RESUMO
OBJECTIVE: Annexins are Ca(2+)-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 H(2)O(2) 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 H(2)O(2) treatment of neonatal cardiomyocytes resulted in significant increases of apoptosis at 24 h, but H(2)O(2) 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 H(2)O(2) induction, the number of annexin A5-positive cells was higher than that of PS-positive cells (p=0.05) and colabeling showed that half annexin A5-positive cells were PS-negative. We further demonstrated by immunoblots that free annexin A5 was absent from the media and could not be released from cardiomyocytes by washes at 1.8 mM Ca(2+). Removing annexin A5 by Ca(2+)-free washes 15 or 30 min after H(2)O(2) 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.
Assuntos
Anexina A5/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Anexina A5/análise , Anexina A5/imunologia , Anticorpos Monoclonais/farmacologia , Apoptose , Caspase 3 , Caspases/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Inibidores Enzimáticos/farmacologia , Peróxido de Hidrogênio/farmacologia , Marcação In Situ das Extremidades Cortadas , Microscopia Confocal , Miócitos Cardíacos/patologia , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos Wistar , Sarcolema/metabolismo , Estaurosporina/farmacologiaRESUMO
Annexin A5 is a Ca2+-dependent phospholipid binding protein well known for its high phosphatidylserine affinity. In vitro, translocation to sarcolemma and externalization of endogenous annexin A5 in the cardiomyocyte has recently been demonstrated to exert a proapoptotic effect. To determine whether these in vitro findings occurred in vivo, we performed myocardial infarction (MI) and studied the time course of apoptosis and annexin A5 localization (0.5 to 8 h) in the border zone around the infarcted area. This zone that was defined as Evans blue unstained and triphenyltetrazolium chloride (TTC) stained, represented 42.3 +/- 5.5% of the area at risk and showed apoptotic characteristics (significant increases in caspase 3 activity 2.3-fold at 0.5 h; P < 0.05), transferase-mediated dUTP nick-end labeling-positive cardiomyocytes (15.8 +/- 0.8% at 8 h), and DNA ladder. When compared with sham-operated rats, we found that in this area, annexin A5 was translocated to the sarcolemma as early as 0.5 h after MI and that translocation increased with time. Moreover, the amount of annexin A5 was unchanged in the border zone and decreased in the infarcted area after 1 h (77.1 +/- 4.8%; P < 0.01 vs. perfused area), suggesting a release in the latter but not in the former. In conclusion, we demonstrated that annexin A5 translocation is an early and rapid event of the whole border zone, likely due to Ca2+ increase. Part of this translocation occurred in areas where apoptosis was later detected and suggests that in vivo as in vitro annexin A5 might be involved in the regulation of early apoptotic events during cardiac pathological situations.