Synthesis of rat myosin light chains in heterokaryons formed between undifferentiated rat myoblasts and chick skeletal myocytes

The control of gene expression during terminal myogenesis was explored in heterokaryons between differentiated and undifferentiated myogenic cells by analyzing the formation of species specific myosin light chains of chick and rat skeletal muscle. Dividing L6 rat myoblasts served as the biochemically undifferentiated parent. The differentiated parental cells were mononucleated muscle cells (myocytes) that were obtained from primary cultures of embryonic chick thigh muscle by blocking myotube formation with EGTA and later incubating the postimitotic cells in cytochalasin B. Heterokaryons were isolated by the selective rescue of fusion products between cells previously treated with lethal doses of different cell poisons. 95-99% pure populations of heterokaryons formed between undifferentiated rat myoblasts and differentiated chick myocytes were obtained. The cells were labeled with [35S]methionine, and whole cell extracts were analyzed on two-dimensional polyacrylamide gels. These heterokaryons synthesize the light chain of chick myosin and both embryonic and adult light chains of rat skeletal myosin. Control homokaryons formed by fusing undifferentiated cells to themselves did not synthesize skeletal myosin light chains. Control heterokaryons formed between undifferentiated rat myoblasts and chick fibroblasts also failed to synthesize myosin light chains. These results indicate that differentiated chick muscle cells provide some factor that induces L6 myoblasts to synthesize rat myosin light chains. This system provides a model for investigating the processes by which differentiated cell functions are induced.     

The suppression of myogenic functions in heterokaryons formed by fusing chick myocytes to diploid rat fibroblasts

Heterokaryons were formed by fusing differentiated chick skeletal myocytes to fibroblasts derived from skin, lung or heart cultures. The heterokaryons were analyzed for the synthesis of skeletal myosin light chains, acetylcholine receptor, total CPK activity and the ability to spontaneously fuse to form myotubes. Whereas all of the above myogenic functions were expressed in control heterokaryons formed between myocytes and myoblasts, all were extinguished in the crosses between myocytes and fibroblasts. These results confirm that the suppression of myogenic functions previously observed in cell hybrids involving fibroblastoid tumor cells also occurs in heterokaryons isolated using biochemical inhibitors between diploid fibroblasts and chick skeletal myocytes.     

Expression of differentiated functions in heterokaryons between skeletal myocytes,adrenal cells,fibroblasts and glial cells

The regulation of both muscle and adrenal functions was examined in heterokaryons formed by fusing differentiated chick skeletal myocytes to Y1 mouse adrenal cells. Mouse fast skeletal myosin light chain one (LC1) synthesis was induced and acetylcholine receptor expression was maintained at muscle control levels. Steroid secretion, although reduced compared with Y1 × Y1 adrenal homokaryon control fusions, was nonetheless maintained at relatively high levels. Steroid secretion in the myocyte × adrenal heterokaryons was constitutively expressed and was not increased by exposure to either adrenocorticotrophic hormone or db-cAMP. The population of heterokaryons was thus simultaneously expressing both muscle and adrenal functions. The steroid secretion in these heterokaryons was compared to that in heterokaryons formed by fusing Y1 adrenal cells to either chick skin fibroblasts or rat C6 glial cells. Both of these sets of heterokaryons exhibited low baseline levels of steroid secretion that were inducible to control values by ACTH. These results extend previous observations showing that heterokaryons are functionally very different than cell hybrids, and exhibit a variety of phenotypic interactions. Although fibroblasts suppress muscle functions in heterokaryons, they are permissive for adrenal functions. C6 glial cells are permissive for both adrenal and muscle functions, and along with several other neurectodermal derivatives contain an inducible skeletal myosin light chain gene. Finally, myocytes and Y1 adrenal cells are mutually permissive for their differentiated functions, and Y1 adrenal cells contain an inducible myosin light chain gene.     

Uncoupling of muscle-specific protein expression in myocyte X myoblast heterokaryons

The inducibility of several rat skeletal muscle proteins was examined in heterokaryons formed by fusing differentiated chick myocytes to undifferentiated rat myoblasts. Chicken and rat proteins were distinguished using species-specific antibodies or by their different migrations in polyacrylamide or agarose gels. Both rat skeletal myosin light chain 1 and rat α-tropomyosin were induced in the heterokaryons. In contrast, neither rat acetylcholine receptors nor creatine kinase could be detected. These results suggest that chick myocytes may contain quantities of regulatory factors that are sufficient for the activation of some but not all of these rat muscle-specific proteins within the cellular context of the heterokaryon.     

Phenotypic expression in chick erythrocyte X rat myoblast hybrids and in chick myoblast X rat myoblast hybrids

Attempts were made to reprogram chick erythrocyte nuclei to specify the synthesis of chick myosin. Chick erythrocytes were fused with rat myogenic cells with the aid of UV-inactivated Sendai virus. In the heterokaryons and hybrid myotubes which resulted from this fusion, the erythrocyte nuclei resumed RNA synthesis and formed nucleoli. Although some new chick antigens developed in those myotubes which contained fully reactivated chick erythrocyte nuclei, accumulation of chick myosin could not be detected by immunological methods. Neither small heterokaryons nor large hybrid myotubes which were actively synthesizing rat myosin reacted with antibodies directed against chick myosin. A small number of mononucleated cells, believed to be synkaryons formed by mitotic division of heterokaryons, did, however, react strongly with antibodies directed against chick myosin and showed a cross striation typical of skeletal muscle. The frequency of such cells was too low, however, to permit karyological analysis or further characterization of the antigen. Hybrids between chick myoblasts and rat myoblasts produced both chick and rat myosin thus indicating that simultaneous translation of chick and rat mRNA for myosin in a common cytoplasm was possible. In summary the evidence obtained suggested that reprogramming of chick erythrocyte nuclei, if it did occur in the present system, was a rare phenomenon.The possibility that hybrids between chick erythrocytes and rat myoblasts expressed markers typical of an erythroid phenotype was examined by immune staining with antibodies directed against chick haemoglobin. The results suggested that haemoglobin was introduced into hybrid cells by erythrocytes which failed to lyse before fusion. The intensity of this immune fluorescence decreased with increasing time after fusion. The rate at which this decrease occurred was not affected by inhibition of RNA synthesis. Thus, there was no evidence for the accumulation of haemoglobin in the hybrid cells.     

Control of differentiation in heterokaryons and hybrids involving differentiation-defective myoblast variants

Clones of differentiation-defective myoblasts were isolated by selecting clones of L6 rat myoblasts that did not form myotubes under differentiation-stimulating conditions. Rat skeletal myosin light chain synthesis was induced in heterokaryons formed by fusing these defective myoblasts to differentiated chick skeletal myocytes. This indicates that the structural gene for this muscle protein was still responsive to chick inducing factors and that the defective myoblasts were not producing large quantities of molecules that dominantly suppressed the expression of differentiated functions. The regulation of the decision to differentiate was then examined in hybrids between differentiation- defective myoblasts and differentiation-competent myoblasts. Staining with antimyosin antibodies showed that the defective myoblasts and homotypic hybrids formed by fusing defective myoblasts to themselves could in fact differentiate, but did so more than a thousand times less frequently than the 64% differentiation achieved by competent L6 myoblasts or homotypic competent X competent L6 hybrids. Heterotypic hybrids between differentiation-defective myoblasts and competent L6 cells exhibited an intermediate behavior of approximately 1% differentiation. A theoretical model for the regulation of the commitment to terminal differentiation is proposed that could explain these results by invoking the need to achieve threshold levels of secondary inducing molecules in response to differentiation-stimulating conditions. This model helps explain many of the stochastic aspects of cell differentiation.     

Differentiation of myogenic cells in micromass cultures of cells from chick facial primordia

Antibodies to the myosin heavy chains of striated muscle were used to trace myogenic differentiation in the developing face and in cultures of cells from the facial primordia of chick embryos. In the intact face, myogenic cells differentiate first in the mandibular primordia and can be detected at stage 28. The early muscle blocks contain both fast and slow classes of myosin heavy chains. At stages 20 and 24, no myogenic cells are found in any of the facial primordia. However, when the cells are placed in micromass (high density) cultures, myogenic cells differentiate, revealing the presence of potentially myogenic cells in all the facial primordia. The number of myogenic cells bears no consistent relationship to the extent and pattern of chondrogenesis. Therefore the ability of the cell populations of the facial primordia to differentiate into cartilage when placed in culture is independent of the muscle cell lineage. The facial primordia represent a mixed cell population of neural crest and mesodermal cells from at least as early as stage 18.     

Actin and myosin expression during development of cardiac muscle from cultured embryonal carcinoma cells

P19 embryonal carcinoma cells are multipotential stem cells that differentiate into striated muscle as well as some other cell types when aggregated and exposed to dimethyl sulfoxide (DMSO). Immunofluorescence experiments using monospecific antibodies indicated that the majority of muscle cells were mononucleate and contained four myosin isoforms normally found in cardiac muscle; atrial and ventricular myosin heavy chains, ventricular myosin light chain 1, and atrial myosin light chain 2. Northern blot analysis of RNA isolated from differentiating cultures indicated that cardiac actin and skeletal actin mRNAs were expressed at similar levels and with identical kinetics during the differentiation of P19-derived myocytes. These results demonstrate that most of the P19-derived myocytes are of the cardiac type and suggest that they closely resemble the cells of the early embryonic myocardium.     

Myosin accumulation in mononucleated cells of chick muscle cultures.

The biosynthesis and accumulation of the myosin heavy chain (MHC) peptide has been examined in embryonic chick skeletal muscle cultures under conditions of normal or arrested cell fusion. When compared with primary chick fibroblasts, the myogenic cells accumulated significantly more MHC, even while mononucleated. Electron microscopy of the fusion-blocked cultures revealed the presence of myosinlike thick filaments in the myoblasts. It is concluded that cell fusion is not a prerequisite for myosin accumulation or myofilament assembly during embryonic chick muscle differentiation.     

Analysis of muscle protein expression in polyethylene glycol-induced chicken: rat myoblast heterokaryons

Heterokaryons derived from polyethylene glycol-mediated fusion of myoblasts at different stages of development were used to investigate the transition of cells in the skeletal muscle lineage from the determined to the differentiated state. Heterokaryons were analyzed by immunofluorescence, using rabbit antibodies against the skeletal muscle isoforms of chicken creatine kinase and myosin, and a mouse monoclonal antibody that cross-reacts with chicken and rat skeletal muscle myosin. When cytochalasin B-treated rat L8(E63) myocytes (Konieczny S.F., J. McKay, and J. R. Coleman, 1982, Dev. Biol., 91:11-26) served as the differentiated parental component and chicken limb myoblasts from stage 23-26 or 10-12-d embryos were used as the determined, undifferentiated parental cell, heterokaryons exhibited a progressive extinction of rat skeletal muscle myosin during a 4-6-d culture period, and no precocious expression of chicken differentiated gene products was detected. In the reciprocal experiment, 85-97% of rat myoblast X chicken myocyte heterokaryons ceased expression of chicken skeletal muscle myosin and the M subunit of chicken creatine kinase within 7 d of culture. Extinction was not observed in heterokaryons produced by fusion of differentiated chicken and differentiated rat myocytes and thus is not due to species incompatibility or to the polyethylene glycol treatment itself. The results suggest that, when confronted in a common cytoplasm, the regulatory factors that maintain myoblasts in a proliferating, undifferentiated state are dominant over those that govern expression of differentiated gene products.     

Turnover of myosin heavy and light chains in cultured embryonic chick cardiac and skeletal muscle

The relative rates of synthesis and breakdown of myosin heavy and light chains were studied in primary cell cultures of embryonic chick cardiac and skeletal muscle. Measurements were made after 4 days in culture, at which time both skeletal and cardiac cultures were differentiated and contracted spontaneously. Following a 4-hr pulse of radioactive leucine, myosin and its heavy and light chains were extracted to 90% or greater purity and the specific activities of the proteins were determined. In cardiac muscle, myosin heavy chains were synthesized approximately 1.6 times the rate of myosin light chains, and in skeletal muscle, heavy chains were synthesized at approximately 1.4 times the rate of light chains. Relative rates of degradation of muscle proteins were determined using a dual-isotope technique. In general, the soluble and myofibrillar proteins of both types of muscle had decay rates proportional to their molecular weights (larger proteins generally had higher decay rates) based on analyses utilizing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A notable exception to this general rule was myosin heavy chains, which had decay rates only slightly higher than the myosin light chains. Direct measurements on purified proteins indicated that the heavy chains of myosin were turning over at a slightly greater rate (approximately 20%) than the myosin light chains in both cardiac and skeletal muscle. The reasons for the apparent discrepancy between these measurements of myosin heavy and light chain synthesis and degradation are discussed.     

The methylation state of 2 muscle-specific genes: restriction enzyme analysis did not detect a correlation with expression

To examine the possible role of DNA methylation in the modulation of expression of genes involved in the differentiation of muscle cells, we compared the methylation state of a number of CpG sites in the rat skeletal muscle actin and myosin light chain 2 genes, in muscle and nonmuscle cells, and in proliferating myoblasts and differentiated myotubes of the myogenic cell line L8. No correlation was detected between the state of methylation of these sites and the expression of the two genes. Essentially the same pattern of DNA methylation was observed, in the sites examined, in DNA from muscle, kidney and stomach. In DNA extracted from cultures of proliferating mononucleated myoblasts, as well as from differentiated multinucleated fibers of the myogenic cell line L8, the two genes were more methylated than in other tissues.     

Induction of myosin light chain synthesis in heterokaryons between normal diploid cells

Summary Quail myoblasts were maintained in an undifferentiated state by first blocking differentiation with 5-bromodeoxyuridine and then reversing the block in the presence of phorbol-12-myristate-13-acetate. The synthesis of quail skeletal myosin light chain 1 is induced in heterokaryons formed by fusing these undifferentiated quail myoblasts to differentiated chick myocytes. These results extend observations previously obtained using an established line of rat myoblasts and indicate that the induction is a result of regulatory interactions present in normal diploid cells. This work was supported by grants from the Muscular Dystrophy Association and the National Institutes of Health.     

Acetylcholinesterase activity in developing skeletal muscle cells

Acetylcholinesterase activity has been demonstrated biochemically and cytochemically in developing chick embryo skeletal muscle cells growing in culture. The enzyme shows the same pattern of drug sensitivity as that of adult skeletal muscle acetylcholinesterase and in present in cultured myogenic cells before the time of cell fusion, the formation of myotubes, and the subsequent increase in rate of myosin synthesis. Myogenic cell fusion is accompanied, however, by a large increase in activity of acetylcholinesterase. The enzyme activity is restricted in these cultures to myogenic cells. Neighboring fibroblasts show no cytochemical responses when challenged with techniques showing intense activity in myoblasts and myotubes. In addition, evidence is presented which strongly suggests that acetylcholinesterase activity in dividing myogenic cells is not constant over the cell cycle.     

Volume occupation, environment, and accessibility in proteins. Environment and molecular area of RNase-S

The myosin light chains of cultured muscle cells and embryonic muscle tissue have been examined by two-dimensional gel electrophoresis. Myosin purified from primary cultures of rat muscle cells or the myogenic cell line L6 contain not only the light chains corresponding to those of fast twitch muscle but also another protein, differing slightly in molecular weight and isoelectric point from the adult LC1 protein. By a number of criteria this additional protein is shown to be a myosin light chain: (1) it is found in highly purified myosin preparations; (2) in L6 myosin it replaces the other LC1-type light chains in stoichiometric amounts; (3) it is part of the subfragment-1 complex of myosin produced by chymotrypsin. as expected for an LC1-type light chain. Total extracts of fused cultured muscle cells, when analyzed by two-dimensional electrophoresis, contain substantial amounts of this additional LC1-type protein, strongly suggesting that it is not a proteolytic fragment produced during myosin isolation. Unfused cultures do not synthesize detectable amounts of the adult light chains or the additional LC1-type light chain. This additional LC1 protein can be detected in embryonic or newborn muscle tissue but it is not present in adult myosin or myofibrils. These results indicate that a novel form of myosin light chain, referred to as an embryonic LC1 or LC1_emb, is characteristic of the early stages of muscle development.     

Conditional conversion of ES cells to skeletal muscle by an exogenous MyoD1 gene.

A variety of differentiated cell types can be converted to skeletal muscle cells following transfection with the myogenic regulatory gene MyoD1. To determine whether multipotent embryonic stem (ES) cells respond similarly, cultures of two ES cell lines were electroporated with a MyoD1 cDNA driven by the beta-actin promoter. All transfected clones, carrying a single copy of the exogenous gene, expressed high levels of MyoD1 mRNA. Surprisingly, although maintained in mitogen-rich medium, this ectopic expression was associated with a transactivation of the endogenous myogenin and myosin light chain 2 gene but not the endogenous MyoD1, MRF4, Myf5, the skeletal muscle actin, or the myosin heavy chain genes. Preferential myogenesis and the appearance of contracting skeletal muscle fibers were observed only when the transfected cells were allowed to differentiate in vitro, via embryoid bodies, in low-mitogen-containing medium. Myogenesis was associated with the activation of MRF4 and Myf5 genes and resulted in a significant increase in the level of myogenin mRNA. Not all cells were converted to skeletal muscle cells, indicating that only a subset of stem cells can respond to MyoD1. Moreover, the continued expression of the introduced gene was not required for myogenesis. These results show that ES cells can respond to MyoD1, but environmental factors control the expression of its myogenic differentiation function, that MyoD1 functions in ES cells even under environmental conditions that favor differentiation is not dominant (incomplete penetrance), that MyoD1 expression is required for the establishment of the myogenic program but not for its maintenance, and that the exogenous MyoD1 gene can trans-activate the endogenous myogenin and MLC2 genes in undifferentiated ES cells.     

Altered synthesis of myosin light chains is associated with contractility in cultures of differentiating chick embryo breast muscle

Cultured chick embryo skeletal muscle cells normally synthesize only the embryonic isoform of mysoin. We have found that aneural muscle cultures that become or are provoked into an extremely contractile state will begin to synthesize a pattern of myosin light chains typical of maturing muscle. Immunoblots with neonatal and adult specific monoclonal antibodies did not reveal a corresponding isozyme transition in myosin heavy chain. These results demonstrate a correlation between contractility and the regulation of myosin light chain maturation, and also suggest that the transitions of heavy and light chain synthesis during development do not appear to be under close coordinate regulation.     

Isolation and characterization of an avian myogenic cell line

Myogenic cell lines have proven extremely valuable for studying myogenesis in vitro. Although a number of mammalian muscle cell lines have been isolated, attempts to produce cell lines from other classes of animals have met with only limited success. We report here the isolation and characterization of seven avian myogenic cell lines (QM1-4 and QM6-8), derived from the quail fibrosarcoma cell line QT6. A differentiation incompetent QM cell derivative was also isolated (QM5DI). The major features of QM cell differentiation in vitro closely resemble those of their mammalian counterparts. Mononucleated QM cells replicate in medium containing high concentrations of serum components. Upon switching to medium containing low serum components, cells withdraw from the cell cycle and fuse to form elongated multinucleated myotubes. Cultures typically obtain fusion indices of 43-49%. Northern blot and immunoblot analyses demonstrate that each differentiated QM cell line expresses a wide variety of genes encoding muscle specific proteins: desmin, cardiac troponin T, skeletal troponin T, cardiac troponin C, skeletal troponin I, alpha-tropomyosin, muscle creatine kinase, myosin light chain 2, and a ventricular isoform of myosin heavy chain. While all QM lines analyzed to date express at least some myosin light chain 2, only one line, QM7, expresses this gene at high levels. Surprisingly, none of the QM lines reported here express any known form of alpha-actin. The absence of sarcomeric actin expression may explain the absence of myofibrils in QM myotubes. These novel features of muscle gene expression in QM cells may prove useful for studying the role of specific muscle proteins during myogenesis. More importantly, however, the isolation of QM cell lines indicates that it may be feasible to isolate other avian myogenic cell lines with general utility for the study of muscle development.