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Updated: Aug 14, 2025
Cell Membrane Repair Assay Using a Two-photon Laser Microscope
Published on: January 2, 2018
Minimal expression of dysferlin prevents development of dysferlinopathy in dysferlin exon 40a knockout mice
Joe Yasa1,2, Claudia E Reed1,3, Adam M Bournazos1,3
1Kids Neuroscience Centre, The Children's Hospital at Westmead, Cnr Hawkesbury Road, Hainsworth Street, Westmead, Sydney, NSW, 2145, Australia.
Abstract:
Dysferlin is a Ca2+-activated lipid binding protein implicated in muscle membrane repair. Recessive variants in DYSF result in dysferlinopathy, a progressive muscular dystrophy. We showed previously that calpain cleavage within a motif encoded by alternatively spliced exon 40a releases a 72 kDa C-terminal minidysferlin recruited to injured sarcolemma. Herein we use CRISPR/Cas9 gene editing to knock out murine Dysf exon 40a, to specifically assess its role in membrane repair and development of dysferlinopathy. We created three Dysf exon 40a knockout (40aKO) mouse lines that each express different levels of dysferlin protein ranging from ~ 90%, ~ 50% and ~ 10-20% levels of wild-type. Histopathological analysis of skeletal muscles from all 12-month-old 40aKO lines showed virtual absence of dystrophic features and normal membrane repair capacity for all three 40aKO lines, as compared with dysferlin-null BLAJ mice. Further, lipidomic and proteomic analyses on 18wk old quadriceps show all three 40aKO lines are spared the profound lipidomic/proteomic imbalance that characterises dysferlin-deficient BLAJ muscles. Collective results indicate that membrane repair does not depend upon calpain cleavage within exon 40a and that ~ 10-20% of WT dysferlin protein expression is sufficient to maintain the muscle lipidome, proteome and membrane repair capacity to crucially prevent development of dysferlinopathy.
Insights
Dysferlin
Area of Science:
- Muscle biology and membrane repair mechanisms.
Background:
- Dysferlin (DYSF) is crucial for muscle membrane repair.
- Mutations in DYSF cause dysferlinopathy, a muscular dystrophy.
- Calpain cleavage of dysferlin releases a C-terminal fragment involved in repair.
Purpose of the Study:
- To investigate the specific role of exon 40a in dysferlin's function.
- To assess the impact of exon 40a knockout on membrane repair and dysferlinopathy development.
- To determine the minimum dysferlin expression level required for muscle health.
Main Methods:
- CRISPR/Cas9 gene editing to create three Dysf exon 40a knockout (40aKO) mouse lines with varying dysferlin levels (90%, 50%, 10-20%).
- Histopathological analysis of skeletal muscles from 12-month-old mice.
- Lipidomic and proteomic analyses of quadriceps from 18-week-old mice.
Main Results:
- All three 40aKO mouse lines showed no significant dystrophic features and maintained normal membrane repair capacity.
- Dysferlin-deficient BLAJ mice exhibited profound lipidomic and proteomic imbalances, which were absent in 40aKO lines.
- Even 10-20% of wild-type dysferlin protein expression was sufficient to prevent muscle pathology.
Conclusions:
- Calpain cleavage within exon 40a is not essential for muscle membrane repair.
- A low level of dysferlin expression (10-20%) is sufficient to maintain muscle integrity and prevent dysferlinopathy.
- These findings challenge the previously understood role of exon 40a in dysferlin function and disease pathogenesis.
