Molecular Control of Cardiomyocyte Mitophagy by the RhoGAP GRAF1

RhoGAP GRAF1 对心肌细胞线粒体自噬的分子控制

• 批准号: 9885284
• 负责人: Joan M Taylor
• 金额: $ 45.29万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-15 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9885284
• 关键词: Actins; Affect; Apoptosis; Autophagocytosis; Autophagosome; Award; Binding; Biogenesis; Cardiac; Cardiac Myocytes; Cardiopulmonary; Cells; Cessation of life; Coupled; Data; Defect; Disease; Exhibits; Extravasation; Foundations; Generations; Genetic; Genomics; Heart; Heart Diseases; Homeostasis; Impairment; Injury; Ischemia; Knockout Mice; Lead; Learning; Link; Lipid Binding; Lipids; Mediating; Membrane; Metabolic; Metabolism; Mitochondria; Modeling; Molecular; Mus; Muscle Cells; Myocardial Infarction; Myocardial Ischemia; Myocardium; Necrosis; Operative Surgical Procedures; Organelles; Outcome; Outer Mitochondrial Membrane; Oxidative Stress; Oxygen; PH Domain; Pathologic; Pathway interactions; Patients; Phospholipids; Phosphorylation; Play; Process; Production; Protein-Serine-Threonine Kinases; Proteins; Quality Control; Reperfusion Therapy; Role; SH3 Domains; Stress; Structure; Time; base; cardiogenesis; cardioprotection; design; experimental study; fatty acid oxidation; fetal; fluorophore; heart cell; loss of function; metabolomics; mitochondrial dysfunction; mitochondrial membrane; novel; novel strategies; novel therapeutics; oxidative damage; parkin gene/protein; polymerization; postnatal; receptor; receptor binding; recruit; response; rho GTPase-activating protein; ubiquitin-protein ligase

Summary Because mitochondrial dysfunction affects ATP production and promotes oxidative damage, these organelles are turned over every 2-3 weeks in healthy cardiomyocytes by a process that involves autophagy. Defects in mitochondrial quality control also enhance the progression of cardiac disease making it critical to identify the mechanisms that regulate this process. Several major pathways have been implicated that involve binding of the cytosolic E3 ubiquitin ligase, Parkin, to the outer mitochondrial membrane followed by the subsequent recruitment of mitochondria into double-membraned autophagosomes. This process is facilitated by the recruitment of the autophagosomal protein, LC3, by LC3-binding receptors that accumulate on damaged mitochondria providing a critical link between the cargo to be degraded and the autophagosome. Although evidence suggests that certain autophagy receptors promote the clearance of specific organelles or organelle components, little is known about the precise LC3-receptors that mediate cardiomyocyte mitophagy. Having previously identified GRAF1 as a critical regulator of cardiac form and function, our current data indicate that GRAF1 plays an important role in regulating cardiomyocyte mitochondrial clearance and metabolism. GRAF1 is expressed at high levels in the heart from E17 onwards and is poised to co-regulate actin- and lipid- dynamics by virtue of its multi-domain structure that includes a lipid binding/bending BAR domain, a phospholipid binding PH domain, a Rho-GAP domain, and a protein-interaction SH3 domain. Importantly, GRAF1 depletion in primary cardiomyocytes led to impaired mitochondrial OXPHOX-mediated ATP generation, mitochondrial membrane depolarization, increased mitochondrial-associated ROS, and increased ischemia/reperfusion-dependent myocyte death. GRAF1 depletion in cultured cardiomyocytes reduced LC3 mediated autophagic flux and led to the accumulation of mitochondria, and we observed similar effects in hearts from genetically modified GRAF1-deficient mice. Mechanistically, we showed that GRAF1 facilitates Parkin-dependent mitophagy by serving as a novel LC3 receptor. Our aims for this award are three-fold: In aim1, we will undertake a step-wise approach using sophisticated pH sensitive fluorophores to identify the precise mechanisms by which GRAF1 regulates cardiomyocyte mitochondrial homeostasis. In aim 2, we will use our newly developed cardiac-restricted GRAF1 knock-out mice to assess GRAF1’s contributions to cardiac metabolic reprogramming and in aim 3 we will use these mice to evaluate a Role of GRAF1-mediated mitophagy in cardioprotection. Results from the experiments proposed herein will provide the scientific foundation for the rational design of strategies to control cardiomyocyte mitophagy and could lead to novel approaches to treat ischemic heart disease.

摘要 由于线粒体功能障碍影响ATP的产生并促进氧化损伤，这些细胞器 在健康的心肌细胞中，每隔2-3周就会通过涉及自噬的过程进行翻转。中的缺陷 线粒体质量控制也促进了心脏病的进展，因此识别 调节这一过程的机制。已经涉及到几个主要的途径，涉及结合 胞浆中的E3泛素连接酶Parkin连接到线粒体膜外膜，随后 线粒体在双膜自噬体内的募集。这一过程是由 自噬蛋白Lc3被累积在损伤表面的Lc3结合受体募集 线粒体在待降解的货物和自噬小体之间提供了关键的联系。虽然 有证据表明，某些自噬受体促进特定细胞器的清除。 对于介导心肌细胞有丝分裂的准确的Lc3受体，我们知之甚少。拥有 以前发现GRAF1是心脏形态和功能的关键调节因子，我们目前的数据表明 GRAF1在调节心肌细胞线粒体清除和代谢方面发挥重要作用。GRAF1 从E17开始在心脏中高水平表达，并准备共同调节肌动蛋白和脂质- 动力学，其多域结构包括脂质结合/弯曲杆域， 磷脂结合的PH结构域、Rho-GAP结构域和蛋白质相互作用SH3结构域。重要的是 原代心肌细胞GRAF1缺失导致线粒体OXPHOX介导的ATP损伤 产生，线粒体膜去极化，增加线粒体相关的ROS，并增加 缺血/再灌流依赖的心肌细胞死亡。培养心肌细胞中GRAF1的耗竭降低了Lc3 介导的自噬通量并导致线粒体的积累，我们在 来自转基因GRAF1缺陷小鼠的心脏。从机制上讲，我们证明了GRAF1促进了 Parkin依赖的有丝分裂吞噬作用作为一种新的LC_3受体。我们的这个奖项有三个目标： AIM 1，我们将采取循序渐进的方法，使用复杂的pH敏感荧光团来识别 GRAF1调节心肌细胞线粒体稳态的精确机制。在目标2中，我们将 利用我们新研制的心脏限制性GRAF1基因敲除小鼠来评估GRAF1对心脏功能的贡献 在目标3中，我们将使用这些小鼠来评估GRAF1介导的作用 有丝分裂在心脏保护中的作用。本文提出的实验结果将提供科学的 为合理设计控制心肌细胞有丝分裂的策略奠定了基础，并可能导致新的 治疗缺血性心脏病的方法。