Modulation of mitochondrial proteins expression after exogenous induction/inhibition of SPARC in proliferating myoblasts.

<p>C2C12 cells were cultured in four different conditions which were kept in GM supplement with/without rSPARC and/or anti-SPARC for 48 h. Extracted cells lysates were prepared to perform western blot and the expression of mitochondrial OXPHOS proteins were measured using 30 μg of whole cell lysate proteins. The experiments were repeated three times (three different passages of C2C12 cells) and a representative western blot image was shown. Data were expressed as a ratio to the positive control (pooled samples). One-way ANOVA with repeated measurements was used to adjust the validations of the different passages and a contrast analysis was performed. P value was set at < 0.05 after the Bonfferoni adjustments. The addition of rSPARC (8 μg/ml) increased SDHB (a) and UQCR2 (b) proteins levels, whereas, anti-SPARC (40 μg/ml) decreased it. Abbreviations: SDHB: succinate dehydrogenase iron-sulfur subunit beta, UQCR2: ubiquinol-cytochrome c reductase core protein 2, PBS: phosphate buffered saline, rSPARC: recombinant SPARC protein, and anti-SPARC: anti-SPARC antibody. *Significant differences between experimental conditions.</p

Implication of SPARC in the modulation of the extracellular matrix and mitochondrial function in muscle cells

<div><p>Secreted protein, acidic and rich in cysteine (SPARC) is differentially associated with cell proliferation and extracellular matrix (ECM) assembly. We show here the effect of exogenous SPARC inhibition/induction on ECM and mitochondrial proteins expression and on the differentiation of C2C12 cells. The cells were cultured in growth medium (GM) supplemented with different experimental conditions. The differentiation of myoblasts was studied for 5 days, the expressions of ECM and mitochondrial proteins were measured and the formation of the myotubes was quantified after exogenous induction/inhibition of SPARC. The results indicate that the addition of recombinant SPARC protein (rSPARC) in cell culture medium increased the differentiation of C2C12 myoblasts and myogenin expression during the myotube formation. However, the treatment with antibody specific for SPARC (anti-SPARC) prevented the differentiation and decreased myogenin expression. The induction of SPARC in the proliferating and differentiating C2C12 cells increased collagen 1a1 protein expression, whereas the inhibition decreased it. The effects on fibronectin protein expression were opposite. Furthermore, the addition of rSPARC in C2C12 myoblast increased the expression of mitochondrial proteins, ubiquinol-cytochrome c reductase core protein II (UQCRC2) and succinate dehydrogenase iron-sulfur subunit (SDHB), whereas the anti-SPARC decreased them. During the differentiation, only the anti-SPARC had the effects on mitochondrial proteins, NADH dehydrogenase ubiquinone 1 beta subcomplex subunit 8 (NADHB8), SDHB and cytochrome c oxidase 1 (MTCO1). Thus, SPARC plays a crucial role in the proliferation and differentiation of C2C12 and may be involved in the link between the ECM remodeling and mitochondrial function.</p></div

Involvement of SPARC in myoblasts fusion.

<p>Myoblasts (4×10⁴ cells/well in 24-well plates) were induced to differentiate in DM with/without the induction/inhibition of SPARC for 5 days. The mediums were changed each 48 h. C2C12 were stained with May-Grünwald and Giemsa solutions, and the stained wells were observed under light microscopy. One-way ANOVA followed by the Tukey's HSD post-hoc test was used. All results are reported as means ± SEM. The addition of rSPARC (2 μg/ml) induced myotube formation, whereas, anti-SPARC (10 μg/ml) inhibited it. (a) Captured microscopic images of C2C12 fusion. The images were captured using 30× objectives under light microscopy (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192714#pone.0192714.s002" target="_blank">S2 Fig</a> for 100× objectives). Examples of multinucleated myotubes were shown by arrows. (b) Fusion index was calculated from 10 different photos per condition. Abbreviations: PBS: phosphate buffered saline, rSPARC: recombinant SPARC protein, and anti-SPARC: anti-SPARC antibody. *Significant differences between experimental conditions (n = 3): p < 0.05.</p

Modulation of mitochondrial OXPHOS proteins expression after exogenous inhibition of SPARC in differentiating myobalsts.

<p>The myogenic differentiation was induced in DM supplemented with the induction/inhibition of SPARC for 5 days. DM with rSPARC (2 μg/ml) and anti-SPARC (10 μg/ml) was replaced each 2 days. Proteins extraction was performed, and extracted cells were prepared to perform western blot and the expression of mitochondrial OXPHOS proteins were measured using 30 μg of whole cell lysate proteins. The experiments were repeated three times (three different passages of C2C12 cells) and a representative western blot image was shown. Data were expressed as a ratio to the positive control (pooled samples). One-way ANOVA with repeated measurements was used to adjust the validations of the different passages and a contrast analysis was performed. P value was set at < 0.05 after the Bonfferoni adjustments. The expression of three different mitochondrial proteins was shown to be reduced after SPARC inhibition. Abbreviations: SDHB: succinate dehydrogenase iron-sulfur subunit beta, NADHB8: NADH dehydrogenase ubiquinone 1 beta subcomplex subunit 8, MTCO1: cytochrome c oxidase 1, PBS: phosphate buffered saline, rSPARC: recombinant SPARC protein, and anti-SPARC: anti-SPARC antibody. *Significant differences between experimental conditions.</p

Effect of SPARC on the modulation of ECM proteins in proliferating C2C12 cells.

<p>Representative western blots for collagen 1a1 and fibronectin expressions in C2C12 myoblasts after induction/inhibition of SPARC. C2C12 myoblasts were cultured in GM. One day before adding anti-SPARC (40 μg/ml) or rSPARC (8 μg/ml), cells were trypsinezed and plated at density of 4×10⁵/well in 12-well plates. Cells were cultured in four different conditions for 48 h. Proteins extraction was performed as described above and collagen 1a1 and fibronectin protein expression levels were measured by western blot. The experiments were repeated three times (three different passages of C2C12 cells) and a representative western blot image was shown. Data were expressed as a ratio to the positive control (pooled samples). One-way ANOVA with repeated measurements was used to adjust the validations of the different passages and a contrast analysis was performed. P value was set at < 0.05 after the Bonfferoni adjustments. (a) Thirty μg of whole cell lysate proteins were loaded to measure collagen 1a1 levels in C2C12 proliferating myoblasts. Addition of SPARC induced collagen 1a1 expression, however, SPARC inhibition decreased it. (b) Five μg of the proteins from proliferating cells were loaded to measure fibronectin levels. Fibronectin expression trended to decrease after rSPARC addition and anti-SPARC increased it. Abbreviations: PBS: phosphate buffered saline, rSPARC: recombinant SPARC protein, and anti-SPARC: anti-SPARC antibody. *Significant differences between experimental conditions.</p

SPARC effect on ECM proteins in differentiating myoblasts.

<p>C2C12 cells were cultured in DM for 5 days with/without exogenous rSPARC (2 μg/ml) and/or anti-SPARC (10 μg/ml). The mediums were changed each 2 days. Proteins extraction was performed, and western blot analysis was done on RIPA soluble lysates to indicate proteins. The experiments were repeated three times (three different passages of C2C12 cells) and a representative western blot image was shown. Data were expressed as a ratio to the positive control (pooled samples). One-way ANOVA with repeated measurements was used to adjust the validations of the different passages and a contrast analysis was performed. P value was set at < 0.05 after the Bonfferoni adjustments. (a): collagen 1a1 levels were measured using the 30 μg proteins and the results showed that the induction of SPARC increased collagen 1a1 expression, however, an opposite effect of anti-SPARC was observed. (b) Five μg of the proteins were loaded to measure fibronectin levels. A decrease of fibronectin expression with rSPARC and an increase after anti-SPARC were observed. Abbreviations: PBS: phosphate buffered saline, rSPARC: recombinant SPARC protein, and anti-SPARC: anti-SPARC antibody. *Significant differences between experimental conditions.</p

Table1_Electrophysiological basis of cardiac arrhythmia in a mouse model of myotonic dystrophy type 1.DOCX

Introduction: Myotonic dystrophy type 1 (DM1) is a multisystemic genetic disorder caused by the increased number of CTG repeats in 3′ UTR of Dystrophia Myotonia Protein Kinase (DMPK) gene. DM1 patients experience conduction abnormalities as well as atrial and ventricular arrhythmias with increased susceptibility to sudden cardiac death. The ionic basis of these electrical abnormalities is poorly understood.Methods: We evaluated the surface electrocardiogram (ECG) and key ion currents underlying the action potential (AP) in a mouse model of DM1, DMSXL, which express over 1000 CTG repeats. Sodium current (INa), L-type calcium current (ICaL), transient outward potassium current (Ito), and APs were recorded using the patch-clamp technique.Results: Arrhythmic events on the ECG including sinus bradycardia, conduction defects, and premature ventricular and atrial arrhythmias were observed in DMSXL homozygous mice but not in WT mice. PR interval shortening was observed in homozygous mice while ECG parameters such as QRS duration, and QTc did not change. Further, flecainide prolonged PR, QRS, and QTc visually in DMSXL homozygous mice. At the single ventricular myocyte level, we observed a reduced current density for Ito and ICaL with a positive shift in steady state activation of L-type calcium channels carrying ICaL in DMSXL homozygous mice compared with WT mice. INa densities and action potential duration did not change between DMSXL and WT mice.Conclusion: The reduced current densities of Ito, and ICaL and alterations in gating properties in L-type calcium channels may contribute to the ECG abnormalities in the DMSXL mouse model of DM1. These findings open new avenues for novel targeted therapeutics.</p

Alternative splicing events co-regulated by MBNL1 and RBFOX1.

<p>Genes in bold are mis-spliced in DM1 tissues.</p><p>Alternative splicing events co-regulated by MBNL1 and RBFOX1.</p

Role of MBNL1 and RBFOX1 in splicing regulation.

<p>(<b>A–C</b>) <b>A.</b> Venn diagram representing the overlap of hits obtained by knocking down MBNL1 and RBFOX1 in the HFN embryonic muscle cell line. In panels <b>B</b> and <b>C</b>, Venn diagrams are presented to illustrate events coregulated by MBNL1 and RBFOX1 that are mis-spliced in embryonic DM1 lines or and DM1 adult samples. The number and identity of the ASEs in each category are indicated. Gene names in bold indicate that the splicing shift for those ASEs occur in the reverse direction to the DM1 mis-splice.</p

Splicing defects in a mouse strain expressing CUG repeats.

<p>Total RNA from muscle tissues of transgenic C57BL6 mice expressing 600 and 1200 CUG-repeats were screened for alternative splicing defects. We interrogated 172 ASEs in genes reported to be susceptible to changes in HSA^LR and MBNL knockout mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107324#pone.0107324-Du1" target="_blank">[14]</a>. Using a false discovery rate threshold (<i>q-</i>value) of 0.05 and |ΔΨ| greater than 5 percentage points, we identified 24 ASEs in CUG1200 (black bars) that are significantly different from WT (white bars). Changes that were also significant in CUG600 (grey bars) are indicated with an asterisk. Results are presented in histograms by order of significance based on <i>q</i>-values.</p