Fig. 5

TPM2 variants disrupt myotube morphogenesis. (A) Transfection efficiency. C2C12 myoblasts were transfected with TPM2.IRES.GFP and imaged 24 hours posttransfection. About 25% of cells were GFP positive. (B) Western blot of TPM2 expression. C2C12 cells were transfected with Flag-TPM2 and collected after 3, 5, and 7 days of differentiation. (C) Western blot of TPM2 variants. C2C12 cells were transfected with Flag-tagged variants and collected after 7 days of differentiation. Protein expression was similar among the variants. (D) C2C12 cells transfected with pathogenic TPM2 variants showed impaired morphology. Confocal micrographs of cells fixed after 7 days in differentiation media and labeled for α-actinin (green) to detect differentiated myotubes and Hoechst to visualize myonuclei. Myotubes that expressed E41K, K49Del, R91G, and E122K appeared shorter than controls (wild-type TPM2 and the benign variant E273K). Variant-expressing myotubes were often rounded (arrows). Scale bars, 20 μm. (E) Myotube length distribution showing Gaussian distribution fit curves (solid lines). The length distribution of myotubes that expressed pathogenic variants skewed toward shorter lengths. (F) Quantification of myoblast fusion. Fusion index represents the number of nuclei in multinucleate myotubes; variant-expressing cells fused less than controls. (G) Roundness score. Individual myotubes were traced to calculate roundness; a score of 1.0 represents complete circularity. Myotubes that expressed K49Del and R91G were more round than controls. Significance was determined by unpaired, 1-tailed Student’s t test (E) or 1-way ANOVA (F and G). n ≥ 10 imaging fields per treatment. *(P < 0.05), **(P < 0.01), ***(P < 0.001), ****(P < 0.0001). Error bars, SEM.