Cardiac hypertrophy and failure are associated with increased mechanical stretch, electrical remodeling and arrhythmogenesis. We studied the effect of pulsatile stretch on connexin (Cx), adherence protein (AP) expression and propagation velocity (v) in a cell culture model of patterned neonatal rat cardiomyocytes. V was measured with optical mapping of voltage-sensitive dye fluorescence. 1--3 hours pulsatile stretch is associated with 2- 3-fold upregulation of Cx43 and N-cadherin, with a concomitant increase in v. Stimuli and signaling pathways responsible for upregulation of electrical and mechanical junctions by acute stretch involved binding of the RGD (arginine-glycine-aspartate) motif of extracellular matrix proteins to beta1-integrins. Either stretch in presence of masked RGD motivs (collagen I) or direct exposure to matrix proteins with unmasked RGD motivs (fibronectin) upregulated Cx43 and AP. Upregulation of both Cx43 and AP were mediated intracellularly by activation of focal adhesion kinase (FAK). Downstream of FAK, stretch-induced secretion of vascular endothelial growth factor was responsible for upregulation of Cx43 via autocrine and paracrine effects, while upregulation of AP's was mediated by a pathway involving src-kinase. Thus, mechanical stretch and extracellular matrix composition in physiological and pathophysiological conditions affect the degree of remodeling of cell-to-cell coupling and electrical propagation velocity.