Slow and Fast Myosin Heavy Three Types of Myotubes in Chain Content Defines Early Muscle Cell Cultures

We prepared monoclonal antibodies specific for fast or slow classes of myosin heavy chain isoforms in the chicken and used them to probe myosin expression in cultures of myotubes derived from embryonic chicken myoblasts. Myosin heavy chain expression was assayed by gel electrophoresis and immunoblotting of extracted myosin and by immunostaining of cultures of myotubes. Myotubes that formed from embryonic day 5-6 pectoral myoblasts synthesized both a fast and a slow class of myosin heavy chain, which were electrophoretically and immunologically distinct, but only the fast class of myosin heavy chain was synthesized by myotubes that formed in cultures of embryonic day 8 or older myoblasts. Furthermore, three types of myotubes formed in cultures of embryonic day 5-6 myoblasts: one that contained only a fast myosin heavy chain, a second that contained only a slow myosin heavy chain, and a third that contained both a fast and a slow heavy chain. Myotubes that formed in cultures of embryonic day 8 or older myoblasts, however, were of a single type that synthesized only a fast class of myosin heavy chain. Regardless of whether myoblasts from embryonic day 6 pectoral muscle were cultured alone or mixed with an equal number of myoblasts from embryonic day 12 muscle, the number of myotubes that formed and contained a slow class of myosin was the same. These results demonstrate that the slow class of myosin heavy chain can be synthesized by myotubes formed in cell culture, and that three types of myotubes form in culture from pectoral muscle myoblasts that are isolated early in development, but only one type of myotube forms from older myoblasts; and they suggest that muscle fiber formation probably depends upon different populations of myoblasts that co-exist and remain distinct during myogenesis. There are two major sets of biochemical differences in developing muscles and in muscle fibers. One set is found between physiologically fast and slow muscles. Fibers in these two muscle types contain different isoforms of myosin and other muscle-specific proteins, although both fibers and muscles are found with mixed properties. A second set is due to the appearance of isoforms specific to different developmental ages within a single muscle. For example, a sequence of myosin heavy chain and myosin light chain isoform expression occurs during the formation of all muscles (reviewed in reference 1). The developmental basis for the formation of different fiber types and sequential expression of isoforms in putative fast and slow fibers is not known, but fiber diversification has been postulated to result from either a single cell lineage or multiple cell lineages (2). The sequential appearance of different isoforms at different THE JOURNAL OF CELL BIOLOGY • VOLUME 101 NOVEMBER 1985 1643-1650 © The Rockefeller University Press • 0021-9525]85111[1643/08 $1.00 stages of avian muscle development is found for a number of proteins, including the fast myosin heavy chain (3-9), myosin light chain (10-14), troponin (15), C-protein (16), tropomyosin (14, 17), and creatine kinase (18). Similar isoform transitions are found in mammals (l 9-22). One particularly wellstudied isoform transition is that of the fast myosin heavy chains of the avian breast muscle. Three fast isoforms that appear sequentially during mid-embryonic to adult development of this muscle have been described (6, 9). The first isoform appears on or before embryonic day (ED) ~ l0 and is expressed through ED 18, and the second is expressed from ED 16 to several weeks after hatching. The first two isoforms Abbreviations used in this paper: ALD, anterior latissimus dorsi; ED, embryonic day; McAb, monoclonal antibody; PM, pectoral muscle. 1643 on A uust 7, 2017 jcb.rress.org D ow nladed fom are shared by several muscles. The third isoform, which is apparently unique to adult pectoralis major, appears several weeks after hatching. Isoform transitions in the mid-embryonic to adult pectoral muscle (PM) are, therefore, well demonstrated. Biochemical differentiation of the PM during very early development (ED 4-6) is, however, comparatively unstudied. As a step toward understanding differentiation in the early PM, we have studied myosin heavy chain expression in cultures o f ea r ly P M cells. M y o b l a s t s w e r e i s o l a t e d f r o m t h e P M o f e m b r y o s o f d i f f e r e n t a g e s a n d a l l o w e d to f o r m m y o t u b e s in c u l t u r e . M y o s i n h e a v y c h a i n e x p r e s s i o n w a s a s s e s s e d u s i n g m o n o c l o n a l a n t i b o d i e s to fas t a n d s l o w c lasses o f i s o f o r m s as p r o b e s in i m m u n o b l o t t i n g a n d i m m u n o c y t o c h e m i c a l p r o c e d u r e s . W e f o u n d t h a t , u n l i k e t h e m y o t u b e s f o r m e d in c u l t u r e s o f E D 8 o r o l d e r d o n o r s , c u l t u r e s o f p e c t o r a l m y o b l a s t s f r o m E D 5 6 f o r m e d b i o c h e m i c a l l y d i s t i n c t p o p u l a t i o n s o f m y o t u b e s . M a n y o f t h e s e m y o t u b e s c o n t a i n e d t h e s l ow c lass o f m y o s i n h e a v y c h a i n a n u n e x p e c t e d f i n d i n g for c u l t u r e d m y o t u b e s . T h e r e s u l t s s h o w t h a t c u l t u r e d m y o b l a s t s c a n f o r m m y o t u b e s t h a t s y n t h e s i z e s l o w m y o s i n h e a v y c h a i n a n d m a i n t a i n t h r e e d i f f e r e n t p h e n o t y p e s in a s ing le c u l t u r e , a n d t h a t t h e s e p r o p e r t i e s d e p e n d u p o n t h e age o f t h e e m b r y o f r o m w h i c h t h e m y o b l a s t s a r e d e r i v e d . MATERIALS AND METHODS Cell Culture: PM cells were isolated and cultured as described by O'Neill and Stockdale (23). The PM was identified and dissected from stage 27-28 (ED 5), stage 29 (ED 6), ED 8, and ED 12 embryos, minced, dissociated with trypsin, and plated at 2.5 x 10 + cells/cm 2 on gelatin-coated dishes. Whereas it was easy to dissect the PM in ED 8 or older embryos, the PM in ED 5-6 embryos was recognized as a linear thickening on the forming chest wall and care was taken to avoid the inclusion of tissue from the trunk muscles, ribs, and upper wing during the dissection. Culture medium, 0.3 ml/cm 2, consisted of Dulbecco's modified Eagle's medium supplemented with 10% horse serum, 2.5% chick embryo extract, penicillin, streptomycin, and fungizone. When used, conditioned medium was prepared from ED 12 PM cultures as described (24). Cultures of these cells contained multinucleated cells after 2-3 d. The number of contaminating cells could be reduced without changing the results by preplating the trypsin-treated cells for 30 min before initial culture and by treating the cultures with cytosine arabinoside at 10 ~g/ml for 48 h beginning on the third day of culture. Myosin Preparations: Myosin was purified from adult white Leghorn chicken by repeated cycles of high salt solubilization and low salt precipitation (25), and stored as described below. The predominantly fast PM and predominantly slow anterior latissimus dorsi (ALD) muscle were used as sources of myosin. Myosin was extracted from cultured myotubes after the cells were removed from culture dishes with trypsin and collected by centrifugation. The cell pellet was extracted on ice for 15 min with a buffer consisting of 0.6 M NaCI, l0 mM sodium phosphate, 1 mM sodium pyrophosphate, 0.5 mM MgC12, 0.1 mM EGTA, 1 mM dithiothreitol, and 0.5% Triton X-100, pH 6.8. The cell pellet was extracted with 1 ml of this buffer per 150 cm 2 of culture area. The extracted cells were centrifuged at 250 g for 10 min, and the supernatant was dialyzed in 500 ml of 5 mM KCI, 0.5 mM sodium phosphate, and 0+25 mM EGTA, pH 6.8, overnight at 4°C. Myosin was collected from the dialysate by centrifugation at 15,000 g for 2 min. The pellet was dissolved in 80 mM sodium pyrophosphate, 2 mM MgCI2, and 2 mM EGTA at one-tenth of the extraction buffer volume, mixed with an equal volume of glycerol, and stored at -20°C until used. Protein was quantitated with the dye-binding assay of Bradford (26). Monoclonal Antibodies: Monoclonal antibodies (McAb's) to chicken myosin heavy chains were prepared as described previously (13). BALB/c mice were immunized with myosin purified from adult PM (the source of McAb F59) or ED 19 upper leg muscle (the source of McAb's $58 and $46), and the spleen cells were hybridized with the myeloma cell line P3-NS1/I Ag 4-1. Immunization, hybridoma formation, and selection were performed as described by Oi and Herzenberg (27). Hybridoma culture supernatants were initially screened with a solid-phase binding assay using purified myosin heavy chain fractions adsorbed to polyvinylchloride plates as the antigen (13). Hybridomas with positive supernatants were recloned by limiting dilution. 1644 THE JOURNAL OF CELL BIOLOGY VOLUME 101, 1985 Immunocytochemistry and Microscopy: Fixed myotube cultures and cryostat sections (8-~m thick) of frozen, unfixed muscle were used to determine the myosin heavy chain content of individual cells. Cryostat sections were prepared as described (13) but the sections were not fixed. Endogenous peroxidase activity was blocked by a 10-min incubation of the sections in methanol and 3% H202 at room temperature. Myotube cultures were fixed for 5 min with 3.7% formaldehyde in phosphate-buffered saline (PBS) and for 5 min more in 100% ethanol. The procedure for myosin visualization was identical for sections and cultures. The samples were incubated in 2% bovine serum albumin (BSA), 2% horse serum in PBS for 30 rain and then with a 1:10 dilution of the hybridoma supernatant in PBS-BSA-horse serum for 1 h. The cells were washed with several changes of PBS and incubated with biotinylated horse anti-mouse lgG at 5 t~g/ml. Bound antibody was visualized with either an avidin-biotin horseradish peroxidase or an avidin-biotin-glucose oxidase complex as described by the manufacturer (Vectastain ABC kit, Vector Laboratories, Inc., Burlingame, CA). For double-label immunofluorescenee, McAb's F59 and $46 were purified from ascites fluid on DEAE Am-gel blue columns (Bio-Rad Laboratories, Richmond, CA) as described (28). The purified McAb's were biotinylated by reaction with N-hydroxysuccinimidobiotin as described (29). Cultures were fixed as above and incubated overnight with biotinylated McAb $46 at 1 ~g/ ml in PBS-BSA-horse serum at 4"C. As was the case between each change of reagents, the cultures were washed with four changes of PBS. The cultures were then incubated with fluorescein-conjugated avidin (Vector Laboratories; Inc.) for 3 h at 25 ~g/ml at room temperature, biotin for 30 min at 25 ~g/ml, biotinylated McAb F59 for 1 h at 1 gg/ml, and rhodamine-conjugated avidin (Vector Laboratories, Inc.) for 1 h at 10 #g/ml. Control experiments showed that the biotin blocking step was sufficient to saturate biotin binding sites on the fluorescein-conjugated avidin, and also that the order in which the biotinylated $46 and F59 were added did not affect the results. The cultures were mounted for microscopy in PBS at pH 8.6 supplemented with 90% glycerol and 2.5% 1,4-diazabicyclo (2, 2, 2) octane to retard fluorescence bleaching (30). Observations were made with a Zeiss photomicroscope equipped for epifluorescence, and Kodak Tri-X film was used for photography. Immunoelectrophoresis and Myosin Quantitation: SDS PAGE was performed as described by Laemmli (31) on 5% gels. Proteins were electrophoretically transferred to nitrocellulose as described by Burnette (32). Myosin heavy chain on the nitrocellulose transfer was detected by incubating the transfers for 1-2 h with hybridoma supernatants diluted l:10 with 2% nonfat powdered milk (33) in PBS. The horseradish peroxidaseor glucose oxidase-linked systems described above were used to visualize McAb binding to myosin heavy chain. A dot blot assay (34) was used to quantitate myosin isoforms in cultured myotubes. Myosin was extracted from cultures as above and diluted with 0.2 M NaCl, 5 mM HEPES, l0 mM 2-mercaptoethanol, and 1% SDS (dot blot buffer). Fast myosin heavy chain from the PM and slow myosin heavy chain from the ALD were extracted as above and subjected to SDS PAGE on 5% gels. The areas of the gel that contained myosin heavy chain were cut out, the myosin heavy chain was electroeluted, and protein was quantitated. These purified heavy chain preparations were diluted with dot blot buffer and used as standards in the assays. 20-~1 samples were applied to nitrocellulose as serial 1:3 dilutions starting with 2 ug of protein in 20 gl. The blots were air dried, incubated with hybridoma supernatants, and visualized with the horseradish peroxidase system as described above. Myosin was quantitated by comparing the size and intensity of dots produced by the standards with those of the samples. Similar results were obtained using a t2Sl-labeled rabbit anti-mouse secondary antibody, but the peroxidase-linked system was used routinely.