Cytokine response of primary human myotubes in an in vitro exercise model

Abstract

Muscle contraction during exercise is a major stimulus for the release of peptides and proteins (myokines) that are supposed to take part in the beneficial 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 molecular response to exercise in a clearly defined model. We applied EPS for 24 h to primary human myotubes and studied the whole genome-wide transcriptional response 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 antioxidant defense. Multiplex immunoassays verified the translation of the transcriptional response of several cytokines into high-secretion levels (IL-6, IL-8, CXCL1, LIF, CSF3, IL-1B, and TNF) and the increased secretion of further myokines such as angiopoietin-like 4. Notably, EPS did not induce the release of creatine kinase. Inhibitor studies and immunoblotting revealed the participation of ERK1/2-, JNK-, and NF-κB-dependent pathways in the upregulation of myokines. To conclude, our data highlight the importance of skeletal muscle cells as endocrine cells. This in vitro exercise model is not only suitable to identify exercise-regulated myokines, but it might be applied to primary human myotubes obtained from different muscle biopsy donors to study the molecular mechanisms of the individual response to exercise.

Myotubes from lean and severely obese subjects with and without type 2 diabetes respond differently to an in vitro model of exercise

“…This may reflect an inherent abnormal IL-6 response in insulin-resistant skeletal muscle (27,47). It has previously been shown that EPS increased gene expression of IL-8 in lean nondiabetic myotubes (48), and our current data indicate that the impact of EPS on gene expression of IL-8 differs in myotubes from severely obese nondiabetic subjects compared with diabetic subjects. Exercise-derived IL-8 is postulated to stimulate angiogenesis in skeletal muscles (39), but the biological role of muscle-derived IL-8 remains to be clarified.…”

“…Gene regulation involved in exercise adaptation is complex (14,48), but genes regulated by PPAR␦ may be involved (15). It has also been proposed that PPAR␦ agonists and exercise training synergistically increase the proportion of type 1 fibers (MHCI) in adult mice (35).…”

“…Human myotubes are considered a valid model for studying metabolic disorders as obesity and T2D because perturbances evident in vivo, such as depressed lipid oxidation and insulin resistance, are retained in myotubes in culture (1,7,19). After EPS, myotubes established from lean nondiabetic subjects showed an increase in glucose and lipid metabolism as well as indications of fast-twitch glycolytic muscle fiber transformation into slow-type oxidative fibers (36) and enhanced expression and release of IL-6 and IL-8 (31,44,48). In the present study, we wanted to explore whether there are differences in EPS response on insulin sensitivity, glucose and lipid metabolism, and gene expression in myotubes from three groups of donors-lean nondiabetic, severely obese nondiabetic, and severely obese subjects with established T2D-and whether these differences reflect the in vivo characteristics of the donor groups.…”

Contracting C2C12 myotubes release CCL2 in an NF-κB-dependent manner to induce monocyte chemoattraction

“…Moreover, IL-6, CCL2, and CXCL1 rise in the circulation during exercise (5,22), but the cellular source is unknown. These cytokines are also secreted from contracted mouse C 2 C 12 myotubes (8,22) and cultured primary human myotubes (32,36,37), suggesting the possibility that muscle cells may be a direct source of circulating chemoattractants in response to exercise. As Pedersen and Febbraio proposed, "myokines" (defined as cytokines and other peptides that are produced and released by muscle fibers) may exert autocrine, paracrine, or endocrine effects (28).…”

Adenosine A2B Receptors - Mediated Induction of Interleukin-6 in Skeletal Muscle Cells

“…1 hour, 3 hours and up to 24 hours), which in line with my main aim was employed to identify the transcriptional events that precede the induction of inflammation under the activation of adenosine A2B receptors. It has shown previously 57 that most of the inflammatory transcripts studied (such as IL-6) in skeletal muscle cells are not translated into protein within that time frame. Therefore my future studies using longer durations of NECA (after 24 hours) will be recommended to investigate the translational changes in response to treatment.…”