Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060, P.R. China.
Department of Cardiology, Yidu People's Hospital, Yidu, Hubei 443000, P.R. China.
Mol Med Rep. 2024 Apr;29(4). doi: 10.3892/mmr.2024.13179. Epub 2024 Feb 9.
The present study was designed to explore the role of M2 macrophage‑derived exosomes (M2‑exos) on the KCa3.1 channel in a cellular atrial fibrillation (AF) model using rapidly paced HL‑1 myocytes. M2 macrophages and M2‑exos were isolated and identified. MicroRNA (miR)‑146a‑5p levels in M2 macrophages and M2‑exos were quantified using reverse transcription‑quantitative PCR (RT‑qPCR). HL‑1 myocytes were randomly divided into six groups: Control group, pacing group, pacing + coculture group (pacing HL‑1 cells cocultured with M2‑exos), pacing + mimic‑miR‑146a‑5p group, pacing + NC‑miR‑146a‑5p group and pacing + pyrrolidine dithiocarbamate (PDTC; a special blocker of the NF‑κB signaling pathway) group. Transmission electron microscopy, nanoparticle tracking analysis, western blotting, RT‑qPCR and immunohistochemistry were performed in the present study. A whole‑cell clamp was also applied to record the current density of KCa3.1 and action potential duration (APD) in each group. The results revealed that miR‑146a‑5p was highly expressed in both M2 macrophages and M2‑exos. Pacing HL‑1 cells led to a shorter APD, an increased KCa3.1 current density and higher protein levels of KCa3.1, phosphorylated (p‑)NF‑κB p65, p‑STAT3 and IL‑1β compared with the control group. M2‑exos, miR‑146a‑5p‑mimic and PDTC both reduced the protein expression of KCa3.1, p‑NF‑κB p65, p‑STAT3 and IL‑1β and the current density of KCa3.1, resulting in a longer APD in the pacing HL‑1 cells. In conclusion, M2‑exos and their cargo, which comprised miR‑146a‑5p, decreased KCa3.1 expression and IL‑1β secretion in pacing HL‑1 cells via the NF‑κB/STAT3 signaling pathway, limiting the shorter APD caused by rapid pacing.
本研究旨在使用快速起搏 HL-1 心肌细胞探讨 M2 巨噬细胞衍生的外泌体（M2-exos）对细胞性心房颤动（AF）模型中 KCa3.1 通道的作用。分离并鉴定 M2 巨噬细胞和 M2-exos。采用逆转录-定量 PCR（RT-qPCR）定量检测 M2 巨噬细胞和 M2-exos 中的 microRNA（miR）-146a-5p 水平。HL-1 心肌细胞随机分为六组：对照组、起搏组、起搏+共培养组（起搏 HL-1 细胞与 M2-exos 共培养）、起搏+miR-146a-5p 模拟物组、起搏+阴性对照（NC）miR-146a-5p 组和起搏+吡咯烷二硫代氨基甲酸盐（PDTC；NF-κB 信号通路的特殊抑制剂）组。本研究采用透射电子显微镜、纳米颗粒跟踪分析、Western blot、RT-qPCR 和免疫组织化学法。还应用全细胞膜片钳记录各组 KCa3.1 电流密度和动作电位时程（APD）。结果显示，M2 巨噬细胞和 M2-exos 中均高表达 miR-146a-5p。与对照组相比，起搏 HL-1 细胞可导致 APD 缩短、KCa3.1 电流密度增加和 KCa3.1 蛋白水平升高，磷酸化（p）NF-κB p65、p-STAT3 和 IL-1β升高。M2-exos、miR-146a-5p 模拟物和 PDTC 均降低了起搏 HL-1 细胞中 KCa3.1、p-NF-κB p65、p-STAT3 和 IL-1β的蛋白表达以及 KCa3.1 的电流密度，导致 APD 延长。综上所述，M2-exos 及其包含的 miR-146a-5p 通过 NF-κB/STAT3 信号通路减少了起搏 HL-1 细胞中 KCa3.1 的表达和 IL-1β 的分泌，限制了快速起搏引起的 APD 缩短。
