Treatment of human muscle myotube cultures with 2 μM-cytochalasin D (CD) for 6 h stimulated synthesis of both the (muscle-specific) a-actin and the (non-muscle) β and γ-actins usually expressed by these cells. In non-muscle (HEp-2) cell cultures, CD enhanced synthesis of β and γ-actin, but did not induce synthesis of α-actin, which is not normally present in these cells. Thus, synthesis of both muscle and non-muscle actins can be increased by CD, but enhancement of actin synthesis results from increases in the isoactins usually present, rather than induction of new isotypes. Comparison of CD-treated (fused) myotube cultures with (unfused) myoblast cultures indicated that β and γ-actin synthesis was similarly enhanced in both cultures, but that α-actin synthesis was stimulated to a greater extent in the myoblast cultures. Desmin synthesis was also stimulated in the myoblasts but not the myotubes, suggesting that the effect of CD on synthesis of these developmentally regulated cytoskeletal proteins (a-actin, desmin) might be modulated by fusion or the state of differentiation of the muscle cell.

In cytochalasin D(CD)-treated HEp-2 cells, the same range of drug concentrations that induces rearrangement of the actin-containing cytoskeleton also produces an increase in the relative content of actin. This increase results from an elevation in the relative rate of synthesis of actin (Tannenbaum & Godman, 1983), rather than from a decrease in its turnover (Tannenbaum, 1985). We have presented the hypothesis that it is the CD-induced perturbation of cytoskeletal structure that generates a signal to elevate actin synthesis (Tannenbaum & Godman, 1983). These changes in actin synthesis and content accompanying CD-induced cytoskeletal reorganization are not restricted to (epithelioid) HEp-2 cells, but have also been observed in a variety of mesenchymal cells, both normal and transformed, including cultured rabbit aortic smooth muscle cells (Brett & Tannenbaum, 1985).

Since previous observations from this laboratory relied on one-dimensional gel electrophoresis, which does not resolve the various isoactins known to exist (summarized by Vandekerckhove & Weber, 1981), it was not possible to establish from these data whether the elevation of actin synthesis by CD reflected enhanced synthesis of the actin isotypes already being expressed or, perhaps, resulted from induction of the synthesis of isoactin(s) not normally present in the cell types under examination. An additional question was raised by our observations on the smooth muscle cells, which would be expected to contain smooth-muscle-specific α and γ-isoactins (Franke et al. 1980) in addition to the non-muscleβ and γ-isoactins normally present in the non-muscle cells tested. If the increase in actin synthesis in response to treatment with CD was proved to involve only those isoactins usually expressed in a given cell type, would this response be limited to non-muscle isoactins, which are present in the cytoskeleton, or would it, in muscle cells, also include muscle-specific isotypes?

In order to determine the effect of CD on the relative rate of synthesis of these various isoactins, we have now used high-resolution two-dimensional gel electrophoresis (O’Farrell, 1975) to separate α, β and γ-actins. Since the smooth-muscle-specific γ-actin and the non-muscle γ-actin are not resolved from each other by this technique (see Vandekerckhove & Weber, 1981), we chose to examine these effects in human skeletal muscle cultures, which contain muscle-specific α-actin and the nonmuscle and γ-actins, rather than in smooth muscle. In order to determine what effect CD has on the synthesis of a muscle-specific isoactin (a) in cells not normally expressing it, we have also examined HEp-2 cells. Part of this work has been published in abstract form (Tannenbaum & Miranda, 1985).

Cell culture, drug treatment and labelling

Muscle cultures from biopsies obtained during the course of diagnostic evaluation were, by routine morphological, histochemical and biochemical criteria, deemed to be free of muscle disease. For all biopsies, the patient’s consent was obtained, as specified by established institutional guidelines.

The muscle samples, cleaned from adhering connective and adipose tissue, were cut into 0·5–1 mm fragments and dissociated with trypsin (0·125%) and EDTA (0·015%) in Earle’s balanced salt solution, with continuous stirring at 37°C. Trypsinization was stopped by addition of foetal bovine serum (FBS), samples were centrifuged, and the cell suspension was planted in 100 mm culture dishes (200 or 300cells/dish) in Earle’s minimal essential medium (MEM) and 15% pretested FBS, enriched with non-essential amino acids, pyruvate and vitamins (Miranda et al. 1979). The dishes were left undisturbed for 14–20 days at saturation humidity in 5% CO2 and 95% air. Cell colonies that began to show early myotube formation in the centre were scored. Cloning rings (Brandel Laboratories, Gaithersburg, MD) were glued over the myogenic colonies with sterile non-toxic silicone grease (Dow Corning Corporation, Midland, MI), the cells were trypsinized, and 5–10 colonies were pooled and planted in 75 cm2 flasks (Coming) for further growth. As the cell layer approached confluency, but before fusion (8–12 days), the cells were trypsinized and planted in the 35 mm wells of Costar tissue culture dishes (5× 104/well). Unfused myoblasts were studied 96 h after planting, and myotube cultures 72 h after optimal fusion, induced by feeding the cells with MEM and 5% FBS without added enrichments. The cultures used for these studies were derived from muscle satellite cells present in the muscle biopsies. Under appropriate conditions, these cells proliferate, fuse and differentiate in vitro, similarly to myoblasts derived from immature (foetal) muscle. Using creatine kinase isozyme patterns as a biochemical marker of differentiation, unfused (myoblast) cultures expressed only the common creatine kinase isozyme (BB), while myotube cultures expressed all three isozymes: BB, the hybrid MB, and the muscle-specific MM form (for a review, see Miranda et al. 1986). Representative cultures, fixed in 5% (v/v) formalin in phosphate-buffered saline, were photographed in a Wild M-40 inverted microscope equipped with phase-contrast optics.

HEp-2 cells (ATCC CCL23) were grown in monolayers in MEM with 10% newborn calf serum (NCS) as previously described (Tannenbaum & Godman, 1983). At 24h before drug treatment, cells were planted in 35 mm plastic Petri dishes (Falcon Plastics), at 2× 105cells/dish.

A stock solution of 1 mg ml−1 of CD (Sigma Chemical Co.) in dimethyl sulphoxide (DMSO) was diluted to a final concentration of 2μM in tissue culture media just before use; control cultures received media with the corresponding concentration of DMSO (0·1%, v/v). For myoblast and myotube cultures, drug solutions were prepared in MEM with 5% FBS; for HEp-2, MEM with 10% NCS was used. After 6h incubation with CD or DMSO, cells were labelled with L-[3SS]methionine (1035 Cimmol−1; Amersham Corporation) in methionine-free MEM with 10% NCS (50μCiml−1 for muscle cells; 20gCiml−1 for HEp-2) for 1 h in the continued presence of CD (or DMSO for controls). The media were aspirated, and the cell monolayers were rinsed with Earle’s balanced salt solution, scraped into solution A of O’Farrell (1975) with 1 mM each of phenylmethylsulphonyl fluoride and L-l-tosylamino-2-phenylethylchloromethyl ketone added immediately before use, sheared in a syringe with a 26-gauge needle, and frozen at — 70°C. Total incorporation of [35S]methionine into proteins was determined as trichloroacetic acid (TCA)-precipitable ctsmin−1 (Tannenbaum & Godman, 1983), and 150000 TCA-precipitable ctsmin−1 of each sample was applied to each isoelectric focusing gel.

Two-dimensional gel electrophoresis

The method of O’Farrell (1975), with previously described modifications (Tannenbaum & Rich, 1979) was used, except that urea was omitted from the stacking gels and the slab gels used for the second dimension were 10% (w/v) polyacrylamide with an acrylamide/bisacrylamide ratio of 120:1 (w/w) (Milstone & McGuire, 1981). The isoelectric focusing gels were cylinders of 2·4mm×220mm containing ampholytes (Bio-Rad) of pH range 5·7 (1·6%, w/v) and 3·10 (0·4%, w/v). A segment that contained the isoactins was cut from each isoelectric focusing gel and applied to the 120 mm× 120 mm slab gels. The labelled material in each protein spot was quantified by scintillation counting of the material eluted from protein spots cut from dried two-dimensional gels after radioautography, as previously described (Tannenbaum & Godman, 1983).

Protein spots from muscle cell lysates (Fig. 1) were identified by comigration (not shown) with previously identified HEp-2 proteins (vimentin, β-tubulin, β, γ, δ5, ε-actins (Tannenbaum, 1986). The muscle a-actin spot was identified by its position at the acidic side of the j3-actin spot. Desmin was located by comparison with a desmin protein marker present in a cytoskeletal preparation of extracted BHK-21 cells (cf. Lazarides, 1980).

Fig. 1.

Effect of CD on synthesis of isoactins. Monolayers of human myotubes (A,B,a,b), unfused myoblasts (C,D,c,d) or HEp-2 cells (E,F,e,f) were incubated for 6h in the presence of 2·0 μM-CD (B,D,F,b,d,f) or 0·1% DMSO for controls (A,C,E,a,c,e), and then labelled with [35S]methionine for 1 h in the continued presence of drug. Samples of cell lysates containing 150000ctsmin−1 of TCA-precipitable material were resolved by two-dimensional gel electrophoresis. In these autoradiograms of the dried gels, the acid end of the pH gradient is to the right; only the portion of each gel pattern containing actin and nearby protein spots is shown. Exposure time for the autoradiography was 122·3 h (A,B,C,D) or 143·5 h (E,F) to permit visualization of less-prominent proteins. Since the actin spots were over-exposed and therefore appear to overlap in these autoradiograms, shorter exposures of the same gel patterns are shown, i.e. a for A, b for B, etc. (a,b,c,d, 21–6h; e,f, 17-6h) to illustrate that the actin isotypes (α, β γ, δ, ε) were resolved as separate spots. Patterns shown correspond to the data of expt 2 in Table 1. Note the absence of α-actin in pattern from HEp-2 cells (E,F,e,f); the pale streak to the right of) β-actin is due to slight overloading. By comparison of the apparent optical density of this streak with the density of the δ andε spots of actin (which each contain ≈3% of the total labelled actin), it is estimated to represent much less than 3% of the total actin synthesis in these cells, α, β, γ, δ ε, actin isotypes; D, desmin; V, vimentin; Tu, fl-tubulin.

Fig. 1.

Effect of CD on synthesis of isoactins. Monolayers of human myotubes (A,B,a,b), unfused myoblasts (C,D,c,d) or HEp-2 cells (E,F,e,f) were incubated for 6h in the presence of 2·0 μM-CD (B,D,F,b,d,f) or 0·1% DMSO for controls (A,C,E,a,c,e), and then labelled with [35S]methionine for 1 h in the continued presence of drug. Samples of cell lysates containing 150000ctsmin−1 of TCA-precipitable material were resolved by two-dimensional gel electrophoresis. In these autoradiograms of the dried gels, the acid end of the pH gradient is to the right; only the portion of each gel pattern containing actin and nearby protein spots is shown. Exposure time for the autoradiography was 122·3 h (A,B,C,D) or 143·5 h (E,F) to permit visualization of less-prominent proteins. Since the actin spots were over-exposed and therefore appear to overlap in these autoradiograms, shorter exposures of the same gel patterns are shown, i.e. a for A, b for B, etc. (a,b,c,d, 21–6h; e,f, 17-6h) to illustrate that the actin isotypes (α, β γ, δ, ε) were resolved as separate spots. Patterns shown correspond to the data of expt 2 in Table 1. Note the absence of α-actin in pattern from HEp-2 cells (E,F,e,f); the pale streak to the right of) β-actin is due to slight overloading. By comparison of the apparent optical density of this streak with the density of the δ andε spots of actin (which each contain ≈3% of the total labelled actin), it is estimated to represent much less than 3% of the total actin synthesis in these cells, α, β, γ, δ ε, actin isotypes; D, desmin; V, vimentin; Tu, fl-tubulin.

To assess the effect of CD on synthesis of muscle (α) and non-muscle (β, γ) isotypes of actin, cultures of fused human myotubes were treated with 2·0) μM-CD (0·1% DMSO for controls) for 6h and then labelled for 1 h with [35S]methionine in the continued presence of the drug. Lysates of cells were analysed by twodimensional gel electrophoresis. As shown in the autoradiogram of the resulting gel patterns (Fig. 1A, a) three major species of actin (α β, γ) and two minor precursor (Garrels & Gibson, 1976; Palmer et al. 1980) species (δ, ε) were resolved. Spots corresponding to desmin, vimentin andβ-tubulin were also identified in these patterns (Fig. 1A). The relative rate of synthesis of each of these proteins was quantified by scintillation counting of the protein spots cut from dried gels (see Materials and Methods).

Table 1.

Effect of cytochalasin D on synthesis of actin

Effect of cytochalasin D on synthesis of actin
Effect of cytochalasin D on synthesis of actin

Treatment of human myotube cultures with 2·0μM-CD for 6h produced comparable increases in the relative rate of synthesis of both the muscle-specific α-actin and the non-muscle γ-actin isotypes, but a smaller increase in non-muscle β-actin (Table 1). Although the level of expression of α-actin varied from one muscle isolate to the other, its enhancement by CD was very reproducible, as was the effect onβ and γ-actins (Table 1, compare experiments 1 and 2). As illustrated in Fig. 2C, the myotube cultures used in experiment 2 consisted of fused, multinucleated myotubes (78–85% of total nuclei present incorporated into myotubes with 10–50 nuclei per myotube). As previously described in cultured myotubes from the mouse (Miranda & Godman, 1973), these human myotubes became highly retracted after treatment with CD (Fig. 2D). The isolate of cells used for experiment 1 (Table 1), although it had differentiated to myotubes, was a somewhat less extensively fused population (65% of total nuclei present in myotubes) with fewer nuclei (3—7) present in each myotube (not shown).

Fig. 2.

Effect of CD on muscle cultures. Human muscle cells in culture derived from myogenic clones were fixed in 5% formalin, wet-mounted, and studied with phasecontrast optics. A. Unfused mononuclear myoblasts treated with 0·1% DMSO for 6h. B. Unfused mononuclear myoblasts exposed to 2μM-CD for 6h show cell rounding and cell retraction. C. Myotube culture, treated with 0·1% DMSO for 6h, contains multinucleated myotubes aligned in parallel (arrowheads). D. Myotube culture, treated with 2μM-CD for 6h, exhibits many severely retracted syncytia (arrowheads) and a few unfused mononuclear cells (arrows). Bar, 20μm.

Fig. 2.

Effect of CD on muscle cultures. Human muscle cells in culture derived from myogenic clones were fixed in 5% formalin, wet-mounted, and studied with phasecontrast optics. A. Unfused mononuclear myoblasts treated with 0·1% DMSO for 6h. B. Unfused mononuclear myoblasts exposed to 2μM-CD for 6h show cell rounding and cell retraction. C. Myotube culture, treated with 0·1% DMSO for 6h, contains multinucleated myotubes aligned in parallel (arrowheads). D. Myotube culture, treated with 2μM-CD for 6h, exhibits many severely retracted syncytia (arrowheads) and a few unfused mononuclear cells (arrows). Bar, 20μm.

In experiment 2, cultures of myoblasts from the same isolate of muscle were also examined. These myoblast cultures consisted primarily of mononucleated, unfused cells (Fig. 2A) with a few, small myotubes; CD triggered retraction of the cells (Fig. 2B). As for the corresponding myotubes (Table 1), the CD-induced increase in synthesis of non-muscleγ-actin was greater than for β. Although these increases were similar in the myotube and myoblast cultures, the increase in α-actin synthesis was much higher for the myoblasts than for the myotubes (Table 1).

Since CD enhanced synthesis of both muscle-specific and non-muscle actins in these human muscle cultures, we wondered whether the ability of CD to stimulate muscle-specific actin synthesis was a general phenomenon or was restricted to cells already expressing muscle proteins. Since the human epithelioid cell line, HEp-2, in which we had previously observed CD-induced enhancement of actin synthesis (Tannenbaum & Godman, 1983), does not normally synthesize a-actin (Fig. 1e; see also Garrels & Gibson, 1976), we examined the synthesis of actin isotypes in CD-treated HEp-2 to answer this question. As previously reported in HEp-2 cells (Tannenbaum & Brett, 1985), the magnitude of the stimulation of actin synthesis by short-term treatment with CD was somewhat variable because of differences in the kinetics of the response in each experiment. Like the cultures of myotubes and myoblasts, CD-treated HEp-2 cells exhibited enhanced synthesis ofβ and γ-actin; but the differential elevation of γ was less marked than in the muscle cultures (Table 1). The increased actin synthesis in these non-muscle cells did not, however, include synthesis of α-actin (Fig. If and Table 1).

The effect of CD on synthesis of some major components of two other cytoskeletal structures, microtubules (β-tubulin) and intermediate filaments (vimentin, desmin) was also determined. In both myotubes and myoblasts, synthesis of β-tubulin was depressed about 40%; in HEp-2 cells the decrease was less (about 10%). Vimentin synthesis in myotubes and HEp-2 cells was not affected by 6h treatment with CD, but it was depressed about 30% in myoblasts, bringing it to the value seen in myotubes. The relative rate of synthesis of desmin in myotube cultures varied between 0·10 ± 0·01 (expt 1) and 0·31 ± 0·02 (expt 2), but was not altered by CD. In myoblasts, though, its rate (initially 0’09 ± 0) increased by 44% after treatment with CD. HEp-2 cells did not synthesize desmin (Fig. 1e), and CD did not induce its synthesis (Fig. If).

The creatine kinase isozyme pattern in CD-treated myoblasts was also determined in order to examine the effect of the drug on a non-cytoskeletal protein. Although the total activity of creatine kinase was depressed in the CD-treated myoblasts, the enzyme profile was unchanged from control cultures (data not shown).

The experiments described in this paper were undertaken to characterize the isotype specificity of the enhancement of actin synthesis in response to treatment with CD. If this response is triggered by a signal dependent on the CD-induced rearrangement of cytoskeletal actin-containing structures, according to our hypothesis (Tannenbaum & Godman, 1983; Brett & Tannenbaum, 1985), the simplest expectation might be that only the synthesis of non-muscle isoactins (β, γ), i.e. those actin species present in the rearranging cytoskeleton, would be stimulated by this mechanism. Since, in cultures of human myotubes and myoblasts, CD was able to elevate the relative rate of synthesis of both (muscle-specific) α-actin and (nonmuscle) β and γ-actins (Table 1), it is clear that the mechanism(s) regulating actin synthesis in response to treatment with CD are more complex than predicted by this simple interpretation of the proposed model. However, despite the apparently greater complexity, several properties of the regulatory mechanism(s) involved can be deduced from the data in Table 1.

The ability of CD to trigger increased synthesis of a muscle-specific actin isotype (a) indicates that the effects of the drug can extend to muscle as well as non-muscle isotypes of actin. However, elevation of α-actin synthesis is apparently restricted to cell types in which this isotype is already being expressed (myotubes and myoblasts). In the human epithelioid HEp-2 cell line, which does not normally synthesize α-actin, this isotype was not induced by CD. Therefore, although the response to CD was observed for all isoactins examined, enhancement of actin synthesis by CD is specific for those isoactins already being expressed and does not involve induction of the synthesis of new isoactins. The data in Table 1 also suggest that CD preferentially enhances synthesis of γ compared to β-actin, but preliminary results of assays on bovine aortic endothelial cells (unpublished observations) indicated similar increases in synthesis of β and γ-actin, suggesting that this differential effect is not a general feature of the response to CD. In addition, as in HEp-2 cells, a-actin synthesis was not induced in the endothelial cells by CD.

As previously reported after 20 h treatment of HEp-2 cells with CD (Tannenbaum, 1985), the stimulatory effects of CD do not extend to all cytoskeletal proteins. The synthesis of β-tubulin was depressed in myotubes, myoblasts and HEp-2 cells. Effects on synthesis of intermediate filament proteins varied with cell type. Synthesis of vimentin and desmin was not affected by exposure of myotubes to 2μM-CD for 6h, but vimentin synthesis was depressed and desmin synthesis enhanced in the myoblasts. In contrast to 20h treatment with CD, which slightly depressed vimentin synthesis in HEp-2 cells (Tannenbaum, 1985), the shorter treatment used in this study had no effect. Desmin is not normally synthesized by HEp-2 and, as for α-actin, its synthesis was not induced by CD.

Since cytochalasins inhibit myoblast fusion (Sanger et al. 1971; Miranda & Godman, 1973), it seemed possible that the effects of CD on actin synthesis in the muscle cultures might merely reflect this inhibition. However, for primary human muscle cultures, the 6h incubation period used in this study would not be long enough to permit a significant level of fusion in the control culture of myoblasts. In addition, any prevention of fusion by CD would be expected to suppress the increase in synthesis of α-actin and desmin that normally occurs upon fusion (Devlin & Emerson, 1978; Gard & Lazarides, 1980; Shani et al. 1981), not to increase it as CD did.

Comparison of the effects of CD on myotubes and on the unfused myoblasts from which they are derived suggests that some responses to CD might be modulated by fusion or the state of differentiation of the muscle cell, as other investigators have proposed for the distinct responses of myoblasts and myotubes to stimuli inducing synthesis of heat-shock proteins (Atkinson, 1981; Atkinson et al. 1983). Thus, while the effect of CD on the synthesis of β-actin, γ-actin and β-tubulin was similar in myoblasts and myotubes, the response of the two types of cultures differed for α-actin, desmin and vimentin. For both α-actin and desmin, the increase in synthesis stimulated by CD was greater in myoblasts than in myotubes. CD decreased vimentin synthesis in myoblasts to the value seen in myotubes, but did not alter the values in myotubes. Although vimentin synthesis was reported to rise upon fusion of chick embryonic myoblasts (Gard & Lazarides, 1980), in our human muscle cell cultures vimentin synthesis was lower in myotubes compared to myoblasts, perhaps reflecting differences between embryonic muscle-derived cultures (Gard & Lazarides, 1980) and cultures from mature muscle cells (see Materials and Methods). More extensive data will be required to reach a definitive conclusion, but these initial observations on myoblast cultures treated with CD raise the possibility that the effect of CD on synthesis of α-actin, desmin and vimentin qualitatively follow the pattern of changes seen upon myotube formation. However, since the isozyme pattern of creatine kinase, which is a biochemical marker of myotube formation and differentiation (Miranda et al. 1986), was not altered in CD-treated myoblasts, CD evidently does not influence the synthesis of all developmentally regulated muscle proteins or completely mimic the effects of fusion on their synthesis.

It should also be noted that the response of a-actin synthesis to CD may not involve the same isotype of a-actin in myotube1 and myoblast cultures. Mouse and human skeletal muscle cultures have been reported to express a high level of cardiac a-actin mRNA upon fusion, which later declines as the expression of skeletal muscle type a-actin rises (Bains et al. 1984). Since two-dimensional gel patterns do not distinguish between these two isotypes of α-actin, our data do not exclude the possibility that the greater response of myoblasts to CD involves the cardiac isotype, while the lesser effect in myotubes reflects synthesis of the skeletal isotype of α-actin.

This work was supported by grants from the National Science Foundation (PCM 80–14072 to J.T.), the National Institutes of Health (NS 18446 and NS 11766 to A.F.M., and HL 15832–12 to G. C. Godman), the Muscular Dystrophy Association (to A.F.M.), and the Council for Tobacco Research (grant 1432R2 to G.C.G.).

We thank Ms C. Squires for assistance with the gel electrophoresis and Mrs J. Johnson for typing the manuscript.

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