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浙江大学学报（医学版）
浙江大学学报（医学版）  2019, Vol. 48 Issue (1): 65-74    DOI: 10.3785/j.issn.1008-9292.2019.02.11
原著     
Rictor对小鼠胚胎干细胞来源心肌细胞线粒体钙信号的调控
邵颖1(),王佳丹1,2(),朱丹雁1,*()
1. 浙江大学药学院药理毒理研究所, 浙江 杭州 310058
2. 浙江萧山老年医院药学部, 浙江 杭州 310006
Rictor regulates mitochondrial calcium signaling in mouse embryo stem cell-derived cardiomyocytes
SHAO Ying1(),WANG Jiadan1,2(),ZHU Danyan1,*()
1. Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
2. Department of Pharmacy, Zhejiang Xiaoshan Geriatric Hospital, Hangzhou 310006, China
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摘要：

目的: 探索Rictor在小鼠胚胎干细胞来源心肌细胞（ESC-CM）中的表达、定位及其对线粒体钙信号的调控作用。方法: 通过经典"悬滴-悬浮-贴壁"三步法建立ESC-CM模型。利用免疫荧光法及蛋白质印迹法观察Rictor在ESC-CM中的定位。慢病毒技术干扰小鼠胚胎干细胞Rictor表达后，采用免疫荧光法考察ESC-CM内质网与线粒体的叠加情况；通过透射电镜观察ESC-CM的超微结构；活细胞工作站测定分化后心肌细胞线粒体钙瞬变；免疫共沉淀法检测ESC-CM中1，4，5-三磷酸肌醇受体（IP3R）、葡萄糖调节蛋白75（Grp75）、线粒体外膜的电压依赖性阴离子通道1（VDAC1）间的相互作用；蛋白质印迹法检测线粒体融合蛋白2（Mfn2）的表达情况。结果: Rictor在ESC-CM中主要定位于内质网及线粒体-内质网结构偶联（MAM）域，且其表达定位与线粒体及内质网有很好的叠加。干扰Rictor后，心肌细胞线粒体部分呈散点状，线粒体与内质网的叠加率降低（P < 0.01）；ESC-CM超微MAM形成减少；ATP刺激引起的ESC-CM线粒体钙瞬变幅度下降，其中钙瞬变斜率和上升峰值均降低（均P < 0.01）；MAM中IP3R、Grp75、VDAC1相互作用明显减弱，且Mfn2蛋白表达降低（P < 0.01）。结论: 干扰小鼠胚胎干细胞中Rictor表达可降低ESC-CM中钙从内质网到线粒体的释放，这可能是通过影响IP3R、Grp75、VDAC1间相互作用，减少Mfn2表达，进而破坏MAM来实现的。
关键词： 胚胎干细胞/细胞学;  肌细胞, 心脏/细胞学;  哺乳动物雷帕霉素靶蛋白;  钙信号;  内质网;  线粒体    
Abstract:

Objective: To explore the expression, localization and regulatory effect on mitochondrial calcium signaling of Rictor in embryonic stem cell-derived cardiomyocytes (ESC-CMs). Methods: Classical embryonic stem cell cardiomyogenesis model was used for differentiation of mouse embryonic stem cells into cardiomyocytes. The location of Rictor in ESC-CMs was investigated by immunofluorescence and Western blot. The expression of Rictor in mouse embryonic stem cells was interfered with lentiviral technology, then the superposition of mitochondria and endoplasmic reticulum (ER) in ESC-CMs was detected with immunofluorescence method; the cellular ultrastructure of ESC-CMs was observed by transmission electron microscope; the mitochondrial calcium transients of ESC-CMs was detected by living cell workstation; immunoprecipitation was used to detect the interaction between 1, 5, 5-trisphosphate receptor (IP3 receptor, IP3R), glucose-regulated protein 75 (Grp75) and voltage-dependent anion channel 1 (VDAC1) in mitochondrial outer membrane; the expression of mitochondrial fusion protein (mitonusin-2, Mfn2) was detected by Western blot. Results: Rictor was mainly localized in the endoplasmic reticulum and mitochondrial-endoplasmic reticulum membrane (MAM) in ESC-CMs. Immunofluorescence results showed that Rictor was highly overlapped with ER and mitochondria in ESC-CMs. After mitochondrial and ER were labeled with Mito-Tracker Red and ER-Tracker Green, it was demonstrated that the mitochondria of the myocardial cells in the Rictor group were scattered, and the superimposition rate of mitochondria and ER was lower than that of the negative control group (P < 0.01). The MAM structures were decreased in ESC-CMs after knockdown of Rictor. The results of the living cell workstation showed that the amplitude of mitochondrial calcium transients by ATP stimulation in ESC-CMs was decreased after knockdown of Rictor (P < 0.01). The results of co-immunoprecipitation showed that the interaction between IP3R, Grp75 and VDAC1 in the MAM structure of the cardiomyocytes in the Rictor group was significantly attenuated (P < 0.01); the results of Western blot showed that the expression of Mfn2 protein was significantly decreased (P < 0.01). Conclusion: Using lentiviral technology to interfere Rictor expression in mouse embryonic stem cells, the release of calcium from the endoplasmic reticulum to mitochondria in ESC-CMs decreases, which may be affected by reducing the interaction of IP3R, Grp75, VDAC1 and decreasing the expression of Mfn2, leading to the damage of MAM structure.
Key words: Embryonic stem cells/cytology    Myocytes, cardiac/cytology    Mammalian target of rapamycin    Calcium signaling    Endoplasmic reticulum    Mitochondrial
收稿日期: 2018-08-01 出版日期: 2019-05-13
:  R966  
基金资助: 国家重点研发计划政府间国际科技创新合作重点专项（中印尼生物技术联合实验室）(2017YFE0102200);国家自然科学基金(81573426);浙江省公益性技术应用研究计划(2016C33157)
通讯作者: 朱丹雁     E-mail: shao940618@zju.edu.cn;304712785@qq.com;zdyzxb@zju.edu.cn
作者简介: 邵颖(1994-), 女, 硕士研究生, 主要从事干细胞生物学研究; E-mail:shao940618@zju.edu.cn; https://orcid.org/0000-0002-9907-2228|王佳丹(1992-), 女, 硕士, 初级药师, 主要从事干细胞生物学研究; E-mail:304712785@qq.com; https://orcid.org/0000-0002-5257-8811
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引用本文:

邵颖,王佳丹,朱丹雁. Rictor对小鼠胚胎干细胞来源心肌细胞线粒体钙信号的调控[J]. 浙江大学学报（医学版）, 2019, 48(1): 65-74.

SHAO Ying,WANG Jiadan,ZHU Danyan. Rictor regulates mitochondrial calcium signaling in mouse embryo stem cell-derived cardiomyocytes. J Zhejiang Univ (Med Sci), 2019, 48(1): 65-74.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2019.02.11    或    http://www.zjujournals.com/med/CN/Y2019/V48/I1/65

图 1  小鼠胚胎干细胞来源的心肌细胞中Rictor与内质网及线粒体的共定位结果
图 2  Rictor在小鼠胚胎干细胞来源的心肌细胞各细胞器中的表达电泳图
图 3  干扰Rictor蛋白表达后小鼠胚胎干细胞来源的心肌细胞中线粒体与内质网的叠加在共聚焦显微镜下所见
图 4  干扰Rictor蛋白表达后小鼠胚胎干细胞来源的心肌细胞中MAM、线粒体及内质网的超微结构透射电镜观察
图 5  干扰Rictor蛋白表达后ATP刺激的小鼠胚胎干细胞来源心肌细胞线粒体钙瞬变免疫荧光图
图 6  干扰Rictor蛋白后小鼠胚胎干细胞来源心肌细胞在ATP刺激后的荧光强度变化
图 7  干扰Rictor蛋白表达后小鼠胚胎干细胞来源心肌细胞中Mfn2蛋白表达电泳图
图 8  干扰Rictor蛋白表达后小鼠胚胎干细胞来源心肌细胞中Grp75、IP3R、VDAC1蛋白共沉淀电泳图
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