Background Recent studies suggest that both endocardial endothelium and coronary vascular endothelium influence myocardial contraction, but the mediators responsible and their mechanisms of action are not well defined.Methods and Results We investigated the effects of cultured endocardial endothelial and vascular endothelial cell superfusate on contraction and intracellular calcium transients of isolated rat cardiac myocytes. Endothelial cell superfusate induced a potent negative inotropic effect, with a rapid reversible decrease in myocyte twitch amplitude, earlier twitch relaxation, and a significant increase in diastolic length. This effect was not associated with significant changes in intracellular calcium or pH; was not attributable to nitric oxide, prostanoids, cGMP, or protein kinase C activation; and did not involve pertussis toxin-sensitive G proteins. The activity was stable at 37 degrees C for several hours, was not destroyed by protease treatment, and was found in low-molecular-weight (<<1 kD) superfusate fractions.Conclusions These data suggest the tonic release by endothelial cells of a novel, stable factor that acts predominantly by reducing the response of cardiac myofilaments to calcium (ie, "desensitizes" them). This "desensitizing factor" could rapidly modulate cardiac contraction-relaxation coupling and diastolic tonus and exert distant effects because of its stability. (Circulation. 1994;89:2492-2497.)