Electric Pulse Stimulation of Myotubes as an In Vitro Exercise Model: Cell-Mediated and Non-Cell-Mediated Effects

Abstract

Regular exercise has emerged as one of the best therapeutic strategies to prevent and treat type-2-diabetes. Exercise-induced changes in the muscle secretome, consisting of myokines and metabolites, may underlie the inter-organ communication between muscle and other organs. To investigate this crosstalk, we developed an in vitro system in which mouse C2C12 myotubes underwent electric pulse stimulation (EPS) to induce contraction. Subsequently the effects of EPS-conditioned media (EPS-CM) on hepatocytes were investigated. Here, we demonstrate that EPS-CM induces Metallothionein 1/2 and Slc30a2 gene expression and reduces Cyp2a3 gene expression in rat hepatocytes. When testing EPS-CM that was generated in the absence of C2C12 myotubes (non-cell EPS-CM) no decrease in Cyp2a3 expression was detected. However, similar inductions in hepatic Mt1/2 and Slc30a2 expression were observed. Non-cell EPS-CM were also applied to C2C12 myotubes and compared to C2C12 myotubes that underwent EPS: here changes in AMPK phosphorylation and myokine secretion largely depended on EPS-induced contraction. Taken together, these findings indicate that EPS can alter C2C12 myotube function and thereby affect gene expression in cells subjected to EPS-CM (Cyp2a3). However, EPS can also generate non-cell-mediated changes in cell culture media, which can affect gene expression in cells subjected to EPS-CM too. While EPS clearly represents a valuable tool in exercise research, care should be taken in experimental design to control for non-cell-mediated effects.

Electrical pulse stimulation: an in vitro exercise model for the induction of human skeletal muscle cell hypertrophy. A proof‐of‐concept study

“…A second event in support of the EPS-mediated phosphorylation of mTOR was the absence of upregulation of AMPK, a key cellular energy sensor (Jäger et al 2007;Lambernd et al 2012;Evers-van Gogh et al 2015). It has been suggested that while resistance exercise is linked with an upregulation of the mTOR pathway, adaptation of skeletal muscle to endurance exercise is promoted through the upregulation of the AMPK pathway.…”

“…Skeletal muscle is an endocrine organ associated with the release of several myokines in response to exercise (Nedachi et al 2008;Raschke et al 2013). Interleukin-6 is known to be an important contraction-regulated myokine released by muscle cells in response to exercise in vivo (Keller et al 2001;Febbraio et al 2004) and in vitro (Scheler et al 2013;Evers-van Gogh et al 2015;Feng et al 2015). In accordance with previous data, increased release of IL-6 was observed in myotubes exposed to EPS (Lambernd et al 2012;Farmawati et al 2013;Scheler et al 2013;Christensen et al 2015;Feng et al 2015), which indicates that in addition to the hypertrophy of muscle cells, our in vitro exercise model recapitulates another important exercise-induced physiological event.…”

“…; Evers‐van Gogh et al . ). It has been suggested that while resistance exercise is linked with an upregulation of the mTOR pathway, adaptation of skeletal muscle to endurance exercise is promoted through the upregulation of the AMPK pathway.…”

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).…”

Exercise-Induced Myokines as Emerging Therapeutic Agents In Colorectal Cancer Prevention and Treatment

“…Thus, unraveling the molecular interplay between myokines and identifying the potentially therapeutic ones necessitates a simplified system. Our laboratory has adapted the in vitro exercise platform (myotubules stimulated with C-Pace system) [19] and used it for the application of discovering specific myokines (individual or combination) which may have therapeutic effects. Our initial focus has been on synergism of myokines with chemotherapy in CRC [20].…”