Fig. 5.

TRIM32 mutations seem to lead to premature senescence of myogenic cells. a Immunohistochemistry staining and quantification of Pax7⁺ satellite cells in skeletal muscle from family A patients (n = 2), family C patient (n = 1), healthy controls (n = 5) and disease controls (LGMD2B, LGMD1C, LGMD2N, NEM6) (n = 4). Immunostaining for collagen type IV (red) to show the muscle fibers, Pax7 (green) to show the satellite cells and with Topro 3 staining for the nuclei (blue). Quantification of Pax7⁺ cells revealed a significant reduction in the number of satellite cells in TRIM32^V591M and TRIM32^C39LfsX17 muscles compared with controls. Data from 8 to 31 independent fields were analyzed per condition. Mean ± SEM; Kruskal-Wallis with Dunn’s multiple comparisons test. Scale bar, 50 μm. b Immunohistochemical staining of MHC-neo of skeletal muscle from family A patients, family B patients, healthy control and disease control (LGMD1B) revealed a large number of positive regenerating fibers in the disease control. In contrast, TRIM32 patients showed no positive cells (patients A/II.2, A/IV.3 and B/II.2) or, at most, few scattered positive cells (patients B/II.3 and C/II.2). Scale bar, 100 μm. c SEM images of myoblasts at 5 days growing in proliferation medium from AIV.3 and BII.3 patients, and healthy controls. TRIM32^V591M and TRIM32^{N217S/F568del} myoblasts were larger than control myoblasts. Higher magnification showed a reduction in the size of projections and number of filopodia of TRIM32^V591M and TRIM32^{N217S/F568del} myoblasts comparing to control myoblasts. Scale bars, 100 μm: lower magnification view; 2 μm: hyper magnification view. d Immunofluorescence staining and quantification of the percentage of SA-β-gal⁺ cells in human myoblasts after 10 days growing in proliferation medium from family A patients (n = 2), family B patients (n = 2) and healthy controls (n = 2). A higher increment of SA-β-gal⁺ cells was observed in TRIM32^V591M and TRIM32^{N217S/F568del} myoblast cultures compared to controls, supporting a premature senescence in the muscles with TRIM32 altered function. Data from 8 independent fields were analyzed per condition. Mean ± SEM; One-way ANOVA with Tukey’s multiple comparisons test. Scale bar, 100 μm