Expression profiling of a cellular exercise model based on the electrical pulse stimulation.
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE176563 | GEO | 2022/12/14
REPOSITORIES: GEO
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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.
2023-04-19 | GSE227887 | GEO
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.
2023-04-19 | GSE227938 | GEO
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 microarray 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.
2023-04-19 | GSE227936 | GEO
Project description:We hypothesized that muscle contraction produces a cellular stress signal capable to increase lipolysis to sustain fuel availability during exercise. The aim of the present study was to identify novel exercise-regulated myokines, aka exerkines, able to promote lipolysis in human adipocytes. To this end, human primary myotubes from lean healthy volunteers were submitted to electrical pulse stimulation to mimic either acute intense or chronic moderate exercise. Conditioned media experiments with hMADS adipocytes were performed. Unbiased proteomic and ELISA analyses were applied in conditioned media and human plasma samples. Real-time qPCR was performed in cultured myotubes and muscle biopsy samples.
2020-05-26 | PXD014126 | Pride
Project description:Muscle contraction during exercise is the major stimulus for the release of peptides and proteins (myokines) that are supposed to take part in the benefical adaptation to exercise. We hypothesize that application of an in vitro exercise stimulus as electric pulse stimulation (EPS) to human myotubes enables the investigation of the human muscle secretome in a clearly defined model. We applied EPS for 24 h to primary human myotubes and studied the whole genome-wide transcriptional response and as well as the release of candidate myokines. We observed 183 differentially regulated transcripts with fold-changes > 1.3. The transcriptional response resembles several properties of the in vivo situation in the skeletal muscle after endurance exercise, namely significant enrichment of pathways associated with interleukin and chemokine signaling, lipid metabolism, and anti-oxidant defense; notably without increased release of creatin kinase. Multiplex immunoassays verified the translation of the transcriptional response of several myokines into high secretion levels (IL6, IL8, CXCL1, LIF, CSF3, IL1B, TNF) and identified an increased secretion of additional cytokines (IL2, IL4, IL13, IL17A). Inhibitor studies and immunoblotting revealed the participation of ERK1/2 and AMPK dependent pathways in the upregulation of myokines. To conclude our data highlight the importance of skeletal muscle cells per se as endocrine cells. This in vitro exercise model is not only suitable to identify known and novel exercise-regulated myokines but it might be applied to primary human myotubes obtained from different muscle biopsy donors to study molecular mechanisms of the individual response to exercise. We performed gene expression microarray analysis of myotubes in vitro stimulated by EPS and control myotubes
2013-10-09 | E-GEOD-44051 | biostudies-arrayexpress
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.
2023-03-17 | PXD040935 | Pride
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).
2022-06-29 | GSE200335 | GEO
Project description:Physical exercise triggers numerous positive adaptations through the regulation of genes controlling muscle structure and function. Epigenetic factors like DNA methylation participate in transcriptional activation by allowing the recruitment of the transcription machinery to gene promoters. Exercise induces dynamic DNA demethylation at gene promoters, but the contribution of the demethylation precursor hydroxymethylcytosine is unknown. Given the evanescent nature of hydroxymethylcytosine, a model of muscle contraction amenable to collection of repeated and acute time series is requested to determine if contraction-induced demethylation is preceded by increased hydroxymethylcytosine levels. Here, we aimed to establish an acute skeletal muscle contraction model that could, at least partly, mimic the effect of acute exercise on gene expression, in order to investigate the effect of muscle contraction in DNA demethylation and hydroxymethylation. We refined an Electrical Pulse Stimulation (EPS) protocol in C2C12 myotubes that we benchmarked to the nuclear receptor subfamily 4 group A member 3 (Nr4a3) gene, a gene selected for having the highest increase in expression in response to acute exercise in humans. Using targeted bisulfite sequencing, we found that a region of the Nr4a3 promoter is rapidly demethylated 60 minutes after EPS and subsequently re-methylated after 120 minutes. Of interest, hydroxymethylation of the differentially methylated region of Nr4a3 promoter after EPS was elevated at 0 minutes and reached lowest levels at 60 minutes after EPS. We established a cell culture-based protocol to mimic acute transcriptional responses to exercise and provide insight into the mechanism by which exercise-responsive genes are demethylated after muscle contraction
2017-02-22 | GSE87748 | GEO
Project description:Physical exercise triggers numerous positive adaptations through the regulation of genes controlling muscle structure and function. Epigenetic factors like DNA methylation participate in transcriptional activation by allowing the recruitment of the transcription machinery to gene promoters. Exercise induces dynamic DNA demethylation at gene promoters, but the contribution of the demethylation precursor hydroxymethylcytosine is unknown. Given the evanescent nature of hydroxymethylcytosine, a model of muscle contraction amenable to collection of repeated and acute time series is requested to determine if contraction-induced demethylation is preceded by increased hydroxymethylcytosine levels. Here, we aimed to establish an acute skeletal muscle contraction model that could, at least partly, mimic the effect of acute exercise on gene expression, in order to investigate the effect of muscle contraction in DNA demethylation and hydroxymethylation. We refined an Electrical Pulse Stimulation (EPS) protocol in C2C12 myotubes that we benchmarked to the nuclear receptor subfamily 4 group A member 3 (Nr4a3) gene, a gene selected for having the highest increase in expression in response to acute exercise in humans. Using targeted bisulfite sequencing, we found that a region of the Nr4a3 promoter is rapidly demethylated 60 minutes after EPS and subsequently re-methylated after 120 minutes. Of interest, hydroxymethylation of the differentially methylated region of Nr4a3 promoter after EPS was elevated at 0 minutes and reached lowest levels at 60 minutes after EPS. We established a cell culture-based protocol to mimic acute transcriptional responses to exercise and provide insight into the mechanism by which exercise-responsive genes are demethylated after muscle contraction
2017-02-22 | GSE87747 | GEO
Project description:Muscle contraction during exercise is the major stimulus for the release of peptides and proteins (myokines) that are supposed to take part in the benefical adaptation to exercise. We hypothesize that application of an in vitro exercise stimulus as electric pulse stimulation (EPS) to human myotubes enables the investigation of the human muscle secretome in a clearly defined model. We applied EPS for 24 h to primary human myotubes and studied the whole genome-wide transcriptional response and as well as the release of candidate myokines. We observed 183 differentially regulated transcripts with fold-changes > 1.3. The transcriptional response resembles several properties of the in vivo situation in the skeletal muscle after endurance exercise, namely significant enrichment of pathways associated with interleukin and chemokine signaling, lipid metabolism, and anti-oxidant defense; notably without increased release of creatin kinase. Multiplex immunoassays verified the translation of the transcriptional response of several myokines into high secretion levels (IL6, IL8, CXCL1, LIF, CSF3, IL1B, TNF) and identified an increased secretion of additional cytokines (IL2, IL4, IL13, IL17A). Inhibitor studies and immunoblotting revealed the participation of ERK1/2 and AMPK dependent pathways in the upregulation of myokines. To conclude our data highlight the importance of skeletal muscle cells per se as endocrine cells. This in vitro exercise model is not only suitable to identify known and novel exercise-regulated myokines but it might be applied to primary human myotubes obtained from different muscle biopsy donors to study molecular mechanisms of the individual response to exercise.
2013-10-09 | GSE44051 | GEO