Research Papers

On Modeling Morphogenesis of the Looping Heart Following Mechanical Perturbations

[+] Author and Article Information
Ashok Ramasubramanian, Nandan L. Nerurkar, Kate H. Achtien, Benjamen A. Filas, Dmitry A. Voronov

Department of Biomedical Engineering, Washington University, St. Louis, MO 63130

Larry A. Taber1

Department of Biomedical Engineering, Washington University, St. Louis, MO 63130lat@wustl.edu

The outer layer of the atria is more properly termed “presumptive myocardium,” because the cells in this layer do not begin to beat until the atria fuse and become part of the HT.


Corresponding author.

J Biomech Eng 130(6), 061018 (Oct 23, 2008) (11 pages) doi:10.1115/1.2978990 History: Received August 27, 2007; Revised December 07, 2007; Published October 23, 2008

Looping is a crucial early phase during heart development, as the initially straight heart tube (HT) deforms into a curved tube to lay out the basic plan of the mature heart. This paper focuses on the first phase of looping, called c-looping, when the HT bends ventrally and twists dextrally (rightward) to create a c-shaped tube. Previous research has shown that bending is an intrinsic process, while dextral torsion is likely caused by external forces acting on the heart. However, the specific mechanisms that drive and regulate looping are not yet completely understood. Here, we present new experimental data and finite element models to help define these mechanisms for the torsional component of c-looping. First, with regions of growth and contraction specified according to experiments on chick embryos, a three-dimensional model exhibits morphogenetic deformation consistent with observations for normal looping. Next, the model is tested further using experiments in which looping is perturbed by removing structures that exert forces on the heart—a membrane (splanchnopleure (SPL)) that presses against the ventral surface of the heart and the left and right primitive atria. In all cases, the model predicts the correct qualitative behavior. Finally, a two-dimensional model of the HT cross section is used to study a feedback mechanism for stress-based regulation of looping. The model is tested using experiments in which the SPL is removed before, during, and after c-looping. In each simulation, the model predicts the correct response. Hence, these models provide new insight into the mechanical mechanisms that drive and regulate cardiac looping.

Copyright © 2008 by American Society of Mechanical Engineers
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Figure 1

Schematic for cardiac rotation hypothesis. Ventral views ((a)–(c)) and cross-sectional views ((a′ )–(c′ )) are shown, with locations of cross sections indicated by dashed lines in (a)–(c). (HT=heart tube, LA=primitive left atrium, RA=primitive right atrium, CJ=cardiac jelly, MY=myocardium, EN=endocardium, SPL=splanchnopleure, and FG=foregut.) ((a) and (a′ )) A straight heart tube before looping. ((b) and (b′ )) Both atria push against the caudal end of the heart tube, and relatively greater force exerted by the left atrium displaces the heart tube slightly toward the right. ((c) and (c′ )) The splanchnopleure pushes the heart tube dorsally, completing torsion.

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Figure 2

Effect of splanchnopleure (SPL) removal on cardiac torsion. Fluorescent labels are used to visualize rotation. SPL was removed at stage 10 ((a)–(a″ )), stage 11 ((b)–(b″ )), and stage 12 ((c)–(c″ )). (a) Stage-10 heart with SPL removed (HT=heart tube, RA=right atrium, and LA=left atrium). (a′ ) The same heart after 6 h culture (stage 11); little rotation occurred. (a″ ) The same heart after 12 h culture (stage 12); rotation was fully restored. (b) Stage-11 heart with SPL intact. (b′ ) The same heart 20 min after the SPL-removal; the heart partially untwisted, as shown by labels moving toward the center of HT. (b″ ) The same heart after 6 h culture (stage 12); rotation was fully restored. (c) Stage-12 heart with SPL intact (CT=conotruncus). (c′ ) The same heart 20 min after the SPL-removal; the heart remained rotated. (c″ ) The same heart after 6 h culture (stage 14); the heart continued to develop normally. Scale bar=200 μm.

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Figure 3

Finite element models (reference geometry). (a) Model for microindentation of primitive atria. Top: ventral view; bottom: caudal view showing cross section. The circle with cross hair denotes the location of the indenter. (b) Ventral view of the 3D model for the looping heart (undeformed configuration) with loads and boundary conditions as indicated; the insert shows the side view. Note the angled atria and the fully bent heart tube. The dotted line in the atria denotes the longitudinal direction. (c) Two-dimensional (cross-sectional) model for cardiac rotation. (HT=heart tube, CT=conotruncus (outflow tract), TA=top atrial region, BA=bottom atrial region, DM=dorsal mesocardium, FG=foregut wall, MY=myocardium, CJ=cardiac jelly, and SPL=splanchnopleure).

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Figure 4

Optical coherence tomography (OCT) images of the embryonic heart showing the effects of splanchnopleure (SPL) removal. ((a)–(d)) Left lateral view; ((a′ )–(d′ )) ventral view; ((a″ )–(d″ )) cross-sectional view of the heart tube (HT) at locations indicated by dotted lines in ventral view. The white arrowheads in (b′ )–(d′ ) mark the position of a cluster of beads used to visualize HT rotation; rotation angle θ is defined by orientation of lumen, as shown in (b″ ). ((a)–(a″ )) Stage-10 heart with SPL intact. ((b)–(b″ )) The heart with SPL removed after 3 h culture. ((c)–(c″ )) The same heart after 6 h culture. Note that the bead cluster has not migrated rightward. ((d)–(d″ )) The same heart after 12 h culture. The bead cluster has migrated to the ventral midline, indicating rotation of the heart. Rotation also can be seen in cross-sectional views (MY=myocardium and CJ=cardiac jelly). The lines in (b)–(d) indicate the orientation of the primitive left atrium. Relative to the embryonic plane, the orientation angle progressively increases (approximately 30 deg, 45 deg, 90 deg, and 90 deg in (a), (b), (c), and (d), respectively). Note that (b)–(b″ ), (c)–(c″ ), and (d)–(d″ ) are images of the same heart. A different heart is used in (a)–(a″ ). Scale bars=200 μm.

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Figure 5

Stiffness measurements in normal stage-11 chick hearts. (a) Stage-11 heart indicating the tested regions (LC=left side of conotruncus, RC=right side of conotruncus, TLA=top of left atrium, OC=outer curvature of the heart tube, and BA=bottom of atria). (b) Measured stiffness for each region at 15 μm indentation depth (mean±SEM). For normal hearts (first five bars), region BA is significantly stiffer (p<0.001) than the other regions, which have similar stiffnesses. (The data for normal OC are from Ref. 8.) Also shown are the data for the following cases: BA exposed to 30 μM blebbistatin (bleb), OC of embryos cultured for 6 h without splanchnopleure (no SPL, from Ref. 5), and OC of embryos cultured for 6 h without splanchnopleure exposed to 12.5 μM Y-27632 (no SPL, Y; from Ref. 5). The cytoskeletal contraction inhibitors blebbistatin and Y-27632 significantly reduced the stiffness (p<0.001).

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Figure 6

Effect of contraction on curvature of primitive atria. The dotted lines trace the shape of the anterior intestinal portal (AIP). (a) Normal heart at stage 10+; AIP curvature is relatively small. (a′ ) The same heart after 20 min exposure to 30 μM blebbistatin; AIP curvature increased dramatically. (b) Computational model of heart at stage 10+, with growth and contraction specified as indicated in atria. (b′ ) The same model with contraction turned off; curvature of the AIP increases as seen experimentally.

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Figure 7

Three-dimensional finite element model for looping without splanchnopleure (ventral view). Nodes on the ventral midline are marked to visualize rotation; the arrow in (b) marks the region where the rotation angle is measured (see schematic in Fig. 4(b″ )) (HT=heart tube and CT=conotruncus). (a) Undeformed configuration with morphogenetic loads indicated. (b) Deformed configuration. The midline nodes move rightward as HT rotates, similar to experiment (see Fig. 2(b″ )). (c) Same model with contraction turned off on the right side of HT and CT; the amount of rotation decreases. (d) Model with contraction turned off everywhere; the rotation decreases further.

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Figure 8

Effect of atria orientation on cardiac rotation. The arrows mark the regions where the lumen rotation angle is measured (see Fig. 4(b″ )). Deformed ventral views are shown with inserts showing the undeformed side views. The filled circles mark the midline nodes. (a) Atria lie in plane of embryo; the rotation angle is 11 deg. (b) Atria are oriented at 45 deg relative to embryonic plane; the rotation angle is 24 deg. (c) Atria are oriented normal to embryonic plane; the rotation angle is 24 deg.

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Figure 9

Experimental and computational effects of atria removal on looping. ((a)–(f)) Initial configuration (stage 10) with splanchnopleure (SPL) and at least one atrium removed (the solid lines indicate the cut locations). ((a′ )–(f′ )) Final configuration (approximately 12 h later). Midline labels (experiment) and nodes (model) are used to visualize rotation. Top row: left atrium is removed; heart loops leftward (11/15 in experiment). Middle row: right atrium is removed; heart loops rightward with abnormal morphology (12/12 in experiment). Bottom row: both atria are removed; rightward rotation occurs (14/14 in experiment). In each case, the model predicts approximately the correct heart shape, as indicated by dotted traces.

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Figure 10

Two-dimensional model for cardiac rotation including mechanical feedback in the myocardium. Simulations show splanchnopleure (SPL) removal at stages 10 ((a)–(a″ )), 11 ((b)–(b″ )), and 12 ((c)–(c′ )). Time t=0 corresponds to these respective stages, and time points shown in each case correspond to the experimental time points shown in Fig. 2. (a) Model geometry for stage-10 heart (MY=myocardium, DM=dorsal mesocardium, CJ=cardiac jelly, and FG=foregut wall). The heart is given a small initial rightward push, and then the load is removed. (a′ ) Little rotation occurs during the first 6 h. (a″ ) After 12 h, the heart has rotated fully. (b) Model for the stage-11 heart with SPL in contact with the myocardium. (b′ ) When the SPL is removed, there is an immediate loss of rotation. (b″ ) Full rotation has occurred 6 h later. (c) Model for the stage-12 heart with full rotation. (c′ ) Little rotation is lost when the SPL is removed.




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