Cellular Localization of α3β1 Integrin Isoforms in Association with Myofibrillogenesis during Cardiac Myocyte Development in Culture

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Abstract
The cellular localization of α3β1 integrin isoforms was examined in cultured neonatal myocytes at selected times during development using double immunofluorescence assays. The distribution of α3A subunits began as diffuse and patternless, but as the cells matured, the distribution assumed a sarcomeric banding pattern, and α3A appeared to be localized in costameres - sarcolemmal regions adjacent to the Z-disks. α-actinin, a component of the Z-disk, was localized in the same intracellular regions. Temporal analysis of the incorporation of the α3A subunit and other myofibrillar proteins into sarcomeres revealed that α3A was integrated into sarcomeres following incorporation of α-actinin and myosin heavy chain (MHC) but prior to that of desmin. This suggests that α3A integrins are incorporated into a pre-existing myofibrillar structure, and it is unlikely that α3A integrins participate in the initial assembly of myofibrillar proteins. The α3B, β1A and β1D subunits were also localized in costameres, where they formed α3Aβ1A, α3Aβ1D and α3Bβ1A heterodimers. The α3Bβ1D heterodimer, however, was not found in cardiac myocytes. The antisera raised against the cytoplasmic domains of α3A, α3B, β1A and β1D caused disruption of sarcomere structure. Thus, the myofibril-extracellular matrix linkages mediated by isoforms of α3β1 integrin may play a crucial role in the stabilization of myofibril assembly and in the maintenance of sarcomere structure. Co-immunoprecipitation experiments revealed that β1A, but not β1D, interacts with the Nck signaling protein, suggesting that Nck participates in downstream signaling triggered by β1A and that the β1A-mediated signaling pathway is distinct from that of β1D.