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1.
Yasuko Manabe Shouta Miyatake Mayumi Takagi Mio Nakamura Ai Okeda Taemi Nakano Michael F. Hirshman Laurie J. Goodyear Nobuharu L. Fujii 《PloS one》2012,7(12)
A cultured C2C12 myotube contraction system was examined for application as a model for acute contraction-induced phenotypes of skeletal muscle. C2C12 myotubes seeded into 4-well rectangular plates were placed in a contraction system equipped with a carbon electrode at each end. The myotubes were stimulated with electric pulses of 50 V at 1 Hz for 3 ms at 997-ms intervals. Approximately 80% of the myotubes were observed to contract microscopically, and the contractions lasted for at least 3 h with electrical stimulation. Calcium ion (Ca2+) transient evoked by the electric pulses was detected fluorescently with Fluo-8. Phosphorylation of protein kinase B/Akt (Akt), 5′ AMP-activated protein kinase (AMPK), p38 mitogen-activated protein kinase (p38), and c-Jun NH2-terminal kinase (JNK)1/2, which are intracellular signaling proteins typically activated in exercised/contracted skeletal muscle, was observed in the electrically stimulated C2C12 myotubes. The contractions induced by the electric pulses increased glucose uptake and depleted glycogen in the C2C12 myotubes. C2C12 myotubes that differentiated after exogenous gene transfection by a lipofection or an electroporation method retained their normal contractile ability by electrical stimulation. These findings show that our C2C12 cell contraction system reproduces the muscle phenotypes that arise in
vivo (exercise), in situ (hindlimb muscles in an anesthetized animal), and in
vitro (dissected muscle tissues in incubation buffer) by acute muscle contraction, demonstrating that the system is applicable for the analysis of intracellular events evoked by acute muscle contraction. 相似文献
2.
Existing models describing sarcomere assembly have arisen primarily from studies using cardiac muscle. In contrast to cardiac muscle, skeletal muscle differentiation is characterised by dramatic changes in protein expression, from non-muscle to muscle-specific isoforms before organisation of the sarcomeres. Consequently, little is understood of the potential influence of non-muscle cytoskeletal proteins on skeletal sarcomere assembly. To address this issue, transfectant (gamma33-B1) and control mouse C2 myoblasts were differentiated to form myotubes, and various stages of skeletal sarcomere assembly were studied. Organisation of non-muscle gamma-actin and co-localisation with sarcomeric alpha-actinin, an early marker of sarcomere assembly and a major component of Z lines, was noted. gamma-Actin was also identified in young myotubes with developing sarcomeric myofibrils in regenerating adult mouse muscle. Localisation of gamma-actin in a different area of the myotube to the muscle-specific sarcomeric alpha-actin also indicated a distinct role for gamma-actin. The effects of aberrant gamma-actin expression in other myoblast lines, further suggested a sequestering role for gamma-actin. These observations make the novel suggestion that non-muscle gamma-actin plays a role in skeletal sarcomere assembly both in vitro and in vivo. Consequently, a modified model is proposed which describes the role of gamma-actin in skeletal sarcomere assembly. 相似文献
3.
《Biophysical journal》2021,120(23):5384-5394
The goal of this project was to validate the functional relevance and utility of mucus produced by an in vitro intestinal cell culture model. This is facilitated by the need to physiologically replicate both healthy and abnormal mucus conditions from native intestinal tissue, where mucus properties have been connected to intestinal disease models. Mucus harvested from colonic cell cultures derived from healthy donors was compared to mucus collected from surgically resected, noninflamed transverse colon tissue. The rheological and biochemical properties of these mucus samples were compared using oscillational rheometry, particle-tracking microrheology, multiangle laser light scattering, refractometry, and immunohistochemical imaging. An air-liquid interface culture of primary human colonic epithelial cells generated a continuous monolayer with an attached mucus layer that displayed increasing weight percent (wt%) of solids over 1 week (1.3 ± 0.5% at 2 days vs. 2.4 ± 0.3% at 7 days). The full range of mucus concentrations (0.9–3.3%) observed during culture was comparable to that displayed by ex vivo mucus (1.3–1.9%). Bulk rheological measurements displayed similar wt%-based complex viscosities between in vitro and ex vivo mucus, with the complex viscosity of both systems increasing with wt% of solids. Particle-tracking microrheology showed higher complex viscosities for ex vivo mucus samples than in vitro mucus which was explained by a greater fraction of water present in in vitro mucus than ex vivo, i.e., in vitro mucus is more heterogeneous than ex vivo. Refractometry, multiangle laser light scattering, and immunostaining showed increased mucus complex size in ex vivo mucus compared with in vitro mucus, which may have been due to the admixture of mucus and cellular debris during ex vivo mucus collection. The air-liquid interface culture system produced intestinal mucus with similar composition and rheology to native human gut mucus, providing a platform to analyze pathological differences in intestinal mucus. 相似文献
4.
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 µm) 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 ~10× 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. Biotechnol. Bioeng. 2010; 106: 482–489. © 2010 Wiley Periodicals, Inc. 相似文献
5.
The assembly of sarcomeres, the smallest contractile units in striated muscle, is a complex and highly coordinated process that relies on spatio-temporal organization of sarcomeric proteins, a process requiring spontaneous Ca(2+) transients. To investigate the relationship between Ca(2+) transients and sarcomere assembly in C2C12 myotubes, we employed electric pulse stimulation (EPS), which allows the frequency of Ca(2+) transients to be manipulated. We monitored contractile activity as a means of evaluating functional sarcomere establishment using the differential image subtraction (DIS) method. C2C12 myotubes initially displayed no contractility with EPS, due to a lack of sarcomere architecture. However, C2C12 myotubes showed remarkable contractile activity with EPS-induced repetitive Ca(2+) transients (1 Hz) within only 2 h. This activity was concurrent with the development of sarcomere structure. Importantly, the period required for the acquisition of contractile activity in response to excitation was dependent upon the frequency of Ca(2+) oscillations, but a sustained increase in intracellular Ca(2+) (not oscillatory) by high-frequency EPS (10 Hz) was incapable of conferring either contractility or sarcomere assembly on the myotubes. The EPS-facilitated de novo functional sarcomere assembly appeared to require calpain-mediated proteolysis. In addition, modulation of integrin signals, by adding collagen IV or RGD-peptide, significantly affected the EPS-induced development of contractility. Taken together, these observations indicate that the frequency of the Ca(2+) oscillation determines the time required to establish functionally active sarcomere assembly and also suggest that the Ca(2+) oscillatory signal may be decoded through reorganization of the integrin-cytoskeletal protein complex via calpain-mediated proteolysis. 相似文献
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8.
Nedachi T Fujita H Kanzaki M 《American journal of physiology. Endocrinology and metabolism》2008,295(5):E1191-E1204
Adequate exercise leads to a vast variety of physiological changes in skeletal muscle as well as other tissues/organs and is also responsible for maintaining healthy muscle displaying enhanced insulin-responsive glucose uptake via GLUT4 translocation. We generated highly developed contractile C(2)C(12) myotubes by manipulating intracellular Ca(2+) transients with electric pulse stimulation (EPS) that is endowed with properties similar to those of in vivo skeletal muscle in terms of 1) excitation-induced contractile activity as a result of de novo sarcomere formation, 2) activation of both the AMP kinase and stress-activated MAP kinase cascades, and 3) improved insulin responsiveness as assessed by GLUT4 recycling. Tbc1d1, a Rab-GAP implicated in exercise-induced GLUT4 translocation in skeletal muscle, also appeared to be phosphorylated on Ser(231) after EPS-induced contraction. In addition, a switch in myosin heavy-chain (MHC) expression from "fast type" to "slow type" was observed in the C(2)C(12) myotubes endowed with EPS-induced repetitive contractility. Taking advantage of these highly developed contractile C(2)C(12) myotubes, we identified myotube-derived factors responsive to EPS-evoked contraction, including the CXC chemokines CXCL1/KC and CXCL5/LIX, as well as IL-6, previously reported to be upregulated in contracting muscles in vivo. Importantly, animal treadmill experiments revealed that exercise significantly increased systemic levels of CXCL1/KC, perhaps derived from contracting muscle. Taken together, these results confirm that we have established a specialized muscle cell culture model allowing contraction-inducible cellular responses to be explored. Utilizing this model, we identified contraction-inducible myokines potentially linked to the metabolic alterations, immune responses, and angiogenesis induced by exercise. 相似文献
9.
Skeletal muscles show a high plasticity to cope with various physiological demands. Different muscle types can be distinguished by the force, endurance, contraction/relaxation kinetics (fast-twitch vs. slow-twitch muscles), oxidative/glycolytic capacity, and also with respect to Ca2+-signaling components. Changes in Ca2+ signaling and associated Ca2+-dependent processes are thought to underlie the high adaptive capacity of muscle fibers. Here we investigated the consequences and the involved mechanisms caused by the ectopic expression of the Ca2+-binding protein parvalbumin (PV) in C2C12 myotubes in vitro, and conversely, the effects caused by its absence in in fast-twitch muscles of parvalbumin null-mutant (PV−/−) mice in vivo. The absence of PV in fast-twitch muscle tibialis anterior (TA) resulted in an increase in the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and of its positive regulator, the deacetylase sirtuin 1 (SIRT1). TA muscles from PV−/− mice also have an increased mitochondrial volume. Mild ionophore treatment of control (PV-devoid) C2C12 myotubes causing a moderate elevation in [Ca2+]c resulted in an increase in mitochondrial volume, together with elevated PGC-1α and SIRT1 expression levels, whilst it increased PV expression levels in myotubes stably transfected with PV. In PV-expressing myotubes the mitochondrial volume, PGC-1α and SIRT1 were significantly lower than in control C2C12 myotubes already at basal conditions and application of ionophore had no effect on either one. SIRT1 activation causes a down-regulation of PV in transfected myotubes, whilst SIRT1 inhibition has the opposite effect. We conclude that PV expression and mitochondrial volume in muscle cells are inversely regulated via a SIRT1/PGC-1α signaling axis. 相似文献
10.
The ultrastructural differentiation of the clonal myogenic cell line L6 in normal and high K+ medium
Cells of the clonal myogenic line L6 were examined electron microscopically at successive stages of growth. L6 cells are developmentally similar to those of chick and rat primary skeletal muscle cultures and skeletal muscle in vivo, with respect to myofibrillogenesis and sarcomere organization. However, the sarcoplasmic reticulum and T systems of L6 myotubes are not as well differentiated as those of primary muscle cultures and adult skeletal muscle. Finally, L6 myotubes show precocious sarcomere differentiation when cultured in medium containing 25 mM potassium. 相似文献
11.
Rachel A. Stern Srinivasan Dasarathy Paul E. Mozdziak 《In vitro cellular & developmental biology. Animal》2017,53(2):99-110
Increased myostatin expression, resulting in muscle loss, has been associated with hyperammonemia in mammalian models of cirrhosis. However, there is evidence that hyperammonemia in avian embryos results in a reduction of myostatin expression, suggesting a proliferative myogenic environment. The present in vitro study examines species differences in myotube and liver cell response to ammonia using avian and murine-derived cells. Primary myoblasts and liver cells were isolated from embryonic day 15 and 17 chick embryos to be compared with mouse myoblasts (C2C12) and liver (AML12) cells. Cells were exposed to varying concentrations of ammonium acetate (AA; 2.5, 5, or 10 mM) to determine the effects of ammonia on the cells. Relative expression of myostatin mRNA, determined by quantitative real-time PCR, was significantly increased in AA (10 mM) treated C2C12 myotubes compared to both ages of chick embryonic myotube cultures after 48 h (P < 0.02). Western blot analysis of myostatin protein confirmed an increase in myostatin expression in AA-treated C2C12 myotubes compared to the sodium acetate (SA) controls, while myostatin expression was decreased in the chick embryonic myotube cultures when treated with AA. Myotube diameter was significantly decreased in AA-treated C2C12 myotubes compared to controls, while avian myotube diameter increased with AA treatment (P < 0.001). There were no significant differences between avian and murine liver cell viability, assessed using 2′, 7′- bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein, acetoxymethyl ester, when treated with AA. However, after 24 h, AA-treated avian myotubes showed a significant increase in cell viability compared to the C2C12 myotubes (P < 0.05). Overall, it appears that there is a positive myogenic response to hyperammonemia in avian myotubes compared to murine myotubes, which supports a proliferative myogenic environment. 相似文献
12.
Daniel Knappe Laszlo Otvos Jr. Francesco De Pascali Marco Cassone Laura Scolaro Giovanni Abbadessa Cynthia Carrillo-Infante Mohammad F. Kiani Fred Donelson Ralf Hoffmann Eva Surmacz 《International journal of peptide research and therapeutics》2008,14(3):247-254
A glycosylated dodecapeptide fragment corresponding to the hypothalamus-active cytokine leptin exhibits agonistic properties
to the leptin receptor (ObR) in vitro and penetrates into the brain in vivo. In order to characterize the drug development
potential of the lead peptide and to optimize it for pharmacological applicability, a series of biochemical screening assays
were custom-tailored to the leptin/ObR system. To identify peptides that bind the extracellular domain of ObR, we characterized
the optimal conditions for an ELISA-type assay where the leptin fragments were immobilized to the plates. With this technology
we could identify low-dose binder peptidomimetics which, according to a comparison of the conventional cell proliferation
assay and a measure of metabolically active cells, revealed that agonists identified by these cellular assays may not necessarily
induce the expected growth characteristics in ObR expressing cells. The original glycopeptide lead displayed a 2 h half life
in 25% diluted mouse serum but poor stability in mouse brain extract. Fifteen percent of the glycopeptide crossed a dual endothelial/astrocyte
cell layer (representing an in vitro model of blood-brain-barrier) in 30 min, and the coexistence of the two cell types appeared
necessary to quantify the level of brain accessibility. Finally, in an in vivo mouse model, a Cy5.5 labeled glycopeptide was
more evenly distributed all over the body, including the brain, than a similarly labeled full-sized leptin protein. 相似文献
13.
Traditionally, muscle cell lines are cultured on glass coverslips and differentiated to investigate myoblast fusion and differentiation. Efficient differentiation of myoblasts produces a dense network of myotubes with the correct organisation for contraction. Here we have tested the ability of artificially generated, precisely controlled peptide surfaces to enhance the efficiency of myoblast differentiation. We focused on specific short peptides from α-laminin-2 (IKVSV, VQLRNGFPYFSY and GLLFYMARINHA) as well as residues 15–155 from FGF1. We tested if these peptides in isolation, and/or in combination promoted muscle differentiation in culture, by promoting fusion and/or by improving sarcomere organisation. The majority of these peptides promoted fusion and differentiation in two different mouse myogenic cell lines and in primary human myoblasts. The additive effects of all four peptides gave the best results for both mouse cell lines tested, while primary human cell cultures differentiated equally well on most peptide surfaces tested. These data show that a mixture of short biomimetic peptides can reliably promote differentiation in mouse and human myoblasts. 相似文献
14.
《Microbes and infection / Institut Pasteur》2020,22(10):567-575
Mycobacterium avium complex is a causative organism for refractory diseases. In this study, we examined the effects of N-acetyl-cysteine on M. avium infection in vitro and in vivo. N-acetyl-cysteine treatment suppressed the growth of M. avium in A549 cells in a concentration-dependent manner. This effect was related to the induction of the antibacterial peptide human β-defensin-2. In a mouse model, N-acetyl-cysteine treatment significantly reduced the number of bacteria in the lungs and induced murine β-defensin-3. In interleukin-17-deficient mice, the effects of N-acetyl-cysteine disappeared, indicating that these mechanisms may be mediated by interleukin-17. Moreover, an additional reduction in bacterial load was observed in mice administered N-acetyl-cysteine in combination with clarithromycin. Our findings demonstrate the potent antimycobacterial effects of N-acetyl-cysteine against M. avium by inducing antimicrobial peptide, suggesting that N-acetyl-cysteine may have applications as an alternative to classical treatment regimens. 相似文献
15.
Humanization of fibroblast growth factor 1 single‐chain antibody and validation for its antitumorigenic efficacy in breast cancer and glioma cells
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Xiao‐Xiao He Shuang Du Shi‐Qian Gao Jing‐Ying Chen Ran‐Juan Cao Zhen‐Kai Xing Alia Rizvi Syeda Kazim Hua‐Li Yu Qing‐Chuan Zheng Xiao‐Juan Zhu 《Journal of cellular and molecular medicine》2018,22(6):3259-3263
Single‐chain variable fragment (scFv) antibodies are the smallest immunoglobulins with high antigen‐binding affinity. We have previously reported that fibroblast growth factor 1 played pivotal roles in cancer development and generated a mouse scFv (mscFv1C9) could effectively prohibit cancer cell proliferation in vitro and in vivo. Here, we further humanized this scFv (hscFv1C9) using a structure‐guided complementarity determining region grafting strategy. The purified hscFv1C9 maintained similar antigen‐binding affinity and specificity as mscFv1C9, and it was capable of inhibiting growth of different tumours in vitro and in vivo. These data strongly suggested that hscFv1C9 has antitumour potentials. 相似文献
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17.
Glucose uptake in human and animal muscle cells in culture 总被引:5,自引:0,他引:5
Human muscle cells were grown in culture from satellite cells present in muscle biopsies and fusion-competent clones were identified. Hexose uptake was studied in fused myotubes of human muscle cells in culture and compared with hexose uptake in myotubes of the rat L6 and mouse C2C12 muscle cell lines. Uptake of 2-deoxyglucose was saturable and showed an apparent Km of about 1.5 mM in myotubes of all three cell types. The Vmax of uptake was about 6000 pmol/(min.mg protein) in human cells, 4000 pmol/(min.mg protein) in mouse C2C12 muscle cells, and 500 pmol/(min.mg protein) in L6 cells. Hexose uptake was inhibited approximately 90% by cytochalasin B in human, rat, and mouse muscle cell cultures. Insulin stimulated 2-deoxyglucose uptake in all three cultures. The hormone also stimulated transport of 3-O-methylglucose. The sensitivity to insulin was higher in human and C2C12 mouse myotubes (half-maximal stimulation observed at 3.5 X 10(-9) M) than in rat L6 myotubes (half-maximal stimulation observed at 2.5 X 10(-8) M). However, insulin (10(-6) M) stimulated hexose uptake to a larger extent (2.37-fold) in L6 than in either human (1.58-fold) or mouse (1.39-fold) myotubes. It is concluded that human muscle cells grown in culture display carrier-mediated glucose uptake, with qualitatively similar characteristics to those of other muscle cells, and that insulin stimulates hexose uptake in human cells. These cultures will be instrumental in the study of human insulin resistance and in investigations on the mechanism of action of antidiabetic drugs. 相似文献
18.
5'-AMP-activated protein kinase phosphorylates IRS-1 on Ser-789 in mouse C2C12 myotubes in response to 5-aminoimidazole-4-carboxamide riboside 总被引:10,自引:0,他引:10
Jakobsen SN Hardie DG Morrice N Tornqvist HE 《The Journal of biological chemistry》2001,276(50):46912-46916
Exercise is known to increase insulin sensitivity and is an effective form of treatment for the hyperglycemia observed in type 2 diabetes. Activation of 5'-AMP-activated protein kinase (AMPK) by 5-aminoimidazole-4-carboxamide riboside (AICAR), exercise, or electrically stimulated contraction leads to increased glucose transport in skeletal muscle. Here we report the first evidence of a direct interaction between AMPK and the most upstream component of the insulin-signaling cascade, insulin receptor substrate-1 (IRS-1). We find that AMPK rapidly phosphorylates IRS-1 on Ser-789 in cell-free assays as well as in mouse C2C12 myotubes incubated with AICAR. In the C2C12 myotubes activation of AMPK by AICAR matched the phosphorylation of IRS-1 on Ser-789. This phosphorylation correlates with a 65% increase in insulin-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity in C2C12 myotubes preincubated with AICAR. The binding of phosphatidylinositol 3-kinase to IRS-1 was not affected by AICAR. These results demonstrate the existence of an interaction between AMPK and early insulin signaling that could be of importance to our understanding of the potentiating effects of exercise on insulin signaling. 相似文献
19.
Bertrand C.W. Tanner Gerrie P. Farman Thomas C. Irving David W. Maughan Bradley M. Palmer Mark S. Miller 《Biophysical journal》2012,103(6):1275-1284
The demembranated (skinned) muscle fiber preparation is widely used to investigate muscle contraction because the intracellular ionic conditions can be precisely controlled. However, plasma membrane removal results in a loss of osmotic regulation, causing abnormal hydration of the myofilament lattice and its proteins. We investigated the structural and functional consequences of varied myofilament lattice spacing and protein hydration on cross-bridge rates of force development and detachment in Drosophila melanogaster indirect flight muscle, using x-ray diffraction to compare the lattice spacing of dissected, osmotically compressed skinned fibers to native muscle fibers in living flies. Osmolytes of different sizes and exclusion properties (Dextran T-500 and T-10) were used to differentially alter lattice spacing and protein hydration. At in vivo lattice spacing, cross-bridge attachment time (ton) increased with higher osmotic pressures, consistent with a reduced cross-bridge detachment rate as myofilament protein hydration decreased. In contrast, in the swollen lattice, ton decreased with higher osmotic pressures. These divergent responses were reconciled using a structural model that predicts ton varies inversely with thick-to-thin filament surface distance, suggesting that cross-bridge rates of force development and detachment are modulated more by myofilament lattice geometry than protein hydration. Generalizing these findings, our results suggest that cross-bridge cycling rates slow as thick-to-thin filament surface distance decreases with sarcomere lengthening, and likewise, cross-bridge cycling rates increase during sarcomere shortening. Together, these structural changes may provide a mechanism for altering cross-bridge performance throughout a contraction-relaxation cycle. 相似文献