Effect of the lipid-raft disorganization on the muscular differentiation in zebrafish model

Abstract

Striated muscle differentiation is based on the construction of a three-dimensional contractile apparatus. Myogenesis has been studied in cell cultures, because of the simpler observation in vitro. But in these cultures, cells are forced to adapt to the two-dimensional environment, and are induced to form structures that they rarely form in vivo, such as stress fibers. To overcome the culture limitations, we have been using the three-dimensional model of zebrafish embryos to study structural aspects of myogenesis. We studied the spatial distribution of myofibrils using Alpha-actinin, Desmin, Troponin, Titin, Actin, the distribution of cell adhesion sites using Paxillin, Dystrophin, Vinculin, and the extracellular proteins Laminin and Fibronectin. Our previous results in cell culture models showed that cholesterol is important to myogenesis. We therefore decided to study the effects of simvastatin, which impairs the synthesis of cholesterol. The embryos at 6 hpf were immersed in embryonic water at a concentration ranging from 0.003 to 12 μM of simvastatin for 18 h. In treated groups, we detected the formation of the same numbers of somites, like in control, but a significant dosedependent somite and septum damage phenotype. We used conventional Zeiss optical microscope, but more commonly we used Zeiss multiphoton laser scanning, and Olympus spinning disk confocal microscope. The three-dimensional analysis, reconstructions and projections were made with ImageJ and we confirm that there are significant differences between the myogenesis in vitro, in twodimensional cultures, from in situ and in three-dimensional models. The characterization of the consequences of simvastatin is important not only to understand myogenesis, but also to explain the collateral effects of its use as a treatment for cholesterol problems in humans.