Chick Embryo Skeletal Muscle Research Articles

Antisense oligonucleotides targeted against protein kinase c alpha inhibit proliferation of cultured avian myoblasts.

Protein kinase C (PKC) has been implicated in the control of proliferation and differentiation of many cell types. There is evidence indicating that it plays a role in signal transduction mechanisms related to myogenesis, but little is known about the individual functions of PKC isoforms in muscle cell development. Data obtained in previous studies using cultured chick embryo skeletal muscle cells suggested that PKC alpha is linked to the regulation of myoblast proliferation. However, this causal relationship could not be definitively established as no experiments based on selective inhibition of this isoform were carried out. In the present work, specific inhibition of the expression of PKC alpha in cultured myoblasts by using antisense oligonucleotide technology resulted in a significant decrease of culture cell density and DNA synthesis, clearly showing that this isoenzyme is involved in signalling pathways which promote muscle cell proliferation.

Effects of calcitriol and its analogues, calcipotriol (MC 903) and 20-Epi-1α,25-dihydroxyvitamin D 3 (MC 1288), on calcium influx and DNA synthesis in cultured muscle cells

The fast actions of the secosteroid hormone 1α,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3; calcitriolJ and the synthetic analogues calcipotriol (MC 903) and 20-epi-1α,25(OH) 2D 3 (MC 1288) on cell calcium influx were compared in rat duodenum enterocytes as well as in cells from chick embryo skeletal muscle (myoblasts) and heart (myocytes), at various concentrations (10 −12 to 10 −8 M) and treatment intervals (1–10 min). In enterocytes, at a concentration of 10 −11 M, MC 1288 was significantly more active than 1,25(OH) 2D 3 in rapidly stimulating 45Ca 2+ uptake by enterocytes (80 vs 38% above controls, respectively), whereas MC 903 was devoid of activity. However, calcipotriol increased Ca 2+ influx in myocytes and myoblasts to a greater extent than the natural hormone, whereas MC 1288 was more active only in myoblasts. Analogously to 1,25(OH) 2D 3, the fast MC 903- and MC 1288-induced stimulation of 45Ca 2+ uptake in enterocytes and muscle cells could be blocked by both verapamil and nifedipine. In addition, MC 903 and MC 1288 were more effective than 1,25(OH) 2D 3 in stimulating DNA synthesis in proliferating myoblasts and in inhibiting DNA synthesis in differentiating myoblasts. The results suggest, therefore, that modifications in the side-chain of the 1,25(OH) 2D 3 molecule increase its ability to modulate muscle cell Ca 2+ metabolism and growth. These findings are potentially relevant for the development of analogues for the treatment of vitamin D-dependent myopathies.

1,25 Dihydroxyvitamin D3 affects calmodulin distribution among subcellular fractions of skeletal muscle.

1,25 Dihydroxyvitamin D3 has been shown to stimulate calcium fluxes across skeletal muscle membranes. The involvement of calmodulin in the effects of the metabolite was investigated. Primary cultures of chick embryo skeletal muscle myoblasts and soleus muscles from vitamin D-deficient or 1,25 (OH)2D3-treated chicks were used. Culture of myoblasts and vitamin D-deficient soleus with 1,25 (OH)2D3 (0.05 ng/ml) for 24 and 1 hour, respectively, significantly increased 45Ca uptake by the preparations. In the presence of the calmodulin antagonists flufenazine or compound 48/80 in the uptake medium, no differences between control and treated cultures were observed. The calmodulin content of myoblasts and soleus homogenates and subcellular fractions derived therefrom was estimated by measuring their capacity to stimulate calmodulin-depleted cAMP phosphodiesterase. No changes in total calmodulin cellular content could be detected in response to 1,25(OH)2D3. However, the sterol produced an increase in calmodulin levels of microsomes, mitochondria, and crude myofibrillar fraction and a proportional decrease in cytosolic calmodulin concentration. The 1,25(OH)2D3-dependent changes in calmodulin distribution among subcellular fractions of soleus muscle were observed either in vivo or in vitro. The effects in vitro were already detectable after 5 minutes of treatment with the sterol and parallel 1,25(OH)2D3-dependent changes in tissue Ca uptake. The results suggest that changes in calmodulin intracellular distribution may underly part of the mechanism by which 1,25(OH)2D3 affects muscle calcium transport.

A low-molecular weight chick brain-derived growth factor is mitogenic for cultured astroglia from the chick embryo

Immunohistochemically defined astrocytes from the 10-day chick embryo were stimulated to incorporate increased levels of [ 3H]thymidine when a low-molecular weight peptide growth factor, chick brain-derived growth factor (CBGF), was added to the cultures. Treatment of these GFAP-positive astrocytes with 10 ng/ml CBGF in medium supplemented with 1% fetal bovine serum resulted in a 3.5–4-fold increase in [ 3H]thymidine incorporation when compared to astrocytes cultured in defined medium supplemented with 1% serum alone. CBGF had no effect on the survival, proliferation or differentiation of a number of other cell types from the 10-day chick embryo brain, including neurons and meningeal fibroblasts. CBGF was also ineffective as a mitogen for chick embryo skeletal muscle myoblasts, primary mouse embryo fibroblasts and one murine teratocarcinoma-derived cell line (STO). We suggest that CBGF might act as a mitogenic signal for astroglia during central nervous system development and repair.

The phospholipid and fatty acid composition of skeletal muscle cells during culture in the presence of vitamin D-3 metabolites

The phospholipid and fatty acid composition of primary cultures (24 h) of chick embryo skeletal muscle myoblasts treated for 4–24 h with physiological concentrations of 1,25-dihydroxyvitamin D-3 and 25-hydroxyvitamin D-3 were analyzed. 25-Hydroxyvitamin D-3 did not alter the relative amounts of individual muscle cell phospholipids whereas 1,25-dihydroxyvitamin D-3 significantly increased phosphatidylcholine content, mainly at the expense of a decrease in phosphatidylethanolamine concentration. The increase in phosphatidylcholine occurred at a faster rate during the first 8 h than in the subsequent 8–24 h treatment period. A similar time course in 1,25-dihydroxyvitamin D 3-dependent changes in myoblast calcium uptake has been observed. In addition, this metabolite markedly increased (100%) the arachidonate content of myoblast phosphatidylcholine near the fusion stage of the cells (24 h of treatment). The levels of docosahexaenoate, a minor polyunsaturated fatty acid, in phosphatidylcholine and phosphatidylethanolamine were also substantially elevated by 1,25-dihydroxyvitamin D-3. No significant changes in fatty acid composition in response to 25-hydroxyvitamin D-3 were observed. Modifications in phospholipids and polyunsaturated fatty acids may play a role in the effects of 1,25-dihydroxyvitamin D-3 on muscle cell calcium transport and differentiation.

Troponin in Cultured Chicken Breast Muscle Cells: (troponin / muscle cell / chicken).

The onset of troponin accumulation and the localization of troponin in cultured chick embryo skeletal muscle cells were studied by means of indrect immunofluorescent microscopy. At 31 hr after plating, troponin components were detected in 54-62% of total mononucleated myogenic cells and in all myotubes as longitudial fibrous structures. 3 H-thymidine incorporation stduy coupled with the immunofluorescent microscopy showed that mononucleated myogenic cells at the mitotic stage did not contain troponin. As myotube maturation proceeds, the troponin-containing fibers were organized into cross-striated structures. At the myotube stage, muscle cells were labeled with 35 S-methionine and proteins synthesized were analyzed by two-dimensional gel electrophoresis. It was found that myotubes in culture synthesized both fast and slow types of troponin-I and-C. Our results suggest that fast and slow types of troponin components are synthesized in cultured skeletal muscle cells before or at the early phase of myofibrillogenesis.

Phosphate Accumulation by Muscle in vitro and the Influence of Vitamin D3 Metabolites

Phosphate accumulation by muscle in vitro and the effects of vitamin D3 metabolites thereupon were studied in cultures of chick embryo skeletal muscle myoblasts and intact chick soleus muscles. A significant proportion of phosphate accumulation by the cells was Na+-dependent, saturable with respect to phosphate, energy-dependent and inhibited by ouabain and arsenate, in agreement with the operation of a Na+-phosphate cotransport system in the muscle cell plasma membrane as has been described for intestine and kidney. This was further supported by the demonstration of substrate-saturable phosphate uptake in sarcolemma vesicles isolated from chick skeletal muscle. Preincubation of myoblast and soleus muscle cultures with physiological levels of 25-hydroxy-vitamin D3 resulted in a significant stimulation of phosphate accumulation by cultures. 1,25-dihydroxy-vitamin D3 had no effects on the differentiated tissue whereas it markedly increased phosphate accumulation by embryonic muscle cells. In addition, it could be shown that 25-hydroxy-vitamin D3 affects the Na+-linked component of cell phosphate uptake through a mechanism dependent on de novo protein synthesis.

An acetylcholinesterase-mediated density shift technique demonstrates that coated vesicles from chick myotubes may contain both newly synthesized acetylcholinesterase and acetylcholine receptors

Coated vesicles isolated from 17 d chick embryo skeletal muscle contain acetylcholine receptors (AChRs) as shown by the presence of specific, latent binding sites for 125I-alpha bungarotoxin (125I-alpha-BTX). Since these coated vesicles also contain AChE (Benson et al., 1985), we hypothesized that a coated vesicle could carry both molecules: one an integral membrane protein, the other a secreted protein. An AChE-mediated density shift technique was used to obtain data that indicate that most isolated coated vesicles contain AChE and that some contain AChRs as well. Similar results were obtained with coated vesicles isolated from cultured chick embryo myotubes treated briefly with diisopropylfluorophosphate (DFP) to inactivate all preexisting AChE and allowed to synthesize AChE for 2 1/2 hr. These data are compatible with the hypothesis that both an integral plasma membrane protein, AChR, and a secretory protein, AChE, traverse the identical pathway after synthesis, as proposed by Rotundo and Fambrough (1980a). We suggest that coated vesicles are important intermediates in the exocytotic pathway, and that the large percentage of coated vesicles utilized for exocytotic transport can explain the rapid net increase in surface area achieved during myotube development. We also discuss the potential utility of the AChE-mediated density shift in studying the exocytotic and endocytotic pathways in other cell types, and possible pitfalls associated with its use.

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.

Biochemical and cytochemical evidence indicates that coated vesicles in chick embryo myotubes contain newly synthesized acetylcholinesterase.

We have isolated highly purified coated vesicles from 17-d-old chick embryo skeletal muscle. These isolated coated vesicles contain acetylcholinesterase (AChE) in a latent, membrane-protected form as demonstrated enzymatically and morphologically using the Karnovsky and Roots histochemical procedure (J. Histochem. Cytochem., 1964, 12:219-221). By the use of appropriate inhibitors the cholinesterase activity can be shown to be specific for acetylcholine. It also can be concluded that most of the AChE represents soluble enzyme since it is rendered soluble by repeated freeze-thaw cycles. To determine the origin of the coated vesicle-associated AChE, we have isolated coated vesicles from cultured chick embryo myotubes which have been treated with diisopropylfluorophosphate, an essentially irreversible inhibitor of both intra- and extracellular AChE, and have been allowed to recover for 3 h. This time is not enough to allow any newly synthesized AChE to be secreted. These coated vesicles also contain predominantly soluble AChE. These data are compatible with the hypothesis that coated vesicles are important intermediates in the intracellular transport of newly synthesized AChE.

Coated vesicles contain a phosphatidylinositol kinase.

When coated vesicles (CVs) are incubated with [gamma-32P]ATP, radioactivity is rapidly incorporated into a compound identified by thin layer chromatography as phosphatidylinositol 4-phosphate. This activity has been identified in CVs isolated from bovine brain as well as from rat liver and chick embryo skeletal muscle. Phosphatidylinositol (PI) kinase is not separated from CVs during agarose electrophoresis, which produces CVs of greater than 95% purity, indicating that the activity present does not derive from contamination. The specific activity of these highly purified CVs was demonstrated to be approximately twice that of synaptic plasma membranes, further ruling out contamination from this source. The PI kinase remains associated with the vesicle upon removal of clathrin and its associated proteins and is solubilized by nonionic detergents, suggesting it is an integral membrane protein. We have been unable to demonstrate the formation of significant amounts of phosphatidylinositol 4,5-bisphosphate in any of our CV preparations. In the presence of exogenous PI, activity is stimulated, with maximal phosphorylation occurring at 0.1 mM. The enzyme appears to be maximally stimulated by 200 mM MgCl2 and 1 mM ATP and is most active at pH 7.25. Calculations indicate that, under optimal conditions, approximately 25 molecules of PIP are produced per CV within 60 s, suggesting that these structures may play an important role in cellular PI metabolism.

Presence of a 1,25-dihydroxy-vitamin D 3 receptor in chick skeletal muscle myoblasts

The presence of a specific receptor for 1,25-dihydroxy-vitamin D 3 was investigated in myoblasts released from chick embryo skeletal muscle by trypsin and collagenase treatment. Density gradient analysis of the cytosol obtained from these muscle cell preparations showed that 1,25-dihydroxy-vitamin D 3 binds specifically to a 3.7 S macromolecule. Scatchard analysis yielded an equilibrium dissociation constant of 2.46 × 10 −10 M and a Nmax of 74 fmol/mg of cytosol protein. The data is in agreement with previous evidence which indicates that the action of the vitamin D metabolite on muscle Ca uptake is mediated by de novo protein and RNA synthesis, and supports the concept that muscle is a target organ for 1,25-dihydroxy-vitamin D 3.

Streptomycin retards the phenotypic maturation of chick myogenic cells.

As part of an effort to optimize conditions required for the complete maturation of muscle cells in vitro, we have investigated the effects of the antibiotics penicillin, streptomycin, and Fungizone (amphotericin B) on the development of cultured chick embryo skeletal muscle. It is shown that even low dosages of streptomycin, but not penicillin or Fungizone, retard protein synthesis and accumulation in these cultures. Myosin accumulation was also reduced and the appearance of striations in fused cells was delayed in myotubes formed in medium containing streptomycin. Additional data suggest that this overall retardation of myogenesis is due to the influence of streptomycin on maturing myotubes rather than early proliferation and cell fusion. These results are discussed with regard to recent efforts to promote the full maturation of muscle cells grown in culture.

Changes in cytoplasmic terminal transferase activities during chick embryo skeletal muscle development

Changes in cytoplasmic terminal transferase activities during chick embryo skeletal muscle development

In situ reconstitution of myosin filaments within the myosin-extracted myofibril in cultured skeletal muscle cells.

We studied the in situ reconstitution of myosin filaments within the myosin-extracted myofibrils in cultured chick embryo skeletal muscle cells using the electron microscope and polarization microscope. Myosin was first extracted from the myofibrils in glycerinated muscle cells with a high-salt solution containing 0.6 M KCl. When rabbit skeletal muscle myosin was added to the myosin-extracted cells in the high-salt solution, thin filaments in the ghost myofibrils were bound with myosin to form arrowhead complexes. Subsequent dilution of KCl in the myosin solution to 0.1 M resulted in the formation of thick myosin filaments within the myofibrils, increasing the birefringence of the myofibrils. When Mg-ATP was added such myosin-reassembled myofibrils were induced either to form supercontraction bands or to restore the sarcomeric arrangement of thick and thin filaments. Under the polarization microscope, vibrational movement of the myofibrils was seen transiently upon addition of Mg-ATP, often resulting in a regular arrangement of myofibrils in register. These myofibrils, with reconstituted myosin filaments, structurally and functionally resembled the native myofibrils. The findings are discussed with special reference to the myofibril formation in developing muscle cells.

Neural cell adhesion molecule is on embryonic muscle cells and mediates adhesion to nerve cells in vitro.

The interaction of nerves with muscles, particularly at neuromuscular junctions, is one of the most extensively studied areas of neurophysiology. From the point of view of developmental biology, however, little is known about the cellular and molecular events that lead to formation of these junctions1,2. In previous investigations of cell-cell adhesion in the chick embryo, we identified a molecule on the surface of essentially all nerve cells, called neural cell adhesion molecule (N-CAM)3,4, that appears to be a ligand in the formation of cell-cell bonds5. N-CAM is involved in a variety of developmental processes including neurite fasciculation and the formation of plexiform and cell layers in retinal tissue6–8. We report here that N-CAM antigenic determinants are also present on chick embryo skeletal muscle cells. This observation raised the possibility that N-CAM is involved in adhesion between nerve and muscle cells. In model systems set up to explore this possibility, we observed that myoblasts will adhere in vitro to nerve cells from retina, and that this adhesion can be inhibited by F(ab′) fragments of antibodies against N-CAM. In addition, membrane vesicles obtained from chick embryo brain or artificial vesicles5 reconstituted from lipid and affinity-purified N-CAM9 bound to the surface of myoblasts and myotubes; this binding was also blocked by anti-N-CAM F(ab′). These results suggest that the N-CAM adhesion system can facilitate nerve-muscle as well as nerve-nerve interactions in vitro.

Endogenous lectin from chick embryo skeletal muscle is not involved in myotube formation in vitro.

Antisera against a beta-D-galactoside-specific lectin purified to homogeneity from embryonic chick thigh muscle and from adult chicken livers were raised in rabbits. The sera contained antibodies which strongly inhibited the hemagglutinin activity of the lectin. Immune and preimmune [125I]IgG and [125I]Fab preparations were tested for ability to bind to monolayers of cultured chick myoblasts and showed no significant differences in binding. Thus, we were unable to detect the lectin on the cell surface. When added to myoblast cultures, the purified lectin had no influence on the process of myoblast fusion. Furthermore, neither anti-lectin IgG nor Fab fragments interfered with myotube formation when present at high concentration in fusing cultures. On the basis of these results, we conclude that intercellular interactions between the lectin and complementary cell surface receptors do not play a role in myoblast adhesion and fusion in vitro.

The contribution of cell death to medium-released fractions of cell cultures.

Cell death was estimated by prelabeling primary chick embryo skeletal muscle cell cultures with [3H]thymidine and by subsequently measuring the release of label into complete culture medium or serum- and embryo-extract-free medium for a 6 h period. Cultures of the established muscle cell line L6 and the fibroblastic cell line 3T3 were used for comparative purposes. Comparison of the nigrosin exclusion test with the thymidine release test shows that the former underestimates cell death because it measures only the instantaneously occurring cell death. The [3H]thymidine release test estimates the cumulative amount of cell death. From cumulative cell death estimates it is calculated that 12.0 and 17.8% of the 3H-fucosylated medium-released fractions from primary cell cultures are the result of cell death contamination when release occurs in complete or macromolecule-free media, respectively. High speed centrifugation is shown to eliminate most contamination from cell death. Evidence is presented that the absence of macromolecules in the culture medium has little effect on the release process. Contamination of the released fraction resulting from cell death is much less in the established cell lines than in the primary cells. It is concluded that the release process can be studied in primary muscle cell cultures and especially in established cell lines if adequate precautions are taken and if corrections for cell death contamination are taken into account.

In Vitro effects of thyroxine and insulin on myoblasts from chick embryo skeletal muscle

The role of insulin and l-thyroxine (L-T 4) in stimulating myoblast proliferation and differentiation was investigated in vitro. A superphysiological concentration of insulin or a physiological concentration of L-T 4 was added to cultures of myoblasts from 11-day-old chick embryo thigh muscle, grown in serum-free DM-153 medium. While the addition of insulin resulted in an increase in the total number of cells, in the extent of fusion, and in the creatine phosphokinase (CPK) activity, myotubes changed into globular structures which tended to degenerate rapidly. On the other hand, while the addition of L-T 4 had less effect on myogenesis, myotubes retained their differentiated state longer. Furthermore, the two hormones exhibited synergistic effects. An increase in the initial cell density resulted in an increase in the amount of protein and CPK activity, irrespective of the presence or absence of the hormones. This suggests that the effect of insulin and L-T 4 on myogenesis is not a differentiation-specific effect, but rather an indirect result of cell proliferation.

Developmental changes in glycosaminoglycans during skeletal muscle cell differentiation in culture

Tissue specificity and developmental changes in glycosaminoglycans were studied in chick embryo skeletal muscle cells differentiating in vitro. Myogenic cells appear to be able to synthesize three main kinds of glycosaminoglycans, namely hyaluronic acid, heparan sulfate and chondroitin sulfate. The patterns of glycosaminoglycans synthesized by terminally differentiated multinucleated myotubes are clearly distinguishable from those synthesized by duplicating mononucleated myogenic cells and by sparse or confluent fibroblasts. These multinucleated myotubes, in particular, exhibit a specific increase of heparan sulfate and a specific decrease of hyaluronic acid in their cell layer-associated fraction of the total synthesized glycosaminoglycans.