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Comparison of initial stages of muscle differentiation in rat and mouse myoblastic and mouse mesodermal stem cell lines
Abstract
1. Electrophysiological and immunohistochemical properties during the early stages of muscle differentiation were studied in two myoblastic cell lines, mouse C2C12 and rat L6, and compared to those in myogenic clonal cells derived from the mouse mesodermal stem cell line C3H10T1/2, studied in the preceding paper. 2. Mouse C2C12 cells were induced to differentiate to muscle by changing from 10% fetal calf serum to 2% horse serum in the medium. Most of the C2C12 cells before serum reduction showed ATP-induced slow K+ current. Twelve per cent showed inward rectifier K+ current. They expressed fibronectin and Neural Cell Adhesion Molecule (NCAM). Small spindle-shaped cells at an early stage of muscle differentiation began to appear 24 h after serum reduction. In contrast to cells before serum reduction, only 13% of these spindle-shaped cells showed an ATP response. Most showed tetrodotoxin (TTX)-resistant Na+ current and outward K+ current. Thirty-eight per cent had inward rectifier K+ current. They expressed NCAM but not fibronectin. The T-type Ca2+ current was not observed up to the latest stage of differentiation investigated. 3. Rat L6 cells in maintaining culture medium showed only infrequent ATP responses, but already showed TTX-resistant Na+ current. No clear T-type Ca2+, inward rectifier K+ or outward K+ currents were observed. About one-third of the cells did not express fibronectin. From these results, L6 cells appear to be at a stage near to but slightly earlier than that of C2C12 cells after serum reduction. 4. The properties of the early stages of muscle differentiation in C3H10T1/2 cells, such as the disappearance of ATP-induced K+ current and fibronectin, and the appearance of NCAM, were also seen in C2C12 and L6. However, T-type Ca2+ and inward K+ currents, which were found in the initial stages of C3H10T1/2 muscle differentiation, were not clearly observed in C2C12 and L6. Instead, C2C12 and L6 showed a TTX-resistant Na+ current which was never observed in C3H10T1/2 cells. 5. The properties of the TTX-resistant Na+ current were investigated. In L6 cells, it was reduced to 60% by 1 microM-TTX. It could be evoked by depolarizations to a level above -50 mV with a maximum amplitude at around -15 mV. Steady-state inactivation was detectable with pre-pulses to -100 mV for 100 ms and reached half at pre-pulses of -78 mV. These parameters of inactivation are clearly different from those of the TTX-sensitive Na+ current observed in C3H10T1/2-derived mature muscle cells in the preceding paper.
... The cells were cultured in 35 mm × 10 mm culture dish (Corning, USA), and the culture medium for cell growth included Dulbecco's Modified Eagle's Medium high glucose culture fluid (DMEM high Glucose Medium) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (all reagents were purchased from Gibco). The growth medium is changed every 2 days (Kubo 1991). ...
... In order to induce C2C12 myoblasts to differentiate into myotube cells, when the cells were grown to 60-70% confluence, the proliferation medium was discarded and the cells were rinsed gently 3 times with Hanks' Balanced Salt Solution (HBSS) and then replaced with differentiation medium (DMEM high glucose medium supplemented with 2% horse serum and 1% penicillin/streptomycin) which induced its differentiation (the horse serum was purchased from Biological Industries). The solution was changed every 2 days for 6 days, and the process of myotube formation was observed with an inverted microscope (Kubo 1991). ...
The aim of this study was to investigate the effects of miR-34c-5p on the main voltage-dependent ion channels in skeletal muscle cells. This study focused on the effects of miR-34c-5p on sodium, potassium, and calcium currents in C2C12 myoblasts. The miR-34c-5p overexpression group, knockdown group, and control group were differentiated for 7 days, fused into myotubes, and used for the whole-cell patch clamp recording. Compared with the control group, the whole-cell sodium current density of the other two groups had no significant changes. In the knockdown group, the delayed rectifier potassium current density was increased (statistically significant), and the whole-cell calcium channel current density did not change. In the overexpression group, the change of rectifier potassium current density was not obvious, while the peak calcium channel current density increased (− 9.23 ± 0.95 pA/pF, n = 6 cells for the overexpression group vs. − 6.48 ± 0.64 pA/pF, n = 7 cells for the control; p < 0.05). Changes in the expression of miR-34c-5p can affect the electrophysiological characteristics of calcium and potassium voltage-gated channels in C2C12 myotubes. Overexpression of miR-34c-5p increased whole-cell L-type calcium channel current (ICa,L), while miR-34c-5p knockdown increased whole-cell delayed rectifier potassium current (IKd).
... Skeletal myoblasts also exhibit different current and ion channel profiles in their proliferating and differentiating states. Murine C 2 C 12 myoblasts undergoing active proliferation express an ATP-induced K + current, a swellingactivated Cl -current, and an IK Ca current868788. Upon initiation of differentiation, these currents are replaced with a tetrodotoxin-sensitive Na + current, an IK DR current, an IK IR current, and an L-type Ca 2+ current87888990. ...
... Murine C 2 C 12 myoblasts undergoing active proliferation express an ATP-induced K + current, a swellingactivated Cl -current, and an IK Ca current868788. Upon initiation of differentiation, these currents are replaced with a tetrodotoxin-sensitive Na + current, an IK DR current, an IK IR current, and an L-type Ca 2+ current87888990. In muscle satellite cell-derived human myoblasts, voltage-gated Na + and Ca 2+ -activated K + channels are expressed during proliferation [91], while hEAG, IK DR , IK IR , T-type Ca 2+ , and L-type Ca 2+ channels are expressed in differentiated fusion-competent myoblasts9293949596. ...
Biophysical signaling, an integral regulator of long-term cell behavior in both excitable and non-excitable cell types, offers enormous potential for modulation of important cell functions. Of particular interest to current regenerative medicine efforts, we review several examples that support the functional role of transmembrane potential (V(mem)) in the regulation of proliferation and differentiation. Interestingly, distinct V(mem) controls are found in many cancer cell and precursor cell systems, which are known for their proliferative and differentiation capacities, respectively. Collectively, the data demonstrate that bioelectric properties can serve as markers for cell characterization and can control cell mitotic activity, cell cycle progression, and differentiation. The ability to control cell functions by modulating bioelectric properties such as V(mem) would be an invaluable tool for directing stem cell behavior toward therapeutic goals. Biophysical properties of stem cells have only recently begun to be studied and are thus in need of further characterization. Understanding the molecular and mechanistic basis of biophysical regulation will point the way toward novel ways to rationally direct cell functions, allowing us to capitalize upon the potential of biophysical signaling for regenerative medicine and tissue engineering.
... In the case of a mesodermal stem cell line, myogenic clones express the development or from the 32-cell to the midgastrula stage. When a-amanitin was injected at the 32-cell stage, the inward rectifier in addition to NCAM and TTX-insensitive Na / channels (146,147). Myocytes of Xenopus embryos amount of inward rectifier expressed was markedly reduced, leaving a small population of channels that appear also possess inward rectifier K / channels (292). Cardiac myocytes also express an inward rectifier that is useful to be maternally derived in both uninduced and induced blastomeres (235). ...
... The cinoma (EC) cell lines, can be established with transformed cells from the primitive ectoderm in embryonic presence of T type is also observed in cells at relatively immature states of differentiation, such as mesodermal 5.5-day mouse embryo (68). If the cloned TC or EC cells are injected into and mixed with the inner cell mass of stem cell lines (146,147), myocytes from the early chick embryo or the neonatal rat (70,124), rat atrial myocytes the normal embryo, chimeric mice are obtained with the expression of all types of cells derived from these cell (324), chick embryonic dorsal root ganglion cells, and neonatal rat hippocampal neurons (24,325). lines (103). ...
In this review we underscore the merits of using voltage-dependent ion channels as markers for neuronal differentiation from the early stages of uncommitted embryonic blastomeres. Furthermore, a fairly large part of the review is devoted to the descriptions of the establishment of a simple model system for neural induction derived from the cleavage-arrested eight-cell ascidian embryo by pairing a single ectodermal with a single vegetal blastomere as a competent and an inducer cell, respectively. The descriptions are focused particularly on the early developmental processes of various ion channels in neuronal and other excitable membranes observed in this extraordinarily simple system, and we compare these results with those in other significant and definable systems for neural differentiation. It is stressed that this simple system, for which most of the electronic and optical methods and various injection experiments are applicable, may be useful for future molecular physiological studies on the intracellular process of differentiation of the early embryonic cells. We have also highlighted the importance of suppressive mechanisms for cellular differentiation from the experimental results, such as epidermal commitment of the cleavage-arrested one-cell Halocynthia embryos or suppression of epidermal-specific transcription of inward rectifier channels by neural induction signals. It was suggested that reciprocal suppressive mechanisms at the transcriptional level may be one of the key processes for cellular differentiation, by which exclusivity of cell types is maintained.
... Given that NLK potently inhibits SRF/MKL signaling, we hypothesized that NLK may serve as a negative regulator of myoblast differentiation. To this end, horse serum and highconfluence cells were employed to induce the differentiation of C2C12 cells [23,24]. We detected mRNA and protein levels during the differentiation course of C2C12 cells. ...
Serum response factor (SRF) regulates differentiation and proliferation by binding to RhoA-actin-activated MKL or Ras-MAPK-activated ELK transcriptional coactivators, but the molecular mechanisms responsible for SRF regulation remain unclear. Here, we show that Nemo-like kinase (NLK) is required for the promotion of SRF/ELK signaling in human and mouse cells. NLK was found to interact with and phosphorylate SRF at serine residues 101/103, which in turn enhanced the association between SRF and ELK. The enhanced affinity of SRF/ELK antagonized the SRF/MKL pathway and inhibited mouse myoblast differentiation in vitro. In a skeletal muscle-specific Nlk conditional knockout mouse model, forming muscle myofibers underwent hypertrophic growth, resulting in an increased muscle and body mass phenotype. We propose that both phosphorylation of SRF by NLK and phosphorylation of ELKs by MAPK are required for RAS/ELK signaling, confirming the importance of this ancient pathway and identifying an important role for NLK in modulating muscle development in vivo.
... All these findings indicate that miR-142a-3p may have an important effect on skeletal muscle ion channels. 7 th day for the subsequent experiments (Kubo, 1991). ...
MicroRNAs can regulate the function of ion channels in many organs. Based on our previous study we propose that miR-142a-39, which is highly expressed in denervated skeletal muscle, might affect cell excitability through similar mechanisms. In this study, we overexpressed or knocked down miR-142a-3p in C2C12 cells using a lentivirus method. After 7 days of differentiation culture, whole-cell currents were recorded. The results showed that overexpression of miR-142a-3p reduced the cell membrane capacitance, increased potassium current density and decreased calcium current density. Knockdown of miR-142a-3p reduced sodium ion channel current density. The results showed that change in miR-142a-3p expression affected the ion channel currents in C2C12 cells, suggesting its possible roles in muscle cell electrophysiology. This study was approved by the Animal Ethics Committee of Peking University in July 2020 (approval No. LA2017128).
... Firstly, mouse C2C12 expressed fibronectin and presented ATP-induced slow K + current, while rat L6 cells did not express fibronectin and demonstrated infrequent ATP responses. [20] Secondly, the glucose uptake in these cell lines was reported to be insulin-stimulated, [21] carrier-mediated and saturable. [19,21,22] Rat L6 cells were reported to have lower level of GLUT-1 and GLUT-3 expression, but higher expression of GLUT-4. ...
Objectives
The myoblast cell line, C2C12, has been utilised extensively in vitro as an examination model in understanding metabolic disease progression. Although it is indispensable in both preclinical and pharmaceutical research, a comprehensive review of its use in the investigation of insulin resistance progression and pharmaceutical development is not available.
Key findings
C2C12 is a well‐documented model, which can facilitate our understanding in glucose metabolism, insulin signalling mechanism, insulin resistance, oxidative stress, reactive oxygen species and glucose transporters at cellular and molecular levels. With the aid of the C2C12 model, recent studies revealed that insulin resistance has close relationship with various metabolic diseases in terms of disease progression, pathogenesis and therapeutic management. A holistic, safe and effective disease management is highly of interest. Therefore, significant efforts have been paid to explore novel drug compounds and natural herbs that can elicit therapeutic effects in the targeted sites at both cellular (e.g. mitochondria, glucose transporter) and molecular level (e.g. genes, signalling pathway).
Summary
The use of C2C12 myoblast cell line is meaningful in pharmaceutical and biomedical research due to their expression of GLUT‐4 and other features that are representative to human skeletal muscle cells. With the use of the C2C12 cell model, the impact of drug delivery systems (nanoparticles and quantum dots) on skeletal muscle, as well as the relationship between exercise, pancreatic β‐cells and endothelial cells, was discovered.
... All cell lines used were checked for mycoplasma contamination intermittently. For myotube formation, C2C12 cells at 80% confluence were induced to differentiate by replacing FBS containing medium with medium containing 2% horse serum for 72-96 h (Kubo, 1991;Sasi Kumar et al., 2015). Lipofectamine Plus, 2000, 3000, LTX, and RNA Imax from Invitrogen were used for transfection, following the manufacturer's instructions. ...
C3G (RapGEF1) plays a role in cell differentiation and is essential for early embryonic development in mice. In this study, we identify C3G as a centrosomal protein colocalizing with cenexin at the mother centriole in interphase cells. C3G interacts through its catalytic domain with cenexin, and they show interdependence for localization to the centrosome. C3G depletion caused a decrease in cellular cenexin levels. Centrosomal localization is lost as myocytes differentiate to form myotubes. Stable clone of cells depleted of C3G by CRISPR/Cas9 showed the presence of supernumerary centrioles. Overexpression of C3G, or a catalytically active deletion construct inhibited centrosome duplication. Cilia length is longer in C3G knockout cells, and the phenotype could be reverted upon reintroduction of C3G or its catalytic domain. Association of C3G with the basal body is dynamic, decreasing upon serum starvation, and increasing upon reentry into the cell cycle. C3G inhibits cilia formation and length dependent on its catalytic activity. We conclude that C3G inhibits centrosome duplication and maintains ciliary homeostasis, properties that may be important for its role in embryonic development.
... 93 Tetrodotoxin-resistant sodium currents were also observed in the mouse C2C12 cell line along with calcium-activated potassium channels, an ATP-induced slow potassium current, an inward rectifier potassium current, and a volume-induced chloride current. 94 There is no data to suggest whether or not these currents are directly involved in cell cycle progression of MPCs or simply related to ''housekeeping'' processes, but it is known that satellite cells (proliferative stage) are measured to have a Vmem around -10 mV and myotubes (nonproliferating, withdrawn from the cell cycle) are around -70 mV. 95,96 These data are consistent with the previous observations made in other cell types and suggest a concomitant relationship between Vmem and MPC proliferation. ...
Modern stem cell research has mainly focused on protein expression and transcriptional networks. However, transmembrane voltage gradients generated by ion channels and transporters have demonstrated to be powerful regulators of cellular processes. These physiological cues exert influence on cell behaviors ranging from differentiation and proliferation to migration and polarity. Bioelectric signaling is a fundamental element of living systems and an untapped reservoir for new discoveries. Dissecting these mechanisms will allow for novel methods of controlling cell fate and open up new opportunities in biomedicine. This review focuses on the role of ion channels and the resting membrane potential in the proliferation and differentiation of skeletal muscle progenitor cells. In addition, findings relevant to this topic are presented and potential implications for tissue engineering and regenerative medicine are discussed.
... In the case of PC12h cells, DMEM supplemented with 5% FBS and 5% horse serum was used. To induce differentiation of C2C12 cells, culture media were replaced with 2% horse serum in DMEM (Kubo, 1991). ...
... After switching culture conditions from GM to differentiation medium (DM), myoblasts differentiation was revealed showing typically morphological changes such as alignment, elongation and fusion of mononucleated cells to multinucleated myotubes whereas a subpopulation of cells remained undifferentiated as reserve cells. In parallel to these morphological changes, the total protein content and specific biochemical markers of muscle differentiation such as creatine kinase, myogenin, tropomiosin and myosin were raised during the first seven days of the progression of differentiation as reported before (data not shown) [13,16,17]. Under these conditions, the role of the natural product Solbone A on myogenesis was studied and compared to synthetic 1a,25(OH) 2 D 3 . ...
The presence of glycoside derivatives of 1α,25(OH)2D3 endows plants to gradual release of the free bioactive form of 1α,25(OH)2D3 from its glycoconjugates by endogenous animal tissue glycosidases. This results in increased half-life of the hormone in blood when purified plant fractions are administered for therapeutic purposes. In this work, we evaluated the role 1α,25(OH)2D3-glycosides enriched natural product (Solbone A) from Solanum glaucophyllum leaf extract compared with synthetic 1α,25(OH)2D3 on myogenic differentiation in C2C12 myoblasts. For these, differentiation markers and myogenic parameters were studied in C2C12 myoblasts. Results showed that Solbone A, likewise the synthetic hormone, increased creatine kinase activity at day 2 after differentiation induction (60%, p <0.05). Solbone A and synthetic 1α,25(OH)2D3 increased vitamin D3 receptor protein expression at 10 nM (50 and 30%, respectively) and the transcription factor myogenin (80%, p<0.05). However, tropomyosin expression was not affected by both compounds). In addition, myosin heavy chain (MHC) protein expression was increased 30% at day 2 of differentiation. Solbone A or synthetic 1α,25(OH)2D3 had no effects on myogenin nor MHC cell localization. Cellular mass increased with myogenesis progression, being Solbone A more effective than synthetic 1α,25(OH)2D3. Finally, Solbone A, as well as synthetic 1α,25(OH)2D3, augmented the index fusion of cultured muscle fibers. In conclusion, these results demonstrated that Solbone A exhibit at least equal or greater effects on early myoblast differentiation as synthetic hormone, suggesting that plant glycosides could be an effective, accessible and cheaper substitute for synthetic 1α,25(OH)2D3 to promote muscle growth.
... Pour différencier les myoblastes en myotubes, le sérum de veau foetal est remplacé par 2% de sérum de cheval (Sigma Aldrich, H1270-500ml) quand les cellules C2C12 atteignent 100% de confluence (Kubo 1991). Le milieu de différenciation est alors changé toutes les 48h pendant une semaine Le Ct (« cycle threshold » ou cycle seuil) est défini comme la valeur du cycle pour laquelle la quantité de fluorescence dépasse de manière significative le seuil de détection de l'appareil. ...
Un concept qui a fait son apparition ces dernières années est l’utilisation de micronutriments comme agents thérapeutiques connus pour leurs propriétés anti-inflammatoires, antioxydantes, cardio-protectrices ou anticancéreuses. Dans notre travail, nous nous sommes intéressés à l’étude des mécanismes d’actions in vivo et in vitro du resvératrol et de deux formulations naturelles, l’une impliquée dans les effets antioxydants (produit « X ») et l’autre (produit « Y ») destinée à corriger les désordres de nature lipidique de sujets présentant un syndrome métabolique. Nous avons caractérisé les effets pro-différenciateurs d’un polyphénol naturel, le resvératrol, sur cellules squelettiques musculaires C2C12 en culture. L’étude a montré que ce polyphénol permet d’induire l’expression de facteurs de la différenciation musculaire (Myogénine, Scrp3) et d’augmenter le taux de la myosine, protéine impliquée dans la contraction musculaire. Enfin, le resvératrol permet de moduler l’expression de microRNA, comme par exemple miR-133b impliqué dans les mécanismes de différenciation musculaire. Les résultats sur le stress oxydant montrent que le produit « X » est capable, à la fois, de moduler de façon modérée l’expression des gènes des défenses anti-radicalaires au niveau du muscle gastrocnémiens (Sirt1, Sod1, Ucp2, Nrf1) mais aussi au niveau des cellules musculaires squelettiques murines C2C12 (Prdx1, Nrf2, Pgc1α) en culture. Les résultats au niveau des gastrocnémiens ont montré également une élévation du potentiel global des défenses anti-radicalaires par dosage KRL. Une diminution du taux de ROS intracellulaires a été observée avec sonde DHE dans les cellules C2C12 en culture. L’étude des mécanismes d’action de « Y » a montré qu’il est capable de moduler de façon modérée l’expression des gènes du métabolisme énergétique (Gs1, Srebp1c, Fabp3, Vlcad, Pparβ) dans les cellules C2C12. Nos résultats montrent également que « Y » induit un effet anti-inflammatoire par la diminution du nombre de lymphocytes sous un régime riche en calorie. Toutefois, « Y » n’est pas un agoniste de PPARα et de PPARγ dans nos conditions. L’ensemble des résultats obtenus au cours de ce travail confirme que les micronutriments sont capables d’induire des effets au niveau génique quoique de façon modérée. Des études à long terme sont nécessaires pour comprendre et confirmer réellement leur impact sur l’organisme
... The dox-induced skeletal muscle cell differentiation consists of three phases: induction, differentiation, and maturation (Fig. 4). Skeletal muscle cell maturation is promoted in 2 % horse serum medium [16]. Administration of IGF-I also boosts maturation and enlarges the size of myocytes [17,18]. ...
Patient-derived induced pluripotent stem cells (iPSCs) have opened the door to recreating pathological conditions in vitro using differentiation into diseased cells corresponding to each target tissue. Yet for muscular diseases, a method for reproducible and efficient myogenic differentiation from human iPSCs is required for in vitro modeling. Here, we introduce a myogenic differentiation protocol mediated by inducible transcription factor expression that reproducibly and efficiently drives human iPSCs into myocytes. Delivering a tetracycline-inducible,myogenic differentiation 1 (MYOD1) piggyBac (PB) vector to human iPSCs enables the derivation of iPSCs that undergo uniform myogenic differentiation in a short period of time. This differentiation protocol yields a homogenous skeletal muscle cell population, reproducibly reaching efficiencies as high as 70-90 %. MYOD1-induced myocytes demonstrate characteristics of mature myocytes such as cell fusion and cell twitching in response to electric stimulation within 14 days of differentiation. This differentiation protocol can be applied widely in various types of patient-derived human iPSCs and has great prospects in disease modeling particularly with inherited diseases that require studies of early pathogenesis and drug screening.
... Both L6 and C2C12 myoblasts exhibit characteristic features of muscle myogenesis, including migration and proliferation in medium containing 10% (v/v) foetal calf serum, followed by elongation and fusion to form myotubes following serum reduction. Interestingly, previous studies have shown that L6 cells differentiate readily into myotubes when maintained in medium containing 2% (v/v) serum, whereas C2C12 differentiation is more pronounced in serum-free medium compared to a serum-containing system [26,27]. Thus, under standard conditions used to differentiate both cell lines (i.e. 2% (w/w) serum), it is possible that L6 myotubes mature more rapidly and/or more efficiently than C2C12 myotubes. ...
Ceramides are known to promote insulin resistance in a number of metabolically important tissues including skeletal muscle, the predominant site of insulin-stimulated glucose disposal. Depending on cell type, these lipid intermediates have been shown to inhibit protein kinase B (PKB/Akt), a key mediator of the metabolic actions of insulin, via two distinct pathways: one involving the action of atypical protein kinase C (aPKC) isoforms, and the second dependent on protein phosphatase-2A (PP2A). The main aim of this study was to explore the mechanisms by which ceramide inhibits PKB/Akt in three different skeletal muscle-derived cell culture models; rat L6 myotubes, mouse C2C12 myotubes and primary human skeletal muscle cells. Our findings indicate that the mechanism by which ceramide acts to repress PKB/Akt is related to the myocellular abundance of caveolin-enriched domains (CEM) present at the plasma membrane. Here, we show that ceramide-enriched-CEMs are markedly more abundant in L6 myotubes compared to C2C12 myotubes, consistent with their previously reported role in coordinating aPKC-directed repression of PKB/Akt in L6 muscle cells. In contrast, a PP2A-dependent pathway predominantly mediates ceramide-induced inhibition of PKB/Akt in C2C12 myotubes. In addition, we demonstrate for the first time that ceramide engages an aPKC-dependent pathway to suppress insulin-induced PKB/Akt activation in palmitate-treated cultured human muscle cells as well as in muscle cells from diabetic patients. Collectively, this work identifies key mechanistic differences, which may be linked to variations in plasma membrane composition, underlying the insulin-desensitising effects of ceramide in different skeletal muscle cell models that are extensively used in signal transduction and metabolic studies.
... The above studies were performed in undifferentiated C2C12 cells. To determine whether CM from tumor cell lines reduce miR486 in myoblasts undergoing differentiation to myotubes, we grew C2C12 cells in media containing 2% horse serum (26). CM from both cell lines reduced the levels of miR486 in the differentiated cells (Fig. 4). ...
Circulating microRNAs are emerging as important biomarkers of various diseases including cancer. Intriguingly, circulating levels of several microRNAs are lower in cancer patients compared with healthy individuals. In this study, we tested the hypothesis that a circulating microRNA might serve as a surrogate of the effects of cancer on microRNA expression or release in distant organs. Here we report that circulating levels of the muscle-enriched miR-486 is lower in breast cancer patients compared with healthy individuals, and that this difference is replicated faithfully in MMTV-PyMT and MMTV-Her2 transgenic mouse models of breast cancer. In tumor-bearing mice, levels of miR-486 were relatively reduced in muscle, where there was elevated expression of the miR-486 target genes PTEN and FOXO1A and dampened signaling through the PI3K/AKT pathway. Skeletal muscle expressed lower levels of the transcription factor MyoD which controls miR-486 expression. Conditioned media (CM) obtained from MMTV-PyMT and MMTV-Her2/Neu tumor cells cultured in vitro was sufficient to elicit reduced levels of miR-486 and increased PTEN and FOXO1A expression in C2C12 murine myoblasts. Cytokine analysis implicated TNFα and four additional cytokines as mediators of miR-486 expression in CM-treated cells. Since miR-486 is a potent modulator of PI3K/AKT signaling and the muscle-enriched transcription factor network in cardiac/skeletal muscle, our findings implicated TNFα-dependent miRNA circuitry in muscle differentiation and survival pathways in cancer.
... Traditionally, differentiation of C2C12 cells into myotubes has been achieved by components, or lack of thereof, in the growth media [8]. For example, the exchange of high (10e20%) with low (2e5%) serum containing media led to muscle differentiation [9]. Alternative approaches include the transfection of cells with various TFs [10] or microRNAs [11], the usage of signaling molecules [12] or extracellular matrix proteins [13], and the physical properties of strata/scaffolds upon which cells are grown [14]. ...
... Myoblast C2C12 of the mouse skeletal muscle origin starts differentiation to a myotube fibrous cell by lowering serum density in culture fluid. Therefore, to differentiate the myoblast into myotube cells, culture fluid was changed for DMEM where a myoblast contained serum 7%HS (House Serum) for differentiation instructions in a subconfluent state (the state that multiplied to around 80% of the dish base) [9]. The culture fluid is changed every two days, and cell cultures were carried out in incubator under the environment of 100% of humidity with 5% CO 2 /95% air at 37℃ for several days. ...
Fibroblast L929 and myoblast C2C12 cells of the mouse connective tissue origin were sown on the surface of conductive polymer films (polypyrrole, PPy and poly(3,4-ethylenedioxythiophene), PEDOT) in the cell culture medium, and the proliferative process of these cells was observed. Without changing the form, fibroblast L929 and myoblast C2C12 cells were observed to proliferate almost similarly to the cell which cultured on a dish on the market and to maintain compatibility. In other word, it has been understood these two kinds of conductive polymers used in this study, the PEDOT films maintain the secretion function of the cell cultured on the surface of these polymers. Therefore, the PPy- and the PEDOT-coated electrode suggested the possibility usable as a nerve stimulation electrode with biocompatibility, because these polymers were effective to culture the cell.
... C2C12 myoblasts were maintained in growth medium (GM) consisting of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin solution. Differentiation medium (DM) was DMEM containing 2% horse serum, 1% L-glutamine and 1% penicillin/streptomycin solution (Kubo, 1991). For differentiation studies, 15×10 4 cells were seeded in a 150mm dish with or without 20nM arsenic as sodium arsenite (NaAsO 2 , certified >99.8%; ...
Arsenic is a toxicant commonly found in water systems and chronic exposure can result in adverse developmental effects including increased neonatal death, stillbirths, and miscarriages, low birth weight, and altered locomotor activity. Previous studies indicate that 20 nM sodium arsenite exposure to C2C12 mouse myocyte cells delayed myoblast differentiation due to reduced myogenin expression, the transcription factor that differentiates myoblasts into myotubes. In this study, several mechanisms by which arsenic could alter myogenin expression were examined. Exposing differentiating C2C12 cells to 20 nM arsenic increased H3K9 dimethylation (H3K9me2) and H3K9 trimethylation (H3K9me3) by 3-fold near the transcription start site of myogenin, which is indicative of increased repressive marks, and reduced H3K9 acetylation (H3K9Ac) by 0.5-fold, indicative of reduced permissive marks. Protein expression of Glp or Ehmt1, a H3-K9 methyltransferase, was also increased by 1.6-fold in arsenic-exposed cells. In addition to the altered histone remodeling status on the myogenin promoter, protein and mRNA levels of Igf-1, a myogenic growth factor, were significantly repressed by arsenic exposure. Moreover, a 2-fold induction of Ezh2 expression, and an increased recruitment of Ezh2 (3.3-fold) and Dnmt3a (~2-fold) to the myogenin promoter at the transcription start site (-40 to +42), were detected in the arsenic-treated cells. Together, we conclude that the repressed myogenin expression in arsenic-exposed C2C12 cells was likely due to a combination of reduced expression of Igf-1, enhanced nuclear expression and promoter recruitment of Ezh2, and altered histone remodeling status on myogenin promoter (-40 to +42).
... To gain insight into microRNA function in skeletal muscle, we used a well-characterized in vitro model of myogenesis: C2C12 mesenchymal stem cells. C2C12 cells differentiate along the myogenic pathway when they are transferred from growth medium into differentiation medium containing reduced serum [29]. Gain-of-function analyses have beenFigure 3. Induction of myogenesis in AdvmiR-206 transfected C2C12 cells. ...
The expression of microRNA-206 (miR-206) is high in skeletal muscle but low in most other tissues. The expression of miR-206 is increased in muscular dystrophy, suggesting its involvement in the pathogenesis of muscle diseases. To determine the role of miR-206 in muscle cell differentiation and explore a possible gene therapy vector, we constructed a miR-206 adenoviral expression vector (AdvmiR-206) and tested for transfection into C2C12 stem cells.
A 355-bp PCR amplicon from C57B6 mouse skeletal muscle genomic DNA was inserted into the adenoviral shuttle vector pAdTrack-CMV, which was then co-transformed with the adenoviral backbone plasmid pAdEasy-1 into competent E. coli BJ5183 bacteria. The specificity and function of this recombinant adenoviral MiR-206 were studied in C2C12 cells by Northern blot, immunofluorescence, Western blot, and flow cytometry.
Increased expression of miR-206 in AdvmiR-206 transfected C2C12 cells (P < 0.001) and resulted in morphological and biochemical changes over time that were similar to serum deprivation, including elongated cells and increased myosin heavy chain proteins. Even in the absence of serum deprivation, miR-206 overexpression accounted for a 50% reduction of S-phase cells (P < 0.01). Moreover, in untransfected C2C12 cells, the introduction of miR-206-specific antisense oligoribonucleotides inhibited the normal response to serum deprivation. Twenty-four hours after lipofection of antisense oligoribonucleotides, the number of elongated cells was reduced by half (P < 0.01).
Collectively, these data support a role for miR-206 in myoblast differentiation. We foresee potential applications for the AdvmiR-206 vector in research and therapy.
... We found Vcam1 and Ncam-180 up-regulated, both of which code for cell adhesion molecules and which contribute to myotube formation through fusion of myoblasts[39,40]. Some studies have shown that for C2C12 myoblasts[67], Ncam expression is associated with the early stages of differentiation, and is concurrent with expression of T-type Ca 2+ channels and inward rectifier K + channels[68]. Our data again confirm the activation of these pathways during several phases of myogenesis. ...
Several purine receptors have been localised on skeletal muscle membranes. Previous data support the hypothesis that extracellular guanosine 5'-triphosphate (GTP) is an important regulatory factor in the development and function of muscle tissue. We have previously described specific extracellular binding sites for GTP on the plasma membrane of mouse skeletal muscle (C2C12) cells. Extracellular GTP induces an increase in intracellular Ca(2+) concentrations that results in membrane hyperpolarisation through Ca(2+)-activated K(+) channels, as has been demonstrated by patch-clamp experiments. This GTP-evoked increase in intracellular Ca(2+) is due to release of Ca(2+) from intracellular inositol-1,4,5-trisphosphate-sensitive stores. This enhances the expression of the myosin heavy chain in these C2C12 myoblasts and commits them to fuse into multinucleated myotubes, probably via a phosphoinositide-3-kinase-dependent signal-transduction mechanism. To define the signalling of extracellular GTP as an enhancer or modulator of myogenesis, we investigated whether the gene-expression profile of differentiated C2C12 cells (4 and 24 h in culture) is affected by extracellular GTP. To investigate the nuclear activity and target genes modulated by GTP, transcriptional profile analysis and real-time PCR were used. We demonstrate that in the early stages of differentiation, GTP up-regulates genes involved in different pathways associated with myogenic processes, including cytoskeleton structure, the respiratory chain, myogenesis, chromatin reorganisation, cell adhesion, and the Jak/Stat pathway, and down-regulates the mitogen-activated protein kinase pathway. GTP also increases the expression of three genes involved in myogenesis, Pp3ca, Gsk3b, and Pax7. Our data suggests that in the myogenic C2C12 cell line, extracellular GTP acts as a differentiative factor in the induction and sustaining of myogenesis.
... The mouse myoblast C2C12 cell line (ATCC, Manassas, VA) was seeded at 860 cells/well in 6 well plates, and cultured with or without 20 nm sodium arsenite in DMEM supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/ streptomycin (growth media) at 37°C in a humidified incubator containing 5% CO 2 . To differentiate the cells, the medium was replaced with DMEM supplemented with 2% horse serum, 1% L-glutamine, and 1% penicillin/streptomycin (differentiation media) (Kubo, 1991) with or without 20nM arsenic. Myotube formation was examined on differentiation days 0, 1, 2, 3, and 4 (n=6 per group per day) by fixing the cells in methanol and incubating them in Giemsa stain. ...
Epidemiological studies have correlated arsenic exposure with cancer, skin diseases, and adverse developmental outcomes such as spontaneous abortions, neonatal mortality, low birth weight, and delays in the use of musculature. The current study used C2C12 mouse myoblast cells to examine whether low concentrations of arsenic could alter their differentiation into myotubes, indicating that arsenic can act as a developmental toxicant. Myoblast cells were exposed to 20 nM sodium arsenite, allowed to differentiate into myotubes, and expression of the muscle-specific transcription factor myogenin, along with the expression of tropomyosin, suppressor of cytokine signaling 3 (Socs3), prostaglandin I2 synthesis (Ptgis), and myocyte enhancer 2 (Mef2), was investigated using QPCR and immunofluorescence. Exposing C2C12 cells to 20 nM sodium arsenite delayed the differentiation process, as evidenced by a significant reduction in the number of multinucleated myotubes, a decrease in myogenin mRNA expression, and a decrease in the total number of nuclei expressing myogenin protein. The expression of mRNA involved in myotube formation, such as Ptgis and Mef2 mRNA, was also significantly reduced by 1.6-fold and 4-fold during differentiation. This was confirmed by immunofluorescence for Mef2, which showed a 2.6-fold reduction in nuclear translocation. Changes in methylation patterns in the promoter region of myogenin (-473 to +90) were examined by methylation-specific PCR and bisulfite genomic sequencing. Hypermethylated CpGs were found at -236 and -126 bp, whereas hypomethylated CpGs were found at -207 bp in arsenic-exposed cells. This study indicates that 20 nM sodium arsenite can alter myoblast differentiation by reducing the expression of the transcription factors myogenin and Mef2c, which is likely due to changes in promoter methylation patterns. The delay in muscle differentiation may lead to developmental abnormalities.
... The use of an established cell line enables easy gene modification and effortless drug screening without ethical issues and guarantees repeat experiments in other laboratories. The properties of C2C12 cells, such as electrophysiological properties or muscle protein expression, had been extensively studied and shown to mimic properties of in vivo skeletal muscle (Kubo, 1991;McMahon et al., 1994;Miller, 1990). Numerous muscle studies were carried out in C2C12 such as muscle differentiation (Andres and Walsh, 1996;Burattini et al., 2004), skeletal muscle tissue engineering (Dennis et al., 2001;Fujita et al., 2009), and effect of exercise (Fujita et al., 2007;Nedachi et al., 2008). ...
We have developed a novel method for measuring active tension generated by cultured myotubes using UV-crosslinked collagen film. Skeletal myoblasts cell line C2C12 or human primary skeletal myoblasts were seeded onto a thin (35 microm) collagen film strip, on which they proliferated and upon induction of differentiation they formed multinucleated myotubes. The collagen film-myotube complex contracted upon electric pulse stimulation which could be observed by light microscope. When collagen film-myotube complex were attached to force transducer, active tension generation was observed upon electric pulse stimulation. Measurement of active tension was possible for multiple times for more than 1 month with the same batch of collagen film-myotube complex. Active tension generation capability of C2C12 myotubes increased with progression of differentiation, reaching maximal value 6 days after induction of differentiation. Using this method, we measured the effect of artificial exercise induced by electric pulse on active tension generation capability of C2C12 myotubes. When the electric pulses of 1 Hz were continuously applied to induce artificial exercise, the active tension augmentation was observed. After 1 week of artificial exercise, the active tension reached approximately 10x of that before the exercise. The increased active tension is attributable to the formation of the sarcomere structure within the myotubes and an increased amount of myotubes on the collagen film. The increased amount of myotubes is possibly due to the suppressed atrophy of myotubes by enhanced expression of Bcl-2.
... All cell lines were grown according to ATCC-recommended protocols. Myoblast C2C12 cells were differentiated with DMEM in low-sodium bicarbonate and 2% horse serum for 6 d (Kubo 1991). ...
The Fox proteins are a family of regulators that control the alternative splicing of many exons in neurons, muscle, and other tissues. Each of the three mammalian paralogs, Fox-1 (A2BP1), Fox-2 (RBM9), and Fox-3 (HRNBP3), produces proteins with a single RNA-binding domain (RRM) flanked by N- and C-terminal domains that are highly diversified through the use of alternative promoters and alternative splicing patterns. These genes also express protein isoforms lacking the second half of the RRM (FoxDeltaRRM), due to the skipping of a highly conserved 93-nt exon. Fox binding elements overlap the splice sites of these exons in Fox-1 and Fox-2, and the Fox proteins themselves inhibit exon inclusion. Unlike other cases of splicing autoregulation by RNA-binding proteins, skipping the RRM exon creates an in-frame deletion in the mRNA to produce a stable protein. These FoxDeltaRRM isoforms expressed from cDNA exhibit highly reduced binding to RNA in vivo. However, we show that they can act as repressors of Fox-dependent splicing, presumably by competing with full-length Fox isoforms for interaction with other splicing factors. Interestingly, the Drosophila Fox homolog contains a nearly identical exon in its RRM domain that also has flanking Fox-binding sites. Thus, rather than autoregulation of splicing controlling the abundance of the regulator, the Fox proteins use a highly conserved mechanism of splicing autoregulation to control production of a dominant negative isoform.
... C2C12 is a skeletal muscle myoblast cell line derived from mouse. C2C12 will be differentiated into myotube in 2 % horse serum (Kubo, 1991). It has been previously shown by my colleague that TSPYL2 maybe involved in C2C12 differentiation (unpublished data). ...
Aging is a complex biological process which can be associated with skeletal muscle degradation leading to sarcopenia. The aim of this study consisted i) to determine the oxidative and inflammatory status of sarcopenic patients and ii) to clarify the impact of oxidative stress on myoblasts and myotubes. To this end, various biomarkers of inflammation (C-reactive protein (CRP), TNF-α, IL-6, IL-8, leukotriene B4 (LTB4)) and oxidative stress (malondialdehyde, conjugated dienes, carbonylated proteins and antioxidant enzymes: catalase, superoxide dismutase, glutathione peroxidase) as well as oxidized derivatives of cholesterol formed by cholesterol autoxidation (7-ketocholesterol, 7β-hydroxycholesterol), were analyzed. Apelin, a myokine which contributes to muscle strength, was also quantified. To this end, a case-control study was conducted to evaluate the RedOx and inflammatory status in 45 elderly subjects (23 non-sarcopenic; 22 sarcopenic) from 65 years old and higher. SARCopenia-Formular (SARC-F) and Timed Up and Go (TUG) tests were used to distinguish between sarcopenic and non-sarcopenic subjects. By using red blood cells, plasma and/or serum, we observed in sarcopenic patients an increased activity of major antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase) associated with lipid peroxidation and protein carbonylation (increased level of malondialdehyde, conjugated dienes and carbonylated proteins). Higher levels of 7-ketocholesterol and 7β-hydroxycholesterol were also observed in the plasma of sarcopenic patients. Significant differences were only observed with 7β-hydroxycholesterol. In sarcopenic patients comparatively to non-sarcopenic subjects, significant increase of CRP, LTB4 and apelin were observed whereas similar levels of TNF-α, IL-6 and IL-8 were found. The increased plasma level of 7-ketocholesterol and 7β-hydroxycholesterol in sarcopenic patients led us to study the cytotoxic effect of these oxysterols on undifferentiated (myoblasts) and differentiated (myotubes) murine C2C12 cells. With the fluorescein diacetate and sulforhodamine 101 assays, an induction of cell death was observed both on undifferentiated and differentiated cells: the cytotoxic effects were less pronounced with 7-ketocholesterol. In addition, IL-6 secretion was never detected whatever the culture conditions, TNF-α secretion was significantly increased on undifferentiated and differentiated C2C12 cells treated with 7-ketocholesterol- and 7β-hydroxycholesterol, and IL-8 secretion was increased on differentiated cells. 7-ketocholesterol- and 7β-hydroxycholesterol-induced cell death was strongly attenuated by α-tocopherol and Pistacia lentiscus L. seed oil both on myoblasts and/or myotubes. TNF-α and/or IL-8 secretions were reduced by α-tocopherol and Pistacia lentiscus L. seed oil. Our data support the hypothesis that the enhancement of oxidative stress observed in sarcopenic patients could contribute, especially via 7β-hydroxycholesterol, to skeletal muscle atrophy and inflammation via cytotoxic effects on myoblasts and myotubes. These data bring new elements to understand the pathophysiology of sarcopenia and open new perspectives for the treatment of this frequent age-related disease.
AAV (adeno-associated virus) gene transfer is a promising treatment for genetic abnormalities. Optimal AAV vectors are showing success in clinical trials. Gene transfer to skeletal muscle and liver is being explored as a potential therapy for some conditions i.e. Alpha-1-antitrypsin (AAT) disorder and hemophilia B. Exploring approaches that enhances transduction of liver and skeletal muscle using these vectors is beneficial for gene therapy. Regulating hormones as an approach to improve AAV transduction is largely unexplored. In this study we tested if an insulin therapy improves liver and skeletal muscle gene transfer. In-vitro studies demonstrated that the temporary co-administration (2hrs, 8hrs and 24hrs) of insulin significantly improves AAV2-CMV-LacZ transduction of cultured liver cells and differentiated myofibers but not lung cells. Additionally, there was a dose response related to this improved transduction. Interestingly, when insulin was not co-administered with the virus but given 24 hours after, there was no increase in the transgene product. Insulin receptor (INSR) gene expression levels were increased 5-13 fold in cultured liver cells and differentiated myofibers when compared to lung cells. Similar INSR gene expression profiles occurred in mouse tissues. Insulin therapy was performed in mice using a subcutaneously implanted insulin pellet or a high carbohydrate diet. Insulin treatment began just prior to intramuscular delivery of AAV1-CMV-schFIX or liver directed delivery of AAV8-CMV-schFIX and continued for 28 days. Both insulin augmentation therapies improved skeletal muscle and liver directed gene transduction in mice as seen by a 3.0 – 4.5 fold increase in human factor IX (hFIX) levels. The improvement was observed even after the insulin therapy ended. Monitoring insulin showed that insulin levels increased during the brief period of rAAV delivery and during the entire insulin augmentation period (28 days). This study demonstrates that AAV transduction of liver or skeletal muscle can be improved by an insulin therapy.
Uncoupling protein 3 (UCP3) and pyruvate dehydrogenase kinase 4 (PDK4) in skeletal muscle are key regulators of the glucose and lipid metabolic processes that are involved in insulin resistance. Medium-chain fatty acids (MCFAs) have anti-obesogenic effects in rodents and humans, while long-chain fatty acids (LCFAs) cause increases in body weight and insulin resistance. To clarify the beneficial effects of MCFAs, we examined UCP3 and PDK4 expression in skeletal muscles of mice fed a MCFA- or LCFA-enriched high-fat diet (HFD). Five-week feeding of the LCFA-enriched HFD caused high body weight gain and induced glucose intolerance in mice, compared with those in mice fed the MCFA-enriched HFD. However, the amounts of UCP3 and PDK4 transcripts in the skeletal muscle of mice fed the MCFA- or LCFA-enriched HFD were similar. To further elucidate the specific effects of MCFAs, such as capric acid (C10:0), on lipid metabolism in skeletal muscles, we examined the effects of various FAs on expression of UCP3 and PDK4, in mouse C2C12 myocytes. Although palmitic acid (C16:0) and lauric acid (C12:0) significantly induced expression of both UCP3 and PDK4, capric acid (C10:0) upregulated only UCP3 expression via activation of peroxisome proliferator-activated receptor-6. Furthermore, palmitic acid (C16:0) disturbed the insulin-induced phosphorylation of Akt, while MCFAs, including lauric (C12:0), capric (C10:0), and caprylic acid (C12:0), did not. These results suggest that capric acid (C10:0) increases the capacity for fatty acid oxidation without inhibiting glycolysis in skeletal muscle.
Recent innovation in the tissue engineering makes it real to construct the biological organ and tissue in vitro using proteins and living cells. This technology is not only for the regeneration of patients’ organ and tissue but also for the actuation of any artificial machine. The living muscles driven by the activation of actin-myosin molecular motors transform biochemical energy of ATP into mechanical energy. They have excellent characteristics of lightweight, high flexibility, and remarkable efficiency for energy conversion compared to the mechanical actuators that require electricity as a power source. Therefore, a muscle cell-based bio-hybrid actuator termed bio-actuator has the potential of being flexible and highly efficient on a micro to macro scale. The aim of this chapter is to describe the bio-actuator made of cultured skeletal muscle cells in vitro based on our investigation. There are still several issues to be solved for getting large and powerful bio-actuator which works long term with proven reliability, however it must be done in the near future by intensive studies of vigorous tissue-engineers.
The influence of differentiation medium (DM) components on C2C12 murine myoblast differentiation has only been studied in monolayer cultures. Serum-free formulations have been applied that omit the use of sera with unknown composition. The goal of the present study was to compare the influence of serum-free media on C2C12 differentiation in 3-dimensional tissue-engineered muscle constructs. Myoblast proliferation and differentiation in media containing Ultroser G (DMU), insulin-like growth factor (IGF)-I (DMI), or both (DMUI) were compared with those induced by more-traditional media containing horse serum (HS) or horse serum and IGF-I (HSI). Effects of the applied media were assessed from gross construct morphology, total protein content, creatine kinase activity, and tissue viability. Addition of IGF-I (HSI) to the standard DM (HS) improved myoblast differentiation in muscle constructs. Even better results were obtained using DMU and DMUI culture conditions. DMI could not induce differentiation or maintain cell viability. Serum-free culture medium supplemented with DMU or DMUI accelerates and improves myoblast differentiation in engineered muscle tissue better than the gold standard HS.
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Fibroblast L929 and myoblast C2C12 cells of the mouse connective tissue origin were sown on the surface of conductive polymer films (polypyrrole, PPy and poly(3,4-ethylenedioxythiophene), PEDOT) in the cell culture medium, and the proliferative process of these cells was observed. Without changing the form, fibroblast L929 and myoblast C2C12 cells were observed to proliferate almost similarly to the cell which cultured on a dish on the market and to maintain compatibility. In other word, it has been understood that these two kinds of conductive polymers used in this study, the PPy and PEDOT films maintain the secretion function of the cell cultured on the surface of these polymers. Therefore, the PPy- and the PEDOT-coated electrode suggested the possibility usable as a nerve stimulation electrode with biocompatibility, because these polymers were effective to culture the cell.
The majority of studies of the functional distribution of purinoceptors have been carried out with mammalian preparations. The objective of this article is to review the disparate literature describing purinoceptor-mediated effects in invertebrates and lower vertebrates and, in view of the concept that ontogeny repeats phylogeny, to review also the evidence for purinoceptor involvement in the complex signaling involved in embryonic development. Even with the limited information currently available, it is clear that purinoceptors are involved in early signalling in vertebrate embryos; one novel G protein-coupled P2Y receptor has already been cloned and characterized in frog embryo and hopefully more will follow. It is also clear that purinoceptors for both adenosine and ATP are present in early evolution and play a number of different roles in most, if not all, invertebrate and lower species. However, until selective agonists and antagonists are identified for the recently cloned purinoceptors subtypes in vertebrates, it will not be possible to resolve questions concerned with the evolution of these subtypes. Molecular cloning of genes encoding receptors for purines and pyrimidines from invertebrates and lower vertebrates represents an alternative approach to advancing knowledge in the area. Drug Dev. Res. 39:204–242, 1996. © 1997 Wiley-Liss, Inc.
Resveratrol is one of the most widely studied bio-active plant polyphenols. While its effect on endothelial blood vessel cells, cancer cells, inflammatory processes and neurodegenerative events is well documented, little is known about the implication of this phytophenol in differentiating processes, particularly in skeletal muscle cells. Here, we report the effects of resveratrol on mouse skeletal muscle-derived cells (C2C12) in either a nondifferentiated (myoblasts) or differentiated state (myotubes) by evaluating resveratrol uptake, cell proliferation, changes in cell shape, and the expression of genes encoding muscle-specific transcription factors or contractile proteins. Resveratrol: (1) rapidly accumulates within cells through passive and facilitated processes; (2) does not strongly affect cell viability, cell cycle and apoptosis; (3) behaves as a pro-differentiating agent as shown by the lengthening of cells, leading to a myotube phenotype; (4) upregulates muscular pro-differentiation markers and transcription factors (myogenin, Scrp3) starting after 12h of exposure and strongly increases heavy chain myosin content after 18h of exposure to resveratrol; (5) increases the Srf transcription factor's transcript level, a target mRNA of the miRNA-133b, which is itself downregulated by this polyphenol. These results put forward new pro-differentiating regulatory properties of resveratrol on skeletal muscles at least partly via modulation of specific miRNAs.
A potassium inward rectifier (Kir) current was previously shown by us to be induced in primitive hematopoietic progenitor cells, stimulated with the combination of interleukin-3 (IL-3) and stem cell factor (SCF). Biophysical features of whole cell currents implicated the involvement of more than one Kir channel type. Employing IL-3 + SCF stimulated human cord blood CD34+38− cells, we isolated and characterized different components of this current. Reverse transcription-polymerase chain reaction (RT-PCR) subcloning identified the expression of a strongly rectifying Kir channel (Kir 4.3) as well as a weakly rectifying Kir channel (Kir 1.1) in these cells. Inhibition of the expression of each of the channels suppressed progenitor cell generation by IL-3 and SCF-stimulated CD34+38− cells in 7-day suspension cultures. The variable expression of two essential inward rectifying potassium channels early in the course of hematopoietic progenitor cell differentiation may play a potentially important role in potassium homeostasis in these cells. J. Cell. Physiol. 177:197–205, 1998. © 1998 Wiley-Liss, Inc.
Excess glucocorticoids induce insulin resistance and reduce glucose uptake although the underlying mechanisms are unclear. Here we demonstrate that Dex (1 μM for 24 h) inhibits basal and insulin (1 nM) stimulated glucose uptake in human and murine adipocytes by 50% with a concomitant reduction in the levels of GLUT1/4 at the plasma membrane but no change in total GLUT1/4 levels. Expression and phosphorylation of proximal insulin signalling molecules (IRS1, PI3K, AKT) was unaffected by Dex as was phosphorylation of mTOR and FOXO1. In contrast, phosphorylation of AKT substrate 160 kDa (AS160) at T642, which is essential for 14-3-3 recruitment and GLUT4 translocation, was reduced by 50% in basal and insulin-stimulated cells and this was mirrored by decreased 14-3-3 association. Co-treatment with the glucocorticoid receptor antagonist RU486 (10 μM) abrogated the Dex effect on AS160-T642 phosphorylation and restored glucose uptake by 80%. These data suggest Dex inhibits glucose uptake in adipocytes, at least in part, by reducing AS160 phosphorylation and interaction with 14-3-3.
We previously reported that 1α,25-dihydroxy-vitamin D(3) [1α,25(OH)(2)D(3)] induces non-transcriptional rapid responses through activation of Src and MAPKs in the skeletal muscle cell line C2C12. In the present study we investigated the modulation of Akt by the secosteroid hormone in C2C12 cells at proliferative stage (myoblasts) and at early differentiation stage. In proliferating cells, 1α,25(OH)(2)D(3) activates Akt by phosphorylation in Ser473 in a time-dependent manner (5-60 min). When these cells were pretreated with methyl-beta-cyclodextrin to disrupt caveolae microdomains, hormone-induced activation of Akt was suppressed. Similar results were obtained by siRNA silencing of caveolin-1 expression, further indicating that hormone effects on cell membrane caveolae are required for downstream signaling. PI3K and p38 MAPK, but not ERK1/2, participate in 1α,25(OH)(2)D(3) activation of Akt in myoblasts. The involvement of p38 MAPK in Akt phosphorylation by the hormone probably occurs through MAPK-activated protein kinase 2 (MK2), which is activated by the steroid. In addition, the participation of Src in Akt phosphorylation by 1α,25(OH)(2)D(3) was demonstrated using the inhibitor PP2 and antisense oligodeoxynucleotides that suppress Src expression. We also observed that PI3K participates in hormone-induced proliferation. During the early phase of C2C12 cell differentiation 1α,25(OH)(2)D(3) also increases Akt phosphorylation and activates Src. Of relevance, Src and PI3K are involved in Akt activation and in MHC and myogenin increased expression by 1α,25(OH)(2)D(3). Altogether, these data suggest that 1α,25(OH)(2)D(3) upregulates Akt through Src, PI(3)K, and p38 MAPK to stimulate myogenesis in C2C12 cells.
The aim of this study was to investigate whether the Kir2.1 gene is expressed by the human placenta throughout pregnancy and in cytotrophoblast cells at different stages of differentiation in culture. RNA was extracted from cytotrophoblast cells isolated from term placentas and maintained in culture for 18, 66 and 114 h and from first, second and third trimester placentas. Using the reverse transcriptase-polymerase chain reaction (RT-PCR) with gene-specific primers, a cDNA product of 1.2 kb, as expected for Kir2.1 gene expression, was detected in all the RNA samples from cytotrophoblast cells and from placentas. The RT-PCR products were verified by sequencing and by detection of the expected transcript size for the Kir2.1 mRNA at 5.6-5.7 kb on Northern blots, using the 1.2 kb cDNA generated by RT-PCR. Northern blot quantification, using a control 28S rRNA probe, showed no significant difference in Kir2.1 mRNA expression between any of the three stages of cytotrophoblast cell differentiation studied (ANOVA; n = 3 RNA samples from each stage). These data demonstrate that the Kir2.1 gene is expressed by the human placenta and, specifically, by cytotrophoblast cells, at all stages of development and differentiation.
Obesity is characterized by the accumulation of triacylglycerol in adipocytes. Coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) is one of two known DGAT enzymes that catalyze the final and only committed step in triacylglycerol synthesis. In this report, we describe the pharmacological effects of a novel selective DGAT1 inhibitor, Compound-A. This compound inhibited triacylglycerol synthesis in both adipocytes and skeletal myotubes, and increased fatty acid oxidation in skeletal myotubes at 1 μM. The repeated administration of Compound-A to diet-induced obese C57BL/6J and genetically obese KKA(y) mice (3-30 mg/kg for 3-4 weeks) significantly decreased the visceral fat pad weights and the hepatic lipid contents compared to controls without affecting food intake. In addition, fatty acid oxidation in skeletal muscle tissues was increased by the treatment of Compound-A in both mice strains. This is the first report demonstrating that a small synthetic DGAT1 inhibitor increases fatty acid oxidation in skeletal muscle in vitro and ex vivo. These results suggest that DGAT1 inhibition is a promising therapeutic approach for the treatment of obesity and lipid abnormalities such as hepatic steatosis.
Intracellular free Ca2+ is one of important biological signals regulating a number of cell functions. It has been discussed widely and extensively in several cell types during the past two decades. Attention has been paid to the Ca2+ transportation in mesenchymal stem cells in recent years as mesenchymal stem cells have gained considerable interest due to their potential for cell replacement therapy and tissue engineering. In this paper, roles of intracellular Ca2+ oscillations and its transporters in mesenchymal stem cells have been reviewed.
To evaluate whether rat fetal brain stem cells can be induced to acquire cell fates outside the nervous system, hypothesising that cell-based replacement therapy with stem cells can aid in the regeneration of penile smooth musculature and might help to attenuate organic erectile dysfunction (ED), as the degeneration of penile smooth muscle cells leading to subsequent impairment of function is important in organic ED.
Fetal brain stem cells (FBSCs) from embryonal 12-day Fisher 344 rats were isolated and characterized. For in vitro studies, undifferentiated FBSCs were cultured for 21 days in either N2 media (control) or N2 media conditioned in rat penile smooth muscle cell culture. These were then subjected to immunocytochemistry for specific markers of neural stem cells (nestin) and penile smooth muscle cells, i.e. alpha-smooth muscle actin (alphaSMA), penis-specific myosin light chain (MLC) desmin, calponin, vimentin, phosphodiesterase-5 (PDE5) and connexin. For in vivo studies, male adult Fisher 344 rats had an intracavernous injection with saline (five rats, control) or FBSCs that were labelled genetically by an expression construct for green fluorescent protein (GFP, nine rats, experimental) and maintained for 6 weeks. The rats were then killed and penile tissue was harvested and subjected to immunocytochemistry for markers of neural stem cells, smooth muscle cells, and sinusoidal endothelium (vascular endothelial growth factor, VEGF).
Undifferentiated cells exposed to N2 media continued to maintain the characteristic morphological and protein marker features of FBSCs, while the cells exposed to the conditioned media acquired the morphological features of smooth muscle cells. In addition, the differentiated cells (30-40%) expressed smooth muscle markers. Rats implanted with FBSCs had cells that showed double-labelling for GFP/alphaSMA, GFP/calponin and GFP/VEGF. The control group had no evidence of such double-labelling.
These results suggest the transdifferentiation of FBSCs into penile smooth muscle cells. Such transdifferentiated cells showed long-term survival when injected into the cavernous tissue, thus raising the possibility of a novel therapeutic option for organic ED.
1. A mesodermal stem cell line C3H10T1/2 was induced to differentiate to muscle by adding 0.3 microM-5-aza-2'-deoxy-cytidine to the medium for 24 h. The changes in membrane currents during differentiation were studied by whole-cell recording and changes in the expression of fibronectin, Neural Cell Adhesion Molecule (NCAM), myosin and desmin were studied immunohistochemically. 2. The stem cells showed the morphology of fibroblastic cells. Most of the stem cells showed ATP-induced slow K+ current. T-type Ca2+ current and inward rectifier K+ current were observed in 19% of the stem cells. The stem cells expressed fibronectin, but not NCAM, myosin or desmin. 3. About 2 weeks after the addition of 5-aza-2'-deoxy-cytidine, large multinucleated skeletal muscle-like cells appeared. Most of the induced muscles showed L-type Ca2+ current, responses to acetylcholine, outward K+ current, inward rectifier K+ current and contraction upon depolarizing stimulation. They expressed NCAM, myosin and desmin, but not fibronectin, and showed no ATP response. 4. In some batches (2/14), the induced muscles showed spontaneous twitches, and possessed tetrodotoxin (TTX)-sensitive Na+ current in addition to the currents described above. Furthermore clear striation was observed in some of the twitching muscles under Nomarski optics. 5. To ascertain the properties of cells at the initial step of muscle differentiation, whose differentiation is determined but not yet evident morphologically or electrophysiologically, subcloning was performed from the heterogeneous cells 10 days after induction. Three myogenic clones were obtained, which proliferated at low cell densities but differentiated to muscle with a high incidence at high cell densities, as well as ten non-myogenic clones. 6. Most myogenic clones still showed ATP-induced K+ current and fibronectin. In addition, most of them showed T-type Ca2+ current and inward rectifier K+ current. They had already expressed NCAM. No other properties observed in muscles had yet been expressed. Most cells of the non-myogenic clones showed ATP-induced K+ current and fibronectin. T-type Ca2+ current was also expressed, but not inward rectifier K+ current or NCAM. 7. The properties of the observed ionic currents were studied. The TTX-sensitive Na+ current could be completely blocked by 0.1 microM-TTX. It could be evoked by depolarizing steps to a level above -40 mV, while steady-state inactivation was detectable around -75 mV and reached half by -52 mV. T-type Ca2+ current could be evoked by a depolarizing pulse to a level above -45 mV, with a maximum amplitude around -15 mV.(ABSTRACT TRUNCATED AT 400 WORDS)
1. ATP was puff applied to cells of a mesodermal stem cell line, C3H10T1/2, and the responses were studied by whole-cell patch clamp recording. 2. In 91% of the cells (90/99), K+ current lasting for tens of seconds was observed after several seconds latency. The current showed outward rectification. In 10% of the cells (9/99), ATP induced Cl- current which also lasted for tens of seconds after several seconds latency, but showed little rectification. In 6% of these cells (5/99), both K+ and Cl- currents were induced by ATP. 3. The K+ current induced by ATP was dose dependent, with a Kd of 0.4 microM. The effects of ATP analogues were tested at a concentration of 20 microM. ADP and ATP-gamma-S induced the K+ current, while AMP and adenosine did not. alpha, beta-Methylene ATP produced a diminished K+ current. 4. The ATP-induced K+ current was not observed when EGTA in the internal solution was raised from 0.1 to 5 mM. In Fluo-3-loaded cells, an increase in intracellular Ca2+ concentration induced by the application of ATP was observed, and the time course was similar to the induced K+ current. Both the increase in intracellular Ca2+ and the K+ current were induced by ATP even in Ca(2+)-free external solution. Ryanodine (50 microM) in the external solution did not affect the ATP response, and application of 10 mM-caffeine alone to the external solution did not induce any response. 5. The variance of the steady-state fluctuations in the course of the ATP-induced slow K+ current was analysed. The single-channel conductance was estimated as 2.7 pS at 0 mV with external and internal K+ concentrations of 5 and 140 mM respectively. The K+ current was not affected by apamin at concentrations of up to 1 microM but was reduced to one-third by 140 mM-tetraethylammonium (TEA). 6. It was concluded that puff-applied ATP has two main effects in the mesodermal stem cells: an increase in the intracellular Ca2+ concentration and a succeeding hyperpolarization due to the Ca(2+)-activated K+ conductance which is present in this cell. The significance of the increase in intracellular Ca2+ caused by ATP is discussed.
Many properties of skeletal muscle are closely regulated by motor nerves. We have shown that nerve stimulation in vivo induced a rapid rise in mRNA for the immediate early gene (IEG) zif268 in stimulated muscle. However, the mechanisms leading to neural regulation of zif268 gene expression in muscle are not yet known. In this study, we used a mammalian skeletal muscle cell line (C2C12) to analyze the role of cholinergic transmission, and calcium flux, in the neural regulation of zif268. Treatment of the C2C12 muscle cells with carbachol, a cholinergic agonist, induced zif268 gene expression rapidly and transiently. This effect was blocked by alpha-bungarotoxin (alpha-BuTx), which specifically blocks nicotinic acetylcholine receptors. Treatment with ryanodine or dantrolene, which block the calcium release channel of the sarcoplasmic reticulum, inhibited the carbachol-induced zif268 response essentially completely. Calcium influx produced by A23187, a calcium ionophore, induced an increase of zif268 gene expression equivalent to the effect of carbachol stimulation. These results suggest that the effect of neural stimulation on zif268 may be attributable to cholinergic transmission, and the subsequent release of calcium from the sarcoplasmic reticulum.
K+ conductances dominate and potentially modulate the resting potential of skeletal muscle cells. The expression and modulation of a major K+ conductance were examined during in vitro differentiation of the mouse myoblast cell line C2C12. The inwardly rectifying K+ conductance (IKi) increased from unmeasurable levels in undifferentiated myoblasts to approximately 1.56 +/- 0.51 nA (n = 17) in myoballs derived from myotubes at 5 days after induction of differentiation. The inward rectifier was subject to modulation by intracellular signals. Exposure of cytoplasm to guanosine 5'-O-(3-thiotriphosphate) during whole cell recording produced a concentration (5-100 microM)- and time (1-20 min)-dependent inhibition of the mean conductance. Elevation of intracellular free Ca2+ (> 200 nM) also inhibited IKi. These findings demonstrate a potential mechanism for modulation of the resting potential of muscle fibers via the control of skeletal muscle IKi.
Primary cultures from enzymatically dissociated satellite cells of newborn rat skeletal muscles enabled developmental in vitro studies of mechanical and electrical properties during the first steps of myogenesis. The present work focused on the appearance, evolution and roles of two types of calcium currents (ICa,T and ICa,L) and of depolarization-induced contractile activity during the early stages of muscle cell development in primary culture. Prefusional mononucleated cells (myoblasts), young myotubes of 1 day (with less than 10 nuclei) or 2-3 days (more than 9 nuclei) and myoballs from 4-6, 7-9, 10-12 and 13-16 days cultures were patch-clamped (whole-cell configuration), and calcium currents and contraction simultaneously recorded. Sodium but not calcium currents could be recorded at the myoblast stage. In young myotubes (1 day), ICa,L was present with high incidence as compared to ICa,T, which was poorly expressed. Contractile responses appeared at the next stage (2-3 days) while the occurrence of ICa,T progressively increased. This developmental evolution of the calcium currents and contraction expression was accompanied by some changes in their characteristics: the ICa,T/ICa,L amplitudes ratio progressively increased and the time-to-peak of contraction progressively decreased with the age of myoballs. Physiological functions for calcium currents in developing muscle are suggested and discussed: ICa,T, which is transiently expressed, could be involved in the pacemaker-like activity while ICa,L could serve as an early contraction triggering mechanism and/or initially to fill and then to maintain the intracellular calcium stores.
We have used the whole cell configuration of the patch-clamp technique to investigate volume-activated Cl- currents in BC3H1 and C2C12 cells, two mouse muscle cell lines that can be switched from a proliferating to a differentiated musclelike state. Reducing the extracellular osmolality by 40% evoked large Cl- currents in proliferating BC3H1 and C2C12 cells. These currents were outwardly rectifying and had an anion permeability sequence as follows: I- > Br- > Cl- > gluconate. They were inhibited by >50% by flufenamic acid (500 microM), niflumic acid (500 microM), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM) but were relatively insensitive to tamoxifen (100 microM). A reduction in the serum concentration in the culture medium induced growth arrest in both cell lines, and the cells started to differentiate into spindle-shaped nonfusing muscle cells (BC3H1) or myotubes (C2C12). This differentiation was accompanied by a drastic decrease in the magnitude of the volume-activated Cl- currents. The close correlation between volume-activated Cl- currents and cell proliferation suggests that these currents may be involved in cell proliferation.
An immortal, cloned cell line (RCMH), obtained from human skeletal muscle was established in our laboratory and shown to express muscle specific proteins. We measured ligand binding to ion channels, ion currents using whole cell patch clamp and intracellular calcium both in cells grown in complete media and in cells grown for 4-40 days in media supplemented with hormones and nutrients (differentiating media). Markers for differentiated muscle, such as the muscle isoform of creatine kinase and the cytoskeletal proteins alpha-actinin, alpha-sarcomeric actin, myosin and titin were present in early stages. Receptors for gamma toxin from Tityus serrulatus scorpion venom, a specific modulator for voltage dependent sodium channels, were present (0.9-1.0 pmol mg-1 protein) during stage 1 (0-6 days in culture with differentiating media) and increased by 50% in stage 3 (more than 10 days in differentiating media). High and low affinity dihydropyridine receptors present in stage 1 convert into a single type of high affinity receptors in stage 3. Both intracellular calcium release and InsP3 receptors were evident in stage 1 but ryanodine receptors were expressed only in stage 3. RCMH cells showed no voltage sensitive currents in stage 1. Between 7 and 10 days in differentiating media (stage 2), an outward potassium current was observed. Small inward currents appeared only in stage 3; we identified both tetrodotoxin sensitive and tetrodotoxin resistant sodium currents as well as calcium currents. This pattern is consistent with the expression of voltage dependent calcium release before appearance of both the action potential and ryanodine receptors.
Cell-to-cell communication plays important roles in development and in tissue morphogenesis. Gap junctional intercellular communication (GJIC) has been implicated in embryonic development of various tissues and provides a pathway to exchange ions, secondary messengers, and metabolites through the intercellular gap junction channels. Although GJIC is absent in adult skeletal muscles, the formation of skeletal muscles involves a sequence of complex events including cell-cell interaction processes where myogenic cells closely adhere to each other. Much experimental evidence has shown that myogenic precursors and developing muscle fibers can directly communicate through junctional channels. This review summarizes current knowledge on the GJIC and developmental events involved in the formation of skeletal muscle fibers and describes recent progress in the investigation of the role of GJIC in myogenesis: evidence of gap junctions in somitic and myotomal tissue as well as in developing muscle fibers in situ, GJIC between perfusion myoblasts in culture, and involvement of GJIC in cytodifferentiation of skeletal muscle cells and in myoblast fusion. A model of intercellular signaling is proposed where GJIC participates to coordinate a multicellular population of interacting myogenic precursors to allow commitment to the skeletal muscle fate.
In myocytes, calcium plays an important role in intracellular signaling and contraction. However, the ability of calcium to
modulate the differentiation of striated muscle cells is poorly understood. To examine this issue we studied C2C12 cells,
which is a myoblast cell line that differentiates in vitro. First, we observed that the L-type calcium channel blockers nifedipine and verapamil effectively inhibited electrically
induced calcium transients. Next, C2C12 cells were exposed to these agents during conditions that induce myocyte differentiation.
In the presence of nifedipine and verapamil, myoblasts failed to form myotubes. Dantrolene and thapsigargin, which decrease
intracellular calcium by different mechanisms, also inhibited differentiation. In addition, nifedipine and verapamil inhibited
the expression of myosin heavy chain and myogenin, two markers of skeletal myoblast differentiation. In contrast, levels of
the transcriptional factor Myf5, which is expressed in undifferentiated myoblasts, did not decline. Calcium channel blockade
also prevented the expression of a reporter driven by the skeletal muscle α-actin promoter. These data demonstrate that lowering
intracellular calcium levels inhibits the differentiation of skeletal myoblasts into mature myotubes.
Human mesenchymal stem cells (HMSC) have the potential to differentiate into many cell types. The physiological properties of HMSCs including their Ca(2+) signaling pathways, however, are not well understood. We investigated Ca(2+) influx and release functions in HMSCs. In Ca(2+) imaging experiments, spontaneous Ca(2+) oscillations were observed in 36 of 50 HMSCs. The Ca(2+) oscillations were completely blocked by the application of 10 micro M cyclopiazonic acid (CPA) or 1 micro M thapsigargin (TG). A brief application of 1 micro M acetylcholine (ACh) induced a transient increase of [Ca(2+)](i) but the application of caffeine (10 mM) did not induce any Ca(2+) transient. When the stores were depleted with Ca(2+)-ATPase blockers (CPA or TG) or muscarinic agonists (ACh), store-operated Ca(2+) (SOC) entry was observed. Using the patch-clamp technique, store-operated Ca(2+) currents (I(SOC)) could be recorded in cells treated with ACh or CPA, but voltage-operated Ca(2+) currents (VOCCs) were not elicited in most of the cells (17/20), but in 15% of cells examined, small dihydropyridine (DHP)-sensitive Ca(2+) currents were recorded. Using RT-PCR, mRNAs were detected for inositol 1,4,5-trisphosphate receptor (InsP(3)R) type I, II, and III and DHP receptors alpha1A and alpha1H were detected, but mRNA was not detected for ryanodine receptor (RyR) or N-type Ca(2+) channels. These results suggest that in undifferentiated HMSCs, Ca(2+) release is mediated by InsP(3)Rs and Ca(2+) entry through plasma membrane is mainly mediated by the SOCs channels with a little contribution of VOCCs.
Sodium currents in cultured rat muscle cells converted to myoballs by treatment with colchicine were recorded using a giga-ohm seal voltage clamp procedure in the whole cell configuration. The mean peak Na+ conductance of the myoballs was 90 pS/microns2 of surface membrane. Half-maximal activation of Na+ currents was observed for test pulses to -31 mV and half-maximal inactivation was observed for prepulses to -74 mV. Titration of the inhibition of Na+ currents by tetrodotoxin (TTX) yielded a biphasic inhibition curve consistent with the presence of two classes of Na+ channels differing in affinity for TTX. The TTX-sensitive channels carried 28% of the Na+ current and had an apparent KD for TTX of 13 nM at 20 degrees C. The TTX-insensitive Na+ channels had an apparent KD for TTX of 3.2 microns. Inhibition of TTX-insensitive Na+ channels by TTX was enhanced by repetitive stimulation of the myoballs at 2 Hz, whereas the inhibition of TTX-sensitive Na+ channels by TTX was not frequency dependent. We conclude that rat muscle cells developing in vitro synthesize physiologically functional, TTX-sensitive Na+ channels in the absence of innervation. These channels, which are characteristic of adult skeletal muscle, function in parallel with TTX-insensitive Na+ channels that are present in embryonic muscle.
Heterokaryons provide a model system in which to examine how tissue-specific phenotypes arise and are maintained. When muscle
cells are fused with nonmuscle cells, muscle gene expression is activated in the nonmuscle cell type. Gene expression was
studied either at a single cell level with monoclonal antibodies or in mass cultures at a biochemical and molecular level.
In all of the nonmuscle cell types tested, including representatives of different embryonic lineages, phenotypes, and developmental
stages, muscle gene expression was induced. Differences among cell types in the kinetics, frequency, and gene dosage requirements
for gene expression provide clues to the underlying regulatory mechanisms. These results show that the expression of genes
in the nuclei of differentiated cells is remarkably plastic and susceptible to modulation by the cytoplasm. The isolation
of the genes encoding the tissue-specific trans-acting regulators responsible for muscle gene activation should now be possible.
Single- (whole-cell patch) and two-electrode voltage-clamp techniques were used to measure transient (Ifast) and sustained (Islow) calcium currents, linear capacitance, and slow, voltage-dependent charge movements in freshly dissociated fibers of the flexor digitorum brevis (FDB) muscle of rats of various postnatal ages. Peak Ifast was largest in FDB fibers of neonatal (1-5 d) rats, having a magnitude in 10 mM external Ca of 1.4 +/- 0.9 pA/pF (mean +/- SD; current normalized by linear fiber capacitance). Peak Ifast was smaller in FDB fibers of older animals, and by approximately 3 wk postnatal, it was so small as to be unmeasurable. By contrast, the magnitudes of Islow and charge movement increased substantially during postnatal development. Peak Islow was 3.6 +/- 2.5 pA/pF in FDB fibers of 1-5-d rats and increased to 16.4 +/- 6.5 pA/pF in 45-50-d-old rats; for these same two age groups, Qmax, the total mobile charge measurable as charge movement, was 6.0 +/- 1.7 and 23.8 +/- 4.0 nC/microF, respectively. As both Islow and charge movement are thought to arise in the transverse-tubular system, linear capacitance normalized by the area of fiber surface was determined as an indirect measure of the membrane area of the t-system relative to that of the fiber surface. This parameter increased from 1.5 +/- 0.2 microF/cm2 in 2-d fibers to 2.9 +/- 0.4 microF/cm2 in 44-d fibers. The increases in peak Islow, Qmax, and normalized linear capacitance all had similar time courses. Although the function of Islow is unknown, the substantial postnatal increase in its magnitude suggests that it plays an important role in the physiology of skeletal muscle.
We have analyzed the biophysical and developmental properties of Ca2+ and Na+ currents in C2 muscle cells, whose morphological and biochemical phenotype closely resembles differentiated skeletal muscle. Both fused and unfused C2 myocytes possessed: (1) membrane capacitance consistent with the presence of complex sarcotubular invaginations, (2) tetrodotoxin-sensitive Na+ channels, and (3) "fast" and "slow" Ca2+ channels that inactivated at holding potentials of -40 and -20 mV, respectively. Thus, the passive electrical properties, Na+ currents, and Ca2+ currents expressed in C2 cells each differed from those found in the nonfusing muscle cell line, BC3H1, and corresponded more precisely to characteristic findings observed in skeletal muscle fibers. In further contrast to BC3H1 cells, C2 muscle also expressed "transient" Ca2+ channels similar to those reported in embryonic or neonatal skeletal muscle, which were detected within 12-24 hr of mitogen withdrawal, up to 60 hr before appearance of "fast" and "slow" currents. Na+ channels also were induced 12-24 hr after mitogen withdrawal. Unlike the "fast" and "slow" Ca2+ currents, which were maximally expressed at 8-14 d of serum withdrawal, "transient" Ca2+ channels became down-regulated upon prolonged differentiation (as found in postnatal skeletal muscle in vivo) and were no longer expressed at 14 d. Despite their divergent kinetic and developmental properties, all components of Ca2+ and Na+ current in C2 myocytes were suppressed reversibly in the presence of transforming growth factor beta-1, a purified growth factor that inhibits the myogenic phenotype. The results indicate that fusion is not essential for skeletal myoblasts to produce developmentally regulated voltage-gated channels that resemble those of intact muscle and demonstrate that the formation of diverse Ca2+ and Na+ channels can be mediated by a single peptide that affects the myogenic pathway.
We have examined the expression of MyoD1, a potential determination factor of myogenic cells, in permissive and inducible C2 myoblasts. These two types of myoblasts exhibit distinct requirements to undergo terminal differentiation. Unlike permissive cells, inducible cells fail to differentiate in the presence of growth medium plus fetal calf serum and require insulin to undergo terminal differentiation. We show that while expression of MyoD1 is constitutive in permissive cells, no trace of MyoD1 transcripts is found in inducible cells at the myoblast stage. In these cells, however, expression of MyoD1 accompanies differentiation. This indicates that MyoD1 may not be required for the maintenance of the myoblast phenotype, and could act as an effector of terminal differentiation in already determined muscle cells. Our results provide new evidence that permissive and inducible cells represent two distinct stages of the progression of determined muscle cells toward terminal differentiation.
5-azacytidine treatment of mouse C3H10T1/2 embryonic fibroblasts converts them to myoblasts at a frequency suggesting alteration of one or only a few closely linked regulatory loci. Assuming such loci to be differentially expressed as poly(A)+ RNA in proliferating myoblasts, we prepared proliferating myoblast-specific, subtracted cDNA probes to screen a myocyte cDNA library. Based on a number of criteria, three cDNAs were selected and characterized. We show that expression of one of these cDNAs transfected into C3H10T1/2 fibroblasts, where it is not normally expressed, is sufficient to convert them to stable myoblasts. Myogenesis also occurs, but to a lesser extent, when this cDNA is expressed in a number of other cell lines. The major open reading frame encoded by this cDNA contains a short protein segment similar to a sequence present in the myc protein family.
There are three stages of differentiation of voltage dependent Na+ channels during the in vitro development of rat skeletal muscle.(i)
Myoblasts which are less than 60 h old in culture have Na+ channels which normally do not give rise to action potentials but do so after treatment of the cells with very low concentrations of sea anemone toxin. These Na+ channels revealed by sea anemone toxin are resistant to TTX.
(ii)
Myoblasts prior to fusion are electrically excitable (\(\dot V\)
max=10 V/s). Electrically activated Na+ channels are only blocked by high concentrations of TTX. Titration of TTX resistant Na+ channels with a tritiated derivative of TTX indicates a dissociation constant of the TTX-Na+ channel complex of 50 nM.
(iii)
Myotubes have both high and low affinity binding sites for TTX (Frelin et al. 1983). Action potentials (\(\dot V\)
max=100−200 V/s) are only inhibited at high concentrations of TTX. Experiments with rat myoballs indicate that only Na+ channels with a low affinity binding site for TTX are functional in voltage-clamp studies. The K0.5 value for TTX inhibition of the peak Na+ current is observed at 70 nM. Spontaneous contractions of myotubes are blocked by TTX with a K0.5 value of 100 nM, suggesting that TTX resistant Na+ channels are also the ones responsible for the spontaneous contractions in rat myotubes in culture.
22Na+ flux studies after activation of the Na+ channel with neurotoxins have been carried out at the different stages of differentiation. Toxin activated Na+ channels have the same high affinity for sea anemone toxins at all stages of development; likewise, the sensitivity for TTX is the same.
The in vitro differentiation of Na+ channels in rat and chick skeletal muscle cells is compared and discussed in relation to the action of neurotrophic influences.
5-AZACYTIDINE (aza-CR)-a s-triazine nucleoside analogue of cytidine-has strong inhibitory effects in various biological systems including neoplasms: it is a powerful bacteriostatic, antitumour and mutagenic agent1. Aza-CR also exhibits immunosuppressive, antimitotic, radioprotective and virostatic effects1. The cytostatic effect of aza-CR has primarily been directed against leukaemia2 and it has been used to treat human solid tumours3. During a study on the morphological transformation of C3H 10T½ C18 cells by cancer chemotherapeutic agents, it was noted that elongated multinucleated cells arose in cultures exposed to aza-CR4. The C3H 10T½ C18 line is a clonal line of mouse embryo cells developed at the McArdle Laboratory for Cancer Research for use in studies on chemical oncogenesis in vitro5-10. The cells are highly sensitive to post-confluence inhibition of cell division, show a remarkably low rate of spontaneous transformation and grow with a fibroblast-like morphology with long cytoplasmic processes in sparse cultures. When confluent, they form flat even monolayers and appear epithelioid. Scanning electron microscopy has shown the confluent cells to be extremely thin and polygonal with smooth surfaces resembling epithelial cells11. Here we describe experiments showing that the multinucleated tubular cells which arise in C3H 10T½ C18 cells treated with aza-CR are in fact functional myotubes.
Three new mesenchymal phenotypes were expressed in cultures of Swiss 3T3 and C3H/10T1/2CL8 mouse cells treated with 5-azacytidine or 5-aza-2'-deoxycytidine. These phenotypes were characterized as contractile striated muscle cells, biochemically differentiated adipocytes and chondrocytes capable of the biosynthesis of cartilage-specific proteins. The number of muscle and fat cells which appeared in treated cultures was dependent upon the concentration of 5-azacytidine used, but the chondrocyte phenotype was not expressed frequently enough for quantitation. The differentiated cell types were only observed several days or weeks after treatment with the analog, implying that cell division was obligatory for the expression of the new phenotypes. Oncogenically transformed C3H/10T1/2CL8 cells also developed muscle cells after exposure to 5-azacytidine, but at a reduced rate when compared to the parent line. Five subclones of the 10T1/2 line which were the progeny of single cells all expressed both the muscle and fat phenotypes following 5-azacytidine treatment. The effects of the analog are therefore not due to the selection of preexisting myoblasts or adipocytes in the cell populations. Rather, it is possible that 5-azacytidine, after incorporation into DNA, causes a reversion to a more pluripotential state from which the new phenotypes subsequently differentiate.
THE muscular dystrophies are a group of hereditary disorders manifested by a progressive wasting of the skeletal muscles. In spite of extensive studies, the nature of the primary lesion is unknown (for review see ref. 1). Because of the complex interaction between tissues, it is difficult to study this question in vivo. Therefore attempts have been made to investigate this question in cultures of dystrophic muscles of human or animal origin. Tissue explants as well as monolayer primary cell cultures contain, in addition to the myogenic cells, a heterogeneous cell population, the composition of which might differ in normal and dystrophic muscle cultures. It is difficult in such experiments to distinguish between properties intrinsic to the myogenic cells and effects exerted by other cell types. Indeed, previous experiments have yielded conflicting conclusions2-6. We therefore tested the possibility of obtaining cell cultures consisting of pure populations of myogenic cells obtained from dystrophic muscles. The present report describes the isolation of a cloned population of such cells, derived from adult dystrophic mouse muscle, that can proliferate and differentiate in cell culture.
1. Developmental changes in action potential properties were studied in a clonal rat skeletal muscle cell line. 2. Small action potentials were evoked in mononucleate myoblasts. No spike was seen in Na-free saline. A similar spike was evoked in a medium where all NaCl was replaced by LiCl. No spike was evoked when NaCl was replaced by CsCl. 3. Action potentials overshot zero membrane potential in multinucleate myotubes. The action potential was composed of two components, an initial fast spike and a hump on the falling phase or in some cases a distinct second peak. 4. Teh overshoot of the initial fast spike decreased when the external Na concentration was decreased. 5. In saline with 10 mM-Ca the second component often formed a distinct peak following the initial fast spike. A slow regenerative potential was evoked in Na-free media with a depolarizing current pulse. 6. In saline containing BaCl-2 instead of CaCl-2 there was always a second peak, the overshoot of which changed with external Ba concentration. A slow regenerative potential was evoked in Na-free, Ba-saline. The membrane conductance at the peak of the Ba-action potential was larger than in the resting state. 7. In adult rat skeletal muscle, the shape of the action potential was not changed when Ca was replaced by Ba. No action potential was evoked in Na-free Ba-saline or Ba-saline with tetrodotoxin (3 times 10-7 M). 8. The significance of the Ca component in the developing muscle is discussed.
1. A mesodermal stem cell line C3H10T1/2 was induced to differentiate to muscle by adding 0.3 microM-5-aza-2'-deoxy-cytidine to the medium for 24 h. The changes in membrane currents during differentiation were studied by whole-cell recording and changes in the expression of fibronectin, Neural Cell Adhesion Molecule (NCAM), myosin and desmin were studied immunohistochemically. 2. The stem cells showed the morphology of fibroblastic cells. Most of the stem cells showed ATP-induced slow K+ current. T-type Ca2+ current and inward rectifier K+ current were observed in 19% of the stem cells. The stem cells expressed fibronectin, but not NCAM, myosin or desmin. 3. About 2 weeks after the addition of 5-aza-2'-deoxy-cytidine, large multinucleated skeletal muscle-like cells appeared. Most of the induced muscles showed L-type Ca2+ current, responses to acetylcholine, outward K+ current, inward rectifier K+ current and contraction upon depolarizing stimulation. They expressed NCAM, myosin and desmin, but not fibronectin, and showed no ATP response. 4. In some batches (2/14), the induced muscles showed spontaneous twitches, and possessed tetrodotoxin (TTX)-sensitive Na+ current in addition to the currents described above. Furthermore clear striation was observed in some of the twitching muscles under Nomarski optics. 5. To ascertain the properties of cells at the initial step of muscle differentiation, whose differentiation is determined but not yet evident morphologically or electrophysiologically, subcloning was performed from the heterogeneous cells 10 days after induction. Three myogenic clones were obtained, which proliferated at low cell densities but differentiated to muscle with a high incidence at high cell densities, as well as ten non-myogenic clones. 6. Most myogenic clones still showed ATP-induced K+ current and fibronectin. In addition, most of them showed T-type Ca2+ current and inward rectifier K+ current. They had already expressed NCAM. No other properties observed in muscles had yet been expressed. Most cells of the non-myogenic clones showed ATP-induced K+ current and fibronectin. T-type Ca2+ current was also expressed, but not inward rectifier K+ current or NCAM. 7. The properties of the observed ionic currents were studied. The TTX-sensitive Na+ current could be completely blocked by 0.1 microM-TTX. It could be evoked by depolarizing steps to a level above -40 mV, while steady-state inactivation was detectable around -75 mV and reached half by -52 mV. T-type Ca2+ current could be evoked by a depolarizing pulse to a level above -45 mV, with a maximum amplitude around -15 mV.(ABSTRACT TRUNCATED AT 400 WORDS)
1. ATP was puff applied to cells of a mesodermal stem cell line, C3H10T1/2, and the responses were studied by whole-cell patch clamp recording. 2. In 91% of the cells (90/99), K+ current lasting for tens of seconds was observed after several seconds latency. The current showed outward rectification. In 10% of the cells (9/99), ATP induced Cl- current which also lasted for tens of seconds after several seconds latency, but showed little rectification. In 6% of these cells (5/99), both K+ and Cl- currents were induced by ATP. 3. The K+ current induced by ATP was dose dependent, with a Kd of 0.4 microM. The effects of ATP analogues were tested at a concentration of 20 microM. ADP and ATP-gamma-S induced the K+ current, while AMP and adenosine did not. alpha, beta-Methylene ATP produced a diminished K+ current. 4. The ATP-induced K+ current was not observed when EGTA in the internal solution was raised from 0.1 to 5 mM. In Fluo-3-loaded cells, an increase in intracellular Ca2+ concentration induced by the application of ATP was observed, and the time course was similar to the induced K+ current. Both the increase in intracellular Ca2+ and the K+ current were induced by ATP even in Ca(2+)-free external solution. Ryanodine (50 microM) in the external solution did not affect the ATP response, and application of 10 mM-caffeine alone to the external solution did not induce any response. 5. The variance of the steady-state fluctuations in the course of the ATP-induced slow K+ current was analysed. The single-channel conductance was estimated as 2.7 pS at 0 mV with external and internal K+ concentrations of 5 and 140 mM respectively. The K+ current was not affected by apamin at concentrations of up to 1 microM but was reduced to one-third by 140 mM-tetraethylammonium (TEA). 6. It was concluded that puff-applied ATP has two main effects in the mesodermal stem cells: an increase in the intracellular Ca2+ concentration and a succeeding hyperpolarization due to the Ca(2+)-activated K+ conductance which is present in this cell. The significance of the increase in intracellular Ca2+ caused by ATP is discussed.
Excitability is generated in developing skeletal muscle by the incorporation of sodium-selective ion channels into the surface
membrane. Whole-cell and patch voltage-clamp recording from myotubes and their embryologic precursors, myoblasts, indicated
that voltage-activated sodium current in myoblasts was more resistant to block by tetrodotoxin (TTX) than that in myotubes.
Single-channel recording from both cell types showed two classes of sodium channels. One class had a lower single-channel
conductance, activated at more hyperpolarized voltages, and was more resistant to TTX than the other. The proportion of TTX-resistant
to TTX-sensitive sodium channels was higher in myoblasts than in myotubes. Thus, the difference in TTX sensitivity between
myoblasts and myotubes can be explained by a difference in the proportion of the two classes of sodium channels. In addition,
the lower conductance of TTX-resistant channels provides insight into the relationship between the TTX binding site and the
external mouth of the sodium channel.
The action potential was recorded from cultured chick skeletal muscle cells in Na-, Ca-, Cl-free saline containing Ba and tetraethylammonium ions (Ba saline). The action potential consisted of two components: a low-threshold, fast inactivating component and a high-threshold, long-lasting component. Both components of the action potential were dependent on external Ba ions and eliminated by Co ions. It is concluded that both components are generated by inward currents carried by Ba ions through Ca channels. The two Ca channel components of the action potential differed with regard to activation and inactivation potential, presence or absence of fast inactivation, sensitivity to an organic Ca channel blocker, and developmental profile. In addition, the failure of occurrence of one or the other components was observed in some cells. These results could be explained by assuming that two components of the action potential in Ba saline were mediated by the two different Ca channels. Furthermore, there was a tendency for younger cells to have more prominent Ca channel components. This may suggest that Ca channels have some function in the early stages of myogenesis.
In this report, we describe the isolation, sequence, and initial characterization of the cDNA for the muscle-specific regulatory factor skeletal myogenin. Transfection of myogenin into the mesenchymal cell line C3H10T1/2 produces cells expressing muscle-specific markers. Myogenin is absent in undifferentiated cells, peaks, and then declines following a stimulus to differentiate, and is overexpressed in myoblasts selected with 5-bromodeoxyuridine for the overproduction of factors that regulate the decision to differentiate. High levels of myogenin transcripts are present in the myotomal region of somites at 8.5 days of gestation in the mouse. Although myogenin and MyoD are different genes, they share the myc homology domain. Myogenin and MyoD thus form part of a gene family regulating myogenesis, and together with myd may constitute a set of factors that interact to regulate the determination and differentiation of muscle cells.
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.
Transfection of cDNA expression vectors encoding either MyoD1 or myogenin into 10T1/2 cells converts them to myogenic cells. We show that transfection of 10T1/2 cells with the MyoD1 cDNA activates expression of endogenous MyoD1 mRNA, indicating that MyoD1 is subject to positive autoregulation. This activation of endogenous MyoD1 mRNA was also observed in Swiss 3T6 cells, but not in several other fibroblast or adipoblast cell lines transfected with the MyoD1 cDNA. In addition, transfection of the MyoD1 cDNA leads to activation of myogenin expression, and transfection of the myogenin cDNA leads to activation of MyoD1 expression. Thus, MyoD1 and myogenin appear to function in a positive autoregulatory loop that could either: account for or contribute to the stability of myogenic commitment; or amplify the level of expression of both MyoD1 and myogenin above a critical threshold that is required for activation of the myogenic program.
MyoD1 and myogenin are muscle-specific proteins which can convert non-myogenic cells in culture to differentiated muscle fibres, implicating them in myogenic determination. The pattern of expression of MyoD1 and myogenin during the early stages of muscle formation in the mouse embryo in vivo and in limb-bud explants cultured in vitro, indicates that they may have different functions in different types of muscle during development.
1. The whole-cell voltage clamp technique was used to record Ba2+ currents in voltage-sensitive Ca2+ channels in mouse flexor digitorum brevis muscles developing in situ from day 1 to 30 after birth. Effects of denervation and tissue culture on the Ca2+ channel currents were also studied. 2. The muscle fibres in newborn mice showed two distinct types of Ca2+ channel currents, a low-threshold transient current and a high-threshold sustained current. 3. The specific amplitude of the transient current was 2.7 +/- 1.7 (S.D.) A/F in response to -30 mV test pulses in medium containing 30 mM-Ba2+ on day 1 after birth. The transient current decreased progressively in the post-natal days and became undetectable by day 17. In contrast, the specific amplitude of the sustained current in response to +20 mV test pulses increased 4-fold from 6.9 A/F on day 1 to 27.7 A/F on day 30. 4. The disappearance of the transient current could not be accounted for by either shifts in voltage dependence of activation and inactivation or changes in activation and inactivation times of the two types of current during development. 5. Denervating muscle fibres on day 8 after birth did not prevent the disappearance of the transient current. Denervating them on day 17 did not allow reappearance of the transient current. However, the increase of the sustained current was suppressed by the denervation either on day 8 or day 17. 6. In muscle fibres isolated on day 8 after birth and cultured thereafter, the transient current did not disappear until day 19 in culture (27 days after birth), while the sustained current was maintained at the level on day 8. 7. In muscle fibres isolated on day 17, when the transient current had become undetectable, and cultured thereafter, the transient current did not reappear until day 15 in culture (32 days after birth), while the sustained current was maintained at a level similar to that on day 17. 8. We conclude that innervation has little influence on the developmental disappearance of the transient Ca2+ channel current in mouse muscle fibres, and suggest that some influencing factors from surroundings other than the nerve may be required for the disappearance of the functional transient channels.
Stable myogenic cell lines have been derived at a high frequency by transfection of a cloned multipotential mouse embryo cell line, C3H 10T1/2, with cloned human DNA linked to a selectable neomycin resistance gene. The myogenic phenotype remains linked to neomycin resistance during secondary transfections. Although proliferative in growth conditions, these cell lines maintain the ability to differentiate and express muscle-specific proteins. We conclude that there is a simple genetic basis for myogenic determination and that a single gene, myd, converts 10T1/2 cells to a myoblast lineage. Southern blot analysis demonstrates nonidentity of myd and the MyoD1 gene. Northern blot analysis shows that myd-transfected myogenic lineages express MyoD1 mRNA while parental 10T1/2 cells do not. These results suggest that a dependent regulatory gene pathway mediates myogenic determination and differentiation.
A line of C3H mouse embryo cells highly sensitive to postconfluence inhibition of cell division, designated C3H/10T1/2, was established, and a clone from this line (clone 8) was characterized at early and late passages (200 to 450 days in culture). The cells of clone 8 are approximately 1730 cu μm in volume, their plating efficiency is 12 to 30%, their average generation time is 15.5 hr, and their saturation density is 2.9 to 3.8 x 10 4 cells/sq cm. The cell survival is 30% after freezing in 10% dimethyl sulfoxide and storage in liquid nitrogen. The cells of this clone are negative with respect to the spontaneous expression of C type RNA murine viruses and viral antigens. Tests for mycoplasma contamination are negative. All the cells of this line are aneuploid with a stable mode of 81 chromosomes (40 to 60% of cells). Tests for tumorigenicity at all passages were negative. No spontaneous transformation in vitro was observed in the stock cultures transferred on a regular schedule.
Sodium and calcium components of the action potential in a developing skeletal muscle cell line
KIDOKORO, Y. (1975). Sodium and calcium components of the action potential in a developing
skeletal muscle cell line. Journal of Physiology 244, 145-159.