RhoA/ROCK/Drp1调控线粒体分裂在糖尿病心肌病中的作用及机制研究

Diabetes mellitus (DM), a common metabolic disease related to many chronic complications, causes great challenge to public health all over the world. Diabetic cardiomyopathy (DCM) is a clinical condition of cardiac dysfunction, independently of hypertension, coronary artery disease and cardiac valvulopathy. DCM is a major risk factor for ill-health among diabetic population and responsible for increased morbidity and mortality. However, there is no effective treatment to prevent cardiac remodeling in DCM at present. Accumulating evidence has demonstrated that mitochondrial dynamics (mitochondrial fusion and mitochondrial fission) played a key role in DCM, which was one of the major components in triggering myocardial changes. Previous evidence showed increased mitochondrial fission in myocardium of DCM, which subsequently induced reactive oxygen species (ROS) generation, apoptosis, and myocardial collagen deposition. Phosphorylation was one of the major post-translational modifications for Drp1, which regulated mitochondrial fission. In the preliminary experiment, DCM was induced by STZ injection, and the protein levels of Drp1 and p-Drp1 (Ser616) were increased in myocardial tissue compared with control group. In addition, another study from our group showed that RhoA/ROCK pathway were activated in high glucose induced cardiac microvascular endothelial cells, which resulted in oxidative stress and cardiac dysfunction. Therefore, in the present study we are going to investigate whether RhoA/ROCK/Drp1 signal pathway mediates mitochondrial fission, and links to myocardial fibrosis and cardiac remodeling in diabetic cardiomyopathy.

引起糖尿病心肌重构的机制尚未完全阐明，目前仍无有效手段抑制糖尿病心肌病发生发展。线粒体动力学稳定是维持心脏功能的前提，糖尿病心肌组织线粒体分裂增加。Drp1磷酸化修饰是线粒体分裂的重要调控方式，并受多种因素调控。我们在前期研究中采用STZ诱导大鼠糖尿病，发现心肌组织中Drp1表达及Drp1（Ser616）磷酸化均增加。此外另一项研究提示糖尿病大鼠心肌微血管内皮细胞RhoA/ROCK通路激活，促进氧化应激反应，引起糖尿病心功能异常。基于此，本课题拟从线粒体动力学的角度，以“ROCK-Drp1信号通路”为靶点，分别从整体动物、组织和细胞水平探讨糖尿病心肌损伤及心室重构的分子调控机制，重点阐明RhoA/ROCK调控Drp1磷酸化在其中的作用，并通过ROCK抑制剂及基因修饰的方式在体验证RhoA/ROCK/Drp1信号通路对糖尿病心肌病的调控作用，为防治糖尿病心肌损伤及心室重构提供新靶点和新策略。

研究背景：引起糖尿病心肌损伤及重构的机制尚未完全阐明，目前仍无有效手段抑制糖尿病心肌病发生发展。线粒体动力学稳定是维持心脏功能的前提，糖尿病状态下心肌组织线粒体分裂增加。Drp1磷酸化修饰是线粒体分裂的重要调控方式，我们在前期研究中发现心肌组织中Drp1（Ser616）磷酸化增加，此外另一项研究提示糖尿病大鼠心肌微血管内皮细胞RhoA/ROCK1通路激活，促进氧化应激反应。基于此，本课题拟以“RhoA/ROCK1/Drp1信号通路”为靶点，分别从整体动物、组织和细胞水平探讨糖尿病心肌损伤及心室重构的分子调控机制。.研究内容：采用高脂高糖喂养联合STZ腹腔注射成功构建糖尿病大鼠模型，明确高剂量STZ（50mg/kg）单次注射后8周即可出现心肌纤维化及心室重构，同时发现糖尿病心肌组织氧化应激程度及凋亡水平显著高于对照组；进一步研究发现糖尿病大鼠心肌组织RhoA、ROCK1信号分子表达明显升高，DRP1（Ser616）磷酸化水平显著升高，给予糖尿病大鼠RhoA/ROCK1抑制剂fasudil干预12周，DRP1（Ser616）磷酸化水平显著降低，线粒体损伤明显改善，心肌组织纤维化程度、氧化应激及凋亡水平显著下降；对糖尿病大鼠心肌进行细胞内钙的光学定位，发现与对照组相比，90%钙瞬变时程（CTD90）显著延长。将人源诱导性多能干细胞定向分化成hiPSC-CMs，构建高糖诱导的细胞损伤模型。高糖作用下，心肌细胞核及细胞面积均增大，细胞氧化应激水平增强，葡萄糖摄取和ATP生成显著抑制，线粒体膜电位显著降低；进一步研究发现细胞内游离钙浓度在高糖糖干预组显著升高；高糖诱导下心肌细胞RhoA、ROCK1信号分子表达明显升高，DRP1（Ser616）磷酸化水平显著升高，采用fasudil、siRhoA、siROCK等抑制RhoA/ROCK1信号通路，DRP1（Ser616）磷酸化水平较高糖处理组明显降低，细胞氧化应激及凋亡水平亦显著下降。.研究意义：本研究围绕线粒体分裂与糖尿病心肌损伤与重构的关系，率先发现RhoA/ROCK1/Drp1信号通路促进线粒体分裂，导致心肌细胞氧化应激和凋亡增加，并进一步明确了在糖尿病心肌组织中RhoA/ROCK信号通路对于Drp1调控的磷酸化位点是Ser616，可能为今后防治糖尿病心肌损伤及心室重构提供新靶点。