Dataset Information

Expression and functional analysis of the hydrogen peroxide biosensors HyPer and HyPer2 in C2C12 myoblasts/myotubes and single skeletal muscle fibres.

ABSTRACT: Hydrogen peroxide (H₂O₂) is generated in cells and plays an important role as a signalling molecule. It has been reported that H₂O₂ is involved in physiological and pathological processes in skeletal muscle. However, H₂O₂ detection in cells with traditional techniques produces frequent artefacts. Currently, the HyPer biosensor detects intracellular H₂O₂ specifically in real time using fluorescence microscopy. The aim of this study was to develop and optimize approaches used to express the HyPer biosensor in different models of skeletal muscle cells, such as the C2C12 myoblast/myotube cell line and mature skeletal muscle fibres isolated from C57BL/6J mice, and to measure intracellular H₂O₂ in real time in these cells. The results show that the expression of the HyPer biosensor in skeletal muscle cells is possible. In addition, we demonstrate that HyPer is functional and that this biosensor detects changes and fluctuations in intracellular H₂O₂ in a reversible manner. The HyPer2 biosensor, which is a more advanced version of HyPer, presents improved properties in terms of sensitivity in detecting lower concentrations of H₂O₂ in skeletal muscle fibres. In conclusion, the expression of the HyPer biosensor in the different experimental models combined with fluorescence microscopy techniques is a powerful methodology to monitor and register intracellular H₂O₂ specifically in skeletal muscle. The innovation of the methodological approaches presented in this study may present new avenues for studying the role of H₂O₂ in skeletal muscle pathophysiology. Furthermore, the methodology may potentially be adapted to yield other specific biosensors for different reactive oxygen and nitrogen species or metabolites involved in cellular functions.

SUBMITTER: Fernandez-Puente E

PROVIDER: S-EPMC6972731 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Expression and functional analysis of the hydrogen peroxide biosensors HyPer and HyPer2 in C2C12 myoblasts/myotubes and single skeletal muscle fibres.

Fernández-Puente Escarlata E Sánchez-Martín Manuel A MA de Andrés Jorge J Rodríguez-Izquierdo Lorena L Méndez Lucía L Palomero Jesús J

Scientific reports 20200121 1

Hydrogen peroxide (H2O2) is generated in cells and plays an important role as a signalling molecule. It has been reported that H2O2 is involved in physiological and pathological processes in skeletal muscle. However, H2O2 detection in cells with traditional techniques produces frequent artefacts. Currently, the HyPer biosensor detects intracellular H2O2 specifically in real time using fluorescence microscopy. ...[more]

PMID: 31965006

Similar Datasets

Project description:In muscle, reactive oxygen species (ROS) generation increases with strenuous activity, chronic unloading, and inflammatory stimuli; skeletal muscle function is very sensitive to ROS; and there are redox-sensitive signaling pathways. Using myogenic cell cultures, we asked whether hydrogen peroxide (H2O2) induces adaptive changes in skeletal muscle gene expression. H2O2 downregulated or failed to induce antioxidant or apoptotic genes in the myotubes. Instead, H2O2 changed the expression of genes for cytosolic and mitochondrial enzymes, and upregulated inflammatory mediators. Finally, H2O2 had a mostly inhibitory effect on the expression of many transcription factors. The results indicate that mild oxidative stress may induce an adaptive response in skeletal muscle without antioxidant upregulation or apoptosis. Experiment Overall Design: Cell Culture: Experiment Overall Design: C2C12 myoblasts (ATCC, Rockville, MD) were cultured in DMEM supplemented with 10% fetal calf serum at 37°C in 5% CO2. Myoblast differentiation was initiated by switching to differentiation medium (DMEM supplemented with 2% heat-inactivated horse serum) and allowed to continue for 96 hrs. Differentiated myotubes were treated for 2 hours with H2O2 (Sigma-Aldrich, St. Louis, MO) 0, 1, 10, 100, or 1000 uM in DMEM supplemented with 0.5% heat-inactivated horse serum. Experiment Overall Design: cDNA microarrays: Experiment Overall Design: Total RNA was obtained from treated C2C12 myotubes with TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's recommended protocol.Oligonucleotide microarray studies with Affymetrix Mouse Genome 430A gene chips (n=20 chips) were conducted as described earlier. Microarrays were washed and stained with a streptavidin-bound marker and scanned. Data were analyzed with Affymetrix Microarray Suite 5.0 software. Only genes with consistent absent/present calls in all four independent replicates per group were considered for further analysis. Treatment comparisons were crossed so that each H2O2 sample was compared to each control. The software uses the one-sided Wilcoxon's signed rank test to estimate increase/no change/ decrease difference calls for each pair-wise comparison. Only difference calls consistent in all pair-wise comparisons and with average changes > 1.75 were considered significant, resulting in a conservative list of genes with changed expression levels. Functional classification of genes was based on an extensive literature review.

Project description:BackgroundSex differences are known to impact muscle phenotypes, metabolism, and disease risk. Skeletal muscle stem cells (satellite cells) are important for muscle repair and to maintain functional skeletal muscle. Here we studied, for the first time, effects of sex on DNA methylation and gene expression in primary human myoblasts (activated satellite cells) before and after differentiation into myotubes.MethodWe used an array-based approach to analyse genome-wide DNA methylation and gene expression in myoblasts and myotubes from 13 women and 13 men. The results were followed up with a reporter gene assay.ResultsGenome-wide DNA methylation and gene expression differences between the sexes were detected in both myoblasts and myotubes, on the autosomes as well as the X-chromosome, despite lack of exposure to sex hormones and other factors that differ between sexes. Pathway analysis revealed higher expression of oxidative phosphorylation and other metabolic pathways in myoblasts from women compared to men. Oxidative phosphorylation was also enriched among genes with higher expression in myotubes from women. Forty genes in myoblasts and 9 in myotubes had differences in both DNA methylation and gene expression between the sexes, including LAMP2 and SIRT1 in myoblasts and KDM6A in myotubes. Furthermore, increased DNA methylation of LAMP2 promoter had negative effects on reporter gene expression. Five genes (CREB5, RPS4X, SYAP1, XIST, and ZRSR2) showed differential DNA methylation and gene expression between the sexes in both myoblasts and myotubes. Interestingly, differences in DNA methylation and expression between women and men were also found during differentiation (myoblasts versus myotubes), e.g., in genes involved in energy metabolism. Interestingly, more DNA methylation changes occur in women compared to men on autosomes.ConclusionAll together, we show that epigenetic and transcriptional differences exist in human myoblasts and myotubes as well as during differentiation between women and men. We believe that these intrinsic differences might contribute to sex dependent differences in muscular phenotypes.

Dataset Information

Expression and functional analysis of the hydrogen peroxide biosensors HyPer and HyPer2 in C2C12 myoblasts/myotubes and single skeletal muscle fibres.

Publications

Expression and functional analysis of the hydrogen peroxide biosensors HyPer and HyPer2 in C2C12 myoblasts/myotubes and single skeletal muscle fibres.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets