Molecular Control of Cardiomyocyte Mitophagy by the RhoGAP GRAF1

总结 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2-3周通过一个涉及自噬的过程被翻转。缺陷 线粒体质量控制也促进了心脏病的进展，这使得识别线粒体质量控制变得至关重要。 调节这一过程的机制。几个主要的途径已经涉及到的结合， 细胞溶质E3泛素连接酶，帕金，到外线粒体膜，随后 线粒体进入双膜自噬体的募集。这一过程由 自噬体蛋白LC 3通过LC 3结合受体的募集， 线粒体在待降解的货物和自噬体之间提供关键联系。虽然 有证据表明，某些自噬受体促进特定细胞器或细胞器的清除， 由于LC 3受体是心肌细胞的重要组成部分，因此对介导心肌细胞线粒体自噬的LC 3受体的精确性知之甚少。具有 我们先前将GRAF 1鉴定为心脏形态和功能的关键调节因子，目前的数据表明， GRAF 1在调节心肌细胞线粒体清除和代谢中起重要作用。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， 动力学凭借其多结构域结构，包括脂质结合/弯曲BAR结构域， 磷脂结合PH结构域、Rho-GAP结构域和蛋白质相互作用SH 3结构域。重要的是， 原代心肌细胞中GRAF 1缺失导致线粒体OXPHOX介导的ATP受损 产生，线粒体膜去极化，增加的线粒体相关的ROS，并增加 缺血/再灌注依赖性心肌细胞死亡。在培养的心肌细胞中，GRAF 1耗竭降低了LC 3 介导的自噬通量，并导致线粒体的积累，我们观察到类似的效果， 来自基因改造的GRAF 1缺陷小鼠的心脏。从机制上讲，我们表明GRAF 1促进了 作为一种新的LC 3受体的帕金森依赖性线粒体自噬。我们对这个奖项的目标有三个方面： aim 1，我们将采取一个逐步的方法，使用复杂的pH敏感的荧光团，以确定 GRAF 1调节心肌细胞线粒体稳态的精确机制。在目标2中，我们将 使用我们新开发的心脏限制性GRAF 1基因敲除小鼠来评估GRAF 1对心脏功能的贡献。 代谢重编程，在目标3中，我们将使用这些小鼠来评估GRAF 1介导的作用 线粒体自噬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线粒体自噬的策略奠定了基础，并可能导致新的 治疗缺血性心脏病的方法。

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.