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Effects of skeletal muscle regeneration on the proliferation potential of satellite cells
Mechanisms of Ageing and Development, 30 (1985) 63-72 Elsevier Scientific Publishers Ireland Ltd.
63
EFFECTS OF SKELETAL MUSCLE REGENERATION ON THE PROLIFERATION POTENTIAL OF SATELLITE CELLS
EDWARD SCHULTZ and DEBRA L. JARYSZAK Department of Anatomy, University of Wisconsin, Madison, WI 53706 (U.S.A.J
(Received July 23rd, 1984) SUMMARY Skeletal muscle satellite cells are myogenic stem cells that function to repair damaged muscle fibers. Participation of satellite cells in a regeneration response following muscle injury results in a significant reduction in their cumulative proliferation potential. The magnitude of the reduction is proportional to the number of regeneration responses in which the cells participate. Key words: Skeletal muscle; Satellite cells; Regeneration; Proliferation potential
INTRODUCTION Recent in vitro studies have demonstrated that satellite cells [1] from mammalian skeletal muscle exhibit a limited or finite proliferative potential that is inversely proportional to the age of the donor animal [2]. The age-related reduction in proliferation potential is similar to that observed in a number of other cell types, but with satellite cells appears to be related to their function of supplying myonuclei to growing myofibers in immature animals [3]. In the rat, the most pronounced reduction in their colony-forming ability /n vitro is coincident with the rapid growth period between birth and 1 - 3 months of age. Satellite cells also function throughout the life of an animal as myogenic stem cells for muscle regeneration and repair [4]. The growth-related reduction in proliferation potential with increased age suggests that the regeneration potential of muscle tissue may also change as an animal matures. Conversely, it follows that a regeneration response in a muscle may significantly alter or diminish the proliferative capacity of the satellite cells that participate in the repair process. The extent of any change in proliferative capacity would likely depend upon the number of stem cells available to supply progeny for regeneration, their cumulative proliferation capacity at the time of injury and the severity or amount of injured muscle. Skeletal muscle regeneration offers a unique opportunity to investigate the extent to which increased proliferative demands might impact on the total proliferative capacity of a select cell population (satellite cells). 0047-6374/85/$03.30 Printed and Published in Ireland
© 1985 Elsevier Scientific Publishers Ireland Ltd.
64 Such an investigation is possible using skeletal muscle because following a regeneration response, the satellite cells in the regenerate are the progeny of the satellite cells that were present in the muscle at the time of injury. The effects of inducing additional proliferations through a "normal" repair process can easily be monitored by removing the satellite cells from the regenerate and examining their colony-forming ability in vitro.
In the present study we examined the effect of single and multiple regeneration cycles on the proliferative capacity of satellite cells in the adult rat extensor digitorum lougus muscle. Because the magnitude of any changes in proliferative capacity of satellite ceils is dependent upon the amount of muscle that is damaged, each degeneration/regeneration cycle was induced by the intramuscular injection of the local anesthetic Marcaine [5]. This drug has been shown both in vivo and in vitro to selectively kill myofibers yet not alter the ability of satellite cells to form new myofibers [6-81 . Following multiple regeneration cycles, satellite cells removed from the regenerates exhibited reduced colonyforming ability when grown in vitro. The magnitude of the reduction was proportional to the number of regeneration cycles the muscles had undergone. MATERIALS AND METHODS Muscle regeneration
A total of 16 male Sprague-Dawley rats were used in this study. Regeneration was induced by injection of the local anesthetic Marcaine into surgically exposed exlensor digitorum longus (EDL) muscles. A needle was introduced at the distal end and passed through the central core of the muscle to the proximal end. As the needle was withdrawn, Marcaine was introduced to the extent that it caused considerable swelling of the muscle. Approximately 1 ml of anesthetic was injected. Excess Marcaine was removed from around the injected muscle, which was then returned to its bed. During the procedure, care was taken not to disrupt the neural and vascular connections to the muscle because of their importance to the success of the regeneralion response. The 2X-Marcaine-treated animals (n = 8) received an initial Marcaine injection at 14 days of age and a second injection 6 weeks later. The animals were killed at 120 days of age. The 4×-treated animals (n = 6) received injections at 30, 44, 58 and 72 days of age and were killed at 90 days of age. Our previous studies indicated that age-related changes in prolilferation potential were insignificant between 90 and 120 days of age [21 so that any differences that were obtained between 2×- and 4×-treated groups would be solely a result of the two additional regeneration responses. In each animal the opposite EDL muscle received no treatment and served as normal control. In vitro assay
The regenerated EDL and its contralateral normal non-injected counterpart were removed from animals anesthetized with 3.5% chloral hydrate and prepared for cell culture using a slight modification of the procedure outlined by Bischoff [g]. Briefly,
65 the muscles were minced, incubated for 1 h in 0.169% trypsin and 0.085% collagenase, washed in fresh medium, then released by trituration into complete medium. The liberated single cells from each muscle were innoculated into nine 100-ram (~3000 cells/dish) gelatin coated culture dishes. In all cases the culture medium consisted of Eagle's minimal essential medium (79%) supplemented with selected horse serum (15%), chick embryo extract (5%) and penicillin (1%). Incubation was carried out in a humidified atmosphere of 5% CO= in air for 4, 6 and 8 days, at which times dishes were fixed and stained. Colony sizes were obtained using a Nikon dissecting microscope at a magnification of 400×. The individual cells in myogenic colonies, identified on the basis of the morphology and staining characteristics [2,10,11] were counted and tabulated. The data were assembled into frequency histograms for comparison.
Effect o f Marcaine on myogenic cells A series of experiments was carried out to ensure that Marcaine had no direct effect on the proliferative behavior of satellite cell-derived myogenic cells. Cultures were first established and allowed to develop into myotubes in order to determine the concentrations at which the drug would kill multinucleated cells. We found that a concentration of 2 and 5 mM Marcaine killed all myofibers in culture within 15 rain, a similar concentration and duration used previously to kill avian myotubes in vitro [8]. Marcaine (2 raM) was presented for a period of 1 5 - 3 0 rain to myoblasts in culture or to freshly dissociated myoblasts prior to plating in culture. The treated satellite cellderived myoblasts were grown in vitro for a period of 4 or 6 days. The cultures were assayed as described above in order to determine if exposure to Marcaine altered the proliferative behavior of the cells. RESULTS
Effects o f Marcaine on the proliferation behavior o f satellite cells in vitro Previous studies demonstrated that Marcaine kills multinucleated myofibers but not myoblasts (satellite cells) [8], however, it was still possible that Marcaine might have some direct effect on the proliferation behavior of treated cells. Because of the importance to this study of any such effect we examined the in vitro growth of normal and Marcaine-treated satellite cell-derived myoblasts. Treatment of myoblasts either prior to plating or after innoculation and attachment in culture dishes produced no measurable alteration in the subsequent growth of the cells. A Student's t-test of the mean colony sizes of treated (19.82 -+ 14.87) and untreated cultures (12.25 -+ 8.07), showed no significant differences. Therefore, any differences in the growth potential of satellite cells from normal and regenerated muscles reported below can be attributed to their involvement in a regeneration response and not to a direct Marcaine influence. Proliferation potentials o f sat~,llite cells from normal 3 month-oM EDL muscles The distribution of colony sizes of satellite cells from EDL muscles of 3-month-old
66 rats, grown for 4, 6 and 8 days in vitro is illustrated in Fig. 1. The frequency histograms are virtually superimposable for each in vitro growth period suggesting that within 4 days of growth, virtually the entire satellite cell population has expressed its full replication potential, Addition of [3HI thymidine into the culture medium (0.4 #Ci/ml) after 4 days showed essentially no incorporation by cells in small colonies. Labeled cells were located only in the largest colonies of the distribution where they constituted only a small percentage of the total cells in the colony. One feature that was noted to change between 4 and 8 days was approximately a 10cA reduction in the proportion of colonies with 16 or more cells and a concurrent increase in the proportion of colonies with less than 16 cells. A possible explanation is that some 8-
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NUMBER
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1. Frequency histograms illustrate the colony size profiles of myogenic (satellite) cells from normal EDL muscles of 90-day-old rats after 4 , 6 a n d 8 d a y s in vitro growth. Analysis of variance indicates no significant change in mean number of ceils per colony between 4 and 8 days growth. Fig.
67 TABLE I MEAN COLONY SIZE AT 4 DAYS IN VITRO a
Normal 2 X-Marcaine 4)< -Marcaine
Mean
S.D.
N
Total colonies counted
19.59 9.44 8.20
9.01 3.37 1.1 t
8 8 6
1054 1433 1503
aN = number of animals/group. S.D. = standard deviation. Analysis of variance indicates that the mean colony size from 2×- and 4X-Marcaine treated cells are significantly reduced from normal (P < 0.05).
o f the dividing cells (or non-dividing) in the larger colonies detached during the 4 - 8 - d a y period and then re-attached at other sites on the dish where, if division continued, they would be counted as small colonies. The same phenomenon was also noted in the colony distributions o f satellite cells from the 2×-Marcaine-treated muscles. Proliferation potentials o f satellite cells f r o m regenerated muscles Following two or more regeneration responses, satellite cells from the regenerates had a mean colony size at 4 days growth in vitro that was significantly reduced from normal (Table I, Fig. 2). The decrease in the mean number o f cells per colony appeared to result mainly from a dramatic reduction in the proportion o f large colonies (Fig. 3). After two regeneration responses only 8.5% of the colonies had ~ 1 6 cells compared to 35% o f the normal colonies. Colonies from the 2×-Marcaine-treated muscles were rarely greater than 50 cells. After four Marcaine treatments only 4% o f the colonies had :>16 cells. In all cases there was a substantial concurrent increase in the proportion of colonies ~ 1 6 cells. Accompanying the changes in colony size distributions were changes in the yield of myogenic cells. Estimates o f myogenic cells suggested a substantial increase in the yield that was most pronounced in the 4×-treated muscles (Table II). These data suggest that the the that the
number o f 4×-treated ranged in 2X-treated
small colony forming cells were increased in all regenerates. In addition, muscles also yielded cells that by 6 days in culture grew into colonies size up to 120 cells. Such large colonies never developed from cells o f muscles and were found only in cultures o f cells from normal muscles.
DISCUSSION
Satellite cells and muscle regeneration This study demonstrates a direct relationship between the number of mitotic divisions through which a cell passes in vivo and the number o f divisions that remain. More specifically, it shows an inverse relationship between the remaining proliferative capacity o f
68
4
DAYS
8765452To0
10 20 50 40 50 60 70 80 90 100 110 120 150 140 550
12 m
2X-MARCAINE
1110987654521
0 10 20 30 40 50 6 0 70 80 90 100 llO 120 1~0
350
16 4X-
~5
MARCAINE
14 13121110987-
6-
0
~0 20 30 4 0 50 60 NUMBER
OF
70 BO 90 100 110 120 150 CELLS/
350
COLONY
Fig. 2. F r e q u e n c y histograms illustrate t h e effects of two or four regeneration responses, induced by Marcaine injections, o n t h e colony size profiles o f satellite cells grown in ~'itro for 4 days. Muscle regeneration increased t h e proportion of small colonies and reduced the proportion of larger colonies.
69 100 9o 8o 70 t.-z 60 L~J 5O Ixl O_
40
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!
,
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10
20
50
NUMBER
-
-
40
OF CELLS/
T
50
-
-"l
I
60
70
>70
COLONY
Fig. 3. Percentage o f colonies capable of attaining at least a specified number of cells per colony after 4 days in ~'itro growth. Solid line illustrates normal cells; ( - - - ) 2 treatments of Marcaine; (. . . . . ) 4 treatments of Marcaine. satellite cells in vitro and the number of regeneration responses in which the cells participated in vivo. It was possible to draw these conclusions because satellite cells are known to be the stem cells of manunalian muscle regeneration and because of some unique features of satellite cell behavior. Recent work (Gibson, M.C. et aL, unpublished observations) has shown that when a single muscle is damaged in a manner similar to the procedure used in this study, there appears to be no recruitment of exogenous myogenic cells from , adjacent muscles. The situation is quite different within a muscle where satellite cells migrate relatively long distances to participate in the repair of a damaged area [ 12]. The absence of exogenous satellite cell recruitment into the Marcaine-treated muscles indicates that satellite cells in the regenerates are solely the progeny of the satellite cell population in the muscle at the time of injection. Therefore, in vitro examination of the colony-forming ability of satellite cells from the regenerates permits a direct observation of the effects of one or more regeneration cycles on the proliferation potential of the cells. Distribution o f colony sizes." normal muscle The colony size distributions from normal muscles indicate that satellite cells are a
TABLE I1 RATIO OF MYOGENIC CELL YIELD: EXPERIMENTAL VS. NORMAL MUSCLES No. of Marcaine injections
Ratio
IX 2× 4X
1.08 1.46 2.30
70 heterogeneous population with respect to proliferation potential and as a result give rise to a spectrum of colonies ranging in size from one cell to over 200 cells. Approximately 35% of the population, or in the order of 1.8 X l0 s cells [13], are capable of more than four population doublings. The Poisson distribution of colony sizes that we obtained is similar to that reported by Quinn and Nameroff (sum of A, B, C, in Fig. 9) [141 of embryonic chick myogenic colonies after 76 h in culture. However, cells derived from young donors exhibit growth in vitro for periods exceeding 7 days so the extent to which the profile of colony-size distribution illustrated by Quinn and Nameroff might change is unknown. The lack of any change in the colony-size profile beyond 4 days in vitro growth of the normal 3-month cells suggests the cells have expressed their full proliferative potential. This lack of change is consistent with previous studies [2] and with the [aH] thymidine incorporation study where only cells of large colonies incorporated label. The profile of colony sizes we obtained may be an age-related characteristic of 3-month old muscle. The large population of small colonies (<216 cells) derived from the satellite cells of mature rat muscle may represent cells that were actively dividing during growth in vivo in order to supply the accretion of myofiber DNA. Alternatively, these cells may be regarded as a source of cells entering terminal differentiation and, therefore, a subpopulation of cells that are readily available to fuse with the growing myofibers [141 . A population of satellite cells that fuse after one or two divisions is consistent with the observations of Moss and Leblond [3] who found 65% of labeled satellite cells in growing muscle had fused with myofibers within 72 h of injection of [3H]thymidine into growing animals. A high fusion rate is also consistent with the fact that the satellite cell population in the EDL appears not to be self-renewing during growth [13]. The large colonies may represent a subpopulation of cells that, under normal conditions, are mitotically quiescent [151 but are activated in diseased or damaged conditions for muscle repair. Distribution o f colony sizes: Marcaine-injected muscles The frequency histograms of colony sizes from cells of the 2X-treated muscles show that virtually all of the colonies above 60 cells have been eliminated as a result of the regeneration responses (Fig. 2). This observation suggests that all cells in the injected muscles participated in the repair process or, at the very least, only those cells participated that were capable of forming large colonies. We have shown previously that virtually the entire satellite cell population is activated even when the portion of muscle that is injured is very small [12]. The lack of larger colonies also supports our suggestion that there was little or no recruitment of cells from exogenous sources because undoubtedly some cells that immigrated into the injected muscle would have had a limited role in repair and consequently produced larger colonies in vitro. The concommitant loss of larger colonies from injected muscles and the increase in proportion of small colonies resulted in a significant reduction in the mean colony size at 4 days in vitro growth. We conclude from these results that regeneration of the injected
71 EDI_ muscles occurred at the expense of the cumulative proliferative potential of the
satellite cell population and, alternatively, that a successful regeneration must be dependent upon not only the size of the stem cell population but also its cumulative proliferation capacity. Likewise, the increase in proportion of small colonies occurs at the expense of the cells with greater proliferative potential. We are unable to explain the increased yield of myogenic cells from the regenerated muscles. An increase in cell numbers would be a natural consequence of activating the large colony forming cells, but why these myogenic cells did not contribute to formation of new myofibers is unknown. The 4×-treated muscles also exhibited the same trends observed in the 2X-treated muscles. The significant reduction in mean colony size when compared to normal, occurred mainly as a result of an increase in the proportion of small colonies. This increased proportion may have been, in part, the result of an increase in their number since the estimated increase in myogenic cell yield was greatest in the 4×-treated muscles. The appearance of cells giving rise to large colonies in these muscles was unexpected. Possibilities that might account for these large colonies include factor(s) in the environment of the multiply-damaged muscle which increased the proliferative capacity of a small but responsive population of cells. Addition of hydrocortisone to cells in vitro, for example, is capable of increasing their proliferative capacity [16,17[ and is first detected as an increase in the size of colonies [18]. The increase in proliferative capacity of cells appears proportional to the duration of treatment and their remaining population doublings. Interestingly, damaged myofibers appear to release substance(s) that specifically stimulate nlyoblast growth [19]. Such substances may be acting to increase the proliferation potential of satellite cells only in the 4X-treated muscles simply because of the duration of e>:posure. A second possible source of the larger colonies is from migration of exogenous cells into the regenerating muscle. The multiple surgical interventions that were required for the Marcaine injections may have resulted in the production of adhesions between muscles. The adhesions would likely have produced "bridges" over which satellite cells could migrate into the damaged muscles, We have recently obtained evidence that direct connnunications between muscles is required for migration of satellite cells to occur between adjacent muscles. Finally, we have not ruled out a direct effect of Marcaine due to repeated administration to the same lineage of satellite cells. An interesting feature of this system is that regeneration changes the normally heterogeneous population of satellite cells with respect to proliferative capacity to a relatively pure population of cells with a rather narrow range of proliferation capacity. This is in distinction to the satellite cells in normal senile muscle where cells continue to be present with large colony-forming abilities [2]. Satellite cells from regenerated muscle appear to be a more suitable population to examine the effects of proliferative aging on the functional abilities [20] without contaminating cells of young proliferative age.
72 ACKNOWLEDGEMENTS
This work was supported
by NSF Grant PCM 8302348.
We t h a n k D o n n a W a s h a f o r
typing the manuscripl.
REFERENCES 1 A. Mauro, Satellite cells o f skeletal muscle fibers. J. Bioph)'s. Biochem. (),tol., 9 ( 1961) 4 9 3 - 4 9 5 . 2 E. Schultz and B.H. Lipton, Skeletal muscle cells: changes in proliferation potential as a function o f a g c . Mech. Ageing Der.. 20 (1982) 377 383. 3 F.P. Moss and C.P. Leblond, Satellite cells as a .source of nuclei in muscles of growing rats. A,zat. Rec.. 170 11971) 421 - 4 3 6 . 4 M.H. Snow, Origin o f regenerating myoblasts in manrmalian skeletal muscle. In A. Mauro ted.), Muscle Regeneratiol~, Raven Press, New York, 1979, pp. 91 - 100. 5 P.W. Benoit and W.D. Belt, Destruction and regeneration of skeletal muscle after treatment with a local anesthetic, bupivicaine (Marcaine). J. Anat.. 10 7 (1970) 547 556. 6 E.C.B. Hall-Craggs, Rapid degeneration and regeneration of a whole skeletal muscle following treatment with Bupivicaine (Marcaine). Exp. Neurol., 43 11974) 349 358. 7 1. Jirmanova and S. Thesloff, Ultrastructural study of experimental muscle degeneration and regeneration in t he adult rat. Z. Zelllorsch.. 131 ( 1972) 77 - 9 7 . 8 E. Schultz and B.H. Lipton, The effect of Marcaine on muscle and non-muscle cells m r#ro. Anat. Rec., 191 11978) 3 5 1 - 3 7 0 . 9 R. Bischoff, Enzymatic liberation of myogenic cells from adult rat inu~'lc. Anat. Rec.. 180 11974) 642-662. 10 I.R. Konigsberg, Clonal analysis of myogenesis. Science, 140 I 1963) 1 2 7 3 - 1 2 8 4 . 11 B.H. Lipton, A fine structure analysis oi normal and modulated cells in myogenic cultures. Der. Biol., 60 11977) 2 6 - 4 7 . 12 E. Schultz, D.L. Jaryszak and C.R. Valliere, Response of satellite cells to focal skeletal muscle injury. Anat. Rec., 208 (1984) 159A. 13 M.C. Gibson and E, Schultz, Age-related differences in the absolute number of satellite cells in rat soleus and extensor digilorum Iongus muscles. Muscle Net're, 6 11983) 574 5 8 0 . 14 L.S. Quinn and M. Nameroff, Analysis o f the myogenic lineage in chick embryos. I11. Quantitative evidence for discrete c o m p a r t m e n t s of precursor cells. D(ljerentiation, 24 (1983) 111 - 1 2 3 . 15 E. Schultz, M.C. Gibson and T. Champion, Satellite cells are mitotically quiescent in mature m o u s e muscle: An EM and radioautographic study. J. Exp. ZooL, 206 (1978) 451 456. 16 V.J. Cristofalo, Hydrocortisone as a modulator of cell division and population life span. In V.J. Cristofalo, J. Roberts and R.C. Adelman (eds.), Explorations in Ageing, Plenum Press, New York, 1975, pp. 5 7 - 7 9 . 17 V.J. Cristofalo and B.M. Stanulis-Praeger, Cellular senescence in ritro. Adr. ('ell Culture (1982) 1-68. 18 J.R. Smith, O.M. Pereira-Smith, K.I. Braunschweiger, T.W. Roberts and R.G. Whitney, A general m e t h o d for determining the replicative age of normal animal cell cultures. Mech. Ageing, Def.. 12 (1980) 3 5 5 - 3 6 5 . 19 R. Bischoff, Activation and proliferation of muscle satellite cells on isolated fibers. J. Cell Biol., 91 (1981) 342A. 20 R.E. Allen, P.K. McAllister, K.C. Masak and G.R. Anderson, Influence of age on accumulatk~n of c~-actin in satellite cell-derived m y o t u b e s in vitro. Mech. Ageing Dev.. 18 (! 982) 8 9 - 9 5 .
63
EFFECTS OF SKELETAL MUSCLE REGENERATION ON THE PROLIFERATION POTENTIAL OF SATELLITE CELLS
EDWARD SCHULTZ and DEBRA L. JARYSZAK Department of Anatomy, University of Wisconsin, Madison, WI 53706 (U.S.A.J
(Received July 23rd, 1984) SUMMARY Skeletal muscle satellite cells are myogenic stem cells that function to repair damaged muscle fibers. Participation of satellite cells in a regeneration response following muscle injury results in a significant reduction in their cumulative proliferation potential. The magnitude of the reduction is proportional to the number of regeneration responses in which the cells participate. Key words: Skeletal muscle; Satellite cells; Regeneration; Proliferation potential
INTRODUCTION Recent in vitro studies have demonstrated that satellite cells [1] from mammalian skeletal muscle exhibit a limited or finite proliferative potential that is inversely proportional to the age of the donor animal [2]. The age-related reduction in proliferation potential is similar to that observed in a number of other cell types, but with satellite cells appears to be related to their function of supplying myonuclei to growing myofibers in immature animals [3]. In the rat, the most pronounced reduction in their colony-forming ability /n vitro is coincident with the rapid growth period between birth and 1 - 3 months of age. Satellite cells also function throughout the life of an animal as myogenic stem cells for muscle regeneration and repair [4]. The growth-related reduction in proliferation potential with increased age suggests that the regeneration potential of muscle tissue may also change as an animal matures. Conversely, it follows that a regeneration response in a muscle may significantly alter or diminish the proliferative capacity of the satellite cells that participate in the repair process. The extent of any change in proliferative capacity would likely depend upon the number of stem cells available to supply progeny for regeneration, their cumulative proliferation capacity at the time of injury and the severity or amount of injured muscle. Skeletal muscle regeneration offers a unique opportunity to investigate the extent to which increased proliferative demands might impact on the total proliferative capacity of a select cell population (satellite cells). 0047-6374/85/$03.30 Printed and Published in Ireland
© 1985 Elsevier Scientific Publishers Ireland Ltd.
64 Such an investigation is possible using skeletal muscle because following a regeneration response, the satellite cells in the regenerate are the progeny of the satellite cells that were present in the muscle at the time of injury. The effects of inducing additional proliferations through a "normal" repair process can easily be monitored by removing the satellite cells from the regenerate and examining their colony-forming ability in vitro.
In the present study we examined the effect of single and multiple regeneration cycles on the proliferative capacity of satellite cells in the adult rat extensor digitorum lougus muscle. Because the magnitude of any changes in proliferative capacity of satellite ceils is dependent upon the amount of muscle that is damaged, each degeneration/regeneration cycle was induced by the intramuscular injection of the local anesthetic Marcaine [5]. This drug has been shown both in vivo and in vitro to selectively kill myofibers yet not alter the ability of satellite cells to form new myofibers [6-81 . Following multiple regeneration cycles, satellite cells removed from the regenerates exhibited reduced colonyforming ability when grown in vitro. The magnitude of the reduction was proportional to the number of regeneration cycles the muscles had undergone. MATERIALS AND METHODS Muscle regeneration
A total of 16 male Sprague-Dawley rats were used in this study. Regeneration was induced by injection of the local anesthetic Marcaine into surgically exposed exlensor digitorum longus (EDL) muscles. A needle was introduced at the distal end and passed through the central core of the muscle to the proximal end. As the needle was withdrawn, Marcaine was introduced to the extent that it caused considerable swelling of the muscle. Approximately 1 ml of anesthetic was injected. Excess Marcaine was removed from around the injected muscle, which was then returned to its bed. During the procedure, care was taken not to disrupt the neural and vascular connections to the muscle because of their importance to the success of the regeneralion response. The 2X-Marcaine-treated animals (n = 8) received an initial Marcaine injection at 14 days of age and a second injection 6 weeks later. The animals were killed at 120 days of age. The 4×-treated animals (n = 6) received injections at 30, 44, 58 and 72 days of age and were killed at 90 days of age. Our previous studies indicated that age-related changes in prolilferation potential were insignificant between 90 and 120 days of age [21 so that any differences that were obtained between 2×- and 4×-treated groups would be solely a result of the two additional regeneration responses. In each animal the opposite EDL muscle received no treatment and served as normal control. In vitro assay
The regenerated EDL and its contralateral normal non-injected counterpart were removed from animals anesthetized with 3.5% chloral hydrate and prepared for cell culture using a slight modification of the procedure outlined by Bischoff [g]. Briefly,
65 the muscles were minced, incubated for 1 h in 0.169% trypsin and 0.085% collagenase, washed in fresh medium, then released by trituration into complete medium. The liberated single cells from each muscle were innoculated into nine 100-ram (~3000 cells/dish) gelatin coated culture dishes. In all cases the culture medium consisted of Eagle's minimal essential medium (79%) supplemented with selected horse serum (15%), chick embryo extract (5%) and penicillin (1%). Incubation was carried out in a humidified atmosphere of 5% CO= in air for 4, 6 and 8 days, at which times dishes were fixed and stained. Colony sizes were obtained using a Nikon dissecting microscope at a magnification of 400×. The individual cells in myogenic colonies, identified on the basis of the morphology and staining characteristics [2,10,11] were counted and tabulated. The data were assembled into frequency histograms for comparison.
Effect o f Marcaine on myogenic cells A series of experiments was carried out to ensure that Marcaine had no direct effect on the proliferative behavior of satellite cell-derived myogenic cells. Cultures were first established and allowed to develop into myotubes in order to determine the concentrations at which the drug would kill multinucleated cells. We found that a concentration of 2 and 5 mM Marcaine killed all myofibers in culture within 15 rain, a similar concentration and duration used previously to kill avian myotubes in vitro [8]. Marcaine (2 raM) was presented for a period of 1 5 - 3 0 rain to myoblasts in culture or to freshly dissociated myoblasts prior to plating in culture. The treated satellite cellderived myoblasts were grown in vitro for a period of 4 or 6 days. The cultures were assayed as described above in order to determine if exposure to Marcaine altered the proliferative behavior of the cells. RESULTS
Effects o f Marcaine on the proliferation behavior o f satellite cells in vitro Previous studies demonstrated that Marcaine kills multinucleated myofibers but not myoblasts (satellite cells) [8], however, it was still possible that Marcaine might have some direct effect on the proliferation behavior of treated cells. Because of the importance to this study of any such effect we examined the in vitro growth of normal and Marcaine-treated satellite cell-derived myoblasts. Treatment of myoblasts either prior to plating or after innoculation and attachment in culture dishes produced no measurable alteration in the subsequent growth of the cells. A Student's t-test of the mean colony sizes of treated (19.82 -+ 14.87) and untreated cultures (12.25 -+ 8.07), showed no significant differences. Therefore, any differences in the growth potential of satellite cells from normal and regenerated muscles reported below can be attributed to their involvement in a regeneration response and not to a direct Marcaine influence. Proliferation potentials o f sat~,llite cells from normal 3 month-oM EDL muscles The distribution of colony sizes of satellite cells from EDL muscles of 3-month-old
66 rats, grown for 4, 6 and 8 days in vitro is illustrated in Fig. 1. The frequency histograms are virtually superimposable for each in vitro growth period suggesting that within 4 days of growth, virtually the entire satellite cell population has expressed its full replication potential, Addition of [3HI thymidine into the culture medium (0.4 #Ci/ml) after 4 days showed essentially no incorporation by cells in small colonies. Labeled cells were located only in the largest colonies of the distribution where they constituted only a small percentage of the total cells in the colony. One feature that was noted to change between 4 and 8 days was approximately a 10cA reduction in the proportion of colonies with 16 or more cells and a concurrent increase in the proportion of colonies with less than 16 cells. A possible explanation is that some 8-
4
7-
DAY
65432 I O-
()
10 20 30 4 0 50 6 0 70 8 0 90 100 110 120 130 1 4 0 3 5 0
98-
I ,
6
DAY
7I--
Z W 0 n," W n
65-
4.L 32I
0 0
I0 20 30 40 50 60 70 80 90 100 110
180 190 EO0
8 DAY
(5543210
I
~
Ilk . . . . .
NUMBER
OF
I "'I 7 ~ "I 80 90 100 II0 120 CELLS/
190 2 0 0
COLONY
1. Frequency histograms illustrate the colony size profiles of myogenic (satellite) cells from normal EDL muscles of 90-day-old rats after 4 , 6 a n d 8 d a y s in vitro growth. Analysis of variance indicates no significant change in mean number of ceils per colony between 4 and 8 days growth. Fig.
67 TABLE I MEAN COLONY SIZE AT 4 DAYS IN VITRO a
Normal 2 X-Marcaine 4)< -Marcaine
Mean
S.D.
N
Total colonies counted
19.59 9.44 8.20
9.01 3.37 1.1 t
8 8 6
1054 1433 1503
aN = number of animals/group. S.D. = standard deviation. Analysis of variance indicates that the mean colony size from 2×- and 4X-Marcaine treated cells are significantly reduced from normal (P < 0.05).
o f the dividing cells (or non-dividing) in the larger colonies detached during the 4 - 8 - d a y period and then re-attached at other sites on the dish where, if division continued, they would be counted as small colonies. The same phenomenon was also noted in the colony distributions o f satellite cells from the 2×-Marcaine-treated muscles. Proliferation potentials o f satellite cells f r o m regenerated muscles Following two or more regeneration responses, satellite cells from the regenerates had a mean colony size at 4 days growth in vitro that was significantly reduced from normal (Table I, Fig. 2). The decrease in the mean number o f cells per colony appeared to result mainly from a dramatic reduction in the proportion o f large colonies (Fig. 3). After two regeneration responses only 8.5% of the colonies had ~ 1 6 cells compared to 35% o f the normal colonies. Colonies from the 2×-Marcaine-treated muscles were rarely greater than 50 cells. After four Marcaine treatments only 4% o f the colonies had :>16 cells. In all cases there was a substantial concurrent increase in the proportion of colonies ~ 1 6 cells. Accompanying the changes in colony size distributions were changes in the yield of myogenic cells. Estimates o f myogenic cells suggested a substantial increase in the yield that was most pronounced in the 4×-treated muscles (Table II). These data suggest that the the that the
number o f 4×-treated ranged in 2X-treated
small colony forming cells were increased in all regenerates. In addition, muscles also yielded cells that by 6 days in culture grew into colonies size up to 120 cells. Such large colonies never developed from cells o f muscles and were found only in cultures o f cells from normal muscles.
DISCUSSION
Satellite cells and muscle regeneration This study demonstrates a direct relationship between the number of mitotic divisions through which a cell passes in vivo and the number o f divisions that remain. More specifically, it shows an inverse relationship between the remaining proliferative capacity o f
68
4
DAYS
8765452To0
10 20 50 40 50 60 70 80 90 100 110 120 150 140 550
12 m
2X-MARCAINE
1110987654521
0 10 20 30 40 50 6 0 70 80 90 100 llO 120 1~0
350
16 4X-
~5
MARCAINE
14 13121110987-
6-
0
~0 20 30 4 0 50 60 NUMBER
OF
70 BO 90 100 110 120 150 CELLS/
350
COLONY
Fig. 2. F r e q u e n c y histograms illustrate t h e effects of two or four regeneration responses, induced by Marcaine injections, o n t h e colony size profiles o f satellite cells grown in ~'itro for 4 days. Muscle regeneration increased t h e proportion of small colonies and reduced the proportion of larger colonies.
69 100 9o 8o 70 t.-z 60 L~J 5O Ixl O_
40
%,\\ ".° \
!
,
""(~'~-"T
10
20
50
NUMBER
-
-
40
OF CELLS/
T
50
-
-"l
I
60
70
>70
COLONY
Fig. 3. Percentage o f colonies capable of attaining at least a specified number of cells per colony after 4 days in ~'itro growth. Solid line illustrates normal cells; ( - - - ) 2 treatments of Marcaine; (. . . . . ) 4 treatments of Marcaine. satellite cells in vitro and the number of regeneration responses in which the cells participated in vivo. It was possible to draw these conclusions because satellite cells are known to be the stem cells of manunalian muscle regeneration and because of some unique features of satellite cell behavior. Recent work (Gibson, M.C. et aL, unpublished observations) has shown that when a single muscle is damaged in a manner similar to the procedure used in this study, there appears to be no recruitment of exogenous myogenic cells from , adjacent muscles. The situation is quite different within a muscle where satellite cells migrate relatively long distances to participate in the repair of a damaged area [ 12]. The absence of exogenous satellite cell recruitment into the Marcaine-treated muscles indicates that satellite cells in the regenerates are solely the progeny of the satellite cell population in the muscle at the time of injection. Therefore, in vitro examination of the colony-forming ability of satellite cells from the regenerates permits a direct observation of the effects of one or more regeneration cycles on the proliferation potential of the cells. Distribution o f colony sizes." normal muscle The colony size distributions from normal muscles indicate that satellite cells are a
TABLE I1 RATIO OF MYOGENIC CELL YIELD: EXPERIMENTAL VS. NORMAL MUSCLES No. of Marcaine injections
Ratio
IX 2× 4X
1.08 1.46 2.30
70 heterogeneous population with respect to proliferation potential and as a result give rise to a spectrum of colonies ranging in size from one cell to over 200 cells. Approximately 35% of the population, or in the order of 1.8 X l0 s cells [13], are capable of more than four population doublings. The Poisson distribution of colony sizes that we obtained is similar to that reported by Quinn and Nameroff (sum of A, B, C, in Fig. 9) [141 of embryonic chick myogenic colonies after 76 h in culture. However, cells derived from young donors exhibit growth in vitro for periods exceeding 7 days so the extent to which the profile of colony-size distribution illustrated by Quinn and Nameroff might change is unknown. The lack of any change in the colony-size profile beyond 4 days in vitro growth of the normal 3-month cells suggests the cells have expressed their full proliferative potential. This lack of change is consistent with previous studies [2] and with the [aH] thymidine incorporation study where only cells of large colonies incorporated label. The profile of colony sizes we obtained may be an age-related characteristic of 3-month old muscle. The large population of small colonies (<216 cells) derived from the satellite cells of mature rat muscle may represent cells that were actively dividing during growth in vivo in order to supply the accretion of myofiber DNA. Alternatively, these cells may be regarded as a source of cells entering terminal differentiation and, therefore, a subpopulation of cells that are readily available to fuse with the growing myofibers [141 . A population of satellite cells that fuse after one or two divisions is consistent with the observations of Moss and Leblond [3] who found 65% of labeled satellite cells in growing muscle had fused with myofibers within 72 h of injection of [3H]thymidine into growing animals. A high fusion rate is also consistent with the fact that the satellite cell population in the EDL appears not to be self-renewing during growth [13]. The large colonies may represent a subpopulation of cells that, under normal conditions, are mitotically quiescent [151 but are activated in diseased or damaged conditions for muscle repair. Distribution o f colony sizes: Marcaine-injected muscles The frequency histograms of colony sizes from cells of the 2X-treated muscles show that virtually all of the colonies above 60 cells have been eliminated as a result of the regeneration responses (Fig. 2). This observation suggests that all cells in the injected muscles participated in the repair process or, at the very least, only those cells participated that were capable of forming large colonies. We have shown previously that virtually the entire satellite cell population is activated even when the portion of muscle that is injured is very small [12]. The lack of larger colonies also supports our suggestion that there was little or no recruitment of cells from exogenous sources because undoubtedly some cells that immigrated into the injected muscle would have had a limited role in repair and consequently produced larger colonies in vitro. The concommitant loss of larger colonies from injected muscles and the increase in proportion of small colonies resulted in a significant reduction in the mean colony size at 4 days in vitro growth. We conclude from these results that regeneration of the injected
71 EDI_ muscles occurred at the expense of the cumulative proliferative potential of the
satellite cell population and, alternatively, that a successful regeneration must be dependent upon not only the size of the stem cell population but also its cumulative proliferation capacity. Likewise, the increase in proportion of small colonies occurs at the expense of the cells with greater proliferative potential. We are unable to explain the increased yield of myogenic cells from the regenerated muscles. An increase in cell numbers would be a natural consequence of activating the large colony forming cells, but why these myogenic cells did not contribute to formation of new myofibers is unknown. The 4×-treated muscles also exhibited the same trends observed in the 2X-treated muscles. The significant reduction in mean colony size when compared to normal, occurred mainly as a result of an increase in the proportion of small colonies. This increased proportion may have been, in part, the result of an increase in their number since the estimated increase in myogenic cell yield was greatest in the 4×-treated muscles. The appearance of cells giving rise to large colonies in these muscles was unexpected. Possibilities that might account for these large colonies include factor(s) in the environment of the multiply-damaged muscle which increased the proliferative capacity of a small but responsive population of cells. Addition of hydrocortisone to cells in vitro, for example, is capable of increasing their proliferative capacity [16,17[ and is first detected as an increase in the size of colonies [18]. The increase in proliferative capacity of cells appears proportional to the duration of treatment and their remaining population doublings. Interestingly, damaged myofibers appear to release substance(s) that specifically stimulate nlyoblast growth [19]. Such substances may be acting to increase the proliferation potential of satellite cells only in the 4X-treated muscles simply because of the duration of e>:posure. A second possible source of the larger colonies is from migration of exogenous cells into the regenerating muscle. The multiple surgical interventions that were required for the Marcaine injections may have resulted in the production of adhesions between muscles. The adhesions would likely have produced "bridges" over which satellite cells could migrate into the damaged muscles, We have recently obtained evidence that direct connnunications between muscles is required for migration of satellite cells to occur between adjacent muscles. Finally, we have not ruled out a direct effect of Marcaine due to repeated administration to the same lineage of satellite cells. An interesting feature of this system is that regeneration changes the normally heterogeneous population of satellite cells with respect to proliferative capacity to a relatively pure population of cells with a rather narrow range of proliferation capacity. This is in distinction to the satellite cells in normal senile muscle where cells continue to be present with large colony-forming abilities [2]. Satellite cells from regenerated muscle appear to be a more suitable population to examine the effects of proliferative aging on the functional abilities [20] without contaminating cells of young proliferative age.
72 ACKNOWLEDGEMENTS
This work was supported
by NSF Grant PCM 8302348.
We t h a n k D o n n a W a s h a f o r
typing the manuscripl.
REFERENCES 1 A. Mauro, Satellite cells o f skeletal muscle fibers. J. Bioph)'s. Biochem. (),tol., 9 ( 1961) 4 9 3 - 4 9 5 . 2 E. Schultz and B.H. Lipton, Skeletal muscle cells: changes in proliferation potential as a function o f a g c . Mech. Ageing Der.. 20 (1982) 377 383. 3 F.P. Moss and C.P. Leblond, Satellite cells as a .source of nuclei in muscles of growing rats. A,zat. Rec.. 170 11971) 421 - 4 3 6 . 4 M.H. Snow, Origin o f regenerating myoblasts in manrmalian skeletal muscle. In A. Mauro ted.), Muscle Regeneratiol~, Raven Press, New York, 1979, pp. 91 - 100. 5 P.W. Benoit and W.D. Belt, Destruction and regeneration of skeletal muscle after treatment with a local anesthetic, bupivicaine (Marcaine). J. Anat.. 10 7 (1970) 547 556. 6 E.C.B. Hall-Craggs, Rapid degeneration and regeneration of a whole skeletal muscle following treatment with Bupivicaine (Marcaine). Exp. Neurol., 43 11974) 349 358. 7 1. Jirmanova and S. Thesloff, Ultrastructural study of experimental muscle degeneration and regeneration in t he adult rat. Z. Zelllorsch.. 131 ( 1972) 77 - 9 7 . 8 E. Schultz and B.H. Lipton, The effect of Marcaine on muscle and non-muscle cells m r#ro. Anat. Rec., 191 11978) 3 5 1 - 3 7 0 . 9 R. Bischoff, Enzymatic liberation of myogenic cells from adult rat inu~'lc. Anat. Rec.. 180 11974) 642-662. 10 I.R. Konigsberg, Clonal analysis of myogenesis. Science, 140 I 1963) 1 2 7 3 - 1 2 8 4 . 11 B.H. Lipton, A fine structure analysis oi normal and modulated cells in myogenic cultures. Der. Biol., 60 11977) 2 6 - 4 7 . 12 E. Schultz, D.L. Jaryszak and C.R. Valliere, Response of satellite cells to focal skeletal muscle injury. Anat. Rec., 208 (1984) 159A. 13 M.C. Gibson and E, Schultz, Age-related differences in the absolute number of satellite cells in rat soleus and extensor digilorum Iongus muscles. Muscle Net're, 6 11983) 574 5 8 0 . 14 L.S. Quinn and M. Nameroff, Analysis o f the myogenic lineage in chick embryos. I11. Quantitative evidence for discrete c o m p a r t m e n t s of precursor cells. D(ljerentiation, 24 (1983) 111 - 1 2 3 . 15 E. Schultz, M.C. Gibson and T. Champion, Satellite cells are mitotically quiescent in mature m o u s e muscle: An EM and radioautographic study. J. Exp. ZooL, 206 (1978) 451 456. 16 V.J. Cristofalo, Hydrocortisone as a modulator of cell division and population life span. In V.J. Cristofalo, J. Roberts and R.C. Adelman (eds.), Explorations in Ageing, Plenum Press, New York, 1975, pp. 5 7 - 7 9 . 17 V.J. Cristofalo and B.M. Stanulis-Praeger, Cellular senescence in ritro. Adr. ('ell Culture (1982) 1-68. 18 J.R. Smith, O.M. Pereira-Smith, K.I. Braunschweiger, T.W. Roberts and R.G. Whitney, A general m e t h o d for determining the replicative age of normal animal cell cultures. Mech. Ageing, Def.. 12 (1980) 3 5 5 - 3 6 5 . 19 R. Bischoff, Activation and proliferation of muscle satellite cells on isolated fibers. J. Cell Biol., 91 (1981) 342A. 20 R.E. Allen, P.K. McAllister, K.C. Masak and G.R. Anderson, Influence of age on accumulatk~n of c~-actin in satellite cell-derived m y o t u b e s in vitro. Mech. Ageing Dev.. 18 (! 982) 8 9 - 9 5 .