Impaired Regulated Exocytosis and GLUT4 Trafficking in Dysferlin-Deficient Human Myoblasts: A Pilot Study

Background/Objectives: Dysferlinopathies are inherited muscular dystrophies caused by DYSF mutations that lead to progressive muscle degeneration. Proteomic analyses of patient muscle reveal altered abundance of proteins involved in regulated exocytosis and energy metabolism, reflecting complex molecular remodeling within these pathways. However, whether dysferlin deficiency impairs GLUT4 exocytosis remains unknown. Methods: Immortalized human myoblasts from a healthy control (C25) and a dysferlinopathy patient (DYSF2) were used. Cytosolic Ca2+ was measured using Fura-2 and Fluo-4; submembrane Ca2+ dynamics were recorded with membrane-targeted sensor pN1-Lck-GCaMP3. Single exocytosis events induced by 10 µM ionomycin or 100 nM insulin were tracked with IRAP-pHluorin and total internal reflection fluorescence microscopy (TIRFM). GLUT4 puncta near the plasma membrane were quantified using immunofluorescence and visualized by TIRFM. Dysferlin-3HA was expressed to test rescue. Results: Both cell lines showed comparable cytosolic and submembrane Ca2+ responses to ionomycin. However, the frequency of IRAP-pHluorin exocytosis events evoked by both ionomycin and insulin was significantly reduced in DYSF2 myoblasts. Expression of dysferlin-3HA restored insulin-induced exocytosis in DYSF2 cells to control levels but failed to fully rescue ionomycin-induced events. Insulin increased the trafficking of GLUT4 puncta near the plasma membrane in C25 cells, whereas DYSF2 cells lacked this insulin-stimulated response. Conclusions: In the patient-derived cell line examined here, dysferlin deficiency impaired stimulus-evoked exocytosis and insulin-induced GLUT4 translocation without altering Ca2+ dynamics. The inability of dysferlin-3HA to significantly rescue ionomycin-induced exocytosis points to additional downstream defects, likely secondary proteomic alterations, that constrain full functional recovery.