MCL-1 is a critical regulator of mitochondrial dynamics and function in myocytes

项目摘要 虽然线粒体功能障碍在衰竭的心脏中很明显，但它在疾病进展中的确切作用尚不清楚。 而其产生的机制(S)也不甚清楚。线粒体在许多细胞过程中起着关键作用， 包括氧化磷酸化、代谢物合成和钙储存。它们也很重要 调节细胞死亡，并监测细胞内环境的变化，如活性氧的存在 物种和DNA损伤。Bcl2蛋白在线粒体膜调节中起关键作用 通透性和细胞凋亡。我们最近发现，抗凋亡的bcl2蛋白mcl1是至关重要的 以维持正常的线粒体功能和心脏内稳态。心肌细胞中MCL-1的丢失导致快速 线粒体功能障碍、心力衰竭的发展和早期死亡。令人惊讶的是，MCL-1缺乏 心肌细胞显示出坏死的迹象，而不是像预期的那样出现凋亡，这表明除了它的抗 MCL-1在维持心肌线粒体功能中起着重要但尚未确定的作用 肌细胞。我们还发现MCL-1既存在于线粒体膜外膜(MCL-1OM)，也存在于 心脏中的线粒体基质(MCL-1基质)。虽然一项研究表明MCL-1OM参与调控细胞凋亡， MCL-1Matrix的功能目前尚不清楚。基于我们的初步数据，我们建议研究这一假说 MCL-1在维持心肌线粒体功能和健康方面具有双重作用，这依赖于其 线粒体定位：MCL-1OM促进线粒体分裂和线粒体有丝分裂 阻止激活不必要的细胞凋亡，而MCL-1Matrix促进线粒体融合以保存 在营养限制条件下，生物能量能力和防止自噬降解。这 假设将通过三个具体目标进行检验。在具体目标1中，我们将描述MCL-1矩阵的作用 调节线粒体的融合、功能、周转和存活。在具体目标2中，我们将界定 MCL-1OM在调控线粒体分裂和翻转中的作用我们将确定MCL-1OM是否与DRP1相互作用以 促进受损线粒体的不对称分裂和移除，以及这是否是其促进生存的一部分 功能。最后，在特定的目标#3中，我们将调查MCL-1OM是否也作为LC3的受体发挥作用 驱动自噬小体选择性地降解线粒体。我们将利用两个分离的心肌细胞 转基因小鼠模型与蛋白质组学、细胞和分子生物学相结合，揭示了 MCL-1OM和MCL-1Matrix在基础状态和应激反应中的作用 (空腹和心肌梗死)。这些研究将为了解两国关系提供重要的新见解。 线粒体动力学、心脏周转和存活之间的关系。更好地了解线粒体如何 在正常情况下，心脏的功能是受调节的，心肌梗死等疾病也会影响心脏功能。 展望未来心脏病的临床治疗。

Project Summary While mitochondrial dysfunction is evident in the failing heart, its precise role in disease progression is unclear and the mechanism(s) of its origin is not well understood. Mitochondria play a key role in many cellular processes, including oxidative phosphorylation, metabolite synthesis and calcium storage. They are also important regulators of cell death and monitor changes in the intracellular environment such as presence of reactive oxygen species and DNA damage. The BCL-2 proteins play a key role in regulating mitochondrial membrane permeabilization and apoptosis. We recently discovered that the anti-apoptotic BCL-2 protein MCL-1 is critical for normal mitochondrial function and cardiac homeostasis. Loss of MCL-1 in cardiac myocytes leads to rapid mitochondrial dysfunction, development of heart failure, and early mortality. Surprisingly, MCL-1 deficient myocytes display signs of necrosis rather than apoptosis as would be expected, suggesting that besides its anti- apoptotic role, MCL-1 has an essential but yet unidentified role in maintaining mitochondrial function in cardiac myocytes. We have also found that MCL-1 exists both in the outer mitochondrial membrane (MCL-1OM) and in the mitochondrial matrix (MCL-1Matrix) in the heart. While a study has implicated MCL-1OM in regulating apoptosis, the function of MCL-1Matrix is still unknown. Based on our preliminary data, we propose to study the hypothesis that MCL-1 has a dual role in maintaining cardiac mitochondrial function and health that is dependent on its mitochondrial location: MCL-1OM facilitates mitochondrial fission and mitophagy of aberrant mitochondria to prevent activation of unnecessary apoptosis, whereas MCL-1Matrix promotes mitochondrial fusion to preserve bioenergetic capacity and protect against autophagosomal degradation during nutrient limiting conditions. This hypothesis will be tested with three specific aims. In Specific Aim 1, we will characterize the role of MCL-1Matrix in regulating mitochondrial fusion, function, turnover and survival. In Specific Aim 2, we will delineate the role of MCL-1OM in regulating mitochondrial fission and turnover. We will determine if MCL-1OM interacts with Drp1 to promote asymmetrical fission and removal of damaged mitochondria and whether this is part of its pro-survival function. Finally, in Specific Aim #3, we will investigate whether MCL-1OM also functions as a receptor for LC3 to drive selective degradation of mitochondrial by autophagosomes. We will utilize both isolated cardiac myocytes and genetically modified mouse models combined with proteomics, cell and molecular biology to uncover the bi- functional roles of MCL-1OM and MCL-1Matrix in myocytes under baseline conditions and in response to challenge (fasting and myocardial infarction). These studies will provide important new insights into the relationship between mitochondrial dynamics, turnover and survival in the heart. A better understanding of how mitochondrial function is regulated in the heart under normal and disease conditions such as myocardial infarct will contribute towards future clinical management of heart disease.