Externalization of endogenous annexin A5 participates in apoptosis of rat cardiomyocytes

Objective: Annexins are Ca2+-dependent phospholipid binding proteins. Externalized annexin A5 has been recently suggested to have a proapoptotic effect. Our aim was to determine whether annexin A5, which is intracellular in cardiomyocytes, could be translocated and/or externalized and play a role during the apoptotic process. Methods: Apoptosis was induced in rat cardiomyocytes by continuous incubation with staurosporine or 30 min treatment with H2O2 and was measured by phosphatidylserine (PS) externalization, TUNEL staining and DNA ladder. Immunofluorescence labeling of annexin A5 was performed on permeabilized or nonpermeabilized cardiomyocytes. Results: Staurosporine or H2O2 treatment of neonatal cardiomyocytes resulted in significant increases of apoptosis at 24 h, but H2O2 treatment led to a faster and higher PS externalization than that observed with ST. In both neonatal and adult cardiomyocytes, annexin A5 was intracellular in control conditions but was found at the external face of sarcolemma during apoptosis. Furthermore, neonatal cardiomyocytes with externalized annexin A5 have apoptotic characteristics and their number increased with time. Interestingly, immediately after H2O2 induction, the number of annexin A5-positive cells was higher than that of PS-positive cells (pless than or equal to0.05) and colabeling showed that half annexin A5-positive cells were PS-negative. We further demonstrated by immumoblots that free annexin A5 was absent from the media and could not be released from cardiomyocytes by washes at 1.8 mM Ca2+. Removing annexin A5 by Ca2+-free washes 15 or 30 min after H2O2 treatment or blocking externalized annexin A5 by antibodies lead to a significant decrease of apoptotic cardiomyocytes, cytochrome c release and caspase 3 activity. Conclusion: This study indicated for the first time that annexin A5 was externalized at a very early stage of apoptosis and could have a proapoptotic effect in cardiomyocytes. (C) 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.