Rab GTPases-mediated mitochondrial clearance in diabetic cardiomyopathy

项目总结 糖尿病心肌病是一种常见的但被低估的心力衰竭和死亡的原因 糖尿病。这种通常致命的综合症的潜在基础尚不清楚，尽管有多条途径汇聚在一起 有一个共同点--功能失调的线粒体。了解线粒体受损程度 清除线粒体是至关重要的，因为受损线粒体的存在会导致快速产生活性氧 物种，然后影响剩下的健康线粒体。这导致广泛分布的线粒体 功能障碍和细胞死亡。尽管受损的线粒体被认为是由线粒体降解的 自噬(自噬的一种特殊形式)，我们最近发现了一种新的内噬-- 高血糖条件下心肌细胞线粒体降解途径的研究。对中国的压制 这一途径可能与糖尿病心肌病的易感性增加有关。这一观察结果 这是因为我们有能力从糖尿病患者中产生针对患者的诱导多能干细胞(IPSCs) 有(T2DCM)和无(T2D)心肌病患者。我们发现只有T2D细胞，而不是T2DCM， 表现出内体介导的线粒体降解增加。然而，无论是分子信号还是分子信号 调节这一新途径，也不确定这种内体途径的功能意义，在 糖尿病心脏的心肌细胞。因此，本项目的目标是展示其功能意义 糖尿病心肌病中内体介导的线粒体降解途径的研究及意义 调节这一途径的潜在机制。目标1将定义内体介导的功能角色 糖尿病心脏线粒体清除通过破坏Rab5和Rab7的功能，这是糖尿病心脏的关键决定因素 内切溶酶体系统。这些研究将使用多种创新试剂，包括CRISPR介导的 Rab基因敲除和过度激活的iPSC系和一种新的可诱导的、心脏特异的Rab7基因敲除小鼠模型， 要询问这些RAB在维持两个不同的功能线粒体降解中的重要性 糖尿病小鼠模型。AIM 2将确定Vps34/UVRAG在生成磷脂酰肌醇3中的作用- 磷酸盐(PI3P)是内体成熟和清除有缺陷的线粒体所必需的。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功能受损，与Vps34形成复合体生成PI3P以形成适当的内体 成熟和线粒体退化。利用遗传和药理学方法，我们将 证明维持稳定的UVRAG/Vps34复合体是PI3P介导转换的先决条件 将Rab5转化为Rab7，用于内体介导的线粒体降解。总体而言，这些创新研究将 阐明一种新的内体介导的线粒体降解途径是一种重要的适应性反应 当暴露在高血糖应激下时，在心肌细胞中的作用，并为未来的可用药靶点铺平道路。

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.