Transcriptome analyses of in vitro exercise by EPS and adrenaline
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE183159 | GEO | 2021/11/06
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: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:Regular physical exercise evokes profound physiological responses which are strongly associated with many health benefits. However, the details of cellular networks and mechanisms underlying the adaptive responses to exercise remain to be elucidated. We have previously shown the usefulness of electrical pulse stimulation (EPS) to investigate metabolic effects of exercise in cultured human myotubes. The aim of the present study was to uncover networks of signaling pathways and regulatory molecules responsible for the metabolic effects of exercise in human skeletal muscle cells exposed to chronic EPS. Differentiated myotubes were subjected to two different EPS protocols (protocol 1; 2 ms, 10 V, 0.1 Hz for 24 h or protocol 2; 2 ms, 30 V, and 1 Hz for 48 h). Fuel handling was assessed using radiolabeled substrates. The transcriptome, cell proteome, and secreted proteins from EPS-treated and untreated myotubes were analyzed using a combination of high-throughput RNA sequencing, microarray, and high-resolution liquid chromatography mass spectrometry. Independent validation of selected putative myokines were measured using ELISA or multiplex assay. Oxidative metabolism was enhanced in human myotubes exposed to EPS protocol 1. A total of 81 differentially regulated proteins and 952 differentially expressed genes (DEGs) were observed in the cells after EPS protocol 1. Gene ontology (GO) analysis indicated that significantly regulated proteins and genes were enriched in biological processes related to glycolytic pathways, positive regulation of fatty acid oxidation, and oxidative phosphorylation, as well as muscle contraction, autophagy/mitophagy, and oxidative stress. Moreover, proteomic identification of secreted proteins revealed extracellular levels of 137 proteins were changed in myotubes subjected to EPS protocol 1. We also found some degree of overlap between the DEGs found in myotubes submitted to EPS protocol 1 and protocol 2. Among these DEGs was a myokine, leukemia inhibitory factor, which showed to enhance the glucose uptake in skeletal muscle cells, indicating autocrine mechanism to maintain metabolic homeostasis. Collectively, our data provides new insight into the transcriptome, proteome and secreted proteins alterations following in vitro exercise and is a valuable resource for understanding the molecular mechanisms and regulatory molecules mediating the beneficial metabolic effects of exercise.
2022-07-27 | GSE201340 | GEO
Project description:To identify miRNAs, a microarray analysis were differentially expressed between C2C12 myotubes stimulated with 1 μM clenbuterol for 1 h and their control myotubes.
2019-04-24 | GSE130181 | GEO
Project description:Skeletal muscle mediates the beneficial effects of exercise, thereby improving insulin sensitivity and reducing the risk for type 2 diabetes. Current skeletal-muscle-models in vitro are incapable of fully recapitulating its physiological functions especially regarding exercise. Supplementation of IGF1, a growth factor secreted by myofibers in vivo, might help to overcome these limitations. Primary human CD56-positive myoblasts were differentiated into myotubes in the presence/absence of IGF1 in serum-free medium for 10 days. Daily collected samples were analyzed by proteomics, qRT-PCR and myotube contractibility via electrical-pulse-stimulation (EPS). Mitochondrial respiration and glucose uptake were measured. IGF1-supported differentiation formed thicker multinucleated myotubes showing physiological contraction upon EPS following day 6 while myotubes without IGF1 were almost incapable of contraction. IGF1-treatment upregulated particularly muscle-specific proteins that contribute to myofibril and sarcomere assembly, striated muscle contraction, and ATP production. Elevated PPARGC1A, MYH7 and reduced MYH1/2 suggest a switch towards a more oxidative phenotype in line with elevated mitochondrial respiration. IGF1-treatment also upregulated expression of GLUT4 and increased insulin-dependent glucose uptake. To conclude, utilizing IGF1, we engineered human myotubes that recapitulate the physiological traits of skeletal muscle in vivo vastly superior to established protocols. This novel model enables investigation of exercise on a molecular level, drug screening and interorgan-crosstalk by transfer to organ-on-chip.
2024-10-17 | PXD043200 | 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.
2013-10-09 | GSE44051 | 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