G.P.48 Affixin is involved in sarcolemmal repair

Abstract

Dysferlin is a sarcolemmal protein that is defective in Miyoshi myopathy and LGMD2B. In the presence of Ca2+, dysferlin accumulates around the damaged membrane site and is suggested to mediate sarcolemmal repair. We previously reported that affixin is a dysferlin-binding protein and co-localizes with dysferlin at the sarcolemma of normal human skeletal muscle. The association of dysferlin with affixin was confirmed by immunoprecipitation study using normal human skeletal muscles and COS-7 transfectants. The immunoreactivity of affixin was reduced in sarcolemma of dysferlinopathy muscles. We also reported that affixin activates Rac1 via GDP/GTP nucleotide exchange factor (GEF) and regulates the reorganization of cytoskeletal actin. The purpose of this study is to examine the effect of calcium on the association of dysferlin with affixin and to test the possibility that affixin is involved in sarcolemmal repair. The calcium-dependency of dysferlin–affixin association was examined by pull-down assay using lysates from COS-7 transfectants expressing human dysferlin and bacterially expressed affixin CH1 domain. To clarify molecular behavior of affixin in sarcolemmal repair, mCherry-tagged human affixin was expressed in FDB of mice (C57BL/6J and dysferlin-deficient A/J) by electroporation. Membrane wound-repair assay of single myofiber was performed using confocal microscope equipped two-photon laser. Pull-down assay revealed that dysferlin associates with affixin in a calcium-dependent manner. However, the association of dysferlin and caveolin-3 was not affected in different calcium concentrations. mCherry–affixin accumulates at the wounded site in C57BL/6J mice, however accumulation of affixin was not observed in A/J mice. These results suggest affixin involvement in sarcolemmal repair. We are analyzing movement of mCherry–affixin and dysferlin–GFP during sarcolemmal repair.