Date Published: April 3, 2019
Publisher: Public Library of Science
Author(s): Guo Dong, Fengying Liang, Bo Sun, Chengcheng Wang, Yangyang Liu, Xiangpeng Guan, Bo Yang, Chunhong Xiu, Ning Yang, Fengyu Liu, Tianyi Lu, Wei Han, Georg Stoecklin.
Stress 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).
Cell 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). SGs 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.
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 . SGs are cytoplasmic contents that lack an envelope and are not present under normal growth conditions but can be induced by stress stimulation. SGs typically include untransformed mRNAs, small 40S ribosomal subunits, mRNA-associated translation initiation complexes and many RNA-binding proteins, some of which are specific to SGs, such as Ras-GAP SH3-binding protein (G3BP1) . A lack of these unique proteins will block the formation of SGs . 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 . A recent study also suggested that approximately 20% of SG diversity is stress- or cell-type-dependent .
In the present study, we demonstrated for the first time that there were SGs in cardiomyocytes following AF. We transfected the exogenous G3BP1 overexpression plasmid into HL-1 cells and observed the formation of SGs and a series of morphological changes in SGs under different pacing times. Then, we demonstrated that G3BP1 overexpression not only promoted the formation of SG, reduced ROS accumulation and calcium overload but also inhibited fibrogenesis induced by AngII stimulation. G3BP1 overexpression can also reduce cell proliferation and inhibit the expression of COLI and FN1. This finding may play an important role in further studies of the pathophysiological mechanisms and treatments of AF.
In summary, we were the first to discover that SGs exist in AF myocytes although the presence was partial and at a low level. At the same time, we also found that G3BP1 overexpression had an inhibitory effect on ROS aggregation and calcium overload, as well as collagen expression under stress conditions in AF. The dual effects of G3BP1 on electrical and structural changes induced by AF in myocardial cells will provide a new perspective for the treatment of AF.