Elevation of components of the GLUT4 trafficking pathway during mouse skeletal muscle regeneration.

<p>A) VAMP2 and CHC22 levels during muscle regeneration after cardiotoxin injection on day 0 were compared between WT and CHC22-mice in muscle samples harvested on the day indicated. One typical set of immunoblots is shown at the left and quantification of VAMP2 relative to GAPDH signals from three experiments generated the ratios plotted at the right (upper plot). There was a statistically significant difference between WT and CHC22-mice in VAMP2 expression on day 14 after cardiotoxin injection (*<i>p</i><0.05), as determined by Student’s t test. Quantification of VAMP2 and CHC22 relative to GAPDH in the CHC22-mice shown at the left was plotted (right, bottom). B) GLUT4 levels during muscle regeneration for wild type and CHC22-mice (n = 3). One typical set of immunoblots is shown at the left and quantification of GLUT4 relative to GAPDH signals is plotted on the right. No significant difference between WT and CHC22-mice was detected. Molecular mass (kilodaltons) of the proteins detected is indicated at the right.</p

Delayed maturation and fiber type analysis of regenerating fibers in CHC22-transgenic mice.

<p>A) Hematoxylin and eosin staining of transverse muscle sections from WT or CHC22 transgenic mice after cardiotoxin injection at day 0, on the indicated days (D). Control (CTRL) sections were prepared from uninjured muscle. On day 28, some CHC22 myofibers were similar in cross-sectional area and diameter to WT myofibers; however, smaller myofibers (<40 µm diameter, marked by asterisks) were more frequently observed in the CHC22-mice compared to WT mice (scale bar, 20 µm; <i>n = </i>3). B) The mean cross-sectional area of myofibers with centrally located nuclei from WT (white bars) and CHC22-mice (black bars) was calculated and plotted for each indicated day after cardiotoxin injection. Control mice for each strain were not injected with cardiotoxin. There was a statistically significant difference in the average fiber cross-sectional area between WT mice and CHC22-mice on days 14 and 28 after injection (<i>n</i> = 3, evaluating 1200–2000 myofibers per mouse; **<i>p</i><0.01), as determined by Student’s t test. C) Transverse sections of muscle from CHC22-mice and WT mice, harvested 28 days after cardiotoxin injection, were stained for NADH-TR in order to determine the myofiber type. Oxidative (red) myofibers appear dark, glycolytic (white) myofibers appear light and intermediate (pink) myofibers appear intermediate in color. D) Fibers in 28-day regenerating muscle from CHC22-mice (black bars) and WT mice (white bars) were classified by type and their cross-sectional area measured in pixels using ImageJ (n = 3, evaluating ∼1500 fibers per mouse, *p<0.05, by Student’s t test). E) The percent of each fiber type in 28-day regenerating muscle from CHC22-mice (black bars) and WT mice (white bars) from the analysis in D is plotted.</p

Co-localization of CHC22 and components of the GLUT4 trafficking pathway in regenerating human muscle.

<p>A, B and C) Transverse sections of skeletal muscle from a patient with PM were immunostained with a polyclonal antibody against GLUT4 (red) and monoclonal antibodies against A) embryonic myosin heavy chain (eMHC, green), B) CHC22 (green) or C) CHC17 (green). Regenerating myofibers were identified by centrally located nuclei stained with DAPI (blue in merge). Boxed regions in the merged images are magnified seven-fold at the right showing overlap of markers in yellow (scale bars, 20 µm). D and E) Transverse sections of skeletal muscle from a patient with PM were immunostained with a polyclonal antibody against CHC22 (red) and a monoclonal antibody against VAMP2 (green). Regenerating myofibers were identified by centrally located nuclei stained with DAPI (blue in merge). Boxed regions in the merged images are magnified seven-fold at the right showing intracellular co-localization (yellow) of CHC22 and VAMP2 in a regenerating fiber of D) small diameter or E) large diameter (scale bars, 20 µm). At the far right, the percent internal staining for the markers indicated on the y-axis was quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077787#pone-0077787-g001" target="_blank">Figure 1F</a>, for fibers that were characterized according to internal staining for the marker indicated on the x-axis. <i>n</i> is the number of fibers analyzed in each staining combination shown at the left. Significant increased internal staining was determined by Student’s t-test (*<i>p</i><0.05, **<i>p</i><0.01).</p

Myoblasts from CHC22-mice undergo fusion but do not exhibit glucose-dependent proliferation.

<p>A and C) Images of primary myoblasts from wild-type (WT) or CHC22-transgenic mice cultured in FM with A) low (5.6 mM) or C) high (25 mM) glucose for the indicated time in hours (h), all seeded at the same density (scale bars, 100 µm). B and D) At the indicated time period for myoblasts cultured as in A and C, the nuclei were quantified for total number (Total), number present in multi-nuclear myotubes (Myotube), and number in mono-nuclear cells (Myoblast). These quantifications are plotted relative to the total nuclei present at the start of differentiation (switch to FM at 0 hours) for cultured myoblasts from CHC22-mice (filled circles) and WT mice (open circles). Two-way ANOVA and Tukey-Kramer post-hoc test showed significant differences between WT and CHC22 in all three panels. (*p<0.05 and **p<0.01) at indicated time points.</p

Effect of cytokines on human myoblast or myotube cultures.

<p>A) LHCNM2 human skeletal muscle myoblasts were cultured under differentiation conditions for the indicated number of days in the presence or absence (−) of the indicated cytokines. Cell lysates prepared on the indicated day were analyzed by immunoblotting for the protein indicated at the left. B) Experiment as in A with addition of indicated cytokines on differentiation day 8 when myotubes had formed. Molecular mass (kilodaltons) of the proteins detected is indicated at the right.</p

Increased internal CHC22 in regenerating muscle fibers in several human myopathies.

<p>A, B, C and D) Transverse sections of human skeletal muscle from a control individual or patients with polymyositis (PM), dermatomyositis (DM) or limb girdle muscular dystrophy (LGMD) were immunostained with polyclonal antibody against CHC22 (red) and a monoclonal antibody against embryonic myosin heavy chain (eMHC, green). Black and white images for each antibody are shown. In the merged color images nuclei were stained with DAPI (blue) and red-green overlap is shown in yellow (scale bars, 20 µm). The boxed regions in the merged images are magnified seven-fold at the far right and show CHC22 staining only. To illustrate the way in which internal fiber staining was quantified, the fiber boundaries drawn are shown in the magnified boxed regions with the thin dashed line representing the fiber border and the line of arrowheads representing the boundary for quantifying internal staining with the arrowheads pointing to the fiber interior. Only a segment of the fiber quantified is shown, but the asterisks in the internal region highlight the increased internal staining in the eMHC-positive fibers compared to the control. E) Quantification of total CHC22 fluorescence intensity (adjusted for patient comparison, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077787#s2" target="_blank">methods</a>) in eMHC-negative (eMHC−) fibers (<i>n</i> = 40) from all patients and eMHC-positive (eMHC+) fibers from patients with PM (<i>n</i> = 17), DM (<i>n</i> = 14) or LGMD (<i>n</i> = 5) in the patients shown in A–D, where <i>n</i> is the number of fibers analyzed for each patient. All patients had statistically significant higher CHC22 fluorescence intensity in eMHC+ fibers (solid grey bars) compared to eMHC− fibers (solid black bar), as determined by one-way ANOVA (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001). F) Quantification of internal CHC22 (internal pixel values/total pixel values with internal staining cut-off 2.5 microns beneath the fiber border, as illustrated in A–D in eMHC-negative (eMHC−) fibers (<i>n</i> = 19) from all patients and eMHC−positive (eMHC+) fibers from patients with PM (<i>n</i> = 8), DM (<i>n</i> = 4) or LGMD (<i>n</i> = 3) in the patients shown in A–D, where <i>n</i> is the number of fibers analyzed for each patient. Internal CHC22 fluorescence was significantly higher in eMHC+ fibers from patients with PM, DM and LGMD as determined by one-way ANOVA (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001). G) The frequency of muscle fibers with internal CHC22 labeling was determined for fibers that were eMHC+ with central nuclei or eMHC− fibers in muscle sections from patients with PM (<i>n = </i>4) and DM (<i>n</i> = 3), immunostained as in A, where n is the number of patients analyzed. All patients analyzed had a statistically significant higher frequency of internal CHC22 in eMHC+ fibers (solid bars) than in eMHC− fibers (open bars) (**<i>p</i><0.01), as determined by Fisher’s exact test.</p

CHC22 and GLUT4 in satellite cells expressing Pax7.

<p>A) Transverse sections of human skeletal muscle from patients diagnosed with dermatomyositis (DM) or necrotizing myopathy (NM) were immunostained with a monoclonal antibody against Pax7, a polyclonal (rabbit) antibody against CHC22 and a polyclonal (goat) antibody against GLUT4. Arrowheads indicate Pax7+ CHC22+ GLUT4+ cells. (scale bars, 50 µm) B) The frequency of Pax7-positive cells with increased CHC22 and/or GLUT4 labeling was quantified for sections from DM (<i>n</i> = 3) and NM (<i>n</i> = 3) patient samples, immunostained as in A.</p

Muscle fiber type switch during aging of CHC22-mice.

<p>Skeletal muscle (<i>gastrocnemius</i>) was harvested from CHC22-mice and WT mice at the indicated ages and tissue homogenate was analyzed by immunoblotting for Type I and Type IIa/b myosin heavy chains, as well as for CHC22 and loading control GAPDH. A typical immunoblot for each age group is shown at the left. Signals for myosin heavy chain (MyHC) type levels relative to GAPDH signals are shown at the right for 4–5 animals from the indicated age groups. The drop in Type I myosin heavy chain in aged CHC22 mice is significant, *p<0.05, by Student’s t test.</p

Mean diameter (microns) of regenerating muscle fibers in wild type mice or CHC22 transgenic mice on indicated days after cardiotoxin injection.

<p>Mean diameter (microns) of regenerating muscle fibers in wild type mice or CHC22 transgenic mice on indicated days after cardiotoxin injection.</p