Rab GTPases-mediated mitochondrial clearance in diabetic cardiomyopathy

项目摘要 糖尿病性心肌病是一种常见但被低估的心力衰竭和死亡的患者， 糖尿病尽管多种途径汇聚，但这种通常致命的综合征的潜在基础尚不清楚 一个共同点功能失调的线粒体了解线粒体如何受损 去除是至关重要的，因为受损线粒体的存在导致活性氧的快速产生 然后影响剩余的健康线粒体。这导致了广泛的线粒体 功能障碍和细胞死亡。虽然受损的线粒体被认为是由线粒体降解， 自噬（自噬的一种特殊形式），我们最近发现了一种新的内体的存在， 介导的线粒体降解途径。制止 该途径可能与糖尿病心肌病易感性增加有关。该观察结果 由于我们能够从糖尿病患者中产生患者特异性诱导多能干细胞（iPSC）， 有（T2 DCM）和无（T2 D）心肌病的患者。我们发现只有T2 D细胞，而不是T2 DCM， 表现出增加的内体介导的线粒体降解。然而，无论是分子线索， 调节这种新途径，也不是这种内体途径的功能意义，是建立在 糖尿病心脏的心肌细胞。因此，本项目的目标是展示功能的重要性 内体介导的线粒体降解途径在糖尿病心肌病，并阐明 调节这一途径的潜在机制。目的1将定义内体介导的 通过破坏Rab 5和Rab 7的功能，糖尿病心脏中的线粒体清除，Rab 5和Rab 7是糖尿病心脏的关键决定因素。 内溶酶体系统这些研究将采用多种创新试剂，包括CRISPR介导的 Rab敲除和过度激活iPSC系和一种新的可诱导的心脏特异性Rab 7敲除小鼠模型， 为了探究这些Rab在维持两种不同的线粒体功能性降解中的重要性， 糖尿病小鼠模型。目的2将定义VPS 34/UVRAG在产生磷脂酰肌醇3-磷酸化中的作用。 内体成熟所需的磷酸盐（PI 3 P），因此清除有缺陷的线粒体。我们有 支持mTOR在选定的糖尿病患者中过度上调导致磷酸化的数据 UVRAG，削弱其与VPS 34形成复合物的功能，产生PI 3 P，用于适当的内体 成熟和线粒体降解。利用遗传学和药理学方法，我们将 证明维持稳定的UVRAG/VPS 34复合物是PI 3 P介导转化的先决条件 将Rab 5转化为Rab 7用于内体介导的线粒体降解。总的来说，这些创新研究将 阐明一种新内体介导的线粒体降解途径作为重要的适应性反应 当暴露于高血糖应激时，心肌细胞中的胰岛素水平，并为未来的药物靶点铺平道路。

PROJECT SUMMARY Diabetic cardiomyopathy is a common yet underestimated cause of heart failure and mortality in patients with diabetes. The underlying basis of this often-fatal syndrome is unknown, although multiple pathways converge on a common denominator - dysfunctional mitochondria. Understanding how damaged mitochondria are removed is critical, as the presence of damaged mitochondria leads to rapid generation of reactive oxygen species which then affect the remaining healthy mitochondria. This leads to wide-spread mitochondrial dysfunction and cell death. Although damaged mitochondria are believed to be degraded by mitochondrial autophagy (a specific form of autophagy), we have recently discovered the existence of a novel endosomal- mediated mitochondrial degradation pathway in cardiomyocytes exposed to hyperglycemia. The suppression of this pathway is potentially linked to increased susceptibility against diabetic cardiomyopathy. This observation was possible due to our ability to generate patient-specific induced pluripotent stem cells (iPSCs) from diabetic patients with (T2DCM) and without (T2D) cardiomyopathy. We found that only T2D cells, but not T2DCM, exhibited increased endosomal-mediated mitochondrial degradation. However, neither the molecular cues regulating this novel pathway, nor the functional significance of this endosomal pathway, is established in cardiomyocytes from a diabetic heart. Thus, the goal of this project is to demonstrate the functional significance of the endosomal-mediated mitochondrial degradation pathway during diabetic cardiomyopathy and to elucidate the underlying mechanisms regulating this pathway. Aim 1 will define the functional role of endosomal-mediated mitochondrial clearance in the diabetic heart by disrupting the function of Rab5 and Rab7, key determinants of the endo-lysosomal system. These studies will employ multiple innovative reagents, including CRISPR-mediated Rab knockout and overactivation iPSC lines and a novel inducible, cardiac-specific Rab7 knockout mouse model, to interrogate the importance of these Rabs in maintaining functional mitochondrial degradation in two distinct diabetic mouse models. Aim 2 will define the role of VPS34/UVRAG in generating phosphatidylinositol 3- phosphate (PI3P) required for endosomal maturation and hence clearance of defective mitochondria. We have supporting data that mTOR is excessively upregulated in selected diabetic patients leading to phosphorylation of UVRAG, impairing its function to form a complex with VPS34 in generating PI3P for proper endosomal maturation and mitochondria degradation. Using both genetic and pharmacological methods, we will demonstrate that maintaining a stable UVRAG/VPS34 complex is a prerequisite for PI3P to mediate conversion of Rab5 into Rab7 for endosomal-mediated mitochondrial degradation. Collectively, these innovative studies will illuminate a novel endosomal-mediated mitochondrial degradation pathway as an important adaptive response in cardiomyocytes when exposed to hyperglycemic stress and pave the way for druggable targets in the future.