高原低氧环境下缺氧心肌自噬流受损的关键作用及机制研究

Heart is a major target organ of hypoxic injury in both acute hypobaric hypoxia at high altitude and severe burn/traumatic hemorrhagic shock. Impairment of autophagic flux triggered by hypoxia can induce cardiac injury, however, the underlying mechanisms remain to be illustrated. Based on our previous findings and the latest advances, we hypothesize that under hypoxic conditions such as high altitude and severe burn injury/traumatic hemorrhagic shock, activation of CD38 consumes NAD, which induces the deficiency of lysosomal acidification and (or) accumulation of intracellular Ca2+, leading to impairment of cardiac autophagic flux. To confirm this hypothesis, hypobaric chamber is applied to mimic hypobaric hypoxia at high altitude.CD38 knockout mouse and cardiomyocyte with up/down-regulation of CD38 are planned to be used, and aimed to explore the upstream mechanisms and downstream effects of NAD consumption, from three different levels of animal (hypobaric hypoxia combined with burn injury/taumatic hemorrhagic shock), isolated heart and cardiomyocytes. The key role of NAD consumption in mediating impairment of autophagic flux under hypoxia will be firstly investigated, and then the mechanisms that contributes to NAD consumption and the key link in inducing impairment of autophagy flux triggered by NAD consumption in hypoxia are to be verificated. At last, the protective effects and its mechanisms of autophagic flux impairment in hypoxic myocardium will be explored. It is hopeful that through this study, the key roles of the new pathway of “CD38 activation-NAD consumption -lysosomal acidification deficiency/intracellular Ca2+ accumulation” in impairment of cardiac autophagic flux under hypoxia will be verificated, which will expand our knowledge on mechanisms of cardiac hypoxia following burn injury/taumatic hemorrhagic shock at high altitude and provide new targets and theory for the clinical treatment of vital organ injuries induced by burn injury/taumatic hemorrhagic shock at high altitude.

心脏是高原低氧环境下多种伤病引起机体缺氧损害的重要靶器官，缺氧通过损伤自噬流导致心肌损害，其机制未明。基于我们前期研究和最新进展，申请者率先提出“CD38激活介导烟酰胺腺嘌呤二核苷酸（NAD）消耗，进而引起溶酶体酸化障碍/胞浆Ca2+蓄积是高原条件下缺氧心肌自噬流受损的关键分子机制”的科学假设。拟模拟高原低氧环境，利用CD38基因敲除/转基因小鼠和CD38高/低表达心肌细胞模型，从整体动物、离体心脏和心肌细胞三个层次，围绕NAD消耗的上游机制和下游环节，首先明确NAD在缺氧心肌自噬流受损中的作用，再揭示缺氧消耗NAD的分子机制及NAD消耗导致自噬流受损的关键环节，最后探讨改善自噬流受损对高原条件下缺氧心肌的保护效应及其机制。通过研究，可望证实“CD38激活-NAD消耗-溶酶体酸化障碍/胞浆Ca2+蓄积”在缺氧心肌自噬流受损中的关键作用，为高原条件下缺氧脏器损伤的临床防治提供新靶点与新思路。

自噬流受损是低氧环境下心肌缺氧损害的重要因素，但机制不清。本项目利用模拟高原缺氧、严重烧伤心肌缺氧、高原复合烧伤、心脏冠脉结扎、体外缺氧处理心肌细胞等多种心肌缺氧模型，以及法洛四联症严重缺氧患者心肌组织，证明自噬流受损是高原等多种缺氧环境心肌存在的普遍现象。模拟5000米高原环境可致心肌自噬流受损，心肌损伤，心功能降低；高原低氧复合严重烧伤可进一步加重自噬流受损，心肌损害和心功能降低更为显著。. 较系统地证明了自噬流受损是缺氧导致心肌损害的重要因素，阐明了缺氧导致自噬流受损及其引起心肌损害的机制，为防治高原和烧伤心肌缺氧损伤提供了新靶点和新思路。证明缺氧心肌CD38高表达通过NAD依赖和非依赖途径导致自噬流和心肌损害，在体敲除CD38基因可改善自噬流和心功能；HuR核浆转运受限和TPC2降低导致溶酶体酸化障碍是缺氧心肌自噬流受损和心肌损害的重要机制；缺氧下调TPC2通过减少钙通道开放，H离子内流减少，引起溶酶体酸化障碍，心肌细胞自噬流受损；发现TRPV1促进心肌细胞Ca2+内流,激活AMPK信号通路，改善缺氧心肌细胞自噬流受损；缺氧下调Lamp2通过CD-M6PR导致心肌细胞自噬流受损，进而引起溶酶体膜通透化和心肌细胞损伤；缺氧引起的自噬体累积通过LC3B与RIPK1、RIPK3相互作用可导致心肌细胞坏死；发现细胞外pH降低可通过HDAC6调控微管乙酰化水平导致心肌细胞自噬体生成障碍和心肌损害。. 首次发现MAP4高磷酸化可损害自噬流，而MAP4去磷酸化可显著降低缺氧诱导的自噬激活与自噬流受损，特异性减少心肌凋亡，为缺氧心肌损害新药开发提供了有潜在临床应用价值的信息。国际著名心脏病专家Masafumi Kitakaze在EBioMedicine专文撰写同期述评，认为该研究系统证明和回答了微管损害的机制和微管损害与心脏疾病关系这一根本问题，有直接临床应用前景。. 已发表标注论文12篇，已俢回论文6篇，包括SCI论文4篇和投寄《中华医学杂志》论文2篇；部分实验结果还在总结，论文在撰写中。主编专著4部，获授权实用新型专利7件。培养博士研究生6名，硕士研究生7名。项目负责人担任三个全国性二级学术机构主任委员。主办全国性学术会议6次，在国内和国际会议大会报告20次。