Embryonic Chick Skeletal Muscle Cells Research Articles

Lysosomes accumulate at the perinuclear region of muscle cells during chick myogenesis.

Lysosomes are involved in a myriad of cellular functions, such as degradation of macromolecules, endocytosis and exocytosis, modulation of several signaling pathways, and regulation of cell metabolism. To fulfill these diverse functions, lysosomes can undergo several dynamic changes in their content, size, pH, and location within cells. Here, we studied some of these parameters during embryonic chick skeletal muscle cells. We used an anti-lysosome-associated membrane protein 2 (LAMP2) antibody to specifically determine the intracellular localization of lysosomes in these cells. Our data shows that lysosomes are highly enriched in the perinuclear region of chick embryonic muscle cells. We also showed that the wingless signaling pathway (Wnt)/β-catenin signaling pathway can modulate the location of LAMP2 in chick myogenic cells. Our results highlight the role of lysosomes during muscle differentiation and particularly the presence of a subcellular population of lysosomes that are concentrated in the perinuclear region of muscle cells.

Regulation by an extract of embryonic chick brain of the densities of voltage-dependent Na + and Ca 2+ channels in embryonic chick skeletal muscle cells during their development in culture

We studied the chronic effects of a brain extract (BE) prepared from chick embryonic brains on voltage-dependent Na + channels (VDNCs) and Ca 2+ channels (VDCCs) during the development of chick skeletal muscle cells in culture. The maximum rates of rise of Na + and Ca 2+ action potentials were measured electrophysiologically in an attempt to determine the effects of BE on the densities of these channels. The basic culture medium was supplemented with chick transferrin instead of whole-embryo extract and skeletal muscle cells were grown in the absence or in the presence of crude BE or fractionated BE. Long-term inclusion of BE to the culture medium increased the densities of both VDNCs and L-type VDCCs. By contrast, BE apparently decreased the density of T-type VDCCs. Our results indicate that BE contains some protein(s) that has a negative effect on the density of T-type VDCCs of skeletal muscle cells at a less differentiated stage and that this effect of BE is closely associated with subsequent regulation of the densities of VDNCs and L-type VDCCs. Possible roles of the influx of Ca 2+ ions through T-type and L-type VDCCs in the control of the densities of VDNCs and L-type VDCCs are discussed.

Ca2+/Calmodulin-Dependent Regulation of the Density of Na+ Channels in Embryonic Chick Skeletal Muscle Cells During Their Development in Culture

Regulation of the density of voltage-dependent Na+ channels (VDNC) was studied in chick myotubes during their development in culture. The density of VDNC was assessed quantitatively in terms of the maximum rate of rise (M.R.R.) of Na+ spikes. Chronic treatment of myotubes, whose density of VDNC had reached a plateau level after 6 days in culture, with the calmodulin (CaM) inhibitor W-7 caused a further increase in the density. A derivative of W-7, known as W-5, which has a lower affinity for CaM than does W-7, was without effect. A selective inhibitor of Ca2+/CaM-dependent protein kinase II (CaM-KII), namely, KN-62, also caused an increase in the density of VDNC when its effect was examined in mature myotubes. A structural analogue of KN-62, namely, KN-04, which is a much less effective inhibitor of CaM-KII, was without effect. These results suggest that density of VDNC in developing myotubes is regulated by Ca2+/CaM and CaM-KII. However, the role of CaM-KII in this regulation is not straightforward since it appears to decrease the density of VDNC in mature myotubes, but to increase their density in immature myotubes.

Calcium channels in embryonic chick skeletal muscle cells after cultivation with calcium channel blocker

The effects of chronic treatment with calcium channel blockers were studied on the expression of voltage-dependent calcium channels (VDCCs) in chick skeletal muscle cells developing in culture. Myotubes were treated after 2 days in culture with either 20 μM D600 or 10 μM nifedipine, and measurements were made of the maximum rate of rise (M.R.R.) of the two components of action potential, operated by T- and L-type VDCCs, respectively. Treatment with either blocker reduced the M.R.R. of the action potential component operated by the L-type VDCC throughout the culture period examined. The M.R.R. of the T-type VDCC component, on the other hand, was unaffected by either treatment. The reduction in the M.R.R. of the L-type component in blocker-treated cells is thought to be due to the down-regulation of the expression of L-type VDCC. Thus, it appears that the expression of L-type VDCC in the chick skeletal muscle cells can be regulated by a function of L-type VDCC, which mediate the entry of Ca 2+ into the cells. The physiological significance of the L-type VDCC, which expressed prominently early in the development of skeletal muscle cells, for the differentiation of excitability is discussed.

Effect of an arginine-specific ADP-ribosyltransferase inhibitor on differentiation of embryonic chick skeletal muscle cells in culture

Primary cultures of embryonic chick skeletal myogenic cells were used as an experimental model to examine the possible role of mono(ADP-ribosyl)ation reactions in myogenic differentiation. Initial studies demonstrated arginine-specific mono(ADP-ribosyl)transferase activity in the myogenic cell cultures. We then examined the effect of a novel inhibitor of cellular arginine-specific mono(ADP-ribosyl)transferases, meta-iodobenzylguanidine (MIBG), on differentiation of cultured embryonic chick skeletal myoblasts. MIBG reversibly inhibited both proliferation and differentiation of embryonic chick myoblasts grown in culture. Micromolar (15–60 μ M) concentrations of MIBG blocked myoblast fusion, the differentiation-specific increase in creatine phosphokinase activity, and both DNA and protein accumulation in myogenic cell cultures. Meta-iodobenzylamine, an analog of MIBG missing the guanidine group, had no effect. Low concentrations of methylglyoxal bis-guanylhydrazone, a substrate for cholera toxin with a higher K m than MIBG, also had no effect, but higher concentrations reversibly inhibited fusion. These findings suggest a possible role for mono(ADP-ribosyl)ation reactions in myogenesis. In addition, the total arginine-specific mono(ADP-ribosyl)transferase activity increased with differentiation in the myogenic cell cultures, and this increase was also blocked by MIBG treatment. Because high levels of activity were found in the membrane fraction derived from later, myotube cultures, the membrane fraction from 96-h cultures was incubated with [ 32P]NAD + and subjected to electrophoresis and autoradiography. Three proteins, migrating at 21, 20, and 17 kDa, that were ADP-ribosylated in the absence, but not the presence, of MIBG were identified. These proteins may be endogenous substrates for this enzyme.

Spatial relationship of nebulin relative to other myofibrillar proteins during myogenesis in embryonic chick skeletal muscle cells in vitro.

The developmental expression of nebulin was studied in embryonic chick skeletal muscle cells in vitro by means of immunofluorescence microscopy. Initially nebulin appeared homogeneously or in a punctate form in the cytoplasm, and then it was assembled into I-Z-I-like complexes containing actin and alpha-actinin but not myosin and connectin (titin). Striated patterns of nebulin ('singlets') in myofibrils appeared simultaneously with those of alpha-actinin (Z-bands), myosin (A-bands) and connectin ('doublets'), but earlier than those of actin. After actin striations were formed as myofibrils matured, each nebulin band started to exhibit 'droplets'. The delayed development of nebulin compared to the I-Z-I brush formation and the myofibril maturation seems to indicate that this giant myofibrillar protein is unnecessary for both the initial (formation of I-Z-I-like structures) and the subsequent (regular alignment of myofibrils) phases of myofibrillogenesis.

Pharmacological properties of two types of calcium channel in embryonic chick skeletal muscle cells in culture

We have studied the effects of organic and inorganic Ca channel blockers on the two-component action potentials (low- and high-threshold components) in cultured chick skeletal muscle cells, which are generated by the T- and L-type voltage-dependent Ca channels (VDCCs), respectively. Nifedipine and D600 effectively blocked the high-threshold component of the action potential, whereas ω-conotoxin and phenytoin had no effect on this component. By contrast, the low-threshold component was insensitive to all of these organic Ca channel blockers. Blocking effects of polyvalent cations were observed with the following rank order of relative potency (mean apparent dissociation constant in μM): La 3+ (14.7) > Ni 2+ (20.7) > Cd 2+ (51.2) > Co 2+ (912) for the low-threshold component, and Cd 2+ (0.7) > La 3+ (29.2) > Co 2+ (431) > Ni 2+ (1241) for the high-threshold component. Taken together, these findings suggest that the pharmacological properties of the T- and L-type VDCCs in cultured chick skeletal muscle cells may differ from those in other preparations.

Three-dimensional electron microscopy of the sarcoplasmic reticulum and T-system in embryonic chick skeletal muscle cells in vitro

The formation of the sarcoplasmic reticulum (SR) and the transverse tubular system (T-system) in embryonic chick skeletal muscle cells in vitro was studied by either the critical point drying-physical rupturing or physical rupturing-freeze drying together with rotary shadowing. In these cells, two membranous systems were observed. One was composed of flattened sacs which were either isolated or were connected to each other with slender processes to form mostly longitudinally oriented strands. Initially, these sacs had small granules at their surface and were found mainly under the sarcolemma. Later, they became smooth at their surface, extending throughout the cytoplasm to form irregular and dense networks. At later phases, the networks tended to be disposed at right angle to nascent myofibrils, exhibiting a characteristic honeycomb appearance. From the similarities in thin section images, they were identified as developing SR.

Three-Dimensional Structure of Intracellular Membranous Organelles in Embryonic Chick Skeletal Muscle Cells <italic>in vitro</italic> Revealed with Scanning Electron Microscope

Using the freeze-polishing and osmium-maceration procedures, the ultrastructure of intracellular membranous organelles in embryonic chick skeletal muscle cells in vitro was investigated three-dimensionally with the scanning electron microscope (SEM). T-system tubules followed a tortuous course and possessed many warts. Some of these tubules could be traced to the sarcolemma. The sarcoplasmic reticulum (SR) had elaborate structures of a hexagonal pattern. By comparing the SEM images with those of ultrathin sections of myotubes impregnated with ferritin particles, the distinction between the T-system and SR was confirmed. Late in the culture, these two intracellular membranous systems were arranged periodically at right angles to the longitudinal axis of the myotubes. Round-shaped mitochondria were often arranged in chains connected with slender rods. Long rod-shaped mitochondria exhibited occasional branchings.

Two components of calcium channel current in embryonic chick skeletal muscle cells developing in culture

The properties of the Ca channel currents in chick skeletal muscle cells (myoballs) in culture were studied using a suction pipette technique which allows internal perfusion and voltage clamp. The Ca channel currents as carried by Ba ions were recorded, after suppression of currents through ordinary Na, K and Cl channels by absence of Na, K and Cl ions, by external TEA, by internal EGTA and by observing the Ba currents instead of the Ca currents. Two components of Ba current could be distinguished. One was present only if the myoballs were held at relatively negative holding potentials below −50 mV. This component first became detectable at clamp potentials of about −50 mV and reached a maximum between −10 and −20 mV. During long clamp steps, it became inactivated completely. The inactivation process of this component at a clamp potential of−30 mV was well fitted to a single exponential with a time constant of about −20 ms. Half-maximal steady-state inactivation was observed at −63 mV. The other component persisted even at relatively positive holding potentials above −40 mV, was observed during clamp pulses to −20 mV and above, and reached a maximum between +10 and +20 mV. This component inactivated very little; a substantial fraction of this component remained at the end of clamp pulses lasting 1 s. The inactivation process of this component at a clamp potential of −10 mV apparently followed a single exponential with a time constant of about 1 s. Half-maximal steady-state inactivation was attained at −33 mV. Both components of Ba current were blocked by Co ions, but organic Ca channel blocker D600 preferentially blocked the high-threshold, slowly inactivating component. The relationship between the current amplitude and the concentration of the external Ba ions was different between the two components. Furthermore, the two components of Ba current also differed in their developmental profile. These findings demonstrate the existence of two distinct types of Ca channels in the early stages of chick muscle cell development.

Verapamil, neuromuscular transmission and the nicotinic receptor

The effect of verapamil on neuromuscular transmission was studied in the frog by analysing ionophoretic endplate current (iEPC) trains and the growth and decay phases of miniature endplate currents (mepcs). In addition, single channel data on the interaction of verapamil with the nicotinic acetylcholine receptor were obtained from cultured embryonic chick skeletal muscle cells. Verapamil caused both open and closed channel blockade in the iEPC trains. Mepc amplitude was decreased at low micromolar concentrations, and at higher concentrations there was also accelerated mepc decay indicating open channel blockade. The latter effect oould not be explained by a sequential channel occlusion mechanism. Analysis of the mepc rising phase showed that low micromolar concentrations of the drug decreased the pool of receptors which could be activated. Single channel data confirmed the specific interaction of verapamil with the nicotinic receptor, showing closed channel blockade at low concentrations, and at higher levels the shortening of open channel lifetime. It is suggested that both forms of blockade may involve desensitization processes.

Ganglioside potentiation of NGF-independent conditioned medium enhancement of neuritic outgrowth from spinal cord and ciliary ganglia explants

Culture medium conditioned (CM) by embryonic chick skeletal muscle or RN22 Schwannoma cells enhanced dramatically the neuritic development of chick embryonic spinal cord slices explanted onto a collagen substratum. The addition of a mixture of bovine brain gangliosides (BBG) or the monosialoganglioside GM1 to this medium potentiated the nerve growth factor (NGF)-independent CM-mediated neuritogenesis. A 3–4 fold increase in spinal cord outgrowth was due to increased neurite number, length and branching. The ability of the gangliosides to potentiate the positive neuritogenic action of CM was not limited solely to spinal cord cultures since similar results were obtained in parallel studies employing organized cultures of embryonic chick ciliary ganglia. These studies demonstrate the ability of gangliosides to enhance the trophic action of factor(s) present in CM. They suggest further that gangliosides may play a modulatory role in the development of the nervous system.

Organization of filaments underneath the plasma membrane of developing chicken skeletal muscle cells in vitro revealed by the freeze-dry and rotary replica method.

Cytoskeletal organization and its association with plasma membranes in embryonic chick skeletal muscle cells in vitro was studied by the freeze-drying and rotary-shadowing method of physically ruptured cells. The cytoskeletal filaments underlying the plasma membranes were sparse in myogenic cells at the stage when cells exhibited great lipid fluidity in plasma membranes (fusion competent mononucleated myoblasts and recently fused young myotubes). Myotubes at more advanced stages of development possessed a highly interconnected dense filamentous network just underneath the cell membrane. This subsarcolemmal network was composed predominantly of 8-10 nm filaments; they were identified as actin filaments because of their decoration with myosin subfragment-1. Fine fibrils having a diameter of 3-5 nm were found on the protoplasmic surface of the plasmalemma at both the early and advanced stages of development. They were associated with the subsarcolemmal cytoskeletal filaments. Short 2-5 nm cross-linking filaments were occasionally seen between filaments in the subsarcolemmal network. We conclude that, although the subsarcolemmal cytoskeletal network contains many actin filaments, this domain appears to play some role in preserving the cell shape in the form of the membrane skeleton rather than membrane mobility.

Maintenance of motility of bull spermatozoa by a low molecular weight factor(s) released from cultured embryonic cells

This study was undertaken to investigate the factor(s) released from cultured embryonic cells that is responsible for the prolonged mortility of bull spermatozoa and to determine some of the properties of the factor(s). Spermatozoa were incubated at 37°C in a supernatant removed from a 4-day culture medium of embryonic chick skeletal muscle cells. Their motility was maintained for 25.2 h. In contrast, spermatozoa incubated in the fresh medium rapidly lost their motility. A filtrate of the 4-day culture medium, obtained by passing the fluid through an Amicon YC-05 ultrafiltration membrane, retained a favorable effect on prolonging motility of spermatozoa. Heating or freezethawing of the filtrate did not interfere with the ability of the spermatozoa to maintain their motility. Oxygen consumption of spermatozoa in the filtrate of the 4-day culture medium was similar to that of spermatozoa in the fresh medium. These results suggest that a low molecular weight factor(s) (Mol. Wt. < 500) supplied by the cultured cells effectively prolonged the survival of the spermatozoa.

Prolonged motility of fowl spermatozoa in vitro due to a low molecular weight factor(s) released from cultured embryonic cells

Fowl spermatozoa were incubated at 41°C in a supernatant removed from a 4-day culture medium of embryonic chick skeletal muscle cells. Their motility, as assessed at room temperature (20–25°C), was maintained for 48 h. Fertilizing ability was also retained for at least 24 h. In contrast, spermatozoa incubated in the fresh culture medium lost their motility and fertilizing ability rapidly. A filtrate of the 4-day culture medium, obtained by passing the fluid through an Amicon PM-10 ultrafiltration membrane, prolonged the motility of spermatozoa. These results suggested that a low molecular weight factor(s) (mol. wt. < 10 000) supplied by the cultured cells effectively prolonged the motility of spermatozoa.

Insulin binding on cultured chick muscle cells: decrease in binding associated with cell fusion.

Binding of 125I-bovine and chicken insulin to cultured embryonic chick skeletal muscle cells was studied. Bovine and chicken insulin bound cultured cells with high affinities of 2.4 X 10(9)M-1 and 4.8 X 10(9)M-1 and low affinities of 2.4 X 10(7)M-1 and 3.7 X 10(7)M-1, respectively. Maximum insulin binding was achieved after 90 min of incubation at 20 degrees C and the maximum value was maintained for an additional 3 hr. Insulin binding increased in a linear manner with increasing nuclei number over a 5-fold range. Maximum insulin binding per nuclei decreased as cell fusion increased between 24 and 72 hr in culture, primarily due to a decrease in the number of low affinity insulin receptors.

Differential expression of sodium channel activities during the development of chick skeletal muscle cells in culture.

The expression of Na+ channels during differentiation of cultured embryonic chick skeletal muscle cells was investigated using saxitoxin (STX) and batrachotoxin (BTX), which previously have been shown to interact with distinct, separate receptor sites of the voltage-sensitive Na+ channel of excitable cells. In the present study, parallel measurements of binding of [3H]-STX (STX) and of BTX-activated 22Na+ uptake (Na influx) were made in order to establish the temporal relationship of the appearance of these two Na+ channel activities during myogenesis. Na influx was clearly measurable in 2-d cells; from day 3 to day 7 the maximum Na influx approximately doubled when measured with saturating BTX concentrations potentiated by Leiurus scorpion toxin, while the apparent affinity of BTX, measured without scorpion toxin, also increased. Saturable STX binding did not appear consistently until day 3; from then until day 7 the STX binding capacity increased about threefold, whereas the equilibrium dissociation constant (KD) decreased about fourfold. Although Na influx in cells of all ages was totally inhibited by STX or tetrodotoxin (TTX) at 10 microM, lower concentrations (2-50 nM) blocked the influx in 7-d cells much more effectively than that in 3-d cells, where half the flux was resistant to STX at 20-50 nM. Similar but smaller differences characterized the block by TTX. In addition, when protein synthesis is inhibited by cycloheximide, both Na influx and STX binding activities disappear more rapidly in 3-d than in 7-d cells, which shows that these functions are less stable metabolically in the younger cells.

Effect of tunicamycin, an inhibitor of protein glycosylation, on the biological properties of acetylcholine receptor in cultured muscle cells.

We have studied the effect of tunicamycin (TM), an antibiotic which inhibits the glycosylation of nascent proteins, on the properties of the acetylcholine receptor (AChR) at the surface of embryonic chick skeletal muscle cells. The use of two separate assays, specific binding of 125I-alpha-bungarotoxin and carbamylcholine-activated 22Na+ uptake, has allowed us to monitor the effects of impaired glycosylation on the metabolic and functional properties of AChR. A significant decrease in the amounts of surface AChR elaborated in the presence of TM is detected by both measurements. This decrease has been found to reflect an enhanced proteolytic degradation of the underglycosylated AChR. The underglycosylated AChR, expressed on the cell surface in the presence of TM, retains the capability of mediating agonist-activated ionic permeability changes, but displays quantitatively altered interactions with receptor ligands. We conclude that the carbohydrate moiety on AChR may play a role in determining the folding of newly synthesized polypeptides to form a conformation compatible with the metabolic properties and ligand interactions characteristic of glycosylated AChR.

The appearance of voltage-sensitive Na+ channels during the in vitro differentiation of embryonic chick skeletal muscle cells.

The properties of Na+ channels at different stages of the in vitro development of embyronic chick skeletal muscle were analyzed using three different classes of toxins that act specifically on Na+ channels: alkaloids like veratridine and batrachotoxin, which stabilize an open form of the Na+ channel; polypeptide neurotoxins like sea anemone and scorpion neurotoxins that prolong the life time of the conducting form of the channel by slowing down the inactivation process; and tetrodotoxin, which inhibits the entry of Na+ through the channel. The rate of 22Na+ uptake, which is activated by a mixture of 10 p~ sea anemone toxin II and 100 pM veratridine and which is inhibited by 0.1 p~ tetrodotoxin, was used as an estimate of the amount of Na+ channels. Toxin-activated rates of “Na+ uptake were very low in unfused cultures; they increased in parallel with the extent of fusion to reach a maximum at 100 h of culture, and then stabilized. Dose-response curves for the action of the different neurotoxins on the initial rate of ‘*Na+ uptake were obtained at different ages of culture between 2 and 7 days. The apparent affinities of the different toxins for their receptors on the Na’ channel remained invariant, indicating that the same type of functional, tetrodotoxin-sensitive Na+ channel is present at all stages of myotube development. Only the number of Na+ channels is increasing during myogenesis. Direct biochemical titration of Na+ channels was carried out with a highly radiolabeled derivative of tetrodotoxin. Specific tetrodotoxin binding could not be de

Neurotrophic effect of nerve extract on development of tetrodotoxin‐sensitive spike potential in skeletal muscle cells in culture

The effect of the presence of nerve extracts on the development of tetrodotoxin (TTX)-sensitive sodium channels in cultures of dissociated embryonic chick skeletal muscle cells was examined by measuring the maximum rate of rise of TTX-sensitive spike potential. The addition of the nerve extract prepared from brain or spinal cord of chick embryos to the culture medium caused an increase in the channel density. Extracts of non-neural tissues, i.e., lung, kidney, and muscle, were ineffective. Liver extract, however, produced an effect similar to the nerve extracts. These results suggest that the TTX-sensitive sodium channels in the muscle cell membrane are regulated by a diffusible chemical substance independently of innervation, and that this substance resides in neural tissues, and perhaps also in liver.