利用iPSC和类器官技术研究线粒体突变在心脏衰老中的作用机制

The accumulation of mitochondrial DNA (mtDNA) mutation with age will induce mitochondrial dysfunction, the major cause of aging and degenerative diseases. High risk of severe cardiac adverse events was reported in Patients with mitochondrial m.3243A>G mutation. However, the mechanisms of mitochondrial mutation-induced cardiac aging and dysfunction are still unknown due to the lack of human cardiomyocytes and mitochondrial disease models. In this project, the applicants will construct a new cardiac organoid model using the established human induced pluripotent stem cells (iPSCs) with m.3243A>G mutation. Basing on the cardiac organoid model, the applicants have proposed three studies: (1) assessing the senescence-associated phenotypes and functions in iPSC-derived cardiomyocytes (iPSC-CMs); (2) illustrating the multi-omic integrated networks in mtDNA mutation-induced cardiac aging by using the single-cell technologies; (3) screening the small molecule drugs to prevent or delay the senescence-associated phenotypes in iPSC-CMs with m.3243A>G mutation, and evaluating the therapeutic effects of these drug-pretreated iPSC-CMs on ischemic myocardial injury. With the success of this project, the applicants will establish an iPSC-based organoid platform for the mechanism study of cardiac disease and drug screening, uncover the regulatory mechanisms in m.3243A>G mutation-associated cardiac aging, and provide new strategies for preventing or delaying mitochondrial dysfunction-induced cardiac aging and degenerative diseases.

线粒体DNA突变累积导致线粒体功能紊乱是机体衰老和退行性病变的重要因素。m.3243A>G突变是临床报道引起衰老性心脏功能障碍的主要线粒体突变，但受限于人源心肌细胞系和线粒体突变模型难以建立，具体调控机制仍不清楚。本项目拟在团队已建立m.3243A>G突变患者来源iPSCs基础上，利用小分子诱导和类器官技术建立m.3243A>G突变心脏衰老模型；比较健康和m.3243A>G突变心肌细胞的衰老和功能；采用单细胞测序、蛋白和代谢组学分析等描绘m.3243A>G突变引发的多组学分子调控网络；筛选预防和延缓心肌衰老的小分子化合物，并评估药物处理m.3243A>G突变心肌细胞的心肌修复能力。本项目预期将建立线粒体突变心脏类器官功能研究和药物筛选平台，阐明m.3243A>G突变在心脏衰老中的功能和分子机制，为延缓线粒体功能紊乱导致的心脏衰老和退行性病变提供理论和技术支持。

线粒体DNA突变累积导致线粒体功能紊乱是机体衰老和退行性病变的重要因素。m.3243A>G突变是临床报道引起衰老性心脏功能障碍的主要线粒体突变，但受限于人源心肌细胞系和线粒体突变模型难以建立，具体调控机制尚不明确。本项目从携带线粒体m.3243A>G突变病人的体细胞出发，经重编程为病人特异性iPSCs，利用小分子诱导分化为稳定携带m.3243A>G突变的人源心肌细胞和类器官。与野生型相比，iPSC-CMm.3243A>G呈衰老表型，具体表现为β-半乳糖苷酶活性增高、溶酶体数量增加和细胞表面积增大等。从线粒体自身而言，iPSC-CMm.3243A>G的氧化磷酸化能力显著下降，表现为耗氧率的降低，细胞基础呼吸水平、最大呼吸能力和储备呼吸能力均有不同程度的下降。借助扫描电镜和机器深度学习，我们发现心肌细胞中线粒体m.33243A>G突变会破坏线粒体相关内质网膜MAM结构，具体表现为线粒体与内质网接触面积和接触数量的减少，心肌细胞钙信号处理能力下降。综上，线粒体m.3243A>G突变诱发衰老与线粒体相关内质网膜MAM结构的破坏有关，而维持线粒体相关内质网膜结构的稳定可能是延缓心肌细胞衰老的关键因素，为延缓线粒体功能紊乱导致的心脏衰老和退行性病变提供理论依据和技术支持。