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47, 123-135 (197%

Isolation of Myosin-Synthesizing Polysomes from Cultures of Embryonic Chicken Myoblasts before Fusion’ RONALD

B. YOUNC,~

DARREL

E. GOLL,

AND

M. H. STROMER

Muscle Biology Group, Departments ofAnimal Science, Biochemistry and Biophysics, Cooperating, Iowa State Uniuesity, Ames 50010

and Food Technology,

Accepted June 6,197s Mononucleated myoblasts and multinucleated myotubes were obtained by culturing embryonic chicken skeletal muscle cells. Comparison of total polysomes isolated from these mononucleated and multinucleated cell cultures by density gradient eentrifugation and electron microscopy revealed that mononucleated myoblasts contain polysomes similar to those contained by multinucleated myotubes and large enough to synthesize the ZOO.OOO-daltonsubunit of myosin. When placed in an in vitro protein-synthesizing assay containing [“Hlleucine. total polysomes from both mononucleated and multinucleated myogcnic cultures were active in synthesizing polypeptides indistinguishable from myosin heavy chains as detected by measurement of radioactivity in slices through the myosin band on sodium dodecyl sulfate (SDS)polyacrylamide gels. Fractionation of total polysomes on sucrose density gradients showed that myosin-synthesizing polysomes from mononucleated myoblasts may be slightly smaller than myosin-synthesizing polysomes from myotubes. Multinucleated myotubes contain approximately two times more myosin-synthesizing polysomes per unit of DNA than mononucleated myoblasts, and the proportion of total polysomes constituted by myosin polysomes is only 1.2 times higher in multinucleated myotubes than it is in mononucleated myoblasts. The results of this study suggest that mononucleated myoblasts contain significant amounts of myosin messenger RNA before the hunt of myosin synthesis that accompanies muscle differentiation and that a portion of this messenger RNA is associated with rihosomes to form polysomes that will actively translate myosin heavy chains in an in vitro protein-synthesizing assay.

tions (Fambrough, 1974; Fambrough and Fusion of mononucleated myoblasts into Rash, 1971; Fambrough et al., 1974; Fluck multinucleated myotubes during muscle and Strohman, 1973; Hartzell and Fambrough, 1973; Lough et al., 1972; Tennyson differentiation is accompanied by extenet al., 1973; Wilson et al., 1973). In addisive alterations in nucleic acid metabolism (Clissold and Cole, 1973; Love et al., 1969; tion, it is clear that rapid synthesis of the Marchok and Wolff, 1968; O’Neill and 200,000-dalton subunit of myosin begins abruptly 4-8 hr after the onset of fusion Strohman, 1969; Paterson and Strohman, 1972; Scholl et al., 1968; Stockdale, 1970; (Coleman and Coleman, 1968; Morris et nl., 1972; Paterson and Strohman, 1972; Thi Man and Cole, 1974), cyclic nucleotide Stockdale and O’Neill, 1972; Yaffe and metabolism (Novak et al., 1972; Wahrman et al., 1973b; Zalin and Montague, 19741, Dym, 1972). The extent to which myosin is synthesized in cultures of mononucleated energy metabolism (Keller and Nameroff, 1974; Shainberg et al., 1971; Tarikas and myoblasts, however, has not been firmly established. Although Coleman and ColeSchubert, 1974; Turner et al., 1974; Wahrman et al., 1973a), and membrane func- man (1968) and Yaffe and Dym (1972) could detect no myosin heavy chain synthesis in ’ Journal Paper No. J-8119 of the Iowa Agriculcultures of mononucleated myoblasts, Pature and Home Economics Experiment Station, Projterson and Strohman (1972) reported a low ects No. 1795, 1796, and 2025. level of myosin synthesis in such cultures. ‘LPresent address: Departments of Biomechanics Paterson and Strohman, however, attriband Food Science and Human Nutrition, Michigan State University, East l,ansing, Mich. 48824. uted this small amount of myosin syntheINTRODUCTION

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sis entirely to the few small myotubes that contaminate myoblast cultures. On the other hand, Morris et al. (1972) and Stockdale and O’Neill (1972) found low levels of myosin heavy chain synthesis in mononucleated muscle cells before the onset of cell fusion. In addition, Rubenstein et al. (1974) have recently detected myosin and actin synthesis in cultures of mononucleated myoblasts as well as in cultures of embryonic fibroblasts and chrondroblasts. The few studies done thus far on appearance of the messenger RNA coding for the heavy chains of myosin during muscle differentiation have also been conflicting. Results of Yaffe and Dym’s experiments with actinomycin D administration at different times during myogenesis (Yaffe and Dym, 1972) suggest that myosin messenger RNA may be present in mononucleated myoblasts; others, in working with extracts of prefusion myoblasts, however, have been unable to isolate a messenger RNA that would direct synthesis of the 200,000-dalton subunit of myosin in a reticulocyte cell-free protein synthesizing assay (Pryzbyla et al., 1973; Pryzbyla and Strohman, 1974; Strohman et al., 1974). Recently, Buckingham et al. (1974) identified in cultures of mononucleated fetal calf muscle cells a 26s polyadenylic acid-containing RNA that hybridized to DNA complementary to myosin messenger RNA with kinetics identical to those of the original myosin messenger RNA. ‘The capacity of this 26s RNA to direct synthesis of myosin heavy chains in a cell-free proteinsynthesizing assay was not demonstrated, however. We have analyzed muscle cell cultures at different stages of differentiation for polysomes capable of synthesizing polypeptides that would migrate with the 200,000-dalton subunit of myosin during SDS-polyacrylamide-gel electrophoresis.” The results of our study have identified such polysomes both in cultures of mononu” Abbreviation used: SDS-polyacrylamide-gel electrophoresis is polyacrylamide-gel electrophoresis done in the presence of sodium dodecyl sulfate.

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cleated myoblasts myotubes. MATERIALS

and in multinucleated AND

METHODS

Preparation of muscle cell cultures. After removal of skin and bones, leg muscle from four to six 12-day-old chicken embryos was suspended in 10 ml of complete medium (85% Eagle’s minimum essential medium, 10% horse serum, 5% chicken embryo extract, 50 units of penicillin/ml, 50 pg of streptomycin/ml, and 2.5 pg of Fungizone/ml). The tissue was dissociated with a Vortex mixer at maximum speed for 20 set and filtered through two sterile Swinny filters, the first one containing 200 x 200mesh nylon cloth and the second a double layer of lens paper. The filtrate was centrifuged at 700g for 5 min, and the cell pellet was resuspended in complete medium by aspiration. Cells were counted with a hemocytometer and plated in l&cm Falcon tissue culture dishes (coated with 1.75 ,ug/cm2 of collagen) such that cell density after 24 hr of incubation was approximately 1.1 x 10” cells/cm2. Cultures were incubated at 37°C in an atmosphere of 95% air and 5% CO, in 15 ml of complete medium, which was changed every 24 hr. Nuclei counts. Cultures to be counted were rinsed two times with an isotonic saline solution at 37”C, fixed in absolute methanol for 5 min, and stained with Giemsa stain for 20 min at room temperature. At least 1000 nuclei were counted in randomly chosen fields, and both the total number of nuclei per dish and the percentage of total nuclei within multinucleated fibers were calculated from these data. Pulse labeling of cultures. The rate of myosin heavy chain synthesis at various stages of differentiation was determined by pulse labeling with [“Hlleucine in a manner similar to that of Paterson and Strohman (1972). Cultures in lo-cm dishes were labeled at 37°C for 4.0 hr in 4.0 ml of complete medium containing 10 @Zi of [“Hlleucineiml (specific radioactivity 5 Ciimmol). At the end of the labeling pe-

YOUNG. GOLL AND STROMER

riod, the dishes were rinsed twice with cold 0.25 M potassium chloride, 0.02 M Tris, pH 7.4, and the cells were scraped from the surface with a plastic spatula into 1.0 ml of the 0.25 M potassium chloride, 0.02 M Tris, pH 7.4, buffer. Cells were homogenized with 30 strokes of a 7-ml Dounce homogenizer (B pestle), and the homogenate was centrifuged at 12,000g for 10 min. Enough cold water was added to lower the potassium chloride concentration to 0.025 M, the tubes were left at 2°C for 2 hr, and myosin-containing material was pelleted at 12,OOOgfor 20 min. The pellet was dissolved in 0.075 ml of 1.5 M 2-mercaptoethanol, 0.01% bromophenol blue, 1.5% sodium dodecyl sulfate, 60 mM sodium phosphate, pH 7.0, and 6% glycerol by heating at 100°C for 10 min. Samples were then electrophoresed according to the procedure of Weber and Osborn (1969) on 7.5% polyacrylamide gels. Gels were stained with 0.1% Coomassie blue and destained electrophoretically in a H,O:methanol:acetic acid mixture (87.5:5:7.5, by volume). Destained gels were frozen in Dry Ice, and a series of 0.8mm slices were taken through the region of the gels containing the 200,000-dalton subunit of myosin. Slices were dissolved in 0.2 ml of 30% hydrogen peroxide by heating at 50°C for 3 hr in polyethylene minivials, 4.5 ml of Aquasol (New England Nuclear) was added to each vial, and the radioactivity was counted (after cooling for at least 24 hr) in a Model 3320 Packard liquid scintillation spectrometer. Counts per minute were converted into disintegrations per minute by using the automatic external standardization method with either chloroform or pyridine as the quenching agent. Isolation of polysomes. Three to four hours before polysome isolation, cultures were fed 15 ml of complete medium at 37°C. Cultures were removed from the incubator, and the medium was poured off and very quickly was replaced with approximaely 25 ml of ice-cold isolation buffer (0.25 M potassium chloride, 0.01 M magne-

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sium chloride, 0.01 M Tris, pH 7.4) to chill the cultures and to rinse out remaining complete medium. All subsequent steps were performed at 2°C. Cells were scraped from the surface of the dishes into cold isolation buffer containing 0.5% Triton X100 and were lysed as described by Morse et al. (1971). For preparation of total polysomes, the supernatant fluid remaining after centrifugation of the lysate at 12,000g for 10 min was layered onto 2.0 ml of isolation buffer containing 1.5 M sucrose and was centrifuged in a Beckman Ti 50 rotor at 18O,OOOg,,,,, for 2.0 hr. Pelleted polysomes from either fractionated or total polysome preparations were resuspended in 0.25 ml of incubation buffer (0.15 M KCl, 5.0 mM MgCL, 6.0 mM 2-mercaptoethanol, 10% glycerol, 20 mA4 Tris, pH 7.6), and the resuspended polysomes were analyzed for their capacity to synthesize myosin heavy chains in an in vitro assay for protein synthesis (Heywood et al., 1967; Rourke and Heywood, 1972) as described in detail below. Generally, total polysomes from eight to twelve 24-hr cultures and three to six 72-hr cultures were assayed in duplicate for myosin-synthesizing polysomes in a single experiment. Polysomes from 14-day-old embryonic muscle were prepared according to the procedure of Heywood et al. (1967). Assay of myosin-synthesizing polysomes. Assay of myosin-synthesizing polysomes was performed at 37°C in the presence of 4.0 mM ATP, 1.0 mM GTP, 6.0 n-&Y 2-mercaptoethanol, 10 PM each of 19 unlabeled amino acids, 10 PM [“Hlleucine (5 Ci/mmol), 2.6 mgiml of phosphocreatine, 0.2 mgiml of creatine phosphokinase, 0.1 mgiml of transfer RNA prepared from embryonic chicken muscle according to von Ehrenstein (1967), 4.0 mgiml of crude aminoacyl tRNA synthetases prepared according to Rourke and Heywood (1972), 150 mM KCl, 5.0 n-J4 MgC12, 10% glycerol, and 10 mM Tris, pH 7.6. Aliquots were removed after various times and were placed into enough cold (2°C) water to

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lower the KC1 concentration to 25 m&f. After thorough mixing, tubes were left at 2°C for 2-4 hr, and the myosin-containing material was pelleted at 2000g for 40 min [crude aminoacyl tRNA synthetases prepared by the procedure of Rourke and Heywood (1972) contain approximately 15 pg of myosin/mg of synthetases, and this myosin is utilized very effectively as carrier myosinl. The pellet was resuspended, electrophoresed on SDS-polyacrylamide gels, and analyzed for incorporation of radioactivity into myosin heavy chains as described under pulse labeling of cultures. All pipets, centrifuge tubes, and water were autoclaved before use in polysome preparation or amino acid incorporation to reduce ribonuclease contamination. Electron microscopy of polysomes. Polysomes from the heavier region of the gradients were fixed at 2°C for 1.5 hr in 1.25% glutaraldehyde, 10 mM MgCL, 10 mM Tris, pH 7.4, and were pelleted onto carbon-coated grids at 150,OOOg for 1.5 hr. Polysomes were stained for 1.0 min with 2% uranyl acetate and examined by electron microscopy. Concentration of polysomes was determined by measuring the absorbance at 260 nm and using an extinction coefficient of 11.2 OD unitsimg of polysomesiml. RESULTS

AND

DISCUSSION

Cultures of embryonic chicken muscle cells have been widely used to study the relationship of myosin synthesis to fusion of mononucleated myoblasts into multinucleated myotubes (Coleman and Coleman, 1968; Morris et al., 1972; Paterson and Strohman, 1972; Stockdale and O’Neill, 1972; Yaffe and Dym, 1972). Under the culure conditions described in Materials and Methods, the percentage of nuclei within myotubes increases from less than 5% after 30 hr in culture to a plateau of 6070% after 55 hr (Fig. 1). Immediately after this burst of fusion, approximately a sevenfold increase in the rate of myosin heavy chain synthesis per unit of DNA occurs

VOLUME 47, 1975

CULTURE

AGE

fhrsl

FIG. 1. Kinetics of fusion and myosin synthesis in myogenic cell cultures. Percent fusion and rate of myosin heavy chain synthesis were determined as described in Materials and Methods. Each point is the mean of duplicate determinations.

(Fig. 1). These results are in general agreement with other reports on the kinetics of myoblast fusion and myosin synthesis (Morris et al., 1972; Paterson and Strohman, 1972; Stockdale and O’Neill, 1972). Heywood et al. (1967) have demonstrated that polysomes containing 50-60 ribosomes in embryonic chicken muscle are responsible for synthesis of the 200,000dalton heavy chain of myosin. Because rate of myosin synthesis per unit of DNA (Fig. 1) increases sevenfold soon after the onset of fusion, cultures of multinucleated myotubes were expected to contain more large 50-60-ribosome polysomes than cultures of mononucleated myoblasts when compared on sucrose density gradients. Polysomes were isolated from 24- and 72-hr cultures of cells that contained similar numbers of nuclei. An optical density tracing at 260 nm of the density gradient separations of the polysome fractions from these 24- and 72-hr cultures is shown in Fig. 2. Although, as expected (Hosick and Strohman, 19711, the 72-hr cultures contained more total polysomes per nucleus than the 24-hr cultures (greater optical density in Fig. 2; also, see left column, Table 3), the distribution of polysome sizes

YOUNG, GOLL AND STROMER

FIG. 2. Polysomes from 24- and 7%hr muscle cell cultures isolated as described in Materials and Methods and displayed on a linear 15-404 sucrose gradient in isolation buffer after centrifugation at for 2.0 hr in a Beckman SW 25.2 rotor. 15wwnE!x Direction of sedimentation is from right to left. Bracket denotes the part of the gradient from which polysomes were isolated for electron microscopy. Both 24- and 72-hr samples were obtained from cultures containing approximately 7 X lo7 nuclei.

from cultures at these two stages of differentiation was qualitatively similar (Fig. 2). The similarity in proportion of polysomes in the bottom one-third of the gradient (at the left side of Fig. 2) is especially interesting because this is the region of the gradient from which myosin-synthesizing polysomes have isolated (Heywood et al., 1967). Polysomes from the region of the gradients designated by the bracket in Fig. 2 were isolated and prepared for electron microscopy as described in Materials and Methods. Figure 3 shows that polysomes in this region of the gradients from both fused and nonfused cultures were large enough to synthesize a peptide chain as large as myosin heavy chains. The presence of polysomes large enough to support synthesis of the 200,000-dalton subunit of myosin in mononucleated muscle cells prompted an investigation into whether these polysomes would incorporate [“Hlleucine into polypeptides that

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127

would migrate with the heavy chains of myosin in SDS-polyacrylamide-gel electrophoresis. To demonstrate that the assay described in Materials and Methods would accurately measure synthesis of myosin heavy chains, total polysomes from leg muscle of 14-day-old embryonic chickens were isolated and analyzed for ability to incorporate amino acids into protein. Proteins in the in vitro protein-synthesizing system were separated by SDS-polyacrylamide-gel electrophoresis, and, after staining, the SDS-polyacrylamide gels were sliced and the slices assayed for radioactivity, as described in Materials and Methods. Radioactivity in the gel slices increased at the distance of migration corresponding to a molecular weight of 200,000

FIG. 3. Electron micrographs of large polysomes isolated from 24- and 72-hr embryonic chicken muscle cell cultures. Polysomes from the region of the gradient designated by the bracket in Fig. 2 were isolated and were stained with uranyl acetate as described in Materials and Methods. Polysomes from 24- and 72-hr muscle cell cultures appear similar in the electron micrograph and are similar to the myosin-synthesizing polysomes shown by Heywood et al. (1967). x 98,470.

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(Fig. 41, and the sum ofradioactivity in the slices making up this peak (indicated in Fig. 4) was used as the 13Hlleucine that had been incorporated into myosin heavy chains, Plotting this sum versus assay time (Fig. 5) showed that completion of myosin heavy chains increased very rapidly for the first 5 min, slowed between 5 and 7 min, and was finished by approximately 10 min. Because the assay contained no ribosomes other than those attached to the mRNA’s of the polysomes and because the KC1 concentration in the polysome isolation-buffer was high enough to extract muscle protein-specific initiation factors (Heywood, 19701, only chaincompletion synthesis of myosin occurred in this system. To alleviate any possible dif-

‘t /

FIG. 4. Synthesis of myosin heavy chains by embryonic chicken leg muscle polysomes. Polysomes isolated from 14-day embryonic chicken leg muscle were incubated for 0 or for 20 min in the assay mixture described in Materials and Methods. The protein in these assay mixtures was dissolved in SDS and analyzed by SDS-polyacrylamide-gel electrophoresis. After staining, the gels were sliced and radioactivity in the slices was measured as described in Materials and Methods. The peak of radioactivity designated by the bracket migrated with a molecular weight of 200,000, and slices included under the bracket were used to calculate the 20-min time points in Fig. 5.

VOLUME 47. 1975 1600 k2 2 z

:: z z

1400

800 600

0

5

10 ASSAY

15 TIME

20

(MIN)

FIG. 5. Time course of synthesis of myosin heavy chains by embryonic chicken muscle polysomes. Polysomes isolated from 14-day embryonic chicken leg muscle were incubated in the cell-free assay medium described in Materials and Methods. The lower curve is endogenous incorporation into myosin that occurs when polysomes are omitted from the assay medium. Such a control was run with each experiment, and this endogenous incorporation was subtracted from total myosin synthesis by polysomes from fused or from nonfused muscle cell cultures.

ferences in ability to initiate specific protein synthesis by ribosomes from 24- and 72-hr cultures, it was necessary that only chain-completion synthesis of myosin be occurring. The results shown in Fig. 5 are compatible with reports that about 4-8 min are required for movement of ribosomes along the entire length of a myosin messenger RNA (Coleman and Coleman, 1968; Herrmann et al., 1970; Morris et al., 1972). When total polysomes isolated as described in Materials and Methods were subjected to sucrose gradient analysis after 20 min of assay time (i.e., after the accumulation of completed polypeptides had subsided), essentially all the ribosomes sedimented as 80s monomers or as subunits (Fig. 61, and no rapidly sedimenting polysomes remained. Hence, polysomes prepared according to the procedures used in this study were capable of actively moving along the messenger RNA’s and completing the synthesis of polypeptides. Because these preliminary experiments

YOUNG,

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AND STROMER

7-

1-

I-

s-

5-

z-

3-

v FIG. 6. Distribution of embryonic chicken muscle polysomes on a 15-409’~ sucrose gradient after these polysomes had been incubated for 0 or for 20 min in the assay mixture described in Materials and Methods. Gradients were centrifuged at EO,OOOg,,,,, for 1.5 hr in a Beckman SW 41 rotor. Direction of sedimentation is from right to left.

estabished that polysomes prepared and assayed according to the procedures used in this study were able to complete the synthesis of peptide chains that had been initiated before their isolation, only 0 and 20-min time points were taken. The difference between the incorporation at 0 and 20 min was used as a measure of synthesis of myosin heavy chains. The lower curve in Fig. 5 represents endogenous incorporation into myosin heavy chains that occurred when polysomes were omitted from the assay mixture. Such a control was run with each experiment, and this control value was always subtracted from total myosin-synthesizing ability of the polysomes being analyzed. The assay described in the preceding par-

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129

Polysomes

agraphs was applied to total polysomes prepared from 24- and 72-hr muscle cell cultures to measure synthesis of myosin heavy chains by these polysomes. Because, as indicated in the preceding paragraph, the assay used in this study measures run-off of peptide chains that had been initiated before isolation of the polysomes, measurement of the amount of myosin heavy chain synthesis in these systems was a measure of amount of myosin polysomes in 24- and 72-hr cultures. Polysomes from both 24- and 72-hr cultures directed synthesis of polypeptides that migrated with a molecular weight of 200,000 on SDS-polyacrylamide-gel electrophoresis (Fig. 7). Furthermore, because the results in Fig. 7 are expressed on the basis of radioactivity incorporated into myosin heavy chains per 10’ nuclei, the total polysomes from nonfused muscle cell cultures support synthesis of approximately half as much myosin as those from myotube cultures. It is possible that some of the myosin-synthesizing polysomes originally present in the cultured cells could have

24

hour

r

72

hour

P ,

* I

. I , I , (

0 246810

0246810 SLICE

NUMBER

7. Synthesis of myosin heavy chains by total polysomes isolated from 24- and 72-hr muscle cell cultures. Total polysomes were isolated, assayed, and the products analyzed by SDS-polyacrylamidegel electrophoresis as described in Materials and Methods. The peak of [:‘H]leucine incorporation supported by polysomes from either 24- or 72-hr cultures migrated exactly the same distance as myosin heavy chains. FIG.

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remained in the pellet from the 12,OOOg centrifugation of the cell lysate in 0.5% Triton X-100 either because of incomplete cell lysis or because of physical entrapment. Any myosin-synthesizing polysomes remaining in these 12,000g pellets would not have been assayed and would therefore make the results shown in Fig. 7 inaccurate. To eliminate this possibility, the 12,000g pellets were reextracted in isolation buffer containing 0.5% Triton X-100 by mild homogenization with a Dounce homogenizer (A pestle). This homogenate was centrifuged at 12,OOOgfor 10 min, and this second 12,000g supernatant, fraction was also assayed for polysomes capable of synthesizing myosin heavy chains as described earlier. The results of these experiments suggest that, although a slightly larger percentage of myosin-synthesizing polysomes was pelleted at 12,OOOgin the myotube lysate (Table 1) than in the lysate from mononucleated myoblasts, this difTABLE MYOSIN

1

SYNTHESIS BY POLYSOMES FROM 24- AND 72HR MUSCLE CULTURES”

Source of polysomes

Percent of myosinsynthesizing polysomes in each supernatant” 24-Hr cultures

First 12,000g supernatant fraction Second 12,OOOgsupernatant fraction

72-Hr cultures

94.9

84.9

5.1

15.1

‘I Polysomes in the first 12,000g supernatant fraction from 24- and 72-hr muscle cultures were prepared and assayed as described in Materials and Methods. The pellet from the first 12,000g centrifugation was reextracted with isolation buffer plus 0.5% Triton X-100 to test for complete extraction of myosin-synthesizing polysomes. This second extract was also centrifuged at 12,OOOgfor 10 min, and the supernatant fluid was assayed for polysomes capable of incorporating r3Hlleucine into myosin heavy chains as described in Materials and Methods. DPercent of myosin-synthesizing polysomes = (myosin dpm by polysomes in each supernatantisum of myosin dpm by polysomes in both supernatants) x 100.

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47. 1975

ference is small, and the data shown in Fig. 7 accurately indicate the relative quantities of myosin-synthesizing polysomes in 24- and 72-hr myogenic cell cultures. The summary of a number of experiments like the one in Fig. 7 indicates clearly that total polysomes from nonfused muscle cultures have about half the capability for incorporating [3H]leucine into a 200,000-dalton polypeptide that polysomes from myotubes have (Table 2, left column). Because a small but consistent level of fusion exists in 24-hr cultures (Fig. 11, it is critical to determine whether myosin-synthesizing polysomes in 24-hr cultures could originate entirely from the small number of fused myotubes present in those cultures. The number of nuclei within myotubes in the 24- and 72-hr cultures was measured by counting the nuclei in stained cultures as described in Materials and Methods and scoring these nuclei as from either fused or nonfused cells. By using this measure of total nuclei present in multinucleated myotubes, and by using the total disintegrations per minute incorporated into myosin heavy chains by polysomes from 24- and 72-hr cultures (left column, Table 2), it is possible to calculate the amount of myosin polysomes that would be present per 10’ nuclei in multinucleated myotubes if it were assumed that the mononucleated myoblasts in the 24and 72-hr cultures contained no myosin TABLE

2

MYOSIN HEAVY CHAIN SYNTHESIS PER 10’ NUCLEI AND PER 10’ FUSED NUCLEI BY POLYSOMES FROM 24AND 72-HR MUSCLE CELL CULTURES”

Culture

24 Hr 72 Hr

age

[“Hlleucine in myosin heavy chain&O’ nuclei (dpm)

[3H]leucine in myosin heavy chains/lo’ fused nuclei (dpm)

498 914

12,475 1,406

(1Incorporation of [3H]leucine into myosin heavy chains and number of nuclei in single cells and in multinucleated myotubes were determined by the procedures described in Materials and Methods.

YOUNG,

GOLL AND STROMER

polysomes. The results of this calculation (right column, Table 2) show that the quantity of myosin polysomes per lo7 fused nuclei would be nearly ninefold greater in myotubes in 24-hr cultures than in myotubes in 72-hr cultures if the small myotubes in 24-hr cultures were the only source of myosin-synthesizing polysomes. Because it seems incongruous to suggest that myotubes in 24-hr myogenic cell cultures should contain ninefold greater amounts of myosin polysomes than myotubes in 72-hr myogenic cultures, these results indicate that mononucleated muscle cells at 24 hr in culture contain significant levels of myosin polysomes. The conclusion that mononucleated myoblasts contain approximately half as many myosin-synthesizing polysomes per lo7 nuclei as multinucleated myotubes (Fig. 7 and Table 2) is partly based on the assumption that the average number of ribosomes per myosin-synthesizing polysome is identical in 24- and 72-hr muscle cell cultures, because total run-off of initiated peptide chains was used to measure amounts of myosin-synthesizing polysomes in the cells in these two cultures. To

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test this assumption, total polysomes from 24- and 72-hr cultures were fractionated on sucrose density gradients. The gradients were collected in eight different fractions, and the polysomes in each fraction were pelleted and then assayed for their ability to incorporate [3H]leucine into myosin heavy chains as described in Materials and Methods (Figs. 8a and b). As expected (Heywood et al., 19671, polysomes capable of synthesizing myosin were located near the bottom of the density gradient of total polysomes prepared from either 24- or 72hr cells. Average size of the myosin-synthesizing polysomes from 24- and 72-hr myogenic cell cultures was similar, although the peak of myosin-synthesizing activity was in density gradient fraction 3 for polysomes from l-day cultures but in density gradient fraction 2 for polysomes from 3day cultures (Figs. Sa and b). These results indicate that mononucleated cells contain at least half as many myosin-synthesizing polysomes per lo7 nuclei as multinucleated cells do. Indeed, if, as the data in Fig. 8 suggest, the average number of ribosomes per myosin-synthesizing polysome is actually slightly less in mononucleated cells

FIG. 8. Fractionation of total polysomes from 24-hr (a) and from 7%hr (b) muscle cell cultures on 15-40s sucrose gradients. Density gradients for the 24- and 72-hr fractions were centrifuged at the same time in identical tubes at 105,OOOg,,, for 2.0 hr in a Beckman SW 25.2 rotor and were fractionated identically into eight different fractions each. Both 24- and 72-hr samples were obtained from cultures containing approximately 7 x 10’ nuclei. Polysomes in each fraction were isolated and assayed for ability to synthesize myosin heavy chains as described in Materials and Methods. Direction of sedimentation is from right to left.

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than it is in multinucleated cells, then our procedure for measuring myosin-synthesizing polysomes quantitatively by measuring run-off of initiated peptide chains would underestimate the number of myosin-synthesizing polysomes in mononucleated cells. As indicated earlier, the 72-hr myogenic cell cultures contained approximately 1.5 times more total polysomes than the 24-hr cell cultures (left column, Table 3), and the results discussed in the preceding paragraph show that 72-hr myogenic cell cultures contain no more than two times more myosin-synthesizing polysomes than 24-hr myogenic cell cultures (left column, Table 2). These data can be used to estimate the change in proportion of total polysomes that is myosin-synthesizing polysomes which occurs between 24 and 72 hr in culture. The results of this calculation show that the proportion of myosin-synthesizing, polysomes relative to total polysomes is only 1.2 times greater in 72-hr, multinucleated myogenic cell cultures than it is in 24hr, mononucleated myogenic cell cultures (right column, Table 3). Yet, rate of myosin heavy chain synthesis is at least seven times higher in 72-hr myogenic cell cultures than in 24-hr cell cultures (Fig. 1). Consequently, cell fusion and the onset of bulk synthesis of myosin heavy chains is not accompanied by a dramatic increase in either the proportion of myosin-synthesizing polysomes relative to total polysomes or the total amount of myosin-synthesizing polysomes. Whether this ostensible disparTABLE

3

TOTAL POLYSOME CONTENT AND MYOSIN HEAVY CHAIN SYNTHESIS PER UNIT OF POLYSOMES BY POLYSOMES FROM 24- AND 7%HR MUSCLE CELL CULTURES

Culture

24 Hr 72 Hr

age

Total polysomes/lO’ nuclei (mg)

[3Hlleucine in myosin heavy chaindmg of total polysomes (dpm)

0.242 0.370

2,058 2,470

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ity between rate of myosin heavy chain synthesis and amount of myosin-synthesizing polysomes indicates that myosin polysomes in multinucleated cells have a greater translation rate is presently unknown. One factor which complicates interpretation of the data presented in this communication is that all muscle cultures are contaminated with fibroblasts which continue to proliferate for several days after extensive myoblast fusion has been completed. Because the proportion of tibroblasts increases somewhat between 24 and 72 hr in culture and because myosin synthesis and myosin-synthesizing polysomes are normalized on the basis of a constant number of nuclei, the values reported for 72-hr cultures are reduced to a greater extent than the values reported for 24-hr cultures. However, preliminary data obtained in this laboratory indicate that both the rate of myosin heavy chain synthesis and the amount of myosin-synthesizing polysomes in pure cultures of proliferating embryonic chicken skin fibroblasts may be similar to those values reported here for mononucleated myoblasts. If this is the case, then the values reported for rate of myosin synthesis and amount of myosin-synthesizing polysomes in mononucleated myoblasts are unaffected by fibroblast contamination, and these values in 72-hr cultures of multinucleated myotubes are not seriously affected. At any rate, the basic conclusion reached from the data presented in this communication that mononucleated myoblasts synthesize significant quantities of a polypeptide that has chemical properties identical to myosin is unaltered. Another assumption on which the conclusion that multinucleated myotubes exhibit approximately seven times the rate of myosin heavy chain synthesis but only twice as many myosin-synthesizing polysomes as mononucleated myoblasts (Figs. 1, 7, Table 2) is based is that the only polypeptide that would survive precipitation in 0.025 M KC1 followed by migration

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with a polypeptide molecular weight of 200,000 on SDS-polyacrylamide-gel electrophoresis is myosin heavy chains. Although muscle contains several large proteins, most of them are easily resolved from myosin heavy chains on SDS-polyacrylamide gels and, in addition, these proteins have solubility properties unlike those of myosin. One possible exception may be the large subunit of nuclear RNA polymerase II (Weaver et al., 1971). The experiments described in the preceding paragraphs suggest not only that myosin messenger RNA is present in cultures of mononucleated myoblasts but that at least a portion of the myosin messenger RNA is associated with ribosomes to form polysomes approximately as large as the myosin-synthesizing polysomes from myotube cultures. These results are in general agreement with the data reported by two earlier groups of workers (Buckingham et al., 1974; Yaffe and Dym, 1972). Yaffe and Dym (1972) found that myosin synthesis continues for several hours after fusion but then eventually decreases when cultures of dividing myoblasts are treated with actinomycin D. This result would be expected if the mononucleated myoblasts already contained myosin messenger RNA as we have found in the present study. Buckingham et al. (1974) demonstrated that a 26S, polyadenylic acid-containing RNA that hybridized to complementary DNA with kinetics identical to those of the myosin messenger RNA from which the complementary DNA was prepared could be isolated from fetal calf mononucleated myoblast cultures. Because our techniques would only detect myosin messenger RNA that was attached to ribosomes, we do not know whether our 24- and 72-hr cultures of embryonic chick myoblasts contained myosin messenger RNA unattached to ribosome in addition to the myosin polysomes that we found in these cells. In contrast to our results with embryonic chick myoblasts, however, Buckingham et al. (1974) did not report the presence of

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myosin polysomes in their fetal calf mononucleated myoblast cultures. Several attempts by other investigators to isolate from mononucleated myoblasts a messenger RNA that would direct the cellfree synthesis of myosin heavy chains have been unsuccessful (Pryzbyla and Strohman, 1974); Pryzbyla et cd., 1973; Strohman et al., 1974). In view of the recent accumulation of a large amount of evidence indicating that myofibrillar proteins are present in many different cell types, the isolation of myosin messenger RNA from mononucleated myoblasts is not surprising. This work was supported in part by grants from the National Institutes of Health (No. AM-12654 and HL-156791, the Muscular Dystrophy Associations of America, the American Heart Association (No. 71679), the Iowa Heart Association, and the American Meat Institute Foundation. We are grateful to Janet Stephenson and to Jacqueline Harvey for expert technical assistance, to Dr. Margaret Schmidt for many helpful suggestions during manuscript preparation, and to Ron Allen for assistance with the electron microscopy REFERENCES BUCKINGHAM, M. E., CAPUT, D., COHEN, A., WHALEN, R. G., and GROS, F. (1974). The synthe-

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