Publications
Grant Duration
10/19 – 02/21
Project Results Summary
NOTE: This project was a collaboration between Dr. Frances Lemckert, Kid’s Neuroscience Centre, The Children’s Hospital at Westmead & Functional Neuromics, The Children’s Medical Research Institute and Professor Noah Weisleder, Dorothy M. Davis Heart and Lung Research Institute, Department of Physiology & Cell Biology, The Ohio State University Wexner Medical Center, Columbus, Ohio, USA
Dysferlin is cleaved by the ubiquitous calpains -1 and -2 that are activated by the influx of extracellular calcium following membrane injury. Calpain cleavage occurs in the amino acid residues coded by the alternatively spliced exon 40a. The cleaved form of dysferlin, mini-dysferlinC72, recruits to sites of membrane injury and we hypothesise it is a specialised module of dysferlin with an important role in membrane repair. Using CRISPR/Cas9 gene-editing, we generated three mouse lines in which exon 40a of dysferlin was targeted for knockout, to examine the importance of exon 40a in membrane repair and the development of muscular dystrophy. Due to the different deletions that occurred in each line, the three mouse lines also expressed varying reduced levels of dysferlin protein compared to WT.
We allowed our mice to age to 12 months to examine the development of muscle disease, comparing our lines to WT and to the dysferlin-deficient BLA/J mice. In collaboration with Professor Noah Weisleder, we examined the membrane repair capacity of isolated flexor digitorum brevis (FDB) myofibres from young adult mice using the laser injury/fluorescent dye uptake assay. We also harvested quadriceps muscle from mice at 18 weeks of age (before the onset of observable disease symptoms in the BLA/J line) and performed proteomics and lipidomics analysis, as it had previously been shown that 18 week BLA/J muscle displays an altered lipidome well before the onset of dystrophic pathology.
Knockout of dysferlin exon40a does not impair membrane repair, and both mildly reduced and low level dysferlin expression allows repair kinetics that are not significantly different from WT.
Dysferlin expression at low levels prevented most of the dystrophic pathology observed in the BLA/J muscle, and muscle with higher level dysferlin expression was almost indistinguishable from WT muscle with regard to the disease parameters of centralised nuclei counts, myofibre size and variability, fibrosis and lipid accumulation.
The muscle lipidome of our dysferlin exon 40a knockout lines closely resembled the lipidome of WT mice and was different from that of BLA/J mice, irrespective of dysferlin levels. Even low level dysferlin expression appeared able to “normalise” the lipidomic changes that precede disease pathology in muscle.
In contrast, the proteome in our mouse lines showed a gradation of response that correlated with dysferlin protein expression. The muscle with lowest level dysferlin expression was more similar to BLA/J muscle, while the muscle with the highest level of dysferlin expression was more similar to WT muscle.
We found no evidence for a requirement for dysferlin exon 40a for membrane repair, prevention of disease pathology, maintenance of the lipidome or proteome in our mice. Mice carrying a repertoire of dysferlin isoforms that do not include those susceptible to calpain cleavage are able to grow and function normally until at least 12 months of age. The mild disease phenotype in our lowest expressing exon 40a KO mouse line is more likely due to the reduced total expression of dysferlin rather than specific loss of exon 40a-containing dysferlin isoforms.
We suggest low level but widespread dysferlin expression may be sufficient to prevent many of the symptoms of muscular dystrophy. This study provides a target for the minimal levels of dysferlin restoration required for normal muscle function, vital for the development of effective gene therapies.
Frozen sperm from the 40aKO mice evaluated in this project are available – https://www.jain-foundation.org/research/access-resources/research-tools/animal-models/
