Caspase-9 as a nodal point connecting necrotic and apoptotic cell death in myocardial infarction

心肌梗死的主要心脏事件是细胞死亡。对小鼠的遗传学研究已经证实，大多数心肌细胞 再灌注MI（MI/R）期间的死亡通过调节的细胞死亡程序发生。这表明， 在概念上可能限制MI/R期间心肌细胞死亡，以获得再灌注在维持 心脏功能然而，实现这一目标的一个重要障碍是，MI/R中的心肌细胞死亡是心肌缺血的一个重要因素。 由几个受调节的凋亡和坏死程序介导，以及连接这些程序的机制 产生一个完整的细胞死亡反应仍然知之甚少。这种知识上的差距一直是 限制MI/R期间心脏损伤的合理治疗设计的关键障碍。因此，一个目标 我们实验室的目标是了解MI/R中细胞死亡程序的分子框架。我们 初步的研究已经确定半胱天冬酶-9是细胞凋亡和坏死程序之间的重要联系 在MI/R期间。caspase-9在细胞凋亡中的典型作用是激活下游的caspase-3和-7。 然而，我们已经确定了一个新的途径，其中caspase-9介导心肌梗死/再灌注期间心肌细胞坏死。 与caspase-9介导的凋亡相反，caspase-3/7不参与caspase-9介导的坏死 这表明了两种途径的不同。然而，在细胞凋亡中，半胱天冬酶-9的酶活性被抑制。 必需的.为了进一步剖析该途径，我们进行了基于蛋白质组学的procaspase-9相互作用的筛选。 在MI/R期间心脏中的蛋白质，这揭示了已知参与坏死的各个方面的蛋白质。 到目前为止，我们已经集中在一个procaspase-9相互作用，SERCA 2a，我们假设功能 caspase-9的下游以介导细胞内在杀伤。我们的数据表明caspase-9切割SERCA 2a 在MI/R期间使其功能丧失，这与先前的遗传研究一致，表明SERCA 2a丢失加剧了心肌梗死/再灌注损伤。 心肌细胞坏死和梗死面积。然而，我们认为，半胱天冬酶原9和 SERCA 2a更复杂。除了caspase-9诱导SERC 2a丢失外，我们的数据表明SERCA 2a 丢失可能通过Ca 2+超载导致caspase-9活化。因此，我们假设一个双向的 caspase-9和SERCA 2a之间存在相互加强的关系，并有助于心肌细胞 坏死和梗死的发生。本项目研究caspase-9介导的 坏死通路包括激活机制、下游信号传导、通路与细胞的关系 死亡程序被认为与MI/R有关，以及是否可以在治疗上抑制该途径以限制 梗死面积。目标1。探讨心肌梗死/再灌注（MI/R）过程中caspase-9坏死轴的基因表达。目标二。划定 caspase-9和SERCA 2A在心肌梗死/再灌注心肌细胞坏死中的双向调节作用。目标3。到 评估在MI/R期间使用细胞渗透性肽特异性抑制半胱天冬酶原-9的治疗益处 来源于内源性半胱天冬酶原-9抑制剂。这些研究定义了一种新的细胞死亡途径， 在MI/R中似乎很重要，并可能为限制梗死面积的新治疗方法提供基础。

The primary cardiac event in MI is cell death. Genetic studies in mice have established that most cardiomyocyte death during reperfused MI (MI/R) occurs through regulated cell death programs. This suggests that it should be possible in concept to limit cardiomyocyte death during MI/R to obtain the full benefit of reperfusion in maintaining cardiac function. A significant obstacle in achieving this goal, however, is that cardiomyocyte death in MI/R is mediated by several regulated apoptosis and necrosis programs, and the mechanisms that link these programs to produce an integrated cell death response remain poorly understood. This gap in knowledge has been a critical impediment for the rational design of therapies to limit cardiac damage during MI/R. Accordingly, a goal of our lab has been to understand the molecular framework that connects cell death programs in MI/R. Our preliminary studies have identified caspase-9 as an important link between apoptosis and necrosis programs during MI/R. The canonical role of caspase-9 in apoptosis is to activate downstream procaspases-3 and -7. However, we have identified a new pathway in which caspase-9 mediates cardiomyocyte necrosis during MI/R. In contrast to caspase-9-mediated apoptosis, caspases-3/7 are dispensable for caspase-9-mediated necrosis indicating the distinctness of the two pathways. However, as in apoptosis, caspase-9 enzymatic activity is required. To further dissect the pathway, we performed a proteomics-based screen for procaspase-9 interacting proteins in the heart during MI/R, which revealed proteins known to be involved in various aspects of necrosis. Thus far, we have focused on one procaspase-9 interactor, SERCA2a, which we hypothesize functions downstream of caspase-9 to mediate cell-intrinsic killing. Our data suggest that caspase-9 cleaves SERCA2a during MI/R disabling its function consistent with prior genetic studies showing that SERCA2a loss exacerbates cardiomyocyte necrosis and infarct size. We believe, however, that the relationship between procaspase-9 and SERCA2a is more complex. In addition to caspase-9 inducing SERC2a loss, our data suggest that SERCA2a loss may contribute to caspase-9 activation through Ca2+ overload. Thus, we hypothesize that a bidirectional mutually-reinforcing relationship exists between caspase-9 and SERCA2a and contributes to cardiomyocyte necrosis and infarct generation in MI/R. This project investigates critical aspects of the caspase-9-mediated necrosis pathway including activation mechanisms, downstream signaling, the relationship of the pathway to cell death programs thought to be involved in MI/R, and whether this pathway can be therapeutically inhibited to limit infarct size. Aim 1. To genetically dissect the caspase-9 necrosis axis during MI/R in vivo. Aim 2. To delineate the bidirectional regulation between caspase-9 and SERCA2A in cardiomyocyte necrosis during MI/R. Aim 3. To assess the therapeutic benefit of specifically inhibiting procaspase-9 during MI/R using a cell permeable peptide derived from an endogenous procaspase-9 inhibitor. These studies define a novel cell death pathway that appears important in MI/R and may provide the basis for a new therapeutic approach to limit infarct size.