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1.
Fig. 4.

Fig. 4. From: Blood flow dynamics of one cardiac cycle and relationship to mechanotransduction and trabeculation during heart looping.

A: peristaltoid map. The S marks the location of end-contraction, and the D of end-relaxation. Black blocks correspond to lumen areas within 25% of end-contraction. Light gray and dark gray blocks correspond to lumen areas that are 50 and 75% of end-contraction, respectively. White corresponds to lumen areas within 25% of end-relaxation. B: area fractional shortening (AFS) measurements depict the percent reductions in area from end-relaxation to end-contraction for the heart and Endo lumen at the six heart levels analyzed. The myocardial AFS averages −46 ± 2.4%, and the AFS of the endocardial lumen averages −86 ± 3.5%.

Barbara Garita, et al. Am J Physiol Heart Circ Physiol. 2011 March;300(3):H879-H891.
2.
Fig. 5.

Fig. 5. From: Blood flow dynamics of one cardiac cycle and relationship to mechanotransduction and trabeculation during heart looping.

Looping heart and relationship to Spl membrane (SPM). A: looping heart (HT) enclosed in SPM (arrows). B: acetylated α-tubulin antibody (bright green signal) localized to monocilia in cardiomyocytes of a stage 17 chick Myo (white arrows). Blue arrows point to a monocilium associated with the endocardial endothelium cells. CF: 4 frames of an OCT movie show the myocardial surface touching the SPM (green arrows) during diastole (C; red arrow indicates membrane contact area), then moving further along the membrane (D and E; red arrows delineate contact region), and then Myo pulls away from membrane during systole (F; contraction). Magnification bar in B = 76 μm. AIP, anterior intestinal portal.

Barbara Garita, et al. Am J Physiol Heart Circ Physiol. 2011 March;300(3):H879-H891.
3.
Fig. 6.

Fig. 6. From: Blood flow dynamics of one cardiac cycle and relationship to mechanotransduction and trabeculation during heart looping.

A: multilevel analysis of the behavior of the CJ (dashed line with solid circles) as the Endo area (dotted line with triangles) changes from end-relaxation to end-contraction. L1–L6 on top of each curve refer to the heart level, whereas T1, T2, T5, T6, T7, T8, T9, T14, and T15 represent time during the cardiac cycle shown in Fig. 2. endRelax, end-relaxation; endCont, end-contraction. Gray lines represent trend lines for the behavior of the CJ. The data were normalized by subtracting the lumen area by the minimum lumen area, and the result was divided by the maximum lumen area. B: normalized quantity of CJ in the heart at the 15 time points during a cardiac cycle analyzed. The solid line is the average (0.87 ± 0.07) for all time points.

Barbara Garita, et al. Am J Physiol Heart Circ Physiol. 2011 March;300(3):H879-H891.
4.
Fig. 2.

Fig. 2. From: Blood flow dynamics of one cardiac cycle and relationship to mechanotransduction and trabeculation during heart looping.

Cross-sectional cuts are specified at 6 different levels along the length of the heart tube for a HH stage 13 embryonic heart during a cardiac cycle of 450-ms duration. Cuts L1 and L2 are through the outflow region. L3, L4, and L5 cuts are in the ventricular region, and L6 is representative of the inflow region. The red fillings represent end-relaxation, and the underlined tracings indicate end-contraction. All outlines are aligned in a consistent orientation and in accordance with the legend on the right corner. At time point 15 (t15) and for all levels, the blue line demonstrates the change in the shape of the heart tube from inflow to outflow during diastole. Orientation of the heart tube relative to the whole embryo is indicated. Magnification bar on bottom left of figure = 300 μm.

Barbara Garita, et al. Am J Physiol Heart Circ Physiol. 2011 March;300(3):H879-H891.
5.
Fig. 1.

Fig. 1. From: Blood flow dynamics of one cardiac cycle and relationship to mechanotransduction and trabeculation during heart looping.

The embryonic heart as captured in real time and under physiological conditions with optical coherence tomography (OCT). A: the dashed white line shows the direction of blood flow on a digitally reconstructed OCT radiograph. B: for all 15 volumes analyzed, the placement of the midcoronal plane (white line) was the same. C: three-dimensional (3D) reconstruction of Hamburger and Hamilton (HH) stage 13 heart depicts endocardium (Endo) in dark gray, and myocardium (Myo) is transparent in light gray. Six cross-sectional cuts [levels 1–6 (L1–L6)] were defined perpendicular to blood flow. D: cross-sectional area measurements of the outer Myo area, endocardial lumen area, and cardiac jelly (CJ) area were performed with MetaMorph 6.1. The outlines are shown in white. Number 18 on figure identifies frame that was used. Magnification bars in all panels = 100 μm.

Barbara Garita, et al. Am J Physiol Heart Circ Physiol. 2011 March;300(3):H879-H891.
6.
Fig. 3.

Fig. 3. From: Blood flow dynamics of one cardiac cycle and relationship to mechanotransduction and trabeculation during heart looping.

Mechanotransducing molecules at myocardial-endocardial attachment areas. On left, diagrams are tracings of the outlines of the Myo and Endo as captured by OCT at the six levels (L1–L6) of the heart tube from anterior to posterior. The arrows are color-coded defining cardiac areas shown in the figures: orange arrows define outer curvature; green arrows, the ventral midline at floor of the foregut (FG). AC: from serial sections (A), a 3D reconstruction of endocardial wall is shown in blue (B). C: endocardial wall is closely associated with the Myo (color-coded pink) in anterior regions (orange arrows). D and E: fibronectin (FN) localization. Top panel is for orientation, with boxes indicating regions shown in D and E panels. In D, FN (bright red signal) localized at attachment area at the FG, and in E (green signal), at the outer curvature where FN tethers the Endo (arrows) to the Myo. F: α-tubulin is present throughout Myo. G: at the outer curvature region shown at higher magnification, α-tubulin is oriented in direction of strain exerted by the Endo at this site. H and I: tenascin-C (TN-C) localization in myocardial basal lamina and within the Endo. I: 4 TN-C-mediated attachments (arrows) facilitate myocardial-endocardial communication at the outer curvature. NT, neural tube; N, notochord; DM, dorsal mesocardium; Spl, splanchnopleural. Magnifications bars = 180 μm (A, B, and C), 100 μm (D and E), 76 μm (F and H), and 38 μm (G and I).

Barbara Garita, et al. Am J Physiol Heart Circ Physiol. 2011 March;300(3):H879-H891.
7.
Fig. 8.

Fig. 8. From: Blood flow dynamics of one cardiac cycle and relationship to mechanotransduction and trabeculation during heart looping.

Initiation of trabeculation is associated with looping stages and NMHC-II expressing cells. A and B: isolated myocardial fragments of HH stage 13/14 hearts show a two-layered Myo with cells extending from the wall, but remaining attached (yellow arrows). The cells in red boxed-in region in A are shown at higher magnification in B. Superimposed images of immunolocalization of NMHC-II with nuclear 4,6-diamidino-2-phenylindole staining (arrows pointing to such a doublet of cells in inset) demonstrate that these are cell structures within the CJ. C: confocal micrograph of a chick looping heart at stage 13 immunostained for NMHC-II shows fine cell extensions in CJ extending from the Myo basal lamina toward the Endo. DG: a cellular organization characteristic of trabeculation becomes apparent between HH stages 12 and 16. D: a cell is present in the CJ near Myo at HH stage 13 with NMHC-IIB in CJ beginning to organize (arrows). E: similarly, cells are observed in the CJ near Endo at HH stages 13/14. F: by day 4 postlooping, distinct trabeculae (arrows) are apparent, showing similar periodicity as “pioneer” cells in A. G: by day 10, the ventricular region is highly trabeculated (arrows), maintaining the spacing that was evident earlier. Bottom row: diagrams of a wedge through the ventricular region of a tubular heart shown at different stages to illustrate our hypothesis: the initial pioneer cells lead to cellular clones of cells, forming a network in the CJ that postlooping has formed the trabeculae. The double-headed arrow depicts the expected direction of strain within the CJ and cellular networks during systole and diastole. Magnification bar in A and C = 100 μm; B = 33 μm.

Barbara Garita, et al. Am J Physiol Heart Circ Physiol. 2011 March;300(3):H879-H891.
8.
Fig. 7.

Fig. 7. From: Blood flow dynamics of one cardiac cycle and relationship to mechanotransduction and trabeculation during heart looping.

Fine nonmuscle myosin heavy chain (NMHC)-IIB-expressing cell processes and fibrils extend between Myo and Endo (En). A and B: adjacent plastic sections through the ventricular region of a looping avian HH stage 13 heart. A: cell processes/fibrils (green arrow) extend radially through CJ between the Myo and Endo. Yellow arrow points to the undulating surface of the Endo. In B, the cell processes/fibrils were color-coded in purple. C and D: optical coherence microscopy (OCM) images of the looping mouse heart (embryonic day 9.5). Endocardial folds are present (yellow arrows), as well as a fine fibrillar network in the CJ. Boxed region in C is shown at a higher magnification in D (green arrows point to cell processes and fibrils in the CJ; yellow arrow, an attachment of the Endo to the Myo). E: red Cy-3 immunostaining of NMHC-IIB shows localization to endocardial associations with the myocardial wall and in finer fibrillar-like material in the CJ. F: endocardial evaginations associate with the mouse Myo via FN-mediated attachments (yellow arrows; green signal for FN). G: 3D reconstruction of the endocardial tube from serial sections as in A and B. The undulating surface of the avian Endo during looping is evident. The ventral FG wall is encoded in gray and white, here defining the embryonic midline. Dark blue denotes where endocardial wall has evaginated toward the Myo, forming folds and outpocketings. H: superimposition of the segmented purple fibrils/processes with the Endo. The cell processes and fibrils colocalized at the endocardial ridges. Magnification bars in A and for B = 100 μm; in C and for D = 150 μm. Sp, splanchnopleural membrane.

Barbara Garita, et al. Am J Physiol Heart Circ Physiol. 2011 March;300(3):H879-H891.

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