Human primary myotubes +/- electrical pulse stimulation
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB46916 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR6465115_1.fastq.gz | Fastqsanger.gz | |||
| ERR6465115_2.fastq.gz | Fastqsanger.gz | |||
| ERR6465116_1.fastq.gz | Fastqsanger.gz | |||
| ERR6465116_2.fastq.gz | Fastqsanger.gz | |||
| ERR6465117_1.fastq.gz | Fastqsanger.gz |
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Project description:Human myogenic progenitor cells (MPCs) were isolated from the gracilis of two patients (1 female and 1 male) undergoing anterior cruciate ligament reconstruction (age 30-34 years) using a human anti-CD56 (1:20, Cat# 355503, Biolegend) antibody for FACS. Cells were passaged 2-3 times in growth media consisting of Ham’s F-10 (Thermofisher), 20% fetal bovine serum (FBS, Atlanta Biological, Minneapolis, MN, USA), 1% penicillin/streptomycin and 10ng/ml basic fibroblast growth-factor (bFGF, Peprotech, Rocky Hill, NJ, USA). Cells were differentiated into myotubes for 5 days using MyoCult differentiation media (Stemcell Technologies, Vancouver, Canada). On the fifth day, fresh media was added just prior to electrical pulse stimulation (E). Cells were then either stimulated at 12 V, 1 Hz, 2 ms for 24 hr (IonOptix C‐Pace EP, Westwood, MA, USA) or had electrodes placed in wells with no stimulation (E(+) or E(-)), followed by immediate RNA extraction using Qiazol (Qiagen, Hilden, Germany).
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Project description:We have used a combined transcriptome-proteome approach to describe how EPS affected the cargo of extracellular vesicles derived from myotubes from morbidly obese patients with T2D, and revealed several new factors, both miRs and proteins, that might act as exercise factors. During exercise, skeletal muscles release signaling factors that communicate with other organs and mediate beneficial effects of exercise. These factors include myokines, metabolites, and extracellular vesicles (EVs). In the present study, we have examined how electrical pulse stimulation (EPS) of myotubes, a model of exercise, affects the cargo of released EVs. Chronic low frequency EPS was applied for 24 h to human myotubes isolated and differentiated from biopsy samples from 6 morbidly obese females with T2D, and EVs, both exosomes and microvesicles (MV), were isolated from cell media 24 h thereafter.Protein content was assessed by high-resolution proteomic analysis (LC-MS/MS), non-coding RNA was quantified by Affymetrix GeneChip Multispecies miRNA-4_0 Array and Flash Tag TM Biotin RNA labelling, Thermo Fisher), and selected microRNAs (miRs) validated by real time RT-qPCR. We found that human myotubes secrete both exosomes and MV. EPS treatment of the myotubes, clearly changed the protein content of both exosomes and MV, whereas the miR content was changed only in exosomes. We suggest that skeletal muscle derived EVs can contribute to circulatory EVs and mediate beneficial effects of exercise in metabolically active organs.
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Project description:We have used a combined transcriptome-proteome approach to describe how EPS affected the cargo of extracellular vesicles derived from myotubes from morbidly obese patients with T2D, and revealed several new factors, both miRs and proteins, that might act as exercise factors. During exercise, skeletal muscles release signaling factors that communicate with other organs and mediate beneficial effects of exercise. These factors include myokines, metabolites, and extracellular vesicles (EVs). In the present study, we have examined how electrical pulse stimulation (EPS) of myotubes, a model of exercise, affects the cargo of released EVs. Chronic low frequency EPS was applied for 24 h to human myotubes isolated and differentiated from biopsy samples from 6 morbidly obese females with T2D, and EVs, both exosomes and microvesicles (MV), were isolated from cell media 24 h thereafter.Protein content was assessed by high-resolution proteomic analysis (LC-MS/MS), non-coding RNA was quantified by Affymetrix microarray GeneChip TM Multispecies miRNA 4.0 and Flash Tag TM Biotin RNA labelling, Thermo Fisher), and selected microRNAs (miRs) validated by real time RT-qPCR. We found that human myotubes secrete both exosomes and MV. EPS treatment of the myotubes, clearly changed the protein content of both exosomes and MV, whereas the miR content was changed only in exosomes. We suggest that skeletal muscle derived EVs can contribute to circulatory EVs and mediate beneficial effects of exercise in metabolically active organs.
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Project description:In this study, we aimed to investigate transcriptomic profile changes in human skeletal muscle cells that are triggered by a well-established in vitro model of exercise using electrical pulse stimulation (EPS).
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Project description:Appropriate amount of exercise is the best way to prevent various diseases and have a healthy life. Skeletal muscles communicate with other organs through myokines, which are secreted by muscle itself during exercise and elicit various effects in the body. However, despite the years of efforts to understand molecular mechanisms of crosstalk between muscle and other organs that mediate the beneficial effects of exercise are not completely understood. To identify novel myokines, we used an in vitro exercise model in C2C12 cells using the electrical pulse stimulation (EPS) that can mimic muscle contraction. We generated RNA-seq data of seven in vitro samples to costruct a prediction model based on differentially expressed genes, showing significantly mimicking in vivo exercise.
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Project description:Lifestyle disorders like obesity, type 2 diabetes (T2D), and cardiovascular diseases can be prevented and treated by regular physical activity. During exercise, skeletal muscles release signaling factors that communicate with other organs and mediate beneficial effects of exercise. These factors include myokines, metabolites, and extracellular vesicles (EVs). In the present study, we have examined how electrical pulse stimulation (EPS) of myotubes, a model of exercise, affects the cargo of released EVs. Chronic low frequency EPS was applied for 24 h to human myotubes isolated and differentiated from biopsy samples from 6 morbidly obese females with T2D, and EVs, both exosomes and microvesicles (MV), were isolated from cell media 24 h thereafter. Size and concentration of EV subtypes were characterized by nanoparticle tracking analysis, surface markers were examined by flow cytometry and Western blotting, and morphology was confirmed by transmission electron microscopy. Protein content was assessed by high-resolution proteomic analysis (LC-MS/MS), non-coding RNA was quantified by Affymetrix microarray, and selected microRNAs (miRs) validated by real time RT-qPCR. The size and concentration of exosomes and MV were unaffected by EPS. Of the 400 miRs identified in the EVs, EPS significantly changed the level of 15 exosome miRs, of which miR-1233-5p showed the highest fold change. The miR pattern of MV was unaffected by EPS. Totally, about 1000 proteins were identified in exosomes and 2000 in MV. EPS changed the content of 73 proteins in exosomes, 97 in MVs, and of these 4 were changed in both exosomes and MV (GANAB, HSPA9, CNDP2, and ATP5B). By matching the EPS-changed miRs and proteins in exosomes, 31 targets were identified, and among these several promising signaling factors. Of particular interest were CNDP2, an enzyme that generates the appetite regulatory metabolite Lac-Phe, and miR-4433b-3p, which targets CNDP2. Several of the regulated miRs, such as miR-92b-5p, miR-320b, and miR-1233-5p might also mediate interesting signaling functions.
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Project description:Cell culture media is collected from mouse myotubes subject to electrical stimulation and control cells with no stimulation.
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