Linking cell death and mitochondrial quality control mechanisms in heart disease

 描述(申请人提供)：线粒体参与多种基本过程，包括生物发生、分裂/融合、有丝分裂和细胞死亡-除了它们在细胞中作为ATP生成器的作用。这些过程是复杂的，因此在很大程度上是孤立地研究的。然而，很明显，它们在分子水平上、在功能上以及在疾病中的作用方面基本上是重叠的。在这一应用中，我们提出了双向连接Bax和Parkin的实验，Bax是心肌细胞死亡的中心介质，Parkin是心肌细胞有丝分裂的重要激活剂。利用基因操作，我们出人意料地发现，健康细胞中内源性水平的Bax抑制了有丝分裂。我们已经在体外成年心肌细胞和活体心脏中观察到了这一点。我们推测，这是一种抑制线粒体不适当或过度消除的生理机制，这种情况对高度依赖ATP的心肌细胞是有害的。我们将阐明其机制，其中可能涉及Bax和Parkin之间的相互作用，并在体外和体内测试其功能意义。巴克斯和帕金的互动引发了一个对等的问题：帕金可能会在内心对巴克斯做什么？先前在非心脏系统中的研究表明，Parkin在细胞死亡过程中抑制Bax向线粒体的移位，但其机制尚未阐明。已知Parkin在体外可以抑制心肌细胞的死亡，而体内Parkin的缺失显著加剧了梗死后的重塑、心力衰竭和死亡率。这些效应被认为完全是通过帕金刺激的有丝分裂来调节的。然而，鉴于Bax与心肌细胞凋亡和坏死之间的直接联系，我们对这种观点和Parkin主要通过抑制Bax来保护心脏的假设提出了质疑。我们将验证这一假说，并描述Parkin抑制Bax的分子机制。因此，该项目的总体假设是，Bax和Parkin为了心肌细胞的利益而相互对抗：Bax与Parkin的结合可能是在细胞死亡不活跃的构象中，抑制Parkin介导的有丝分裂，以防止线粒体枯竭。相反，Bax-Parkin相互作用抑制Bax，可能是通过干扰Bax向其活性构象的转变，从而抑制心肌细胞的凋亡和坏死。这是一个MPI应用程序，汇集了Richard Kitsis(心肌细胞死亡)和Gerald Dorn(心脏有丝分裂症)的专业知识。这项研究具有很高的意义，因为它将刺激该领域 考虑和研究个体线粒体过程之间的联系，如细胞死亡和有丝分裂。此外，这将导致对帕金保护心脏的主要机制的重新考虑。这项工作也具有很高的创新性，因为大多数概念和关系以前都没有考虑过。通过这项研究，我们将更好地了解急性心肌缺血/再灌注损伤的发病机制和梗死后重构。

 DESCRIPTION (provided by applicant): Mitochondria engage in multiple fundamental processes including biogenesis, fission/fusion, mitophagy, and cell death - in addition to their role as ATP generators in the cell. These processes are complex, and for this reason, have been largely studied in isolation. Yet, it is clear that they overlap substantially at the molecula level, functionally, and with respect to their roles in disease. In this application, we propose experiments that bi-directionally connect Bax, a central mediator of cardiomyocyte death, with Parkin, an important activator of mitophagy in cardiomyocytes. Using genetic manipulations, we have found unexpectedly that endogenous levels of Bax in healthy cells suppress mitophagy. We have observed this in adult cardiomyocytes in vitro and in the heart in vivo. We postulate that this is a physiological mechanism that restrains inappropriate or excessive elimination of mitochondria, a situation that would be deleterious to cardiomyocytes, which are highly dependent on ATP. We will elucidate the mechanism, which may involve an interaction between Bax and Parkin, and test the functional significance in vitro and in vivo. The fact that Bax and Parkin interact raises the reciprocal question: What might Parkin be doing to Bax in the heart? Previous work in non-cardiac systems has shown that Parkin inhibits Bax translocation to mitochondria during cell death, but the mechanism has not been elucidated. Parkin is known to inhibit cardiomyocyte death in vitro, and deletion of Parkin in vivo markedly exacerbates post-infarct remodeling, heart failure, and mortality. These effects have been assumed to be mediated solely through Parkin-stimulated mitophagy. Given the directness of the connection between Bax and cardiomyocyte apoptosis and necrosis, however, we challenge that view and hypothesize that Parkin protects the heart primarily through its inhibition of Bax. We will test ths hypothesis and delineate the molecular mechanism by which Parkin inhibits Bax. Accordingly, the over-arching hypothesis of this project is that Bax and Parkin antagonize each other for the benefit of cardiomyocytes: The binding of Bax, likely in its cell death-inactive conformation, to Parkin inhibits Parkin-mediated mitophagy to prevent mitochondrial depletion. Conversely, the Bax-Parkin interaction inhibits Bax, likely through interfering with its transition to its active conformation, thereby inhibiting cardiomyocyte apoptosis and necrosis. This is an MPI application that brings together the expertise of Richard Kitsis (cardiomyocyte death) and Gerald Dorn (cardiac mitophagy). This research is highly significant as it will stimulate the field to consider and investigate connections between individual mitochondrial processes, such as cell death and mitophagy. In addition, it will lead to a reconsideration of the primary mechanism by which Parkin protects the heart. The work is also highly innovative because most of the concepts and relationships have not been previously considered. Through this research, we will achieve a better understanding of the pathogenesis of acute cardiac injury during ischemia/reperfusion and post-infarct remodeling.