Adult fast myosin pattern and Ca2+-induced slow myosin pattern in primary skeletal muscle culture (original) (raw)
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Adult fast myosin pattern and Ca 2+ -induced slow myosin pattern in primary skeletal muscle culture
Proceedings of the National Academy of Sciences of the United States of America, 1997
A primary muscle cell culture derived from newborn rabbit muscle and growing on microcarriers in suspension was established. When cultured for several weeks, the myotubes in this model develop the completely adult pattern of fast myosin light and heavy chains. When Ca 2؉ ionophore is added to the culture medium on day 11, raising intracellular [Ca 2؉ ] about 10-fold, the myotubes develop to exhibit properties of an adult slow muscle by day 30, expressing slow myosin light as well as heavy chains, elevated citrate synthase, and reduced lactate dehydrogenase. The remarkable plasticity of these myotubes becomes apparent, when 8 days after withdrawal of the ionophore a marked slow-to-fast transition, as judged from the expression of isomyosins and metabolic enzymes, occurs. EXPERIMENTAL PROCEDURES Culture and Harvesting of Muscle Cells. Newborn White New Zealand rabbits were killed by decapitation. Hindlimb muscles were cut in small pieces and incubated in BSS, pH 7.0 (4.56 mM KCl͞0.44 mM KH 2 PO 4 ͞0.42 mM Na 2 HPO 4 ͞25 mM NaHCO 3 ͞ 119.8 mM NaCl͞50 mg/liter penicillin͞100 mg/liter streptomycin) with 0.125% trypsin under stirring at 37ЊC for 1 h. The suspension was centrifuged at 800 ϫ g for 5 min, the pellet resuspended in DMEM with 10% neonatal calf serum (NCS), and the entire procedure repeated once. The final pellet was suspended in DMEM͞ 10% NCS and then filtered through a sieve with 0.4-mm pores. The filtrate was transferred into culture bottles where the fibroblasts were allowed to settle and attach themselves to the bottom for 30 min. The supernatant suspension was decanted and diluted to a final cell density of 8 ϫ 10 5 ͞ml in DMEM with 10% NCS. A total of 15 ml of this suspension were filled into one 260-ml culture flask and 0.04 g cross-linked gelatin beads with a diameter of 100-300 m (CultiSpher-GL; Percell Biolytica, Astorp, Sweden) were added per flask. The flasks were kept at 37ЊC in 8% CO 2 in air and 95% humidity while being shaken gently to ensure adequate O 2 supply to the cells and to prevent cells and beads from settling down. Twenty-four hours later the cell suspension was diluted to a cell concentration of 4 ϫ 10 5 cells͞ml. Myoblasts attached themselves to the gelatin beads and began to fuse after 3 days in culture. After 2 weeks fusion appeared to be complete and only myotubes were detectable. To collect the myotubes after 3-5 weeks of culture, cell-covered beads were allowed to sediment, washed twice in BSS (pH 7.0) with 0.02% EDTA, and resuspended in BSS (pH 7.9) containing 0.35% trypsin, 1.8 mM CaCl 2 , and 0.8 mM MgSO 4. After incubation for 30 min at 37ЊC under shaking, the isolated cells were spun down at 800 ϫ g for 5 min, washed twice in BSS (pH 7.0) with 0.02% EDTA, and then suspended in BSS (pH 7.0). This suspension was sonicated 6 ϫ 5 s with 60 W at 0ЊC. Scanning Electron Microscopy. The carrier suspension is pipetted onto a collagen-coated glass slide and incubated for 3 hr in a chamber saturated with water vapor. Thereafter the carriers are firmly attached to the slide and are washed two times with 0.1 M cacodylate (pH 7.3). Glutardialdehyde (2.5%) is pipetted onto the carriers, and the slides are incubated again for 2 hr in a humid chamber for fixation, then again rinsed in cacodylate buffer. This is followed by standard treatment for scanning electron microscopy (6). MLC Electrophoresis. Cell culture homogenates were centrifuged at 100,000 ϫ g for 1 hr and the pellets were incubated in extraction buffer with 0.6 M KCl, 10 mM EGTA, 0.5 mM dithiotreitol, 1 mM phenylmethylsulfonyl fluoride, 10 mM phosphate (pH 6.8) (1:7 vol͞vol) for 1 hr at 4ЊC. After centrifugation at 10,000 ϫ g for 10 min, the supernatant was diluted 1:10 with ice-cold water to precipitate actomyosin over night at 0ЊC. After centrifugation at 20,000 ϫ g the pellet was solubilized with extraction buffer, mixed 1:1 with glycerol, and stored at Ϫ20ЊC The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Cell Biology International, 2016
A primary skeletal muscle cell culture, in which myoblasts derived from newborn rabbit hindlimb muscles grow on gelatin bead microcarriers in suspension and differentiate into myotubes, has been established previously. In the course of differentiation and beginning spontaneous contractions, these multinucleated myotubes do not detach from their support. Here, we describe the development of the primary myotubes with respect to their ultrastructural differentiation. Scanning electron microscopy reveals that myotubes not only grow around the surface of one carrier bead but also attach themselves to neighboring carriers, forming bridges between carriers. Transmission electron microscopy demonstrates highly ordered myofibrils, T‐tubules, and sarcoplasmic reticulum. The functionality of the contractile apparatus is evidenced by contractile activity that occurs spontaneously or can be elicited by electrostimulation. Creatine kinase activity increases steadily until day 20 of culture. Regar...
Journal of Muscle Research and Cell Motility, 1995
We investigated the myogenic properties of rabbit fast or slow muscle satellite cells during their differentiation in culture, with a particular attention to the expression of myosin heavy chain and myogenic regulatory factor genes. Satellite cells were isolated from Semimembranosus proprius (slow-twitch muscle; 100% type I fibres) and Semimembranosus accessorius (fast-twitch musde; almost 100% type II fibres) muscles of 3-month-old rabbits. Satellite cells in culture possess different behaviours according to their origin. Cells isolated from slow muscle proliferate faster, fuse earlier into more numerous rnyotubes and mature more rapidly into striated contractile fibres than do cells isolated from fast muscle. This pattern of proliferation and differentiation is also seen in the expression of myogenic regulatory factor genes. Myf5 is detected in both fast or slow 6-day-old cell cultures, when satellite cells are in the exponential stage of proliferation. MyoD and myogenin are subsequently detected in slow satellite cell cultures, but their expression in fast cell cultures is delayed by 2 and 4 days respectively. MRF4 is detected in both types of cultures when they contain striated and contractile myofibres. Muscle-specific myosin heavy chains are expressed earlier in slow satellite cell cultures. No adult myosin heavy chain isoforms are detected in fast cell cultures for 13 days, whereas cultures from slow cells express neonatal, adult slow and adult fast myosin heavy chain isoforms at that time. In both fast and slow satellite cell cultures containing striated contractile fibres, neonatal and adult myosin heavy chain isoforms are coexpressed. However, cultures made from satellite cells derived from slow muscles express the slow myosin heavy chain isoform, in addition to the neonatal and the fast isoforms. These results are further supported by the expression of the mRNA encoding the adult myosin heavy chain isoforms. These data provide further evidence for the existence of satellite cell diversity between two rabbit muscles of different fibre-type composition, and also suggest the existence of differently preprogrammed satellite cells.
Histochemistry of Cultured, Embryonic and Regenerating Rat Muscle
Journal of Histochemistry & Cytochemistry, 1973
Histochemical features of cultured rat muscle were compared to those of cultured chicken muscle, as well as of young embryonic and regenerating rat muscle. Primary cultures were established from trypsin-dissociated myoblasts of thigh muscle from 17- to 19-day-old rat embryos and of breast muscle from 10- to 12-day-old chicken embryos. Histochemical reactions for myofibrillar adenosine triphosphatase (ATPase) performed at pH 9.4 and for ATPase after acid pH 4.35 preincubation, phosphorylase, succinic dehydrogenase, reduced diphosphopyridine nucleotide dehydrogenase and myoglobin were applied to examine cultured muscle, gastrocnemius muscle of 15-17 day-old embryos and regenerating muscle fibers in cold-injured gastrocnemius muscle of adult rats. Cultured rat and chicken muscles could be best distinguished by the ATPase reaction following acid preincubation (positive in cultured rat muscle, negative in cultured chicken muscle) and by a different pattern of phosphorylase staining. Howe...
Relative contribution of different classes of myogenic cells to muscle fiber formation in culture
Experimental Cell Research, 1973
Mitotically active cells were labelled in the explant with SH-thymidine for 3 h and then chased with an excess of cold precursor for 12 h before plating in culture. In these conditions no further incorporation of radioactivity occurs in culture. The participation of labelled cells in the fiber formation was followed by autoradiography. The data reported show that the formation of muscle fibers in culture occurs preferentially by fusion of myogenic cells that are actively duplicating their DNA in the primary explant. The participation of cells already differentiated and unable to divide mitotically in the explant appears to be less relevant.
Biomaterials, 2009
The use of defined in vitro systems to study the developmental and physiological characteristics of a variety of cell types is increasing, due in large part to their ease of integration with tissue engineering, regenerative medicine, and high-throughput screening applications. In this study, myotubes derived from fetal rat hind limbs were induced to develop several aspects of mature muscle including: sarcomere assembly, development of the excitation-contraction coupling apparatus and myosin heavy chain (MHC) class switching. Utilizing immunocytochemical analysis, anisotropic and isotropic band formation (striations) within the myotubes was established, indicative of sarcomere formation. In addition, clusters of ryanodine receptors were colocalized with dihydropyridine complex proteins which signaled development of the excitation-contraction coupling apparatus and transverse tubule biogenesis. The myotubes also exhibited MHC class switching from embryonic to neonatal MHC. Lastly, the myotubes survived significantly longer in culture (70-90 days) than myotubes from our previously developed system (20-25 days). These results were achieved by modifying the culture timeline as well as the development of a new medium formulation. This defined model system for skeletal muscle maturation supports the goal of developing physiologically relevant muscle constructs for use in tissue engineering and regenerative medicine as well as for high-throughput screening applications.
Myosin thick filaments from adult rabbit skeletal muscles
Biochimica et Biophysica Acta (BBA) - General Subjects, 1999
Myosin subfragment 1 (S1) forms dimers in the presence of Mg 2 or MgADP or MgATP. The entire myosin molecule forms head^head dimers in the presence of MgATP. The angle between the two subunits in the S1 dimer is 95³. Assuming that the length of the globular part of S1 is W12 nm and that the S1/S2 joint (lever arm W7 nm) is clearly bent, the cylinder tangent to this dimer should have a diameter of W18 nm, close to the W16^20 nm suggested by many studies for the diameter of thick filaments in situ. These conclusions led us to reexamine our previous model, according to which two heads from two opposite myosin molecules are inserted into the filament core and interact as dimers. We studied synthetic filaments by electron microscopy, enzyme activity assays, controlled digestion and filament^filament interaction analysis. Synthetic filaments formed by rapid dilution in the presence of 1 mM EDTA at room temperature (W22³C) had all their myosin heads outside the backbone. These filaments are called superfilaments (SF). Synthetic filaments formed by slow dilution, in the presence of either 2 mM Mg 2 or 0.5 mM MgATP and at low temperature (W0³C) had one myosin head outside the backbone and one head inside. These filaments are called filaments (F). Synthetic filaments formed by slow dilution, in the presence of 4 mM MgATP at low temperature (W0³C) had most of their heads inserted in the filament core. These filaments are called antifilaments (AF). These experimental results provide important new information about myosin synthetic filaments. In particular, we found that myosin heads were involved in filament assembly and that filament^filament interactions can occur via the external heads. Native filaments (NF) from rabbit psoas muscle were also studied by enzyme assays. Their structure depended on the age of the rabbit. NF from 4-month-old rabbits were three-stranded, i.e. six myosin heads per crown, two of which were inside the core and four outside. NF from 18-month-old rabbits were two-stranded (similar to F).
Comparative characteristic ofMytilus muscle cells developed in vitro and in vivo
Journal of Experimental Zoology, 2003
The mussel cells from premyogenic larval stages are capable of differentiation into smooth muscle cells in vitro. However, the behavior and protein composition of these cells are not completely identical to those of smooth muscle cells of adult mussels. In this study we compared some properties of mussel muscle cells forming from cells of trochophore (premyogenic larval stage) in vitro with those of muscle cells of veliger and adult mussel. We found a substantial difference between the contractile apparatus protein composition of veliger muscle and cultivated cells. Myorod, one of the molecular markers of the phenotype of mollusc smooth muscle cells (Shelud'ko et al., 1999, Comp Biochem Physiol 122:277-285), is not a constituent of the contractile apparatus of veliger muscle. At the same time the protein composition of contractile apparatus in cultivated cells was similar to that of adult Mytilus muscles. There were only few quantitative differences between them. The contractile activity of cultivated cells was changing in time. The kinetic parameters of first spontaneous contractions were similar to those of phasic contractions, while their period was close to that of tonic contractions. After 50-55 hrs cultivation the cells produced both phasic and tonic contractions, but the character of contractile activity of cultivated cells was regulated after six days of cultivation only. However, there were no muscle cells in vitro, whose contractile activity was similar to that of veliger muscle cells. So, we concluded that properties of muscle cells forming from premyogenic larval mussel cells in culture are similar to those of muscle cells of the adult mussel, but not of veliger.