Fig 1.
Collagen I in the atrial myocardium.
A-D: Immunohistochemical reaction shows collagen I in endomysial extracellular matrix. In atria of both patient groups (SR in A, B and AF in C, D) it is possible to detect higher (A, C) as well as lower (B, D) amount of collagen I-positive ECM. Immunoperoxidase reaction with DAB as a substrate (brown precipitate). No nuclear counterstaining. For all images scale bar = 50μm. (E) A graph showing the result of quantification of collagen I volume fraction (CIVF) in atrial myocardial samples. A comparison between different anatomical locations is shown: right appendage–RA (n = 37), left appendage–LA (n = 18), left atrium—LAt (n = 8), LA+LAt (n = 26).
Fig 2.
Collagen III in the atrial myocardium.
A-D: Immunohistochemical reaction shows collagen III in endomysial and partly also perimysial extracellular matrix. In atria of both patient groups (SR in A, B and AF in C, D) it is possible to detect higher (A, C) as well as lower (B, D) amount of collagen III-positive ECM. Immunoperoxidase reaction with DAB as a substrate (brown precipitate). No nuclear counterstaining. For all images scale bar = 50μm. (E) A graph showing the result of quantification of collagen III volume fraction (CIIIVF) in atrial myocardial samples. A comparison between different anatomical locations is shown: right appendage–RA (n = 37), left appendage–LA (n = 19), left atrium—LAt (n = 9), LA+LAt (n = 28).
Fig 3.
Elastin in the atrial myocardium.
Immunohistochemical detection of elastin in samples from patients with sinus rhythm (A, B) and in samples from patients with atrial fibrillation (C, D). EVF (elastin volume fraction) is the percentage of elastin fibers in morphometrically evaluated myocardial area. Representative images of left appendage myocardium with low EVF (A, C) and images from right appendage show high EVF (B, D) Immunoperoxidase reaction with DAB as a substrate (brown precipitate). No nuclear counterstaining. Scale bar in A-D = 100μm. E: A graph showing atrial EVF of all patients with sinus rhythm and atrial fibrillation dependent on localization. RA–right appendage (n = 26), LA–left appendage (n = 12), LAt–left atrium (n = 4), LA + LAt (n = 16).
Fig 4.
Expression of VEGF in atrial myocardium from patients with sinus rhythm (SR) and atrial fibrillation (AF).
A, B: Atrial samples from patients with SR. (A) A strong immunoreactivity for VEGF is localized to the capillaries, while cardiomyocytes display rather low level of VEGF expression. (B) High level of VEGF immunoreactivity in mesothelial cells and in adipocytes of epicardium. C, D: Atrial samples from patients with AF. (C) A strong immunoreactivity for VEGF is localized to mesothelium. There are VEGF-positive capillaries and moderately positive cardiomyocytes in the atrial myocardium. (D) A strong VEGF immunoreactivity in the myocardium (mainly cardiomyocytes) and in mesothelial cells. Scale bar in A-D = 50μm. (E) A graph showing a result of semiquantitative analysis of VEGF immunoreactivity in the atrial samples from all patients as described in Methods (score 0–3). A comparison between different structures from different anatomical locations is shown: RA–right appendage, LA–left appendage, LAt–left atrium.
Fig 5.
Quantification of microvascular density.
Histochemical visualization of microvasculature using UEA-lectin binding in atrial myocardium from patients with sinus rhythm (SR) (A) and atrial fibrillation (AF) (B). Peroxidase reaction with DAB as a substrate (brown precipitate). No nuclear counterstaining. Scale bar in A-B = 50μm. (C) A graph showing results of quantification of microvascular density (MVD) in atrial samples of all patients as described in Methods. RA–right appendage (n = 24), LA–left appendage (n = 12), LAt–left atrium (n = 10), LA + LAt (n = 22).
Fig 6.
Microvessel pericyte coverage in atria of patients with sinus rhythm (SR) and atrial fibrillation (AF).
(A-D) Confocal images of human atria. (A) Immunodetection of desmin (green) shows its expression in cardiomyocytes. Capillaries labeled with UEA-lectin (red) are devoid of desmin immunoreactivity (arrowheads). Scale bar = 50μm. (B) Desmin is present in cardiomyocytes, while SMA-positive capillary pericyte (arrowhead) does not display desmin signal. Scale bar = 10μm. (C) A representative section of myocardium used for the quantitative analysis of microvessel pericyte coverage index. Blood vessel endothelium is labeled using UEA lectin (red) and pericytes are visualized using anti-SMA antibody (green). Scale bar = 100μm. (D) A high magnification image shows SMA-positive smooth muscle cells surrounding a small arteriole and also SMA-positive pericytes in association with the capillaries (arrowheads). Scale bar = 25μm. (A, B, D) Nuclei are stained with DAPI (blue). (E) A graph showing results of quantification of microvessel pericyte coverage index (MPI) in atrial samples of all patients as described in Methods. RA–right appendage (n = 8), LA–left appendage (n = 9), LAt–left atrium (n = 7), LA + LAt (n = 16).