Attenuation of the upregulation of NF-κB and AP-1 DNA-binding activities induced by tunicamycin or hypoxia/reoxygenation in neonatal rat cardiomyocytes by SERCA2a overexpression

The present study aimed to investigate the effects of the overexpression of sarco/endoplasmic reticulum Ca(2+)-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 in vitro 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.