Dysferlinopathy Fibroblasts Are Defective in Plasma Membrane Repair

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.

Immunoblot analysis of human and mouse fibroblasts using an anti-dysferlin antibody

Normal 237-kDa dysferlin was detected in control human and wild-type mouse fibroblasts. Dysferlin was expressed at a very low level in fibroblasts from a dysferlinopathy patient, whereas a smaller 229-kDa dysferlin was detected in SJL/6J mice.

Immunostaining of human and mouse fibroblasts using anti-dysferlin

Cytoplasmic staining of dysferlin is seen in fibroblasts from control humans, wild-type mice, and SJL/6J mice, whereas dysferlin staining was barely detectable in fibroblasts from the patients. Scale bars, 20 µm.

Membrane repair assay of human and mouse fibroblasts

The panel shows entry of the FM 1-43 dye after laser wounding in human and mouse fibroblasts. Scale bars, 20 µm.

Quantification of FM1-43 dye influx after laser injury

Time course relative fluorescence intensity of FM1-43 at wounded site. Data are presented as means ± S.D.(error bars). Number of laser-induced injuries; n=5.

Membrane blebbing assay of human and mouse fibroblasts

Hypotonic shock using Tyrode buffer diluted by 50% with water was performed in the presence of FM1-43. Membrane blebs are not observed in the mutant fibroblasts from a patient. Both wild-type and mutant fibroblasts with endocytic uptake of FM dye showed increased fluorescence intensity in the intracellular space. Scale bars, 20 μm.

Quantification of membrane bleb formation in response to hypotonic shock

Bar graph depicting the percentage of cells manifesting blebs after osmotic shock (n=30). Data are presents as means ±s.d.

Immunoblot analysis of human and mouse fibroblasts after the treatment with proteosomal inhibitor

Semiconfluent culture of human and mouse fibroblasts were treated with increasing concentration of the proteasomal inhibitor MG-132. Anti-dysferlin and anti-GAPDH antibodies were used for immunoblotting.

Relative dysferlin levels upon MG-132 treatment

Relative dysferlin protein expression levels upon MG-132 treatment. Protein levels were measured by densitometric analysis. GAPDH was used as a loading control, and y axis shows the ratio between dysferlin and GAPDH (n =5). Data are presents as the means ± s.d.

Membrane blebbing assay using MG-132-treated fibroblasts from the patients and SJL mice

Bar graph depicting the percentage of cells manifesting blebs after osmotic shock (n=30). Data are presents as means ±s.d. Mis-sense mutated dysferlin recovered membrane blebbing as similar to truncated dysferlin in SJL mice.