Characterization of an Acute Muscle Contraction Model Using Cultured C2C12 Myotubes

Abstract

A cultured C2C12 myotube contraction system was examined for application as a model for acute contraction-induced phenotypes of skeletal muscle. C2C12 myotubes seeded into 4-well rectangular plates were placed in a contraction system equipped with a carbon electrode at each end. The myotubes were stimulated with electric pulses of 50 V at 1 Hz for 3 ms at 997-ms intervals. Approximately 80% of the myotubes were observed to contract microscopically, and the contractions lasted for at least 3 h with electrical stimulation. Calcium ion (Ca2+) transient evoked by the electric pulses was detected fluorescently with Fluo-8. Phosphorylation of protein kinase B/Akt (Akt), 5′ AMP-activated protein kinase (AMPK), p38 mitogen-activated protein kinase (p38), and c-Jun NH2-terminal kinase (JNK)1/2, which are intracellular signaling proteins typically activated in exercised/contracted skeletal muscle, was observed in the electrically stimulated C2C12 myotubes. The contractions induced by the electric pulses increased glucose uptake and depleted glycogen in the C2C12 myotubes. C2C12 myotubes that differentiated after exogenous gene transfection by a lipofection or an electroporation method retained their normal contractile ability by electrical stimulation. These findings show that our C2C12 cell contraction system reproduces the muscle phenotypes that arise in vivo (exercise), in situ (hindlimb muscles in an anesthetized animal), and in vitro (dissected muscle tissues in incubation buffer) by acute muscle contraction, demonstrating that the system is applicable for the analysis of intracellular events evoked by acute muscle contraction.

Mechanical stretch activates mammalian target of rapamycin and AMP-activated protein kinase pathways in skeletal muscle cells

“…In the present study, 4-h exposure to 15 % cyclic uniaxial stretch at the frequency of 1 Hz significantly increased the phosphorylation of p70S6K in myotubes, suggesting that mechanical stretch activates the initiation of protein translation. In contrast, mechanical stimulus-induced activation of AMPK has also been demonstrated in skeletal muscle cell in vivo [38], ex vivo [39,40], and in vitro [41]. In the present study, phosphorylation of AMPK at T172 of the catalytic a subunit was significantly higher in stretched cells, suggesting that mechanical stretch activated AMPK.…”

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

“…C 2C12 myoblasts were seeded on six-well plates at a density of 3 ϫ 10 5 cells/well with 2 ml of growth medium consisting of DMEM (25 mM glucose) supplemented with 10% FBS and 1% penicillin-streptomycin until confluence. Differentiation of myoblasts into myotubes was induced by switching cultures to DMEM supplemented with 2% calf serum and 1% penicillin-streptomycin, as described (19,20). At day 6 or 7 days after the initiation of differentiation, myotubes were used for experiments.…”

“…Exercise-or ES-induced membrane depolarization is accompanied by cytosolic calcium increases brought about by calcium efflux from sarcoplasmic reticulum,in both skeletal muscles (1) and C 2 C 12 myotubes (19). To investigate the effect of cytosolic calcium elevation on CCL2 expression, myotubes were preincubated for 1 h with 15 M BAPTA-AM, a membrane-permeant calcium chelator, and then electrically stimulated for 4 h in the continued presence of BAPTA-AM.…”

A fragmented form of annexin A1 is secreted from C2C12 myotubes by electric pulse-induced contraction

“…We thus conducted contraction of C2C12 myotubes by electric pulse stimulation and measured the calpain activity. We found that cell contraction increased the phosphorylation of AMP-activated protein kinase (AMPK; Thr172) as an indicator of muscle contraction [17]. We therefore showed that our contraction system could reproduce in vivo muscle contraction (Fig.…”

“…The cell contraction method used here was as described in [17], with a slight modification. Briefly, the medium was changed to 2 mL of serum-free DMEM before the contraction study.…”