mGluR5/mTORC2介导ES细胞定向分化心肌细胞中线粒体-内质网结构偶联分子事件及药物调控机制

The mitochondria-endoplasmic reticulum structure coupling forms a new platform between organelles, which affects calcium signal pathway, regulates mitochondrial morphology, and so on. The molecular mechanisms and biological significance of the structure coupling as intracellular signal transduction have not been elucidated. The applicant had clarified that mGluR5/PI3K mediated the embryonic stem (ES) cells differentiation into cardiomyocytes, which was associated with the shuttle of Ca2+. The mTORC2, as a downstream signal molecule of PI3K signaling pathway, localizes in the mitochondria-endoplasmic reticulum structure coupling site, which is related to the structural integrity and function coupling. This project aims to demonstrate the mitochondria-endoplasmic reticulum structure coupling platform is the important regulating center of mGluR5/mTORC2 signaling pathway during the differentiation of ES cells into cardiomyocytes. The platform related molecular events will be evaluated regulated by mGluR5/mTORC2 signaling pathway, in order to clarify the shuttle of Ca2+, localization and function of Cx43 expression, and mitochondrial energy activity. The results will provide the important academic value for finding the new targets in the regulation of ES cell differentiation. The project will also explore the intervention effects of icariin inducing cardiomyocyte differentiation on mGluR5/mTORC2 signaling regulation mechanism. The results will reveal the new mechanism of icariin as an inducing differentiation agent, provide a useful reference for the follow-up clinical study and potential application of the induced pluripotent stem cells.

线粒体-内质网结构偶联构成细胞器间功能偶联新平台，影响钙信号通路、调控线粒体形态等。该偶联作为细胞内信号转导分子机制和生物学意义尚未阐明。申请者前期研究已明确mGluR5/PI3K介导胚胎干（ES）细胞定向分化心肌细胞，参与细胞内Ca2+活动。mTORC2作为PI3K下游信号分子之一，定位于线粒体-内质网结构偶联，与该结构偶联完整性和功能密切相关。本项目基于ES细胞定向分化心肌细胞体系，进一步深入阐明mGluR5/mTORC2调控线粒体–内质网结构偶联中的分子事件在Ca2+定位穿梭、Cx43表达功能、线粒体发生和能量活动中的生物学意义，为发现干细胞分化新靶点提供重要学术价值。本项目还将探索淫羊藿苷诱导分化时，经由mGluR5/mTORC2信号对线粒体–内质网结构偶联分子事件的调控作用，揭示其促心肌分化作用的新机制。利用ES细胞研究结果为诱导多能干细胞后续研究与临床潜在应用提供有益借鉴。

线粒体-内质网结构偶联（MAM）构成细胞器间功能偶联新平台，影响钙信号通路、调控线粒体形态等。该偶联作为细胞内信号转导分子机制和生物学意义尚未阐明。项目负责人团队前期研究已明确mGluR5/PI3K介导胚胎干（ES）细胞定向分化心肌细胞，参与细胞内Ca2+活动。Rictor/mTORC2作为PI3K下游信号分子之一，定位于线粒体-内质网结构偶联，与该结构偶联完整性和功能密切相关。本项目主要利用ES细胞体外定向心肌细胞分化模型，重点评价了Rictor/ mTORC2介导线粒体-内质网结构偶联相关分子事件。结果发现，ES细胞干扰Rictor能特异性抑制心室样细胞定向分化，导致具有心律失常电生理现象的心肌细胞数量增加。干扰Rictor会破坏ES细胞衍生心肌细胞（ESC-CMs）MAM结构，降低MFN2表达和减弱内质网-线粒体钙流动相关复合物IP3R-Grp75-VDAC1相互作用，致ESC-CMs线粒体损伤，显著降低线粒体钙瞬变幅度。该损伤机制与干扰Rictor致HDAC6表达减少，Hsp90乙酰化程度增加，Cx43与转运蛋白Hsp90和TOM20结合减少，从而抑制Cx43转运至线粒体内膜，减少线粒体Cx43表达，从而降低线粒体呼吸链酶复合物I、IV活性有关；同时干扰Rictor也抑制Akt-GSK3通路，减弱Cx43与mPTP蛋白ANT、CypD结合作用，由此增加mPTP开放程度。本项目还评价了miRNA-218、药物淫羊藿苷及环境污染物全氟辛烷磺酸对ES细胞定向分化为心肌细胞过程中Rictor/mTORC2信号的调控研究。研究结果为阐明Rictor/mTORC2调控与心肌细胞分化密切相关的Ca2+活动及细胞线粒体发生和能量活动之间的关系提供实验依据，同时揭示调控心肌分化的新靶点，也为 iPS 细胞后续研究与临床潜在应用提供有益借鉴。