Department of Cardiology, Second Affiliated Hospital of Anhui Medical University, No. 678 of Furong Road, Hefei, Anhui, China.
Department of Geriatrics Medicine, Shanghai Municipal Hospital of Traditional Chinese Medicine, No. 274 of Middle Zhijiang Road, Jingan District, Shanghai, China.
Biosci Rep. 2017 Nov 23;37(6). doi: 10.1042/BSR20171249. Print 2017 Dec 22.
Previous studies have demonstrated Stromal interaction molecule 1 (STIM1)-mediated store-operated Ca entry (SOCE) contributes to intracellular Ca 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 Ca accumulation and apoptosis in H9c2 cardiomyocytes subjected to hypoxia/reoxygenation (H/R).
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 Ca 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 CoCl, while ROS generation was evaluated using the dye DCFH-DA in H9C2 cardiomyocytes.
Expression of STIM1/Orai1/TRPC1 significantly increased in transcript and translation level after MIRI and H/R In H9C2 cardiomyocytes subjected to H/R, intracellular Ca 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 Ca 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.
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.
先前的研究表明基质相互作用分子 1（STIM1）介导的储存操纵钙内流（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）背景下 STIM1 及其下游分子 Orai1/TRPC1 的表达情况，以及 STIM1 抑制对缺氧/复氧（H/R）诱导的 H9c2 心肌细胞钙积累和凋亡的影响。
采用 RT-PCR 和 Western blot 检测 MIRI 小鼠和 H/R 诱导的 H9C2 心肌细胞中 STIM1/Orai1/TRPC1 的表达。用 Stealth SiRNA 转染 H9C2 心肌细胞以敲低 STIM1。通过流式细胞术和 TUNEL 分析检测细胞凋亡。通过 MTT 测定法测量细胞活力。通过 Fluo-3/AM 探针的激光扫描共聚焦显微镜检测细胞内 Ca 浓度。此外，通过用 calcein AM 和 CoCl 负载评估线粒体通透性转换孔（mPTP）的开放，并用 DCFH-DA 染料在 H9C2 心肌细胞中评估 ROS 生成。
MIRI 和 H/R 后，STIM1/Orai1/TRPC1 的表达在转录和翻译水平上均显著增加。与对照组相比，H/R 诱导的 H9C2 心肌细胞内 Ca 积累显著增加，同时伴有 mPTP 开放增加和 ROS 生成升高。然而，SiRNA 抑制 STIM1 可显著减少 H/R 诱导的 H9C2 心肌细胞凋亡和细胞内 Ca 积累，同时减弱 mPTP 开放和降低 ROS 生成。此外，抑制 STIM1 增加了 Bcl-2/Bax 比值，降低了 Orai1/TRPC1 和 cleaved caspase-3 的表达。
抑制 STIM1 可降低 H9C2 心肌细胞内钙水平并减轻缺氧/复氧诱导的凋亡。我们的研究结果为理解 MIRI 中 STIM1 介导的钙超载提供了新的视角。
