The Role of Cyclic AMP in Myogenesis CHARLES J. EPSTEIN,' LUIS JIMINEZ ENRIQUE ROZENGURT
DE
ASUA
AND
Imperial Cancer Research Fund Laboratories, P. 0 . Box N o . 123, Lincoln's Inn Fields, London WC2A 3PX, England
ABSTRACT The effect of exogenously administered cyclic AMP derivatives and of endogenously elevated cyclic AMP levels on the spontaneous fusion of skeletal muscle myoblasts has been investigated. Contrary to earlier reports, cAMP does not appear to have a direct inhibitory effect on the fusion of an established line (L8)of rat myoblasts. Similarly, CAMP did not block the fusion of primary chick myoblasts. However, fusion of the rat myoblasts was prevented when the cAMP induced inhibition of growth prevented the cells from reaching the "critical" cell density necessary for fusion.
The availability of cell systems which differentiate in vitro offers an excellent opportunity to explore the factors involved. Cyclic AMP has been implicated as one of the signals for differentiation. When agents which increase the endogenous level of cAMP are added to the media of adrenal, neuroblastoma, melanoma, and fibroblastic cell cultures, there are striking changes of cell morphology toward that of the "normal" differentiated cell and the appearance of differentiated cell products (Masui and Garren, '71; Furmanski et al., '71; Prasad and Hsie, '71; Johnson and Pastan, '72; Hsie and Puck, '71; Johnson et al., '71). In addition, cell proliferation is reduced. In sharp contrast, the addition of cAMP to a line of rat myoblasts (Yaffe, '68) which are capable of undergoing extensive spontaneous fusion to form myotubes did inhibit differentiation (as represented by fusion) (Wahrmann et al., '73). Further, since adenyl cyclase activity and cAMP concentration were found to decrease at the time of fusion of primary or established rat myoblasts (Wahrmann et al., '73; Reporter and Raveed, '73) it was proposed that a drop in cAMP was somehow correlated with myotube formation. Likewise, the observation of a sharp but only transient increase of cAMP just prior to fusion of primary chick myoblasts has led to the similar conclusion that an elevated level of CAMP, while necessary for initiation of the fusion process, is incompatible with fusion itself (Zalin and Montague, '74). J. CELL. PHYSIOL.,86: 83-90
In this communication we present an alternative interpretation of the inhibitory effect of cAMP on the fusion of established rat myoblasts. Our experiments indicate that cAMP acts by preventing the cells from reaching the density required for cell fusion rather than by imposing a block at a specific stage of myoblast differentiation. MATERIALS A N D METHODS
Myoblast cultures L8 cells, an established line of rat myoblasts, were obtained from Dr. D. Yaffe. The line was cloned twice, shown to be negative for PPLO contamination, and then carried at low densities in a medium composed of Dulbecco's modiKed Eagle's medium (71.2%), M 199 (17.8%), horse serum (10% , Biocult Laboratories), and chick embryo extract (1 % , Flow Laboratories) (Yaffe, '73). Fusion was assessed by examination of plates fixed for ten minutes with Bouin's fixative and stained with the Leishmann stain. Primary cultures of chick myoblasts were prepared (O'Neill and Stockdale, '72) from pectoral muscle of 12-day chick embryos and plated, after preplating, at densities of 1.5 to 2 X 10"cells per 50 mm gelatin coated (Yaffe, '73) (swine skin gelatin type I, Sigma) plastic plates in 3 ml of a medium composed of Hams F-12 (88%), horse serum ( l o % ) , andchickembryoextract (2%). Received Nov. 13, '74. Accepted Jan. 15, '75. Present address: Department of Pediatrics, University of California, S a n Francisco, Ca. 94143.
83
84
C. J. EPSTEIN, L. J. DE ASUA AND E. ROZENGURT
The cultures were grown at 37" in 8-10% C 0 2 in air, and the medium was changed at 24 hours. The plates were fixed and stained as described above. The proportion of cells fusing was determined by counting 200 nuclei in randomly selected fields and scoring the number present in myotubes. To block fusion of chick myoblasts by reduction in calcium concentration, the cultures were made 0.58 mM in ethyleneglycolbis (p-amino ethyl ether) N,N'-tetraacetic acid (EGTA, Sigma) (Paterson and Strohman, '72) at 48 hours. The "block" was released at 72 hours by addition of CaClz to 0.83 mM. Cyclic AMP assays The levels of intracellular cyclic AMP were determined as previously described (Rozengurt and de Asua, '73). Source of compounds Prostaglandin El (PGEI) was obtained from Dr. J. E. Pike of the Upjohn Company, Kalamazoo, Michigan and SQ 20006, a phosphodiesterase inhibitor, from Dr. M. Chasin of Squibb. NG-monobutyryl adenosine 3': 5'-cyclic monophosphoric acid (mbc AMP) and NG,O2'-dibutyryl adenosine 3':5'cyclic monophosphoric acid (dbcAMP) were obtained from Sigma. RESULTS
I
4
I
I
5 6 Days
7
Fig. 1 Effect of single administration of cAMP elevating compounds on growth and fusion of L8 cells. Cells were plated at a density of 0.5 X 105 cells per 50 mm uncoated plastic dish (Nunc) and grown at 37" in an atmosphere of &lo% COz in air. The compounds were added without a change of medium, and the following final drug concentrations were used: theophylline, 1 mM; SQ 20006 0.2 mM; prostaglandin El (PGEI), 33 pg/ml; monobutyryl cyclic AMP (mbcAMP) 0.4 mM. The time of generalized cell fusion is denoted by an asterisk (*); the interrupted line just prior to this time indicates an intervening undetermined rise and fall in cell number. Cell counts were done in a hemocytometer on duplicates of the plates used for staining, and each point represents the average of two plates. In no instance did fusion occur at a density of less than 1 0 6 cells per plate. A. Theophylline plus PGEl at four days. B. Theophylline plus mbc AMP at four days. C. SQ 20006 plus PGEl at four days. D. Theophylline plus PGEl at three days. E. Theophylline plus mbcAMP at three days. F. SQ 20006 plus PGEl at three days.
Effect of CAMP on fusion of L8 cells Since the reported decreases in adenyl cyclase activity and cAMP concentration occurred about the time of fusion, we expected that the inhibitory effects of cAMP would be maximal at or shortly prior to this time. However, neither the phosphodiester- nificant reductions in the final cell density ase inhibitors, theophylline and SQ 20006, achieved. the adenyl cyclase activator, prostaglandin It thus appeared possible that cAMP eleEl (PGE1), nor dibutyryl cAMP (dbcAMP), vating agents were primarily affecting myoalone or in combination, prevented fusion blast growth, and cell fusion only secondwhen introduced one or two days before the arily. To test this interpretation, cultures expected tjme of myotube formation. TOthe were monitored for a period of ten days durcontrary, fusion was not prevented unless ing which time the medium was changed the agents were added at least three days and drugs added 4, 6 and 8 days after inibefore the expected time (fig. 1). However, tiation of the culture (to rule out the posdirect estimation of cell number showed sibility of drug inactivation). Myotube forthat the inhibited cells were less dense mation ultimately took place when the than the fusing ones (fig. 1). Reexamina- treated cells reached a density equivalent tion of the data of Wahrmann et al. (fig. 2, to that of the control cells at the time of '73) indicated that all treatments which their fusion. Apparently, the block imposed blocked fusion in their experiments also by these compounds was not permanent if interfered with cell growth and led to sig- growth could occur.
85
THE ROLE OF CYCLIC AMP IN MYOGENESIS
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Fig. 2 Effect of repeated administration of cAMP elevating compounds on growth and fusion of L8 cells. Cells were plated at a density of 1 0 5 per 50 mm plate. The compounds were added in fresh medium at four hours and 3, 5, and 7 days after plating. The following concentrations were used; theophylline, 1 mM, SQ 20006, 0.2 mM; dibutyryl cyclic AMP, 0.1 and 0.3 mM. Fusion did not occur at a density below 1.3 X 1 0 6 cells per plate and probably not below 2 X 106 cells. Photomicrographs of the control, SQ 20006 plus dbcAMP, and theophylline plus dbcAMP (0.3 mM) cultures are shown in figure 3.
and the formation of long processes (Wahrmann et al., ’73). Coupled with the effects on growth, these morphological changes indicated that the cells were sensitive to the drugs, and their failure to inhibit fusion cannot be attributed to their failure to act on or in the cells. This was further corroborated by direct measurements of intracellular cAMP levels following administration of PGEl and SQ 20006 (table 1). Exposure of growing, non-fusing cells to these agents resulted in a 14-fold increase in cAMP concentration within ten minutes. When cells just about to fuse were similarly treated, a 2-fold elevation of cAMP was observed after 24 hours and fusion was not prevented. Further, the concentration of CAMP, expressed per unit DNA, was higher in fusing than in non-fusing cells, although this apparent elevation might be the result of the claimed decrease in protein content per cell associated with myoblast proliferation (Tarikas and Schubert, ’74) rather than a true increase.
Effects of cell density on fusion of L8 cells Our results indicated that CAMP,whether exogenously administered or endogenously generated, does not inhibit fusion of the rat myoblasts unless profound growth retardation is produced. With less severe growth retardation, the time of fusion is delayed until a “critical” cell density is TABLE 1
Despite these findings, the results of Wahrmann et al. (‘73) raised the possibility that an early increase in cAMP might inhibit myotube formation by interfering with a developmental program initiated at the start of the culture. Accordingly, various combinations of compounds were added within four hours of cell plating, and the medium changed and the drugs replaced every two or three days (fig. 2). Except for the combination of theophylline (1 mM) plus dbcAMP (0.3 mM) which profoundly inhibited proliferation (fig. 3c), fusion, although delayed, was not blocked by any of the agents used, and, once it occurred, myotube formation was qualitatively and quantitatively indistinguishable from normal (figs. 3a,b). All of the pharmacologic agents used in these experiments induced cell spreading
Cyclic AMP concentrations in L8 cells before and after drug administration Cell stage
Treatment
cAMP concentration Pmolesl mg
Moderate density, non-fusing
None
PGEl (30 kg/ml) plus SQ 20006 (0.2 mM) for 10 minutes Early fusion
DNA 367 319 378
5379 4675
None
520 430 458
PGE, (30 pg/ml) plus SQ 20006 (0.2 mM) for 24 hours
909 893
86
C. J. EPSTEIN, L. J . DE ASUA AND E. ROZENGURT
Fig. 3 a. Photomicrograph of control plate (fig. 2) at six days after initiation of culture, showing extensive myotube formation. Original, X 85. b. Myotube formation after ten days in culture of cells treated with SQ 20006 (0.2 mM) plus dbcAMP (0.1 mM) (fig. 2). Original, X 85. c. Decreased cell density, cell flattening, and formation of large atypical cells after ten days in theophylline (1 mM) plus dbcAMP (0.3 mM) (fig. 2). Original, X 85.
on fusion, the effect of cAMP on the fusion of primary chick myoblasts was investigated. Myotube formation in this system is much less density dependent than with rat myoblasts (ONeill and Stockdale, ’72), and previous work on the effects of cAMP on chick myotube formation indicated that cAMP might delay but would not reduce the ultimate extent of fusion (Zalin, ’73). Under normal culture conditions we found that although the cells showed morphological evidence of drug effect, fusion was not reduced, even when the compounds were added at the time of release from an EGTA imposed fusion block (table 2a). Furthermore, when the fusion system was functioning “suboptimally” because of difficulties with the medium, presumably because of a Effect of CAMP on fusion of chick marginal calcium concentration in the semyoblasts rum used (Molinaro, personal communicaTo establish the generality of our findings tion), fusion was generally improved (table with regard to the lack of an effect of cAMP 2b). A similar enhancing effect of dbcAMP
reached. If this interpretation is correct and a “critical” density is required for myotube Formation, it should be possible to simulate the effects of drug induced growth and fusion retardation by plating the cells at different initial densities. The experiment illustrated in figure 4 confirmed this conclusion. Clearly, the time of myotube formation is inversely related to initial density and a minimal density has to be attained before fusion can occur. While the absolute value of this cell density can vary from experiment to experiment by as much as 2-fold (compare figs. 1 and 4), a minimal density of lo6 cells per 50 mm plate under the defined experimental conditions appears to be necessary.
T H E ROLE OF CYCLIC AMP IN MYOGENESIS
Figure 3
87
88
C.
,J.
EPSTEIN, L. J . DE ASUA AND E. ROZENGURT TABLE 2
m e c t of CAMP elevating compounds on fusion of chick e m b r y o myoblasts Fusion Additions to cultures
.,;i_., OOl0
1
a. Normal s y s t e m Control (no additions) PGE, Thedphylline PGEl plus SQ 20006 Control (EGTA, then CaC12) 1 PGEl plus SQ 20006 after release from EGTA block
, 2
3
,
,
L
5
I
6
L
7
L
-
8
Days
Fig. 4 Effect of variation of plating density on cell growth and time of fusion of L8 cells. Cells were plated a t densities between 0.1 and 10 X 105 cells per plate and cultured without change of medium. Although cell counts at fusion of cultures plated at 0.1 and 10 X 1 0 5 cells were not determined, the time of fusion, at eight and three days respectively, are compatible with the results of the other cultures and are indicated by asterisks and vertical lines. Over the range of 0.5 to 5 X 1 0 5 cells per plate, fusion did not occur below a density of 2.2 x 106 cells per plate.
has recently been reported for a spontaneously fusing rhabdomyosarcoma line and for chick myoblasts in low concentrations of calcium (Aw et al., ’73). DISCUSSION
In none of the experiments reported here was exogenous or endogenous elevation of cAMP associated with inhibition of myoblast fusion. Taken together, the present findings do not support the hypothesis that a decrease in intracellular cAMP concentration is a necessary prerequisite for myoblast fusion in either the established rat or primary chick myoblast system. As has already been noted, our results are not essentially different from those of Wahrmann et al. (’73) in that all of their growth curves do show inhibition of cellular proliferation. The one difference between their observations and ours is the concentrations of drugs required to produce an effect on growth and fusion. They obtained inhibitory effects at concentrations as low as 5 x lo-“ M theophylline and 5 X 10 - 5 M dbc
b. “Suboptimal” system Control (no additions) PGE, plus SQ 20006 PGEl plus theophylline dbcAMP plus theophylline PGEl plus SQ 20006 PGEl plus theophylline dbcAMP plus theophylline
Time of addition
72hrs
96hrs
75% (6)
64% (9) 64% (2) 53% (2)
71% (1)
67% (2)
48 hrs 48 hrs 48hrs
10% (1)
2
61% (2)
22% (6)
33% ( 8 )
28hrs
36% ( 1 )
49% (2)
28 hrs
64% (4)
53% (3)
28 hrs
339 ( 3 )
37% (3)
52hrs
46% (1)
55% (2)
52 hrs
33% (3)
44% (3)
52 hrs
34% (3)
46% (3)
EGTA was added at 48 hours and the “block’ released at 72 hours. The following concentrations of drugs were used: theophylline, 1 mM; SQ 20006, 0.2 mM: PGEl, 33 pglml; dbcAMP, 1 mM. Several experiments have been combined, and the numbers in parentheses indicate the number of individual plates (200 cells each) scored. 2 Scored at 76 hours.
AMP, concentrations which had no effect in our hands and which are considerably lower than are effective in other systems. Similarly, our data on the fusion of chick primary myoblasts do not contradict those of Zalin (’73). While she did observe a delay (which we cannot explain) in the onset of fusion with dbcAMP and a theophylline analog, alone or in combination, the final extent of fusion was not significantly reduced. This was true even when there was severe inhibition of cell proliferation as with the dbcAMP plus 3-isobutyl-1-methylxanthine combination. However, while the lack of a direct and independent effect of CAMP on chick myoblast fusion is similar to what we observed in the rat cells, the
THE ROLE OF CYCLIC AMP IN MYOGENESIS
relation of proliferation to fusion clearly is not. Fusion of the rat cells is clearly density dependent while thqt of the chick cells is much less so. This indicates, therefore, that the two systems are not directly comparable to one another and may differ in other essential details. Evidence of such differences already exists with regard to the relation of adenylcyclase activity to fusion. Thus, while Wahrmann et al. (’73) observed a 20 % decrease immediately following fusion in rat myoblast adenylcyclase activity, and the activity decreased even further as fusion proceeded, Zalin and Montague (’74) found just the opposite with primary chick myoblasts. Chick myoblast adenylcyclase began to increase in activity prior to the onset of fusion and continued to increase linearly thereafter. Furthermore, the intracellular concentration of cAMP fell even though the cyclase activity was rising. It seems, therefore, that the relationship between adenylcyclase and fusion is not the same in all systems and that changes in its activity cannot be directly and causally related to the fusion process. These differences between the rat and chick myoblast systems also indicate that results in one in vitro system cannot be directly extrapolated to another and may not be directly applicable to what occurs in vivo. Therefore, any conclusion about the role of cAMP in natural myogenesis must ultimately be based in experiments carried out at least in organ culture (with the neural elements intact) and preferably in situ. Finally, consideration must be given to the enhancing effect of cAMP on the fusion of chick myoblasts in our own experiments and of both chick myoblasts and a MSV transformed myoblast tumor line in the experiments of Aw et al. (’73). The latter authors have demonstrated that this enhancing effect is related to the concentration of calcium in the medium. The enhancement of fusion of the transformed myoblasts increased in absolute amount with increasing calcium concentration but essentially represented a 2-3-fold increase of the baseline amount of fusion which was itself calcium concentration dependent. Although Aw et al. (’73) did not observe any enhancement of chick myoblast fusion when the calcium concentration was optimal ( 5 1.4 mm), definite enhancement was noted at lower levels of calcium. We presume that a similar
89
situation was operative in our “suboptimal” conditions. The important conclusions from these experiments are that cAMP did not inhibit fusion even when the system was not functioning at maximum capacity when it might be more susceptible to inhibition but, to the contrary, acted to restore the system toward the maximum. In no case did elevation of cAMP produce a greater extent of fusion than was obtained under optimal culture conditions, and enhancement or stimulation of fusion must therefore be considered as an artefact of the system rather than an inherent property of this agent. ACKNOWLEDGMENTS
We thank Miss Moira Schearer for technical assistance. Charles J. Epstein was on leave from the University of California, San Francisco, and was a Fellow of the John Simon Guggenheim Memorial Foundation. Luis Jimez de Asua is a Fellow of the Leukemia Society of America, Inc. LITERATURE CITED Aw, E. J., P. G. Holt and P. J . Simons 1973 Myogenesis in vitro. Enhancement by dibutyryl CAMP.Exp. Cell Res., 83: 436438. Furmanski, P., D. J . Silverman and M. Lubin 1971 Expression of differentiated functions in mouse neuroblastoma mediated by dibutyryl-cyclic adenosine monophosphate. Nature, 233: 413415. Hsie, A. W., and T. T. Puck 1971 Morphological transformation of Chinese hamster cells by dibutyryl adenosine cyclic 3’:5’-monophosphate and testosterone. Proc. Nat. Acad. Sci. (U.S.A.), 68: 358-361. Johnson, G. S., R. M. Friedman and I. Pastan 1971 Restoration of several morphological characteristics of normal fibroblasts in sarcoma cells treated with adenosine-3’ :5’-cyclic monophosphate and its derivatives. Proc. Nat. Acad. Sci. (U.S.A.), 68: 42-29. Johnson, G . S., and I. Pastan 1972 N6,02’-dibutyryl adenosine 3‘,5’-monophosphate induces pigment production in melanoma cells. Nature New Biology, 237: 267-268. Masui, H., and L. D. Garren 1971 Inhibition of replication in functional adrenal tumor cells by adrenocorticotropic hormone mediated by adenosine 3’:5’-cyclic monophosphate. Proc. Nat. Acad. Sci. (U.S.A.), 68: 32063210. O’Neill, M . C., and F. E. Stockdale 1972 A kinetic analyses of myogenesis in vitro. J. Cell Biol., 52 : 52-65. Paterson, B., and R. C. Strohman 1972 Myosin synthesis in cultures of differentiating chicken embryo skeletal muscle. Develop. Biol., 29; 113138. Prasad, K. N., and A . W. Hsie 1971 Morphologic differentiation of mouse neuroblastoma cells induced in vitro by dibutyryl adenosine 3’:5’-cyclic
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monophosphate. Nature New Biology, 2 3 3 : 141142. Reporter, M., and D. Raveed 1973 Plasma membranes: isolation from naturally fused and lysolecithin-treated muscle cells. Science, 181 : 863865. Rozengurt, E.. and L. Jimenez de Asua 1973 Role of cyclic 3’:5’-adenosine monophosphate in the early transport changes induced by serum and insulin in quiescent fibroblasts. Proc. Nat. Acad. Sci. (U.S.A.), 70: 3609-3612. Tarikas, H . , and D. Schubert 1974 Regulation of adenylate kinase and creatine kinase activities in myogenic cells. Proc. Nat. Acad. Sci. (U.S.A.), 71: 2377-2381. Wahrmann, J . P., D. Luzzati and R. Winand 1973 Changes i n adenyl cyclase specific activity during differentiation of a n established myogenic cell line. Biochem. Biophys. Res. Comm., 5 2 : 57% 581.
Wahrmann, J . P., R. Winand and D. Luzzati 1973 Effect of cyclic AMP on growth and morphological differentiation of a n established myogenic cell line. Nature New Biology, 2 4 5 : 112-1 13. Yaffe, D. 1968 Retention of differentiation potentialities during prolonged cultivation of myogenic cells. Proc. Nat. Acad. Sci. (U.S.A.), 61: 474483. 1973 Rat skeletal muscle cells. In: Tissue Culture. Methods and Applications. P. F. Kruse, Jr. and M. K. Patterson, Jr., eds. Academic Press, New York, pp. 10&114. Zalin, R. J. 1973 The relationship of the level of cyclic AMP to differentiation i n primary cell tissues of muscle cells. Exp. Cell Res., 78: 152158. Zalin, R. J., and W. Montague 1974 Changes in adenylate cyclase, cyclic AMP, and protein kinase levels in chick myoblasts, and their relationship to differentiation. Cell, 2 : 103.
DE
ASUA
AND
Imperial Cancer Research Fund Laboratories, P. 0 . Box N o . 123, Lincoln's Inn Fields, London WC2A 3PX, England
ABSTRACT The effect of exogenously administered cyclic AMP derivatives and of endogenously elevated cyclic AMP levels on the spontaneous fusion of skeletal muscle myoblasts has been investigated. Contrary to earlier reports, cAMP does not appear to have a direct inhibitory effect on the fusion of an established line (L8)of rat myoblasts. Similarly, CAMP did not block the fusion of primary chick myoblasts. However, fusion of the rat myoblasts was prevented when the cAMP induced inhibition of growth prevented the cells from reaching the "critical" cell density necessary for fusion.
The availability of cell systems which differentiate in vitro offers an excellent opportunity to explore the factors involved. Cyclic AMP has been implicated as one of the signals for differentiation. When agents which increase the endogenous level of cAMP are added to the media of adrenal, neuroblastoma, melanoma, and fibroblastic cell cultures, there are striking changes of cell morphology toward that of the "normal" differentiated cell and the appearance of differentiated cell products (Masui and Garren, '71; Furmanski et al., '71; Prasad and Hsie, '71; Johnson and Pastan, '72; Hsie and Puck, '71; Johnson et al., '71). In addition, cell proliferation is reduced. In sharp contrast, the addition of cAMP to a line of rat myoblasts (Yaffe, '68) which are capable of undergoing extensive spontaneous fusion to form myotubes did inhibit differentiation (as represented by fusion) (Wahrmann et al., '73). Further, since adenyl cyclase activity and cAMP concentration were found to decrease at the time of fusion of primary or established rat myoblasts (Wahrmann et al., '73; Reporter and Raveed, '73) it was proposed that a drop in cAMP was somehow correlated with myotube formation. Likewise, the observation of a sharp but only transient increase of cAMP just prior to fusion of primary chick myoblasts has led to the similar conclusion that an elevated level of CAMP, while necessary for initiation of the fusion process, is incompatible with fusion itself (Zalin and Montague, '74). J. CELL. PHYSIOL.,86: 83-90
In this communication we present an alternative interpretation of the inhibitory effect of cAMP on the fusion of established rat myoblasts. Our experiments indicate that cAMP acts by preventing the cells from reaching the density required for cell fusion rather than by imposing a block at a specific stage of myoblast differentiation. MATERIALS A N D METHODS
Myoblast cultures L8 cells, an established line of rat myoblasts, were obtained from Dr. D. Yaffe. The line was cloned twice, shown to be negative for PPLO contamination, and then carried at low densities in a medium composed of Dulbecco's modiKed Eagle's medium (71.2%), M 199 (17.8%), horse serum (10% , Biocult Laboratories), and chick embryo extract (1 % , Flow Laboratories) (Yaffe, '73). Fusion was assessed by examination of plates fixed for ten minutes with Bouin's fixative and stained with the Leishmann stain. Primary cultures of chick myoblasts were prepared (O'Neill and Stockdale, '72) from pectoral muscle of 12-day chick embryos and plated, after preplating, at densities of 1.5 to 2 X 10"cells per 50 mm gelatin coated (Yaffe, '73) (swine skin gelatin type I, Sigma) plastic plates in 3 ml of a medium composed of Hams F-12 (88%), horse serum ( l o % ) , andchickembryoextract (2%). Received Nov. 13, '74. Accepted Jan. 15, '75. Present address: Department of Pediatrics, University of California, S a n Francisco, Ca. 94143.
83
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C. J. EPSTEIN, L. J. DE ASUA AND E. ROZENGURT
The cultures were grown at 37" in 8-10% C 0 2 in air, and the medium was changed at 24 hours. The plates were fixed and stained as described above. The proportion of cells fusing was determined by counting 200 nuclei in randomly selected fields and scoring the number present in myotubes. To block fusion of chick myoblasts by reduction in calcium concentration, the cultures were made 0.58 mM in ethyleneglycolbis (p-amino ethyl ether) N,N'-tetraacetic acid (EGTA, Sigma) (Paterson and Strohman, '72) at 48 hours. The "block" was released at 72 hours by addition of CaClz to 0.83 mM. Cyclic AMP assays The levels of intracellular cyclic AMP were determined as previously described (Rozengurt and de Asua, '73). Source of compounds Prostaglandin El (PGEI) was obtained from Dr. J. E. Pike of the Upjohn Company, Kalamazoo, Michigan and SQ 20006, a phosphodiesterase inhibitor, from Dr. M. Chasin of Squibb. NG-monobutyryl adenosine 3': 5'-cyclic monophosphoric acid (mbc AMP) and NG,O2'-dibutyryl adenosine 3':5'cyclic monophosphoric acid (dbcAMP) were obtained from Sigma. RESULTS
I
4
I
I
5 6 Days
7
Fig. 1 Effect of single administration of cAMP elevating compounds on growth and fusion of L8 cells. Cells were plated at a density of 0.5 X 105 cells per 50 mm uncoated plastic dish (Nunc) and grown at 37" in an atmosphere of &lo% COz in air. The compounds were added without a change of medium, and the following final drug concentrations were used: theophylline, 1 mM; SQ 20006 0.2 mM; prostaglandin El (PGEI), 33 pg/ml; monobutyryl cyclic AMP (mbcAMP) 0.4 mM. The time of generalized cell fusion is denoted by an asterisk (*); the interrupted line just prior to this time indicates an intervening undetermined rise and fall in cell number. Cell counts were done in a hemocytometer on duplicates of the plates used for staining, and each point represents the average of two plates. In no instance did fusion occur at a density of less than 1 0 6 cells per plate. A. Theophylline plus PGEl at four days. B. Theophylline plus mbc AMP at four days. C. SQ 20006 plus PGEl at four days. D. Theophylline plus PGEl at three days. E. Theophylline plus mbcAMP at three days. F. SQ 20006 plus PGEl at three days.
Effect of CAMP on fusion of L8 cells Since the reported decreases in adenyl cyclase activity and cAMP concentration occurred about the time of fusion, we expected that the inhibitory effects of cAMP would be maximal at or shortly prior to this time. However, neither the phosphodiester- nificant reductions in the final cell density ase inhibitors, theophylline and SQ 20006, achieved. the adenyl cyclase activator, prostaglandin It thus appeared possible that cAMP eleEl (PGE1), nor dibutyryl cAMP (dbcAMP), vating agents were primarily affecting myoalone or in combination, prevented fusion blast growth, and cell fusion only secondwhen introduced one or two days before the arily. To test this interpretation, cultures expected tjme of myotube formation. TOthe were monitored for a period of ten days durcontrary, fusion was not prevented unless ing which time the medium was changed the agents were added at least three days and drugs added 4, 6 and 8 days after inibefore the expected time (fig. 1). However, tiation of the culture (to rule out the posdirect estimation of cell number showed sibility of drug inactivation). Myotube forthat the inhibited cells were less dense mation ultimately took place when the than the fusing ones (fig. 1). Reexamina- treated cells reached a density equivalent tion of the data of Wahrmann et al. (fig. 2, to that of the control cells at the time of '73) indicated that all treatments which their fusion. Apparently, the block imposed blocked fusion in their experiments also by these compounds was not permanent if interfered with cell growth and led to sig- growth could occur.
85
THE ROLE OF CYCLIC AMP IN MYOGENESIS
(OlrnM)
/‘. ..
SQ20006.dbCAMP (OlrnM)
/ 0050
t
’
‘
2
t
,
t
, 6
4
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8
10
Days
Fig. 2 Effect of repeated administration of cAMP elevating compounds on growth and fusion of L8 cells. Cells were plated at a density of 1 0 5 per 50 mm plate. The compounds were added in fresh medium at four hours and 3, 5, and 7 days after plating. The following concentrations were used; theophylline, 1 mM, SQ 20006, 0.2 mM; dibutyryl cyclic AMP, 0.1 and 0.3 mM. Fusion did not occur at a density below 1.3 X 1 0 6 cells per plate and probably not below 2 X 106 cells. Photomicrographs of the control, SQ 20006 plus dbcAMP, and theophylline plus dbcAMP (0.3 mM) cultures are shown in figure 3.
and the formation of long processes (Wahrmann et al., ’73). Coupled with the effects on growth, these morphological changes indicated that the cells were sensitive to the drugs, and their failure to inhibit fusion cannot be attributed to their failure to act on or in the cells. This was further corroborated by direct measurements of intracellular cAMP levels following administration of PGEl and SQ 20006 (table 1). Exposure of growing, non-fusing cells to these agents resulted in a 14-fold increase in cAMP concentration within ten minutes. When cells just about to fuse were similarly treated, a 2-fold elevation of cAMP was observed after 24 hours and fusion was not prevented. Further, the concentration of CAMP, expressed per unit DNA, was higher in fusing than in non-fusing cells, although this apparent elevation might be the result of the claimed decrease in protein content per cell associated with myoblast proliferation (Tarikas and Schubert, ’74) rather than a true increase.
Effects of cell density on fusion of L8 cells Our results indicated that CAMP,whether exogenously administered or endogenously generated, does not inhibit fusion of the rat myoblasts unless profound growth retardation is produced. With less severe growth retardation, the time of fusion is delayed until a “critical” cell density is TABLE 1
Despite these findings, the results of Wahrmann et al. (‘73) raised the possibility that an early increase in cAMP might inhibit myotube formation by interfering with a developmental program initiated at the start of the culture. Accordingly, various combinations of compounds were added within four hours of cell plating, and the medium changed and the drugs replaced every two or three days (fig. 2). Except for the combination of theophylline (1 mM) plus dbcAMP (0.3 mM) which profoundly inhibited proliferation (fig. 3c), fusion, although delayed, was not blocked by any of the agents used, and, once it occurred, myotube formation was qualitatively and quantitatively indistinguishable from normal (figs. 3a,b). All of the pharmacologic agents used in these experiments induced cell spreading
Cyclic AMP concentrations in L8 cells before and after drug administration Cell stage
Treatment
cAMP concentration Pmolesl mg
Moderate density, non-fusing
None
PGEl (30 kg/ml) plus SQ 20006 (0.2 mM) for 10 minutes Early fusion
DNA 367 319 378
5379 4675
None
520 430 458
PGE, (30 pg/ml) plus SQ 20006 (0.2 mM) for 24 hours
909 893
86
C. J. EPSTEIN, L. J . DE ASUA AND E. ROZENGURT
Fig. 3 a. Photomicrograph of control plate (fig. 2) at six days after initiation of culture, showing extensive myotube formation. Original, X 85. b. Myotube formation after ten days in culture of cells treated with SQ 20006 (0.2 mM) plus dbcAMP (0.1 mM) (fig. 2). Original, X 85. c. Decreased cell density, cell flattening, and formation of large atypical cells after ten days in theophylline (1 mM) plus dbcAMP (0.3 mM) (fig. 2). Original, X 85.
on fusion, the effect of cAMP on the fusion of primary chick myoblasts was investigated. Myotube formation in this system is much less density dependent than with rat myoblasts (ONeill and Stockdale, ’72), and previous work on the effects of cAMP on chick myotube formation indicated that cAMP might delay but would not reduce the ultimate extent of fusion (Zalin, ’73). Under normal culture conditions we found that although the cells showed morphological evidence of drug effect, fusion was not reduced, even when the compounds were added at the time of release from an EGTA imposed fusion block (table 2a). Furthermore, when the fusion system was functioning “suboptimally” because of difficulties with the medium, presumably because of a Effect of CAMP on fusion of chick marginal calcium concentration in the semyoblasts rum used (Molinaro, personal communicaTo establish the generality of our findings tion), fusion was generally improved (table with regard to the lack of an effect of cAMP 2b). A similar enhancing effect of dbcAMP
reached. If this interpretation is correct and a “critical” density is required for myotube Formation, it should be possible to simulate the effects of drug induced growth and fusion retardation by plating the cells at different initial densities. The experiment illustrated in figure 4 confirmed this conclusion. Clearly, the time of myotube formation is inversely related to initial density and a minimal density has to be attained before fusion can occur. While the absolute value of this cell density can vary from experiment to experiment by as much as 2-fold (compare figs. 1 and 4), a minimal density of lo6 cells per 50 mm plate under the defined experimental conditions appears to be necessary.
T H E ROLE OF CYCLIC AMP IN MYOGENESIS
Figure 3
87
88
C.
,J.
EPSTEIN, L. J . DE ASUA AND E. ROZENGURT TABLE 2
m e c t of CAMP elevating compounds on fusion of chick e m b r y o myoblasts Fusion Additions to cultures
.,;i_., OOl0
1
a. Normal s y s t e m Control (no additions) PGE, Thedphylline PGEl plus SQ 20006 Control (EGTA, then CaC12) 1 PGEl plus SQ 20006 after release from EGTA block
, 2
3
,
,
L
5
I
6
L
7
L
-
8
Days
Fig. 4 Effect of variation of plating density on cell growth and time of fusion of L8 cells. Cells were plated a t densities between 0.1 and 10 X 105 cells per plate and cultured without change of medium. Although cell counts at fusion of cultures plated at 0.1 and 10 X 1 0 5 cells were not determined, the time of fusion, at eight and three days respectively, are compatible with the results of the other cultures and are indicated by asterisks and vertical lines. Over the range of 0.5 to 5 X 1 0 5 cells per plate, fusion did not occur below a density of 2.2 x 106 cells per plate.
has recently been reported for a spontaneously fusing rhabdomyosarcoma line and for chick myoblasts in low concentrations of calcium (Aw et al., ’73). DISCUSSION
In none of the experiments reported here was exogenous or endogenous elevation of cAMP associated with inhibition of myoblast fusion. Taken together, the present findings do not support the hypothesis that a decrease in intracellular cAMP concentration is a necessary prerequisite for myoblast fusion in either the established rat or primary chick myoblast system. As has already been noted, our results are not essentially different from those of Wahrmann et al. (’73) in that all of their growth curves do show inhibition of cellular proliferation. The one difference between their observations and ours is the concentrations of drugs required to produce an effect on growth and fusion. They obtained inhibitory effects at concentrations as low as 5 x lo-“ M theophylline and 5 X 10 - 5 M dbc
b. “Suboptimal” system Control (no additions) PGE, plus SQ 20006 PGEl plus theophylline dbcAMP plus theophylline PGEl plus SQ 20006 PGEl plus theophylline dbcAMP plus theophylline
Time of addition
72hrs
96hrs
75% (6)
64% (9) 64% (2) 53% (2)
71% (1)
67% (2)
48 hrs 48 hrs 48hrs
10% (1)
2
61% (2)
22% (6)
33% ( 8 )
28hrs
36% ( 1 )
49% (2)
28 hrs
64% (4)
53% (3)
28 hrs
339 ( 3 )
37% (3)
52hrs
46% (1)
55% (2)
52 hrs
33% (3)
44% (3)
52 hrs
34% (3)
46% (3)
EGTA was added at 48 hours and the “block’ released at 72 hours. The following concentrations of drugs were used: theophylline, 1 mM; SQ 20006, 0.2 mM: PGEl, 33 pglml; dbcAMP, 1 mM. Several experiments have been combined, and the numbers in parentheses indicate the number of individual plates (200 cells each) scored. 2 Scored at 76 hours.
AMP, concentrations which had no effect in our hands and which are considerably lower than are effective in other systems. Similarly, our data on the fusion of chick primary myoblasts do not contradict those of Zalin (’73). While she did observe a delay (which we cannot explain) in the onset of fusion with dbcAMP and a theophylline analog, alone or in combination, the final extent of fusion was not significantly reduced. This was true even when there was severe inhibition of cell proliferation as with the dbcAMP plus 3-isobutyl-1-methylxanthine combination. However, while the lack of a direct and independent effect of CAMP on chick myoblast fusion is similar to what we observed in the rat cells, the
THE ROLE OF CYCLIC AMP IN MYOGENESIS
relation of proliferation to fusion clearly is not. Fusion of the rat cells is clearly density dependent while thqt of the chick cells is much less so. This indicates, therefore, that the two systems are not directly comparable to one another and may differ in other essential details. Evidence of such differences already exists with regard to the relation of adenylcyclase activity to fusion. Thus, while Wahrmann et al. (’73) observed a 20 % decrease immediately following fusion in rat myoblast adenylcyclase activity, and the activity decreased even further as fusion proceeded, Zalin and Montague (’74) found just the opposite with primary chick myoblasts. Chick myoblast adenylcyclase began to increase in activity prior to the onset of fusion and continued to increase linearly thereafter. Furthermore, the intracellular concentration of cAMP fell even though the cyclase activity was rising. It seems, therefore, that the relationship between adenylcyclase and fusion is not the same in all systems and that changes in its activity cannot be directly and causally related to the fusion process. These differences between the rat and chick myoblast systems also indicate that results in one in vitro system cannot be directly extrapolated to another and may not be directly applicable to what occurs in vivo. Therefore, any conclusion about the role of cAMP in natural myogenesis must ultimately be based in experiments carried out at least in organ culture (with the neural elements intact) and preferably in situ. Finally, consideration must be given to the enhancing effect of cAMP on the fusion of chick myoblasts in our own experiments and of both chick myoblasts and a MSV transformed myoblast tumor line in the experiments of Aw et al. (’73). The latter authors have demonstrated that this enhancing effect is related to the concentration of calcium in the medium. The enhancement of fusion of the transformed myoblasts increased in absolute amount with increasing calcium concentration but essentially represented a 2-3-fold increase of the baseline amount of fusion which was itself calcium concentration dependent. Although Aw et al. (’73) did not observe any enhancement of chick myoblast fusion when the calcium concentration was optimal ( 5 1.4 mm), definite enhancement was noted at lower levels of calcium. We presume that a similar
89
situation was operative in our “suboptimal” conditions. The important conclusions from these experiments are that cAMP did not inhibit fusion even when the system was not functioning at maximum capacity when it might be more susceptible to inhibition but, to the contrary, acted to restore the system toward the maximum. In no case did elevation of cAMP produce a greater extent of fusion than was obtained under optimal culture conditions, and enhancement or stimulation of fusion must therefore be considered as an artefact of the system rather than an inherent property of this agent. ACKNOWLEDGMENTS
We thank Miss Moira Schearer for technical assistance. Charles J. Epstein was on leave from the University of California, San Francisco, and was a Fellow of the John Simon Guggenheim Memorial Foundation. Luis Jimez de Asua is a Fellow of the Leukemia Society of America, Inc. LITERATURE CITED Aw, E. J., P. G. Holt and P. J . Simons 1973 Myogenesis in vitro. Enhancement by dibutyryl CAMP.Exp. Cell Res., 83: 436438. Furmanski, P., D. J . Silverman and M. Lubin 1971 Expression of differentiated functions in mouse neuroblastoma mediated by dibutyryl-cyclic adenosine monophosphate. Nature, 233: 413415. Hsie, A. W., and T. T. Puck 1971 Morphological transformation of Chinese hamster cells by dibutyryl adenosine cyclic 3’:5’-monophosphate and testosterone. Proc. Nat. Acad. Sci. (U.S.A.), 68: 358-361. Johnson, G. S., R. M. Friedman and I. Pastan 1971 Restoration of several morphological characteristics of normal fibroblasts in sarcoma cells treated with adenosine-3’ :5’-cyclic monophosphate and its derivatives. Proc. Nat. Acad. Sci. (U.S.A.), 68: 42-29. Johnson, G . S., and I. Pastan 1972 N6,02’-dibutyryl adenosine 3‘,5’-monophosphate induces pigment production in melanoma cells. Nature New Biology, 237: 267-268. Masui, H., and L. D. Garren 1971 Inhibition of replication in functional adrenal tumor cells by adrenocorticotropic hormone mediated by adenosine 3’:5’-cyclic monophosphate. Proc. Nat. Acad. Sci. (U.S.A.), 68: 32063210. O’Neill, M . C., and F. E. Stockdale 1972 A kinetic analyses of myogenesis in vitro. J. Cell Biol., 52 : 52-65. Paterson, B., and R. C. Strohman 1972 Myosin synthesis in cultures of differentiating chicken embryo skeletal muscle. Develop. Biol., 29; 113138. Prasad, K. N., and A . W. Hsie 1971 Morphologic differentiation of mouse neuroblastoma cells induced in vitro by dibutyryl adenosine 3’:5’-cyclic
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monophosphate. Nature New Biology, 2 3 3 : 141142. Reporter, M., and D. Raveed 1973 Plasma membranes: isolation from naturally fused and lysolecithin-treated muscle cells. Science, 181 : 863865. Rozengurt, E.. and L. Jimenez de Asua 1973 Role of cyclic 3’:5’-adenosine monophosphate in the early transport changes induced by serum and insulin in quiescent fibroblasts. Proc. Nat. Acad. Sci. (U.S.A.), 70: 3609-3612. Tarikas, H . , and D. Schubert 1974 Regulation of adenylate kinase and creatine kinase activities in myogenic cells. Proc. Nat. Acad. Sci. (U.S.A.), 71: 2377-2381. Wahrmann, J . P., D. Luzzati and R. Winand 1973 Changes i n adenyl cyclase specific activity during differentiation of a n established myogenic cell line. Biochem. Biophys. Res. Comm., 5 2 : 57% 581.
Wahrmann, J . P., R. Winand and D. Luzzati 1973 Effect of cyclic AMP on growth and morphological differentiation of a n established myogenic cell line. Nature New Biology, 2 4 5 : 112-1 13. Yaffe, D. 1968 Retention of differentiation potentialities during prolonged cultivation of myogenic cells. Proc. Nat. Acad. Sci. (U.S.A.), 61: 474483. 1973 Rat skeletal muscle cells. In: Tissue Culture. Methods and Applications. P. F. Kruse, Jr. and M. K. Patterson, Jr., eds. Academic Press, New York, pp. 10&114. Zalin, R. J. 1973 The relationship of the level of cyclic AMP to differentiation i n primary cell tissues of muscle cells. Exp. Cell Res., 78: 152158. Zalin, R. J., and W. Montague 1974 Changes in adenylate cyclase, cyclic AMP, and protein kinase levels in chick myoblasts, and their relationship to differentiation. Cell, 2 : 103.
