Minimal expression of dysferlin prevents development of dysferlinopathy in dysferlin exon 40a knockout mice

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.

Tetraspanin CD82 Associates with Trafficking Vesicle in Muscle Cells and Binds to Dysferlin and Myoferlin

“…While several reports concluded that the 72 kDa mini-dysferlin might be essential for membrane repair and assembly of proteins involved in this process, recent work reported that mice with mutated exon 40a retain membrane repair capacity, thus suggesting a complex regulation of membrane repair via mechanisms that are still not entirely clear. [43] In summary, our study provides evidence of an interaction between dysferlin and myoferlin proteins with tetraspanin CD82 in muscle cells. While the precise function of this interaction is not entirely clear, myogenic cells from some dysferlinopathy patients exhibit a dramatic loss of expression of CD82 at the cell membrane.…”

“…While several reports concluded that the 72 kDa mini‐dysferlin might be essential for membrane repair and assembly of proteins involved in this process, recent work reported that mice with mutated exon 40a retain membrane repair capacity, thus suggesting a complex regulation of membrane repair via mechanisms that are still not entirely clear. [ 43 ]…”

Gene-edited primary muscle stem cells rescue dysferlin-deficient muscular dystrophy

“…Restoring dysferlin protein levels as little as 10% may be sufficient to rescue the disease phenotype. 56,57 Prime editing could repair the sequence to wild-type 58 and recent developments in this technology highlight interesting prospects. [59][60][61] However, prime editing is yet to be reported in skeletal muscle in vivo and, although smaller variants have been engineered, the prime editor is a substantially larger protein for in vivo delivery with a viral component that raises some additional immunological concerns.…”