Removal of damaged mitochondria by alternative autophagy

自噬是受损线粒体降解的主要机制。没有消除损坏的 线粒体、去极化线粒体和活性氧(ROS)迅速影响健康 线粒体，导致广泛的线粒体功能障碍和细胞死亡。了解损害程度 去除线粒体将为获得更健康的心肌细胞线粒体(CMS)和 开发治疗心力衰竭的新疗法。尽管人们认为受损的线粒体会被降解 主要通过PINK1-Parkin介导的有丝分裂，我们发现CMS通过一种 ATG7非依赖和Ulk1依赖的自噬形式，与“替代”自噬同源 Nishida之前报道过，这种形式的自噬在消除 饥饿导致线粒体受损。然而，无论是分子机制还是功能 在CMS中，通过替代自噬介导的有丝分裂吞噬的意义已经明确。 因此，该项目的目标是证明替代自噬的功能意义 清除心脏中受损的线粒体以应对相关压力并阐明潜在的 分子机制。假设1：作为对心肌缺血的反应，心脏激活ATG7- 独立/Ulk1依赖的替代自噬，它在调节 破坏线粒体，保护心脏免受心肌缺血的影响。假设2：Ulk1被磷酸化 At Ser555作为支架诱导Rab9相互作用形成自噬小体并使其磷酸化 线粒体分裂基因Ser616上的Drp1，这两个基因在介导线粒体自噬中都起着重要作用。 对心肌缺血的反应。我们将：目标1：证明ATG7独立和ulk1依赖 另一种自噬由心肌缺血激活。评估替代自噬是否有保护作用 心肌缺血期间的心脏。为此，我们将使用心脏特异的ATG7和ulk1基因敲除小鼠， 电子显微镜，交替自噬和线粒体自噬的特异性报告，以及功能 线粒体的分析。我们将展示受损的线粒体主要通过替代方式降解 心肌缺血时的自噬。目的2：评估Ulk1在Ser555位的磷酸化是否在 在心肌缺血时介导选择性自噬和心脏保护中的重要作用 刺激与Rab9和Ser616磷酸化的Drp1的相互作用。为此，我们将使用功能损失 和敲入小鼠模型以及替代自噬和溶酶体的独特而可靠的记者 线粒体的降解。从这一目标获得的知识应该导致制定具体的 在心肌缺血期间调节有丝分裂吞噬的干预。总之，我们的研究将证明 替代自噬是线粒体降解的一种新的和主要的机制，这是必不可少的 用于在缺血期间维持心脏线粒体的质量。

Autophagy is a major mechanism of degradation of damaged mitochondria. Without elimination of damaged mitochondria, depolarized mitochondria and reactive oxygen species (ROS) rapidly affect healthy mitochondria, leading to wide-spread mitochondrial dysfunction and cell death. Understanding how damaged mitochondria are removed will provide a key to achieving healthier mitochondria in cardiomyocytes (CMs) and developing novel treatments for heart failure. Although it is believed that damaged mitochondria are degraded primarily by Pink1-Parkin-mediated mitophagy, we have discovered that CMs degrade mitochondria through an Atg7-independent and Ulk1-dependent form of autophagy that is homologous to the “alternative” autophagy previously reported by Nishida, and that this form of autophagy plays a significant role in the elimination of damaged mitochondria in response to starvation. However, neither the molecular mechanism nor the functional significance of mitophagy mediated through alternative autophagy has been clearly established in CMs yet. Thus, the goal of this project is to demonstrate the functional significance of alternative autophagy in eliminating damaged mitochondria in the heart in response to relevant stresses and to elucidate the underlying molecular mechanisms. Hypothesis 1: In response to myocardial ischemia, the heart activates Atg7- independent/Ulk1-dependent alternative autophagy, which plays an essential role in mediating the clearance of damaged mitochondria and protects the heart from myocardial ischemia. Hypothesis 2: Ulk1 phosphorylated at Ser555 acts as a scaffold to induce Rab9 interaction for autophagosome formation and phosphorylation of Drp1 at Ser616 for mitochondrial fission, both of which are important in mediating mitochondrial autophagy in response to myocardial ischemia. We will: Aim 1: Demonstrate that the atg7-independent and ulk1-dependent alternative autophagy is activated by myocardial ischemia. Evaluate whether alternative autophagy protects the heart during myocardial ischemia. To this end, we will use cardiac-specific atg7- and ulk1-knockout mice, electron microscopy, specific reporters of alternative autophagy and mitochondrial autophagy, and functional analyses of mitochondria. We will show that damaged mitochondria are degraded primarily through alternative autophagy during myocardial ischemia. Aim 2: Evaluate whether phosphorylation of Ulk1 at Ser555 plays an essential role in mediating alternative autophagy and cardioprotection during myocardial ischemia by stimulating interaction with Rab9 and Ser616-phosphorylated Drp1. To this end, we will use loss-of-function and knock-in mouse models and unique and reliable reporters for alternative autophagy and lysosomal degradation of mitochondria. The knowledge obtained from this aim should lead to development of specific interventions to modulate mitophagy during myocardial ischemia. In summary, our study will demonstrate that alternative autophagy is a novel and predominant mechanism of mitochondrial degradation, which is essential for the maintenance of mitochondrial quality in the heart during ischemia.