Suppression of Stim1 reduced intracellular calcium concentration and attenuated hypoxia/reoxygenation induced apoptosis in H9C2 cells
Suppression of Stim1 reduced intracellular calcium concentration and attenuated hypoxia/reoxygenation induced apoptosis in H9C2 cells
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DOI：
10.1042/bsr20171249
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发表时间：
2017-12-22
影响因子：
4
通讯作者：
Cheng, Jing
中科院分区：
文献类型：
--
作者：
He, Fei;Wu, Qianfu;Cheng, Jing
目的：先前的研究表明基质相互作用分子1（STIM 1）介导的钙库操纵的钙内流（SOCE）有助于细胞内钙积累。本研究旨在探讨心肌缺血/再灌注损伤（MIRI）时STIM 1及其下游分子Orai 1/TRPC 1的表达以及STIM 1抑制对缺氧/复氧（H/R）H9 c2心肌细胞Ca 2+蓄积和凋亡的影响。RT-PCR和Western blot检测MIRI小鼠和H/R小鼠H9 C2心肌细胞中STIM 1/Orai 1/TRPC 1的表达。为了敲低STIM 1，用Stealth SiRNA转染H9 C2心肌细胞。流式细胞术和TUNEL法检测细胞凋亡。MTT法测定细胞活力。用Fluo-3/AM探针激光扫描共聚焦显微镜检测细胞内Ca ~（2+）浓度。结果：MIRI和H/R分别使H9 C2心肌细胞STIM 1/Orai 1/TRPC 1的转录和翻译水平明显升高，而H9 C2心肌细胞线粒体通透性转换孔（mPTP）的开放程度则明显降低。H9 C2心肌细胞缺氧/复氧后，细胞内Ca 2+积累明显增加，沿着增加mPTP开放和ROS产生。然而，通过SiRNA抑制STIM 1显著降低H/R诱导的H9 C2心肌细胞凋亡和细胞内Ca 2+积累，伴随着减弱的mPTP开放和减少的ROS产生。另外，抑制STIM 1可增加Bcl-2/Bax比值，降低Orai 1/TRPC 1，并切割caspase-3的表达。结论：抑制STIM 1可降低细胞内钙水平，减轻缺氧/复氧诱导的H9 C2心肌细胞凋亡。我们的研究结果提供了一个新的视角，了解STIM 1介导的钙超载的设置MIRI。
Objective: Previous studies have demonstrated Stromal interaction molecule 1 (STIM1)-mediated store-operated Ca2+ entry (SOCE) contributes to intracellular Ca2+ accumulation. The present study aimed to investigate the expression of STIM1 and its downstream molecules Orai1/TRPC1 in the context of myocardial ischemia/reperfusion injury (MIRI) and the effect of STIM1 inhibition on Ca2+ accumulation and apoptosis in H9c2 cardiomyocytes subjected to hypoxia/reoxygenation (H/R).Methods: Expression of STIM1/Orai1/TRPC1 was determined by RT-PCR and Western blot in mice subjected to MIRI and H9C2 cardiomyocytes subjected to H/R. To knock-down STIM1, H9C2 cardiomyocytes was transfected with Stealth SiRNA. Apoptosis was analyzed by both flow cytometry and TUNEL assay. Cell viability was measured by MTT assay. Intracellular Ca2+ concentration was detected by laser scanning confocal microscopy using Fluo-3/AM probe. Furthermore, the opening of mitochondrial permeability transition pore (mPTP) was assessed by coloading with calcein AM and CoCl2, while ROS generation was evaluated using the dye DCFH-DA in H9C2 cardiomyocytes.Results: Expression of STIM1/Orai1/TRPC1 significantly increased in transcript and translation level after MIRI in vivo and H/R in vitro. In H9C2 cardiomyocytes subjected to H/R, intracellular Ca2+ accumulation significantly increased compared with control group, along with enhanced mPTP opening and elevated ROS generation. However, suppression of STIM1 by SiRNA significantly decreased apoptosis and intracellular Ca2+ accumulation induced by H/R in H9C2 cardiomyocytes, accompanied by attenuated mPTP opening and decreased ROS generation. In addition, suppression of STIM1 increased the Bcl-2/Bax ratio, decreased Orai1/TRPC1, and cleaved caspase-3 expression.Conclusion: Suppression of STIM1 reduced intracellular calcium level and attenuated hypoxia/reoxygenation induced apoptosis in H9C2 cardiomyocytes. Our findings provide a new perspective in understanding STIM1-mediated calcium overload in the setting of MIRI.