Abstract:

BACKGROUND: Dysferlin is a sarcolemmal protein that is defective in Miyoshi myopathy and limb-girdle muscular dystrophy type 2B, and is involved in sarcolemmal repair. Primary cultured myoblasts and myotubes established from patient muscle biopsies have been widely utilized to explore the molecular mechanism of dysferlinopathy.
OBJECTIVES: The purpose of this study was to explore the possible utility of dermal fibroblasts from dysferlin-deficient patients and SJL mice as a tool for studying dysferlinopathy.
METHODS: Dysferlin protein expression in fibroblasts from dysferlin-deficient patients and SJL mice was analyzed by immunoblotting and immunocytochemistry. The membrane wound-repair assay was performed on the fibroblasts using a confocal microscope equipped with a UV-laser. The membrane blebbing assay using hypotonic shock, in which normal membrane blebbing is detected only in the presence of dysferlin, was also performed using human and mouse fibroblasts.
RESULTS: Mis-sense mutated dysferlin was expressed at a very low level in fibroblasts from a dysferlinopathy patient, and lower expression level of truncated dysferlin was observed in SJL mouse fibroblast. Fibroblasts from patients with dysferlinopathy and SJL mice showed attenuated membrane repair and did not form membrane blebs in response to hypoosmotic shock. Proteosomal inhibitior increased mis-sense mutated or truncated dysferlin levels, and restored membrane blebbing, however, proteosomal inhibition failed to improve levels of dysferlin with non-sense or frame-shift mutation.
CONCLUSION: Fibroblasts from dysferlinopathy patients and SJL mice showed attenuated plasma membrane repair, and could be a tool for studying dysferlinopathy.

日本語要旨:

dysferlinは骨格筋細胞膜のタンパク質であり、その異常はdysferlinopathyを引き起こす。dysferlinopathyの分子病態の解明には、患者生検筋由来の筋芽、筋管細胞が使用されてきたが、我々は線維芽細胞の有用性を検討した。患者、SJLマウスの線維芽細胞では変異型dysferlinの発現レベルは低下しており、細胞膜修復に異常が観察されることを見出した。この結果から患者、SJLマウスの線維芽細胞はdysferlinopathyの研究ツールとして有用であると考えられる。