Loss of fibronectin fiber tension is inherent to ECM remodeling in human myocarditis and post-inflammatory fibrosis

Figures

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  Fig. 1. Immuno-histochemical and clinical histopathological evaluation of left ventricular biopsies across patient groups: a) IHC images show CD68+ macrophages (green) in biopsies from the left ventricle of patients from acute myocarditis (MC), dilated cardiomyopathy (DCM), inflammatory dilated cardiomyopathy (DCMi) and COVID-19 patient groups, N = 39; MC-17, DCM-6, DCMi-12, COVID-19–4; refer Suppl. Fig. S1 for further images of macrophage infiltration in myocardial tissues across patient groups. b) Enzymatic immunostained images of formalin-fixed paraffin embedded (FFPE) sections from the corresponding patient biopsies, but from different cross-sections, showing CD68+ macrophages (brown), N = 30; MC-13, DCM-5, DCMi-11, COVID-19–1. c) IHC images showing CD3+ T cells (green) in biopsies from patients across the patient groups, N = 37; MC-16, DCM-5, DCMi-12, COVID-19–4; refer Suppl. Fig. S2 for further examples of T cells infiltration in myocardial tissues across patient groups d) Enzymatic immunostained images of FFPE sections from the corresponding patient biopsies, but from different cross-sections, showing CD3+ T cells (brown) in tissues across the patient groups, N = 30; MC-13, DCM-5,…

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  Fig. 2. Clinical histopathology and cardiac function analyses. a) Plot depicting CD68+ macrophage counts across the patient groups. b) Plot depicting CD3+ T cell counts across the patient groups. Both CD68 + and CD3 + cells were counted manually based on immunohistochemistry stainings as shown in Fig. 1. The cells were normalized to cells per sq. mm as tissue sizes are variable. c) Plot depicting the ejection fractions (EF, % of blood volume ejected from the ventricle for each contraction) across the patient groups. d) Plot depicting the left ventricular end diastolic diameters (LVEDD, in mm, measure of left ventricular enlargement) across the different patient groups. e) Plot depicting blood Troponin T (TnT) peak levels in patients across the groups. f) Plot depicting blood N-terminal prohormone of brain natriuretic peptide (NTproBNP) peak levels in patients across the groups. g) Plot depicting creatine kinase (CK) peak levels across the patient groups. h) Plot depicting creatine kinase-myocardial band (CK-MB) peak levels across the patient groups. i) Plot depicting high sensitivity C-reactive protein peak (hsCRP) levels across the patient groups. The threshold level for diagnosis is…

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  Fig. 3. Loci in which fibronectin fibers had lost their tension in inflamed myocardial tissues. a) Immunofluorescence images of fibronectin (polyclonal antibody, red) within myocardial tissues present throughout the tissue cross-section area. b) Corresponding stains from the same tissue sections showing loci of FnBPA5 peptide signal (magenta) indicating the locations of untensed fibronectin fibers; Scale bars: Tile images: 200 µm, enlarged images: 50 µm.; refer Suppl. Fig. S3 and S4 for more images showing relaxed fibronectin fibers in inflamed myocardial tissues across patient groups and corresponding zoom-in images c) Plot depicting the ratio of pixels positive in untensed fibronectin fibers versus total fibronectin fiber pixels in the whole tissue for samples from the different patient groups and healthy donors. d) The histological stainings revealed varying levels of untensed fibronectin fiber pixel content within each patient group: (left) images of tissues with the highest content of untensed to total fibronectin content (%) (shown within green boxes in Fig. 3c) and (right) images of tissues with lowest content of untensed to total fibronectin content (%) (shown within red boxes in…

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  Fig. 4. Fibrillar Collagen deposits (SHG-positive) in inflamed myocardial tissues. a) Second harmonic generation (SHG) microscopy images show thick collagen fiber deposits (grey) in myocardial tissues across the patient groups. b) Clinical histopathological Masson’s trichrome stainings of cardiac muscle fibers in red and collagen fibers in blue from all patient groups. The FFPE tissues are from the same patients but display different cross-sections. Scale bars: Tile images: 200 µm, enlarged images: 50 µm. c) Plot depicting the pixel ratios of fibrillar collagen deposits (SHG) versus the total tissue areas for samples from the patient groups and healthy donors (N = 39). Patient tissues with the highest collagen fiber content versus the lowest collagen fiber content are shown within green and red boxes respectively for individual groups. d) SHG microscopy images revealed varying levels of collagen fiber content within each patient group: (left) images of tissues with the highest content of collagen fibers in each patient group (within green boxes in Fig. 4c) and (right) images of tissues with low/no collagen fiber content (within red boxes in Fig. 4c). It is unclear whether some of these…

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  Fig. 5. Spatial proximity analysis of macrophages and untensed fibronectin fibers. a) Histological stainings and SHG images from two acute myocarditis (MC) patient tissues show macrophage crowded areas (Patient #13) and those without macrophage crowding (Patient #7). Both tissues contained areas rich in untensed fibronectin (Fn) fibers while the deposition of SHG-positive collagen fiber deposits was not pronounced. b) Enlarged ROI shown in images a of macrophage-crowded loci, providing an overlay of macrophages (green) and untensed fibronectin fibers (magenta). c) Enlarged ROI shown in a for patients #7 and #13 together with the corresponding heatmaps quantifying macrophage density (refer Methods section 5.5.1 for methods set the threshold value for defining tissues that contain macrophage-crowded loci versus macrophage non-crowded tissues). d) Plots depict the percentage of pixels within a distance ≤ 5.4 µm relative to each other: (left) fraction of macrophage pixels near untensed fibronectin fiber pixels, and (right) fraction of untensed fibronectin fiber pixels near macrophage pixels (N = 5 for macrophage crowded tissues and N = 30 for macrophage non-crowded tissues, Mann-Whitney…

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  Fig. 6. Myofibroblasts, untensed fibronectin fibers and fibrillar collagen deposits (SHG) are localized in close proximity to each other in inflamed myocardial tissues from MC and DCMi patient groups. (a) In a tissue from an acute myocarditis (MC) patient, immunostaining together with SHG imaging show the presence of αSMA+ myofibroblasts (green) in areas with untensed fibronectin fibers (magenta) and fibrillar collagen deposits (grey (above). Scale bars: 200 µm. Corresponding enlarged images of the region-of-interest (below). Scale bars: 50 µm. (b) In a tissue from a chronic inflammatory dilated cardiomyopathy patient, immunostaining together with SHG imaging show the presence of αSMA+ myofibroblasts in loci with untensed fibronectin fibers and fibrillar collagen deposits (above). Scale bars: 200 µm. corresponding enlarged images of the region-of-interest (below). Scale bars: 50 µm. Refer to Suppl. Fig. S8 for a high-resolution image with myofibroblasts in proximity to areas rich in collagen fibers (SHG) and relaxed fibronectin fibers. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 7. Ranked correlation matrices of clinical diagnostic and experimental data for individual patient groups. (a-d) Spearman rank correlation matrices showing relationships between multiple variables from clinical diagnostic data and immunohistochemical analysis data from the acute MC, DCM and DCMi and COVID-19 groups. The correlation values described in the text are shown as yellow boxes. The correlation coefficients (r) range between −1 and + 1 and the positive/negative signs follow harmonized variable polarity. Only correlations ≥ +0.5 and ≤ -0.5 are shown in red and blue respectively. The most clinically relevant correlations are highlight with a yellow square and are explained below in the table (e) Table with correlation coefficient (r) values of the most relevant correlations between clinical and experimental parameters across all the patient groups (N: MC ≥ 13, DCM-5, DCMi ≥ 10, COVID-19 = 4; Statistical analysis: Spearman rank correlation; Statistical significance level set to α = 0.05, p values for all patient groups are provided in Suppl. Table 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)