New Insights on the Formation of the Mitral Valve Chordae Tendineae in Fetal Life

Figures

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  Figure 1. Representative image of an excised adult bovine anterior mitral valve. Arrows denote (in order from top to bottom) the strut, basal, and marginal chordae.

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  Figure 2. Methods for measuring perforation dimensions in the two modalities. The perimeter was measured as an approximation of perforation size. Representative image of these measurements in a Movat Pentachrome stained section from 219 gestational days (A). Representative image of these measurements in 2D still image from micro-CT from 163 gestational days (B). Note the scale bar varies between images.

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  Figure 3. Diagram depicting the location of samples for transmission electron microscopy. Trunk sections were taken from above the papillary muscle connection to just below the first bifurcation. Branch sections were taken from above the bifurcation to below their next bifurcation.

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  Figure 4. Determination of full width at half maximum (FWHM) and mode calculation from collagen fibril diameter distributions. The FWHM was measured by first identifying the maximum value of the distribution (y max) and then dividing that by 2 (y max/2). To find x1 and x2, the x value that corresponded with the maximum y value was defined. This was then used as a threshold to find the minimum (x1) and maximum value (x2) of x that corresponded with y max/2. FWHM was calculated by subtracting x2 minus x1 (blue double-sided arrow). The mode was defined as the most common value in the distribution (red dotted line).

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  Figure 5. Representative images of the “transition zone” found between the collagenous portions of the mitral anterior leaflet and chordae tendineae throughout gestation. (A,D,G,J) Overview of the “transition zone” in the anterior leaflet. Dashed boxes show where subsequent magnified panel images were taken from. (B,E,H,K) Magnified images of the cell-lined perforations present in this area. (C) Magnified image of the “transition zone” demonstrating a lack of elastic fiber presence at earlier time points. (F,I,L) Magnified images showing the pattern of elastic fiber organization in this transition zone at later developmental stages. (A–C) valve in the early second trimester (111 days into gestation). (D–F) third trimester (212 days into gestation). (G–I) third trimester (219 days into gestation). (J–L) full term (270 days). The long axes of perforations were often in the direction of the chordae tendineae. (Figure 5G,J and Figure 6B,C). Collagen is stained yellow-orange, elastic fibers are dark purple, muscle tissue, and blood cells are red, glycosaminoglycans are blue-green, and cell nuclei are dark red-purple. The scale bar varies per image.

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  Figure 6. Elastin and collagen fibers first appear as short, sparse fragments before beginning to align in parallel with the chordae. (A) Movat pentachrome stained section of a perforation (P) with short elastic fragments (red arrows) and longer fragments (green arrows) beginning to align around it. Image taken at 14X magnification using the Pannoramic MIDI II. (B,C) Picrosirius red and hematoxylin-stained sections of a perforation (P) with shorter sparse collagen fibrils and collagen beginning to align parallel to the chordae under brightfield (B) and polarized light (C). Images were taken on a Nikon Eclipse E600 light microscope (Nikon Instruments Inc., Melville, NY, USA) equipped with a polarizer and an AmScope 10MU1400 digital camera (AmScope, Irvine, CA, USA). All photos were taken from sections from the same third trimester valve (219 days into gestation). The scale bar varies per image.

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  Figure 7. Representative images of CD-31 (endothelial cell marker) expression throughout the mitral valve anterior apparatus. (A,E,I,M) Overview of the CD-31-stained sections. Boxes show where subsequent magnified panel images were taken from. (B,F,J,N) Magnified images of the outer edge of the anterior leaflet showing CD-31 staining in the cells lining the leaflet. (C,G,K,O) Magnified images of the outer edge of the chordae tendineae showing CD-31 staining in the cells lining them. (D,H,L,P) Magnified images showing CD-31 staining in the cells lining the perforations in the transition zone. (A–D) Valve in the early second trimester (111 days into gestation); (E–H) late second trimester (163 days into gestation); (I–L) third trimester (219 days into gestation); (M–P) full term (270 days). CD-31 positive cell staining is shown in brown. Cell nuclei are stained blue. Scale bar and magnification vary per image.

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  Figure 8. Representative images of MMP-1 expression throughout the mitral valve anterior apparatus. (A,E,I,M) Overview of the MMP-1-stained sections. Boxes show where subsequent magnified panel images were taken from. (B,F,J,N) Magnified images of the body of the anterior leaflet showing diffuse MMP-1 staining throughout. (C,G,K,O) Magnified images of the chordae tendineae showing diffuse MMP-1 staining within the chordae and dense expression in the outer chordal edges. (D,H,L,P) Magnified images showing MMP-1 staining in the cells lining the perforations in the transition zone as well as the tissue around them. (A–D) Valve in the early second trimester (111 days into gestation); (E–H) late second trimester (163 days into gestation); (I–L) third trimester (219 days into gestation); (M–P) full term (270 days). MMP-1 positive staining is shown in brown. Cell nuclei are stained blue. Scale bar and magnification vary per image.

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  Figure 9. Representative images of MMP-2 expression throughout the mitral valve anterior apparatus. (A,E,I,M) Overview of the MMP-2-stained sections. Boxes show where subsequent magnified panel images were taken from. (B,F,J,N) Magnified images of the body of the anterior leaflet showing diffuse MMP-2 staining throughout. (C,G,K,O) Magnified images of the chordae tendineae showing diffuse MMP-2 staining within the chordae and dense expression in the outer chordal edges. (D,H,L,P) Magnified images showing MMP-2 staining in the cells lining the perforations in the transition zone as well as the tissue around them. (A–D) Valve in the early second trimester (111 days into gestation); (E–H) late second trimester (163 days into gestation); (I–L) third trimester (219 days into gestation); (M–P) full term (270 days). MMP-2 positive staining is shown in brown. Cell nuclei are stained blue. Scale bar and magnification vary per image. Note that to preserve tissue integrity, the epitope retrieval time was reduced for MMP-2 staining; therefore, differences in intensity cannot be interpreted as differences in the levels of MMP expression between MMP-1 and MMP-2 sections.

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  Figure 10. Representative images of Ki-67 expression throughout the mitral valve anterior apparatus. (A,E,I,M) Overview of the Ki-67-stained sections. Boxes show where subsequent magnified panel images were taken from. (B,F,J,N) Magnified images of the body of the anterior leaflet showing Ki-67 positive cells. (C,G,K,O) Magnified images of the chordae tendineae showing Ki-67 positive cell staining. (D,H,L,P) Magnified images showing Ki-67 staining in the cells lining the perforations in the transition zone as well as in the tissue around them. (A–D) Valve in the early second trimester (113 days into gestation); (E–H) late second trimester (163 days into gestation); (I–L) third trimester (219 days into gestation); (M–P) full term (270 days). Ki-67 positive cells are shown in brown. Ki-67 negative cell nuclei are stained blue. Scale bar and magnification vary per image.

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  Figure 11. Both total cell density and total Ki-67 positive cell density decreased over time but were highest within the transition zone. (A) Total cell density (number of cells per mm2) in the anterior leaflet (purple triangles), the chordae tendineae (blue circles), and the transition zone (green diamonds) plotted as a function of gestational age, showing a significant correlation between these variables for each area. (B) Total Ki-67 positive cell density (number of cells per mm2) in the anterior leaflet (purple triangles), the chordae tendineae (blue circles), and the transition zone (green diamonds) plotted as a function of gestational age, showing a significant correlation between these variables for each area. Data are shown from five animals of five gestational ages. For each animal, data are shown for the anterior leaflet, the transition zone, and chordae tendineae, where three grids were analyzed in each region. Note the x-axis break on both graphs. * Denotes a slope significantly greater than zero.

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  Figure 12. Representative images of the cell clusters present within the transition zone throughout gestation. (A,E,I,M) CD-31staining is present in cells within the clusters. (B,F,J,N) MMP-1 staining is found within and around the cell clusters. (C,G,K,O) MMP-2 staining is found within and around the cell clusters. (D,H,L,P) Some cells within the clusters are positive for Ki-67 staining. (A–D) Valve in the early second trimester (113 days into gestation); (E–H) late second trimester (163 days into gestation); (I–L) third trimester (219 days into gestation); (M–P) full term (270 days). Positive marker staining is shown in brown. Cell nuclei are stained blue. The scale bar varies per image.