RESUMEN
BACKGROUND: The relationship between local epicardial adipose tissue (EAT) macrophages and atrial fibrillation (AF) remains unclear. The purpose of this study was to investigate the role of KCa3.1 in the migration of macrophages from EAT to adjacent atrial tissue during rapid pacing. METHODS: Part 1: Eighteen beagles were randomly divided into the sham group, pacing group, and pacing + clodronate liposome (CL) group. Part 2: Eighteen beagles were randomly divided into the sham group, pacing group, and pacing + TRAM-34 group. HL-1 cells and RAW264.7 cells were cocultured to explore the specific migratory mechanism of macrophages. RESULTS: Depleting EAT macrophages significantly reduced macrophage infiltration in the adjacent atrium and the induction of AF in canines with rapid atrial pacing. TRAM-34 significantly inhibited the migration of macrophages from EAT to the adjacent atrium and electrical remodeling in canines with rapid atrial pacing. Compared with those of the control HL-1 cells, the secretion of CCL2 and the number of migrating macrophages in pacing HL-1 cells were significantly increased, which could be reversed by TRAM-34. Further in vitro experiments showed that KCa3.1 regulated CCL2 secretion through the p65/STAT3 signaling pathway. CONCLUSION: Inhibiting myocardial KCa3.1 reduced the migration of EAT macrophages to adjacent atrial muscles caused by rapid atrial pacing, thereby decreasing vulnerability to AF. The mechanism by which KCa3.1 regulates CCL2 may be related to the p65/STAT3 signaling pathway.
RESUMEN
Background: Fibroblast-derived exosomes can regulate the electrical remodeling of cardiomyocytes, and the intermediate-conductance calcium-activated potassium channel (KCa3.1) is important in atrial electrical remodeling. However, the underlying molecular mechanisms remain unclear. This study aimed to investigate the regulation of cardiac electrophysiology by exosomes linked to KCa3.1. Methods: Atrial myocytes (AMs) and atrial fibroblasts were isolated from Sprague-Dawley suckling rats and cultured individually. The cellular atrial fibrillation (AF) model was established via electrical stimulation (1.0 v/cm, 10 Hz), and fibroblast-derived exosomes were isolated via ultracentrifugation. Exosomes were co-cultured with AMs to investigate their influences on KCa3.1 and the underlying mechanisms. Nanoparticle tracking analysis and transmission electron microscopy were used to measure exosome particle sizes and concentrations. Whole-cell patch clamp was applied to record the current density of KCa3.1 and action potential duration (APD). The expression of miR-21-5p was detected by reverse-transcription polymerase chain reaction (RT-PCR). Western blotting or immunofluorescence was used to measure the expression of exosomal markers, Akt phosphorylation, and KCa3.1. Results: Rapid pacing promoted the secretion of exosomes from atrial fibroblasts and miR-21-5p expression in atrial fibroblasts and exosomes. KCa3.1 protein expression and current density significantly increased, and APD50 and APD90 were sharply shortened after rapid pacing in AMs. TRAM-34 (KCa3.1 blocker) extended APD and reduced susceptibility to AF. KCa3.1 and P-AKT expressions were amplified after co-culturing AMs with exosomes secreted by atrial fibroblasts. In contrast, the increase in KCa3.1 expression was reversed after the cells were co-cultured with exosomes secreted by atrial fibroblasts that were transfected with miR-21-5p inhibitors or after the use of LY294002, a PI3K/Akt pathway inhibitor. Conclusions: Rapid pacing promoted the secretion of exosomes from fibroblasts, and miR-21-5p was upregulated in exosomes. Moreover, the miR-21-5p-enriched exosomes upregulated KCa3.1 expression in AMs via the PI3K/Akt pathway.
