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

Prognostic Model for Hepatocellular Carcinoma with Time-Dependent Factors

The purpose of this study was to build a prognostic model of hepatocellular carcinoma (HCC) using time-dependent covariates to re-evaluate the prognosis at any stage of the disease. The subjects were consecutive HCC patients who were treated at our institute between 1995 and 2007. We constructed time-fixed and time-dependent prognostic models with a training group (n＝336) and compared the prognostic abilities between conventional Cancer of the Liver Italian Program (CLIP) scores, Japan Integrated Staging (JIS) scores, an Okuda classification, and our prognostic models in the testing group (n＝227) with the c-index. The time-dependent prognostic model consisted of main tumor size, tumor number, portal vein invasion, distant metastasis, alpha-fetoprotein, des-gamma-carboxy prothrombin (DCP), bilirubin, and albumin and the weighted scores were set for each factor depending on the hazard ratio for the prognosis. The prognostic index was determined by summing the scores. The c-index values for the CLIP scores, JIS scores, Okuda classification, and our time-dependent model were 0.741, 0.727, 0.609, and 0.870, respectively. These results indicate that our time-dependent model can estimate the prognosis of HCC more precisely than traditional time-fixed models and can be used to re-predict the prognosis of HCC

Fine tuning of phosphorothioate inclusion in 2'-O-methyl oligonucleotides contributes to specific cell targeting for splice-switching modulation

Splice-switching antisense oligonucleotide- (SSO-) mediated correction of framedisrupting mutation-containing premessenger RNA (mRNA) transcripts using exon skipping is a highly promising treatment method for muscular diseases such as Duchenne muscular dystrophy (DMD). Phosphorothioate (PS) chemistry, a commonly used oligonucleotide modification, has been shown to increase the stability of and improve the pharmacokinetics of SSOs. However, the effect of PS inclusion in 2'-O-methyl SSOs (2OMe) on cellular uptake and splice switching is less well-understood. At present, we demonstrate that the modification of PS facilitates the uptake of 2OMe in H2k-mdx myoblasts. Furthermore, we found a dependency of SSO nuclear accumulation and high splice-switching activity on PS inclusion in 2OMe (2OMePS), as tested in various reporter cell lines carrying pLuc/705. Increased exon-inclusion activity was observed in muscle, neuronal, liver, and bone cell lineages via both the gymnotic uptake and lipofection of 2OMePS. Using the photoactivatable ribonucleoside-enhanced crosslinking and a subsequent proteomic approach, we identified several 2OMePS-binding proteins, which are likely to play a role in the trafficking of 2OMePS to the nucleus. Ablation of one of them, Ncl by small-interfering RNA (siRNA) enhanced 2OMePS uptake in C2C12 myoblasts and upregulated luciferase RNA splicing in the HeLa Luc/705 reporter cell line. Overall, we demonstrate that PS inclusion increases nuclear delivery and splice switching in muscle, neuronal, liver, and bone cell lineages and that the modulation of 2OMePS-binding partners may improve SSO delivery

Scavenger Receptor Class A1 Mediates Uptake of Morpholino Antisense Oligonucleotide into Dystrophic Skeletal Muscle

Exon skipping using phosphorodiamidate morpholino oligomers (PMOs) is a promising treatment strategy for Duchenne muscular dystrophy (DMD). The most significant limitation of these clinically used compounds is their lack of delivery systems that target muscles; thus, cell-penetrating peptides are being developed to enhance uptake into muscles. Recently, we reported that uptake of peptide-conjugated PMOs into myofibers was mediated by scavenger receptor class A (SR-A), which binds negatively charged ligands. However, the mechanism by which the naked PMOs are taken up into fibers is poorly understood. In this study, we found that PMO uptake and exon-skipping efficiency were promoted in dystrophin-deficient myotubes via endocytosis through a caveolin-dependent pathway. Interestingly, SR-A1 was upregulated and localized in juxtaposition with caveolin-3 in these myotubes and promoted PMO-induced exon skipping. SR-A1 was also upregulated in the skeletal muscle of mdx52 mice and mediated PMO uptake. In addition, PMOs with neutral backbones had negative zeta potentials owing to their nucleobase compositions and interacted with SR-A1. In conclusion, PMOs with negative zeta potential were taken up into dystrophin-deficient skeletal muscle by upregulated SR-A1. Therefore, the development of a drug delivery system targeting SR-A1 could lead to highly efficient exon-skipping therapies for DMD. Keywords: phosphorodiamidate morpholino oligomer, exon skipping, oligonucleotide uptake, scavenger receptor, zeta potential, Duchenne muscular dystrophy, endocytosis, splice switching, mdx52, deliver

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

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

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

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