 Frn from one block from the middle region of a muscle that had been stretched for 5 days to examine if the degree of fiber degeneration varied along the length of the muscle. Statistical Analysis
FIG. 6. Myofiber is from the middle region of a muscle that had been stretched for 7 days. Macrophage has infiltrated the basal lamina (arrow) of the myofiber. Magnification, x9800; bar, 1 pm.
each block. The first section, stained with toluidine blue, was used for identification and quantitative analysis of injury in the control and stretched ALD muscles. The second section was mounted on a subbed glass slide with a section of the gut. After drying, the slides were dipped in a 50% solution of Kodak NTB2 emulsion (EastmanKodak, Rochester, NY) and distilled water. The slides were dried and placed in light-tight boxes and exposed for 4 weeks at 4°C. Exposed slides were developed and stained with toluidine blue or methylene azure blue. The autoradiographic sections were viewed under an oil immersion lens (X600) for identification of labeled nuclei. Serial thin sections (600-900 A thick) were taken and stained with uranyl acetate and lead citrate and analyzed in a JEOL 1OOCXor JEOL 1200 electron microscope. Labeled nuclei were identified in the serial ultrathin sections using photographic montages of the adjacent thick section for orientation. Fiber Injury For quantitative analysis of muscle fiber injury, two sections were randomly selected from each third of the control and stretched ALD muscles and designated as coming from the proximal, middle, or distal region of
Descriptive statistics included means f SE. Change in stretched muscle mass was expressed as a percentage of the contralateral control muscle. The percentage of change was tested for differences due to duration of stretch using the Kruskal-Wallis one-way analysis of variance by ranks. To determine if differences existed in the number of injured muscle fibers in the proximal, middle, and distal regions of the control and stretched muscles, a repeated measures analysis of variance was conducted. These results warranted multiple pairwise comparisons to test for significant differences between the number of injured myofibers in control and stretched muscles at each time interval. RESULTS
Stretched muscle mass was significantly greater than the intra-animal control after 5,7,10,14,21, and 30 days of stretch (Fig. 1). Mass of the 5-day stretched ALD muscle was 33.6 + 7.1% greater than the contralateral control muscles. Mass of the stretched muscles continued to increase at all subsequent time intervals studied, so that by Day 30 of stretch, mass of the stretched ALD muscle was 115.3 + 8.0% greater than the contralateral control muscles. Fiber Injury Figure 2 illustrates the percentage of total fibers counted that were injured in the proximal, middle, and distal regions of the control and stretched ALD muscles at nine different intervals of stretch. Very few fibers were classified as injured in any of the control muscles; therefore, the control data from the nine intervals of stretch was pooled and plotted at Day 0 of stretch. The
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FIG. 7. Electron micrograph of longitudinally sectioned ALD muscle stretched for 3 days. (A) Focal areas of sarcomere hypercontraction, and (B) Z band streaming are noted. Magnification, X5300; bar, 2 pm.
mean percentage of injured fibers was 0.3 -t 0.4% in the proximal, 0.2 f 0.2% in the middle, and 0.1 ? 0.1% in the distal regions of the contralateral control muscles. The percentage of injured myofibers in the stretched muscle was significantly greater than control values in the middle region at Days 5 (15.8 k 15.1%) and 7 (25.4 t 20.4%) and in the distal region at Days 5 (49.0 f 24.8%), 7 (30.4 + 14.2%), and 10 (26.9 * 13.6%) of stretch. At later times of stretch, the percentage of injured fibers declined, such that by Day 30 the mean percentage of injured fibers was 0.6 + 0.3% in the proximal, 0.5 f 0.2% in the middle, and 1.9 f 1.2% in the distal regions of the stretched muscles. Signs of myofiber degeneration, such as phagocytic cell infiltration, pyknotic nuclei, and loss of contractile elements, were only observed in the middle and distal regions of the stretched ALD muscle. Sections taken for light microscopic analysis frequently demonstrated normal muscle architecture in a l-mm block of tissue and extensive areas of disruption and interstitial edema in the adjacent l-mm block of muscle. Figure 3 shows a group of fibers followed at 200-pm intervals through the
middle region of a muscle that had been stretched for 5 days. One fiber has been followed and shows a spectrum of degenerative changes. This fiber appears relatively normal (Fig. 3A); its plasmalemma appears to be intact and nearly all contractile elements are organized. In the next section (Fig. 3B), 200 pm away, the fiber shows vacuolation. In Fig. 3C, the contractile protein is disrupted and disorganized. Only a basal lamina tube remains in Fig. 3D. Electron microscopy was used to confirm the light microscopic analysis of stretch-induced muscle fiber injury of cross sections of degenerating fibers in stretched ALD muscles. Frequently, extensive areas of sarcolemma disruption was observed but the basal lamina was intact. In these degenerating fibers, contractile protein was foamy with membrane-bound vacuoles, and often pyknotic nuclei were observed (Fig. 4). Infiltrating cells were often observed in the injured and necrotic fibers of the stretched muscle. Figure 5 demonstrates a degenerating fiber that had been infiltrated by red blood cells and Fig. 6 shows a macrophage that has infiltrated the basal lamina of a necrotic myofiber. Electron
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cell was widened and contained radiolucent membranous inclusions (Figs. 12 and 13). Occasionally, these activated satellite cells were no longer contained entirely within the basal lamina of the myofiber. These satellite cells were often associated with other cells located in the interstitium that had a similar morphology to satellite cells but that were not contained within a basal lamina of a myofiber (Fig. 13). In some instances, these interstitial cells were labeled (Fig. 13, inset). These cells were not observed in the control muscles. Terminal
D$erentiation
of Myogenic
Cells
Evidence of myofiber regeneration was seen, particularly from Days 5 through 10 of stretch (Fig. 14A). Clusters of myoblasts were observed within a persisting
FIG. 8. Electron micrograph of a cross section through a muscle that had been stretched for 1 day. An activated satellite cell (arrow) is beneath the basal lamina of the muscle fiber. Its cytoplasm contains numerous mitochondria and extensive Golgi complex. Magnification, x5194; bar, 2 pm. The inset is a serial section of the same area in a light microscopic autoradiograph. This bird received a single injection of [3H]TdR 2 hr prior to surgery. Note the satellite cell is labeled.
micrographs of longitudinally sectioned stretched ALD muscles revealed focal areas of sarcomere hypercontraction (Fig. ‘7A) and Z band streaming (Fig. ‘7B) in muscles that had been stretched for l-5 days. Satellite Cell Activation
As early as Day 1 of stretch, labeled, activated satellite cells were observed between the basal lamina and plasmalemma of stretched myofibers (Fig. 8). These cells contained a higher cytoplasmic-to-nuclear ratio with a euchromatic nucleus. Satellite cell activation was often observed to be associated with fiber injury in the first week of stretch. Figure 9 demonstrates an activated satellite cell in one myofiber from an ALD muscle that had been stretched for 5 days. In an adjacent myofiber, a labeled leucocyte that had infiltrated the myofiber was observed. Caveolae were often noted both on the external and internal plasma membrane of the activated satellite cells (Fig. 10). In the first week of stretch, the basal lamina of the myofiber was observed in the space between the myofiber and the satellite cell (Fig. 11). Beginning with Day 7 of stretch, activated satellite cells were often observed to be closely apposed to a cytoplasmic tail of another myogenic cell (Fig. 12). In many cases, the space between the myofiber and the satellite
FIG. 9. Electron micrograph of a cross section through a muscle that had been stretched for 5 days. An activated satellite cell (SC)is beneath the basal lamina of one of the muscle fibers. In an adjacent myofiber a leucocyte (lc) has invaded the fiber. Magnification, x2700; bar, 4 pm. The inset is a serial section of the same area in a light microscopic autoradiograph. This bird received two injections of [aHITdR 1 and 2 days prior to surgery. Note that the satellite cell and leucocyte are labeled (arrowheads).
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peared to have normal organization of their contractile elements with no necrotic signs. Furthermore, examination revealed only one basal lamina that was closely ap: posed to the sarcolemma. These large hypertrophic fibers often demonstrated a peripheral halo of myoplasm that was rich in organelles, but lacked any contractile protein. DISCUSSION
This study examined satellite cell activation and the terminal differentiation in the chronically stretch-enlarged ALD muscles of adult quail. The avian stretch model produces rapid and substantial gains in muscle mass (Sola et ab, 1973; Gollnick et ah, 1983; Kennedy et ab, 1988; Alway et ah, 1989; Winchester et al, 1991a). The mass increases observed in this study were similar to those described in an earlier study (Winchester et al., 1991a), except that significant increases in muscle mass were not achieved until Day 5 of stretch compared to Day 3 of stretch in the prior study. This could be due to the smaller sample size used in this study. Muscle mass continued to increase throughout the duration of this study.
FIG. 10. Electron micrograph of a satellite cell in an ALD muscle that had been stretched for days. The nucleus is euchromatic with a prominent nucleolus and caveolae are noted (arrow) on the plasma membrane of the cell. Magnification, ~20,000; bar, 500 nm.
basal lamina tube that was adjacent to the new basal lamina (Fig. 14B). These regenerating myofibers characteristically contained a high incidence of central and eccentric nuclei, some of which were labeled (Fig. 14, inset). Small myotubes or myofibers were observed in the interstitial spaces of the stretched muscles. In contrast to the morphology of regenerating fibers, these fibers were surrounded by only one basal lamina closely adhered to the plasmalemma. Frequently, these new myotubes contain a series of central nuclei, surrounded by loosely organized contractile protein (Fig. 15). Groups of myogenic cells contained within a common basal lamina were also observed in the interstitial spaces of the stretched muscles. In some cases, some of the myogenic cells contained loosely organized contractile protein (Fig. 16). Labeled central or eccentric myonuclei were observed in some of the newly formed small myofibers FIG. 11. Electron micrograph of an activated satellite cell in a mus(Fig. 17). cle that had been stretched for 7 days. Basal lamina has invaded the Labeled peripheral myonuclei were observed within space between the myofiber and the satellite cell (arrows). Magnificalarge hypertrophic fibers (Fig. 18). These fibers ap- tion, x9800; bar, 1 pm.
WINCHESTER AND GONYEA
FIG. 12. An activated satellite cell closely apposed to a cytoplasmic tail of another muscle cell (asterisk). The satellite cell demonstrates a high nuclear-to-cytoplasmic ratio with a euchromatic nucleus. The space between the myofiber and the cell is widened and contains radiolucent membranous inclusions. Part of a myonuclei is noted in the left lower hand corner. Magnification, X6499; bar, 2 pm.
In order to evaluate the role of the satellite cell in an enlarging muscle, it was first important to determine whether chronic stretch resulted in fiber injury because satellite cell activation has been associated with muscle fiber degeneration and regeneration in the adult animal (Schmalbruch, 1976; Snow, 1977a,b). Other investigators have examined morphological changes in the avian stretched muscle, but their assay was either limited to the light microscopic level (Ashmore et al, 1988) or not quantified in different regions of the stretched muscle (Khan, 1986; Kennedy et ah, 1988). Myofibers exhibiting signs of injury were significantly greater in the stretched muscles compared to the contralateral control muscles in the middle region at Days 5 and 7 and in the distal region at Days 5, 7, and 10 days of stretch. Since the extent of injury was quantified at the light level, it is possible that structural changes at earlier days were missed due to the resolution limits of light microscopy. In those muscles that were examined at the ultrastructural level, we did observe some minor degree of injury at the earlier time intervals of stretch. These changes included 2 band streaming and myofilament disruption.
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Regardless of the onset of muscle fiber injury, we concluded that significant injury does occur in the overloaded muscle in the first 10 days of stretch. Many of the morphological changes observed in this study were typical of those described with compensatory hypertrophy (James, 1976; Snow, 1990), eccentric activity (Armstrong et ab, 1983; Friden, 1984), marathon running (Hikida et ah, 1983), and prolonged high-resistance exercise (Giddings et aZ.,1985, in press). Z band alterations previously described in the stretched teres minor muscle of adult chicken (Khan, 1986) and stretched ALD muscle of young chicken (Kennedy et al, 1988) were also observed in this study (Fig. 7). Additional morphological changes included true signs of muscle fiber necrosis, as described in the stretched PAT muscle of adult chicken (Ashmore et ab, 1988). Disruption of the sarcolemma, presumably caused leakage of muscle cell contents which appeared to have activated macrophages (Fig. 6) and attracted neutrophils and leucocytes (Fig. 9) to participate in phagocytosis. Erythrocytes were noted in the extravascular spaces, but more unusual, they were also observed within the injured muscle fibers (Fig. 5). This phenomena has been reported in the gastrocnemius muscle in marathon runners (Hikida et ah, 1983). Other signs of true muscle degeneration included pyknotic nuclei, swollen and disrupted mitochondria, and dilation of the sarcoplasmic reticulum and T-tubule system (Fig. 4). In contrast, Kennedy et al. (1988) reported few signs of myofiber necrosis and degeneration in the stretched ALD muscle of young chicken. The response of young chickens may be different than in the adult animals used in this study. Alternatively, since the muscle fiber injury observed in this study was limited to the middle and distal regions of the ALD muscle, depending on where they sampled, muscle fiber necrosis may have gone undetected. Signs of myofiber injury or necrosis were rarely observed in the proximal region of the stretched ALD muscle. This regional injury and focal necrosis is evident in the series of micrographs in Fig. 3, where the fiber appears relatively normal in one micrograph, and then 600 pm away, only a basal lamina tube remains. Segmental necrosis and focal injury has been previously described in habitually exercised cat muscle (Giddings et al, 1985, in press), rat muscle injured by micropuncture (Carpenter and Karpati, 1989), and in mouse muscle injured by aldehyde fixative (Papadimitriou et aZ., 1990). Carpenter and co-workers (1989) first described the formation of new demarcating membranes between the necrotic region and the viable stump of an injured fiber. Although we did not observe the formation of new membrane that separated the viable portion of the stretched muscle fiber from the injured area, we assume that this process must have occurred or fiber necrosis
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FIG. 13. Electron micrograph of an ALD muscle that had been stretched for 10 days. The activated satellite cell is no longer contained entirely underneath the basal lamina of the myofiber, with the basal lamina left intact only on the left side (white arrowhead) of the cell. Radiolucent membranous inclusions are contained in the widened intercellular space between the satellite cell and the myofiber. Interstitial cells are located in close proximity, one is labeled (arrow). Magnification, X2500; bar, 2 pm. The inset is a serial section of the same area in a light microscopic autoradiograph. This bird received two injections of [3H]TdR at Days 8 and 9 of stretch. Note that one of the interstitial cells is labeled (black arrowhead).
would have been observed along the whole length of the fiber resulting in fiber death. The lack of injury to the proximal region of the stretched muscles supports the concept that fibers are capable of repair. This suggests that fibers injured focally rarely undergo complete degeneration and subsequent removal. A detailed ultrastructural study of longitudinally sectioned muscle needs to be done to confirm this mechanism in stretched muscle.
Signs of muscle fiber regeneration were noted as evidenced by myoblasts located within persisting basal laminas (Fig. 14). The source of these myoblasts was most likely the daughter cells of activated satellite cells (Snow, 1977b). During the early periods of stretch, activation is probably secondary to muscle fiber lysis and release of a mitogenic substance (Bischoff, 1986). In a recent study, satellite cells were shown to be activated as early as Day 1 of stretch and that the peak of satellite
FIG. 14. Photographic montage of a cross section of the distal region of a muscle that had been stretched for 10 days. (A) The regenerating myotubes contains myogenic cells and several central and eccentric nuclei. Magnification, x2910; bar, 4 Nrn. A myogenic cell nucleus (arrow) is labeled in a serial section of the same area in a light microscopic autoradiograph (inset). This bird received two injections of [3H]TdR at days 8 and 9 of stretch. Note that the regenerating cell nucleus is labeled (black arrowhead). (B) A higher magnification of part of the basal laminae of the regenerating myotube. The basal lamina from an originally degenerating fiber surrounds the myotube (white arrowhead). Magnification, x29,100; bar, 500 nm.
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commonly appeared to be leaving the basal lamina of their respective myofibers (Figs. 11, 12, and 13). We noted an increased incidence of interstitial cells, as first described by Kennedy and co-workers (1988), and this observation supports the concept that satellite cells migrate into the interstitium. It has not been determined if caveolae play a role in the migration of satellite cells; however, this study noted a higher incidence of caveolae on both the internal and external plasmalemma of the satellite cells in stretched muscles (Figs. 10). Thymidine-labeled nuclei were observed in small fibers located in the extrafascicular spaces (Fig. 17). This study does not exclude that the source of these labeled nuclei is from nonmuscle tissue as opposed to satellite cells; however, at this time there is no evidence to support nonsatellite stem cell involvement. Labeled peripheral myonuclei were observed in stretched, hypertrophic fibers (Fig. 18). It is well accepted that satellite cells serve as the source of myonuclei in growing muscle (Moss and Leblond, 1971). We have recently demonstrated that the nuclear-to-cyto-
FIG. 15. Electron micrograph of a longitudinal section of a newly formed myotube taken from a muscle that had been stretched for 7 days. Central nuclei are surrounded by loosely organized contractile protein. Two unidentified cells are located in the interstitium, one closely apposed to the myotube. Magnification, ~2000; bar, 4 pm.
cell activation was from Days 3 to 7 of stretch (Winchester et al, 1991a). The time course of injury corresponds to the peak of satellite cell proliferation in this model, but the earlier periods of stretch (Days 1, 2, and 3) resulted in satellite cell activation without detectable injury to the stretched muscle. It is possible that the injury to the muscle is subtle and is not recognized at the light microscopic level. Satellite cells remain activated in the stretched ALD muscle at Days 10, 14, and 21 of stretch (Winchester et al, 1991a), at a time when the majority of myofibers have recovered. New fiber formation occurs in the stretched ALD muscle (Sola et al., 1973; Kennedy et ak, 1988; Alway et al, 1989). Kennedy and co-workers (1988) suggested that satellite cells may leave the basal lamina and migrate into the interstitial spaces to contribute to the formation of new fibers. It has been shown that myogenic cells derived from the satellite cell population are free to move within a muscle and can even penetrate the basal lamina (Schultz et al, 1985). In this study, satellite cells
FIG. 16. Electron micrograph of a cross-section through 3 myogenic cells in the interstitial space of a muscle that had been stretched for 10 days. All 3 cells are contained within a common basal lamina. One ceil contains loosely organized contractile protein (arrow). Magnification, x6566; bar, 2 pm.
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Bischoff (1990b) has demonstrated that a mitogenic muscle extract stimulates satellite cell proliferation on killed myofibers but not on uninjured myofibers. Bischoff (1990b) concluded that satellite cells not in contact with the plasmalemma are more easily activated when compared to satellite cells in contact with the plasmalemma. The extent of satellite cell activation observed in this model may be due to the early disruption of the plasmalemma in the stretched fibers that was undetected at the light microscopic level of resolution. It is probable that satellite cell activation does not require muscle fiber degeneration observed in this study. This is supported by the early activation of satellite cells (l-day stretch) seen in this model (Winchester et al, 1991a) and significant degeneration not occurring until Day 5 of stretch. The plasmalemma disruption observed at Day 1 of stretch in this study may be a significant trigger to initiate the activation sequence for satellite cells. Hence, it is possible that the early activation of satellite cells is due to plasmalemma disruption, and as the degree of muscle fiber injury progresses, the stimulus for satellite cell activation intensifies. This would explain
FIG. 17. Electron micrograph of a cross section through a small, newly formed fiber in a muscle that had been stretched for 7 days. The myofiber contains two eccentric nuclei (arrows) with sparse myofibrils. Magnification, ~2700; bar, 4 pm. The inset is a serial section of the same area in a light microscopic autoradiograph. This bird received an injection of 13H]TdR at Day 5 of stretch. Note that both of the myonuclei are labeled.
plasmic ratio is maintained in the enlarged myofibers of stretched ALD muscles of quail (Winchester and Gonyea, 1992). As in the growing muscle, it is likely that the daughter cells of activated satellite cells fuse to the enlarging fibers to maintain the nuclear-to-cytoplasmic ratio in this stretched adult muscle. Snow (1990) has recently proposed that there are two distinct phases in the enlargement of skeletal muscle following surgical ablation of its synergists. He suggests that during the initial phase, satellite cells are activated in response to the fiber injury, but in the second phase they may play a role in the hypertrophic response of the muscle. A similar response may be occurring in this model, where satellite cells remain activated after the muscle appears to have recovered. In the later time periods of stretch when fibers are enlarging, hypertrophy may play a role in satellite cell activation. Extracts from hypertrophied ALD muscles contain a mitogenic factor that is capable of promoting muscle cell proliferation, as well as differentiation in culture (Summers et al, 1985).
FIG. 18. Electron micrograph through a cross section of a myofiber from a muscle that had been stretched for 14 days. A peripheral myonuclei is noted (arrow). Magnification, ~4000; bar, 2 pm. The inset is a serial section of the same area in a light microscopic autoradiograph. This bird received an injection of [‘H]TdR 1 day prior to surgery. Note that the myonucleus is labeled.
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the peak of satellite cell activation corresponding to the time of peak muscle fiber degeneration. In the avian stretch model, it can be confirmed that activated satellite cells give rise to a population of daughter cells that differentiate to contribute to (1) the regeneration and repair of injured and necrotic fibers, (2) additional myonuclei in the hypertrophic fibers to maintain the nuclear-to-cytoplasmic ratio, and (3) the formation of a new population of fibers that result in a significant increase in total muscle fiber number. The authors thank Ken W. Bourell for assistance with the thin sectioning for electron microscopy. The graphs in this paper were generated by the authors using the SOLO scientific graphics software developed by Epidemiology Division, University of Texas Southwestern Medical Center at Dallas. This work was supported in part by the National Institute of Arthritis and Musculoskeletal and Skin Disease Grant AR 40056 and Physical Therapy Foundation Grant. REFERENCES Alway, S. E., Winchester, P. K., Davis, M. E., and Gonyea, W. J. (1989). Regionalized adaptations and muscle fiber proliferation in stretchinduced enlargement. J Appl. Physiol. 66,771-781. Alway, S. E., Davis, M. E., and Gonyea, W. J. (1990). Muscle fiber formation and fiber hypertrophy during the onset of stretch-overload. Am. J. Physiol 259, C92-C102. Armstrong, R. B., Ogilvie, R. W., and Schwane, J. A. (1983). Eccentric exercise induced injury to rat skeletal muscle. J. Appl. PhysioL 54, 80-93.
Ashmore, C. R., Hitchcock, L., and Lee, Y. B. (1988). Passive stretch of adult chicken muscle produces a myopathy remarkably similar to hereditary muscular dystrophy. Exp. NeuroL 100,341-353. Bischoff, R. (1986). A satellite cell mitogen from crushed adult muscle. Dev. BioL 115.140-147.
Bischoff, R. (1990a). Cell cycle commitment of rat muscle satellite cells. J. Cell Biol. 111, 201-207. Bischoff, R. (1990b). Interaction between satellite cells and skeletal muscle fibers. Development 109,943-952. Campion, D. R. (1984). The muscle satellite cell: a review. Znt. Rev. CytoL 87,225-251.
Carpenter, S., and Karpati, G. (1989). Segmental necrosis and its demarcation in experimental micropuncture injury of skeletal muscle fibers. J. Neuropath. Exp. New. 48,154-170. Ebbeling, C. B., and Clarkson, P. M. (1989). Exercise-induced muscle damage and adaptation. Sports Med. 7,207-234. Friden, J. (1984). Changes in human skeletal muscle induced by long term eccentric exercise. Cell Tissue Res. 236, 365-372. Giddings, C. J., and Gonyea, W. J. (in press). Morphological observations supporting muscle fiber hyperplasia following weight-lifting exercise in cats. Anat. Rec. Giddings, C. J., Neaves, W. B., and Gonyea, W. J. (1985). Muscle fiber necrosis and regeneration induced by prolonged weight lifting exercise in the cats. Anat. Rec., 211, 133-141. Gollnick, P. D., Parsons, D., Riedy, M., and Moore, R. L. (1983). Fiber
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number and size in overloaded chicken anterior latissimus dorsi muscle. J. AppL PhysioL 54, 1292-1297. Grounds, M. D. (1991). Towards understanding skeletal muscle regeneration. Pathol. Res. Pratt. 187,1-22. Hikida, R. S., Staron, R. S., Hagerman, F. C., Sherman, W. M., and Costill, D. L. (1983). Muscle fiber necrosis associated with human marathon runners. J. Neural. Sci. 59,185-203. James, N. T. (1976). Compensatory muscular hypertrophy in the extensor digitorum longus muscle of the mouse. J. Anat. 122,121-131. Kennedy, J. M., Eisenberg, B. R., Kamel, S., Sweeney, L. J., and Zak, R. (1988). Nascent muscle fibers appearance in overloaded chicken slow tonic muscle. Am. J. Anat. W&203-215. Khan, M. A. (1986). An ultrastructural study of the stretch-induced hypertrophy of skeletal muscle. Cell Biol. Znt. Rep. 10, 955-962. McGeachie, J. K., and Grounds, M. D. (1987). Initiation and duration of muscle precursor replication after mild and severe injury to skeletal muscle of mice-An autoradiographic study. Cell Tissue Res. 248, 125-130. Moss, F. P., and Leblond, C. P. (1971). Satellite cells as the source of nuclei in muscles of growing rats. Anat. Rec. 170,421-436. Papadimitriou, J. M., Robertson, T. A., Mitchell, C. A., and Grounds, M. D. (1990). The process of new plasmalemma formation in focally injured skeletal muscle fibers. J. Struct. BioL 103, 124-134. Salleo, A., LaSpada, G., Falzea, G., Kenaro, M. G., and Cicciarello, R. (1983). Response of satellite cells and muscle fibers to long-term compensatory hypertrophy. J. Submicrosc. CytoL 15,929-940. Schmalbruch H. (1976). The morphology of regeneration of skeletal muscles in the rat. Tissue Cell 8, 673-692. Schultz, E., Jaryszak, D. L., and Valliere, C. R. (1985). Response of satellite cells to focal skeletal muscle injury. Muscle Nerve 8,217222.
Snow, M. H. (1977a). Myogenic cell formation in regenerating rat skeletal muscle injured by mincing. I. A fine structural study. Anat. Rec. 188,181-200.
Snow, M. H. (1977b). Myogenic cell formation in regenerating rat skeletal muscle injured by mincing. II. An autoradiographic study. Anat. Rec. 188,201-218.
Snow, M. H. (1990). Satellite cell response in rat soleus muscle undergoing hypertrophy due to surgical ablation of synergists. Anat. Rec. 227,437-446.
Sola, 0. M., Christensen, D. L., and Martin, A. W. (1973). Hypertrophy and hyperplasia of adult chicken anterior latissimus dorsi muscles following stretch with and without denervation. Exp. NeuroL 41, 76-100.
Summers, P. J., Ashmore, C. R., Lee, Y. B., and Ellis, S. (1985). Stretchinduced growth in chicken wing muscles. Role of soluble growthpromoting factors. J. Cell Physiol. 125,288-294. Winchester, P. K., Davis, M. E., Alway, S. E., and Gonyea, W. J. (1991a). Satellite cell activation in the stretch-enlarged anterior latissimus dorsi muscle of the adult quail. Am. J. Physiol. 260, C206c212. Winchester, P. K., Davis, M. E., Alway, S. E., and Gonyea, W. J. (1991b). Fiber necrosis and regional injury in the stretch-enlarged anterior latissimus dorsi muscle of the adult quail (Abstract). Med. Sci. Sports Exercise
Suppl. 23, S148.
Winchester, P. K., and Gonyea, W. J. (1992). A quantitative study of satellite cells and myonuclei in stretched avian slow tonic muscle. Anat. Rec. 232,369-377.
BIOLOGY
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Regional Injury and the Terminal Differentiation of Satellite Cells in Stretched Avian Slow Tonic Muscle P.K. WINCHESTERAND Department
of Cell Biology and Neuroscience,
The University
W.J. GONYEA~ of Texas Southwestern
Accepted February
Medical
Center, Dallas, Texas 75235
25, 1992
In the avian stretch model, the application of a weight overload to the humerus induces enlargement of the anterior latissimus dorsi (ALD) muscle and an increase in muscle fiber number which is accompanied by satellite cell activation. Myofiber injury may be an important stimulus to muscle fiber hyperplasia; therefore, light and electron microscopic evaluation was undertaken to determine if myofiber injury occurs in the stretch-enlarged ALD muscle of the adult quail. Autoradiographic studies were used to determine the terminal differentiation of labeled myogenic cells. A weight equal to 10% of body mass was attached to one wing of 2’7adult quail and 3 birds were euthanized at 9 intervals of stretch, from 1 to 30 days. Birds were injected with tritiated thymidine at intervals ranging from 1 hr to 3 days prior to euthanization. Labeled nuclei were detected by light microscopic examination and identified by electron microscopy of a serial section. Three regions of the muscle wore examined for disorganization of contractile elements, presence of cytoplasmic vacuoles, and/or phagocytic cell infiltration. The percentage of fibers exhibiting one or more of these criterion was significantly greater in the stretched ALD by Days 5 and 7 and declined at Day 10, reaching near control values by Day 14. Myofiber necrosis and phagocytic cell infiltration were only observed in the middle and distal regions of the stretched ALD muscle. Traditional signs of regeneration and repair were observed, including clusters of labeled myoblast-like cells and myotube formation within an existing basal lamina. New myotube formation with labeled central nuclei was also noted in the interstitial space, outside of basal lamina of persisting fibers. Labeled myonuclei were observed in the stretched fibers. These results demonstrate that chronic stretch produces regional injury and fiber degeneration and resultant regeneration in the ALD muscle of the adult quail. This may be an important stimulus for new fiber formation in this model. 0 15x12 Academic Press, Inc.
In the avian stretch model, the application of a weight overload to the humerus induces new fiber formation and fiber hypertrophy of the anterior latissimus dorsi (ALD) muscle (Sola et al, 1973; Kennedy et ok, 1988; Alway et ah, 1989,199O). Satellite cells have been shown to be activated in the stretched-enlarged ALD muscle from young chicken (Kennedy et ah, 1988) and adult quail (Winchester et al, 1991a), It has been proposed that satellite cells proliferate and differentiate to form new fibers in the stretched avian muscle (Kennedy et ah, 1988; Alway et ak, 1989, 1990; Winchester et ak, 1991a). Satellite cells play an important role during postnatal muscle growth by fusing with growing myofibers to increase the number of true myonuclei (Moss and Leblond, 1971). This mechanism may also occur in the hypertrophic fibers of the stretched adult muscle as suggested in the compensatory hypertrophy model (Salleo et al, 1983).
1To whom reprint requests should be sent at Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical School, 5323 Harry Hines Blvd., Dallas, TX 75235.
Activation and proliferation of satellite cells are well known to be associated with injury to the myofibers. The progenies of these activated satellite cells are assumed to give rise to a population of myogenic precursor cells that replaces necrotic myofibers in the regenerating muscle (reviewed in Campion, 1984). Although certain growth factors and hormones have been shown to have a mitogenic effect in vitro (reviewed in Grounds, 1991), little is known about the factors that directly or indirectly control the activation and proliferation of satellite cells in viva. Bischoff (1990a) has recently demonstrated that satellite cells can be stimulated to proliferate on rat myofibers killed with Marcaine, but not on viable myofibers. This suggests that the proliferative response of satellite cells to mitogens is depressed in the presence of an intact plasmalemma. In view of this, we hypothesized that stretch would result in muscle fiber injury and necrosis in the enlarged ALD muscle of the adult quail and this may be an important stimulus for muscle fiber hyperplasia observed in this model. Other investigators have examined structural changes seen in the stretched avian muscle (Khan, 1986; Ashmore et al, 1988; Kennedy et al, 1988). Kennedy and
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(D 1892 by Academic Press, Inc. of reproduction in any form reserved.
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FIG. 1. Percentage of change in muscle mass in the stretched ALD muscle relative to the intra-animal control muscle at nine different intervals of stretch. Bars represent means f SE. Stretched muscle mass was significantly greater than the contralateral muscle mass after 5, 7, 10, 14, 21, and 30 days of stretch. *P < 0.05.
colleagues (1988) reported myofibril disruption and Z band alterations in the stretched ALD muscle of young chicken. They suggested that this type of injury did not lead to muscle fiber death but could play a role in the activation and proliferation of myogenic precursor cells and resultant formation of nascent fibers. In contrast to this, Ashmore and co-workers (1988) reported the necrosis, vacuolation, and infiltration of phagocytic cells in the stretched patagialis (PAT) muscle of adult chicken. If muscle fiber death is observed in the stretched avian muscle, then the progeny of activated satellite cells would play an important role in the regeneration of the necrotic fibers. Before the role of satellite cells can be determined in the stretched avian muscle, autoradiographic studies following the terminal differentiation of labeled myogenic precursor cells would need to be done before conclusions could be drawn concerning their contribution to muscle enlargement in this model. The present study was undertaken to quantify pathological changes in the stretch-enlarged ALD muscles of adult quail. Ultrastructural studies using autoradiographic techniques of thymidine-labeled cells were conducted to trace the terminal differentiation of satellite cells in the stretched ALD muscle. Part of this work has appeared in abstract form (Winchester et al., 1991b).
50 * 1 II i 0123
5
7
10
14
21
30
DAYS OF STRETCH FIG. 2. Percentage of total fibers counted that were injured in the proximal, middle, and distal regions of the control and stretched ALD muscles at nine different intervals of stretch. The control data from the nine intervals of stretch has been pooled and plotted at Day 0 of stretch. Bars represent means + SE. Percentage of injured myofibers in the stretched muscle is significantly greater than control values in the middle region at Days 5 and 7 and in the distal region at Days 5,7, and 10 days of stretch. *P < 0.05.
MATERIALS
Animal
AND METHODS
Procedures
Six-week-old adult male Corturnix quail (Truslow Farms, MA) were obtained for this study. The quail were housed one per cage with 12-hr cycles of light. Quail were fed Purina 18% protein turkey starter and water ad l&turn. The birds were weighed daily for a minimum of 2 weeks to ensure stable body weights prior to the experimental period. Hypertrophy was induced in the ALD muscles by the application of a cuff weight
FIG. 3. Light micrographs of a group of fibers in cross section followed at 200-pm intervals through the middle region of an ALD muscle that had been stretched for 5 days. Several of the fibers show degenerative changes. Toluidine blue stain was used. One fiber (asterisks) has been followed and shows a spectrum of degenerative changes. (A) The fiber appears relatively normal. (B) The fiber shows vaeuolation. (C) Contractile protein is disrupted and disorganized. (D) Only a basal lamina tube remains. Magnification, ~588; bar, 50 pm.
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at the same lengths. The apex of the ventricle was cannulated and the bird was perfused with 50 ml minimal essential medium (MEM, GIBCO Industries, NY), followed by 50 ml 2% glutaraldehyde (Ladd Research Industries, VT) in 0.1 M cacodylate buffer (pH 7.2-7.4). The ALD muscles were removed, cleaned of excessive connective tissue, weighed, and then stored in fresh fixative at 4°C. Autoradiography
and Electron Microscopy
Bundles of fibers (200-500 fibers) were gently teased from each muscle, rinsed in cacodylate buffer, postfixed in 1% osmium tetroxide, and stained overnight in 0.5% uranyl acetate. Muscle bundles were dehydrated in graded alcohols and embedded in Epon (Ladd Research Industries). The embedded muscle fiber bundles were cut transversely into 1 mm segments and mounted in cross section on Epon blocks. In some muscles, additional bundles were mounted longitudinally for sectioning. Two thick sections (0.5 and 1.0 pm) were cut from
FIG. 4. A degenerating fiber from the distal region of a muscle that had been stretched for 5 days. Contractile protein is foamy; membrane bound vacuoles (v) and a pyknotic nuclei (pn) can be seen. MagniAcation, x 3920; bar, 4 am.
filled with lead pellets secured proximal to the humeroulnar joint of the right wing of each bird. The mass of the cuff weight corresponded to IO% of the bird’s body mass. The wing weight was maintained for 1, 2, 3, 5, 7, 10,14,21, or 30 days, The unweighted left wing served as an intra-animal control. The animals tolerated the wing weight well, without change in their daily activity. Body weight did not change over the experimental period (pre, 162.9 + 14.9 g vs post, 153.4 f 15.3 g). Birds received either a single injection of tritiated thymidine ([*HlTdR) (sp act 5 Ci/mmole, ICN Radiochemicals, Irvine, CA) at a dosage of 2 &i/g body weight or two injections each at a dosage of 1.0 &i/g body weight. Birds survived up to 3 days postinjection before being euthanized. By limiting the time between injection and euthanization to 3 days, problems with label dilution was minimized (McGeachie and Grounds, 198’7). Three birds from each of the nine intervals of stretch were anesthetized by an intramuscular injection of Nembutal (0.15 mg/g body weight), Each bird was injected with heparin (1.0 IU/g body weight) and wings were pinned to ensure that the ALD muscles were fixed
FIG. 5. Two degenerating fibers from the middle region of a muscle that had been stretched for 3 days. One fiber has been infiltrated by red blood cells. Magnification, x2000; bar, 4 Grn.
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the muscle. A minimum of 500 fibers were counted in three regions of the control and stretched ALD muscles. Samples from three control and experimental muscles at nine different intervals of stretch were analyzed under an oil immersion lens (x600). Criteria for myofiber injury included the following: loss of organization of the contractile elements; foamy, vacuolated cytoplasm; and/or presence of phagocytic cell infiltration. The percentage of injured myofibers was expressed as a percentage of total fibers counted in each region of the control and stretched ALD muscles. Electron microscopy was also used for ultrastructural examination of fibers identified as injured with light microscopy. In one muscle, serial sections were taken every 8 Frn from one block from the middle region of a muscle that had been stretched for 5 days to examine if the degree of fiber degeneration varied along the length of the muscle. Statistical Analysis
FIG. 6. Myofiber is from the middle region of a muscle that had been stretched for 7 days. Macrophage has infiltrated the basal lamina (arrow) of the myofiber. Magnification, x9800; bar, 1 pm.
each block. The first section, stained with toluidine blue, was used for identification and quantitative analysis of injury in the control and stretched ALD muscles. The second section was mounted on a subbed glass slide with a section of the gut. After drying, the slides were dipped in a 50% solution of Kodak NTB2 emulsion (EastmanKodak, Rochester, NY) and distilled water. The slides were dried and placed in light-tight boxes and exposed for 4 weeks at 4°C. Exposed slides were developed and stained with toluidine blue or methylene azure blue. The autoradiographic sections were viewed under an oil immersion lens (X600) for identification of labeled nuclei. Serial thin sections (600-900 A thick) were taken and stained with uranyl acetate and lead citrate and analyzed in a JEOL 1OOCXor JEOL 1200 electron microscope. Labeled nuclei were identified in the serial ultrathin sections using photographic montages of the adjacent thick section for orientation. Fiber Injury For quantitative analysis of muscle fiber injury, two sections were randomly selected from each third of the control and stretched ALD muscles and designated as coming from the proximal, middle, or distal region of
Descriptive statistics included means f SE. Change in stretched muscle mass was expressed as a percentage of the contralateral control muscle. The percentage of change was tested for differences due to duration of stretch using the Kruskal-Wallis one-way analysis of variance by ranks. To determine if differences existed in the number of injured muscle fibers in the proximal, middle, and distal regions of the control and stretched muscles, a repeated measures analysis of variance was conducted. These results warranted multiple pairwise comparisons to test for significant differences between the number of injured myofibers in control and stretched muscles at each time interval. RESULTS
Stretched muscle mass was significantly greater than the intra-animal control after 5,7,10,14,21, and 30 days of stretch (Fig. 1). Mass of the 5-day stretched ALD muscle was 33.6 + 7.1% greater than the contralateral control muscles. Mass of the stretched muscles continued to increase at all subsequent time intervals studied, so that by Day 30 of stretch, mass of the stretched ALD muscle was 115.3 + 8.0% greater than the contralateral control muscles. Fiber Injury Figure 2 illustrates the percentage of total fibers counted that were injured in the proximal, middle, and distal regions of the control and stretched ALD muscles at nine different intervals of stretch. Very few fibers were classified as injured in any of the control muscles; therefore, the control data from the nine intervals of stretch was pooled and plotted at Day 0 of stretch. The
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FIG. 7. Electron micrograph of longitudinally sectioned ALD muscle stretched for 3 days. (A) Focal areas of sarcomere hypercontraction, and (B) Z band streaming are noted. Magnification, X5300; bar, 2 pm.
mean percentage of injured fibers was 0.3 -t 0.4% in the proximal, 0.2 f 0.2% in the middle, and 0.1 ? 0.1% in the distal regions of the contralateral control muscles. The percentage of injured myofibers in the stretched muscle was significantly greater than control values in the middle region at Days 5 (15.8 k 15.1%) and 7 (25.4 t 20.4%) and in the distal region at Days 5 (49.0 f 24.8%), 7 (30.4 + 14.2%), and 10 (26.9 * 13.6%) of stretch. At later times of stretch, the percentage of injured fibers declined, such that by Day 30 the mean percentage of injured fibers was 0.6 + 0.3% in the proximal, 0.5 f 0.2% in the middle, and 1.9 f 1.2% in the distal regions of the stretched muscles. Signs of myofiber degeneration, such as phagocytic cell infiltration, pyknotic nuclei, and loss of contractile elements, were only observed in the middle and distal regions of the stretched ALD muscle. Sections taken for light microscopic analysis frequently demonstrated normal muscle architecture in a l-mm block of tissue and extensive areas of disruption and interstitial edema in the adjacent l-mm block of muscle. Figure 3 shows a group of fibers followed at 200-pm intervals through the
middle region of a muscle that had been stretched for 5 days. One fiber has been followed and shows a spectrum of degenerative changes. This fiber appears relatively normal (Fig. 3A); its plasmalemma appears to be intact and nearly all contractile elements are organized. In the next section (Fig. 3B), 200 pm away, the fiber shows vacuolation. In Fig. 3C, the contractile protein is disrupted and disorganized. Only a basal lamina tube remains in Fig. 3D. Electron microscopy was used to confirm the light microscopic analysis of stretch-induced muscle fiber injury of cross sections of degenerating fibers in stretched ALD muscles. Frequently, extensive areas of sarcolemma disruption was observed but the basal lamina was intact. In these degenerating fibers, contractile protein was foamy with membrane-bound vacuoles, and often pyknotic nuclei were observed (Fig. 4). Infiltrating cells were often observed in the injured and necrotic fibers of the stretched muscle. Figure 5 demonstrates a degenerating fiber that had been infiltrated by red blood cells and Fig. 6 shows a macrophage that has infiltrated the basal lamina of a necrotic myofiber. Electron
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cell was widened and contained radiolucent membranous inclusions (Figs. 12 and 13). Occasionally, these activated satellite cells were no longer contained entirely within the basal lamina of the myofiber. These satellite cells were often associated with other cells located in the interstitium that had a similar morphology to satellite cells but that were not contained within a basal lamina of a myofiber (Fig. 13). In some instances, these interstitial cells were labeled (Fig. 13, inset). These cells were not observed in the control muscles. Terminal
D$erentiation
of Myogenic
Cells
Evidence of myofiber regeneration was seen, particularly from Days 5 through 10 of stretch (Fig. 14A). Clusters of myoblasts were observed within a persisting
FIG. 8. Electron micrograph of a cross section through a muscle that had been stretched for 1 day. An activated satellite cell (arrow) is beneath the basal lamina of the muscle fiber. Its cytoplasm contains numerous mitochondria and extensive Golgi complex. Magnification, x5194; bar, 2 pm. The inset is a serial section of the same area in a light microscopic autoradiograph. This bird received a single injection of [3H]TdR 2 hr prior to surgery. Note the satellite cell is labeled.
micrographs of longitudinally sectioned stretched ALD muscles revealed focal areas of sarcomere hypercontraction (Fig. ‘7A) and Z band streaming (Fig. ‘7B) in muscles that had been stretched for l-5 days. Satellite Cell Activation
As early as Day 1 of stretch, labeled, activated satellite cells were observed between the basal lamina and plasmalemma of stretched myofibers (Fig. 8). These cells contained a higher cytoplasmic-to-nuclear ratio with a euchromatic nucleus. Satellite cell activation was often observed to be associated with fiber injury in the first week of stretch. Figure 9 demonstrates an activated satellite cell in one myofiber from an ALD muscle that had been stretched for 5 days. In an adjacent myofiber, a labeled leucocyte that had infiltrated the myofiber was observed. Caveolae were often noted both on the external and internal plasma membrane of the activated satellite cells (Fig. 10). In the first week of stretch, the basal lamina of the myofiber was observed in the space between the myofiber and the satellite cell (Fig. 11). Beginning with Day 7 of stretch, activated satellite cells were often observed to be closely apposed to a cytoplasmic tail of another myogenic cell (Fig. 12). In many cases, the space between the myofiber and the satellite
FIG. 9. Electron micrograph of a cross section through a muscle that had been stretched for 5 days. An activated satellite cell (SC)is beneath the basal lamina of one of the muscle fibers. In an adjacent myofiber a leucocyte (lc) has invaded the fiber. Magnification, x2700; bar, 4 pm. The inset is a serial section of the same area in a light microscopic autoradiograph. This bird received two injections of [aHITdR 1 and 2 days prior to surgery. Note that the satellite cell and leucocyte are labeled (arrowheads).
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peared to have normal organization of their contractile elements with no necrotic signs. Furthermore, examination revealed only one basal lamina that was closely ap: posed to the sarcolemma. These large hypertrophic fibers often demonstrated a peripheral halo of myoplasm that was rich in organelles, but lacked any contractile protein. DISCUSSION
This study examined satellite cell activation and the terminal differentiation in the chronically stretch-enlarged ALD muscles of adult quail. The avian stretch model produces rapid and substantial gains in muscle mass (Sola et ab, 1973; Gollnick et ah, 1983; Kennedy et ab, 1988; Alway et ah, 1989; Winchester et al, 1991a). The mass increases observed in this study were similar to those described in an earlier study (Winchester et al., 1991a), except that significant increases in muscle mass were not achieved until Day 5 of stretch compared to Day 3 of stretch in the prior study. This could be due to the smaller sample size used in this study. Muscle mass continued to increase throughout the duration of this study.
FIG. 10. Electron micrograph of a satellite cell in an ALD muscle that had been stretched for days. The nucleus is euchromatic with a prominent nucleolus and caveolae are noted (arrow) on the plasma membrane of the cell. Magnification, ~20,000; bar, 500 nm.
basal lamina tube that was adjacent to the new basal lamina (Fig. 14B). These regenerating myofibers characteristically contained a high incidence of central and eccentric nuclei, some of which were labeled (Fig. 14, inset). Small myotubes or myofibers were observed in the interstitial spaces of the stretched muscles. In contrast to the morphology of regenerating fibers, these fibers were surrounded by only one basal lamina closely adhered to the plasmalemma. Frequently, these new myotubes contain a series of central nuclei, surrounded by loosely organized contractile protein (Fig. 15). Groups of myogenic cells contained within a common basal lamina were also observed in the interstitial spaces of the stretched muscles. In some cases, some of the myogenic cells contained loosely organized contractile protein (Fig. 16). Labeled central or eccentric myonuclei were observed in some of the newly formed small myofibers FIG. 11. Electron micrograph of an activated satellite cell in a mus(Fig. 17). cle that had been stretched for 7 days. Basal lamina has invaded the Labeled peripheral myonuclei were observed within space between the myofiber and the satellite cell (arrows). Magnificalarge hypertrophic fibers (Fig. 18). These fibers ap- tion, x9800; bar, 1 pm.
WINCHESTER AND GONYEA
FIG. 12. An activated satellite cell closely apposed to a cytoplasmic tail of another muscle cell (asterisk). The satellite cell demonstrates a high nuclear-to-cytoplasmic ratio with a euchromatic nucleus. The space between the myofiber and the cell is widened and contains radiolucent membranous inclusions. Part of a myonuclei is noted in the left lower hand corner. Magnification, X6499; bar, 2 pm.
In order to evaluate the role of the satellite cell in an enlarging muscle, it was first important to determine whether chronic stretch resulted in fiber injury because satellite cell activation has been associated with muscle fiber degeneration and regeneration in the adult animal (Schmalbruch, 1976; Snow, 1977a,b). Other investigators have examined morphological changes in the avian stretched muscle, but their assay was either limited to the light microscopic level (Ashmore et al, 1988) or not quantified in different regions of the stretched muscle (Khan, 1986; Kennedy et ah, 1988). Myofibers exhibiting signs of injury were significantly greater in the stretched muscles compared to the contralateral control muscles in the middle region at Days 5 and 7 and in the distal region at Days 5, 7, and 10 days of stretch. Since the extent of injury was quantified at the light level, it is possible that structural changes at earlier days were missed due to the resolution limits of light microscopy. In those muscles that were examined at the ultrastructural level, we did observe some minor degree of injury at the earlier time intervals of stretch. These changes included 2 band streaming and myofilament disruption.
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Regardless of the onset of muscle fiber injury, we concluded that significant injury does occur in the overloaded muscle in the first 10 days of stretch. Many of the morphological changes observed in this study were typical of those described with compensatory hypertrophy (James, 1976; Snow, 1990), eccentric activity (Armstrong et ab, 1983; Friden, 1984), marathon running (Hikida et ah, 1983), and prolonged high-resistance exercise (Giddings et aZ.,1985, in press). Z band alterations previously described in the stretched teres minor muscle of adult chicken (Khan, 1986) and stretched ALD muscle of young chicken (Kennedy et al, 1988) were also observed in this study (Fig. 7). Additional morphological changes included true signs of muscle fiber necrosis, as described in the stretched PAT muscle of adult chicken (Ashmore et ab, 1988). Disruption of the sarcolemma, presumably caused leakage of muscle cell contents which appeared to have activated macrophages (Fig. 6) and attracted neutrophils and leucocytes (Fig. 9) to participate in phagocytosis. Erythrocytes were noted in the extravascular spaces, but more unusual, they were also observed within the injured muscle fibers (Fig. 5). This phenomena has been reported in the gastrocnemius muscle in marathon runners (Hikida et ah, 1983). Other signs of true muscle degeneration included pyknotic nuclei, swollen and disrupted mitochondria, and dilation of the sarcoplasmic reticulum and T-tubule system (Fig. 4). In contrast, Kennedy et al. (1988) reported few signs of myofiber necrosis and degeneration in the stretched ALD muscle of young chicken. The response of young chickens may be different than in the adult animals used in this study. Alternatively, since the muscle fiber injury observed in this study was limited to the middle and distal regions of the ALD muscle, depending on where they sampled, muscle fiber necrosis may have gone undetected. Signs of myofiber injury or necrosis were rarely observed in the proximal region of the stretched ALD muscle. This regional injury and focal necrosis is evident in the series of micrographs in Fig. 3, where the fiber appears relatively normal in one micrograph, and then 600 pm away, only a basal lamina tube remains. Segmental necrosis and focal injury has been previously described in habitually exercised cat muscle (Giddings et al, 1985, in press), rat muscle injured by micropuncture (Carpenter and Karpati, 1989), and in mouse muscle injured by aldehyde fixative (Papadimitriou et aZ., 1990). Carpenter and co-workers (1989) first described the formation of new demarcating membranes between the necrotic region and the viable stump of an injured fiber. Although we did not observe the formation of new membrane that separated the viable portion of the stretched muscle fiber from the injured area, we assume that this process must have occurred or fiber necrosis
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FIG. 13. Electron micrograph of an ALD muscle that had been stretched for 10 days. The activated satellite cell is no longer contained entirely underneath the basal lamina of the myofiber, with the basal lamina left intact only on the left side (white arrowhead) of the cell. Radiolucent membranous inclusions are contained in the widened intercellular space between the satellite cell and the myofiber. Interstitial cells are located in close proximity, one is labeled (arrow). Magnification, X2500; bar, 2 pm. The inset is a serial section of the same area in a light microscopic autoradiograph. This bird received two injections of [3H]TdR at Days 8 and 9 of stretch. Note that one of the interstitial cells is labeled (black arrowhead).
would have been observed along the whole length of the fiber resulting in fiber death. The lack of injury to the proximal region of the stretched muscles supports the concept that fibers are capable of repair. This suggests that fibers injured focally rarely undergo complete degeneration and subsequent removal. A detailed ultrastructural study of longitudinally sectioned muscle needs to be done to confirm this mechanism in stretched muscle.
Signs of muscle fiber regeneration were noted as evidenced by myoblasts located within persisting basal laminas (Fig. 14). The source of these myoblasts was most likely the daughter cells of activated satellite cells (Snow, 1977b). During the early periods of stretch, activation is probably secondary to muscle fiber lysis and release of a mitogenic substance (Bischoff, 1986). In a recent study, satellite cells were shown to be activated as early as Day 1 of stretch and that the peak of satellite
FIG. 14. Photographic montage of a cross section of the distal region of a muscle that had been stretched for 10 days. (A) The regenerating myotubes contains myogenic cells and several central and eccentric nuclei. Magnification, x2910; bar, 4 Nrn. A myogenic cell nucleus (arrow) is labeled in a serial section of the same area in a light microscopic autoradiograph (inset). This bird received two injections of [3H]TdR at days 8 and 9 of stretch. Note that the regenerating cell nucleus is labeled (black arrowhead). (B) A higher magnification of part of the basal laminae of the regenerating myotube. The basal lamina from an originally degenerating fiber surrounds the myotube (white arrowhead). Magnification, x29,100; bar, 500 nm.
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commonly appeared to be leaving the basal lamina of their respective myofibers (Figs. 11, 12, and 13). We noted an increased incidence of interstitial cells, as first described by Kennedy and co-workers (1988), and this observation supports the concept that satellite cells migrate into the interstitium. It has not been determined if caveolae play a role in the migration of satellite cells; however, this study noted a higher incidence of caveolae on both the internal and external plasmalemma of the satellite cells in stretched muscles (Figs. 10). Thymidine-labeled nuclei were observed in small fibers located in the extrafascicular spaces (Fig. 17). This study does not exclude that the source of these labeled nuclei is from nonmuscle tissue as opposed to satellite cells; however, at this time there is no evidence to support nonsatellite stem cell involvement. Labeled peripheral myonuclei were observed in stretched, hypertrophic fibers (Fig. 18). It is well accepted that satellite cells serve as the source of myonuclei in growing muscle (Moss and Leblond, 1971). We have recently demonstrated that the nuclear-to-cyto-
FIG. 15. Electron micrograph of a longitudinal section of a newly formed myotube taken from a muscle that had been stretched for 7 days. Central nuclei are surrounded by loosely organized contractile protein. Two unidentified cells are located in the interstitium, one closely apposed to the myotube. Magnification, ~2000; bar, 4 pm.
cell activation was from Days 3 to 7 of stretch (Winchester et al, 1991a). The time course of injury corresponds to the peak of satellite cell proliferation in this model, but the earlier periods of stretch (Days 1, 2, and 3) resulted in satellite cell activation without detectable injury to the stretched muscle. It is possible that the injury to the muscle is subtle and is not recognized at the light microscopic level. Satellite cells remain activated in the stretched ALD muscle at Days 10, 14, and 21 of stretch (Winchester et al, 1991a), at a time when the majority of myofibers have recovered. New fiber formation occurs in the stretched ALD muscle (Sola et al., 1973; Kennedy et ak, 1988; Alway et al, 1989). Kennedy and co-workers (1988) suggested that satellite cells may leave the basal lamina and migrate into the interstitial spaces to contribute to the formation of new fibers. It has been shown that myogenic cells derived from the satellite cell population are free to move within a muscle and can even penetrate the basal lamina (Schultz et al, 1985). In this study, satellite cells
FIG. 16. Electron micrograph of a cross-section through 3 myogenic cells in the interstitial space of a muscle that had been stretched for 10 days. All 3 cells are contained within a common basal lamina. One ceil contains loosely organized contractile protein (arrow). Magnification, x6566; bar, 2 pm.
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Bischoff (1990b) has demonstrated that a mitogenic muscle extract stimulates satellite cell proliferation on killed myofibers but not on uninjured myofibers. Bischoff (1990b) concluded that satellite cells not in contact with the plasmalemma are more easily activated when compared to satellite cells in contact with the plasmalemma. The extent of satellite cell activation observed in this model may be due to the early disruption of the plasmalemma in the stretched fibers that was undetected at the light microscopic level of resolution. It is probable that satellite cell activation does not require muscle fiber degeneration observed in this study. This is supported by the early activation of satellite cells (l-day stretch) seen in this model (Winchester et al, 1991a) and significant degeneration not occurring until Day 5 of stretch. The plasmalemma disruption observed at Day 1 of stretch in this study may be a significant trigger to initiate the activation sequence for satellite cells. Hence, it is possible that the early activation of satellite cells is due to plasmalemma disruption, and as the degree of muscle fiber injury progresses, the stimulus for satellite cell activation intensifies. This would explain
FIG. 17. Electron micrograph of a cross section through a small, newly formed fiber in a muscle that had been stretched for 7 days. The myofiber contains two eccentric nuclei (arrows) with sparse myofibrils. Magnification, ~2700; bar, 4 pm. The inset is a serial section of the same area in a light microscopic autoradiograph. This bird received an injection of 13H]TdR at Day 5 of stretch. Note that both of the myonuclei are labeled.
plasmic ratio is maintained in the enlarged myofibers of stretched ALD muscles of quail (Winchester and Gonyea, 1992). As in the growing muscle, it is likely that the daughter cells of activated satellite cells fuse to the enlarging fibers to maintain the nuclear-to-cytoplasmic ratio in this stretched adult muscle. Snow (1990) has recently proposed that there are two distinct phases in the enlargement of skeletal muscle following surgical ablation of its synergists. He suggests that during the initial phase, satellite cells are activated in response to the fiber injury, but in the second phase they may play a role in the hypertrophic response of the muscle. A similar response may be occurring in this model, where satellite cells remain activated after the muscle appears to have recovered. In the later time periods of stretch when fibers are enlarging, hypertrophy may play a role in satellite cell activation. Extracts from hypertrophied ALD muscles contain a mitogenic factor that is capable of promoting muscle cell proliferation, as well as differentiation in culture (Summers et al, 1985).
FIG. 18. Electron micrograph through a cross section of a myofiber from a muscle that had been stretched for 14 days. A peripheral myonuclei is noted (arrow). Magnification, ~4000; bar, 2 pm. The inset is a serial section of the same area in a light microscopic autoradiograph. This bird received an injection of [‘H]TdR 1 day prior to surgery. Note that the myonucleus is labeled.
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the peak of satellite cell activation corresponding to the time of peak muscle fiber degeneration. In the avian stretch model, it can be confirmed that activated satellite cells give rise to a population of daughter cells that differentiate to contribute to (1) the regeneration and repair of injured and necrotic fibers, (2) additional myonuclei in the hypertrophic fibers to maintain the nuclear-to-cytoplasmic ratio, and (3) the formation of a new population of fibers that result in a significant increase in total muscle fiber number. The authors thank Ken W. Bourell for assistance with the thin sectioning for electron microscopy. The graphs in this paper were generated by the authors using the SOLO scientific graphics software developed by Epidemiology Division, University of Texas Southwestern Medical Center at Dallas. This work was supported in part by the National Institute of Arthritis and Musculoskeletal and Skin Disease Grant AR 40056 and Physical Therapy Foundation Grant. REFERENCES Alway, S. E., Winchester, P. K., Davis, M. E., and Gonyea, W. J. (1989). Regionalized adaptations and muscle fiber proliferation in stretchinduced enlargement. J Appl. Physiol. 66,771-781. Alway, S. E., Davis, M. E., and Gonyea, W. J. (1990). Muscle fiber formation and fiber hypertrophy during the onset of stretch-overload. Am. J. Physiol 259, C92-C102. Armstrong, R. B., Ogilvie, R. W., and Schwane, J. A. (1983). Eccentric exercise induced injury to rat skeletal muscle. J. Appl. PhysioL 54, 80-93.
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Carpenter, S., and Karpati, G. (1989). Segmental necrosis and its demarcation in experimental micropuncture injury of skeletal muscle fibers. J. Neuropath. Exp. New. 48,154-170. Ebbeling, C. B., and Clarkson, P. M. (1989). Exercise-induced muscle damage and adaptation. Sports Med. 7,207-234. Friden, J. (1984). Changes in human skeletal muscle induced by long term eccentric exercise. Cell Tissue Res. 236, 365-372. Giddings, C. J., and Gonyea, W. J. (in press). Morphological observations supporting muscle fiber hyperplasia following weight-lifting exercise in cats. Anat. Rec. Giddings, C. J., Neaves, W. B., and Gonyea, W. J. (1985). Muscle fiber necrosis and regeneration induced by prolonged weight lifting exercise in the cats. Anat. Rec., 211, 133-141. Gollnick, P. D., Parsons, D., Riedy, M., and Moore, R. L. (1983). Fiber
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