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

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

Dammarane-type triterpene extracts of <i>Panax notoginseng</i> root ameliorates hyperglycemia and insulin sensitivity by enhancing glucose uptake in skeletal muscle

<p>Skeletal muscle is an important organ for controlling the development of type 2 diabetes. We discovered <i>Panax notoginseng</i> roots as a candidate to improve hyperglycemia through <i>in vitro</i> muscle cells screening test. Saponins are considered as the active ingredients of ginseng. However, in the body, saponins are converted to dammarane-type triterpenes, which may account for the anti-hyperglycemic activity. We developed a method for producing a dammarane-type triterpene extract (DTE) from <i>Panax notoginseng</i> roots and investigated the extract’s potential anti-hyperglycemic activity. We found that DTE had stronger suppressive activity on blood glucose levels than the saponin extract (SE) did in KK-<i>A</i>^<i>y</i> mice. Additionally, DTE improved oral glucose tolerance, insulin sensitivity, glucose uptake, and Akt phosphorylation in skeletal muscle. These results suggest that DTE is a promising agent for controlling hyperglycemia by enhancing glucose uptake in skeletal muscle.</p> <p>DTM improved hyperglycemia.</p

Reproducible myogenic differentiation with the optimized protocol.

<p>(<b>a</b>) A schematic of our muscle differentiation protocol beginning with MyoD-hiPSCs. (<b>b</b>) Immunohistochemistry of differentiated MyoD-hiPSCs for MHC (red). Scale bar = 100 µm. (<b>c</b>) Percentage of MHC positive cells per total cells following MyoD-induced differentiation of 6 MyoD-hiPSC clones. (n = 3 for each clone). Data are listed as mean ±S.D.</p

Modeling Miyoshi Myopathy (MM) by patient derived-hiPSCs.

<p>(<b>a</b>) Morphology of patient derived MM-hiPSC clones, expanded following G418 selection for Tet-MyoD1 vector transposition. Scale bar = 200 µm. (<b>b</b>) RT-PCR analysis of endogenous pluripotent stem cell markers in MyoD-MM hiPSCs. (<b>c</b>) Efficient myogenic differentiation of MyoD-MM hiPSCs according to the protocol defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061540#pone-0061540-g003" target="_blank">Figure 3a</a>. MHC positive (left), or Myogenin positive (right) cells were observed dominantly. Scale bars = 100 µm. (<b>d</b>) DYSFERLIN expression of the myofibers from MyoD-MM hiPSCs (lane 1, 2), rescued MyoD-MM hiPSCs which expressed full-length <i>DYSF</i> cDNA driven by EF1α promoter (lane 3, 4), and control non-diseased MyoD-hiPSCs (lane 5) confirmed by western blotting. ACTB = β-actin. (<b>e</b>) Entry of FM1-43 green fluorescent dye into differentiated myofibers from MyoD-MM #5 (left), rescued MyoD-MM #5 with <i>DYSF</i> expression (middle), or control MyoD-hiPSC clone B7 #9 (right), before (0 s) and 20 s after (20 s) two photon laser-induced damage of the sarcolemmal membrane (arrow). Scale bars = 20 µm. (<b>f</b>) Summary time course data of accumulation of FM1-43 dye in laser-damaged myofibers derived from B7 #9 (black circles), MyoD-MM hiPSCs (red or blue triangles) and rescued MyoD-hiPSCs with DYSFERLIN expression (red or blue circles). n = 5 for each clone. Data are listed as mean ±S.E.</p

Functional assay for differentiated MyoD-hiPSCs.

<p>(<b>a, b</b>) Electron microscopy of differentiated MyoD-hiPSCs (<b>a</b>) and differentiated human myoblast Hu5/E18 cells (<b>b</b>). Red arrows indicate myofibrils. Black arrowheads indicate future Z lines. Black arrows indicate myosin fibers. Scale bars = 500 nm. (<b>c</b>) Serial photographs of differentiated MyoD-hiPSCs co-cultured with C2C12 cells. A hiPSC-derived mCherry+ cell (white arrow) fused with a mouse-derived GFP+ cell (white arrowhead) resulting in a yellow cell (red arrow). Time increments between images = TIME. Scale bar = 100 µm. (<b>d</b>) Immunohistochemistry of MyoD-hiPSCs co-cultured with C2C12 cells. White arrows indicate human nuclei in a GFP+ murine myofiber. Scale bar = 100 µm.</p

Optimization of Differentiation Conditions.

<p>(<b>a</b>) Percentage of MHC positive myogenic cells derived from MyoD-hiPSCs during 9 days differentiation with various timing of medium replacement. *<i>p</i><0.05 (<b>b</b>) The average number of nuclei of myofibers in each condition of 5% KSR or 5% HS containing media after 7 days differentiation. (<b>c</b>) Flow cytometric analysis of MyoD-hiPSCs with 24 h Dox treatment in different start points. Dox addition at differentiation d1 promoted higher percentage of mCherry expression in MyoD-hiPSCs than Dox addition at differentiation d4. (<b>d</b>) A merged image of phase-contrast and mCherry images in differentiated MyoD-hiPSCs which were administrated Dox at differentiation d4. Some MyoD-hiPSCs turned to be unresponsive with Dox, indicating no mCherry expression area (dotted line). Scale bar = 100 µm. (<b>e</b>) MHC positive myogenic cell number derived from MyoD-hiPSCs during 11 days differentiation with various administration periods of Dox. *<i>p</i><0.05.</p