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Cellular proteome and secretome analysis of electrically pulse-stimulated human myotubes

ABSTRACT: In this study, we aimed to investigate alterations in secreted proteins as well as expression profile changes in cellular proteome in human skeletal muscle cells that are triggered by a well-established in vitro model of exercise using electrical pulse stimulation (EPS). Here, skeletal muscle cells isolated from muscle biopsies from seven donors were used. The cells from each donor were then exposed to prolonged EPS (i.e. 10 V, 2 ms, 0.1 Hz for 24 h), and non-stimulated cells from the same donors were used as a control.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Tuula Nyman

LAB HEAD: Tuula Nyman

PROVIDER: PXD033025 | Pride | 2022-08-12

REPOSITORIES: Pride

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Publications

Insight Into the Metabolic Adaptations of Electrically Pulse-Stimulated Human Myotubes Using Global Analysis of the Transcriptome and Proteome.

Mengeste Abel M AM Nikolić Nataša N Dalmao Fernandez Andrea A Feng Yuan Z YZ Nyman Tuula A TA Kersten Sander S Haugen Fred F Kase Eili Tranheim ET Aas Vigdis V Rustan Arild C AC Thoresen G Hege GH

Frontiers in physiology 20220706

Electrical pulse stimulation (EPS) has proven to be a useful tool to interrogate cell-specific responses to muscle contraction. In the present study, we aimed 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 from young male subjects were exposed to EPS protocol 1 (i.e. 2 ms, 10 V, and 0.1 Hz for 24 h), whereas myotubes from middle-aged women and men w ...[more]

PMID: 35874526

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Effects of benfotiamine in cultured human myotubes exposed to both normal and high glucose cencentrations

Project description:The aim of the present work was to study the effects of benfotiamine (S-benzoylthiamine O-monophosphate) upon glucose and lipid metabolism and gene expression in differentiated human skeletal muscle cells (myotubes) incubated for 4 days under normal (5.5 mM glucose) and hyperglycemic (20 mM glucose) conditions. Myotubes established from lean, healthy volunteers were treated with benfotiamine for 4 days. Glucose and lipid metabolism were studied with labeled precursors. Gene expression was measured using real time polymerase chain reaction (qPCR) and microarray technology. Cell experiment, pared observations treated vs control, cells from 3 different donors

2011-08-21 | E-GEOD-31553 | biostudies-arrayexpress

Chronic treatment with terbutaline increases glucose and oleic acid oxidation and protein synthesis in cultured human myotubes

Project description:Objective: In vivo studies have reported several beneficial metabolic effects of β-adrenergic receptor agonist administration in skeletal muscle, including increased glucose uptake, fatty acid metabolism, lipolysis and mitochondrial biogenesis. Although these effects have been widely studied in vivo, the in vitro data are limited to mouse and rat cell lines. Therefore, we sought to discover the effects of the β2-adrenergic receptor agonist terbutaline on metabolism and protein synthesis in human primary skeletal muscle cells. Results: The results showed that 4 h treatment with terbutaline was sufficient to increase glucose uptake in human myotubes, but also decreased both glucose and oleic acid oxidation along with oleic acid uptake. Moreover, administration of terbutaline for 96 h increased glucose uptake and oxidation, as well as oleic acid oxidation, leucine incorporation into cellular protein and upregulated several pathways related to mitochondrial metabolism. Conclusion: These results suggest that β2-adrenergic receptor have direct effects in human skeletal muscle affecting fuel metabolism and net protein synthesis, effects that might be favourable for both type 2 diabetes and muscle wasting disorders.

2022-02-14 | PXD024063 | Pride

Cytokines dominate the secretome of primary human myotubes in an in vitro exercise model

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

Gene expression profiles of electrically pulse-stimulated human myotubes

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

Effect of ATF4 overexpression on mRNA levels in differentiated C2C12 myotubes.

Project description:ATF4 is a bZIP transcription factor that that promotes skeletal muscle atrophy. The goal of these studies was to determine the effects of ATF4 overexpression on mRNA levels in differentiated C2C12 myotubes. For additional details see Ebert et al, Stress-Induced Skeletal Muscle Gadd45a Expression Reprograms Myonuclei and Causes Muscle Atrophy. JBC epub. June 12,2012 C2C12 myotubes were infected with adenovirus co-expressing eGFP and ATF4-FLAG. Control myotubes were infected with adenovirus co-expressing eGFP and a transcriptionally inactive ATF4 construct (ATF4∆bZIP).

2012-09-15 | E-GEOD-39194 | biostudies-arrayexpress

Eicosapentaenoic acid improves metabolic switching in human myotubes

Project description:Metabolically healthy skeletal muscle is characterized by the ability to switch easily between glucose and fat oxidation, whereas loss of this ability seems to be related to insulin resistance. The aim of this study was to investigate whether different fatty acids (FAs) and the LXR ligand T0901317 affected metabolic switching in human skeletal muscle cells (myotubes). Pretreatment of myotubes with eicosapentaenoic acid (EPA) increased suppressibility, the ability of glucose to suppress FA oxidation, and metabolic flexibility, the ability to increase FA oxidation when changing from “fed” to “fasted” state. Adaptability, the capacity to increase FA oxidation with increasing FA availability, was increased after pretreatment with EPA, linoleic acid (LA) and palmitic acid (PA). T0901317 counteracted the effect of EPA on suppressibility and adaptability, but did not affect these parameters alone. EPA itself accumulated less, however, EPA, LA, OA and T0901317 increased the number of lipid droplets (LDs) in myotubes, whereas LD size and mitochondria amount were independent of pretreatment. Microarray analysis showed that EPA regulated more genes than the other FAs. Some pathways involved in carbohydrate metabolism were induced only by EPA. The present study suggests a possible favorable effect of EPA on skeletal muscle metabolic switching and glucose utilization. Keywords: Analysis of target gene regulation by using microarrays. Primary human myotubes, derived from 3 healthy, female donors, were preincubated with different fatty acids (oleic acid [OA], palmitic acid [PA], eicosapentaenoic acid [EPA] or linoleic acid [LA], each at 100 µM) or bovine serum albumin [BSA] (40 µM) for 24 h.

2010-06-23 | E-GEOD-18589 | biostudies-arrayexpress

TGFβ1 impairs the transcriptomic response to contraction in myotubes from women with polycystic ovary syndrome

Project description:Polycystic ovary syndrome (PCOS) is characterised by a hormonal imbalance affecting the reproductive and metabolic health of reproductive-aged women. Exercise is often recommended as a first-line therapy for women with PCOS to help improve their overall health however, women with PCOS are resistant to the metabolic benefits of exercise training. Here, we aimed to gain insight into the mechanisms responsible for such resistance to exercise in PCOS. We employed an in vitro approach with electrical pulse stimulation (EPS) of cultured skeletal muscle cells to explore whether muscle cells from women with PCOS have an altered gene expression signature in response to muscle contraction. Following EPS, 4,719 genes were differentially expressed (FDR < 0.05) in myotubes from women with PCOS compared to only 173 in healthy control women. Both groups included genes involved in skeletal muscle contraction. We also determined the effect of two transforming growth factor-beta ligands that are elevated in plasma of women with PCOS, the transforming growth factor beta-1 (TGFβ1) and the anti-müllerian hormone (AMH), alone and on the EPS-induced response. While AMH (30 ng/ml) had no effect, TGFβ1 (5 ng/ml) induced the expression of extracellular matrix genes and impaired the exercise-like gene expression signature in myotubes from women with and without PCOS in response to EPS by interfering with key processes related to muscle contraction, calcium transport and actin filament. Collectively, our findings suggest that while the fundamental gene expression responses of skeletal muscle to contraction is intact in PCOS, elevated circulating factors like TGFβ1 may be responsible for the impaired adaptation to exercise intervention in women with PCOS.

2022-10-12 | GSE199225 | GEO

Cytokines dominate the secretome of primary human myotubes in an in vitro exercise model

2013-10-09 | GSE44051 | GEO

Engineering of human myotubes towards a mature metabolic and contractile phenotype

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

Expression data from C2C12 myotubes infected with adenovirus expressing Gli1

Project description:We sought to determine the effects of over-expression of Gli1 on gene expression in C2C12 myotube cultures. C2C12 myoblasts were induced to differentiate for 4 days. At that time, when >80% of nuclei were incorporated into multi-nucleated syncitial myotubes, we infected the cultures with recombinant adenovirus expressing GFP alone or GFP and a full length human Gli1. Media was changed 12 hours later. Cultures were lysed 60 hours after the initial infection. Gli1 over-expression induces de-differentiation of myotubes and proliferation of myoblasts. Results provide insight into the molecular basis of SHH signaling on skeletal muscle cells.

2013-12-01 | E-GEOD-37702 | biostudies-arrayexpress

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