Fig. 1.

NETs are increased in AF patients and cytotoxic to cardiomyocytes. a Complex enzyme-linked immunosorbent assay (ELISA) experiment comparing NETs in the peripheral blood and coronary blood of patients with AF or SR (n = 4). b Comparison of NETs concentration in left atrial appendage homogenates of patients with AF and SR (n = 4). c NETs formed in the coronary vessels of AF patients. LAA section was stained for DNA (blue), NE (green) and MPO (red). Scale bar: 100 μm. MPO and NE double positive structure was defined as NETs. d NETs formed outside the coronary vessels of AF patients. LAA section was stained for DNA (blue), cit-H3 (green) and CD31 (red). Scale bar: 100 μm. Coronary vessels were labelled with CD31 and NETs were identified as cit-H3 positive. e Cardiomyocytes incubated with NETs underwent atrophy and perinuclear granule increase. Yellow arrows indicate NETs binding to the surface of cardiomyocytes. Scale bar: 20 μm. f, g Death rate of cardiomyocytes measured by Celigo analysis in the presence or absence of NETs and supernatants of PMN (n = 4). Cells in 96well plate were stained for all DNA (blue) and nucleuses of dead cells (red). Scale bar: 150 μm. Hoechst single positive cell was defined as the living, while Hoechst and PI double-positive cell was defined as the dead. h, i Increased cleaved caspase3/caspase 3 ratio in cardiomyocytes incubated with NETs analyzed through WB (n = 3). SR sinus rhythm, AF atrial fibrillation, PB peripheral blood, LAA left atrial appendage, CB coronary blood, NE neutrophil elastase, MPO myeloperoxidase, PMN polymorphonuclear leukocytes, PI propidium iodide. *P < 0.05, **P < 0.01, ***P < 0.001, ns (not significant). Data were presented as mean ± SD