Characterization of an Acute Muscle Contraction Model Using Cultured C2C12 Myotubes

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 $(Ca^{2+})$ 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

Contractile activity of C2C12 myotubes.

<p>(A) The average of contractile activity is shown. The contractile activity of the cells was evaluated as the distance shortened between specified points on a myotube using a motion analyzer. Change in the distance was tracked during contraction. Contraction of the myotubes is synchronized with the intermittent electrical stimulation (see Movie S1). The myotubes were stimulated with electrical pulses of 50 V at 1 Hz for 3 ms at 997-ms intervals. The onset of the electric pulses is shown by solid arrows and their cessation is shown by dashed arrows. Data are mean ± S.E.M., n = 5. (B) Integrated values for 5-s change in the distance at 0, 1, 2, and 3 h after the onset of electrical stimulation. The video is shown in Movies S2, S3, S4 and S5 (0–3 h). The change in the distance at 0, 1, 2, and 3 h after the onset of stimulation are shown as the integral values of the areas above the curves for 5 s (the values are converted to positive number). The integrated values 1, 2, and 3 h after stimulation onset are comparable with that at the onset (0 h). Data are shown as mean ± S.E.M., n = 3.</p

Ca²⁺ fluorescence with and without electrical stimulation.

<p>(A) Ca²⁺ fluorescence with and without electrical stimulation. Myotubes were treated with Fluo-8 dye loading solution 30 min before electrical stimulation. The images are shown at 200× magnification. The upper panel shows the bright-field image. The middle panel shows the myotubes with electric pulses, and the lower panel shows the myotubes without electric pulses. (B) Changes in Ca²⁺ fluorescence intensity with electrical stimulation. The fluorescence intensity was analyzed at 5 arbitrary points. Each line shows the raw fluorescence intensity data at each point. (C) The average fluorescence intensity for 11 s is shown. The average fluorescence intensity with electric pulses is significantly higher than that without electric pulses (<i>p</i><0.01, Student’s t-test).</p

Immunoblotting of phosphoproteins after electrical stimulation for 1 h.

<p>C2C12 myotubes were stimulated with electric pulses (50 V, 1 Hz, 3 ms) for 60 min at 37°C. Representative blots of the phosphorylation of Akt (Ser 308), p-38 (Thr180/Tyr182), AMPK (Thr172), and JNK1/2 (Thr183/Tyr185) and their expression levels induced by electrical stimulation for 60 min in C2C12 myotubes are shown. The phosphorylation ratios were calculated by dividing the phosphorylation levels by the protein expression levels. Significant increases in phosphorylated Akt (Ser 308), AMPK (Thr172), p-38 (Thr180/Tyr182), and JNK1/2 (Thr183/Tyr185) were detected after 1 h of contraction. Data are shown as mean ± S.E.M, n = 6–14.</p

The process of myotube formation in C2C12 cultured cells.

<p>The medium was switched to 2% calf serum differentiation medium when the cells reached 90–100% confluence (day 0). Days 2 and 5 indicate the days after switching to differentiation medium. C2C12 myoblasts started to fuse after induction of differentiation, and formed multinucleated myotubes by day 5. All images are shown at 200× magnification.</p

LDH activity in the culture medium after 1 h of contraction in C2C12 myotubes.

<p>C2C12 myotubes were stimulated by electric pulses (50 V, 1 Hz, 3 ms) for 1 hr. There was no significant difference between non-contracted control and the contraction group (n = 6). LDH release (%) was calculated by dividing the amount of LDH in medium by the total amount of LDH in the medium and lysate (Materials and Methods).</p