Text

Objectives and questions: Skeletal muscles provide stability and enable movement. During development, mononucleated myoblasts differentiate into multinucleated myotubes, a process regulated by myogenic regulatory factors [1]. Myotubes, which are elongated structures containing multiple nuclei [1], serve as precursors to mature muscle fibres. The transmembrane protein tetraspanin CD9 is present on the cell surface and contributes to cell motility, fusion and adhesion [2]. A deeper understanding of the mechanisms of myogenesis and syncytia formation could aid the development of therapies for skeletal muscle diseases such as sarcopenia.

Material and methods: Two model systems were used for analyzing the effect of CD9 on mononucear myoblast differentiation into multinuclear myotubes: C2C12 murine myoblasts and human skeletal muscle myoblasts. In these mode systems stable CD9 overexpression using a lentiviral vector system and a transient CD9 knockdown using siRNA were applied. To demonstrate overexpression, tetraspanin CD9 was tagged with green fluorescent protein. The morphology of the myotubes after differentiation was analyzed using DAPI immunostaining and myosin heavy chain immunostaining. In addition, qPCR and Western blot analysis were used to measure expression levels of CD9 and regulatory factors of differentiation.

Results: Murine and human myoblasts differentiated under low serum conditions (1% or 2% horse serum) to multinuclear elongated myotubes. C2C12 murine myoblasts were successfully modified using a lentiviral approach to overexpress human tetraspanin CD9. In murine myoblasts, siRNA-mediated knockdown resulted in significantly lower relative gene expression of CD9. In experiments using C2C12, siRNA-mediated downregulation of CD9 resulted in smaller myotubes.

Discussion and conclusions: C2C12 murine myoblasts and human myoblasts differentiated under low serum conditions to multinuclear myotubes. Subsequent fluorescence microscopy demonstrated the overexpression of CD9 in murine myoblasts due to the presence of the green fluorescent protein (GFP). Knockdown of CD9 in murine myoblasts leads into smaller multinuclear myotubes. Further studies will assess the effects on myoblast differentiation and functional contractility of myotubes under both overexpression and knockdown conditions in both model systems. The role of CD9 in myogenesis may provide new insights into the development of therapeutic strategies for musculoskeletal disorders.

References