Chick Embryo Skeletal Muscle Research Articles

Calcium and cyclic nucleotide dependent regulatory mechanisms during development of chick embryo skeletal muscle

Calcium and cyclic nucleotide dependent regulatory mechanisms during development of chick embryo skeletal muscle

Diazepam induces relaxation of chick embryo muscle fibers in vitro and inhibits myosin synthesis

Diazepam (Valium/Roche) causes an immediate cessation of spontaneous contraction in chick embryo skeletal muscle fibers growing in vitro. Between 24–48 h later in the presence of 100 μM diazepam the relaxed muscle fibers no longer accumulate myosin as measured by the total amount of myosin heavy-chain peptide extracted from the cell cultures and identified by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The myosin heavy chain assay procedure was standardized by quantitative precipitation of myosin with antibody to column purified chicken skeletal muscle myosin. Failure to accumulate myosin is related to a progressive inhibition of myosin synthesis. Diazepam-treated cultures showed an 80% inhibition of myosin heavy-chain synthesis over a period of 4 days. At the same time the rate of myosin heavy-chain degradation increases in diazepam-treated cultures relative to matched control cultures. Total protein synthesis was only marginally affected suggesting that diazepam may differentially inhibit myofibrillar protein synthesis. All of the observed effects of diazepam were reversible if drug exposure was limited to 48 h. The apparent specificity and reversibility of diazepam suggests that the drug will be useful in probing the mechanisms of terminal skeletal muscle cell differentiation and the hypotrophic relationship between chronic relaxation and inhibition of accumulation of myosin and perhaps other myofibrillar proteins.

DNA-dependent RNA polymerases in skeletal muscle cells differentiating in vitro

The main classes of RNA polymerase in cultured chick embryo skeletal muscle cells were identified and their levels determined during differentiation in vitro. Cell cultures contained a high proportion of muscle cells (>80%) and exhibited a high degree of fusion (>80%). The levels of RNA polymerases I and II increased by 1.8 and 1.5 times, respectively, from prefusion myoblasts (24 hr) to postfusion myotubes (120 hr). RNA polymerase III, a single peak on DEAE-Sephadex chromatography, was less than 7% of the total activity. The results suggest that the rates of synthesis of the main RNA types, which reportedly decline during myogenesis, are not determined by the relative levels of the main RNA polymerases and are more likely a reflection of chromatin template availability.

Effects of innervation on acetylcholine sensitivity of developing muscle in vitro.

1. Chick embryo skeletal muscle fibres were grown in culture. The acetylcholine (AACh) sensitivity of non-innervated fibres was compared with that of fibres innervated in vitro by chick embryo ciliary ganglion neurones. 2. The general pattern of ACh sensitivity was unchanged by innervation: ACh hot spots were superimposed on a background of uniform ACh sensitivity. 3. Quantitative comparisons revealed two differences between non-innervated and innervated fibres. First, hot spots were encountered about one third more often on innervated fibres. Secondly, about one-third of the hot spots on innervated fibres had significantly higher ACh sensitivities than the remainder, which were similar to those on control fibres. 4. Apossible explanation of these results is that nerves which form synapses induce the appearanceof end-plates which have higher ACh sensitivities than the pre-existing ACh hot spots.

Neurotrophic control of cyclic nucleotide levels during muscle differentiation in cell culture

The effects of chick brain-spinal cord extract on morphological development and cyclic nucleotide levels of cultured chick embryo skeletal muscle cells were determined. It had previously been shown that the extract stimulated morphological differentiation, protein synthesis, and choliniesterase activity of muscle cells. Myoblasts fused earlier and an increase in number as well as diameter of myotubes were seen in the extract treated cultures. Cyclic nucleotides levels were higher (almost twice the controls for both adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate) and preceded their occurrence in the control cultures. It was suggested that factor(s) in the extract interact with membrane receptor(s) to alter nucleotide levels which, in turn, allow the effects to be expressed.

CAMP and cGMP changes associated with the differentiation of cultured chick embryo muscle cells

Changes in the intracellular concentration of cAMP and cGMP were examined in primary cultures of chick embryo skeletal muscle cells. The reciprocal changes, increase in cAMP and decrease in cGMP level, were observed during the early stage of myogenesis. Consequently, the ratio of cAMP to cGMP increased significantly during that period reaching the maximum level on day 3. The most marked change in the nucleotide ratio, an approx. 2-fold increase, was observed between the 2nd and 3rd day of culture which coincided with the period of cell fusion. After the 3rd day, the value of nucleotide ratio remained constant throughout the period examined. These findings pose the possibility that the intracellular levels of both cyclic nucleotides are involved in the regulation in myoblast differentiation.

The formation of synapses between chick embryo skeletal muscle and ciliary ganglia grown in vitro.

1. Chick embryo ciliary ganglia (explanted) and skeletal muscle (dissociated) were grown together in vitro for up to 3 weeks. Nerve processes sprouted from the ganglia and contacted neighbouring myotubes and striated muscle fibres. 2. Spontaneous action potentials and subthreshold e.p.p.s. were recorded from muscle fibres with intracellular micropipettes. Similar potentials could be evoked by electrical stimulation of the ganglion. The pharmacological effects of curare and tetrodotoxin were identical to those observed at adult vertebrate neuromuscular junctions. 3. The amplitude, but not the frequency, of the spontaneous potentials was affected by changing the muscle fibre membrane potential. The reversal potential of evoked synaptic potentials occurred at a membrane potential of about 0 mV.

Functional and non-functional contacts between ciliary neurones and muscle grown in vitro.

1. In cultures of chick embryo skeletal muscle and ciliary ganglia, muscle fibres near a ganglion were contacted by many individual nerve processes. Experiments were performed to determine if these muscle fibres were multiply innervated, and if any of the nerve-muscle contacts were non-synaptic. 2. Synaptic potentials evoked by electrical stimulation of a ganglion were graded with stimulus strength. When two ganglia were plated near each other, synaptic potentials could be evoked in some muscle fibres by stimulation of either ganglion. These observations suggest that muscle fibres were multiply innervated. 3. Spontaneous synaptic potentials recorded from single muscle fibres with two widely spaced micropipettes varied in a manner which suggested that the synapses were distributed at different points on the surface of the muscle fibres. 4. Stimulation of some nerve processes failed to evoke synaptic potentials in muscle fibres contacted by those processes. Such nerve-muscle contacts were not strongly adhesive, and the nerves were peeled easily from the surface of the muscle with a micropipette. On the other hand, nerve processes which formed synaptic contacts with muscle fibres seemed to be tightly adherent to the muscle. 5. Electron microscopic observations of nerve-muscle contacts revealed that the vast majority of such contacts lacked morphological specializations characteristic of mature neuromuscular synapses.

Studies on the pharmacology of skeletal muscle in culture: Specificity of receptors

The specificity of the receptors of chick embryo skeletal muscle fibres in culture was examined. Noradrenaline, histamine, dopamine and γ-aminobutyric acid had no effect on resting membrane potential or on depolarization induced by carbachol. Adrenaline and 5-hydroxytryptamine did reduce the carbachol response but were without direct effect on membrane potential. Muscarinic agonists had no action but the muscarinic antagonists, atropine and hyoscine, reduced the response to carbachol. High concentration of atropine and hyoscine produced a well-maintained depolarization of the muscle fibres and this depolarization was not inhibited by tubocurarine, α-bungarotoxin or prior cholinoreceptor desensitization. The cultured muscle fibres were depolarized by a wide range of nicotinic agonists, and gallamine acted as a competitive antagonist. It is concluded that the receptors on cultured skeletal muscle are similar to normal nicotinic receptors and that atropine and hyoscine depolarize by an action at a site other than the cholinoreceptor, possibly at the ‘ion conductance modulator’.

Depolarization, desensitization and the effects of tubocurarine and neostigmine in cultured skeletal muscle

The actions of acetylcholine, carbachol, d-tubocurarine and neostigmine were studied in cultures of chick embryo skeletal muscle. Both agonists produced a rapid, concentration-dependent depolarization. The depolarization to a given concentration was greater in older cells with higher resting membrane potentials, but the response percent of resting potential was constant throughout development. Despite continued presence of the agonists, depolarization was not maintained, the membrane potential returning to control level in about 15 min. The rate of recovery increased with age of cells and concentration of agonist. Specific desensitization of acetylcholine receptors was demonstrated. Cross-desensitization between acetylcholine and carbachol was observed, but receptor desensitization was unaffected by potassium-induced depolarization. d-Tubocurarine competitively antagonised the cholinomimetic response. Neostigmine did not potentiate responses to low concentrations of acetylcholine, but did, itself, cause some desensitization. The results indicate that the acetylcholine receptors of cultured skeletal muscle are similar in many respects to adult nicotinic receptors.

Acetylcholinesterase activity in developing skeletal muscle cells in vitro

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.

Development of Acetylcholine Receptor Clusters on Cultured Muscle Cells

Clusters of acetylcholine receptors were formed in the absence of neurons during the development of cultured chick-embryo skeletal muscle cells. The average concentration of receptors in clusters was estimated to be 9000 per mum(2). At other regions of the cell, receptor concentration was 900 per mum(2). The possibility that receptor clusters may participate in synapse formation is suggested.

The morphological development of chick embryo skeletal muscle grown in tissue culture as studied by electron microscopy

The understanding of events in normal myogenesis is essential before the examination of disordered muscle development can begin. In this study observations on the development of muscle explants from chick embryo muscle grown in tissue culture have been made. The growth of muscle in vitro appears to coincide with or at least is very similar to that of myogenesis in the embryo although the rate of development is accelerated in the culture environment. Actin filaments appear to be the first component of the myofibril that is synthesized and they appear randomly oriented in the sarcoplasm until the myosin filaments are formed. The larger filaments are almost always seen in a myofibrillary pattern with ribosomes often occurring along their length. At 4 days, 2–4 electron-dense masses appeared in the middle of the I-band and later these coalesced to form the Z-band. The M-band could also be identified for the first time in cells from the 4-day cultures and as this band evolved, the A-band achieved a mature appearance. The problems imposed by attempting to interpret sequential events by studying a series of electron micrographs made it difficult to follow the formation of the sarco-tubular systems, mitochondria and other cellular organelles. In these instances supplementary information accumulated from studies reported by many investigators was utilized and integrated with our own findings.

Inclusion bodies in human neuromuscular disorder.

IN 1962, Engel 1 first described a body in pathologic human skeletal muscle fibers and in chick embryo skeletal muscle fibers in vitro. Since then, several reports 2-5 dealing with the ultrastructure of human skeletal muscle have referred to the cytoplasmic body, but without detailed description. Recently, we examined a patient suffering from late-onset atrophy, with weakness of the hands, peroneal muscles, and shoulder girdle. Skeletal muscle fibers contained numerous inclusions resembling the cytoplasmic body. This article describes the ultrastructure of various types of inclusions and the possible origin of these inclusions. Report of a Case In January 1966, a 53-year-old man noted a heavy feeling in his legs after slight exertion, which gradually increased. After six months he had difficulty walking and holding his arms overhead. At age 54, in May 1967, he was admitted to Nagoya National Hospital. His health otherwise had been good except for

Growth and respiration of monolayer cell cultures of chick embryo heart and skeletal muscle: action of malathion and malaoxon.

SummaryThe respiration of chick embryo heart and pectoral muscle cell cultures was determined using a polarographic respiration chamber designed specifically to measure the oxygen consumption of monolayers of cells. Heart cultures had a mean respiration rate of 215 ± 84 mμmoles O2/hr/106 cells; pectoral muscle cultures had a mean respiration rate of 163 ± 46 mμmoles Oa/hr/106 cells. Malathion 3 × 10−4M reduced the respiration of pectoral muscle cultures to 26% of normal and the respiration of heart cultures to 50% of normal. Malaoxon 3 × 10−4M had no effect upon the respiration of either cell type. Growth studies indicated that malathion was more toxic than malaoxon to the growth of both cell types.

The fine structure of the ventral intersegmental abdominal muscles of the insect Rhodnius prolixus during the molting cycle. II. Muscle changes in preparation for molting.

The development of the ventral intersegmental abdominal muscles of Rhodnius prolixus is triggered by feeding. The early muscle (1 day after feeding) contains essentially nonstriated fibrils. However, in cross-sections, areas indicating early I bands, Z lines, and A bands can be recognized. Interdigitating thick and thin myofilaments do not assemble into a precise lattice until sometime between 4 and 5 days after feeding. As development continues, the number of fibrils increases, the region corresponding to the Z line increases in density, and the fibrils contain more recognizable striations. The newly formed fibrils broaden as myofilaments are added peripherally. At all stages throughout development, the ratio of thin to thick myofilaments is always 6:1. The formation of fibrils in the abdominal muscles of Rhodnius is different from that in chick embryo skeletal muscle. The major differences are that at all stages in Rhodnius there are (1) a constant ratio of thin to thick myofilaments, and (2) detectable Z-line material. Other findings in Rhodnius suggest (1) that fusion of mononucleated cells with the multinucleated muscle cell occurs, (2) that microtubules develop in the tendon cell concomitantly with development of myofibrils in the associated muscle cell, and (3) that filaments 55A in diameter aggregate into microtubules.

CYTOLOGICAL LOCALIZATION OF CHOLINESTERASE IN CULTURED SKELETAL MUSCLE CELLS

The location of acetyl and butyryl thiocholinesterase activity was studied in cultured chick embryo skeletal muscle cells which were growing without being innervated. Muscle cells in all three stages of development, i.e., the single myoblast, the plurinucleated myoblastic strap, and the multinucleated myocytic strap, showed sites of thiocholinesterase activity as small brown granules located diffusely in the cytoplasm and accentuated in the perinuclear areas. In the well differentiated myocytic straps, thiocholinesterase was localized along the Z lines. No enzyme activity was found at the sarcolemma or in the nuclei. No thiocholinesterase-containing motor end-plate structures were found in any stage of development.

Cytological localization of cholinesterase in developing chick embryo skeletal muscle.

Cytological localization of cholinesterase in developing chick embryo skeletal muscle.

CYTOLOGICAL LOCALIZATION OF GLYCOGEN IN CULTURED SKELETAL MUSCLE

The periodic acid Schiff stain was used to demonstrate the cellular location of glycogen formed and stored by chick embryo skeletal muscle cells newly grown in vitro without innervation. No glycogen was found in single myoblasts. In the myoblastic and less mature myocytic straps, there were irregular-sized granules of glycogen in the cytoplasm, especially in the perinuclear areas. Much glycogen was formed in the more mature cultured myocytic straps, and in a pattern like that of innervated adult skeletal muscle cells in vivo. There was glycogen in the perinuclear and subsarcolemmal cytoplasm and in alternating wide and narrow bands, tentatively interpreted as I and M bands respectively.