Calsequestrin 1 Is an Active Partner of Stromal Interaction Molecule 2 in Skeletal Muscle

Figures

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  Figure 1. Coimmunoprecipitation of CASQ1 with STIM2 and the binding assay of CASQ1 regions to STIM2: (A) Triad samples obtained from rabbit skeletal muscle were subjected to a coimmunoprecipitation assay with anti-CASQ1 antibody. ‘Triad sample’ indicates a simple immunoblot of the triad sample. ‘Without anti-CASQ1 Ab’ indicates a reaction without an anti-CASQ1 antibody. Three independent experiments were conducted, and a representative result is presented. The degree of coimmunoprecipitated STIM2 to total STIM2 is presented as histograms. STIM2 was successfully coimmunoprecipitated with CASQ1. IB, IP, or Ab indicates immunoblot, immunoprecipitation, or antibody, respectively. * Significant difference was compared with ‘without anti-CASQ1 Ab’ (p < 0.05). (B,E) Schematic diagrams of mouse CASQ1 regions are presented. Numbers indicate the sequence of amino acids. The S1B region is presented according to a previous report [35]. (C,D,F) The bound proteins that were obtained from the binding assays of GST-CASQ1 or GST-CASQ1 regions with triad samples were subjected to immunoblot assays with anti-GST (to detect GST-CASQ1 or GST-CASQ1 regions) or anti-STIM2 antibody. GST was used as a negative…

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  Figure 2. SOCE, Ca2+ amount in the SR, and cytosolic Ca2+ level in WT CASQ1 or C1-expressing mouse skeletal myotubes: (A) Mouse primary skeletal myotubes that were transfected with cDNA of empty vector (vector control), full-length wild-type CASQ1 (WT CASQ1), or C1 region (C1) were subjected to immunocytochemistry using anti-GFP antibodies. The bar represents 100 µm. (B) The lysate of WT CASQ1 or C1-expressing myotubes was subjected to a coimmunoprecipitation assay with anti-CASQ1 antibody, and the immunoprecipitate was subjected to immunoblot analysis with anti-CASQ1 or anti-STIM2 antibody. ‘Myotube lysate’ indicates a simple immunoblot of the myotube lysate. ‘Without anti-CASQ1 Ab’ indicates a reaction without an anti-CASQ1 antibody. Three independent experiments were conducted, and a representative result is presented. The degree of coimmunoprecipitated STIM2 to total STIM2 is presented as histograms. STIM2 was successfully coimmunoprecipitated with CASQ1. IB, IP, or Ab indicates immunoblot, immunoprecipitation, or antibody, respectively. * Significant difference was compared with ‘without anti-CASQ1 Ab’ (p < 0.05). (C) Myotube widths were measured. The normalized mean values of each to…

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  Figure 3. Intracellular Ca2+ release for skeletal muscle contraction, expression levels of various proteins, and coimmunoprecipitation of Orai1 with STIM2: KCl (A) or caffeine (B) was applied to the myotubes, and intracellular Ca2+ release from the SR to the cytosol through RyR1 was measured. A representative trace for each group is shown. Histograms show the normalized peak amplitude (left-hand side) or the slope in the rising phase of the peak to the mean value of the vector control (right-hand side). There was no significant difference in the slopes of the myotubes. The results are presented as the mean ± SEM for the number of myotubes shown in parentheses in Table 1. (C) The myotube lysate was subjected to immunoblot assays with antibodies against fifteen proteins. α-actin was used as a loading control. Three independent experiments per protein were conducted. CASQ1, endogenous CASQ1; JP, junctophilin. The expression level of each protein normalized to the mean value of the vector control is presented as histograms (mean ± SEM for three independent experiments, Supplementary Table S4). * Significant difference compared with vector control (p < 0.05). # Significant difference compared…

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  Figure 4. Abnormal mitochondria in the shape and the expression level of Drp-1 or Mfn-1: (A) Mitochondria in the myotubes were observed using TEM. Areas in the numbered boxes (from 1 to 3) were enlarged. Swelling mitochondria were found in C1-expressing myotubes. The bar represents 2 μm. (B) The length of the swelling mitochondria was measured, and the results are summarized as histograms. The values were normalized to the mean values of the vector control. The values are presented as the mean ± SEM for the number of mitochondria in Table 2. (C) The myotube lysate was subjected to immunoblot assays with antibodies against Drp-1 or Mfn-1. α-actin was used as a loading control. Three independent experiments per protein were conducted. The expression level of each protein normalized to the mean value of the vector control is presented as histograms (Table 2). * Significant difference compared with vector control (p < 0.05). # Significant difference compared with WT CASQ1 (p < 0.05).

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  Figure 5. 3D structure of human CASQ1 and an adaptation model to situations with repetitive and/or long-term contractions of skeletal muscle: (A) 3D structure of human CASQ1 (PDB ID: 3UOM, starting from 3 to 387 amino acids) is presented as a ribbon diagram. The C1 region of CASQ1, which is involved in binding to STIM2, is colored yellow. N or C indicates the N- or C-terminus, respectively. The right images are the 180° rotated images of the left images along the vertical axis. Images in the lower panel are the top view of the images in the upper panel. (B) The overall results of this study are summarized as schematic diagrams in the upper panel. In the lower panel, ‘an adaptation model to situations with repetitive and/or long-term contractions of skeletal muscle’, such as tetanic stimulation or fatigue, is presented.