KCa3.1 promotes exosomes secretion by activating on AKT/Rab27a in atrial myocytes during rapid pacing

KCa3.1 promotes exosomes secretion by activating on AKT/Rab27a in atrial myocytes during rapid pacing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article KCa3.1 promotes exosomes secretion by activating on AKT/Rab27a in atrial myocytes during rapid pacing Dishiwen Liu, Huiyu Chen, Yuntao Fu, Mei Yang, Yajun Yao, Shanqing He, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2029745/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Mar, 2023 Read the published version in Cardiovascular Therapeutics → Version 1 posted You are reading this latest preprint version Abstract Purpose the aim of this study was to investigate the role of intermediate-conductance Ca 2+ -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. KCa3.1 exosomes atrial fibrillation structure remodeling macrophage Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Atrial fibrillation (AF) is a major public health problem worldwide with high morbidity and mortality [ 1 ] . At present, there are many evidences that AF is closely related to electrical remodeling, structural remodeling and neural remodeling [ 2 – 4 ] . However, the mechanism of electrical-substrate remodeling for AF induction and maintenance has not been fully elucidated. Medium-conductance calcium-activated potassium channel (KCNN4, KCa3.1) is one of the family members of calcium-activated potassium channel. In cardiomyocytes induced by pluripotent stems from patients with catecholamine-sensitive polymorphic ventricular tachycardia, KCa3.1 blocker significantly reduced the occurrence of arrhythmias, and affects delayed post-depolarization and calcium transients [ 5 ] . Our previous research showed that KCa3.1 was involved in the early post-depolarization and triggering activities of the myocardium, and the KCa3.1 inhibitor TRAM-34 significantly inhibited electrical remodeling and completely inhibited the induction of acute AF [ 6 ] . Our recent study further showed that TRAM-34 abrogated atrial fibrosis and inflammation in rapid atrial pacing canine [ 7 ] . But the mechanism of how KCa3.1 drives structural remodeling through ion channel remodeling needs further study. Exosomes are extracellular vesicles carried a variety of signaling molecules from donor cells (DNA, mRNA, microRNA, lncRNA and protein) [ 8 ] . Rab27a, a member of the Rab family, affects exosomes secretion by regulating the docking and fusing multiple vesicles bodies with the cell membrane [ 9 ] . Inhibition of Rab27a significantly down-regulates the secretion of MMP9 and platelet-derived growth factor A [ 10 ] . Studies have reported that PI3K/AKT affected Rab27a-related vesicles secretion by regulating the phosphorylation of Rab27a-binding protein JFC1 [ 11 ] . In another study, their results suggested that KCa3.1 is closely related to the PI3K/AKT signaling pathway [ 12 ] . Based on these studies, we hypothesized that KCa3.1 may regulate Rab27a through the AKT signaling pathway to affect exosomes secretion. The purpose of this study is to explore whether the abnormal expression of KCa3.1 leads to exosomes secretion and the downstream signaling pathways involved. Materials And Methods This study was approved by the animal studies subcommittee of our institutional review board and was in accordance with the guidelines of the National Institutes of Health for the care and use of laboratory animals. Animal Model Preparation The rapid atrial pacing canine model was prepared as we previously described [13] . Briefly, eighteen beagles were randomly divided into Sham group (n=6), Pacing group (n=6), and Pacing+TRAM-34 group (n=6). Three days after implanting the pacemaker, the Pacing+TRAM-34 group was given an intravenous injection of TRAM-34 (10 mg/kg/d, tid, MCE, United States). The Pacing group and Pacing+TRAM-34 group were paced at 450 bpm for 7 days. Electrophysiological measurements After seven consecutive days of intravenous administration TRAM-34, the canines were anaesthetized and bilateral thoracotomy was performed. Procedural stimulation was performed after placing multi-lead electrodes in various parts of the atrium. In brief, we measured the atrial effective refractory period (ERP), and the inducibility and duration of AF [13] . Histopathology Analysis For histological analysis, canine atrial tissues were fixed, embedded, and sectioned into 5 µm thick slices. Sections were used to stain hematoxylin & eosin and Masson’s trichrome to assess inflammatory infiltration and collagen deposition, respectively. To detect the expression of KCa3.1 and Rab27a, sections were incubated with KCa3.1 antibody (Affinity, DF4132; 1:100) and Rab27a antibody (Proteintech, 17817-1-AP; 1:300). Immunohistochemistry and Immunofluorescence images were captured with fluoroscope and analyzed by Image J. Cell culture and Treatment The mouse atrial HL-1 cell line (Procell, China) or mouse macrophage cell line RAW264.7 (Wuhan University), was cultured using F12/DMEM medium (Gibico, United States) or DMEM (High glucose, Gibico, United States) containing 10% fetal bovine serum and 1% penicillin/streptomycin, intervening when the cells grew to 60-70%. The rapid pacing HL-1 cell model was prepared as we previously described [13] . In brief, the HL-1 cells were paced by electric field stimulation (600 times/min), intensity 1.5 V/cm, continuous stimulation for 48 h. Lentivirus and Cell Infection Lenti-KCNN4 and lenti-GFP viruses were produced at Genechem (Shanghai, China). HL-1 cells were infected with lenti-KCNN4 and lenti-GFP viruses at a multiplicity of infection of 30 MOI for 24 hours. Subsequently, pressure screening was performed using puromycin at 3 µg/ml for 72 h. Then, the dose of puromycin adjust to 1 µg/ml for routine pressure screening. Expression of KCa3.1 was evaluated by western blotting. siRNA Transfection siRNA duplexes targeting KCNN4 and a negative control siRNA were designed and synthesized by GenePharma (Shanghai, China). The KCNN4 siRNA sequences were as follows: sense 5’-GCUAGGAAGCUUCAGUUAA-3’ and anti-sense 5’-UUAACUGAAGCUUCCUAGC-3’. Rab27a siRNA sequences were as follows: 5’-CGGAUCAGUUAAGUGAAGAAA-3’ and anti-sense 5’-UUCUCCGAACGUGUCACGU-3’. For cell transfections, cells were grown to 60-80% confluency in 6-well dishes. For each well, serum-free F12/DMEM was mixed separately with siRNA and Lipofectamine 6000 (Beyotime, China) for 5 min. Then, the siRNA and Lipofectamine 6000 were mixed gently and were incubated at room temperature for 10 min. Subsequently, the diluted siRNA/Lipofectamine 6000 complex was added to the 6-well dish for 5 h, after which the complex was replaced with normal cell culture medium. Exosome Isolation Cell debris and large vesicles were separated by differential centrifugation at 300 g, 3000 g, 10000 g. Subsequently, the supernatant was concentrated using ultrafiltration tubes (Millipore, 10000 MW, United States) at 4000 g centrifugal force. Finally, the supernatant was ultracentrifuged at 120000 g for 90 min to obtain exosomes. All manipulations were carried out at 4 °C. Exosomes were dissolved in PBS and stored at -80 °C for subsequent detection. Transmission Electron Microscopy The exosome solution was fixed with 4% paraformaldehyde. Exosomes were then adsorbed onto formvar-carbon-coated copper grids. Subsequently, grids were rinsed in PBS and negatively stained with 2% uranyl acetate for 5 min at room temperature, and then photographed with a JEM-1100 transmission electron microscope at an accelerating voltage of 80 kV. Western Blotting Take 30 mg of canine atrial tissue or cultured cells for protein extraction. Protein concentration was determined by a BCA Protein Assay Kit (Aspen, as1086) according to the manufacturer’s instruction. Proteins were separated by 10% SDS-polyacrylamide gel and transferred to 0.45 µm PVDF membrane by semi-dry transfer. PVDF membranes were blocked with 1% polyvinylpyrrolidone-40 and 0.05% Tween-20 for 30 min. The expression of the target protein was determined by incubating with the following primary antibodies overnight at 4°C: GAPDH (Service bio, China, 1:1000), CD81 (Abmart, China, 1:1000), Rab27a (Service bio, China, 1:1000), KCa3.1 (Protein tech, China, 1:1000), TSG101 (Service bio, China, 1:1000), AKT (Service bio, China, 1:1000), p-AKT (Abmart, China, 1:1000). Visualization was performed on a chemiluminescence system after incubation with horseradish peroxidase-conjugated secondary antibodies (Proteintech, China, 1:3000) at room temperature. Quantitative real-time PCR Total RNA was extracted from cells using TRIzol® reagent (Takara, Japan). Isolated RNA (2 μg) was converted into complementary DNA using RT First Strand cDNA Synthesis Kit (Servicebio, China). The cDNA templates were amplified by qRT-PCR system (Applied Biosystem, United States) using SYBR Green PCR Mix (Servicebio, China) with the corresponding primers (IL-6: 5’-TGTGCAATGGCAATTCTGAT-3’, 5’-GGTACTCCAGAAGACCAGAGGA-3’; iNOS: 5’-GCTCATGACATCGACCAGAA-3’, 5’-TGTTGCATTGGAAGTGAAGC-3’). The 2 -ΔΔCt comparative quantification method was used to analyze the semilog amplification curves, and the expression of gene was normalized to GAPDH. Statistics Results are represented as mean ± SD. The comparison among the different groups was performed with one-way ANOVA and post hoc Tukey test. p <0.05 is regarded as statistically significant. All data were analyzed using GraphPad Prism 8 (GraphPad, United States) or SPSS 25.0 (IBM, United States). Result TRAM-34 inhibits the electrical remodeling in canines with rapid atrial pacing Compared with the sham group, the ERP at the recording sites was significantly shortened in the pacing group, and RIPV, LSPV and LIPV increased when treated with TRAM-34 (113.7 ± 5.8 vs. 101.2 ± 10.4 ms in the RIPV, P < 0.05; 121.3 ± 3.5 vs. 109.3 ± 8.0 ms in the LSPV, P < 0.05; 118.0 ± 6.1 vs. 105.7 ± 6.3 ms in the LIPV) (Figure 1 A). Obviously, AF was more easily to be induced in the pacing group, and the administration of TRAM-34 reduced the AF induction by 1.8 times (3.5 ± 1.0 vs. 5.3 ± 1.6 times, P < 0.05) (Figure 1 B). The effect of TRAM-34 on electrophysiology is also reflected in shortening the duration of AF. Compared with pacing group, TRAM-34 reduced the mean duration of AF by at least 10 s (22.2 ±7.3 vs. 36.3 ± 5.9 s, P < 0.01) (Figure 1 C). We did not observe significant differences in disperse ERP among the three groups (Figure 1 D). Blockade KCa3.1 inhibits exosomes secretion in rapid pacing canine atrial tissue We explored in vitro whether rapid pacing leads to increased exosomes secretion and the KCa3.1 specific blocker TRAM-34 on exosomes secretion. Our results found that KCa3.1 expression was increased in the canine atrial tissue of rapid pacing, which was supported by both western blotting and immunohistochemical results. As expected, compared with pacing group, TRAM-34 significantly reduced KCa3.1 level (Figure 2 A, B, C). Rab27a, which is related to exosomes secretion, was also significantly increased in the pacing group. Consistent with the trend of KCa3.1, the administration of TRAM-34 also reduced the expression of Rab27a in the pacing group, which was supported by the results of immunoblotting and immunohistochemistry (Figure 2 A, B, E). Besides, immunoblotting results showed that the expressions of exosome-related markers such as CD81 and TSG101 were increased in pacing group, but these anomalies could be blocked by TRAM-34 (Figure 2 B, F, G). Compared with the sham group, the expression of phosphorylated AKT was significantly increased in the pacing group, and TRAM-34 could reverse these changes (Figure 2 B, D). At the histochemical level, TRAM-34 could significantly reduce inflammatory infiltration and the deposition of collagen in the atrial tissue of the pacing group (Figure 2 A). Taken together, TRAM-34 may reduce structural remodeling in AF through the AKT/Rab27a signaling pathway. Rapid pacing leads to HL-1 cells increased secretion To further confirm whether KCa3.1 is overexpressed in atrium myocytes, we paced HL-1 cells for 48 hours. Immunofluorescence demonstrated that KCa3.1 in the pacing group was significantly higher than that in the control group, while TRAM-34 reduced the expression of KCa3.1 (Figure 3 A, B). In order to qualitatively and quantitatively explore the effect of rapid pacing on exosomes secretion, we extracted exosomes from HL-1 cell supernatants cultured at the same density and examined their morphology and quantity. Transmission electron microscopy indicated that there were no obvious changes in the morphology of exosomes (Figure 3 C). Nanoparticle tracking analysis result demonstrated that the size of the vesicles ranged between 33 and 230 nm, most of which were 90-190 nm in diameter. After rapid pacing, the concentration of cellular supernatant exosomes rose to (6.97 ± 0.42) × 10 6 particles/ml from (6.17 ± 0.15) ×10 6 particles/ml, which was decreased to (6.10 ± 0.10) × 10 6 particles/ml by TRAM-34 treatment (Figure 3 D, E). The results suggested that rapid pacing increases the release of exosomes, which was dampened by TRAM-34 treatment. The levels of TNF-α, IL-1β, IL-6 and TGF-β1 in the cell supernatants were significantly higher in the pacing group when compared with the control group (Figure 3 F-I). Furthermore, compared with the pacing group, BAPTA decreased the levels of TNF-α, IL-1β, IL-6 (Figure 3 F-H), while GSK690693 decreased IL-1β, IL-6 (Figure 3 F, G), and si-Rab27a decreased TGF-β1 (Figure 3 I). Taken together, these results suggest that blocking the KCa3.1/AKT/Rab27a signaling pathways could reduce the exocrine function of HL-1 cells. Rapid pacing HL-1 cell exosomes promote macrophage polarization By co-incubating the extracted HL-1 cell exosomes with macrophages, we found that these exosomes have pro-inflammatory effects. Immunofluorescence results showed that CD68 and iNOS were significantly increased in RAW264.7 macrophages at the translation level (Figure 4 A, B, C). Moreover, it was also verified by RT-PCR that iNOS and IL-6 were significantly increased at the transcription level in macrophages (Figure 4 D, E). These results suggest that macrophages are polarized towards M1. Blockade intracellular calcium reduces AKT activation To determine whether KCa3.1 affects AKT and downstream signaling via calcium ions, we used the calcium chelator BAPTA, AKT inhibitor GSK690693 and si-Rab27a to intervene in rapid pacing HL-1 cells. Our result demonstrated that the expression of KCa3.1 did not change significantly (Figure 5 A, C), indicating that the blockade of downstream cascade does not affect the expression of KCa3.1 through feedback regulation. By intervening intracellular calcium with BAPTA, we found that the phosphorylation level of AKT was significantly reduced in pacing group (Figure 5 A, D). Besides, the downstream signaling pathways, including Rab27a, TSG101, and CD81, were significantly increased in pacing group, while BAPTA alleviated their expression (Figure 5 A, E, F, G). These results support that KCa3.1 affects downstream signaling pathways mainly by affecting intracellular calcium concentration. Activation of the AKT affects the expression of Rab27a We found that p-AKT was elevated in a dose-dependent manner by activating AKT using SC79 ((Figure 5 B, H). When the concentration of SC79 reached 20 µM, the HL-1 cells showed massive death. The expression of Rab27a also increased with the increase of p-AKT (Figure 5 B, I). The expression of the downstream exosome markers CD81 and TSG101 were significantly increased (Figure 5 B, J, L). In contrast, GSK690693, an inhibitor of AKT, significantly reduced the level of p-AKT and affected the expression of Rab27a (Figure 5 A, E) and the downstream signaling pathway (Figure 5 A, F, G). Undoubtedly, by Knocking down the expression of Rab27a, the expression of exosome markers CD81 and TSG101 were significantly reduced (Figure 5 A, E, F). However, knockdown of Rab27a significantly affected the expression of p-AKT (Figure 5 A, C). Hence, Rab27a may regulate the expression of AKT through feedback mechanism. Through immunofluorescence, we could more intuitively observe the effect of BAPTA, GSK1690693, si-Rab27a on the expression of downstream effector molecule Rab27a. Compared with the pacing group, administration of BAPTA, GSK690693 and si-Rab27a could significantly reduce the fluorescence intensity of Rab27a (Figure 5 K, M). These results demonstrated that intervening KCa3.1/AKT/Rab27a signaling pathway reduces exosomes secretion. Overexpression KCNN4 affects AKT/Rab27a signaling pathways By transfecting lentivirus to construct a stable KCNN4 overexpressing HL-1 cell line, we found that the cell growth was significantly increased, confirming the properties of the proto-oncogene of KCNN4. Compared with KCNN4 group, TRAM-34 significantly inhibited the expression of KCa3.1 (Figure 6 A, B). Certainly, TRAM-34 also affects the downstream signaling pathways to varying degrees. The expressions of p-AKT (Figure 6 A, C), Rab27a (Figure 6 A, D) and exosome markers (Figure 6 A, E, F) were significantly decreased after administration TRAM-34. Further exploration found that the use of calcium chelator, AKT inhibitors and si-Rab27a could significantly reduce the expression of AKT (Figure 6 G, H), Rab27a (Figure 6 G, I), and exosome markers (Figure 6 G, J, K). Therefore, circular experiments demonstrated that KCa3.1 overexpression increases exosomes secretion. Discussion This study explored the role of KCa3.1 in exosomes secretion in atrial myocytes during rapid pacing in vivo and vitro. We provide evidence for the following: (1) Overexpression of KCa3.1 promotes atrial structural remodeling in canine with rapid atrial pacing. (2) KCa3.1 promotes exosomes secretion by affecting the intracellular calcium concentration and activating the AKT/Rab27a signaling pathway. (3) These pro-inflammatory exosomes promote the polarization of macrophages towards M1. The KCa3.1 channel phosphorylation and channel activity are considered to have a vital arrhythmogenesis in patients with arrhythmogenic right ventricular cardiomyopathy [ 14 ] . It has been reported that clotrimazole or TRAM-34 reduce the occurrence of catecholaminergic polymorphic ventricular tachycardia [ 5 ] . Our previous study found that blockade of KCa3.1 overexpressed by macrophages in canine atrial tissue using TRAM-34 reduced the susceptibility of AF [ 7 ] . These evidences support the pro-arrhythmic role of KCa3.1, but the mechanism of how KCa3.1 maintains the progression of AF has not been clearly elucidated. Some studies have addressed how ionic remodeling drives structural remodeling. Wang et al. reported that and Ang II stimulates cell proliferation mediated by upregulating KCa3.1 channel through ERK1/2, p38-MAPK, and PI3K/AKT signaling pathways in cultured adult rat cardiac fibroblasts [ 15 ] . Oxidative stress also promotes myocardial fibrosis by upregulating KCa3.1 [ 16 ] . KCa3.1 was found to be overexpressed in diseases such as idiopathic pulmonary fibrosis [ 17 ] , renal fibrosis [ 18 ] , and postburn hypertrophic scar formation [ 19 ] . Using its specific blocker TRAM-34 inhibits the proliferation of fibroblasts and reduces the deposition of extracellular matrix by inhibiting the TGF-β1 signaling [ 19 ] . These results unveiled that the changes in ion channels have profound effects on cell proliferation and differentiation. The hyperproliferation and chemotaxis of fibroblasts and macrophages in myocardial tissue have an indelible effect on local structural remodeling. Structural remodeling provides an anatomical basis for reentry on the basis of ion remodeling of initiating factor. KCa3.1 channel is voltage independent and Ca 2+ sensitive, and its activation preserves the negative membrane potential required for sustained Ca 2+ influx via store-operated Ca 2+ channels. Ca 2+ handling abnormity promotes the secretion of exosomes has been reported earlier. Olivero et al. found that the depolarization-evoked release of exosomes from cortical synaptosomes occurred in a Ca 2+ -dependent fashion [ 20 ] . Interestingly, physical stimuli such as high frequency acoustic stimulation also leads to calcium-dependent exosomes release [ 21 ] . Our study found that calcium antagonist BAPTA reduced the expression of proteins related to exosomes secretion. Thus, these evidences also support increased exosomes secretion due to calcium handle abnormity in AF. Current studies have shown that overexpression of KCa3.1 indicates poor prognosis in patients, promotes tumor cell proliferation and differentiation, and may activate the PI3K/AKT signaling pathway mediated by calcium ions [ 22 , 23 ] . Indeed, our study also verified that overexpression of KCa3.1 leads to HL-1 cells hyperproliferation. As the downstream signaling of KCa3.1, that excessive activation of AKT promotes fibrosis. In cardiac fibroblasts, treatment with angiotensin II promotes extracellular matrix deposition by upregulating PI3K/AKT expression [ 24 ] . In acetylcholine and calcium chloride-induced mouse models of AF, AKT was also highly expressed in atrial tissue [ 25 ] . However, the corresponding degree of activation of the AKT signaling pathway in different cells is inconsistent. Different from Zhou et al.’s study [ 26 ] , we found that overactivation of AKT signaling pathway could be lethal to cardiomyocytes. We found that 20 µM of SC79 could kill all HL-1 cells. So, overexpression of AKT may have adverse effect on cardiomyocytes to some extent. So far, studies have not clearly defined how the activation of KCa3.1/AKT signaling pathway affects its downstream to play a biological regulatory role in cardiovascular disease. We found that either overexpression of KCa3.1 or rapid pacing resulted in enhanced exocrine function. This is mainly reflected in the increased expression of Rab27a, a protein related to exosomes secretion, and the increase of exosome markers. Interestingly, we found that TNF-α, IL-1, IL-6, and TGF-β1 in supernatants were significantly increased in the pacing group, which indicates that inferior stimulation may lead to enhanced cell secretion. This abnormal secretion phenomenon may change the surrounding microenvironment, including local inflammation or fibrosis and drives structural remodeling of the atrial tissue. Rab27a knockout impairs myeloperoxidase secretion of permeabilized neutrophils by interfering with the JFC1/Slp1-Rab27a secretion mechanism [ 27 ] . Jennifer et al. reported that AKT regulates JFC1/Slp1 function through phosphorylation and affects the secretion of Rab27a-containing vesicles [ 11 ] . Recent studies have shown that platelet-rich plasma promotes mesenchymal stem cells exosomal paracrine repair of acute kidney injury via the AKT/Rab27 pathway [ 28 ] . These findings support that AKT is the upstream signaling pathway of Rab27a. However, Zhou et al. found that Rab27a could affect the expression of AKT through feedback regulation by constructing Rab27a knockout mice [ 29 ] . Consistent with their observations, our study confirmed that silencing of the Rab27a affects the phosphorylation of AKT. The role of exosomes on macrophage polarization has been extensively studied. It has been reported that exosomes derived from inflammatory myoblasts or adipocytes promoted M1 polarization [ 30 , 31 ] . In addition to affecting cells in the surrounding microenvironment, exosomes also play a regulatory role through long-distance transport. Majumdar et al. reported exosomes mediate LTB4 release during neutrophil chemotaxis [ 32 ] . Besides, Eiji et al. verified that pro-inflammatory exosomes from lipotoxic hepatocytes activated macrophage chemotaxis. Our previous study found that TRAM-34 reduced the polarization and infiltration of macrophages in canine model with rapid atrial pacing. The current study hints that TRAM-34 may reduce M1 polarization by blocking KCa3.1 involved pro-inflammatory exosome release. In conclusion, our present studies showed that overexpression of KCa3.1 promotes proinflammatory exosomes secretion through the AKT/Rab27a signaling pathway. Inhibition KCa3.1/AKT/Rab27a signal pathway reduces myocardial tissue structure remodeling in AF. These results indicate that might provide a new therapeutic target for blocking the incidence of AF. Declarations Funding This work was supported by National Natural Science Foundation of China (81970277, 82170312). Author Contributions Dishiwen Liu: Conceptualization, Methodology, Validation, Investigation, Writing-original draft, review & editing. Qingyan Zhao: Investigation, Supervision, Writing-review & editing. Huiyu Chen, Yuntao Fu, Mei Yang: Conceptualization, Methodology. Yajun Yao, Shanqing He, Youcheng Wang: Validation, Investigation. Zhen Cao, Xuewen Wang: Investigation, Formal analysis. Data Availability All data involved in this study are available from the corresponding author upon reasonable request. 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Ranolazine improves oxidative stress and mitochondrial function in the atrium of acetylcholine-CaCl2 induced atrial fibrillation rats [J]. Life Sci, 2016, 156: 7-14; DOI: 10.1016/j.lfs.2016.05.026 Munafó D B, Johnson J L, Ellis B A, et al. Rab27a is a key component of the secretory machinery of azurophilic granules in granulocytes [J]. Biochem J, 2007, 402(2): 229-39; DOI: 10.1042/bj20060950 Ji C, Zhang J, Zhou Z, et al. Platelet-rich plasma promotes MSCs exosomes paracrine to repair acute kidney injury via AKT/Rab27 pathway [J]. Am J Transl Res, 2021, 13(3): 1445-57; DOI: Zhou W, Zheng X, Cheng C, et al. Rab27a deletion impairs the therapeutic potential of endothelial progenitor cells for myocardial infarction [J]. Mol Cell Biochem, 2021, 476(2): 797-807; DOI: 10.1007/s11010-020-03945-x Luo Z W, Sun Y Y, Lin J R, et al. Exosomes derived from inflammatory myoblasts promote M1 polarization and break the balance of myoblast proliferation/differentiation [J]. World J Stem Cells, 2021, 13(11): 1762-82; DOI: 10.4252/wjsc.v13.i11.1762 Song M, Han L, Chen F F, et al. Adipocyte-Derived Exosomes Carrying Sonic Hedgehog Mediate M1 Macrophage Polarization-Induced Insulin Resistance via Ptch and PI3K Pathways [J]. Cell Physiol Biochem, 2018, 48(4): 1416-32; DOI: 10.1159/000492252 Majumdar R, Tavakoli Tameh A, Arya S B, et al. Exosomes mediate LTB4 release during neutrophil chemotaxis [J]. PLoS Biol, 2021, 19(7): e3001271; DOI: 10.1371/journal.pbio.3001271 Cite Share Download PDF Status: Published Journal Publication published 29 Mar, 2023 Read the published version in Cardiovascular Therapeutics → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2029745","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":136077347,"identity":"51b2e675-4ec7-4beb-8b71-6b4552b41e64","order_by":0,"name":"Dishiwen Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIie3QsQrCMBCA4QuFdLnaNUV9h0pBB8U+Syj0BVwcI4FO4i76EHVzrGTo4gNYXHRxchCcOtWKTiJt3Rzyj8d9cByATveHtQwARgSgbUrxHBBRR+ibdJz5rimBFxm5B96UmNjL8q1CyM7yjjDqxolxOVUfht7Q2iskKy48hNCLEzpwa0i/TaIjGm0uAgTF4wQpqyNOXhLq7ER5XtGMMKskyMhMIiRNCJ0MrahAhlyStRt4S0X7lcS21SbLo9D3U6Vu1+m4u0jlpZJ89HyV8cO+TqfT6b73AGzwQJI13U71AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-5269-5754","institution":"Renmin Hospital of Wuhan University: Wuhan University Renmin Hospital","correspondingAuthor":true,"prefix":"","firstName":"Dishiwen","middleName":"","lastName":"Liu","suffix":""},{"id":136077348,"identity":"2d3d416b-75eb-4bbc-bda8-2b15ad365eb0","order_by":1,"name":"Huiyu Chen","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University: Wuhan University Renmin Hospital","correspondingAuthor":false,"prefix":"","firstName":"Huiyu","middleName":"","lastName":"Chen","suffix":""},{"id":136077349,"identity":"0dff1e1b-9c69-4e1f-bc0d-debf49176dd2","order_by":2,"name":"Yuntao Fu","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University: Wuhan University Renmin Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuntao","middleName":"","lastName":"Fu","suffix":""},{"id":136077350,"identity":"667babad-8ef9-477b-9125-5eb1ea6c192f","order_by":3,"name":"Mei Yang","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University: Wuhan University Renmin Hospital","correspondingAuthor":false,"prefix":"","firstName":"Mei","middleName":"","lastName":"Yang","suffix":""},{"id":136077351,"identity":"ee24f920-2378-40f1-b682-0fbaff395bd8","order_by":4,"name":"Yajun Yao","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University: Wuhan University Renmin Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yajun","middleName":"","lastName":"Yao","suffix":""},{"id":136077352,"identity":"66f06eeb-cdf5-4fcc-a800-7032b366ec99","order_by":5,"name":"Shanqing He","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University: Wuhan University Renmin Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shanqing","middleName":"","lastName":"He","suffix":""},{"id":136077353,"identity":"60a7a4d2-bdc6-41d8-a789-422e549c86c1","order_by":6,"name":"Youcheng Wang","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University: Wuhan University Renmin Hospital","correspondingAuthor":false,"prefix":"","firstName":"Youcheng","middleName":"","lastName":"Wang","suffix":""},{"id":136077354,"identity":"14c78dea-e502-466e-9bbb-f58ea917057f","order_by":7,"name":"Zhen Cao","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University: Wuhan University Renmin Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhen","middleName":"","lastName":"Cao","suffix":""},{"id":136077355,"identity":"cbccbf0b-3157-4906-a055-d6a8bba0fcd0","order_by":8,"name":"Xuewen Wang","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University: Wuhan University Renmin Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xuewen","middleName":"","lastName":"Wang","suffix":""},{"id":136077356,"identity":"891589fc-0a45-4466-889e-f61f6aaae816","order_by":9,"name":"Qingyan Zhao","email":"","orcid":"https://orcid.org/0000-0002-9610-2452","institution":"Renmin Hospital of Wuhan University: Wuhan University Renmin Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qingyan","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2022-09-04 04:01:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2029745/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2029745/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1155/2023/3939360","type":"published","date":"2023-03-30T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":26470707,"identity":"e556b771-c13a-4fb5-8e6b-38638a840533","added_by":"auto","created_at":"2022-09-14 20:52:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":166806,"visible":true,"origin":"","legend":"\u003cp\u003eCanines electrophysiological examination by programmed stimulation\u003c/p\u003e\n\u003cp\u003eA. Effective refractory period of different parts of the atrium. TRAM-34 increased the ERP of RIPV, LSPV and LIPV after rapid atrial pacing. B. Difference in AF inducibility, shown by the number of episodes. C. Difference in mean AF durations. D. difference in disperse effective refractory period.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SD of six biological replicates. *P \u0026lt; 0.05, **P \u0026lt; 0.01, and ***P \u0026lt; 0.001 vs. Sham group;#P \u0026lt; 0.05, ##P \u0026lt; 0.01, and ###P \u0026lt; 0.001 vs. Pacing group.\u003c/p\u003e\n\u003cp\u003eAbbreviations: RA, right atrium; RSPV, right superior pulmonary vein; RIPV, right inferior pulmonary vein; LA, left atrium; LSPV, left superior pulmonary vein; LIPV, left inferior pulmonary vein; ERP, effective refractory period)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2029745/v1/d52d757ffd3139755388fb08.png"},{"id":26470708,"identity":"37fbd375-f43e-4a64-a131-1f0d563c6afb","added_by":"auto","created_at":"2022-09-14 20:52:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4177134,"visible":true,"origin":"","legend":"\u003cp\u003eTRAM-34 attenuates structure remodeling and the secretion of exosomes in atrial tissue of rapid pacing\u003c/p\u003e\n\u003cp\u003eA. Representative images of inflammation, fibrosis, KCa3.1 and Rab27a as reflected by H\u0026amp;E staining, Masson staining and Immunohistochemistry.\u003c/p\u003e\n\u003cp\u003eB. Representative gel bands depicting after TRAM-34 administration in rapid atrial pacing of canines.\u003c/p\u003e\n\u003cp\u003eC, D, E, F, G. Levels of KCa3.1, p-AKT/AKT, Rab27a, TSG101 and CD81.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SD of six biological replicates.\u003c/p\u003e\n\u003cp\u003e*, **, and *** indicate p\u0026lt;0.05, 0.01, and 0.001, respectively.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2029745/v1/32745201e694df04e0bef30e.png"},{"id":26471767,"identity":"f1b87765-ce82-47bc-a4fa-8bec2394dfed","added_by":"auto","created_at":"2022-09-14 20:57:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1471553,"visible":true,"origin":"","legend":"\u003cp\u003eRapid pacing stimulates HL-1 cells secretion\u003c/p\u003e\n\u003cp\u003eA, B. Representative images of KCa3.1 as reflected by immunofluorescence.\u003c/p\u003e\n\u003cp\u003eC. Morphology of isolated exosomes from HL-1 cells using transmission electron microscopy.\u003c/p\u003e\n\u003cp\u003eD, E. Mean exosomes diameter and concentration shown by ZetaView System.\u003c/p\u003e\n\u003cp\u003eF, G, H, I. TNF-α, IL-β, IL-6, TGF-β1 concentration in HL-1 cells supernatant.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SD of three biological replicates.\u003c/p\u003e\n\u003cp\u003e*P \u0026lt; 0.05, **P \u0026lt; 0.01, and ***P \u0026lt; 0.001 vs. control group;#P \u0026lt; 0.05, ##P \u0026lt; 0.01, and ###P \u0026lt; 0.001 vs. pacing group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2029745/v1/783570d33931b2abb07e8987.png"},{"id":26470711,"identity":"6b2a3a87-2c57-4f9e-886b-5ead34ba1428","added_by":"auto","created_at":"2022-09-14 20:52:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":694770,"visible":true,"origin":"","legend":"\u003cp\u003eExosomes derived from rapid pacing HL-1 cells promotes macrophage polarization\u003c/p\u003e\n\u003cp\u003eA, B, C. Representative images of CD68 and iNOS as reflected by immunofluorescence.\u003c/p\u003e\n\u003cp\u003eD, E. RT-PCR analysis of iNOS and IL-6 expression normalized with GAPDH in macrophage.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SD of six biological replicates.\u003c/p\u003e\n\u003cp\u003e*, **, and *** indicate p\u0026lt;0.05, 0.01, and 0.001, respectively.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2029745/v1/49f0a3d54492795953fba8a6.png"},{"id":26470712,"identity":"5ca06080-c5d9-42ec-8083-8789b5b36eb5","added_by":"auto","created_at":"2022-09-14 20:52:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1058921,"visible":true,"origin":"","legend":"\u003cp\u003eRapid pacing activates KCa3.1/AKT/Rab27a signal pathway\u003c/p\u003e\n\u003cp\u003eA. Representative gel bands depicting after intervention with BAPTA, GSK690693 and si-Rab27a in rapid pacing HL-1 cells.\u003c/p\u003e\n\u003cp\u003eB. Representative gel bands depicting in different concentration of SC79.\u003c/p\u003e\n\u003cp\u003eC, D, E, F, G. Levels of KCa3.1, p-AKT/AKT, Rab27a, TSG101 and CD81 after intervention with BAPTA, GSK690693 and si-Rab27a.\u003c/p\u003e\n\u003cp\u003eH, I, J, L. Levels of p-AKT/AKT, Rab27a, TSG101 and CD81 in different concentration of SC79.\u003c/p\u003e\n\u003cp\u003eK, M. Representative images of Rab27a after intervention with BAPTA, GSK690693 and si-Rab27a as reflected by immunofluorescence.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SD of three biological replicates.\u003c/p\u003e\n\u003cp\u003e*P \u0026lt; 0.05, **P \u0026lt; 0.01, and ***P \u0026lt; 0.001 vs. control group;#P \u0026lt; 0.05, ##P \u0026lt; 0.01, and ###P \u0026lt; 0.001 vs. pacing group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2029745/v1/ed7648ab8a0b8bbab21030ab.png"},{"id":26470709,"identity":"41ed5e48-4589-4a11-9358-b5a43a0a7a3e","added_by":"auto","created_at":"2022-09-14 20:52:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":519761,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression KCNN4 promotes exosome secretion\u003c/p\u003e\n\u003cp\u003eA. Representative gel bands depicting after TRAM-34 administration in overexpression KCNN4 HL-1 cells.\u003c/p\u003e\n\u003cp\u003eB, C, D, E, F. Levels of KCa3.1, p-AKT/AKT, Rab27a, TSG101 and CD81 after TRAM-34 administration in overexpression KCNN4 HL-1 cells.\u003c/p\u003e\n\u003cp\u003eG. Representative gel bands depicting after intervention with BAPTA, GSK690693 and si-Rab27a in overexpression KCNN4 HL-1 cells.\u003c/p\u003e\n\u003cp\u003eH, I, J, K. Levels of p-AKT/AKT, Rab27a, TSG101 and CD81 after intervention with BAPTA, GSK690693 and si-Rab27a in overexpression KCNN4 HL-1 cells.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SD of three biological replicates.\u003c/p\u003e\n\u003cp\u003e*, **, and *** indicate p\u0026lt;0.05, 0.01, and 0.001, respectively.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2029745/v1/1620861f6a9152f3213db7b1.png"},{"id":35887070,"identity":"653cb355-0a01-415c-88ce-2747b9dc2339","added_by":"auto","created_at":"2023-04-17 17:04:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3694871,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2029745/v1/7e480113-5e8f-46ca-a4ab-373ce7b9d4ab.pdf"}],"financialInterests":"","formattedTitle":"KCa3.1 promotes exosomes secretion by activating on AKT/Rab27a in atrial myocytes during rapid pacing","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAtrial fibrillation (AF) is a major public health problem worldwide with high morbidity and mortality\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. At present, there are many evidences that AF is closely related to electrical remodeling, structural remodeling and neural remodeling\u003csup\u003e[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. However, the mechanism of electrical-substrate remodeling for AF induction and maintenance has not been fully elucidated.\u003c/p\u003e \u003cp\u003eMedium-conductance calcium-activated potassium channel (KCNN4, KCa3.1) is one of the family members of calcium-activated potassium channel. In cardiomyocytes induced by pluripotent stems from patients with catecholamine-sensitive polymorphic ventricular tachycardia, KCa3.1 blocker significantly reduced the occurrence of arrhythmias, and affects delayed post-depolarization and calcium transients\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Our previous research showed that KCa3.1 was involved in the early post-depolarization and triggering activities of the myocardium, and the KCa3.1 inhibitor TRAM-34 significantly inhibited electrical remodeling and completely inhibited the induction of acute AF\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Our recent study further showed that TRAM-34 abrogated atrial fibrosis and inflammation in rapid atrial pacing canine\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. But the mechanism of how KCa3.1 drives structural remodeling through ion channel remodeling needs further study.\u003c/p\u003e \u003cp\u003eExosomes are extracellular vesicles carried a variety of signaling molecules from donor cells (DNA, mRNA, microRNA, lncRNA and protein)\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Rab27a, a member of the Rab family, affects exosomes secretion by regulating the docking and fusing multiple vesicles bodies with the cell membrane\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Inhibition of Rab27a significantly down-regulates the secretion of MMP9 and platelet-derived growth factor A\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Studies have reported that PI3K/AKT affected Rab27a-related vesicles secretion by regulating the phosphorylation of Rab27a-binding protein JFC1\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. In another study, their results suggested that KCa3.1 is closely related to the PI3K/AKT signaling pathway\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Based on these studies, we hypothesized that KCa3.1 may regulate Rab27a through the AKT signaling pathway to affect exosomes secretion. The purpose of this study is to explore whether the abnormal expression of KCa3.1 leads to exosomes secretion and the downstream signaling pathways involved.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003eThis study was approved by the animal studies subcommittee of our institutional review board and was in accordance with the guidelines of the National Institutes of Health for the care and use of laboratory animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Model Preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rapid atrial pacing canine model was prepared as we previously described\u003csup\u003e[13]\u003c/sup\u003e.\u0026nbsp;Briefly, eighteen beagles were randomly divided into Sham group (n=6), Pacing group (n=6), and Pacing+TRAM-34 group (n=6). Three days after implanting the pacemaker, the Pacing+TRAM-34 group was given an intravenous injection of TRAM-34 (10 mg/kg/d, tid, MCE, United States). The Pacing group and Pacing+TRAM-34 group were paced at 450 bpm for 7 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElectrophysiological measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eAfter seven consecutive days of intravenous administration TRAM-34, the canines were anaesthetized and bilateral thoracotomy was performed.\u0026nbsp;Procedural stimulation was performed after placing multi-lead electrodes in various parts of the atrium. In brief, we measured the atrial effective refractory period (ERP), and the inducibility and duration of AF\u003csup\u003e[13]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathology Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor histological analysis, canine atrial tissues were fixed, embedded, and sectioned into 5 \u0026micro;m thick slices. Sections were used to stain hematoxylin \u0026amp; eosin and Masson\u0026rsquo;s trichrome to assess inflammatory infiltration and collagen deposition, respectively. To detect the expression of KCa3.1 and Rab27a, sections were incubated with KCa3.1 antibody (Affinity,\u0026nbsp;DF4132; 1:100) and Rab27a antibody (Proteintech, 17817-1-AP; 1:300). Immunohistochemistry and Immunofluorescence images were captured with fluoroscope and analyzed by Image J.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture and Treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mouse atrial HL-1 cell line (Procell, China) or mouse macrophage cell line RAW264.7 (Wuhan University), was cultured using F12/DMEM medium (Gibico, United States) or DMEM (High glucose, Gibico, United States) containing 10% fetal bovine serum and 1% penicillin/streptomycin, intervening when the cells grew to 60-70%. The rapid pacing HL-1 cell model was prepared as we previously described\u003csup\u003e[13]\u003c/sup\u003e. In brief, the HL-1 cells were paced by electric field stimulation (600 times/min), intensity 1.5 V/cm, continuous stimulation for 48 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLentivirus and Cell Infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLenti-KCNN4 and lenti-GFP viruses were produced at Genechem (Shanghai, China). HL-1 cells were infected with lenti-KCNN4 and lenti-GFP viruses at a multiplicity of infection of 30 MOI for 24 hours. Subsequently, pressure screening was performed using puromycin at 3 \u0026micro;g/ml for 72 h. Then, the dose of puromycin adjust to 1 \u0026micro;g/ml for routine pressure screening. Expression of KCa3.1 was evaluated by western blotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003esiRNA Transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003esiRNA duplexes targeting KCNN4 and a negative control siRNA were designed and synthesized by GenePharma (Shanghai, China). The KCNN4 siRNA sequences were as follows: sense 5\u0026rsquo;-GCUAGGAAGCUUCAGUUAA-3\u0026rsquo; and anti-sense 5\u0026rsquo;-UUAACUGAAGCUUCCUAGC-3\u0026rsquo;. Rab27a siRNA sequences were as follows: 5\u0026rsquo;-CGGAUCAGUUAAGUGAAGAAA-3\u0026rsquo; and anti-sense 5\u0026rsquo;-UUCUCCGAACGUGUCACGU-3\u0026rsquo;. For cell transfections, cells were grown to 60-80% confluency in 6-well dishes. For each well, serum-free F12/DMEM was mixed separately with siRNA and Lipofectamine 6000 (Beyotime, China) for 5 min. Then, the siRNA and Lipofectamine 6000 were mixed gently and were incubated at room temperature for 10 min. Subsequently, the diluted siRNA/Lipofectamine 6000 complex was added to the 6-well dish for 5 h, after which the complex was replaced with normal cell culture medium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExosome Isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell debris and large vesicles were separated by differential centrifugation at 300 g, 3000 g, 10000 g. Subsequently, the supernatant was concentrated using ultrafiltration tubes (Millipore, 10000 MW, United States) at 4000 g centrifugal force. Finally, the supernatant was ultracentrifuged at 120000 g for 90 min to obtain exosomes. All manipulations were carried out at 4 \u0026deg;C. Exosomes were dissolved in PBS and stored at -80 \u0026deg;C for subsequent detection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission Electron Microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe exosome solution was fixed with 4% paraformaldehyde. Exosomes were then adsorbed onto formvar-carbon-coated copper grids. Subsequently, grids were rinsed in PBS and negatively stained with 2% uranyl acetate for 5 min at room temperature, and then photographed with a JEM-1100 transmission electron microscope at an accelerating voltage of 80 kV.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTake 30 mg of canine atrial tissue or cultured cells for protein extraction. Protein concentration was determined by a BCA Protein Assay Kit (Aspen, as1086) according to the manufacturer\u0026rsquo;s instruction. Proteins were separated by 10% SDS-polyacrylamide gel and transferred to 0.45 \u0026micro;m PVDF membrane by semi-dry transfer. PVDF membranes were blocked with 1% polyvinylpyrrolidone-40 and 0.05% Tween-20 for 30 min. The expression of the target protein was determined by incubating with the following primary antibodies overnight at 4\u0026deg;C: GAPDH (Service bio, China, 1:1000), CD81 (Abmart, China, 1:1000), Rab27a (Service bio, China, 1:1000), KCa3.1 (Protein tech, China, 1:1000), TSG101 (Service bio, China, 1:1000), AKT (Service bio, China, 1:1000), p-AKT (Abmart, China, 1:1000). Visualization was performed on a chemiluminescence system after incubation with horseradish peroxidase-conjugated secondary antibodies (Proteintech, China, 1:3000) at room temperature.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from cells using TRIzol\u0026reg; reagent (Takara, Japan). Isolated RNA (2 \u0026mu;g) was converted into complementary DNA using RT First Strand cDNA Synthesis Kit (Servicebio, China).\u0026nbsp;The cDNA templates were amplified by qRT-PCR system (Applied Biosystem, United States) using SYBR Green PCR Mix (Servicebio, China) with the corresponding primers (IL-6: 5\u0026rsquo;-TGTGCAATGGCAATTCTGAT-3\u0026rsquo;, 5\u0026rsquo;-GGTACTCCAGAAGACCAGAGGA-3\u0026rsquo;; iNOS: 5\u0026rsquo;-GCTCATGACATCGACCAGAA-3\u0026rsquo;, 5\u0026rsquo;-TGTTGCATTGGAAGTGAAGC-3\u0026rsquo;). The 2\u003csup\u003e-\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e comparative quantification method was used to analyze the semilog amplification curves, and the expression of gene was normalized to GAPDH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResults are represented as mean \u0026plusmn; SD. The comparison among the different groups was performed with one-way ANOVA and post hoc Tukey test. \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 is regarded as statistically significant. All data were analyzed using GraphPad Prism 8 (GraphPad, United States) or SPSS 25.0 (IBM, United States).\u003c/p\u003e"},{"header":"Result","content":"\u003cp\u003e\u003cstrong\u003eTRAM-34 inhibits the electrical remodeling in canines with rapid atrial pacing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompared with the sham group, the ERP at the recording sites was significantly shortened in the pacing group, and RIPV, LSPV and LIPV increased when treated with TRAM-34 (113.7 \u0026plusmn; 5.8 vs. 101.2 \u0026plusmn; 10.4 ms in the RIPV, P \u0026lt; 0.05; 121.3 \u0026plusmn; 3.5 vs. 109.3 \u0026plusmn; 8.0 ms in the LSPV, P \u0026lt; 0.05; 118.0 \u0026plusmn; 6.1 vs. 105.7 \u0026plusmn; 6.3 ms in the LIPV) (Figure 1 A). Obviously, AF was more easily to be induced in the pacing group, and the administration of TRAM-34 reduced the AF induction by 1.8 times (3.5 \u0026plusmn; 1.0 vs. 5.3 \u0026plusmn; 1.6 times, P \u0026lt; 0.05) (Figure 1 B). The effect of TRAM-34 on electrophysiology is also reflected in shortening the duration of AF. Compared with pacing group, TRAM-34 reduced the mean duration of AF by at least 10 s (22.2 \u0026plusmn;7.3 vs. 36.3 \u0026plusmn; 5.9 s, P \u0026lt; 0.01) (Figure 1 C). We did not observe significant differences in disperse ERP among the three groups (Figure 1 D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlockade KCa3.1 inhibits exosomes secretion in rapid pacing canine atrial tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe explored in vitro whether rapid pacing leads to increased exosomes secretion and the KCa3.1 specific blocker TRAM-34 on exosomes secretion. Our results found that KCa3.1 expression was increased in the canine atrial tissue of rapid pacing, which was supported by both western blotting and immunohistochemical results. As expected, compared with pacing group, TRAM-34 significantly reduced KCa3.1 level (Figure 2 A, B, C). Rab27a, which is related to exosomes secretion, was also significantly increased in the pacing group. Consistent with the trend of KCa3.1, the administration of TRAM-34 also reduced the expression of Rab27a in the pacing group, which was supported by the results of immunoblotting and immunohistochemistry (Figure 2 A, B, E). Besides, immunoblotting results showed that the expressions of exosome-related markers such as CD81 and TSG101 were increased in pacing group, but these anomalies could be blocked by TRAM-34 (Figure 2 B, F, G). Compared with the sham group, the expression of phosphorylated AKT was significantly increased in the pacing group, and TRAM-34 could reverse these changes (Figure 2 B, D). At the histochemical level, TRAM-34 could significantly reduce inflammatory infiltration and the deposition of collagen in the atrial tissue of the pacing group (Figure 2 A). Taken together, TRAM-34 may reduce structural remodeling in AF through the AKT/Rab27a signaling pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRapid pacing leads to HL-1 cells increased secretion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further confirm whether KCa3.1 is overexpressed in atrium myocytes, we paced HL-1 cells for 48 hours. Immunofluorescence demonstrated that KCa3.1 in the pacing group was significantly higher than that in the control group, while TRAM-34 reduced the expression of KCa3.1 (Figure 3 A, B). In order to qualitatively and quantitatively explore the effect of rapid pacing on exosomes secretion, we extracted exosomes from HL-1 cell supernatants cultured at the same density and examined their morphology and quantity. Transmission electron microscopy indicated that there were no obvious changes in the morphology of exosomes (Figure 3 C). Nanoparticle tracking analysis result demonstrated that the size of the vesicles ranged between 33 and 230 nm, most of which were 90-190 nm in diameter. After rapid pacing, the concentration of cellular supernatant exosomes rose to (6.97 \u0026plusmn; 0.42) \u0026times; 10\u003csup\u003e6\u003c/sup\u003e particles/ml from (6.17 \u0026plusmn; 0.15) \u0026times;10\u003csup\u003e6\u003c/sup\u003e particles/ml, which was decreased to (6.10 \u0026plusmn; 0.10) \u0026times; 10\u003csup\u003e6\u003c/sup\u003e particles/ml by TRAM-34 treatment (Figure 3 D, E). The results suggested that rapid pacing increases the release of exosomes, which was dampened by TRAM-34 treatment.\u003c/p\u003e\n\u003cp\u003eThe levels of TNF-\u0026alpha;, IL-1\u0026beta;, IL-6 and TGF-\u0026beta;1 in the cell supernatants were significantly higher in the pacing group when compared with the control group (Figure 3 F-I). Furthermore, compared with the pacing group, BAPTA decreased the levels of TNF-\u0026alpha;, IL-1\u0026beta;, IL-6 (Figure 3 F-H), while GSK690693 decreased IL-1\u0026beta;, IL-6 (Figure 3 F, G), and si-Rab27a decreased TGF-\u0026beta;1 (Figure 3 I). Taken together, these results suggest that blocking the KCa3.1/AKT/Rab27a signaling pathways could reduce the exocrine function of HL-1 cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRapid pacing HL-1 cell exosomes promote macrophage polarization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBy co-incubating the extracted HL-1 cell exosomes with macrophages, we found that these exosomes have pro-inflammatory effects. Immunofluorescence results showed that CD68 and iNOS were significantly increased in RAW264.7 macrophages at the translation level (Figure 4 A, B, C). Moreover, it was also verified by RT-PCR that iNOS and IL-6 were significantly increased at the transcription level in macrophages (Figure 4 D, E). These results suggest that macrophages are polarized towards M1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlockade intracellular calcium reduces AKT activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether KCa3.1 affects AKT and downstream signaling via calcium ions, we used the calcium chelator BAPTA, AKT inhibitor GSK690693 and si-Rab27a to intervene in rapid pacing HL-1 cells. Our result demonstrated that the expression of KCa3.1 did not change significantly (Figure 5 A, C), indicating that the blockade of downstream cascade does not affect the expression of KCa3.1 through feedback regulation. By intervening intracellular calcium with BAPTA, we found that the phosphorylation level of AKT was significantly reduced in pacing group (Figure 5 A, D). Besides, the downstream signaling pathways, including Rab27a, TSG101, and CD81, were significantly increased in pacing group, while BAPTA alleviated their expression (Figure 5 A, E, F, G). These results support that KCa3.1 affects downstream signaling pathways mainly by affecting intracellular calcium concentration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eActivation of the AKT affects the expression of Rab27a\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe found that p-AKT was elevated in a dose-dependent manner by activating AKT using SC79 ((Figure 5 B, H). When the concentration of SC79 reached 20 \u0026micro;M, the HL-1 cells showed massive death. The expression of Rab27a also increased with the increase of p-AKT (Figure 5 B, I). The expression of the downstream exosome markers CD81 and TSG101 were significantly increased (Figure 5 B, J, L). In contrast, GSK690693, an inhibitor of AKT, significantly reduced the level of p-AKT and affected the expression of Rab27a (Figure 5 A, E) and the downstream signaling pathway (Figure 5 A, F, G). Undoubtedly,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eby Knocking down the expression of Rab27a, the expression of exosome markers CD81 and TSG101 were significantly reduced (Figure 5 A, E, F). However, knockdown of Rab27a significantly affected the expression of p-AKT (Figure 5 A, C). Hence, Rab27a may regulate the expression of AKT through feedback mechanism. Through immunofluorescence, we could more intuitively observe the effect of BAPTA, GSK1690693, si-Rab27a on the expression of downstream effector molecule Rab27a. Compared with the pacing group, administration of BAPTA, GSK690693 and si-Rab27a could significantly reduce the fluorescence intensity of Rab27a (Figure 5 K, M). These results demonstrated that intervening KCa3.1/AKT/Rab27a signaling pathway reduces exosomes secretion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOverexpression KCNN4 affects AKT/Rab27a signaling pathways\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBy transfecting lentivirus to construct a stable KCNN4 overexpressing HL-1 cell line, we found that the cell growth was significantly increased, confirming the properties of the proto-oncogene of KCNN4. Compared with KCNN4 group, TRAM-34 significantly inhibited the expression of KCa3.1 (Figure 6 A, B). Certainly, TRAM-34 also affects the downstream signaling pathways to varying degrees. The expressions of p-AKT (Figure 6 A, C), Rab27a (Figure 6 A, D) and exosome markers (Figure 6 A, E, F) were significantly decreased after administration TRAM-34. Further exploration found that the use of calcium chelator, AKT inhibitors and si-Rab27a could significantly reduce the expression of AKT (Figure 6 G, H), Rab27a (Figure 6 G, I), and exosome markers (Figure 6 G, J, K). Therefore, circular experiments demonstrated that KCa3.1 overexpression increases exosomes secretion.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study explored the role of KCa3.1 in exosomes secretion in atrial myocytes during rapid pacing in vivo and vitro. We provide evidence for the following: (1) Overexpression of KCa3.1 promotes atrial structural remodeling in canine with rapid atrial pacing. (2) KCa3.1 promotes exosomes secretion by affecting the intracellular calcium concentration and activating the AKT/Rab27a signaling pathway. (3) These pro-inflammatory exosomes promote the polarization of macrophages towards M1.\u003c/p\u003e \u003cp\u003eThe KCa3.1 channel phosphorylation and channel activity are considered to have a vital arrhythmogenesis in patients with arrhythmogenic right ventricular cardiomyopathy\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. It has been reported that clotrimazole or TRAM-34 reduce the occurrence of catecholaminergic polymorphic ventricular tachycardia\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Our previous study found that blockade of KCa3.1 overexpressed by macrophages in canine atrial tissue using TRAM-34 reduced the susceptibility of AF\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. These evidences support the pro-arrhythmic role of KCa3.1, but the mechanism of how KCa3.1 maintains the progression of AF has not been clearly elucidated.\u003c/p\u003e \u003cp\u003eSome studies have addressed how ionic remodeling drives structural remodeling. Wang et al. reported that and Ang II stimulates cell proliferation mediated by upregulating KCa3.1 channel through ERK1/2, p38-MAPK, and PI3K/AKT signaling pathways in cultured adult rat cardiac fibroblasts\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Oxidative stress also promotes myocardial fibrosis by upregulating KCa3.1\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. KCa3.1 was found to be overexpressed in diseases such as idiopathic pulmonary fibrosis\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, renal fibrosis\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, and postburn hypertrophic scar formation\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Using its specific blocker TRAM-34 inhibits the proliferation of fibroblasts and reduces the deposition of extracellular matrix by inhibiting the TGF-β1 signaling\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. These results unveiled that the changes in ion channels have profound effects on cell proliferation and differentiation. The hyperproliferation and chemotaxis of fibroblasts and macrophages in myocardial tissue have an indelible effect on local structural remodeling. Structural remodeling provides an anatomical basis for reentry on the basis of ion remodeling of initiating factor.\u003c/p\u003e \u003cp\u003eKCa3.1 channel is voltage independent and Ca\u003csup\u003e2+\u003c/sup\u003e sensitive, and its activation preserves the negative membrane potential required for sustained Ca\u003csup\u003e2+\u003c/sup\u003e influx via store-operated Ca\u003csup\u003e2+\u003c/sup\u003e channels. Ca\u003csup\u003e2+\u003c/sup\u003e handling abnormity promotes the secretion of exosomes has been reported earlier. Olivero et al. found that the depolarization-evoked release of exosomes from cortical synaptosomes occurred in a Ca\u003csup\u003e2+\u003c/sup\u003e-dependent fashion\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Interestingly, physical stimuli such as high frequency acoustic stimulation also leads to calcium-dependent exosomes release\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Our study found that calcium antagonist BAPTA reduced the expression of proteins related to exosomes secretion. Thus, these evidences also support increased exosomes secretion due to calcium handle abnormity in AF.\u003c/p\u003e \u003cp\u003eCurrent studies have shown that overexpression of KCa3.1 indicates poor prognosis in patients, promotes tumor cell proliferation and differentiation, and may activate the PI3K/AKT signaling pathway mediated by calcium ions\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Indeed, our study also verified that overexpression of KCa3.1 leads to HL-1 cells hyperproliferation. As the downstream signaling of KCa3.1, that excessive activation of AKT promotes fibrosis. In cardiac fibroblasts, treatment with angiotensin II promotes extracellular matrix deposition by upregulating PI3K/AKT expression\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. In acetylcholine and calcium chloride-induced mouse models of AF, AKT was also highly expressed in atrial tissue\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. However, the corresponding degree of activation of the AKT signaling pathway in different cells is inconsistent. Different from Zhou et al.\u0026rsquo;s study\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e, we found that overactivation of AKT signaling pathway could be lethal to cardiomyocytes. We found that 20 \u0026micro;M of SC79 could kill all HL-1 cells. So, overexpression of AKT may have adverse effect on cardiomyocytes to some extent.\u003c/p\u003e \u003cp\u003eSo far, studies have not clearly defined how the activation of KCa3.1/AKT signaling pathway affects its downstream to play a biological regulatory role in cardiovascular disease. We found that either overexpression of KCa3.1 or rapid pacing resulted in enhanced exocrine function. This is mainly reflected in the increased expression of Rab27a, a protein related to exosomes secretion, and the increase of exosome markers. Interestingly, we found that TNF-α, IL-1, IL-6, and TGF-β1 in supernatants were significantly increased in the pacing group, which indicates that inferior stimulation may lead to enhanced cell secretion. This abnormal secretion phenomenon may change the surrounding microenvironment, including local inflammation or fibrosis and drives structural remodeling of the atrial tissue.\u003c/p\u003e \u003cp\u003eRab27a knockout impairs myeloperoxidase secretion of permeabilized neutrophils by interfering with the JFC1/Slp1-Rab27a secretion mechanism\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Jennifer et al. reported that AKT regulates JFC1/Slp1 function through phosphorylation and affects the secretion of Rab27a-containing vesicles\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Recent studies have shown that platelet-rich plasma promotes mesenchymal stem cells exosomal paracrine repair of acute kidney injury via the AKT/Rab27 pathway\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. These findings support that AKT is the upstream signaling pathway of Rab27a. However, Zhou et al. found that Rab27a could affect the expression of AKT through feedback regulation by constructing Rab27a knockout mice\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Consistent with their observations, our study confirmed that silencing of the Rab27a affects the phosphorylation of AKT.\u003c/p\u003e \u003cp\u003eThe role of exosomes on macrophage polarization has been extensively studied. It has been reported that exosomes derived from inflammatory myoblasts or adipocytes promoted M1 polarization\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. In addition to affecting cells in the surrounding microenvironment, exosomes also play a regulatory role through long-distance transport. Majumdar et al. reported exosomes mediate LTB4 release during neutrophil chemotaxis\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Besides, Eiji et al. verified that pro-inflammatory exosomes from lipotoxic hepatocytes activated macrophage chemotaxis. Our previous study found that TRAM-34 reduced the polarization and infiltration of macrophages in canine model with rapid atrial pacing. The current study hints that TRAM-34 may reduce M1 polarization by blocking KCa3.1 involved pro-inflammatory exosome release.\u003c/p\u003e \u003cp\u003eIn conclusion, our present studies showed that overexpression of KCa3.1 promotes proinflammatory exosomes secretion through the AKT/Rab27a signaling pathway. Inhibition KCa3.1/AKT/Rab27a signal pathway reduces myocardial tissue structure remodeling in AF. These results indicate that might provide a new therapeutic target for blocking the incidence of AF.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis work was supported by National Natural Science Foundation of China (81970277, 82170312).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003eDishiwen Liu: Conceptualization, Methodology, Validation, Investigation, Writing-original draft, review \u0026amp; editing. Qingyan Zhao: Investigation, Supervision, Writing-review \u0026amp; editing. Huiyu Chen, Yuntao Fu, Mei Yang: Conceptualization, Methodology. Yajun Yao, Shanqing He, Youcheng Wang: Validation, Investigation. Zhen Cao, Xuewen Wang: Investigation, Formal analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003eAll data involved in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003eAll applicable international, national, and/or institutional guidelines for the care and use of animals were followed.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eK\u0026ouml;nig S, Ueberham L, Schuler E, et al. 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PLoS Biol, 2021, 19(7): e3001271; DOI: 10.1371/journal.pbio.3001271\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"KCa3.1, exosomes, atrial fibrillation, structure remodeling, macrophage","lastPublishedDoi":"10.21203/rs.3.rs-2029745/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2029745/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003ethe aim of this study was to investigate the role of intermediate-conductance Ca\u003csup\u003e2+\u003c/sup\u003e-activated K\u003csup\u003e+\u003c/sup\u003e (KCNN4, KCa3.1) in exosomes secretion of atrial myocytes.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eeighteen beagles were randomly divided into Sham group (n\u0026thinsp;=\u0026thinsp;6), Pacing group (n\u0026thinsp;=\u0026thinsp;6), and Pacing\u0026thinsp;+\u0026thinsp;TRAM-34 group (n\u0026thinsp;=\u0026thinsp;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 \u0026amp; Eosin and Masson\u0026rsquo;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.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTRAM-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.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis 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.\u003c/p\u003e","manuscriptTitle":"KCa3.1 promotes exosomes secretion by activating on AKT/Rab27a in atrial myocytes during rapid pacing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-09-14 20:52:52","doi":"10.21203/rs.3.rs-2029745/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c43a105e-1d31-47f1-b78f-1cd3856c9ef0","owner":[],"postedDate":"September 14th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-04-17T17:03:49+00:00","versionOfRecord":{"articleIdentity":"rs-2029745","link":"https://doi.org/10.1155/2023/3939360","journal":{"identity":"cardiovascular-therapeutics","isVorOnly":true,"title":"Cardiovascular Therapeutics"},"publishedOn":"2023-03-30 00:00:00","publishedOnDateReadable":"March 30th, 2023"},"versionCreatedAt":"2022-09-14 20:52:52","video":"","vorDoi":"10.1155/2023/3939360","vorDoiUrl":"https://doi.org/10.1155/2023/3939360","workflowStages":[]},"version":"v1","identity":"rs-2029745","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2029745","identity":"rs-2029745","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}