Ca2+-线粒体RISP-ROS-RYR2-MCU正反馈环路介导缺氧PASMCs收缩的机制研究

Until now, the mechanism of hypoxic pulmonary hypertension（PH） has still not been fully elucidated. Reactive oxygen species (ROS) play an important role in signal transduction in the formation of the PH. Our previous studies have shown that hypoxia could directly affect ROS production in mitochondria. Rieske iron–sulfur protein (RISP) in the mitochondrial electron transport chain complex III may serve as an essential, primary molecule that mediates the hypoxic ROS generation. ROS may directly cause release of Ca2+ from sarcoplasmic reticulum through ryanodine receptor2 (RyR2), leading to an increase in intracellular Ca2+ concentration ([Ca2+]i) and hypoxic pulmonary vasoconstriction. There are elaborate regulatory mechanisms of Ca2+ between sarcoplasmic reticulum and mitochondrial, Ca2+ release from sarcoplasmic reticulum can be uptaked by mitochondrial calcium uniporter (MCU) to mitochondrial and play an important role. However, the mechanism that how can RISP-dependent mitochondrial ROS generation regulate the Ca2+ release from sarcoplasmic reticulum has not completely understood. At present, the interaction between endoplasmic reticulum Ca2+ release and mitochondrial ROS generation have not been reported. The objective of this project is to study the effect and the mechanism of Ca2+ - mitochondrial RISP - ROS - RYR2 - MCU positive feedback loop in regulating the contraction of pulmonary artery smooth muscle cells in hypoxia conditions. The study will provide new direction and measures for hypoxic PH.

缺氧肺动脉高压(PH)的发病机制尚未完全揭示。活性氧（ROS）在诱导缺氧PH的形成中起着重要的信号转导作用。本组的前期研究发现，肺动脉平滑肌细胞（PASMCs）线粒体电子传递链复合体可以感受缺氧，引起ROS生成增加，其中复合体III的铁硫蛋白（RISP）起关键的作用；同时，ROS增加可以开放理阿诺碱受体2（RyR2）使内质网的Ca2+释放增加，进而使细胞质游离钙离子浓度（[Ca2+]i）升高，引起缺氧肺血管收缩；线粒体与内质网间的Ca2+存在精细的调控，内质网释放的Ca2+可被线粒体膜上的钙单向转运体（MCU）摄回而发挥作用。然而，RISP依赖的线粒体ROS对内质网Ca2+释放和内质网Ca2+对线粒体ROS 生成之间的相互作用机制还不清楚。所以本题基于先前的研究，观察Ca2+-线粒体RISP-ROS-RYR2-MCU正反馈环路对缺氧PASMCs收缩的调控作用，进一步揭示缺氧PH的机制。

项目背景：缺氧肺动脉高压(PH)的发病机制尚未完全揭示。活性氧（ROS）在诱导缺氧PH的形成中起着重要的信号转导作用。本组的前期研究发现，肺动脉平滑肌细胞（PASMCs）线粒体电子传递链复合体可以感受缺氧，引起ROS生成增加，其中铁硫蛋白（RISP）起关键的作用 ；同时，ROS增加可以开放兰尼碱受体2（RYR2）使内质网的Ca2+释放增加，进而使[Ca2+]i升高，引起缺氧肺血管收缩；线粒体与内质网间的Ca2+存在精细的调控，内质网释放的Ca2+可被线粒体膜上的钙单向转运体（MCU）摄回。然而，RISP依赖的线粒体ROS对内质网Ca2+释放和内质网Ca2+对线粒体ROS生成之间的相互作用机制还不清楚。所以本题基于先前研究，观察Ca2+-线粒体RISP-ROS-RYR2-MCU正反馈环路对缺氧PASMCs收缩的调控作用，进一步揭示缺氧PH的机制。为探索缺氧PH预防和治疗新靶点提供依据。.研究内容：①检测急、慢性缺氧时人PASMCs线粒体ROS生成的位点，明确RISP是否在线粒体ROS生成中起关键性的作用。②检测急、慢性缺氧线粒体ROS对内质网RYR2钙通道调控的影响。③检测急、慢性缺氧时Ca2+对线粒体ROS生成的影响。 .主要研究成果：1.发现：缺氧诱发RyR介导的的Ca 2+ 释放会导致MCU的显着增加，而增加的MCU随后会诱发RISP [ROS] m浓度上升。2.发现：缺氧导致了细胞内的[Ca2+]i增加，线粒体等复合物I、II、III Qo前泛半醌位点位点抑制剂，可以降低[Ca2+]i、[ROS]i及肺血管收缩程度。3.FKBP12.6和CaMK- II可能在COPD相关的肺动脉增殖中起一定作用。.重要结果：1缺氧导致线粒体ROS依赖的内质网上RyR2介导Ca2+ 释放，增加PASMC中的MCU，进而增加RISP依赖的[ROS] m，形成的正反馈机制与PH的发病有关。线粒体ETC复合物I、II和IIQo部位与PH有关。.关键数据及其科学意义：.缺氧导致线粒体ROS-内质网RyR2介导Ca2+释放，增加MCU，进而增加RISP依赖的[ROS] m，形成正反馈机制可能与PH的发病有关。.证实线粒体ETC复合物I、II和IIIQo部位与缺氧性PH有关。为研究不同线粒体ETC受体的在PH发病中机制做出了贡献。