The cholesterol synthesis inhibitor simvastatin, which is used to treat cardiovascular diseases, has severe collateral effects. We decided to comprehensively study the effects of simvastatin in zebrafish development and in myogenesis, because zebrafish has been used as a model to human diseases, due to its handling easiness, the optical clarity of its embryos, and the availability of physiological and structural methodologies. Furthermore, muscle is an important target of the drug. We used several simvastatin concentrations at different zebrafish developmental stages and studied survival rate, morphology, and physiology of the embryos. Our results show that high levels of simvastatin induce structural damage whereas low doses induce minor structural changes, impaired movements, and reduced heart beating. Morphological alterations include changes in embryo and somite size and septa shape. Physiological changes include movement reduction and slower heartbeat. These effects could be reversed by the addition of exogenous cholesterol. Moreover, we quantified the total cell number during zebrafish development and demonstrated a large reduction in cell number after statin treatment. Since we could classify the alterations induced by simvastatin in three distinct phenotypes, we speculate that simvastatin acts through more than one mechanism and could affect both cell replication and/or cell death and muscle function. Our data can contribute to the understanding of the molecular and cellular basis of the mechanisms of action of simvastatin.
In vitro studies show that cholesterol is essential to myogenesis. We have been using zebrafish to overcome the limitations of the in vitro approach and to study the sub-cellular structures and processes involved during myogenesis. We use simvastatin—a drug widely used to prevent high levels of cholesterol and cardiovascular disease—during zebrafish skeletal muscle formation. Simvastatin is an efficient inhibitor of cholesterol synthesis that has various myotoxic consequences. Here, we employed simvastatin concentrations that cause either mild or severe morphological disturbances to observe changes in the cytoskeleton (intermediate filaments and microfilaments), extracellular matrix and adhesion markers by confocal microscopy. With low-dose simvastatin treatment, laminin was almost normal, and alpha-actinin was reduced in the myofibrils. With high simvastatin doses, laminin and vinculin were reduced and appeared discontinuous along the septa, with almost no myofibrils, and small amounts of desmin accumulating close to the septa. We also analyzed sub-cellular alterations in the embryos by electron microscopy, and demonstrate changes in embryo and somite size, septa shape, and in myofibril structure. These effects could be reversed by the addition of exogenous cholesterol. These results contribute to the understanding of the mechanisms of action of simvastatin in muscle cells in particular, and in the study of myogenesis in general.
The GARSA system is freely available under GPL license (http://www.biowebdb.org/garsa/). For download requests visit http://www.biowebdb.org/garsa/ or contact Dr Alberto Dávila.
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