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证实，大多数心肌细胞 再灌注心肌梗死(MI/R)期间的死亡是通过调节细胞死亡程序发生的。这表明它应该是 在概念上可能限制MI/R期间的心肌细胞死亡，以在维持中获得再灌注的全部好处 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 由几个受调控的凋亡和坏死程序以及这些程序之间的联系机制所介导 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一个 限制MI/R期间心脏损害的合理治疗设计的关键障碍。 我们实验室的主要任务是了解连接MI/R细胞死亡程序的分子框架。 初步研究已确定caspase-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Proaspase-3和-7。 然而，我们已经发现了一条新的途径，即caspase-9在MI/R期间介导心肌细胞坏死。 与caspase-9介导的细胞凋亡相比，caspase-3/7在caspase-9介导的坏死中是必不可少的。 这表明了这两条路径的区别。然而，正如在细胞凋亡中一样，caspase-9的酶活性是 必填项。为了进一步剖析这一途径，我们进行了基于蛋白质组学的Proaspase-9相互作用筛查 MI/R期间心脏中的蛋白质，这揭示了已知的与坏死的各个方面有关的蛋白质。 到目前为止，我们主要关注一个proaspase-9交互作用因子，SERCA2a，我们假设它的功能 位于caspase-9下游，介导细胞内源性杀伤。我们的数据表明caspase-9裂解SERCA2a 在MI/R失活期间，其功能与先前的遗传学研究一致，表明SERCA2a的丢失加剧了 心肌细胞坏死和梗塞面积。然而，我们认为Proaspase-9和Proaspase-9的关系 SERCA2a更为复杂。除了caspase-9导致SERC2a丢失外，我们的数据还表明SERCA2a 丢失可能通过钙超载导致caspase-9的激活。因此，我们假设一个双向的 Caspase-9和SERCA2a之间存在相互促进的关系，并对心肌细胞有贡献 MI/R中的坏死和梗塞的产生。本项目研究caspase-9介导的关键方面。 坏死途径包括激活机制、下游信号、途径与细胞的关系 被认为与MI/R有关的死亡程序，以及这一途径是否可以被治疗抑制以限制 心肌梗死面积。目的1.对心肌缺血再灌注过程中caspase-9坏死轴进行遗传学解剖。目标2.描绘 Caspase-9和SERCA2a在MI/R心肌细胞坏死中的双向调节 评价应用细胞通透性多肽特异性抑制心肌梗死/再灌注期间原天冬氨酸酶-9的疗效 源自一种内源性前天冬氨酸酶-9抑制剂。这些研究定义了一种新的细胞死亡途径 在MI/R中显得重要，并可能为限制梗塞范围的新的治疗方法提供基础。

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.