Chaperone-Mediated Autophagy in Normal Cardiac Biology and Heart Failure

心血管系统中的自噬研究几乎完全集中在宏观自噬上， 双膜囊泡将分子和细胞器输送到溶酶体进行降解。在……里面 相比之下，这个项目的主题是一个独特的过程，称为伴侣介导的自噬(CMA)。在……里面 CMA，细胞质蛋白选择性地被降解，其机制是Hsc70和Hsc70 辅助伴侣结合了目标蛋白上的识别基序。然后这个复合体移位到溶酶体上 当它被LAMP2A(L2a)输入管腔时，LAMP2A(L2a)是一种必需的溶酶体跨膜蛋白， CMA的特定和速率限制。我们的信息学分析表明，有大约7000个潜在的CMA 心脏中的底物，这些蛋白质中的很大一部分是动态调节的。然而， 直到现在才能评估CMA在健康或患病心脏中的功能意义。 最近，当我们用可诱导的、心肌细胞特异性的LAMP2A(ICS-)基因敲除小鼠时， L2AKO)。虽然这些小鼠在基线状态下是正常的，但当它们出现时，会出现意想不到的表型 压力超负荷或心肌梗死后心力衰竭：每一位患者的收缩功能障碍 这些模型通过对CMA的抑制而减弱，而不是像传统角色所预期的那样恶化 自噬在改善细胞压力方面的作用。机械调查揭示了另一个意想不到的 关系：抑制CMA诱导有丝分裂，这是一个维持整体健康的过程 通过消除有缺陷的细胞器来建立线粒体池。我们提出了一个新的范式，在这个范式中， 在心脏应激反应中被激活，通过耗尽心肌细胞来介导心脏功能障碍 正常情况下会通过有丝分裂促进线粒体质量控制的蛋白质。我们将对此进行测试 模拟和描绘CMA激活和抑制有丝分裂的分子机制。目标1， 将使用压力过载和心肌梗死模型来定义CMA在心力衰竭中的功能作用。这些 研究将使用多种创新试剂，包括可诱导的、心肌细胞特异性的小鼠模型 新开发的CMA小分子激活剂L2a缺失和过表达及新的CMA 记者老鼠。我们还将定义CMA和心脏巨噬细胞之间的功能关系。 目标2将确定介导CMA抑制有丝分裂吞噬的分子。我们将调查一个角色 一个强有力的候选者：吞丝分裂激活剂Parkin，我们的研究表明它是CMA底物。此外, 为了识别新的介体，我们提出了一种结合溶酶体蛋白质组学的无偏见的方法(以识别 直接CMA底物)和RNA-seq(以识别CMA间接调节的介体)。建议数 实验具有非常重要的意义和创新性，因为它们将提供对 CMA在心脏中的作用，并定义了一种新的心力衰竭途径，该途径由先前未知的 CMA与有丝分裂之间的联系。

Autophagy research in the cardiovascular system has focused almost exclusively on macroautophagy, in which double-membrane vesicles transport molecules and organelles to lysosomes for degradation. In contrast, the subject of this project 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 (L2A), 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, and a significant proportion of these proteins are dynamically regulated. However, there has been no means to assess the functional significance of CMA in healthy or diseased hearts until recently, when we generated mice with an inducible, cardiomyocyte-specific knockout of LAMP2A (iCS- L2AKO). 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 attenuated by inhibition of CMA – not worsened, as might be expected 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. We propose a new 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. We will test this model and delineate molecular mechanisms that link activation of CMA with suppression of mitophagy. Aim 1, will define the functional role of CMA in heart failure, using both pressure overload and MI models. These studies will employ multiple innovative reagents including mouse models of inducible, cardiomyocyte-specific L2A deletion and overexpression, a recently developed small molecule activator of CMA, and a new CMA reporter mouse. We will also define functional relationships between CMA and macroautophagy in the heart. Aim 2 will identify molecules that mediate the suppression of mitophagy by CMA. We will investigate a role for a strong candidate: the mitophagy activator Parkin, which our studies suggest is a CMA substrate. In addition, to identify novel mediators, we propose an unbiased approach that combines lysosomal proteomics (to identify direct CMA substrates) and RNA-seq (to identify mediators regulated indirectly by CMA). The proposed experiments are highly significant and innovative in that they will provide the first assessment of the role of CMA in the heart and define a novel heart failure pathway that is mediated by previously unrecognized connections between CMA and mitophagy.