Dysferlinopathies (LGMD2B) are autosomal recessive muscular dystrophies caused by mutations in the dysferlin gene leading to a loss or a severe reduction of dysferlin, a protein normally associated to membranes and involved in muscle membrane trafficking and repair. Muscle inflammation is a distinct feature of dysferlinopathies and may be explained by an attenuated regenerative response and/or homeostasis in skeletal muscle. Failure to successfully conclude muscle regeneration leads to persistent necrotic fibres and inflammation. The human muscle cells do release not only soluble secreted proteins through conventional secretory pathway but also two types of membrane-derived vesicles: microparticles generated from membrane blebs; and exosomes. Here, we hypothesize that changes in the skeletal muscle secretome, may contribute to the regenerative defects observed in dysferlinopathies. Such alteration in secretion/signalling could also participateto the persistence of an inflammatory microenvironmement. Therefore, the general objective of this proposal is to determine how the secretory profile of differentiating human myoblasts is altered in dysferlinopathies. We will employ advanced mass spectrometrybasedquantitative proteomics approach, Stable Isotope Labelling by Amino acids in Cell culture (SILAC) for analysis of “soluble” factors and microvesicles secreted from human satellite cell lines, isolated from healthy donors and dysferlinopathy patients. The original proteomics profiling will be complemented by mRNA and tissue expression studies. The mRNA expression profiles of control and dysferlin-deficient cells will be used to construct"virtual secretomes" and will be compared to the “experimental secretomes” as determined by SILAC. The major potential of this work lays in the identification of the detailed “signals”sent/or not by muscle cells in the absence of dysferlin that may influence the behaviours of neighbouring cells thereby providing the foundation for generating new theories of the underlying pathophysiological mechanism for dysferlinopathies.
|Effective start/end date||01/01/2012 → 31/12/2014|