Monceau Virginie, Belikova Yulia, Kratassiouk Gueorgui, Charue Dominique, Camors Emmanuel, Communal Catherine, Trouvé Pascal, Russo-Marie Françoise, Charlemagne Danièle
INSERM U572, IFR Circulation, Hopital Lariboisière, 41 Boulevard de la Chapelle, 75475 Paris Cedex 10, France.
Cardiovasc Res. 2004 Dec 1;64(3):496-506. doi: 10.1016/j.cardiores.2004.08.003.
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.
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.
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.
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.
膜联蛋白是依赖钙离子的磷脂结合蛋白。最近有研究表明，外化的膜联蛋白A5具有促凋亡作用。我们的目的是确定心肌细胞内的膜联蛋白A5是否会发生转位和/或外化，并在凋亡过程中发挥作用。
用星形孢菌素持续孵育或用H₂O₂处理30分钟诱导大鼠心肌细胞凋亡，通过磷脂酰丝氨酸（PS）外化、TUNEL染色和DNA梯状条带检测凋亡情况。对通透或未通透的心肌细胞进行膜联蛋白A5的免疫荧光标记。
用星形孢菌素或H₂O₂处理新生心肌细胞24小时后凋亡显著增加，但H₂O₂处理导致PS外化比星形孢菌素更快、更高。在新生和成年心肌细胞中，膜联蛋白A5在对照条件下位于细胞内，但在凋亡过程中出现在肌膜外表面。此外，外化膜联蛋白A5的新生心肌细胞具有凋亡特征，且其数量随时间增加。有趣的是，H₂O₂诱导后立即观察到，膜联蛋白A5阳性细胞数量高于PS阳性细胞（p≤0.05），共标记显示一半膜联蛋白A5阳性细胞为PS阴性。我们通过免疫印迹进一步证明，培养基中不存在游离的膜联蛋白A5，在1.8 mM钙离子浓度下洗涤不能使膜联蛋白A5从心肌细胞中释放。H₂O₂处理15或30分钟后用无钙洗涤去除膜联蛋白A5或用抗体阻断外化的膜联蛋白A5会导致凋亡心肌细胞、细胞色素c释放和半胱天冬酶3活性显著降低。
本研究首次表明膜联蛋白A5在凋亡的早期阶段发生外化，并可能在心肌细胞中具有促凋亡作用。
