Communication between the extracellular matrix and the intracellular signal transduction and cytoskeletal system is mediated by integrin receptors. alpha(5)beta(1)-Integrin and its cognate ligand fibronectin are essential in development of mesodermal structures, myocyte differentiation, and normal cardiac development. To begin to explore the potential roles of alpha(5)beta(1)-integrin specifically in cardiomyocytes, we used a transgenic expression strategy. We overexpressed two forms of the human alpha(5)-integrin in cardiomyocytes: the full-length wild-type alpha(5)-integrin and a putative gain-of-function mutation created by truncating the cytoplasmic domain, designated alpha(5-1)-integrin. Overexpression of the wild-type alpha(5)-integrin has no detectable adverse effects in the mouse, whereas expression of alpha(5-1)-integrin caused electrocardiographic abnormalities, fibrotic changes in the ventricle, and perinatal lethality. Thus physiological regulation of integrin function appears essential for maintenance of normal cardiomyocyte structure and function. This strengthens the role of inside-out signaling in regulation of integrins in vivo and suggests that integrins and associated signaling molecules are important in cardiomyocyte function.