Proteasome Inhibitors Reduce Thrombospondin-1 Release in Human Dysferlin-Deficient Myotubes

Abstract

Background: Dysferlin is a type-II transmembrane protein and the causative gene of dysferlinopathies, which are characterized by absence or marked reduction in dysferlin protein and muscle weakness. Dysferlin is implicated in vesicle fusion, trafficking, and membrane repair. The muscle biopsy of patients with dysferlinopathy is characterized by the presence of inflammatory infiltrates. Release of thrombospondin-1 (TSP-1) by dysferlin deficient muscle has been reported as a possible factor of the inflammation observed in the muscle of both human and mouse models of dysferlinopathy. It has also been reported that treatment with vitamin D3 enhances dysferlin expression. The ubiquitin-proteasome system recognizes and removes proteins that fail to fold or assemble properly and previous studies suggest that its inhibition could have a therapeutic implication in muscle dystrophies. Here we assessed whether inhibition of the ubiquitin proteasome system prevented degradation of dysferlin in immortalized myoblasts from a patient carrying two missense mutationsMethods: Dysferlin deficient myotubes were treated with EB1089, a vitamin D3 analog, oprozomib and ixazomib to assess proteasome inhibition. Western blot was performed to analyze the effect of the different treatments on the recovery of dysferlin and myogenin expression. TSP-1 was quantified using Enzyme Linked Immunosorbent Assay to analyze the effect of these drugs on its release.A membrane repair assay was designed to assess the ability of treated myotubes to recover after membrane injury. Data were analyzed using a one-way ANOVA test followed by by Tukey post hoc test and analysis of variance. Ap≤0.05 was considered statistically significant. Results : Treatment with proteasome inhibitors and EB1089 resulted in a slight increase of dysferlin expression which was accompanied by a low increase of myogenin expression. Also, EB1089 and proteasome inhibitors reduced the release of TSP-1 in myotubes from a dysferlinopathy patient. However, the increase of dysferlin had no effect on the repair of muscle membrane after injury. Conclusions: Our findings indicate that the ubiquitin-proteasome system might not be the main mechanism of mutant dysferlin degradation. However, its inhibition could help to improve muscle inflammation by reducing TSP-1 release.