The LIM-Only Protein FHL2 is involved in Autophagy to Regulate the Development of Skeletal Muscle Cell

: Scope: Four and a half LIM domain protein 2 (FHL2) is a LIM domain protein expressed in muscle tissue whose deletion is causative of myopathies. Although FHL2 has a confirmed important role in muscle development, its autophagy-related function in muscle differentiation has not been fully determined. Methods: C2C12 cells were treated with FHL2-konwdown or FHL2-overexpression. The morphology of C2C12 cells was observed by transmission electron microscopy. The mRNA and protein abundances of muscle related genes and autophagy related genes were measured by RT-PCR and western blot. Immunofluorescence and co-immunoprecipitation assay were used to verify the interaction between FHL2 and LC3 protein. Results: FHL2 silencing reduced LC3-Ⅱ protein expression and the amount of LC3 that co-immunoprecipitated with FHL2, indicating that FHL2 interacts with LC3-Ⅱ in the formation of autophagosomes. Moreover, the expression of muscle development marker genes such as MyoD1 and MyoG was lower in FHL2-silenced C2C12 cells but not in FHL2-overexpressing C2C12 cells. Electron microscopy analysis revealed large empty autophagosomes in FHL2-silenced myoblasts, while flow cytometry suggested that FHL2 silencing made cells more vulnerable to staurosporine-induced cell death. Conclusion: These results suggest that FHL2 interacts with LC3-Ⅱ in autophagosome formation to regulate the development of muscle cells.