DEVELOPMENTAL

BIOLOGY

Differentiation

57, 330-344 (1977)

of Creatine Phosphokinase during Myogenesis: Quantitative Fractionation of lsozymes JOHN LOUGH AND RICHARD BISCHOFF

Department

of Anatomy

and Neurobiology, Washington University St. Louis, Missouri 63110

Received May 3,1976;

accepted in revised form February

School of Medicine,

3,1977

A technique is described for the quantitative measurement of creatine phosphokinase (CPK) isozymes in extracts of chick muscle. The isozymes are fractionated by stepwise elution with increasing salt concentrations from DEAE-Sephadex minicolumns. Isozyme separation was confirmed by polyacrylamide gel electrophoresis followed by enzyme staining. We used this method to determine changes in CPK isozymes during the course of myogenesis in culture. The total specific activity of CPK increases about 20-fold during myogenesis. Quantitative analysis of isozyme changes shows that the muscle-specific form (MM) accounts for virtually all of this increase. Activity of MM-CPK is undetectable in l-day cultures, increases rapidly after myoblast fusion, and comprises more than 70% of total CPK in mature cultures. In contrast, the specific activity of the brain-specific isozyme (BB) remains constant throughout myogenesis. This was interpreted as indicating that the B subunit is expressed in both mononucleated cells and myotubes. We confirmed this by analyzing CPK isozymes in fibroblast cultures and in myotube-enriched cultures. Elimination of most of the mononucleated cells in the cultures produced an increase in the specific activity of CPK, but had no effect on the isozyme pattern and did not decrease the relative amount of the BB isozyme. Pure tibroblast cultures contained very low CPK activity, predominantly the BB isozyme. INTRODUCTION

The enzyme creatine phosphokinase (EC 2.7.3.2) (CPK) is present in various tissues, but it is found at the highest concentrations in striated muscles. Thus, CPK activity has been used widely to assess the degree of differentiation of skeletal and cardiac muscle tissue (Reporter et al., 1963; Coleman and Coleman, 1968; Shainberg et al., 1971; Turner et al., 1974; Ziter, 1974). The active form of the enzyme is a dimer composed of two types of enzymatically inactive subunits, designated M and B. These subunits associate to form three isozymes, MM, MB, and BB (Eppenberger et al., 1967a; Dawson and Eppenberger, 1970). Mature skeletal muscle of birds and mammals contains only the MM isozyme, although traces of MB have been reported (Turner and Eppenberger, 1973; Allard and Cabrol, 1970; Belousova and Mesh-

kova, 1973; Ziter, 1974). CPK of the mature myocardium in mammals consists of about 80% MM isozyme, with the remainder being the MB form (Ziter, 1974; Hall and DeLuca, 1975). In birds, on the other hand, the predominant myocardial isozyme is the BB form (Turner and Eppenberger, 1973; Hall and DeLuca, 1975). Neural tissue contains only the BB isozyme. The three CPK isozymes can be fractionated by electrophoresis in a variety of supports (Eppenberger et al., 1965; Sjovall and Jergil, 1966; Traugott and Massaro, 1973). The BB isozyme is the most rapidly migrating anodal form, whereas MB is intermediate in mobility, and MM remains nearest the cathode. Electrophoretic studies of the proportions of the various CPK isozymes during muscle development both in uivo (Eppenberger et al., 1964; Ziter, 1974) and in vitro (Turner et al., 1974) have 330

Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0012-1606

LOUGH AND BISCHOFF

Creatine

shown that tissue maturation is accompanied by changes in the isozyme pattern. The BB isozyme is the predominant form in embryonic tissue and is foilowed by the appearance of the MB isozyme, in addition, as development proceeds. The MM isozyme is the last to appear during development and eventually becomes the major, if not the sole, form of CPK found in adult skeletal muscle. The techniques used in these studies involve electrophoretic separation of isozymes, often followed by densitometric scanning of stained electropherograms. These procedures are laborious and may provide unreliable estimates of quantitative isozyme activity (Murone and Ogata, 1973; Roberts et al., 1974). Recently, anion-exchange chromatography utilizing stepwise salt elution from small columns has been devised for the separation of human serum CPK isozymes (Mercer, 1974; Nealon and Henderson, 1975). These procedures, which are based on the gradient elution behavior of human CPK isozymes reported by Takahashi et al. (1972), were devised to obviate the limitations of electrophoresis in providing quantitative measurements of isozyme activities for the diagnosis of myocardial infarction and neuromuscular disorders. In this communication, we describe modifications of these ion-exchange techniques to achieve good resolution of chicken CPK isozymes. These techniques were applied to obtain quantitative measurements of the specific activities of CPK isozymes during chick skeletal muscle differentiation in culture. MATERIALS

AND

METHODS

Cell Culture Myogenic cells were prepared according to the method of Bischoff and Holtzer (1968). Chick breast muscle was dissected from ll- or 1Zday embryos, teased into small pieces, and incubated for 30 min in 0.1% trypsin (Nutritional Biochemical Co., Cleveland, Ohio, 44128; 1:300) in Earle’s balanced salt solution (BSS). Tryp-

Phosphokinase

Isozymes

331

sin was removed by centrifugation, and the cell pellet was resuspended in a culture growth medium consisting of 8 parts Eagle’s MEM, 1 part selected horse serum, 1 part chick embryo extract, and 0.1 part antibiotic anti-mycotic solution. After trituration with a Pasteur pipet, the cells were passed through a lo-pm-mesh nylon filter (Tobler, Ernst and Traber, New York, N. Y. 10523; Nitex HClO) to ensure removal of myotubes and clumps of mononucleated cells. The suspension was diluted to 5 x lo5 cells/ml of growth medium and was plated in collagen-coated culture dishes (Falcon Plastics, Oxnard, Calif. 93030; 3000 series) at a cell density of 7.5 x lo4 cells/cm2. Cultures were incubated at 37°C in a water-saturated atmosphere of 5% C02-95% air. The culture medium was replaced daily. Embryo extract was prepared from 12-day chick embryos after removal of the eyes; all other culture reagents were purchased from Grand Island Biological Corporation, Grand Island, N. Y. 14072. The collagen-treated culture dishes were prepared according to O’Neill and Stockdale (1972). Fifty milligrams of collagen (Worthington, Freehold, N. J. 07728; CL8EA) was autoclaved in 100 ml of DW, and the insoluble residue was removed by centrifugation. The resulting solution was poured in and out of a series of culture dishes which were then allowed to dry before use. This treatment contributes a negligible amount of protein (about 0.5 PgI cm? to the cultures but greatly improves differentiation. Fibroblast cultures were prepared in two ways: (1) Muscle-derived flbroblasts were obtained by subculturing primary myogenic cultures twice at 5-day intervals. Subculturing was carried out by incubating the cultures in 0.1% trypsin in BSS and removing the formed myotubes by passing the resultant suspension through a Nitex HC-10 filter. Tertiary cultures were assayed for enzyme activity at 7 days. (2) Cultures were established from

332

DEVELOPMENTAL BIOLOGY

cells of loose connective tissue in the groin area of 12-day embryos and were allowed to grow for 7 days before assay. Two methods were also used to prepare myotube-enriched cultures: (1) Myogenic cultures were incubated in medium containing 5fluorodeoxyuridine (FUdR; 1 fl) and cystosine arabinoside (ara C; 10 fi) from Day 4 to 7 with daily medium replacement. (2) Four-day cultures were exposed to 5bromodeoxyuridine (BUdR; 160 fl) for 48 hr, and then the medium and dish cover were removed and the cells were irradiated with uv light for 5 set at a distance of 16 in. (General Electric germicidal lamp, T30T8). Both types of cultures were washed thoroughly with BSS before being harvested on Day 7. The extent of myotube formation was estimated by determining the number of nuclei in multinucleated cells as a percentage of the total nuclei scored. Cells were coverslips grown on collagen-coated (Lough and Bischoff, 19761, fixed at intervals with ethanol, formalin, and acetic acid (20:2:1), and stained with Ehrlich’s hematoxylin. A nucleus was considered to be fused only if at least three nuclei were clearly observed to lie within common cytoplasm. A minimum of lo3 nuclei in random fields was scored at a magnification of 400x for each determination.

VOLUME 57, 1977

Sigma Chemical Co., St. Louis, MO. 631701, 10 mM magnesium acetate, 3.3 mM glucose, 1 mM ADP (Grade 1, Sigma), 10 mM AMP (Type II, Sigma), 1.33 unit/ml each of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (Type XV, Sigma) and hexokinase (EC 2.7.1.1.) (Type F-300, Sigma), and 100 r&4 glycylglycine, pH 6.75, in a final volume of 0.225 ml. Tissue samples containing CPK were incorporated into the reaction mixture after sufficient dilution so that the reaction rate was linear with respect to enzyme concentration. The reaction was started by adding creatine phosphate to a concentration of 15 mM. The production of NADPH was monitored continuously at 340 nm with an Acta dual-beam spectrophotometer (Beckman Instruments Co., Irvine, Calif. 92664). The rate of product formation was calculated after the reaction rate had become linear. The reference cuvette contained the same reaction mixture but without creatine phosphate. One milliunit of CPK activity is defined as the amount of enzyme catalyzing the production of one nanomole of NADPH per minute at 30°C. The specific activity is expressed as the units of CPK per milligram of soluble protein. Protein was estimated by the method of Lowry et al. (1951) using crystalline bovine serum albumin as a standard. We have also used commercial ampoules of lyophilized reaction mixture (Sigma, UV-45) to measure Creatine Phosphokinase Assay CPK activity. This method is based upon Cultures were harvested in ice-cold 50 the same reactions and gave identical acvalues when compared with the mM Tris-HCI buffer, pH 7.5, and were tivity Nielsen and Ludvigsen (1963) method. sonicated with a Biosonic II-A microprobe Since CPK has been reported to bind to (Bronwill Sci., Rochester, N. Y. 14602) for myofibrils (Turner et al., 1973; Scholte, 30 set at 60% maximum power. Tissue 1973), a variable proportion of enzyme actaken from embryos or mature chickens was first ground in a Ten Broeck homoge- tivity might appear in the insoluble fraction during development. To test for this nizer before sonication. After centrifugapossibility we rehomogenized the 20,OOOg tion (30 min at 2O,OOOg),the supernatants were assayed for CPK by the method of pellet with the Tris-HCl buffer containing 0.5 M KC1 to dissolve the myosin filaNielsen and Ludvigsen (19631, but with ments. After extraction and centrifugathe addition of AMP to suppress myokition, the high-salt supernatant was asnase (Rosalki, 1967). The reaction mixture sayed for CPK. We found that solubilizacontained 0.8 mM NADP (sodium salt,

LOUGH AND BISCHOFF

Creatine

Phosphokinase

Isozymes

333

tion of myosin released little additional CPK (less than 4% of that found in the first low-salt supernatant) from cultures at 2, 5, or 8 days in vitro. Hence, this procedure was not used with the cultures. In contrast, significant amounts of activity were recovered by high-salt extraction of 20,OOOg pellets from mature tissues, including brain, skeletal muscle, and cardiac muscle. The CPK activity for these tissues therefore, is the sum of activities in both the low- and high-salt extracts. Because of reports of low molecular weight serum factors affecting CPK activity (Snehalatha et al., 1973), we tested the effects of dilution and dialysis of culture homogenates on enzyme activity. These procedures gave no evidence of dissociable activators or inhibitors of CPK activity. Unused culture medium contained approximately 500 mU of CPK/ml. Virtually all of this was derived from the embryo extract, with only a trace from the horse serum. The activity in the medium rapidly decreased during incubation, presumably owing to thermal denaturation of the enzyme. Cultures were washed in four changes of BSS before being harvested to remove any medium-derived CPK from the cells.

Stepwise Separation

Gradient

To establish the isozyme identity of the enzyme activities eluting at various salt concentrations, polyacrylamide gel electrophoresis as described by Davis (1964) was employed, followed by localization of the separated isozyme bands (Sjovall and Jergil, 1966). To obtain sufficient enzyme activity for electrophoretic identification, a 0.9 x 20-cm column DEAE-Sephadex A50 was loaded with 18,000 mU of CPK from a 5-day culture and was eluted using stepwise concentrations of 50, 100, and 150 mM NaCl. Fractions belonging to the three peaks were pooled and concentrated with a PM-30 Diaflo filter (Amicon Corp., Lexington, Mass. 02173). To obtain satisfactory staining of the CPK isozyme bands in 5% polyacrylamide

Separation

of CPK Activity

The procedure of Takahashi et al. (1972) was used to determine the ion-exchange elution pattern of chick muscle CPK activity. Five-day skeletal muscle cultures were harvested in 50 m&f Tris-HCl buffer, pH 7.5, as described. An aliquot of the 20,OOOgsupernatant containing about 12 U of CPK activity was applied to a 0.9 x 15cm column of DEAE-Sephadex (Pharmacia Fine Chemicals, Piscataway, N. J. 08854) which had been equilibrated with the same buffer. After 40 ml was eluted, a gradient of O-O.2 M NaCl in 50 mM TrisHCl (pH 7.5) was applied. The CPK activity in each of 40 5-ml fractions was determined as described.

of CPK Activity

Separation of CPK activity by stepwise salt elution was performed using columns consisting of 0.5 X 12.5-cm Pasteur pipets filled to a bed height of 6 cm (minicolumns). The gel bed was supported by a plug of glass wool. Before applying the sample, several milliliters Tris-HCl buffer (50 mM, pH 7.5) were passed through the column. Samples of 5-day supernatants in the Tris-HCl buffer were diluted to 400500 mU of CPK/ml, and a sample volume of 1 ml was applied to the bed. The sample was allowed to flow into the bed, and the effluent was collected as the first fraction. Five subsequent l-ml elutions were performed using the starting buffer. This was followed by stepwise elution using six l-ml aliquots each of 50 mM, 100 mM, and 150 mM NaCl in the Tris-HCl buffer. Each fraction was assayed for CPK activity. Salt concentrations greater than 150 mM failed to release additional activity from the column. No effects on total CPK activity were detected at any of the chloride ion concentrations employed. Recovery of CPK following minicolumn fractionation was 91 t 11% (n = 18) for the experiment shown in Fig. 4. Isozyme Identity

of Resolved Fractions

334

DEVELOPMENTAL BIOLOGY

gels, 100 unit each of the linking enzymes, hexokinase and glucose-g-phosphate dehydrogenase, were placed in the cathode buffer compartment (400 ml), and the gels were electrophoretically preflushed for 2 hr at a constant current of 2 mA/gel. Samples of each of the three peaks from the DEAE-Sephadex column containing approximately 500 mU of CPK activity were placed on each of three gels, and a mixture of the three samples was placed on a fourth gel. After electrophoresis for 2 hr at 2 mA/ gel, the gels were placed in a reaction mixture of the following composition: 200 mM triethanolamine-HCl buffer (pH 6.8), 20 n-ii!4 glucose, 20 mM magnesium acetate, 1.3 mg/ml of ADP, 0.7 mg/ml of NADP, 1.33 unit/ml of hexokinase, 0.67 unit/ml of glucose-g-phosphate dehydrogenase, 0.3 mg/ml of phenazine methosulfate, 0.3 mgl ml of nitroblue tetrazolium, and 4 mg/ml of phosphocreatine. Following incubation for 30 min at room temperature in total darkness, the gels were fixed with 10% acetic acid and were scanned at 490 nm using the Beckman spectrophotometer equipped with a GS-2 scanner. RESULTS Chicken CPK from &day myogenic cultures failed to exhibit the same fractionation pattern as reported for the human enzyme when tested with procedures designed for human CPK. The MM isozyme from human serum elutes from DEAESephadex with the starting buffer (50 mM Tris, no added NaCl), whereas the MB isozyme elutes at 200 m&f NaCl, and the BB isozyme requires approximately 300 m&f NaCl for desorption (Takahashi et al., 1972). In contrast to the behavior of human CPK, the chick enzyme does not elute with the starting buffer, and all three isozymes desorb from the column over a narrower range of salt concentration than that required for the human enzyme (Fig. 1). The first peak of CPK activity is almost completely eluted by 50 miV NaCl, the second by 100 mM, and the third by 150 mM.

VOLUME57. 1977

Continued elution with salt concentrations up to 500 mit4 NaCl did not release additional CPK from the column (data not shown). To facilitate simultaneous fractionation of CPK from replicate cultures, these results were adapted to the stepwise elution of isozymes from DEAE-Sephadex minicolumns. The optimum elution volumes were determined by adding tissue extract to a of DEAE-Sepha0.5 x 6-cm minicolumn dex and eluting with a series of successive l-ml aliquots of buffer containing 0, 50, 100, and 150 mM NaCl. The CPK activity was measured in each resulting l-ml fraction (Fig. 2). No activity was eluted with six aliquots of the starting buffer (no NaCl), thus confirming the continuous elution behavior (Fig. 1). The next six aliquots containing 50 mM NaCl, and each successive elution with six aliquots at 100 mM and 150 mM NaCl, released a distinct peak of enzyme activity, indicating that the isozymes desorb in discrete units. From these data, it is apparent that elution volumes of 5 ml for each salt concentration will efficiently release each of the isozymes from the minicolumn. Under the assay conditions used for these experiments, the limit of detection of individual isozymes with 5-ml fractions is about 4% of the total column input (400-500 mu). Added sensitivity could be achieved by concentrating the fractions. From a comparison of the data in Figs. 1 and 2 with the elution pattern of human CPK isozymes previously reported (Takahashi et al., 1972; Mercer, 1974) and from consideration of the fact that the B subunit is more electronegative than the M subunit in both chick and human (Allard and Cabrol, 1970; Eppenberger et al., 1964), we predicted that the activities eluted at 50, 100, and 150 mM NaCl correspond to the MM, MB, and BB forms of the enzyme, respectively. To test this and to determine the purity of the separated isozymes, pooled fractions from each peak were analyzed by polyacrylamide gel electrophoresis. Densi-

Creatine

LOUGH AND BISCHOFF

Phosphokinase

335

Isozymes

,100 -

-0. 2

.050 -

: 3 - 0. I i-

:

i G s

40 PRACTlON

NUMBER

FIG. 1. Separation of CPK isozymes by ion-exchange chromatography. An extract of 5-day cultures containing a total of 15 U of CPK was loaded onto a 0.9 x 20-cm column of DEAE-Sephadex A-50 and was eluted with a gradient of O-O.2 M NaCl in 50 n&f Tris-HCl buffer, pH 7.5. Fractions of approximately 5 ml were collected and assayed for CPK activity as described in Materials and Methods. Putative MM, MB, and BB isozymes eluted in the first, second, and third peaks, respectively. ~0 5Or

I

NaCl -

2

3

4

50mM

5

6

7

6

NaCl .blooEnM

9

FRACTION

I6

ll

12

Naa-

13

14

IS

I6

15omM

I7

19

19

20

Nlsa+

21

22

23

24

NUMRER

FIG. 2. Distribution of CPK isozymes after stepwise elution from a DEAE-Sephadex minicolumn. The column was loaded with about 400 mU of CPK from a B-day culture and was eluted with aliquots of 50 mM Tris, pH 7.5, containing NaCl as indicated. Fractions of 1 ml were collected and assayed for CPK activity.

tometric scans of the gels following histochemical enzyme localization demonstrate that the electrophoretic migration of each chick isozyme is in accordance with its expected behavior (Dawson et al., 1967). Also, almost complete resolution of the chick isozymes is achieved by ion-exchange chromatography (Fig. 3). Although there is a small amount of crosscontamination between the BB and MB

fractions (Fig. 31, measurement of the areas under the peaks shows that contamination is only about 9% for the BB fraction and 4% for the MB fraction. There is no detectable contamination of the MM fraction. The electropherograms also show a distinct double band for CPK-BB. The two bands are of approximately equal intensity and are present in both the total enzyme and the BB gels (Fig. 3, c and d).

336

DEVELOPMENTAL BIOLOGY

VOLUME 57, 1977

FIG. 3. Verification of CPK isozyme identity by polyacrylamide gel electrophoresis. Stepwise ion-exchange chromatography was performed, and effluents containing CPK activity were concentrated by ultrafiltration. Extracts of 5-day cultures containing 500 mU of CPK activity were electrophoresed on 0.5 x fl-cm 5% gels which had been preflushed with linking enzymes. The isozymes were reacted for formazan production, and the stained gels were scanned at 490 nm using a Beckman GS-2 gel scanner. (a) 50 mM NaCl effluent containing MM-CPK; (b) 100 mM NaCl effluent containing MB-CPK; (c) 150 mM NaCl effluent containing BB-CPK; (d) a mixture of a, b, and c.

lsozyme Changes during

Development

To determine the quantitative changes in CPK isozymes during development in vitro, duplicate cultures were sacrificed at intervals and were assayed for CPK activity. The activity of the individual isozymes was measured after their fractionation using the DEAE-Sephadex minicolumn technique described before. Total CPK specific activity increases about 20-fold between Days 1 and 8, then reaches a plateau of about 12 U/mg of pro-

tein from Day 8 to 10 (Fig. 4A). In other experiments, cultures maintained for up to 2 weeks showed only a moderate additional increase in CPK activity to a maximum of about 14 U/mg. Although the CPK activities are expressed in terms of soluble protein in the extract supernatants, the shape of the curves would be the same even if normalized to DNA content, since the soluble protein:DNA ratio remains constant during the period studied (Bischaff, unpublished observations). Each of the CPK isozymes displays a

LOUGH 11 t

A

Days 100 90

Creatine

AND BISCHOFF

in vitro

B

1

12

3 Days

4

5 in

6

1

a

9

lo

vitro

FIG. 4. Creatine phosphokinase isozyme changes during myogenesis in uitro. Duplicate cultures were sacrificed at the indicated times and were assayed for total CPK activity and CPK isozymes using DEAE-Sephadex minicolumns as described in Materials and Methods. (A) Specific activity of CPK normalized to soluble protein: (-0-j total activity; C-A-1 MM isozyme; (-A-) MB isozyme; (-0-j BB isozyme. (B) Activity of each isozyme expressed as a percentage of the total activity: (--0-j MM isozyme; (-0-j MB isozyme; (-A-) BB isozyme.

different developmental pattern (Fig. 4A). The MM isozyme is undetectable at Day 1 but increases rapidly from Day 3 onward and accounts for most of the observed increase in total CPK activity in the older cultures. Both MB- and BB-CPK are present at 1 day, but undergo only modest increases in specific activity during myo-

Phosphokinase

Isozymes

337

genesis. Activity of the BB form increases slightly between Days 1 and 3, but remains essentially constant thereafter. Changes in the enzymatic composition appear more dramatic when the activity of each isozyme is viewed as a percentage of the total (Fig. 4B). Thus, a major differentiative event during myogenesis is a switch in the proportion of the MM and BB isozymes. In l-day cultures, about 84% of the activity consists of the BB isozyme, and the remainder is MB. The BB isozyme drops to about 10% by Day 6, whereas the MM form increases from 0 to about 70% during the same period. Since column fractionation of isozymes may be subject to a small amount of crosscontamination, particularly between the BB and MB isozymes (Fig. 3), it seemed desirable to confirm the presence of MB in l-day cultures by electrophoretic separation. Eight cultures in loo-mm dishes were grown for 1 day, and the cells were harvested as before. Part of the supernatant was used for polyacrylamide gel electrophoresis as described in Materials and Methods, except that the gels were loaded with about 400 mU of CPK activity. The remainder of the supernatant was subjected to fractionation on DEAE-Sephadex minicolumns. The isozyme distribution revealed by the column separation was 86% BB and 14% MB. Electrophoretic fractionation produced similar values with 84% BB, 16% MB, and no trace of MM (Fig. 5). Since both results agree with those obtained earlier (Fig. 4B), the presence of the MB isozyme in l-day cultures seems firmly established. To correlate changes in CPK with growth and myotube formation, the degree of myoblast fusion during development is shown in Fig. 6, along with the total soluble protein per culture. The protein determinations are from the same cultures used in Fig. 4, whereas the fusion measurements were made from cells grown on collagen-coated coverslips under otherwise identical conditions. High cell density pre-

338

DEVELOPMENTAL

BIOLOGY

FIG. 5. Electrophoretic fractionation of isozymes in l-day cultures. Polyacrylamide gels were loaded with 400 mU of CPK activity and were electrophoresed as described in Materials and Methods. The gels were stained for enzyme activity, and the percentage of each isozyme was estimated by cutting out and weighing the peaks from densitometric scans as indicated in the figure. Duplicate gels gave the same results.

eludes accurate fusion determinations beyond about 6 days in culture. The greater part of myoblast fusion is complete before the second day in culture, and, thereafter, the percentage of nuclei in myotubes approaches a plateau. Although CPK is present in l-day cultures, the sharp increase in CPK activity, particularly of the MM isozyme, does not begin until after Day 3 when most of the myotube formation is complete. A small percentage of multinucleated cells is present in the l-day cultures, but the MM isozyme is undetectable at that time. Source of Isozymes in Myogenic

VOLUME

57, 1977

1969), the predominant mononucleated cell is probably the fibroblast. Fibroblasts were obtained from dermal connective tissue and were grown in primary culture for 7 days before being harvested for enzyme assay. Since some traits of fibroblasts may vary with the source of tissue, we also attempted to provide cells more characteristic of muscle fibroblasts. Myogenic cultures were subcultured twice with trypsin, a procedure that tends to select against myoblasts and myotubes. The resulting tertiary cultures were grown for 7 days before assay. Both types of fibroblast preparations were free of multinucleated myotubes (Table 1). Creatine phosphokinase activity was very low in both types of fibroblast-enriched cultures (Table 1). The specific activity in the fibroblast cultures is about 1% of that found in the 7-day control cultures and about 20% of that in l-day untreated cultures (Fig. 4A). The dermal fibroblasts produce only the BB isozyme, whereas the muscle fibroblast cultures contain about 80% BB and 20% MB. Myotube-enriched cultures were also prepared in two ways. First, we took advantage of the observation that the thymi-

Cultures

Since cultured muscle, as well as muscle in vivo, is a heterogeneous tissue, various cell populations could account for the isozyme changes just described. Of particular interest is the contribution, if any, of the nonmyogenic cells to the total CPK activity and the isozyme pattern observed in standard muscle cultures. To approach this point, we measured CPK activities in a series of fibroblast-enriched and myotube-enriched cultures (Table 1). Since 7day muscle cultures contain very few presumptive myoblasts (Bischoff and Holtzer,

FIG. 6. Growth and myotube formation as a function of time in culture. Duplicate cultures of cells grown on collagen-coated coverslips placed in 35-mm dishes were fixed, stained, and examined at a magnification of 400x to determine the percentage of nuclei in myotubes (-0-L For protein measurement, the cells were grown in 35-mm collagencoated dishes and were assayed for soluble protein after sonication in 50 mA4 Tris-HCl buffer (-0-j.

LOUGH

AND BISCHOFF

Creatine TABLE

CREATINE

Cultured

cells@

Standard cultures Fibroblasts From muscle From dermis Myotube-enriched BUdR + uv light FUdR + ara C

Percentage of nuclei in myotubes

PHOSPHOHINASE

Phosphokinase

339

Isozymes

1

ISOZYMES

IN ~-DAY

CULTURES=

CPK Activity

Protein/ dish (mg)

Percentage

U/dish

of each isozyme

~ZZf

45

2.1

0 0

0.99 0.98

86 69

1.36 1.50

20.8 0.116 0.133 15.6 17.8

9.9 0.117 0.136 11.5 11.9

BB

MM

MB

70

20

10

0 0

19 0

81 100

75 72

17 19

8 9

a Cells were grown on coverslips for determination of the percentage of nuclei in myotubes. For CPK measurements, fibroblasts were cultured in loo-mm dishes; all other cultures were in 60-mm dishes. Each value represents the average from duplicate cultures, except that three dishes were pooled for the frbroblast determinations. b See Materials and Methods.

dine analog, 5-bromodeoxyuridine (BUdR) makes cells more susceptible to the lethal effects of ultraviolet radiation when incorporated into cellular DNA (Djordjevic and Szybalski, 1960; Okada, 1970). Accordingly, cultures were exposed to BUdR beginning at 4 days, after most myoblast fusion is complete, and were irradiated with uv light 2 days later. Large numbers of degenerating cells and cell fragments were observed floating in the culture medium at the time of harvest on Day 7. Since myotube nuclei do not synthesize DNA (Stockdale and Holtzer, 19611, only the proliferating mononucleated cells incorporate BUdR and are subsequently killed by uv light. Second, the proportion of myotubes was increased by exposing cultures, from Day 4-7, to a combination of 5fluorodeoxyuridine (FUdR) and cytosine arabinoside (ara C). Both drugs inhibit DNA synthesis and, thus, can be used to suppress the continued growth of mononucleated cells after myotube formation has occurred (Coleman and Coleman, 1968; Fischbach and Cohen, 1973). These procedures increased the percentage of nuclei in myotubes from 45% in the control cultures to 86% in the BUdR + uv light cultures and 69% in the FUdR + ara C cultures (Table 1).

The myotube-enriched cultures contained less CPK per dish than the control cultures, probably as a result of the fact that the treatments designed to reduce fibroblasts also eliminated some dividing presumptive myoblasts and, hence, reduced the number of myotubes somewhat. A small amount of myoblast fusion continues after Day 4 (Fig. 6; also see Bischoff and Holtzer, 1969). The specific activity of CPK was elevated somewhat in the myotube-enriched cultures as a result of the lower protein content (Table 1). The deliciency in mononucleated cells, however, produced little change in the isozyme pattern. The proportion of MM was increased slightly from 70% in the control cultures to 72 and 75% in the myotube-enriched cultures, whereas the percentage of the BB isozyme was essentially unchanged. To summarize, mononucleated cells that are presumed to be mostly fibroblasts contain very low CPK activity. This activity is predominantly the BB isozyme with none of the MM form. A 1.5- to 2-fold increase in the frequency of myotube nuclei produced by killing mononucleated cells or blocking their proliferation results in a modest increase in the specific activity of CPK, but does not substantially change the isozyme pattern. Thus, the myotubes themselves

340

DEVELOPMENTAL

BIOLOGY

probably synthesize both the M and B subunits. Comparison

with in Vito Tissue

To provide some measure of comparison between myogenesis in vitro and in vivo, we measured the CPK activity and isozyme distribution in tissues from chickens of the same strain used for culture (Table 2). The specific activity of CPK in skeletal muscle from a 3-month hen was about four-fold greater than the maximum activity observed in cultured muscle. All activity was in the form of the MM isozyme. In contrast, heart muscle and brain tissue had lower CPK activities, none of which eluted at the position of the MM isozyme. Breast muscle from 11-day embryos represents the starting material used to initiate myogenic cultures. The specific activity is quite low at this stage, less than 10% of that found in the mature breast muscle (Table 2). The CPK activity is composed of a mixture of all isozymes, including about 50% of the BB form. In terms of isozyme pattern, cultured muscle reaches a state comparable to that of the tissue of origin, 11-day breast muscle, at about 3 days in vitro (Table 2, Fig. 4B). At this time the cultures have approximately the same CPK specific activity and isozyme distribution as 11-day in vivo muscle. Since the changes in CPK isozymes are essentially complete by 10 days in vitro TABLE CREATINE

2 IN IN VZVO

PHOSPHOKINASE ISOZYMES TISSUES

CPK Activity

Source of tissue $%Ef

Percentage of each isozyme MM

3-Month hen Breast muscle Heart ventricle Cerebral cortex ll-Day embryo breast muscle 21-Day embryo breast muscle

MB

BB

43.0 8.9 9.5 3.6

100

0

0

0 0 14

14 2 37

86 98 49

11.9

100

0

0

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(Fig. 4A), it seemed of interest to compare mature cultures with in uiuo muscle of the same total chronological age. Accordingly, we measured the CPK activity in 21-day embryos at hatching (Table 2). Although the specific activity in 21-day embryos is approximately the same as that found in lo-day cultures, only the MM isozyme is detectable in the in viuo muscle. Cultured muscle at 10 days contains a mixture of isozymes including about 70% of the MM form. Thus, although the early differentiation of CPK in vitro is comparable to that found in viuo, development in culture appears to be arrested at approximately the condition found in uivo around the time of hatching. Cultured muscle does not attain the specific activity or isozyme pattern found in adult muscle. DISCUSSION

lsozyme Fractionation Creatine phosphokinase is a useful index of differentiation in the experimental analysis of myogenesis. Of the three CPK isozymes however, only the MM form is specific for skeletal muscle. Therefore, measurement of the individual isozymes is necessary for reliable interpretation of enzyme changes during development. Electrophoretic separation of isozymes has been used widely but suffers from several limitations. Most prominent among these is the difficulty in obtaining quantitative data. As an alternative to electrophoresis, isozyme fractionation has been achieved by column chromatographic procedures (Takahashi et al., 1972; Mercer, 1974). These methods were designed for human serum CPK, however, and fail to give adequate separation when applied to CPK from chickens, a widely used source of muscle for developmental studies. The ion-exchange technique described in the present study is capable of rapid separation of the three CPK isozymes from chick tissue in culture and in vivo. The technique facilitates quantitative measurement of indi-

LOUGH AND BISCHOFF

Creatine

vidual isozyme activity and is capable of resolving small amounts of the musclespecific isozyme in the presence of an excess of the other isozymes. Additional speed and efficiency could be achieved by batch adsorption with the use of the DEAE-glass bead method developed recently (Henry et al., 1975). As was first observed by Takahashi et al. (1972), the basic feature that allows efficient fractionation of CPK is the fact that each isozyme displays a different elution characteristic from a column of DEAE-Sephadex (Fig. 1). With human enzyme, the MM isozyme elutes in the starting buffer (50 mM Tris), whereas the other isozymes desorb with increasing salt concentrations (Takahashi et al., 1972). The chicken isozymes, however, all remain bound to the column in the starting buffer, but desorb sequentially in sharp peaks when the column is eluted with a salt gradient (Fig. 1). Since there is little overlap between isozyme peaks, a stepwise gradient of appropriately chosen salt concentrations also achieves good separation (Figs. 2 and 3). Polyacrylamide gel electrophoresis, used to confirm the identity of isozymes following column fractionation, revealed two closely spaced bands of equal intensity in the BB fraction (Fig. 3). This peculiar double band was not investigated further and may be an artifact; however, Eppenberger et al. (196713) have reported the presence of a double BB band using starch gel electrophoresis. In this case, the double band was present in a variety of avian species, but was not found in chicken tissue. The authors suggest that the two bands may represent enzymes with the same primary structure but with different conformations. Developmental

Changes

The increase in specific activity of CPK during myogenesis in vivo and in culture has been documented previously many times (Reporter et al., 1963; Coleman and

Phosphokinase

Isozymes

341

Coleman, 1968; Shainberg et al., 1971; Tzvetanova, 1971; Turner et al., 1974; Ziter, 1974). Although an increase in activity is only circumstantial evidence for differential synthesis, fluorescent antibody staining suggests that there is also an increase in enzyme protein (Turner et al ., 1976). In the present study, myoblast fusion was found to occur during a relatively brief period in the early cultures and is followed about a day later by an increase in CPK activity, particularly of the MM isozyme (Fig. 4A). The temporal correlation between fusion and CPK increase agrees in general with that found in other studies using comparable material, although, as Turner et al. (1974) point out, sequential temporal appearance of specific traits need not imply obligatory coupling. Indeed, in the case of fusion and CPK, it has been demonstrated recently that fusion is not a prerequisite for the increase in CPK during myogenesis (Holtzer et al., 1972; Keller and Nameroff, 1974; Lough, 1975; Turner et al., 1976). Quantitative measurements of isozyme changes in cultured muscle have not been reported previously; therefore, our data may provide additional insight into the processes of myogenesis. The isozyme transitions during myogenesis have often been interpreted as indicating that the BB isozyme is replaced by the MM isozyme as differentiated myotubes form (Morris et al., 1972; Turner et al., 1974; Turner, 1975), and, by implication, that the expression of one gene (B subunit) is replaced by that of another (M subunit). Indeed, examination of the isozyme percentages during development (Fig. 4B) appears to support this concept. When the specific activity of each isozyme is examined, however, a different interpretation emerges (Fig. 4A). Although the specific activity of MM increases dramatically from Day 3 onward, the specific activity of BB remains constant during the same period. If enzyme turnover remains constant, this suggests

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that the gene for the B subunit continues to be expressed at the same rate relative to tissue mass throughout myogenesis, whereas the expression of the M subunit is greatly enhanced shortly after myotube formation occurs. Another point of interest is whether the M subunit is present in l-day cultures, before significant myotube formation has occurred. There is some disagreement in previous studies based upon electrophoretie separation of isozymes from chick tissue. Turner et al. (1974) report that all three isozymes occur in l-day cultures, although BB predominates. Morris et al. (1972) found only the BB isozyme in l-day cultures. Their source of tissue, however, was thigh muscle instead of breast muscle. Keller and Nameroff (1974) failed to detect any CPK at all in l-day cultures. Our results show that, although the MM isozyme is undetectable at l-day using either electrophoretic or chromatographic fractionation, the other two forms are clearly present. The MB isozyme comprises about 15% of the total activity in lday cultures. Carryover of differentiated myotubes from the in vivo tissue of origin seems unlikely since 11-day breast muscle contains the MM isozyme (Table 21, yet none of this is detectable in the l-day cultures. On the other hand, a small amount of fusion has occurred by 1 day (Fig. 6), and, hence, the presence of the M subunit may represent the onset of differentiation in a few multinucleated cells. Since there is no evidence that the M subunit is synthesized by replicating presumptive myoblasts, we would conclude that the capacity to synthesize the M subunit is probably first acquired by postmitotic myoblasts or myotubes. The relative amounts of each isozyme change during myogenesis. The heterodimerit MB form, however, undergoes relatively minor changes during the period studied. The specific activity of MB increases until about Day 6 and then approaches a plateau (Fig. 4A). The percent-

VOLUME 57, 1977

age of MB increases from Day 1 to 3 as MM is increasing, then declines slightly between Days 3 and 7 as MM continues to rise and BB declines (Fig. 4B). Similar isozyme changes have been observed in rat muscle developing in vivo (Ziter, 1974). Source of Zsozymes In mixed cultures, there are several possible cellular sources for each isozyme, including mononucleated myogenic cells, multinucleated myotubes, and fibroblasts. Since the specific activity of the BB isozyme is relatively constant throughout the period studied (Fig. 4A), it might be expected that a constant fraction of fibroblasts in the cultures contributes the major portion of this isozyme. Indeed, our analysis of fibroblast-enriched cultures shows that BB is the predominant, if not the only, isozyme in these cells (Table 1). The 19% of MB found in the fibroblasts from muscle probably results from a small percentage of contaminating myogenic cells in these cultures (Turner et al., 1974). The relative amount (specific activity) of CPK present in the flbroblasts, however, is too low to account for even the small amount of BB in the l-day myogenic cultures. The activity of BB is about 0.4 U/mg in l-day myogenic cultures, whereas the highest activity found in the fibroblasts is only about 0.1 U/mg. Thus, although flbroblasts contain some BB isozyme and perhaps MB, the major fraction of these isozymes comes from either the myogenic cells or the myotubes. The myotubes are the most probable source since older cultures contain very few mononucleated cells (Bischoff and Holtzer, 19691, yet the total activity per dish of BB and MB increases somewhat as the cultures mature (Figs. 4A and 6). This interpretation was confirmed in the myotube-enriched cultures (Table 1). Elimination of most of the mononucleated cells produced an increase in the specific activity of CPK, but had little effect on the isozyme pattern and did not decrease the

LOUGH AND BISCHOFF

CIpeatine Phosphokinase

relative amount of the BB isozyme. Our results on the source of isozymes agree with those of Turner et al. (1976) who used fluorescent antibody staining to show that cultured myotubes contain both the M and B subunits of CPK. Keller and Nameroff (1974) also observed little difference in CPK electropherograms between control and ara C-treated cultures. No quantitative measurements were made, however. The demonstration that embryonic myotubes contain the BB and MB isozymes, whereas these forms do not occur in adult skeletal muscle, may explain why these isozymes are found in the serum of individuals with certain types of muscle disease (Schapira, 1970; Tzvetanova, 1971; Allard and Cabrol, 1970; Goto, 1974). The regeneration that occurs in some myopathies (Walton, 1973) involves the production of generations of immature myotubes, some of which may degenerate and release BB and MB isozymes into the circulation. This work was supported by a Muscular Dystrophy Association. ported by a predoctoral traineeship (GM02025). We thank Ms. Barbara technical assistance.

grant J. L. from Pelle

from the was supthe N.I.H. for expert

REFERENCES

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rum creatine kinase isoenzymes by ion-exchange column chromatography. Clin. Chem. 20,36-40. MORRIS, G. E., COOKE, A., and COLE, R. J. (1972). Isoenzymes of creatine phosphokinase during myogenesis in vitro. Exp. Cell Res. 74, 582-584. MURONE, I., and OGATA, K. (1973). Studies on creatine kinase of skeletal muscle and brain with special reference to subcellular distribution and isoenzymes. J. Biochem. 74, 41-48. NEALON, D. A., and HENDERSON, A. R. (1975). Separation of creatine kinase isoenzymes in serum by ion-exchange chromatography. Clin. Chem. 21, 392-397. NIELSEN, L., and LUDVIGSEN, B. (1963). Improved method for determination of creatine kinase. J. Lab. Clin. Med. 62, 159-168. OKADA, S. (1970). “Radiation Biochemistry,” pp. 136-144. Academic Press, New York. O’NEILL, M., and STOCKDALE, F. E. (1972). Differentiation without cell division in cultured skeletal muscle. Deuelop. Biol. 29, 410-418. REPORTER, M. C., KONIGSBERG, I. R., and STREHLER, B. L. (1963). Kinetics of accumulation of creatine phosphokinase activity in developing embryonic skeletal muscle in uiuo and in monolayer culture. Exp. Cell Res. 30, 410-417. ROBERTS, R., HENRY, P. D., WIITEEVEEN, S. A. G. J., and SOBEL, B. E. (1974). Quantification of serum creatine phosphokinase isoenzyme activity. Amer. J. Cardiol. 33, 650-654. ROSALKI, S. B. (1967). An improved procedure for serum creatine phosphokinase determination. J. Lab. Clin. Med. 69, 696-705. SCHAPIRA, F. (1970). Les isozymes de la creatinekinase et de l’aldolase. Pathol. Biol. 18, 295-301. SCHOLTE, H. R. (1973). On the triple localization of creatine kinase in heart and skeletal muscle cells of the rat: Evidence for the existence of myofibrillar and mitochondrial isoenzymes. Biochim. Biophys. Acta 305, 414-427. SHAINBERG, A., YAGIL, C., and YAFFE, D. (1971). Alterations of enzymatic activities during muscle differentiation in vitro. Develop. Biol. 25, l-29. SJOVALL, L., and JERGIL, B. (1966). Coupled enzyme reactions in polyacrylamide gels: Isoenzymes of serum ATP: Creatine phosphotransferase. &and. J. Clin. Lab. Inuest. 18, 550-552. SNEHALATHA, C., VALMIKINATHAN, K., SRINIVAS, K., and JAGANNAJHAN, K. (1973). Creatine phosphokinase level in neuromuscular disorders. Ef-

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fects of dilution and dialysis. Clin. Chim. Acta 44, 229-235. STOCKDALE, F. E., and HOLTZER, H. (1961). DNA synthesis and myogenesis. Exp. Cell Res. 24, 508520. TAKAHASHI, K., USHIKUBO, S., OIMOMI, M., and SHINKO, T. (1972). Creatine phosphokinase isozymes of human heart muscle and skeletal muscle. Clin. Chim. Acta 38, 285-290. TRAUGOTT, C., and MASSARO, E. J. (1973). The molecular basis of the heterogeneity of the MM isozyme of rabbit muscle creatine phosphokinase. Biochim. Biophys. Acta 295, 549-554. TURNER, D. C. (19751. Isozyme transitions of creatine kinase and aldolase during muscle differentiation in vitro. In “Isozymes” (C.L. Markert, ed.), Vol. 3, pp. 145-158. Academic Press, New York. TURNER, D. C., and EPPENBERGER, H. M. (1973). Developmental changes in creatine kinase and aldolase isoenzymes and their possible function in association with contractile elements. Enzyme 15, 224-238. TURNER, D. C., WALLIMANN, T., and EPPENBERGER, H. M. (1973). A protein that binds specifically to the M-line of skeletal muscle is identified as the muscle form of creatine kinase. Proc. Nat. Acad. Sci. USA 70, 702-705. TURNER, D. C., MAIER, V., and EPPENBERGER, H. M. (1974). Creatine kinase and aldolase isoenzyme transitions in cultures of chick skeletal muscle cells. Develop. Biol. 37, 63-89. TURNER, D. C., GM~R, R., LEBHERZ, H. G., SIEGRIST, M., WALLIMANN, T., and EPPENBERGER, H. M. (1976). Differentiation in cultures derived from embryonic chicken muscle. II. Phosphorylase histochemistry and fluorescent antibody staining for creatine kinase and aldolase. Develop. Biol. 48, 284-307. TZVETANOVA, E. (1971). Creatine kinase isoenzymes in muscle tissue of patients with neuromuscular disease and human fetuses. Enzyme 12,279-288. WALTON, J. N. (1973). Progressive muscular dystrophy: Structural alterations in various stages and in carriers of Duchenne dystrophy. In “The Striated Muscle” (C.M. Pearson and F.K. Mostofi, eds.), pp. 263-291. Williams and Wilkins, Baltimore. ZITER, F. A. (1974). Creatine kinase in developing skeletal and cardiac muscle of the rat. Exp. Neurol. 43, 539-546.

Differentiation of creatine phosphokinase during myogenesis: quantitative fractionation of isozymes.

DEVELOPMENTAL BIOLOGY Differentiation 57, 330-344 (1977) of Creatine Phosphokinase during Myogenesis: Quantitative Fractionation of lsozymes JOHN...
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