Role of epicardial adipose tissue in human atrial fibrillation

Figures

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  FIGURE 1. EAT infiltration toward the atrial myocardium. (A) Concept of our study. (B) Macroscopic findings of the excised LAA. The EAT was commonly observed in human LAA samples. Scale bar = 1 cm. (C) Infiltration of the subepicardial area reached the endocardium in some cases. Scale bar = 100 μm. (D) Massive interstitial fibrosis was observed in the atrial myocardium at the junction with the EAT. Scale bar = 1000 μm. EAT, epicardial adipose tissue; LAA, left atrial appendage. (Figure adapted with permission 10 ).

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  FIGURE 2. Fibrotic EAT remodeling and its relationship with atrial myocardial fibrosis. (A) Representative Masson's trichrome‐stained images of mildly (a), moderately (b), and severely (c) fibrotic EAT. (B) Hematoxylin and eosin‐stained images of the corresponding EAT. Scale bar = 100 μm. (Figure adapted with permission 10 ).

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  FIGURE 3. Comparative transmission electron microscopy findings between severely fibrotic EAT and mildly fibrotic EAT. (A) Macrophage and myofibroblast infiltration was observed around adipocytes in severely fibrotic EAT. (B) Stromal cells and myofibrillar disarray were observed at the junction with the adipocytes in severely fibrotic EAT. EAT, epicardial adipose tissue; M, myocardium. (Figure adapted with permission 10 ).

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  FIGURE 4. Expression of Angptl2 and HIF‐1α in the EAT. (A) Positive correlation between total collagen in the atrial myocardium and Angptl2 in the EAT. (B) Protein expression of HIF‐1α, phospho‐IκBα, phospho‐p38 MAPK, p38 MAPK, and GAPDH in the EAT of the high and low Angptl2 groups (n = 5 per group). The data are presented as the mean ± SD. *p < .05, **p < .01 vs. low Angptl2 group. (C) Representative immunohistochemical staining for HIF‐1α in the mildly and severely fibrotic EAT groups. Angptl2, angiopoietin‐like protein‐2; EAT, epicardial adipose tissue; HIF‐1α, hypoxia‐inducible factor‐1α; p‐p38 MAPK, phospho‐p38 mitogen‐activated protein kinase; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase. (Figure adapted with permission 10 ).

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  FIGURE 5. Organo‐culture system. See text for details. (Figure adapted with permission 11 ).

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  FIGURE 6. Effects of human EAT and SAT on organo‐cultured rat atrial fibrosis. (A) (a) Representative results of Masson's trichrome and immunohistochemical staining using α‐SMA antibody. Scale bar = 550 mm. (b) Quantification of epicardial fibrosis. The data are presented as the mean ± SEM (n = 6 for day 0; n = 9 for days 3 and 7). **p < .01. (c) Quantification of α‐SMA‐positive cells (n = 6 for day 0; n = 9 for days 3 and 7). **p < .01. (B) (a) Representative results of Masson's trichrome staining treated with vehicle‐, SAT‐, or EAT‐conditioned medium for 7 days. Scale bar = 50 μm. (b) Quantification of epicardial fibrosis (n = 6 for the vehicle group; n = 9 for the SAT and EAT groups). (C) (a) Representative results of Masson's trichrome staining treated with the EAT obtained from patients with or without AF for 7 days. Scale bar = 50 μm. (b) Quantification of epicardial fibrosis (n = 6 for the AF (−) group; n = 3 for the AF (+) group). **p < .01. α‐SMA, α‐smooth muscle actin; AF, atrial fibrillation; Epi, epicardium; peri‐LA EAT, peri‐left atrial epicardial adipose tissue; MT, Masson's trichrome; SAT, subcutaneous adipose tissue. (Figure adapted with permission 11 ).

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  FIGURE 7. Transmission electron microscopy findings. (A) Transmission electron microscopy findings of organo‐cultured atria treated with the EAT‐conditioned medium for 7 days. A cell with a well‐developed granular endoplasmic reticulum was observed (black arrow (a)). A cell with Golgi body (white arrow) and actin fibers produced from this cell (black arrow) was observed (b). Scale bar for (a) and left panel in (b) = 10 μm. Scale bars for center and right panels = 2 μm and 500 nm, respectively. (B) Scanning electron microscopy images of organo‐cultured atria treated with the EAT‐conditioned medium for 7 days. Spindle‐shaped cells (white arrows) were specifically observed. (C) Myofibroblasts were rarely seen in the atria treated with the SAT‐conditioned medium. Scale bar = 10 μm. (Figure adapted with permission 11 ).

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  FIGURE 8. Effects of human recombinant Angptl2 and anti‐Angptl2 antibody. (A) Angptl2 protein expression with exposure to the human EAT and SAT‐conditioned media. The data are presented as the mean ± SEM (n = 9 for each group). *p < .05. (B) (a) Representative results for Masson's trichrome staining and immunohistochemical staining using α‐SMA antibody. Scale bar = 50 μm. (b) Quantification of epicardial fibrosis (n = 5 for each group). The data are presented as the mean ± standard error of the mean. *p < .05, **p < .01. (c) Quantification of α‐SMA‐positive cells (n = 5 for each group). The data are presented as the mean ± SEM. *p < .05, **p < .01. (C) (a) Representative images of left atrial sections from rats stained with Masson's trichrome. Scale bar = 50 μm. (b) Quantification of epicardial fibrosis (n = 5 for each group). The data are presented as the mean ± SEM. *p < .05. (D) (a) Representative images of left atrial sections from rats incubated with the EAT‐conditioned medium with or without anti‐Angptl2 antibody and stained with Masson's trichrome. Scale bar = 50 μm. (b) Quantification of epicardial fibrosis (n = 9 for each group). The data are presented as the mean ± SEM. **p <…

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  FIGURE 9. Relationship of adipocyte diameter with BMI and EAT volume. (A) (a,b) Representative examples. (B) (a,b) Correlation of adipocyte diameter with BMI and EAT volume. BMI, body mass index; EAT, epicardial adipose tissue. (Figure adapted with permission 12 ).

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  FIGURE 10. Micro‐dissection of the C‐EAT and M‐EAT for microarray analysis. (A) Laser micro‐dissection was performed on each adipose tissue in sections (C‐EAT: blue, M‐EAT: red), and used for the microarray analysis. (B) (a–c) Downregulation (green) and upregulation (yellow) of inflammation‐, fibrosis‐, and adipogenesis‐related genes in the C‐EAT compared with the M‐EAT. C, central; M, marginal (Figure adapted with permission 12 ).

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  FIGURE 11. Quantification of EAT volume and percent change in EAT fat attenuation on CT. (A) Representative measurement image and three‐dimensional image of the EAT obtained with the Synapse Vincent system. (B) Representative measurement of EAT fat attenuation on CT. (a–c) CT images in the axial plane, including the maximum depiction of the EAT area above the origin of the left coronary artery and below the pulmonary artery bifurcation. (d–f) ROI determination. The starting point was determined as the point of intersection of the EAT edge close to the LAA and the perpendicular line from the midpoint between each closest point of the LAA to the aorta and the LAA to the pulmonary artery. As the ROI, 9 × 9 pixels were drawn from the edge line centered on the starting point to the center of the EAT. The yellow number is the EAT fat attenuation satisfying the window of −195 to −45 HU. (C) (a) Representative three‐dimensional heatmap of EAT fat attenuation within the ROI. (b) The curve derived from the EAT fat attenuation from the LAA toward the center of the EAT. The percent change in EAT fat attenuation was calculated as follows: percent change in EAT fat attenuation = 100 × (maximum CT fat…

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  FIGURE 12. Percent change in EAT fat attenuation on CT imaging. (A) Representative heatmap of EAT fat attenuation in cases of mild (a) and severe (b) fibrotic EAT remodeling. (B) Correlation between the percent change in EAT fat attenuation and EAT fibrosis. (C) Comparison of the percent change in EAT fat attenuation between PAF and PeAF. (D) Correlation between the percent change in EAT fat attenuation and C/M diameter ratio. PAF, paroxysmal atrial fibrillation; PeAF, permanent AF. (Figure adapted with permission 12 ).