A Novel Assay Reveals the Early Setting-Up of Membrane Repair Machinery in Human Skeletal Muscle Cells.

Abstract

Defect in membrane repair contributes to the development of muscular dystrophies such as limb girdle muscular dystrophy (LGMD) type R2 or R12. Nevertheless, many other muscular dystrophies may also result from a defect in this process. Identifying these pathologies requires the development of specific methods to inflict sarcolemma damage on a large number of cells and rapidly analyze their response. We adapted a protocol hitherto used to study the behavior of cancer cells to mechanical constraint. This method is based on forcing the passage of cells through a thin needle, which induces shear stress. Due to size considerations, this method requires working with mononuclear muscle cells instead of myotubes or muscle fibers. Although functional sarcolemma repair was thought to be restricted to myotubes and muscle fibers, we show here that 24h-differentiated myoblasts express a complete machinery capable of addressing membrane damage. At this stage, muscle cells do not yet form myotubes, revealing that the membrane repair machinery is set up early throughout the differentiation process. When submitted to the shear-stress assay, these cells were observed to repair membrane damage in a Ca²⁺-dependent manner, as previously reported. We show that this technique is able to identify the absence of membrane resealing in muscle cells from patient suffering from LGMDR2. The proposed technique provides therefore a suitable method for identifying cellular dysregulations in membrane repair of dystrophic human muscle cells.