Abstract
AimsStress granules (SGs) are transient cytoplasmic mRNA and protein complexes that form in eukaryotic cells under stress. SGs are related to multiple diseases, but there are no reports of the existence of SGs in atrial fibrillation (AF).Methods and resultsCell models of AF were established by field stimulation at 600 times per minute. HL-1 cells, cardiomyocytes and cardiac fibroblasts were transfected with G3BP1-cDNA plasmid by Lipofectamine 2000. The presence of SGs was detected by immunofluorescence analysis against GTPase-activating protein SH3 domain binding protein 1 (G3BP1) and poly(A)-binding protein 1 (PABP-1) and electron microscopy. Stable HL-1 cell lines transfected with lentivirus overexpressing G3BP1were constructed to further induce the formation of SGs in AF. Reactive oxygen species (ROS) and calcium overload in tachypaced HL-1 cells were studied by flow cytometry. The effects of G3BP1 overexpression on cardiac fibroblast proliferation and the protein expression level of collagen I/III and fibronectin-1 were also studied. Additionally, we detected protein synthesis in general and in single cells by puromycin incorporation in paced HL-1 cells. Here, we first showed that SGs are present in both tachypaced mouse cardiomyocytes and HL-1 atrial cells, although the presence is partial and at a low level. G3BP1 overexpression promoted SG formation, inhibited the rapid pacing-induced increase in ROS level, and attenuated calcium overload in HL-1 cells (all P<0.05). Furthermore, G3BP1 overexpression inhibited cardiac fibroblast proliferation (P<0.05) and decreased the protein expression level of collagen I and fibronectin-1 in cardiac fibroblasts stimulated by angiotensin II (all P<0.05). The bulk puromycin incorporation analyzed by Western blot did not show a global reduction in protein synthesis. However, puromycin incorporation in single cells detected by immunofluorescence analysis showed that protein synthesis in SG-containing cells significantly reduced (P<0.01).ConclusionsSGs are rapidly induced and present partially in AF, and G3BP1 overexpression promotes SG formation and confers cytoprotection against oxidative stress, calcium overload and atrial fibrosis in AF.
Highlights
In response to a stress environment, such as oxidative stress, heat shock, ultraviolet radiation and viral infection, one of the main defense mechanisms of eukaryotic cells is the formation of stress granules (SGs)
We detected the expression of endogenous poly(A)-binding protein 1 (PABP-1) protein by red fluorescent antibody and exogenous GTPase-activating protein SH3 domain binding protein 1 (G3BP1) by green fluorescent antibody to locate SGs
SGs were observed at 1 h after pacing as large particles scattered in the cytoplasm
Summary
In response to a stress environment, such as oxidative stress, heat shock, ultraviolet radiation and viral infection, one of the main defense mechanisms of eukaryotic cells is the formation of stress granules (SGs). Cells either activate protective mechanisms or initiate apoptosis, depending on the type and level of stress [1]. SGs are cytoplasmic contents that lack an envelope and are not present under normal growth conditions but can be induced by stress stimulation. Previous studies have shown that the composition of the SGs is not immutable, and SGs have distinct components under different stress conditions; for example, SGs induced by heat shock contain HSP27, which is not present in the granules induced by arsenite [3]. A recent study suggested that approximately 20% of SG diversity is stress- or cell-type-dependent [4]
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