Chaperone Mediated Autophagy in Normal Cardiac Biology and Heart Failure

心脏自噬的研究几乎完全集中在宏自噬上，其中双自噬作用是一种重要的自噬机制。 膜囊泡将分子和细胞器转运到溶酶体进行降解。相反，主题 在亲本R 01中，存在一个独特的过程，称为分子伴侣介导的自噬（CMA）。在CMA中，细胞质 蛋白质通过Hsc 70和辅助分子伴侣结合的机制被选择性地靶向降解 靶蛋白上的识别基序。这种复合物然后易位到溶酶体，在那里它被输入 LAMP 2A是一种溶酶体跨膜蛋白，对细胞增殖和凋亡是必需的、特异的和限速的。 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，在这方面， 没有方法来评估CMA在健康或患病心脏中的功能意义，直到我们 通过创建可诱导的心肌细胞特异性LAMP 2A敲除小鼠，在心肌细胞中灭活CMA。 虽然这些小鼠在基线时是正常的，但当它们受到应激时， 压力超负荷或心肌梗死后心力衰竭：在这些模型中， 通过抑制CMA而明显减弱-而不是像人们可能从传统作用中预期的那样恶化， 自噬在缓解细胞应激中的作用。机械研究揭示了另一个意想不到的 关系：抑制CMA诱导线粒体自噬，这是一个维持细胞整体健康的过程。 线粒体池通过消除有缺陷的细胞器。基于这些观察，我们提出了一个新的 在这种模式中，CMA响应于心脏应激而被激活，通过消耗 心肌细胞的蛋白质，通常会促进线粒体质量控制通过线粒体自噬。在 本研究拟对该模型进行验证，并进一步阐明其分子机制。目标1： CMA在使用压力超负荷和MI模型的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CMA底物。一个有希望的候选人是线粒体自噬诱导剂，帕金， 我们认为它是CMA底物然而，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，以确定CMA底物， 心肌细胞，随后测试这些蛋白质诱导线粒体自噬的能力。的 本多样性补充中提出的研究解决了同样的问题，但使用了一种新的方法， 在写入父R 01时不可用。这种新方法利用了一个 全基因组CRISPR/Cas9筛选新型线粒体自噬诱导剂，由我们的合作者Dr. 佐尔坦·阿拉尼我们建议使用信息学（多样性补充目标1）和生物化学方法 （多样性补充目标2），以确定CMA底物之间的线粒体自噬诱导剂揭示了 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（Diversity Supplement Aim 3）和完整小鼠抑制CMA时， （补充目标4）。因此，在该多样性补充中提出的实验补充和 加快父母补助金中提出的方法。两者结合起来，将推动我们的 了解这种新的途径，其中应激激活的CMA损害线粒体自噬和线粒体 质量控制导致心力衰竭。

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.