Annexin VI is a member of a family of Ca(2+)-dependent phospholipid-binding proteins that is expressed in many tissues, including the heart. It is a regulator of membrane-associated events, including the skeletal muscle ryanodine-sensitive Ca2+ release channel and the cardiac Na+/Ca2+ exchanger. The potential roles of annexin VI in Ca2+ signaling in cardiac myocytes were evaluated by targeting its overexpression to the hearts of transgenic mice. Expression of full-length human annexin VI cDNA was targeted to the heart using the alpha-myosin heavy chain gene promoter (Subramaniam, A., W. K. Jones, J. Gulick, S. Wert, J. Neumann, and J. Robbins. J. Biol. Chem. 266: 24613-24620, 1991). Five transgenic lines exhibited at least 10-fold overexpression of annexin VI protein in both atria and ventricles. Pathological evaluation indicated mice overexpressing annexin VI had enlarged dilated hearts, acute diffuse myocarditis, lymphocytic infiltration, moderate to severe fibrosis throughout the heart, and mild fibrosis around the pulmonary veins of the lungs. Contractile mechanics of cardiomyocytes isolated from hearts of transgenic animals showed frequency-dependent reduced percent shortening and decreased rates of contraction and relaxation compared with control animals. Cardiomyocytes isolated from transgenic animals had lower basal levels of intracellular free Ca2+ and a reduced rise in free Ca2+ following depolarization. After stimulation, intracellular free Ca2+ returned to basal levels faster in transgenic cells than in cells from control animals. These data demonstrate that the overexpression of annexin VI in the heart disrupts normal Ca2+ homeostasis and suggests that this dysfunction may be due to annexin VI regulation of pumps and/or exchangers in the membranes of cardiomyocytes.