Subcellular Regulation of Autophagic Flux in Cardiomyocytes and the Heart

描述(申请人提供)：自噬是包括细胞器在内的细胞质成分被双层膜结构吞噬，并通过与溶酶体融合而被破坏的过程。这一生理过程是正常家务的一部分，是回收蛋白质的一种方式，也是对伤害的一种反应。它可以作为一种机制来防止细胞凋亡，但也可以用来在细胞凋亡被阻断时完成细胞死亡。自噬在心脏对缺血再灌注、肥大和心力衰竭的反应中起着重要作用。我们假设自噬是选择性的，而不是非特异性的，在一种情况下，受损的线粒体可能是自噬的首选目标，而在其他情况下，肌浆网、收缩元件或侵袭体可能是选择性的目标。我们将开发分析细胞器选择性自噬通量的新方法。Beclin1是自噬的关键调控因子，含有一个Bcl2结合域。我们建议研究Bc l-2家族成员在调节自噬中的作用。我们发现，Bcl2对内质网钙库的调节也调节自噬，我们建议更详细地研究这一点。线粒体是自噬的目标，但在被吞噬之前必须经历碎裂。线粒体分裂和融合的调节是通过一小部分蛋白质实现的，这些蛋白质也调节细胞凋亡。我们将检验裂变蛋白调节线粒体自噬的假设。现有的研究体内自噬的工具相当有限。因此，我们将开发更多的体内工具，包括可以通过TAT介导的蛋白质转导引入的自噬调节器。自噬是先天免疫反应的一部分，由细菌脂多糖(LPS)上调。脂蛋白和由此导致的肿瘤坏死因子α的上调已被证明会加剧心力衰竭。我们将检验这一假说，即内毒素介导的自噬上调有助于心力衰竭。自噬对于细胞回收受损的细胞器和胞浆成分是必不可少的，因此可能在心肌梗死后发挥重要的保护作用，但也可能有助于病理重塑。彻底了解心脏中的自噬是很重要的，因为它可能导致新的治疗药物的发展，以改善心功能障碍。公共卫生相关性：该项目将使用细胞系、原代培养的心脏细胞和转基因动物模型来研究自噬在心脏中的作用。自噬是细胞用来清除受损细胞器和蛋白质聚集体的过程。人们对它在心脏中的作用知之甚少。由于感染和炎症增加了自噬并加剧了心力衰竭，我们假设自噬在这种情况下会导致损伤。

DESCRIPTION (provided by applicant): Autophagy is the process whereby cytoplasmic components including organelles are engulfed by a double membrane structure and targeted for destruction by fusion with a lysosome. This physiologic process occurs as part of normal housekeeping, as a way to recycle proteins, and as a response to injury. It can be used as a mechanism to prevent apoptosis but can also be used to accomplish cell death when apoptosis is blocked. Autophagy plays an important role in the heart's response to ischemia and reperfusion, hypertrophy, and heart failure. We hypothesize that autophagy is selective rather than nonspecific, and that damaged mitochondria may be the preferred target for autophagy in one setting while sarcoplasmic reticulum, contractile elements, or aggresomes may be selective targets in others. We will develop new methods to analyze organelle-selective autophagic flux. Beclin1 is a key regulator of autophagy and contains a Bcl-2-binding domain. We propose to examine the role of Bcl-2 family members in regulating autophagy. We show that Bcl-2 regulation of ER calcium stores also modulates autophagy and we propose to investigate this in greater detail. Mitochondria are targets of autophagy but must undergo fragmentation before they can be engulfed. The regulation of mitochondrial fission and fusion is achieved through a small set of proteins that also modulate apoptosis. We will test the hypothesis that fission proteins modulate autophagy of mitochondria. The existing tools for study of autophagy in vivo are rather limited. Accordingly, we will develop additional in vivo tools, including regulators of autophagy that can be introduced via Tat mediated protein transduction. Autophagy is part of the innate immune response and is upregulated by bacterial lipopolysaccharide (LPS). LPS and the resulting upregulation of TNFalpha have been shown to exacerbate heart failure. We will test the hypothesis that LPS mediated upregulation of autophagy contributes to heart failure. Autophagy is essential for cells to recycle damaged organelles and cytoplasmic components and therefore may play an important protective role after myocardial infarction, but may also contribute to pathologic remodeling. It is important to thoroughly understand autophagy in the heart as it may lead to the development of new therapeutic agents for amelioration of cardiac dysfunction. Public Health Relevance: This project will examine the role of autophagy in the heart using cell lines, primary culture heart cells, and transgenic animal models. Autophagy is the process used by cells to eliminate damaged organelles and protein aggregates. Its role in the heart is poorly understood. Since infection and inflammation increase autophagy and exacerbate heart failure, we hypothesis that autophagy contributes to injury in this context.