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

Abstract

What is the central question of this study? Is electrical pulse stimulation (EPS) an in vitro exercise model able to elicit the hypertrophy of human muscle cells? What is the main finding and its importance? The addition of a restitution period of 8 h after EPS induces the enlargement of human muscle cells, a major physiological end-point to resistance exercise. This is supported by downregulation of myostatin, a negative regulator of muscle mass, and increased phosphorylated mTOR and 4E-BP1, key factors in the growth cascade. This proof-of-concept study provides a model of physiologically mediated muscle growth, which will be the basis for future studies aiming to depict molecular events governing the hypertrophy of human muscle cells. Electrical pulse stimulation (EPS) of muscle cells has previously been used as an in vitro exercise model. The present study aimed to establish an EPS protocol promoting the hypertrophy of human muscle cells, which represents a major physiological end-point to resistance exercise in humans. We hypothesized that adding a resting period after EPS would be crucial for the occurrence of the morphological change. Myoblasts obtained from human muscle biopsies (n = 5) were differentiated into multinucleated myotubes and exposed to 8 h of EPS consisting of 2 ms pulses at 12 V, with a frequency of 1 Hz. Myotube size was assessed using immunohistochemistry immediately, 4 and 8 h after completed EPS. Gene expression and phosphorylation status of selected markers of hypertrophy were assessed using RT-PCR and Western blotting, respectively. Release of the myokine interleukin-6 in culture medium was measured using enzyme-linked immunosorbent assay. We demonstrated a significant increase (31 ± 14%; P = 0.03) in the size of myotubes when EPS was followed by an 8 h resting period, but not immediately or 4 h after completion of EPS. The response was supported by downregulation (P = 0.04) of the gene expression of myostatin, a negative regulator of muscle mass, and an increase in phosphorylated mTOR (P = 0.03) and 4E-BP1 (P = 0.01), which are important factors in the cellular growth signalling cascade. The present work demonstrates that EPS is an in vitro exercise model promoting the hypertrophy of human muscle cells, recapitulating a major physiological end-point to resistance exercise in human skeletal muscle.

In vitro experimental models for examining the skeletal muscle cell biology of exercise: the possibilities, challenges and future developments

“…Applications While the use of EPS dates back to the 1970s, its specific use as an exercise-like treatment in vitro is a more recent development. In particular, EPS has been applied to study contraction-mediated changes in skeletal muscle signal transduction [37,83], metabolism [67,85], myokine regulation/release [99,100,112] and morphology [119]. Additionally, contraction-mediated cross-talk between skeletal muscle cells and other cell types (e.g.…”

“…enhanced glucose uptake and oxidation, and insulin sensitivity [67,85] and increased mitochondrial content [85]), myokine expression and release [7,67,99,100,112,123] and morphological changes (e.g. sarcomeric structure formation [37,67] and myotube hypertrophy [119]). Such EPSinduced sarcomeric structure formation reportedly relies upon calpain-mediated proteolysis [37].…”

“…It is likely that timedependent activation also likely occurs following cessation of exercise-like treatment in vitro. Indeed, serial sample harvests have demonstrated that PFI-induced changes in IL-6 mRNA expression [25], and EPS-induced GLUT4 recycling [83], IL-6 mRNA expression [7], PPARδ mRNA expression [93] and myotube hypertrophy [119] exhibit significant time effects over the period following treatment cessation. However, most studies harvest samples at a single time point following treatment cessation, which can vary across studies, meaning the time-course for changes following each of these treatments is not well characterised.…”

Electrical pulse stimulation of skeletal myoblasts cell cultures with simulated action potentials

“…It also showed an increase of myosin heavy chain and tropomyosin and striation patterns. A recent work has also shown the positive effects of an EPS protocol consisting of 2‐ms pulses at 12 V, with a frequency of 1 Hz, for the induction of human skeletal muscle cell hypertrophy (Tarum, Folkesson, Atherton, & Kadi, ).…”

Electrical Pulse Stimulation of Primary Human Skeletal Muscle Cells

“…However, differentiated human skeletal muscle cells (myotubes) are quiescent in cultures, and do not typically contract spontaneously, unless being innervated or electrically stimulated [4,5]. Motor neuron activation can be replaced by electrical pulse stimulation (EPS) in culture [1,2], and this model is widely applied as an in vitro model of contractions [6][7][8][9][10][11][12].…”