The muscular dystrophies are devastating diseases of progressive weakness due to apoptotic and necrotic death of muscle cells. The normal cellular mechanisms regulating cell survival that are disrupted in these diseases are not well understood. Several forms of muscular dystrophy are due to abnormalities of membrane proteins and protein complexes, such as integrins and caveolins, that are known to regulate cellular signaling pathways in general, and cell survival signaling in particular, in different cell types. Others, such as those due to dystrophin mutations, are due to abnormalities of protein complexes that are postulated to transduce signals from the extracellular matrix into the cell. We will focus on three proteins/protein complexes that cause muscular dystrophies when a component of the complex is deficient or defective - alpha5beta1 integrin, caveolin-3, and the dystrophin-glycoprotein complex (DGC). The experiments of this proposal are designed to explore the cellular signaling processes the promote cell survival via these membrane protein complexes, and the mechanisms of cell death when these complexes are disrupted. For studies of integrin signaling, we will use cells genetically deficient in alpha5 integrin to test which isoforms of protein kinase C are important in alpha5 integrin mediated muscle cell survival (based on our previous finding of the importance of protein kinase C in this process). We will explore how alpha5 integrin deficiency leads to muscle cell death by testing for dysregulation of cell survival/cell death pathways involving the Bcl family of proteins, cytochrome c release from mitochondria, and activation of the caspase cascade. We will also examine the role of activation of the PI3 kinase/Akt pathway in alpha5 integrin- mediated muscle cell survival. For studies of the DGC, we will investigate how disruption (genetically, by antibody inhibition, or by antisense expression) of the association of the complex with the extracellular matrix may lead to cell death. In these studies, we will also examine cells for dysregulation of cell survival mechanisms involving Bcl family proteins since apoptosis has been shown to be the earliest change in muscle associated with dystrophin deficiency. For studies of dystrophies due to caveolin-3 mutations, we will render muscle cells functionally deficient in caveolin-3 using both antisense methods and dominant negative inhibitors. We will study the mechanisms by which caveolin-3 deficiency lead to muscle cell death, and we will test whether these mechanisms involve the disruption of either normal integrin signaling or signaling through the DGC.
