To elucidate mechanisms responsible for mechanotransduction in the heart and define the effects of remodeling of the extracellular matrix, we cultured neonatal rat ventricular myocytes on native type I collagen, fibronectin, or denatured collagen and subjected them to uniaxial, pulsatile stretch. Changes in expression of the cardiac gap junction protein, Cx43, were measured by confocal microscopy and immunoblotting. Cells grown on fibronectin or denatured collagen exhibited significantly greater Cx43 expression than cells grown on native collagen. Stretch induced a approximately 2-fold increase in Cx43 expression in cells grown on native collagen but no increase in cells grown on fibronectin or denatured collagen. Incubation of cells on native collagen with a peptide containing the arginine-glycine-aspartate (RGD) motif upregulated Cx43 expression equivalent to that induced by stretch. Nonselective activation of integrin signaling with MnCl2 also upregulated Cx43 expression in cells grown on native collagen. This effect was blocked completely by pretreatment with anti-beta1 integrin antibody but not by anti-beta3 integrin antibody. Stretch led to a marked increase in beta1 integrin immunofluorescent signal in cells grown on native collagen but not in cells grown on fibronectin or denatured collagen. Stretch-induced upregulation of Cx43 was also blocked by anti-beta1 integrin antibody. Thus, matrix protein-myocyte interactions regulate Cx43 expression via beta1 integrin signaling initiated by mechanical stimulation in cells grown on native type I collagen, or by RGD-integrin signaling independent of mechanical stress in cells grown on fibronectin or denatured collagen. Changes in the composition of the extracellular matrix may affect electrical coupling in cardiac myocytes.