Histology-validated quantitative MRI characterization of collagen-rich Purkinje fiber-associated pathways in the ex vivo porcine heart

Figures

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  Fig. 1. Schematic illustration of the anatomical organization of the CCS in the left ventricle. The enlarged view indicates the approximate location of the subendocardial myocardium from which tissue samples were collected. This region includes the subendocardial connective tissue layer and a representative FT spanning the ventricular cavity. PFs are depicted (a) within the cross section of FT and (b) along the subendocardial surface, demonstrating their close spatial relationship with the surrounding connective tissue and adjacent working myocardium. This diagram provides anatomical context for the subsequent MRI and histological analyses

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  Fig. 2. Schematic of the histology analysis pipeline from RGB images to quantitative OD comparison among tissue types. RGB images were decomposed into red and blue channels, converted into OD maps, and segmented to identify myocardium, PFs, and collagen. Mean OD values were statistically compared across tissue types

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  Fig. 3. Masson’s trichrome-stained cross-section illustrating PF networks in the left ventricle (LV). a Low-magnification view of a transverse section from the LV apical region near the base of the papillary muscles, corresponding to the anatomical region highlighted in Fig. 1. Boxed areas indicate the locations of the enlarged views shown in b and c. b Transverse section of a free-running FT, showing a central PF bundle embedded within collagen-rich strands and surrounded by an endothelial layer. c Cross-section of the subendocardial connective tissue layer, showing PF bundles (pale red) interwoven with collagen-rich strands beneath the endocardium and superficial to the working myocardium. ECM, Extracellular matrix; EnC, Endocardium; EpC, Epicardium; LV, Left ventricle

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  Fig. 4. Histology-based tissue segmentation and OD comparison. a Example Masson’s trichrome-stained section containing an FT, showing myocardium, collagen, and PFs. b Corresponding OD maps obtained from red (OD_R) and blue channel (OD_B) decomposition, illustrating distinct staining profiles across tissue types. c Final segmentation mask classifying myocardium, PFs, and collagen using threshold-based separation of OD_R and OD_B. These segmented regions were used for quantitative analysis. d Boxplots of mean OD_R and OD_B for myocardium, PFs, and collagen. Measurements were obtained from two histological sections in five cardiac samples (N = 10). Statistical comparisons were performed using independent t-tests with Benjamini–Hochberg correction. Significance levels: n.s., p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001. ECM, Extracellular matrix

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  Fig. 5. a Photograph of the tissue block used for the histological analyses shown in Figs. 2–4. b, c OD maps obtained from red (OD_R) and blue channel (OD_B) decomposition, illustrating distinct staining profiles across tissue types. d Final segmentation mask classifying myocardium, PFs, and collagen using threshold-based separation of OD_R and OD_B. e Corresponding RAFF relaxation time map. f RAFF relaxation time map overlaid with histology-derived ROIs, showing myocardium (white shaded area) and high-collagen regions (magenta shaded area)

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  Fig. 6. a Relaxation times measured from different qMRI parameters between myocardium control (MC) and high-collagen (HC) regions. b RRTD between HC and remote myocardium for each qMRI parameter. Statistical significance from pairwise comparison is indicated as *p < 0.05, **p < 0.01 and ***p < 0.001 for differences in relaxation times between HC and myocardium, and as #p < 0.05, ##p < 0.01, and ###p < 0.001 for differences in RRTD among qMRI parameters. n.s., not significant