Figure 2.
Analysis of sarcomeres in WT and R2509C-RYR1 primary myocytes. (A) TEM images of thin epoxy resin sections of primary cultured myocytes at 3 d after differentiation. Right panels show higher magnification of the area depicted by white-outlined squares in left panels. Sarcomere structure was preserved in WT (top), R2509C-Het (middle), and R2509C-Hom (bottom) myocytes. SL was measured as the distance between Z-lines (shown by yellow bidirectional arrows). A, I, and Z indicate the A-band, I-band, and Z-line, respectively. (B) TEM images of thin epoxy resin sections of primary cultured myocytes at 3 d after differentiation in the presence of Cpd1. Cpd1 was added at 2 µM from the onset of differentiation. Sarcomere structure was preserved in R2509C-Hom myocytes. SL was measured as the distance between Z-lines (shown by yellow bidirectional arrows). A, I, and Z indicate the A-band, I-band, and Z-line, respectively. (C) Graph showing SL in WT, R2509C-Het, R2509C-Hom, and R2509C-Hom (Cpd1) myocytes and the A-band length in WT and R2509C-Het myocytes. SL was significantly shorter in R2509C-Hom (1.7 ± 0.3 µm, n = 106 from 18 cells) myocytes than in WT (2.2 ± 0.3 µm, n = 95 from 15 cells) or R2509C-Het (2.3 ± 0.4 µm, n = 68 from 11 cells) myocytes. No significant difference was observed in the A-band length between groups (WT, 1.6 ± 0.1 µm, n = 95 from 15 cells; Het, 1.6 ± 0.1 µm, n = 68 from 11 cells; see Fig. S2). As indistinct I-bands were coupled with marked myofibrillar shortening in R2509C-Hom myocytes (see A), the A-band length was not measured to avoid errors. Treatment with Cpd1 significantly increased SL of R2509C-Hom (2.0 ± 0.3 µm, n = 60 from 14 cells) myocytes. Values are shown as mean ± SD. Statistical significance was determined using a one-way ANOVA with Tukey’s test. Primary myocytes were cultured from three mice (N = 3) in each group. (D) Frequency histograms of SL in C. Gaussian fittings showed peaks at 2.2 and 2.3 µm for WT and R2509C-Het, respectively. Histograms for R2509C-Hom and R2509C-Hom+Cpd1 were analyzed by two Gaussians, with one of the peaks fixed at 2.2 µm. Shorter peaks for R2509C-Hom and R2509C-Hom+Cpd1 were 1.6 and 1.8 µm, respectively.