MFN-2介导的线粒体内质网偶联在低氧肺动脉高压中的作用及机制研究

Pulmonary hypertension (PH), characterized by persistent pulmonary vascular contraction and pathological remodeling, shows high mortality and limited therapeutic means. Mitochondria-endoplasmic reticulum (ER) coupling regulating intracellular Ca2+ transport and other biological processes, plays critical roles in the maintenance of cell homeostasis, but its role in PH is unknown. In this study, we took the coupling between the organelles as our starting point to investigate the pathogenesis of PH. Our preliminary data showed that in hypoxic PH: 1. The expression of MFN-2 who is the crucial protein mediated mitochondria-ER coupling decreased and the distance between the organelles increased; 2. MFN-2 siRNA could inhibit the proliferation of pulmonary artery smooth muscle cells. Based on the point that mitochondria-ER uncoupling may lead to abnormal regulation of intracellular Ca2+, we hypothesized that the down-regulation of MFN-2 will lead to mitochondria-ER uncoupling and the abnormal regulation of intracellular Ca2+ will lead to pulmonary vascular contraction and remodeling, and which will eventually promote the development of PH. This study intends to use novel microscopic imaging and chemical biomarker techniques to detect the distance between mitochondria and ER, combining with pulmonary vascular smooth muscle specific MFN-2 knockdown and overexpression experiments, to clarify the mechanisms of MFN-2-mediated mitochondrial-ER coupling in pulmonary vascular contraction and remodeling in PH. This study would provide a new thought on the pathogenesis of PH and facilitate new strategies to seek for new drugs directing at MFN-2 target.

肺动脉高压（PH）以肺动脉持续收缩及病理性重构为主要特征，死亡率高但治疗手段有限。线粒体内质网偶联调控胞内Ca2+转运等过程，对细胞稳态维持至关重要，但其在PH中的作用未知。本研究以细胞器间相互偶联为出发点探讨PH发病机制，预实验发现低氧PH时：1.线粒体内质网偶联关键蛋白MFN-2表达下降，细胞器间距离增加；2.敲低MFN-2表达促肺动脉平滑肌增殖。鉴于线粒体内质网结构失偶联可能导致胞内Ca2+调控异常，本研究因此假设：MFN-2表达下调导致线粒体内质网失偶联，胞内Ca2+调控异常导致肺血管收缩及重构，进而促PH发生发展。本研究拟采用新型显微成像及化学生物学标记等技术检测线粒体内质网距离，结合肺血管平滑肌特异性MFN-2敲减及过表达小鼠实验，阐明MFN-2介导的线粒体内质网偶联在PH肺血管收缩及重构的机制，为寻找防治PH的新靶点奠定基础。

肺动脉高压（PH）以肺动脉持续性收缩及病理性重构为主要特征，死亡率高但治疗手段有限。线粒体内质网偶联调控胞内Ca2+转运等过程，对细胞稳态维持至关重要，但其在PH中的作用未知。我们发现：（1）MFN-2降低与低氧PH及与PASMCs线粒体内质网距离变化相关，低氧情况下肺动脉中膜及PASMCs MFN-2表达降低，线粒体内质网距离变大。（2）内质网MFN-2敲低对PASMCs线粒体内质网之间距离的影响有显著性差异，内质网MFN-2敲低可显著增加线粒体与内质网之间的距离，而线粒体MFN-2敲低无明显差异。（3）内质网MFN-2敲低可显著促进PASMCs的收缩，内质网MFN-2敲低可显著增加细胞质中Ca2+浓度，p-MLC、CaM及MLCK表达显著增加。（4）内质网敲减MFN-2可显著促进PASMCs增殖抑制凋亡，CaMKⅡ、p-CaMKⅡ及p-ERK表达显著增加，PDH及α-KG水平显著降低。（5）动物水平确证了内质网MFN-2对肺血管收缩及重构的作用。通过本课题的研究，我们解析了内质网MFN-2降低可通过直接将线粒体内质网距离增大，抑制线粒体内质网之间的Ca2+转运，进而引起PASMCs收缩及增殖。确定了内质网MFN-2可作为治疗低氧PH的重要靶点。