Effect of Electrical Pulse Stimulation on Anabolic Signaling in L6 Myotubes

Abstract

The mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase with a central role in controlling cellular anabolism. By phosphorylating two downstream targets, p70‐S6 kinase (p70‐S6K) and eIF4E binding protein 1 (4EBP1), mTOR induces an increase in mRNA translation, leading to an upregulation of protein synthesis. In muscle tissue, mTOR activation in response to an exercise stimulus is a key signaling event in the anabolic response to physical activity. As studying the signaling response to exercise at the cellular level is challenging in whole organisms due to difficulty in isolating muscle cell level effects, development of a cell culture model mimicking exercise would provide an avenue for understanding the intracellular cellular signaling response to exercise in skeletal muscle. The aim of this study was to determine whether 12 hours of electrical pulse stimulation (EPS) would alter the anabolic response in L6 myotubes. METHODS Murine L6 myoblasts were cultured on 10 cm plates in standard culture medium supplemented with 1% penicillin/streptomycin, and 10% fetal bovine serum until 60% confluence. At this point, differentiation was induced by switching to medium without sodium pyruvate supplemented with 1% penicillin/streptomycin, 2% horse serum, and 0.5% insulin‐transferrin‐selenium (ITS). After 7 days of differentiation, myotubes were stimulated, in culture, for 12 hours at a low frequency (1 Hz, 2ms, 60V). Differentiation media was changed in all plates immediately before stimulation and immediately after stimulation in half of the plates. Half of the cells were harvested immediately after stimulation to assess the acute effects and the remaining plates were harvested 24 hours post stimulation and re‐feeding. Protein expression of common anabolic markers was measured using western blot techniques. RESULTS Our results indicate an increase in phosphorylated 4EBP1 immediately after stimulation (EPS‐Ea) compared to control (CON‐Ea) (p = 0.05). Additionally, phosphorylation levels return to baseline 24 hours post stimulation. In contrast, phosphorylation levels of p70‐S6K immediately after stimulation (EPS‐Ea) were not different from control (CON‐Ea) but were elevated 24 hours post stimulation (EPS‐La) (p = 0.09). Finally, we determined the ratio of phosphorylated to total levels of 4EBP1 were two‐fold greater immediately after stimulation (EPS‐Ea) compared to 24 hours post stimulation (EPS‐La) (p = 0.09). Conversely, the ratio of phosphorylated to total levels of p70‐S6K were two times greater 24 hours post stimulation (EPS‐La) compared to immediately after stimulation (EPS‐Ea) (p = 0.09). CONCLUSION We were able to determine that electrical pulse stimulation of L6 myotubes could be used as a model to better understand anabolic signaling strategies following EPS in culture. This study represents a first step in using electrical pulse stimulation as an in culture exercise mimetic and elucidating the role of mTOR and downstream targets in cell growth. Support or Funding Information This work was...