Abstract 18584: Filamin-A Mutations Causing Mitral Valve Prolapse Alters Integrin Expression and Causes Breakdown of Mechanotransduction

Introduction: Mutations in the gene encoding Filamin-A, a cytoskeletal protein, were identified in patients with familial mitral valve prolapse(MVP). Mitral valves in these patients were floppy and appeared myxomatous, but the underlying mechanisms governing such changes are unclear. In this study, we sought to investigate the impact of these mutations on cultured cells in comparison with wild type Filamin-A, to delineate the mechanisms underlying MVP. Methods: Four immortal cell lines were established - Filamin-A KO, Filamin-A WT, G288R mutated Filamin-A, and P637Q mutated Filamin-A. Cells were cultured on plastic petri-dishes for a week, and at confluence the cells were imaged under a microscope, then their 3D morphology and mechanical stiffness measured with scanning ion conductance microscopy, and then the cells were assayed for integrins and counted using flow cytometry. Results: Filamin-A KO cells developed blebs on the surface, were flat and did not sufficiently attach to the cell culture substrate, while WT cells demonstrated excellent attachment and formed extensive lamellapodi and filipodia (Fig 1A). Both G288R and P637Q mutated cells had reduced extensions, with a cell structure that is small in circumference but large in height(Fig 1B). α1, α2, α3, α5, β1, β3, β4, α5β3, α2β1 integrin expression was drastically reduced in both G288R and P637Q mutations, compared to WT (Fig 1C1-C2). A 24 hour substrate attachment assay demonstrated 30% attachment of G288R cells were attached and viable, 50% of the P637Q cells were attached and viable, and 98% of WT cells were attached and viable, with similar results seen in gel contraction studies (Fig 1D). Conclusions: Filamin-A mutations associated with mitral valve prolapse seem to alter fundamental cellular mechanisms essential for cell attachment to matrix, motility and mechanotransduction.