scholarly journals Early Changes in Skeletal Muscle of Young C22 Mice, A Model of Charcot-Marie-Tooth 1A

2021 ◽  
pp. 1-17
Author(s):  
Friederike Deres ◽  
Stephanie Schwartz ◽  
Karin Kappes-Horn ◽  
Cornela Kornblum ◽  
Jens Reimann
Keyword(s):  
Skeletal Muscle ◽  
Western Blotting ◽  
Acetylcholine Receptors ◽  
Small Diameter ◽  
Integral Membrane Protein ◽  
Representational Difference Analysis ◽  
Initiation Factor ◽  
Charcot Marie Tooth ◽  
Immuno Histochemistry ◽  
Phosphatidylinositol Transfer

Background: The C22 mouse is a Charcot-Marie-Tooth 1A transgenic model with minimal axonal loss. Objective: To analyse early skeletal muscle changes resulting from this dysmyelinating neuropathy. Methods: Histology of tibialis anterior muscles of C22 mice and wild type litter mate controls for morphometric analysis and (immuno-)histochemistry for known denervation markers and candidate proteins identified by representational difference analysis (RDA) based on mRNA from the same muscles; quantitative PCR and Western blotting for confirmation of RDA findings. Results: At age 10 days, morphometry was not different between groups, while at 21 days, C22 showed significantly more small diameter fibres, indicating the onset of atrophy at an age when weakness becomes detectable. Neither (immuno-)histochemistry nor RDA detected extrajunctional expression of acetylcholine receptors by age 10 and 21 days, respectively. RDA identified some mRNA up-regulated in C22 muscles, among them at 10 days, prior to detectable weakness or atrophy, integral membrane protein 2a (Itm2a), eukaryotic initiation factor 2, subunit 2 (Eif2s2) and cytoplasmic phosphatidylinositol transfer protein 1 (Pitpnc1). However, qPCR failed to measure significant differences. In contrast, Itm2a and Eif2s2 mRNA were significantly down-regulated comparing 21 versus 10 days of age in both groups, C22 and controls. Western blotting confirmed significant down-regulation of ITM2A protein in C22 only. Conclusion: Denervation-like changes in this model develop slowly with onset of atrophy and weakness at about three weeks of age, before detection of extrajunctional acetylcholine receptors. Altered Itm2a expression seems to being early as an increase, but becomes distinct as a decrease later.

scholarly journals Phosphorylation of Initiation Factor IF-2 from Escherichia coli with Skeletal Muscle Kinase

1972 ◽  
Vol 247 (19) ◽  
pp. 6365-6367
Author(s):  
John L. Fakunding ◽  
Jolinda A. Traugh ◽  
Robert R. Traut ◽  
John W.B. Hershey
Keyword(s):  
Escherichia Coli ◽  
Skeletal Muscle ◽  
Initiation Factor

Lipocortin 1 (annexin 1) in patches associated with the membrane of a lung adenocarcinoma cell line and in the cell cytoplasm

1998 ◽  
Vol 111 (10) ◽  
pp. 1405-1418 ◽  
Author(s):  
V. Traverso ◽  
J.F. Morris ◽  
R.J. Flower ◽  
J. Buckingham
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Lung Adenocarcinoma ◽  
Western Blotting ◽  
Signal Sequence ◽  
Membrane Surface ◽  
Integral Membrane Protein ◽  
Subcellular Fractionation ◽  
Cell Fractionation ◽  
Electron Microscopic

Lipocortin 1 (annexin I) is a calcium- and phospholipid-binding annexin protein which can be externalised from cells despite the lack of a signal sequence. To determine its cellular distribution lipocortin 1 in A549 human lung adenocarcinoma cells was localised by light- and electron-microscopic immunocytochemistry and by cell fractionation and western blotting. Lipocortin 1 immunoreactivity is concentrated in prominent patches associated with the plasma membrane. The intensity of these patches varied with the confluence and duration of the culture and was not detectably diminished by an EDTA wash before fixation. Tubulin and cytokeratin 8 were colocalized with lipocortin 1 in the patches. Within the cells lipocortin 1 was distributed throughout the cytoplasm. Electron microscopy revealed prominent immunoreactivity along the plasma membrane with occasional large clusters of gold particles in contact with the membrane surface of the cells; within the cytoplasm the membrane of some vesicle/vacuole structures and some small electron-dense bodies was immunoreactive, but no immunogold particles were associated with the multilamellar bodies. Subcellular fractionation, extraction and western blotting showed that lipocortin 1 in the membrane pellet was present as two distinct fractions; one, intimately associated with the lipid bilayer, which behaved like an integral membrane protein and one loosely attached which behaved like a peripheral membrane protein. The results show that a substantial amounts of lipocortin 1 is concentrated in focal structures associated with and immediately beneath the plasma membrane. These might form part of the mechanism by which lipocortin 1 is released from the cells.

Trophic effect of iron-bound transferrin on acetylcholine receptors in rat skeletal muscle in vivo

1983 ◽  
Vol 38 (3) ◽  
pp. 303-307 ◽  
Author(s):  
Keiji Wada ◽  
Satoshi Ueno ◽  
Takanori Hazama ◽  
Hiro-O Yoshikawa ◽  
Saburo Ogasahara ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Acetylcholine Receptors ◽  
Rat Skeletal Muscle ◽  
Trophic Effect

scholarly journals Adiponectin improves insulin sensitivity via activation of autophagic flux

2017 ◽  
Vol 59 (4) ◽  
pp. 339-350 ◽  
Author(s):  
Penny Ahlstrom ◽  
Esther Rai ◽  
Suharto Chakma ◽  
Hee Ho Cho ◽  
Palanivel Rengasamy ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Insulin Sensitivity ◽  
Er Stress ◽  
Western Blotting ◽  
Muscle Cells ◽  
Dominant Negative ◽  
Skeletal Muscle Cells ◽  
Dominant Negative Mutant ◽  
Autophagic Flux

Skeletal muscle insulin resistance is known to play an important role in the pathogenesis of diabetes, and one potential causative cellular mechanism is endoplasmic reticulum (ER) stress. Adiponectin mediates anti-diabetic effects via direct metabolic actions and by improving insulin sensitivity, and we recently demonstrated an important role in stimulation of autophagy by adiponectin. However, there is limited knowledge on crosstalk between autophagy and ER stress in skeletal muscle and in particular how they are regulated by adiponectin. Here, we utilized the model of high insulin/glucose (HIHG)-induced insulin resistance, determined by measuring Akt phosphorylation (T308 and S473) and glucose uptake in L6 skeletal muscle cells. HIHG reduced autophagic flux measured by LC3 and p62 Western blotting and tandem fluorescent RFP/GFP-LC3 immunofluorescence (IF). HIHG also induced ER stress assessed by thioflavin T/KDEL IF, pIRE1, pPERK, peIF2α and ATF6 Western blotting and induction of a GRP78-mCherry reporter. Induction of autophagy by adiponectin or rapamycin attenuated HIHG-induced ER stress and improved insulin sensitivity. The functional significance of enhanced autophagy was validated by demonstrating a lack of improved insulin sensitivity in response to adiponectin in autophagy-deficient cells generated by overexpression of dominant negative mutant of Atg5. In summary, adiponectin-induced autophagy in skeletal muscle cells alleviated HIHG-induced ER stress and insulin resistance.

scholarly journals MyoScreen, a High-Throughput Phenotypic Screening Platform Enabling Muscle Drug Discovery

2018 ◽  
Vol 23 (8) ◽  
pp. 790-806 ◽  
Author(s):  
Joanne Young ◽  
Yoran Margaron ◽  
Mathieu Fernandes ◽  
Eve Duchemin-Pelletier ◽  
Joris Michaud ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
High Throughput ◽  
Acetylcholine Receptors ◽  
Calcium Release ◽  
Screening Strategy ◽  
Adult Skeletal Muscle ◽  
New Chemical Entities ◽  
Drug Treatments ◽  
Screening Platform

Despite the need for more effective drug treatments to address muscle atrophy and disease, physiologically accurate in vitro screening models and higher information content preclinical assays that aid in the discovery and development of novel therapies are lacking. To this end, MyoScreen was developed: a robust and versatile high-throughput high-content screening (HT/HCS) platform that integrates a physiologically and pharmacologically relevant micropatterned human primary skeletal muscle model with a panel of pertinent phenotypic and functional assays. MyoScreen myotubes form aligned, striated myofibers, and they show nerve-independent accumulation of acetylcholine receptors (AChRs), excitation–contraction coupling (ECC) properties characteristic of adult skeletal muscle and contraction in response to chemical stimulation. Reproducibility and sensitivity of the fully automated MyoScreen platform are highlighted in assays that quantitatively measure myogenesis, hypertrophy and atrophy, AChR clusterization, and intracellular calcium release dynamics, as well as integrating contractility data. A primary screen of 2560 compounds to identify stimulators of myofiber regeneration and repair, followed by further biological characterization of two hits, validates MyoScreen for the discovery and testing of novel therapeutics. MyoScreen is an improvement of current in vitro muscle models, enabling a more predictive screening strategy for preclinical selection of the most efficacious new chemical entities earlier in the discovery pipeline process.

Regulation of translation factors during hindlimb unloading and denervation of skeletal muscle in rats

2001 ◽  
Vol 281 (1) ◽  
pp. C179-C187 ◽  
Author(s):  
Troy A. Hornberger ◽  
R. Bridge Hunter ◽  
Susan C. Kandarian ◽  
Karyn A. Esser
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Muscle Atrophy ◽  
Protein Translation ◽  
Hindlimb Unloading ◽  
Initiation Factor ◽  
Skeletal Muscle Atrophy ◽  
Α Subunit ◽  
Translation Factors ◽  
Ribosomal S6 Kinase

In the rat, denervation and hindlimb unloading are two commonly employed models used to study skeletal muscle atrophy. In these models, muscle atrophy is generally produced by a decrease in protein synthesis and an increase in protein degradation. The decrease in protein synthesis has been suggested to occur by an inhibition at the level of protein translation. To better characterize the regulation of protein translation, we investigated the changes that occur in various translation initiation and elongation factors. We demonstrated that both hindlimb unloading and denervation produce alterations in the phosphorylation and/or total amount of the 70-kDa ribosomal S6 kinase, eukaryotic initiation factor 2 α-subunit, and eukaryotic elongation factor 2. Our findings indicate that the regulation of these protein translation factors differs between the models of atrophy studied and between the muscles evaluated (e.g., soleus vs. extensor digitorum longus).

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