Beta(3)-integrin-mediated focal adhesion complex formation: Adult cardiocytes embedded in three-dimensional polymer matrices

In vivo studies show that beta(3)-integrin-mediated focal adhesion formatio n (FAF) causes recruitment of nonreceptor tyrosine kinases to the cytoskele ton in pressure-overloaded myocardium. To define the mechanism of beta(3)-i ntegrin-mediated signaling, we developed a cell culture model (adult feline cardiocytes embedded in a 3-dimensional matrix of native type 1 collagen, fibronectin, and vitronectin) wherein beta(3)-integrin-mediated focal adhes ion kinase occurs. Focal adhesion kinase was analyzed immunocytochemically using confocal microscopy. Initial studies suggested that cardiocytes cultu red in a 3-dimensional matrix formed focal adhesions consisting of both bet a(3)-integrin and the muscle-specific isoform, beta(1)-integrin (beta(1D)). The focal adhesions were associated with focal adhesion kinase on both cos tameres and intercalated disks. To determine the cause of beta(1D)-integrin -mediated focal adhesion kinase in this model, time course studies were don e. Beta(3)-integrin-mediated focal adhesion kinase occurred within 30 minut es after embedding cardiocytes and persisted for >24 hours, whereas beta(1D )-integrin-mediated focal adhesion kinase was present from the outset. Beca use confocal microscopy showed that laminin was present on the surface of f reshly isolated cardiocytes, we hypothesized that this was causative of bet a(1D)-integrin-mediated focal adhesion kinase. Freshly isolated cardiocytes washed with acidic medium (2 minutes, pH 3.0) to remove laminin and then e mbedded in a 3-dimensional matrix showed complete absence of beta(1D)-integ rin-mediated focal adhesion kinase, but beta(3)-integrin-mediated focal adh esion kinase occurred with a time course similar to that seen in cultured, unwashed cardiocytes. Acid washing did not alter the binding ability of bet a(1D)-integrin, because acid-washed cardiocytes in the presence of laminin showed beta(1D)-integrin-mediated focal adhesion kinase. Thus, cardiocytes embedded in a 3-dimensional matrix show beta(3)-integrin-mediated focal adh esion kinase and provide an in vitro model to study beta(3)-integrin-mediat ed signaling in response to hemodynamic cardiac loading. (C)1999 by Excerpt a Medica, Inc.