Medical School of Chinese PLA, Beijing 100853, P.R. China.
Department of Cardiology, The Second Medical Center, Chinese PLA General Hospital, Beijing 100853, P.R. China.
Int J Mol Med. 2021 Jun;47(6). doi: 10.3892/ijmm.2021.4946. Epub 2021 Apr 28.
The present study aimed to investigate the effects of the overexpression of sarco/endoplasmic reticulum Ca‑ATPase (SERCA2a) on endoplasmic reticulum (ER) stress (ERS)‑associated inflammation in neonatal rat cardiomyocytes (NRCMs) induced by tunicamycin (TM) or hypoxia/reoxygenation (H/R). The optimal multiplicity of infection (MOI) was 2 pfu/cell. Neonatal Sprague‑Dawley rat cardiomyocytes cultured were infected with adenoviral vectors carrying SERCA2a or enhanced green fluorescent protein genes, the latter used as a control. At 48 h following gene transfer, the NRCMs were treated with TM (10 µg/ml) or subjected to H/R to induce ERS. The results of electrophoretic mobility shift assay (EMSA) revealed that overexpression of SERCA2a attenuated the upregulation of nuclear factor (NF)‑κB and activator protein‑1 (AP‑1) DNA‑binding activities induced by TM or H/R. Western blot analysis and semi‑quantitative RT‑PCR revealed that the overexpression of SERCA2a attenuated the activation of the inositol‑requiring 1α (IRE1α) signaling pathway and ERS‑associated apoptosis induced by TM. The overexpression of SERCA2a also decreased the level of phospho‑p65 (Ser536) in the nucleus, as assessed by western blot analysis. However, the overexpression of SERCA2a induced the further nuclear translocation of NF‑κB p65 and higher levels of tumor necrosis factor (TNF)‑α transcripts in the NRCMs, indicating the occurrence of the ER overload response (EOR). Therefore, the overexpression of SERCA2a has a 'double‑edged sword' effect on ERS‑associated inflammation. On the one hand, it attenuates ERS and the activation of the IRE1α signaling pathway induced by TM, resulting in the attenuation of the upregulation of NF‑κB and AP‑1 DNA‑binding activities in the nucleus, and on the other hand, it induces EOR, leading to the further nuclear translocation of NF‑κB and the transcription of TNF‑α. The preceding EOR may precondition the NRCMs against subsequent ERS induced by TM. Further studies using adult rat cardiomyocytes are required to prevent the interference of EOR. The findings of the present study may enhance the current understanding of the role of SERCA2a in cardiomyocytes.
本研究旨在探讨肌浆网/内质网 Ca2+-ATP 酶（SERCA2a）过表达对衣霉素（TM）或缺氧/复氧（H/R）诱导的新生大鼠心肌细胞（NRCMs）内质网应激（ERS）相关炎症的影响。最佳感染复数（MOI）为 2 个病毒颗粒/细胞。将携带 SERCA2a 或增强型绿色荧光蛋白基因的腺病毒载体感染培养的新生 Sprague-Dawley 大鼠心肌细胞，后者用作对照。基因转移后 48 小时，用 TM（10μg/ml）处理 NRCMs 或进行 H/R 以诱导 ERS。电泳迁移率变动分析（EMSA）的结果表明，SERCA2a 的过表达可减弱 TM 或 H/R 诱导的核因子（NF）-κB 和激活蛋白-1（AP-1）DNA 结合活性的上调。Western blot 分析和半定量 RT-PCR 显示，SERCA2a 的过表达可减弱 TM 诱导的肌醇需求酶 1α（IRE1α）信号通路的激活和 ERS 相关凋亡。Western blot 分析还表明，SERCA2a 的过表达可降低细胞核中磷酸化 p65（Ser536）的水平。然而，SERCA2a 的过表达可诱导 NF-κB p65 的进一步核转位，并使 NRCMs 中肿瘤坏死因子-α（TNF-α）转录本水平升高，表明内质网过载反应（EOR）的发生。因此，SERCA2a 的过表达对 ERS 相关炎症具有“双刃剑”效应。一方面，它减弱了 TM 诱导的 ERS 和 IRE1α 信号通路的激活，导致核中 NF-κB 和 AP-1 DNA 结合活性的上调减弱，另一方面，它诱导了 EOR，导致 NF-κB 的进一步核转位和 TNF-α 的转录。先前的 EOR 可能使 NRCMs 对 TM 诱导的后续 ERS 具有预适应能力。需要使用成年大鼠心肌细胞进一步研究以防止 EOR 的干扰。本研究的结果可能增强对 SERCA2a 在心肌细胞中作用的现有认识。
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