Three-dimensional alignment of microvasculature and cardiomyocytes in the developing ventricle

Figures

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  Figure 1. Methods summary. (a) Quail hearts were perfused with DiI, (b) optically cleared, (c) and imaged using confocal microscopy to detect both coronary microvasculature and cardiac nuclei. (d) Vessels were segmented, (e) and automatically skeletonized resulting in vessel-nodes, -segments and -points exported for analysis. A vessel-vector (red arrow) was defined for each vessel by connecting the start and end nodes. (f) The helical angle (αH) was calculated for each vessel-vector based on its position with respect to the heart’s surface. (g) Cardiac nuclei were imaged with DAPI. (h) A Fourier transform (FFT) was applied to each image and the resulting spectrum was multiplied by (i) a series of masks with different axial angles. (j) The resulting angular amplitude A(θi) was plotted as a function of angle, (k) and fit to a von Mises distribution.

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  Figure 2. Highly developed, dense and organized quail coronary vasculature in the day 9 and 13 embryo. Vessels stained with DiI and color-coded based on depth in the 3D confocal image set. (a) Embryonic day 9. (b) Embryonic day 13. Posterior heart wall from the epicardium to the middle of the ventricular lumen. Two representative hearts shown with n = 4 hearts each for ED9 and ED13. Images colored with FIJI based on z-slice depth from confocal image stack. (c) Diameter of a vessel cross section after DiI perfusion. (d) Short diameter of blood cell nuclei as seen in DAPI images.

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  Figure 3. Mean coronary microvasculature orientation in the left ventricular posterior wall. Hearts at two developmental stages (left: ED9, right: ED13) with vessels color-coded based on the local mean helical angle αH, as defined by the coordinate vectors g1–g2–g3. In gray, cross-section views for each heart. Two horizontal cross-sections (g1–g3 plane) at different locations between the apex and the base. One vertical cross-section (g1–g2 plane) intersecting the ventricle from the apex to the middle of the left ventricular base. Volumetric heart surface images (orange) created from the DAPI images with the location of each cross-section indicated (black planes). Two representative hearts shown with n = 4 hearts each for ED9 and ED13. Epi Epicardium, Endo Endocardium. Images rendered in Amira with angles calculated in MATLAB.

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  Figure 4. Quantifying vessel orientation as a function of depth at embryonic day 9. (a) Heart surface created from DAPI images with region-of-interest (ROI) indicated with black box. (b) Mean helical angle (red line, left y-axis) calculated at each depth with a moving window (+ /− 15 μm). Standard uncertainty shown as error bars. The concentration parameter κ was obtained from a von Mises fit performed at each depth (black dashed line, right y-axis). Layers of interest indicated with grey/white shaded regions. Helical angle of 0° indicated with a dashed red line. (c) Vessel images with color coding for helical angle at increasing depths (panels 1–5, depth range indicated in b). White arrows indicate large vessels. (d) Helical angle distribution matched to above panels in c (same color-scale). (e) Transmural cross-section of vessels from the epicardium (Epi) to the endocardium (Endo). Black tick marks separate the five regions in depth shown in b. One representative heart shown with n = 4 hearts for ED9. Images rendered in Amira with angles calculated in MATLAB.

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  Figure 5. Quantifying vessel orientation as a function of depth at embryonic day 13. (a) Heart surface created from DAPI images with region-of-interest (ROI) indicated with black box. (b) Mean helical angle (red line, left y-axis) calculated at each depth with a moving window (+ /− 15 μm), with the standard uncertainty as error bars. The concentration parameter κ was obtained from a von Mises fit performed at each depth (black dashed line, right y-axis). Layers of interest indicated with grey/white shaded regions. Helical angle of 0° indicated with a dashed red line. (c) Vessel images with color coding for helical angle at increasing depths (panels 1–5, depth range indicated in b). White arrows indicate large vessels. (d) Helical angle distribution matched to above panels in (c) (same color scale used). (e) Transmural cross-section of vessels from the epicardium (Epi) to the endocardium (Endo). Black tick marks separate the five regions in depth shown in (b). One representative heart shown with n = 4 hearts for ED13. Images rendered in Amira with angles calculated in MATLAB.

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  Figure 6. Quantifying cardiac nuclei orientation as a function of depth at embryonic day 9. (a) Frontal section of the heart created from DAPI images with region-of-interest (ROI) indicated with the black box. Image colorized in FIJI. (b) Mean cardiac nuclei orientation μ calculated at each depth z (black line) + /− standard uncertainty u with five representative points selected at different depths. Black dashed line indicates 0° (horizontal). (c) Heart ventricle diagram showing the depth axis direction into the left ventricular wall. (d) Five DAPI images corresponding to points in b showing individual cardiac nuclei at different orientations (images used for analysis are 360 × 360 μm). (e) Polar plot showing the corresponding angular distribution of the 2D Fourier transform spectrum (black dots), the von Mises fit (black line), and the mean cardiomyocyte angle (red line) for each depth shown in (d). One representative heart shown with n = 4 hearts for ED9.

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  Figure 7. Cardiac nuclei and coronary vessels have the same orientation throughout the left ventricular wall. Mean projection angle for the vessels (red line) and mean orientation of cardiomyocyte nuclei (blue line) as a function of depth into the posterior left ventricular wall for (a) ED9 and (b) ED13, with standard uncertainty u as error bars. ρ is the Pearson’s correlation coefficient. (c) Pearson’s correlation coefficient between the mean vessel angle and the mean cardiomyocyte angle as a function of depth for all n = 4 ED9 hearts and all n = 4 ED13 hearts. Red data points indicate the hearts represented in (a) and (b). ρ = 1 is a perfect linear correlation. (d) Confocal microscopy image acquired at 20 × magnification in an ED13 heart with DAPI (blue) and DiI (red) signal. The DAPI signal was acquired at depth z = 496 μm, and the DiI signal was averaged over the range z = 482 μm to z = 511 μm. Image processed in FIJI.

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  Figure 8. Comparison of mean vessel orientation, vessel alignment and vessel density across hearts. Mean coronary vessel helical angle as a function of depth in the posterior left ventricular wall for n = 4 hearts at (a) embryonic day (ED) 9, and (b) ED13. Depth across the ventricle wall is normalized for all hearts from 0 (closest vessels to epicardium) to 1 (closest vessels to endocardium). A.u. is arbitrary units. Black dashed line indicates the horizontal at 0°. (c) Concentration parameter κ averaged over the first 60% of the tissue starting from the epicardium (left) and the last 40% of the tissue until the endocardium (right) for all hearts at ED9 and ED13. Mean for each group indicated by gray line. (d) Vessel volume fraction (left) and vessel length density (right) for all hearts at ED9 and ED13. Mean for each group indicated by gray line. Groups compared using a two-tailed T-test assuming unequal variance.