Chaperone Mediated Autophagy in Normal Cardiac Biology and Heart Failure

正常心脏生物学和心力衰竭中伴侣介导的自噬

• 批准号: 9905205
• 负责人: Richard N Kitsis
• 金额: $ 8.27万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9905205
• 关键词: Address; Adult; Attenuated; Autophagocytosis; Binding; Biochemical; Biology; CRISPR screen; Cardiac; Cardiac Myocytes; Cellular Stress; Complement; Complex; Cytoplasmic Protein; Functional disorder; Funding; Health; Heart; Heart Diseases; Heart failure; Impairment; In Vitro; Informatics; Integral Membrane Protein; Investigation; Knockout Mice; Link; Lysosomes; Mediating; Membrane; Mitochondria; Modeling; Molecular; Molecular Chaperones; Mus; Myocardial Infarction; Myocardial dysfunction; Organelles; Parents; Pathogenesis; Pathway interactions; Phenotype; Process; Proteins; Proteomics; Quality Control; Research; Role; Stress; Systolic heart failure; Testing; Time; Transport Vesicles; Vesicle Transport Pathway; attenuation; base; experimental study; genome-wide; novel; novel strategies; parent grant; parkin gene/protein; pressure; response

Autophagy research in the heart has focused almost exclusively on macroautophagy, in which double membrane vesicles transport molecules and organelles to lysosomes for degradation. In contrast, the subject of the parent R01 is a distinct process termed chaperone mediated autophagy (CMA). In CMA, cytoplasmic proteins are selectively targeted for degradation through a mechanism in which Hsc70 and co-chaperones bind a recognition motif on the target protein. This complex then translocates to the lysosome where it is imported into the lumen by LAMP2A, a lysosomal transmembrane protein that is necessary, specific, and rate-limiting for CMA. Our informatics analyses suggest that there are ~7000 potential CMA substrates in the heart. However, there was no means to assess the functional significance of CMA in healthy or diseased hearts until we inactivated CMA in cardiomyocytes by creating inducible, cardiomyocyte-specific LAMP2A knockout mice. While these mice are normal at baseline, an unanticipated phenotype emerges when they are stressed with pressure overload or post-myocardial infarction heart failure: Systolic dysfunction in each of these models is markedly attenuated by inhibition of CMA – not worsened as one might expect from the traditional role of autophagy in ameliorating cellular stresses. Mechanistic investigations revealed another unexpected relationship: Inhibition of CMA induces mitophagy, a process that maintains the overall health of the mitochondrial pool by eliminating defective organelles. Based on these observations, we proposed a novel paradigm in which CMA, activated in response to cardiac stress, mediates cardiac dysfunction by depleting cardiomyocytes of proteins that would normally promote mitochondrial quality control through mitophagy. In the parent grant, we proposed to test this model and further delineate molecular mechanism. Aim 1 addresses the functional role of CMA in heart failure using both pressure overload and MI models; while Aim 2 seeks to identify CMA substrates that induce mitophagy. One promising candidate is the mitophagy inducer, Parkin, which we believe is a CMA substrate. However, Aim 2 also sought to identify additional candidates using an unbiased approach. This involved first performing lysosomal proteomics to define CMA substrates in cardiomyocytes, following which these proteins would be test for the abilities to induce mitophagy. The research proposed in this diversity supplement addresses the same question, but using a novel approach that was not available at the time the parent R01 was written. This new approach makes use the results of a genome-wide CRISPR/Cas9 screen for novel mitophagy inducers that was conducted by our collaborator Dr. Zoltan Arany. We propose to use informatics (Diversity Supplement Aim 1) and biochemical approaches (Diversity Supplement Aim 2) to identify CMA substrates among the mitophagy inducers revealed by the CRISPR screen. We will then test whether these candidates are responsible for the induction of mitophagy when CMA is inhibited using isolated adult cardiomyocytes (Diversity Supplement Aim 3) and intact mice (Diversity Supplement Aim 4). Thus, the experiments proposed in this diversity supplement complement and accelerate the approach proposed in the parent grant. Taken together, the combination will advance our understanding of this novel pathway in which stress-activated CMA impairs mitophagy and mitochondrial quality control resulting in heart failure.

心脏的自噬研究几乎完全集中在巨自噬上，其中双 膜囊泡将分子和细胞器转运至溶酶体进行降解。相比之下，主题 亲本 R01 的分离是一个称为伴侣介导的自噬 (CMA) 的独特过程。在 CMA 中，细胞质 通过 Hsc70 和共伴侣结合的机制选择性地靶向降解蛋白质 目标蛋白上的识别基序。然后该复合物转移到溶酶体并在那里被导入 LAMP2A 进入管腔，LAMP2A 是一种溶酶体跨膜蛋白，对于 CMA。我们的信息学分析表明心脏中有约 7000 种潜在的 CMA 底物。然而， 在我们研究之前，没有办法评估 CMA 在健康或患病心脏中的功能意义 通过创建可诱导的心肌细胞特异性 LAMP2A 敲除小鼠，使心肌细胞中的 CMA 失活。 虽然这些小鼠在基线时是正常的，但当它们受到压力时会出现意想不到的表型。 压力超负荷或心肌梗塞后心力衰竭：这些模型的收缩功能障碍是 CMA 的抑制显着减弱——并没有像人们预期的传统作用那样恶化 自噬改善细胞应激。机制研究揭示了另一个意想不到的现象 关系：抑制 CMA 会诱导线粒体自噬，这是一个维持细胞整体健康的过程 通过消除有缺陷的细胞器来恢复线粒体池。基于这些观察，我们提出了一种新颖的 CMA 响应心脏应激而激活，通过消耗介导心脏功能障碍的范例 心肌细胞中的蛋白质通常会通过线粒体自噬促进线粒体质量控制。在 在家长资助下，我们建议测试该模型并进一步描绘分子机制。目标1地址 使用压力超负荷和 MI 模型研究 CMA 在心力衰竭中的功能作用；而目标 2 旨在 鉴定诱导线粒体自噬的 CMA 底物。一种有希望的候选者是线粒体自噬诱导剂 Parkin， 我们认为这是 CMA 基材。然而，目标 2 还试图使用 公正的方法。这涉及首先进行溶酶体蛋白质组学来定义 CMA 底物 心肌细胞，随后将测试这些蛋白质诱导线粒体自噬的能力。这 本多样性补充中提出的研究解决了同样的问题，但使用了一种新颖的方法 在编写父 R01 时尚不可用。这种新方法利用了 新型线粒体自噬诱导剂的全基因组 CRISPR/Cas9 筛选由我们的合作者 Dr. 佐尔坦·阿兰尼.我们建议使用信息学（多样性补充目标 1）和生化方法 （多样性补充目标 2）鉴定线粒体自噬诱导剂中的 CMA 底物 CRISPR 筛选。然后我们将测试这些候选者是否负责诱导线粒体自噬 当使用分离的成年心肌细胞（多样性补充目标 3）和完整小鼠抑制 CMA 时 （多样性补充目标 4）。因此，在这种多样性补充中提出的实验补充和 加速母基金中提出的方法。总而言之，这种结合将推动我们的发展 了解应激激活的 CMA 损害线粒体自噬和线粒体的这一新途径 质量控制导致心力衰竭。