Annexin V in the adult rat heart: isolation, localization and quantitation

Abstract

Annexins are a family of proteins with calcium- and phospholipid-binding properties. The present study was performed to identify which members of the annexin family are present in rat heart and to determine the cellular and subcellular distribution of annexin V, the most prominent annexin in rat cardiac tissue, in isolated ventricular myocytes and cultured endothelial and fibroblast-like cells. The presence of annexin I plus II, III, IV, V and VI in rat cardiac tissue was positively established with western blot analysis. Immunohistochemistry and western blot analysis revealed that annexin V is present in both cardiomyocytes and non-myocytal cells of the heart. In endothelial cells and fibroblast-like cells annexin V is predominantly localized in the cytoplasm and in cardiac myocytes in close vicinity of the sarcolemma. This last finding is confirmed by electron microscopy. Northern blot analysis demonstrated that all cell types investigated showed expression of annexin V. Annexin V mRNA levels were highest in the fibroblast-like cells, followed by the endothelial cells, and a weak signal was observed in the cardiomyocytes. By means of a sandwich-type enzyme-linked immunosorbent assay (ELISA) annexin V content in intact adult rat heart, isolated myocytes, cultured cardiac endothelial cells and fibroblast-like cells was found to be 0.70, 0.17, 1.63 and 3.84 micrograms/mg total protein, respectively. The differences in subcellular localization of annexin V in myocytes and non-myocytes suggest differences in biological function of annexin V in the various cell types.