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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE44051 | GEO | 2013/10/09

SECONDARY ACCESSION(S): PRJNA188449

REPOSITORIES: GEO

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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.

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

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