iPSC来源外泌体miRNAs靶向调控Egr-1/Agrin通路抑制增龄性心房重构和心房颤动作用机制研究

Aging is the independent determinant of atrial fibrillation (AF) occurrence, and the prevalence of AF is sharply increasing with the aggravating trend of aging population. Prevention and treatment of AF is so difficult. Consequently, it is so urgent to explore new therapeutic approaches for age-related AF. Apoptosis and atrial fibrosis are the initial factors of age-related AF. Stem cell transplantation contributes to cardiomyocytes proliferation and inhibits fibrosis, but the challenges of immune rejection and the tumorigenesis consideration remain unsolved. Pilot study revealed that induced pluripotent stem cells-derived exosomes (iPSCexo) considerably inhibit age-related atrial fibrosis and AF vulnerability. We identified the iPSCexo miRNAs related to fibrosis and AF and then recognized Egr-1/Agrin pathway as the target genes for the miRNAs by high-throughput sequencing and bioinformatics. And iPSCexo could modulate the expression level of egr-1 and agrin in aged mouse. So, in the present project, conditional knockout and transgenic lentiviral vector of Egr-1 and Agrn, electrophysiology, bioinformatics, morphology and molecular biology will be used to explore the roles and mechanisms of iPSC-derived exosomal miRNAs on age-related atrial remodeling and AF via targeting Egr-1/Agrin pathway.The present project will further help reveal the new target in the prevention and treatment of age-related AF.

增龄是心房颤动（AF）发生的独立危险因素。老龄化导致AF发病率急剧上升，但治疗困难，新的防治靶点亟待挖掘。细胞凋亡和心房纤维化是增龄性AF始动因素。干细胞移植促进心肌细胞再生、抑制纤维化，但存在免疫排斥和致瘤等诸多问题。预实验显示iPSC来源外泌体（iPSCexo）显著抑制增龄性心房纤维化和AF易损性。联合高通量测序和生物信息学筛选出调控纤维化和AF的关键iPSCexo miRNAs，并预测Egr-1/Agrin为其靶基因显著性富集信号通路。而且，iPSCexo可以调控小鼠心房组织Egr-1和Agrin增龄性改变。因此，本课题拟采用心肌Egr-1和AGRN条件性敲除和慢病毒载体技术，应用电生理学、生物信息学、形态学及分子生物学等，深入探讨iPSCexo miRNAs通过调控Egr-1/Agrin通路抑制增龄性心房重构和AF作用机制，为进一步探索防治增龄性AF新靶点提供实验依据和理论指导。

心房颤动（Atrial Fibrillation, AF）是最常见且难治的心律失常，其发病机制不明，且治疗困难。在众多危险因素中，增龄是导致AF发病率急剧上升的一个独立危险因素。本研究探索了诱导多能干细胞外泌体（Induced pluripotent stem cells-derived exosome, iPSC-Exo） 对增龄性心房重构和 AF 易损性的作用和机制，并研究了CXCL12-CXCR4轴、运动和程序性坏死在心房颤动发生发展中的作用及其机制。研究发现： iPSC-Exo可以显著降低老年小鼠心肌细胞衰老和凋亡，抑制心肌纤维化，降低老年小鼠的AF 诱发率和持续时间。其作用机制，一方面是通过上调Agrin，促进了自噬的起始，启动自噬相关膜的合成与自噬体的聚集来增强自噬，抑制心肌细胞衰老实现的。另一方面则是通过增加Agrin水平，促进了Dag1与Agrin的结合，削弱Dag1和Yap结合，进而诱导了Cdc42激活，促进心肌细胞增殖而实现的。除此之外，本研究还发现抑制CXCL12/CXCR4轴可以通过下调CD3+T淋巴细胞与F4/80+巨噬细胞的浸润和促炎因子的释放，并抑制AKT/mTOR和ERK1/2信号转导来减少心房结构重塑，降低小鼠房颤诱发率及房颤整体持续时间。最后本研究发现，心房程序性坏是促发AF的关键致病机制，中等强度运动可能通过激活AMPK依赖的自噬通路，抑制心房程序性坏死，从而逆转心房结构重构，减轻房颤。