Enhanced monocyte migratory activity in the pathogenesis of structural remodeling in atrial fibrillation

Fig 1. Proportions of monocyte subsets in non-AF and AF patients, and AF patients subdivided into normal and enlarged LA groups. Percentages of CD14++CD16- classical (A), CD14+CD16++ non-classical (B) and CD14++CD16+ intermediate (C) monocyte subsets were determined in flow cytometry (non-AF: n = 22, normal LA: n = 83, enlarged LA: n = 78, Tukey’s test).

Fig 2. Higher CCR2 mRNA levels in monocytes from AF patients in enlarged LA group. Monocytes were isolated from PBMCs by a magnetic bead-based negative isolation method and then subjected to RNA isolation. Gene products were measured by real-time qPCR. mRNA levels of CCR2 are shown as relative to the normal LA group normalized by Rplp0 (normalLA: n = 25, enlarged LA: n = 38, unpaired t-test).

Fig 3. Increased monocyte CCR2 protein levels in whole cells, but not on the cell surface, in patients with enlarged LA. A) Cell surface levels of CCR2 in monocytes were determined by flow cytometry. After being classified into three subsets by flow cytometry, the median fluorescence intensity (MFI) of CCR2 was determined in each population. (normal LA: n = 9, enlarged LA: n = 9, mean ± SD, N.S.: not significant, unpaired t-test) B) Total CCR2 protein levels in monocytes were determined by Western blotting (upper panel). The arrowhead indicates the bands corresponding to CCR2. Band intensity of CCR2 was quantified and then normalized with cyclophilin B (CypB) levels (lower panel) (normal LA: n = 7, enlarged LA: n = 7, mean ± SD, unpaired t-test).

Fig 4. Enhanced migratory activity of monocytes toward MCP-1 in patients with enlarged LA. Monocytes were isolated and subjected to migratory activity assay. A) Representative fluorescence images of migrated cells after incubation with or without 5 ng/ml of MCP-1 for 90 minutes (Scale bar: 100 μm, 20x objective). B) Number of nuclei was counted in fluorescence images obtained from five random fields of view per well at 10x objective magnification. Chemotaxis index was calculated as described in the Methods section (normal LA: n = 20, enlarged LA: n = 18, mean ± SD, unpaired t-test).

Fig 5. Increased number of monocytes/macrophages in the wall of left atrial appendages in AF patients with enlarged LA. Immunofluorescence staining of left atrial appendages of AF patients who underwent cardiac surgery. A) Representative image of CD68 (green) and CCR2 (red) double-positive cells (monocytes/macrophages) at a high-power field magnification (63x objective) in a left atrial appendage. Scale bar: 5 μm. B) Representative images at low-power magnification (20x objective) in normal and enlarged LA patients. Scale bar: 10 μm. C) The number of CD68-positive cells (monocytes/macrophages) and CCR2-positive monocytes/macrophages, and percentage of CCR2-positive macrophages per randomly selected field of view at 20x objective magnification (normal LA: n = 10, enlarged LA: n = 10, mean ± S.D., unpaired t-test).