Detailed quantification of cardiac ventricular myocardial architecture in the embryonic and fetal mouse heart by application of structure tensor analysis to high resolution episcopic microscopic data

FIGURE 1. Whole-heart volume-rendered three-dimensional high-resolution episcopic microscopy reconstruction of the developing murine heart at (A) E14.5, (B) E15.5, (C) E16.5, (D) E17.5 and (E) E18.5 days, together with black/white inverted original episcopic microscopy 2D image.

FIGURE 2. (A) Example of anisotropic vs. isotropic myocardium sub-volumes with their corresponding eigenvector system ( v→i , i = 1…3) and ellipsoids obtained with structure tensor analysis, showing high anisotropy represented by a flat ellipsoid when myocytes have a more elongated shape, vs. low anisotropy represented by a more spherical ellipsoid, when myocytes have a more spherical shape. (B) Schematic representation of the region of interest (ROI) defined to calculate the structure tensor in a given voxel, formed by the voxel itself (in the centre of the cube—red square in left panel) and their eight nearest voxels. In this drawing, each cylinder represents a single cardiomyocyte. The right panel illustrates the calculation of the myocardial disarray index (MDI) in the same ROI used to calculate the structure tensor. In this drawing, we have plotted the tertiary eigenvectors of the 2D plane indicated in grey in the right panel. MDI in a given voxel quantifies the angular uniformity between the tertiary eigenvectors of the voxel itself (red square) and the tertiary eigenvectors of their nearest neighbours. In an organised myocardium, all the eigenvectors have similar orientations and…

FIGURE 3. Histograms of (A) helical (HA) and (B) intrusion angle (IA) across the apical, mid-ventricular and basal LV and RV of murine hearts at E14.5, E15.5, E16.5, E17.5 and E18.5 days of development. Solid lines: age-group mean. Dotted lines: ± standard deviation.

FIGURE 4. Quantification of myocyte aggregates orientation [via helical (HA) and intrusion angle (IA)] and myocyte aggregates organisation [via fractional anisotropy (FA) and myocardial disarray index (MDI)]. Local HA (A), IA (B), FA (C) and MDI (D) at mid-ventricular level, at E14.5, E15.5, E16.5, E17.5 and E18.5 days of development.

FIGURE 5. Left ventricular (LV) transmural profiles of helical angle (HA) across anterior, septal, posterior and lateral walls, from endocardium (negative transmural depth values) towards the epicardium (positive transmural depth values), at basal (top), mid (middle) and apical myocardium (bottom), at E14.5, E15.5, E16.5, E17.5 and E18.5 days of development. Solid lines represent age-group mean. Transmural depth of 0 µm represents the position where myocyte aggregates have a HA of 0°.

FIGURE 6. Histograms of (A) fractional anisotropy (FA) and (B) myocardial disarray index (MDI) across the apical, mid-ventricular and basal LV and RV of murine hearts at E14.5, E15.5, E16.5, E17.5 and E18.5 days of development. Solid lines: age-group mean. Dotted lines: ± standard deviation.

FIGURE 7. Seventeen-segment AHA bull’s eye plots quantifying fractional anisotropy (FA) (A) and myocardial disarray index (MDI) (B) in developing murine hearts at E14.5, E15.5, E16.5, E17.5 and E18.5 days of gestation.

FIGURE 8. Three-dimensional representation of the tertiary eigenvector representing the longitudinal direction of aggregated myocytes for a single mid-LV slice of the developing murine heart at (A) E14.5, (B) E15.5, (C) E16.5, (D) E17.5 and (E) E18.5 days of gestation. The insets represent magnified areas within the septum, illustrating well how cardiomyocytes show different angles of intrusion throughout the septal wall, and how the myocardium becomes more organised with gestational age.