Cellular localization of alpha 3 beta 1 integrin isoforms in association with myofibrillogenesis during cardiac myocyte development in culture

The cellular localization of alpha 3 beta 1 integrin isoforms was examined in cultured neonatal myocytes at selected times during development using do uble immunofluorescence assays. The distribution of alpha 3A subunits began as diffuse and patternless, but as the cells matured, the distribution ass umed a sarcomeric banding pattern, and alpha 3A appeared to be localized in costameres - sarcolemmal regions adjacent to the Z-disks. alpha-actinin, a component of the Z-disk, was localized in the same intracellular regions. Temporal analysis of the incorporation of the alpha 3A subunit and other my ofibrillar proteins into sarcomeres revealed that alpha 3A was integrated i nto sarcomeres following incorporation of alpha-actinin and myosin heavy ch ain (MHC) but prior to that of desmin. This suggests that alpha 3A integrin s are incorporated into a pre-existing myofibrillar structure, and it is un likely that alpha 3A integrins participate in the initial assembly of myofi brillar proteins. The alpha 3B, beta 1A and beta 1D subunits were also loca lized in costameres, where they formed alpha 3A beta 1A, alpha 3A beta 1D a nd alpha 3B beta 1A heterodimers. The alpha 3B beta 1D heterodimer, however , was not found in cardiac myocytes, The antisera raised against the cytopl asmic domains of a3A, alpha 3B, beta 1A and beta 1D caused disruption of sa rcomere structure. Thus, the myofibril-extracellular matrix linkages mediat ed by isoforms of alpha 3 beta 1 integrin may play a crucial role in the st abilization of myofibril assembly and in the maintenance of sarcomere struc ture. Co-immunoprecipitation experiments revealed that beta 1A, but not bet a 1D, interacts with the Nck signaling protein, suggesting that Nck partici pates in downstream signaling triggered by beta 1A and that the beta 1A-med iated signaling pathway is distinct from that of beta 1D.