Calreticulin translocation aggravates endoplasmic reticulum stress-associated apoptosis during cardiomyocyte hypoxia/reoxygenation.
Calreticulin translocation aggravates endoplasmic reticulum stress-associated apoptosis during cardiomyocyte hypoxia/reoxygenation.
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DOI：
10.4103/0366-6999.150103
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发表时间：
2015-02-05
影响因子：
6.1
通讯作者：
Liu XH
中科院分区：
文献类型：
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作者：
Xu FF;Liu XH
钙网蛋白 (CRT) 是主要的 Ca2+ 结合伴侣，主要驻留在内质网 (ER) 腔中。最近，研究表明非 ER CRT 调节多种细胞反应。我们之前发现CRT在缺氧/复氧（H/R）期间上调，本研究旨在探讨CRT核易位是否会加重新生大鼠心肌细胞H/R损伤期间内质网应激（ERS）相关的细胞凋亡。测量细胞凋亡率和培养基中乳酸脱氢酶（LDH）泄漏作为细胞损伤的指标。免疫荧光染色显示ER形态变化和CRT细胞内易位。采用Western blotting或逆转录聚合酶链反应检测目的分子的表达。与对照相比，H/R 增加了细胞凋亡率和 LDH 活性。 ER 变得凝结并起泡，CRT 转移到细胞核。 Western blotting 显示 H/R 后 CRT、Nrf2、激活转录因子 4 (ATF4)、CHOP 和 caspase-12 表达上调。 H/R 之前质粒转染诱导的外源 CRT 过表达增加了细胞凋亡、LDH 渗漏、ER 紊乱、CRT 核易位和 ERS 相关分子的表达。然而，施用 ERS 抑制剂、牛磺酸或 CRT siRNA 可减轻细胞损伤、ER 紊乱，并抑制 ERS 相关的细胞凋亡。我们的结果表明，在 H/R 应激过程中，CRT 易位会增加细胞凋亡和 LDH 渗漏，加剧 ER 紊乱，上调核转录因子 Nrf2 和 ATF4 的表达，并激活 ERS 相关的细胞凋亡。
Calreticulin (CRT) is major Ca2+-binding chaperone mainly resident in the endoplasmic reticulum (ER) lumen. Recently, it has been shown that non-ER CRT regulates a wide array of cellular responses. We previously found that CRT was up-regulated during hypoxia/reoxygenation (H/R) and this study was aimed to investigate whether CRT nuclear translocation aggravates ER stress (ERS)-associated apoptosis during H/R injury in neonatal rat cardiomyocytes. Apoptosis rate and lactate dehydrogenase (LDH) leakage in culture medium were measured as indices of cell injury. Immunofluorescence staining showed the morphological changes of ER and intracellular translocation of CRT. Western blotting or reverse transcription polymerase chain reaction was used to detect the expression of target molecules. Compared with control, H/R increased apoptosis rate and LDH activity. The ER became condensed and bubbled, and CRT translocated to the nucleus. Western blotting showed up-regulation of CRT, Nrf2, activating transcription factor 4 (ATF4), CHOP and caspase-12 expression after H/R. Exogenous CRT overexpression induced by plasmid transfection before H/R increased cell apoptosis, LDH leakage, ER disorder, CRT nuclear translocation and the expression of ERS-associated molecules. However, administration of the ERS inhibitor, taurine, or CRT siRNA alleviated cell injury, ER disorder, and inhibited ERS-associated apoptosis. Our results indicated that during H/R stress, CRT translocation increases cell apoptosis and LDH leakage, aggravates ER disorder, up-regulates expression of nuclear transcription factors, Nrf2 and ATF4, and activates ERS-associated apoptosis.