Dysferlin deficiency compromises the repair of injured muscle, but the underlying cellular mechanism remains elusive. To study this phenomenon, we have developed mouse and human myoblast models for dysferlinopathy. These dysferlinopathic myoblasts undergo normal differentiation but have a deficit in their ability to repair focal injury to their cell membrane. Imaging cells undergoing repair showed that dysferlin-deficit decreased the number of lysosomes present at the cell membrane, resulting in a delay and reduction in injury-triggered lysosomal exocytosis. We find repair of injured cells does not involve formation of intracellular membrane patch through lysosome–lysosome fusion; instead, individual lysosomes fuse with the injured cell membrane, releasing acid sphingomyelinase (ASM). ASM secretion was reduced in injured dysferlinopathic cells, and acute treatment with sphingomyelinase restored the repair ability of dysferlinopathic myoblasts and myofibers. Our results provide the mechanism for dysferlin-mediated repair of skeletal muscle sarcolemma and identify ASM as a potential therapy for dysferlinopathy.

Recessive mutations in the Anoctamin 5 gene (ANO5) cause limb-girdle muscular dystrophy type 2L (LGMD 2L) and Miyoshi myopathy type MMD3. These conditions have been called “anoctaminopathies” and they are clinically similar to dysferlinopathies, another group of muscular dystrophies which include LGMD 2B and Miyoshi myopathy. The role of ANO5 in muscle is not known, but it has been hypothesized that it works with dysferlin to repair sarcolemmal injuries. We reviewed muscle biopsy data from a group of patients with known ANO5 genetic status to validate a diagnostic strategy to direct gene testing. Using immunohistochemistry and immunoblot techniques we examined the expression profile of a panel of proteins involved in various types of muscular dystrophies including dystrophinopathies, myofibrillar myopathies and other forms of LGMDs. Muscle histology in these patients showed some degree of inflammation and a myopathic/dystrophic pattern of variable severity that correlated with the site of the muscle biopsy. Non specific abnormalities such as reduction of laminin β1 on sections and laminin α2 band present on blots as a doublet around 80 kDa were seen in ANO5 patients. Interestingly, a variable degree of sarcolemmal dysferlin deficiency was detected in a large number of these biopsies, whereas expression of dysferlin in the cytoplasm and a band of normal intensity on immunoblot were seen. All the remaining proteins showed a pattern similar to normal controls. These results indicate that in our population of patients, mutations in the ANO5 gene are associated with secondary muscle protein abnormalities and mislocalization of dysferlin. Further characterization of possible interactions between these proteins is required to fully elucidate their roles in causing muscular dystrophy.

The ferlins are mammalian homologs of the Caenorhabditis elegans sperm vesicle fusion protein FER-1 implicated in membrane fusion. To date three ferlin proteins have been characterized, dysferlin, myoferlin and otoferlin. Dysferlin and myoferlin have been implicated in muscle membrane fusion in vivo, sarcolemmal repair and myoblast fusion respectively. Through bioinformatic analysis we have previously reported the existence of three novel ferlin genes which have been designated FER1L4, FER1L5 and FER1L6, respectively. Homology modeling and sequence analysis of ferlin C2 domains has allowed the in-silico functional subgrouping of the ferlin proteins into two groups, dysferlin-like and otoferlin-like. Since the dysferlin-like ferlin subgroup comprises dysferlin, myoferlin and FER1L5 we hypothesized that FER1L5 may have a role in muscle membrane fusion. Using a FER1L5 specific antibody we have initiated the characterization of FER1L5 in muscle which has in part involved examining FER1L5 expression and distribution during C2C12 myoblast fusion. In C2C12 myotubes we have identified cytoplasmic ring shaped FER1L5 free regions which we demonstrate arise by shedding from the myotube membrane of ball shaped structures containing FER1L5 stained vesicles. We have confirmed the formation of the cytoplasmic ring shaped regions in C2C12 myotubes and membrane shedding by (i) confocal analysis of myotubes immunolabelled with a range of cytoskeletal and membrane proteins (ii) demonstrating using live cell imaging that the formation of the cytoplasmic ring shaped regions and membrane shedding can be enhanced by cholesterol efflux (iii) demonstrating that the shed membrane arises from’lipid raft’ like membrane microdomains resembling multivesicular body (MVB) like structures which contain FER1L5 vesicles. Our results indicate that vesicle shedding is active in muscle cells and highlight that FER1L5 is present in muscle exosomes, which we have designated “myosomes”.

The SJL mouse strain has been widely used as an animal model for experimental autoimmune encephalitis (EAE), inflammatory muscle disease and lymphomas and has also been used as a background strain for the generation of animal models for a variety of diseases including motor neurone disease, multiple sclerosis and atherosclerosis. Recently the SJL mouse was shown to have myopathy due to dysferlin deficiency, so that it can now be considered a natural animal model for limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM). We have cloned the mouse dysferlin cDNA and analysis of the sequence shows that the mouse dysferlin gene is characterized by six C2 domain sequences and a C-terminal anchoring domain, with the human and the mouse dysferlin genes sharing > 90% sequence homology overall. Genomic analysis of the SJL mutation confirms that the 171 bp RNA deletion has arisen by exon skipping resulting from a splice site mutation. The identification of this mutation has implications for the various groups using this widely available mouse stock.