Apoptosis inducing factor in cardiac apoptosis

本研究项目的长期目标是研究AIF在心脏细胞凋亡中的作用。虽然caspase被认为是凋亡程序的中枢介质，但我们的初步研究表明，成年心肌细胞既对caspase依赖的凋亡具有抵抗力，又在凋亡刺激下表现出明显的凋亡诱导因子(AIF)激活，AIF是参与caspase非依赖性凋亡的典型因子。半胱氨酸天冬氨酸氨基转移酶非依赖性细胞凋亡在心脏中的作用尚不清楚。然而，我们发现，心脏特异表达caspase抑制物CrmA的转基因小鼠在缺血/再灌注(I/R)后表现出显著的AIF激活。因此，尽管caspase活性被完全抑制，但对心肌损伤只有部分抑制。然而，同时抑制PARP-1抑制了AIF的激活，并在CrmA小鼠I/R后提供了更有效的心脏保护。这些发现表明，AIF诱导的细胞凋亡可能在导致心血管损伤中的细胞死亡中起着重要作用。我们的假设是，AIF是caspase非依赖性心肌细胞凋亡途径的主要介导者，抑制AIF的激活对于实现完整而有效的心脏抗凋亡治疗将是重要的。在特定的目标1中，我们将在体外检测AIF诱导的心脏细胞凋亡的存在和条件。首先，我们将研究已知的导致caspase依赖的细胞凋亡的凋亡刺激是否也激活了AIF。接下来，我们将确定在caspase抑制的设置下，AIF的激活是否增加。在特定的目标2中，我们将在体外研究AIF诱导的心肌细胞凋亡的调控和抑制策略。首先，我们将研究AIF的亚细胞定位与其凋亡特性之间的关系。在这些实验中，我们将通过过度表达针对大鼠AIIF的siRNA序列来产生AIF缺陷的成年心肌细胞，并利用它们来检测重组具有特定亚细胞特性的AIF突变体的效果。然后，我们将确定PARP-1激活是否是心肌细胞释放AIF所必需的。在特定的目标3中，我们将确定AIF诱导的心脏细胞凋亡的作用，并在体内寻找抑制AIF诱导的细胞凋亡的有效策略。我们将首先研究在体小鼠心脏I/R过程中AIF诱导的细胞凋亡的作用。然后，我们将探索通过抑制PARP-1在体内I/R诱导的心脏细胞凋亡中抑制AIF激活的机制和策略。

The long-term goal of this research project is to study the role of AIF in cardiac apoptosis. Although the caspases are thought to be central mediators of the apoptotic program, our preliminary studies have demonstrated that adult cardiomyocytes are both resistant to caspase-dependent apoptosis, and exhibit a significant activation of apoptosis inducing factor (AIF), a prototypical factor involved in caspase-independent apoptosis, under apoptotic stimulation. The role of caspase-independent apoptosis in heart is poorly understood. However, we have found that transgenic mice with cardiac-specific expression of the caspase inhibitor, CrmA, exhibit significant AIF activation after ischemia/reperfusion (I/R). As a result, despite the complete inhibition of caspase activity, there is only a partial inhibition of myocardial damage. However, the concurrent inhibition of PARP-1 inhibits AIF activation and provides more effective cardioprotection after I/R in CrmA mice. These findings suggest that AIF-induced apoptosis may have a significant role in causing cell death in cardiovascular insults. Our hypothesis is that AIF is a principal mediator of caspase-independent pathways in cardiac apoptosis, and that the inhibition of AIF activation will be important for achieving a complete and effective anti-apoptotic therapy in heart. In Specific Aim 1, we will examine the presence of and conditions for AIF-induced cardiac apoptosis in vitro. First, we will investigate whether apoptotic stimuli known to cause caspase-dependent apoptosis also activate AIF. Next, we will determine if the activation of AIF is increased in the setting of caspase inhibition. In Specific Aim 2, we will investigate the regulation of AIF-induced cardiac apoptosis and inhibitory strategies in cardiomyocytes in vitro. First we will examine how the subcellular localization of AIF is related to its apoptotic properties. In these experiments, we will generate AIF deficient adult cardiomyocytes by overexpressing siRNA sequence specific for rat AIIF, and use them to examine the effect of reconstituting AIF mutants that possess specific subcellular characteristics. Then, we will determine whether PARP-1 activation is necessary for AIF release in cardiomyocytes. In Specific Aim 3, we will identify the role of AIF-induced apoptosis in heart, and identify effective strategies for inhibiting AIF-induced apoptosis in vivo. We will first examine the contribution of AIF-induced apoptosis during I/R in mouse heart in vivo. We will then explore a mechanism and a strategy for inhibiting AIF activation using PARP-1 inhibition in I/R-induced cardiac apoptosis in vivo.