Roubille François, Combes Stéphane, Leal-Sanchez Juani, Barrère Christian, Cransac Frédéric, Sportouch-Dukhan Catherine, Gahide Gérald, Serre Isabelle, Kupfer Elodie, Richard Sylvain, Hueber Anne-Odile, Nargeot Joël, Piot Christophe, Barrère-Lemaire Stéphanie
Department of Physiology CNRS UMR5203, INSERM U661, University of Montpellier I and II, 141 rue de la Cardonille, 34094 Montpellier, Cedex 5, France.
Circulation. 2007 Dec 4;116(23):2709-17. doi: 10.1161/CIRCULATIONAHA.107.694844. Epub 2007 Nov 19.
Apoptosis has been described extensively in acute myocardial infarction and chronic heart failure. Because Daxx (death-associated protein) appears to be essential for stress-induced cell death and acts as an antisurvival molecule, we tested the hypothesis that Daxx is involved in myocardial ischemia/reperfusion-induced cell death in vivo.
Transgenic mice overexpressing a dominant-negative form of Daxx (Daxx-DN) under the control of the beta-actin promoter and control wild-type mice underwent an ischemia/reperfusion protocol: 40 minutes of left coronary artery occlusion and 60 minutes of reperfusion. Area at risk and infarct size were measured after dual staining by triphenyltetrazolium chloride and phthalocyanine blue dye. Apoptosis was measured in the ischemic versus the nonischemic part of the left ventricle by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling staining, enzyme-linked immunosorbent assay, and Western blotting of caspase-3, caspase-8, and poly(ADP-ribose) polymerase. The mitogen-activated protein kinase status was investigated by Western blot analysis. Comparison between groups was assessed by ANOVA or Student t test (statistical significance: P<0.05). Left ventricle tissues from transgenic mice expressed Daxx-DN at the protein level. Area at risk/left ventricle values were comparable among groups. Infarct size/area at risk was 45% reduced in Daxx-DN versus wild-type mice (P<0.001). This cardioprotection was maintained for a 4-hour reperfusion. Ischemia/reperfusion-induced apoptosis was significantly decreased and ERK1/2 prosurvival pathway was activated in ischemic Daxx-DN hearts.
Our study clearly indicates that Daxx participates in myocardial ischemia/reperfusion proapoptotic signaling in vivo.
细胞凋亡在急性心肌梗死和慢性心力衰竭中已有广泛描述。由于死亡相关蛋白（Daxx）似乎是应激诱导细胞死亡所必需的，并且作为一种抗生存分子发挥作用，我们检验了Daxx参与体内心肌缺血/再灌注诱导的细胞死亡这一假说。
在β-肌动蛋白启动子控制下过表达显性负性形式Daxx（Daxx-DN）的转基因小鼠和对照野生型小鼠接受缺血/再灌注方案：左冠状动脉闭塞40分钟，再灌注60分钟。通过氯化三苯基四氮唑和酞菁蓝染料双重染色后测量危险区域和梗死面积。通过末端脱氧核苷酸转移酶介导的dUTP生物素缺口末端标记染色、酶联免疫吸附测定以及半胱天冬酶-3、半胱天冬酶-8和聚（ADP-核糖）聚合酶的蛋白质印迹法，测量左心室缺血部分与非缺血部分的细胞凋亡。通过蛋白质印迹分析研究丝裂原活化蛋白激酶状态。组间比较采用方差分析或学生t检验（统计学显著性：P<0.05）。转基因小鼠的左心室组织在蛋白质水平表达Daxx-DN。各组间危险区域/左心室值相当。与野生型小鼠相比，Daxx-DN小鼠的梗死面积/危险区域减少了45%（P<0.001）。这种心脏保护作用在再灌注4小时后仍持续存在。缺血/再灌注诱导的细胞凋亡在缺血的Daxx-DN心脏中显著减少，并且ERK1/2促生存途径被激活。
我们的研究清楚地表明，Daxx在体内参与心肌缺血/再灌注促凋亡信号传导。
