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:
PROVIDER: PRJNA347343 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR4381360_1.fastq.gz | Fastqsanger.gz | |||
| SRR4381360_2.fastq.gz | Fastqsanger.gz | |||
| SRR4381370_1.fastq.gz | Fastqsanger.gz | |||
| SRR4381370_2.fastq.gz | Fastqsanger.gz | |||
| SRR4381380_1.fastq.gz | Fastqsanger.gz |
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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.
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Project description:Human primary myotubes +/- electrical pulse stimulation
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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: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:Purpose: The goal of this study was to determine the gene expression changes that occur over 7 days in parralyzed muscle in response to isometric contraction elicited by electrical stimulation initiated 4 months after spinal cord injury and to compare such changes to those observed in a normal muscle subjected to overload. Methods: Electrical stimulation of the soleus and plantaris muscle was stimulated in female rats with complete transection of the spinal cord at the interspace between the 9th and 10th thoracic vertebrae. Stimulation was begun 16 weeks after spinal cord transection and produced near-isometric contraction of soleus, plantaris and tibialis anterior. Muscle was analyzed at 1, 2 and 7 days after starting exercise with electrical stimulation. To provide a baseline reference for gene expression at 16 weeks after spinal cord injury, muscle was also analysed from an additional group of spinal cord transected animals. One additional group of animals with a sham-spinal cord injury was included to provide information about gene expression in neurologically intact animals of similar age. In parallel studies, rats underwent bilateral gastrocnemius ablation to overload soleus and plantaris, or a sham ablation as a control. Muscle was analyzed at 1, 3 and 7 days after gastrocnemius ablation or sham-ablation. Gene expression was determined using Affymetrix Rat Exon microarrays. For each group of animals, microarray analysis was performed for soleus muscle for each of 3 separate animals, using one array per animal. Control sammples for the spinal cord injured groups included a group of animals with a Sham-spinal cord injury, and a group of spinal cord injured animals that did not get electrical stimulation. The comparator for determining fold-change expression values was the spinal cord injured group that did not receive electrical stimulation. For each day after gastrocnemius ablation, a control was included that received all procedures needed for this ablation except cutting the distal insertion of the gastrocnemius into the Achilles tendon to control for effects of the surgery on gene expression.
2013-03-30 | E-GEOD-37476 | biostudies-arrayexpress
Project description:We used a novel approach to study the acute effect of three physiologic stressors (active contractions, vibration, and systemic heat stress) in human skeletal muscle. Three hours after the completion of a dose of physiologic stress, we sampled the soleus (contraction and vibration) or vastus lateralis (heat) muscle and developed a unique gene expression signature for each stressor. We discovered repetitive active muscle contractions up regulated metabolic transcription factors NR4A3 (12.45 fold change), PGC-1α (5.46 fold change), and ABRA (5.98 fold change); and repressed MSTN (0.56 fold change). Heat stress repressed PGC-1α (0.74 fold change); while vibration induced FOXK2 (2.36 fold change). Vibration similarly caused a down regulation of MSTN (0.74 fold change), but to a lesser extent than active muscle contraction. Vibration induced FOXK2 while heat stress repressed PGC-1α (0.74 fold change) and ANKRD1 genes (0.51 fold change). These findings support a distinct gene regulation in response to heat stress, vibration, and muscle contractions. Understanding these responses may assist in developing regenerative rehabilitation interventions to improve muscle cell development, growth, and repair.
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