The exact mechanism of atrial fibrillation (AF) has been not well elucidated. Ferroptosis is an iron-dependent cell death due to excessive accumulation of peroxidized polyunsaturated fatty acids. However, the molecular mechanism underlying AF and ferroptosis has never been reported. Here, we established the rapid pacing model in vivo and vitro to investigate the relationship between AF and ferroptosis. In canine model of rapid atrial pacing, the content of malondialdehyde and total ions in the atrial tissue of the Pacing group was significantly increased and the exosome inhibitor GW4869 reduced ferroptosis, fibrosis, and inflammation and improved histological and electrophysiological remodeling. In rapid pacing h9c2 cells, the expression of antioxidative stress genes associated with ferroptosis presented sequential changes and proteins involved in ferroptosis such as FTH1, SLC7A11, and GPX4 were gradually depleted. Furthermore, pacing cardiac fibroblast-derived exosomes (CF-exos) exacerbated ferroptosis in h9c2 cells and pretreated pacing-CF-exos with GW4869 alleviated injury to h9c2 cells. In mechanism, our results demonstrated that pacing-CF-exos highly expressed miR-23a-3p by informatics analysis and experimental verification. Inhibitor-miR-23a-3p protected h9c2 cells from ferroptosis accompanying with upregulation of SLC7A11. In addition, SLC7A11 was shown to be the target gene of miR-23a-3p. In conclusion, our results suggest that CF-exos-miR-23a-3p may promote ferroptosis. The development of AF in a persistent direction could be prevented by intervening with exosomal miRNAs to reduce oxidative stress injury and ferroptosis.
Purpose the aim of this study was to investigate the role of intermediate-conductance Ca2+-activated K+ (KCNN4, KCa3.1) in exosomes secretion of atrial myocytes. Methods eighteen beagles were randomly divided into Sham group (n = 6), Pacing group (n = 6), and Pacing + TRAM-34 group (n = 6). The in vivo electrophysiological data such as effective refractory period, atrial fibrillation (AF) induction, and AF duration were collected by programmed stimulation. Atrial tissues were stained with Hematoxylin & Eosin and Masson’s trichrome. The expression of KCa3.1 and Rab27a were accessed by immunohistochemistry and western blot. The downstream signaling pathways involved in KCa3.1 were explored by rapid pacing and overexpressing KCNN4 in HL-1 cells. Results TRAM-34 (KCa3.1 blocker) significantly inhibits electrical remodeling, inflammation, fibrosis, and exosomes secretion in rapid atrial pacing canines. More importantly, the vitro experiments demonstrated that KCa3.1 regulates the exosomes secretion through AKT/Rab27a signaling pathways. The use of calcium chelator, AKT inhibitor and si-Rab27a also significantly inhibit the exosomes secretion. Moreover, exosomes derived from rapid pacing HL-1 cells promote M1 polarization. Conclusions This study found that KCa3.1 promotes pro-inflammatory exosome secretion through the AKT/Rab27a signaling pathway. Inhibition KCa3.1/AKT/Rab27a signal pathway reduces myocardial tissue structure remodeling in AF.
Background: We previously found that intermediate conductance Ca 2+ -activated K + channel (SK4) might be an important target in atrial fibrillation (AF). Objective: To investigate the role of SK4 in AF maintenance. Methods: Twenty beagles were randomly assigned to the sham group (n=6), pacing group (n=7), and pacing+TRAM-34 group (n=7). Rapid atrial pacing continued for 7 days in the pacing and TRAM-34 groups. During the pacing, the TRAM-34 group received TRAM-34 intravenous injection (10 mg/Kg) 3 times per day. Atrial fibroblasts isolated from canines were treated with angiotensin II or adenovirus carrying the SK4 gene (Ad-SK4) to overexpress SK4 channels. Results: TRAM-34 treatment significantly suppressed the increased intra-atrial conducting time (CT) and AF duration in canines after rapid atrial pacing (P<0.05). Compared with the sham group, the expression of SK4 in atria was higher in the pacing group, which was associated with an increased number of myofibroblasts and levels of extracellular matrix in atrium (all P<0.05), and this effect was reversed by TRAM-34 treatment (all P<0.05). In atrial fibroblasts, the increased expression of SK4 induced by angiotensin II stimulation or Ad-SK4 transfection contributed to higher levels of P38, ERK1/2 and their downstream factors c-Jun and c-Fos, leading to the increased expression of α-SMA (all P<0.05), and all these increases were markedly reduced by TRAM-34 treatment. Conclusion: SK4 blockade suppressed AF by attenuating cardiac fibroblast activity and atrial fibrosis, which was realized through not only a decrease in fibrogenic factors but also inhibition of fibrotic signaling pathways.
Purpose. The aim of this study was to investigate the role of the medium-conductance calcium-activated potassium channel (KCNN4, KCa3.1) in the secretion of proinflammatory exosomes by atrial myocytes. Methods. Eighteen beagles were randomly divided into the sham group ( n = 6 ), pacing group ( n = 6 ), and pacing+TRAM-34 group ( n = 6 ). Electrophysiological data, such as the effective refractory period, atrial fibrillation (AF) induction, and AF duration, were collected by programmed stimulation. Atrial tissues were subjected to hematoxylin and eosin, Masson’s trichrome, and immunofluorescence staining. The expression of KCa3.1 and Rab27a was assessed by immunohistochemistry and western blotting. The downstream signaling pathways involved in KCa3.1 were examined by rapid pacing or overexpressing KCNN4 in HL-1 cells. Results. Atrial rapid pacing significantly induced electrical remodeling, inflammation, fibrosis, and exosome secretion in the canine atrium, while TRAM-34 (KCa3.1 blocker) inhibited these changes. Compared with those in control HL-1 cells, the levels of exosome markers and inflammatory factors were increased in pacing HL-1 cells. Furthermore, the levels of CD68 and iNOS in macrophages incubated with exosomes derived from HL-1 cells were higher in the pacing-exo group than in the control group. More importantly, KCa3.1 regulated exosome secretion through the AKT/Rab27a signaling pathway. Similarly, inhibiting the downstream signaling pathway of KCa3.1 significantly inhibited exosome secretion. Conclusions. KCa3.1 promotes proinflammatory exosome secretion through the AKT/Rab27a signaling pathway. Inhibiting the KCa3.1/AKT/Rab27a signaling pathway reduces myocardial tissue structural remodeling in AF.
Background: Fibroblast-derived exosomes can regulate the electrical remodeling of cardiomyocytes, and the KCa3.1 channel is an important factor in atrial electrical remodeling; however, the underlying molecular mechanisms that influence the fibroblast-derived exosomes on the electrical remodeling of cardiomyocytes are blurry. Therefore, our objective is to study the regulation of cardiac electrophysiology by exosomes linked to KCa3.1. Methods: Atrial myocytes (AMs) and atrial fibroblasts collected from Sprague-Dawley suckling rats were isolated and cultured individually. The cellular atrial fibrillation (AF) model was then established via electrical stimulation (1.0 v/cm, 10 Hz), and fibroblast-derived exosomes were isolated via ultracentrifugation. Moreover, these exosomes were co-cultured with AMs to investigate their influences on KCa3.1 and its potential mechanism. Various techniques, such as nanoparticle tracking analysis, transmission electron microscopy, whole-cell patch clamp technique, reverse-transcription polymerase chain reaction (RT-PCR), Western blot, and immunofluorescence, were used. Results: Rapid pacing promoted the secretion of exosomes from atrial fibroblasts ( P < 0.05), along with elevating the miR-21-5p expression level in atrial fibroblasts and exosomes ( P < 0.01). The expression of protein and current density of the KCa3.1 channel significantly increased after rapid pacing in AMs (0.190 ± 0.010 vs. 0.513 ± 0.057, P < 0.001). The KCa3.1 channel expression and PI3K/AKT pathway were further amplified after co-culturing of AMs with exosomes secreted by atrial fibroblasts (0.513 ± 0.057 vs. 0.790 ± 0.020, P < 0.001). However, the KCa3.1 expression was reversed after the cells were co-cultured with exosomes secreted by atrial fibroblasts transfected with miR-21-5p inhibitors (0.790 ± 0.020 vs. 0.570 ± 0.056, P < 0.001) or after the use of LY294002, a PI3K/AKT pathway inhibitor (0.676 ± 0.025 vs. 0.480 ± 0.043, P < 0.001). Conclusions: Rapid pacing promoted the secretion of exosomes from fibroblasts, and the miR-21-5p was upmodulated in exosomes. Moreover, the miR-21-5p enriched in exosomes up-regulated the KCa3.1 channel expression in AMs via the PI3K/AKT pathway.
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