An electrical pulse stimulation protocol to study acute epigenetic response to muscle cell contraction uncovers acute hydroxymethylation of the exercise-responsive gene Nr4a3
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ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens
PROVIDER: GSE87749 | GEO | 2017/02/22
SECONDARY ACCESSION(S): PRJNA347343
REPOSITORIES: GEO
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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:Exercise training triggers numerous positive adaptations through the regulation of genes controlling muscle structure and function. Epigenetic modifications, including 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; however, the contribution of the demethylation precursor hydroxymethylcytosine is unknown. Given the evanescent nature of hydroxymethylcytosine, a muscle contraction model that allows for the collection of samples that are repeatedly stimulated over time is required to determine whether contraction-induced demethylation is preceded by changes in the hydroxymethylcytosine level. Here, we established an acute skeletal muscle contraction model to mimic the effects of acute exercise on gene expression. We used this model to investigate the effect of muscle contraction on DNA demethylation and hydroxymethylation. First, we performed an acute exercise study in healthy humans to identify an exercise-responsive gene that we could study in culture. We identified the nuclear receptor subfamily 4 group A member 3 (Nr4a3) gene with the highest fold-expression increase after acute exercise. We then refined an electrical pulse stimulation (EPS) protocol that could induce expression of the Nr4a3 gene in C2C12 myotubes. Using targeted bisulfite sequencing, we found that in response to EPS, a region of the Nr4a3 promoter is rapidly demethylated at 60?min and re-methylated at 120?min. Of interest, hydroxymethylation of the differentially methylated region of Nr4a3 promoter after EPS was elevated immediately after EPS, with lowest levels reached at 60?min after EPS. In conclusion, we have established a cell culture-based protocol to mimic the acute transcriptional responses to exercise. Furthermore, we provide insight into the mechanism by which the exercise-responsive gene Nr4a3 is demethylated after muscle contraction.
| S-EPMC5179501 | biostudies-literature
Project description:An electrical pulse stimulation protocol to study acute epigenetic response to muscle cell contraction uncovers acute hydroxymethylation of the exercise-responsive gene Nr4a3
| PRJNA347343 | ENA
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.
2022-12-14 | GSE176563 | GEO
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).
2021-08-20 | E-MTAB-10833 | biostudies-arrayexpress
Project description:Skeletal muscle communicates with other organs via myokines, which are secreted by muscle during exercise and exert various effects. Despite much investigation of the exercise, the underlying molecular mechanisms are still not fully understood. Here, we applied an in vitro exercise model in which cultured C2C12 myotubes were subjected to electrical pulse stimulation (EPS), which mimics contracting muscle. Based on the significantly up- and down-regulated genes in EPS, we constructed an in silico model to predict exercise responses at the transcriptional level. The in silico model revealed similarities in the transcriptomes of the EPS and exercised animals. Comparative analysis of the EPS data and exercised mouse muscle identified putative biomarkers in exercise signaling pathways and enabled to discover novel exercise-induced myokines. Biochemical analysis of selected exercise signature genes in muscle from exercised mice showed that EPS mimics in vivo exercise, at least in part, at the transcriptional level. Consequently, we provide a novel myokine, Amphiregulin (AREG), up-regulated both in vitro and in vivo, that would be a potential target for exercise mimetics.
| S-EPMC9731977 | biostudies-literature
Project description:Physical exercise leads to substantial adaptive responses in skeletal muscles and plays a central role in a healthy life style. Since exercise induces major systemic responses, underlying cellular mechanisms are difficult to study in vivo. It was therefore desirable to develop an in vitro model that would resemble training in cultured human myotubes.Electrical pulse stimulation (EPS) was applied to adherent human myotubes. Cellular contents of ATP, phosphocreatine (PCr) and lactate were determined. Glucose and oleic acid metabolism were studied using radio-labeled substrates, and gene expression was analyzed using real-time RT-PCR. Mitochondrial content and function were measured by live imaging and determination of citrate synthase activity, respectively. Protein expression was assessed by electrophoresis and immunoblotting.High-frequency, acute EPS increased deoxyglucose uptake and lactate production, while cell contents of both ATP and PCr decreased. Chronic, low-frequency EPS increased oxidative capacity of cultured myotubes by increasing glucose metabolism (uptake and oxidation) and complete fatty acid oxidation. mRNA expression level of pyruvate dehydrogenase complex 4 (PDK4) was significantly increased in EPS-treated cells, while mRNA expressions of interleukin 6 (IL-6), cytochrome C and carnitin palmitoyl transferase b (CPT1b) also tended to increase. Intensity of MitoTracker®Red FM was doubled after 48 h of chronic, low-frequency EPS. Protein expression of a slow fiber type marker (MHCI) was increased in EPS-treated cells.Our results imply that in vitro EPS (acute, high-frequent as well as chronic, low-frequent) of human myotubes may be used to study effects of exercise.
| S-EPMC3310863 | biostudies-literature
Project description:Human primary myotubes +/- electrical pulse stimulation
| PRJEB46916 | ENA
Project description:Optimization of resistance exercise (RE) remains a hotbed of research for muscle building and maintenance. However, the interactions between the contractile components of RE (i.e. concentric (CON) and eccentric (ECC)) and age, are poorly defined. We used transcriptomics to compare age-related molecular responses to acute CON and ECC exercise. Eight young (21±1 y) and eight older (70±1 y) exercise-naïve male volunteers had vastus lateralis biopsies collected at baseline and 5 h post unilateral CON and contralateral ECC exercise. RNA was subjected to next-generation sequencing and differentially expressed (DE) genes tested for pathway enrichment using Gene Ontology (GO). The young transcriptional response to CON and ECC was highly similar and older adults displayed moderate contraction-specific profiles, with no GO enrichment. Age-specific responses to ECC revealed 104 DE genes unique to young, and 170 DE genes in older muscle, with no GO enrichment. Following CON, 15 DE genes were young muscle-specific, whereas older muscle uniquely expressed 147 up-regulated genes enriched for cell adhesion and blood vessel development, and 28 down-regulated genes involved in mitochondrial respiration, amino acid and lipid metabolism. Thus, older age is associated with contraction-specific regulation often without clear functional relevance, perhaps reflecting a degree of stochastic age-related dysregulation.
| S-EPMC6503873 | biostudies-literature