Study of cavin expression in a cellular model of FacioScapuloHumeral muscular Dystrophy (FSHD)

PhD student: Ms Armelle WAUTERS

Promoter : Dr. Frédérique COPPEE

Co-Promoter : Prof. Alexandra BELAYEW

 Summary

Caveolae are specialized lipid rafts that form 50 to 100 nm flask-shaped invaginations of the plasma membrane. They are involved in many cellular pathways such as cell signalling and mechanical stress regulation. Caveolae change their shape or exit from the membrane to regulate its size and density thus acting as regulators of tension stresses. Cavins and caveolins form the caveola protein coat, and enhance their formation. When myoblasts differentiate into myotubes they deal with many  embrane fusion events, a significant size expansion and the building of the contractile apparatus. Moreover, little is known about either caveola involvement or cavin expression levels and intracellular location during differentiation. In addition, several myopathies were associated to caveolar disturbances. Our study stems from the differential proteomics study of Tassin et al. 2012, which has highlighted deregulations of caveola proteins in FacioScapuloHumeral Dystrophy (FSHD) myotubes as compared to healthy ones. In this study, we investigated changes in cavin (PTRF, SDPR, SRBC, MURC) expression during in vitro differentiation of healthy and FSHD immortalized human myoblast lines derived from biceps and deltoid muscles.

We measured the abundance of the four cavins messenger RNAs (RT-qPCR) and proteins (immunodetection on western blots) in healthy proliferating myoblasts and myotubes after 4 days of differentiation. We then performed the same study on cell lines derived from patients affected with FSHD and compared the cavin abundances to the healthy controls. The cavin intracellular location was evaluated by co-immunofluorescence with caveolin-3 (CAV3) a muscle-specific caveola marker. We also immunodetected cavins in lipid rafts isolated by Adam et al. 2008 method that we adapted for these muscle cells. The laboratory of Interfaces and Complex Fluids developed a microprinting technique to standardize the study of myoblast fusion and differentiation. Myoblasts were seeded onto precisely printed micropatterns and the traction forces developed by myoblasts on their substrate were compared between FSHD and control myoblasts.

During myoblast differentiation PTRF/Cavin-1 immunostaining decreased in myonuclei while it increased in total myotube extracts but not in lipid rafts, thus corresponding to a cytoplasmic accumulation. SDPR/Cavin-2 and SRBC/Cavin-3 immunodetections were significantly reduced during differentiation while MURC/Cavin-4 became more abundant. At the proliferation stage, cavin mRNAs were globally decreased in FSHD myoblasts compared to healthy ones while they were generally increased in FSHD myotubes.

We found that the expression of cavin mRNAs and proteins was modulated during differentiation of healthy myoblasts to myotubes, and these changes were disturbed in FSHD myoblasts and myotubes. Our study suggests that alterations in cellular processes mediated by caveolins and cavins could contribute to the FSHD pathology.