Identification and immunolocalisation of annexins V and VI, the major cardiac annexins, in rat heart

Abstract

Objective: The heart contains proteins of the annexin family, a unique group of calcium binding proteins. This study was aimed at identifying the major cardiac annexins and determining their distribution in the rat heart.

Methods: Annexins were isolated by affinity chromatography and purified by ion exchange high pressure liquid chromatography. Identification of isolated proteins by immunoblotting was confirmed by partial amino acid sequence determination. Antisera raised against the isolated proteins were used for immunohistochemistry by the avidin-biotin-peroxidase technique.

Results: Two annexins were isolated and purified. Amino acid sequencing confirmed their identities as annexin V and VI. Immunohistochemistry showed that both annexins were present in cardiac myocytes and non-myocytes, but a distinct pattern of distribution was seen for each annexin. Annexin V immunoreactivity was enhanced in the atria compared with the ventricles, whereas annexin VI was more uniformly distributed. In individual cardiac myocytes annexin V was distributed throughout the cell by contrast with annexin VI, which localised to the sarcolemma. Intercalated discs displayed immunoreactivity for both annexins, most prominently for annexin VI. The most striking immunoreactivity for annexin V occurred in vascular endothelial cells, both in the microcirculation and in the major coronary vessels. Immunoreactivity for annexin VI in vascular structures was localised to the nuclei of endothelial and smooth muscle cells.

Conclusions: Annexins V and VI are the major cardiac annexins. The localisation of these annexins to different components of cardiac myocytes will serve to direct the search for their functions in the heart. The striking immunoreactivity for annexins, particularly annexin V, in the coronary vessels indicates that the functions of cardiac annexins include a role in the coronary circulatory system.