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THE ANATOMICAL RECORD 292:652–660 (2009)

Dependence of Aortic Arch Morphogenesis on Intracardiac Blood Flow in the Left Atrial Ligated Chick Embryo NORMAN HU,1,2,5* DOUGLAS A. CHRISTENSEN,3,4 AMIT K. AGRAWAL,3,4 CHARITY BEAUMONT,1 EDWARD B. CLARK,2,4,5 AND JOHN A. HAWKINS1 1 Division of Cardiothoracic Surgery, Department of Surgery, University of Utah, Salt Lake City, Utah 2 Department of Pediatrics, University of Utah, Salt Lake City, Utah 3 Department of Electrical Engineering, University of Utah, Salt Lake City, Utah 4 Department of Bioengineering, University of Utah, Salt Lake City, Utah 5 Primary Children’s Medical Center, Salt Lake City, Utah

ABSTRACT Partial left atrial ligation before cardiac septation redistributes intracardiac blood flow and produces left ventricular hypoplasia in the chick. We hypothesized that redistributed intracardiac blood flow adversely alters aortic arch development. We ligated the left atrial appendage with a 10-0 nylon suture at stage 21 chick embryos, then reincubated up to stage 34. Sham embryos had a suture tied adjacent to the atrial wall, and normal controls were unoperated. We measured simultaneous atrioventricular (AV) and dorsal aortic (DAo) blood velocities from stage 24 embryos with an ultrasound pulsed-Doppler flow meter; and the left and right third and fourth aortic arch blood flow with a laser-Doppler flow meter. Ventricular and atrial cross-sectional areas were measured from sequential video fields for planimetry. Intracardiac flow patterns were imaged on video by injecting India ink into the vitelline vein. In separate embryos, radiopaque microfil was injected into the cardiovascular system for l-CT scanning. We analyzed the morphologic characteristics of the heart at stage 34. Active AV and DAo stroke volume (mm3), right third and fourth aortic arch blood flow (mm3/s) were all decreased in ligated embryos (P < 0.05) when compared with normal and sham embryos. Ventricular end-diastolic volume versus normal and sham embryos decreased by 45% and 46%, respectively (P < 0.05). India ink injection revealed altered right aortic arch flow patterns in the ligated embryos compared with normal embryos. l-CT imaging confirmed altered arch morphogenesis. Alterations in intracardiac blood flow disrupt both early cardiac morphogenesis and aortic arch selection. Anat Rec, 292:652– C 2009 Wiley-Liss, Inc. 660, 2009. V

Key words: chick embryo; left atrial ligation; aortic arch; blood flow; atrioventricular function; blood flow pattern

INTRODUCTION Function and structure are integrally related in most organ systems. The early embryonic heart, though morphologically simple, adjusts to small changes in hemodynamic load (Clark, 1990). Various experimental manipulations of blood volume, course, or rate of intracardiac blood flow produce cardiovascular anomalies and C 2009 WILEY-LISS, INC. V

*Correspondence to: Norman Hu, Division of Cardiothoracic Surgery, University of Utah, 3C145 SOM, 30 North, 1900 East, Salt Lake City, UT 84132. Fax: 801-585-3936. E-mail: [email protected] Received 28 September 2008; Accepted 21 January 2009 DOI 10.1002/ar.20885 Published online 25 March 2009 in Wiley InterScience (www. interscience.wiley.com).

AORTIC ARCH SELECTION IN THE LEFT ATRIAL LIGATED HEART

alter cardiomyocyte assembly (Clark et al., 1984; Hogers et al., 1997; le Noble et al., 2004). In the chick embryo, left atrial ligation before cardiac septation redistributes intracardiac blood flow and produces hypoplasia of left heart structures (Rychter et al., 1979; Sedmera et al., 1999; Tobita and Keller, 2000). Reducing blood flow through one side of the embryonic heart by diverting it to the other side results in underdevelopment or hypoplasia of all structures on the affected side, with a corresponding enlargement or hyperplasia of the side into which the flow is diverted. Associated abnormalities also include abnormal development of the interatrial septum (Rychter and Rychterova, 1981). The right atrioventricular valve loses its typical muscular flap-like morphology resembling a bicuspid fibrous valve, similar to the left atrioventricular (mitral) valve. These alterations affect development of the pumping heart, and the flow pattern of aortic arches well beyond the original site of altered flow. Embryonic blood vessels form from the coalescence of plexiform vascular channels in the developing mesenchyme. The right and left primitive aortae extend from the primitive endocardial heart tube, and fuse ventrally to form the aortic sac, whereas the dorsal aortae remain separate on either side of the notochord. Vascular connections between the ventral aorta sac and dorsal aorta course bilaterally through the mesenchyme of the first pharyngeal arch, termed the first aortic arch. With each successive aortic arch formation, similar vessels bridge the aortic sac to the right and left dorsal aorta. A total of six paired arches form successively from anterior to posterior. The initial formation of the six-paired aortic arches in avian development is similar to mammalian species. The involution pattern of the six-paired arches, however, is different. In chick embryo, the first and second arches persist as portions of the maxillary and stapedial arteries. The third arch pair forms the brachiocephalic arteries from which the common carotid and secondary subclavian arteries are derived. The right fourth arch pair persists as the definitive aortic arch, and the left involutes. The fifth arch pair is transient with no known derivative. The proximal portion of the sixth paired arches forms the pulmonary artery, and the distal portion remains as the paired ductus arteriosi. The true pulmonary arteries arise from the midpoint of the sixth arches and join the developing lung bud. The truncus arteriosus and aortic sac are divided cranially to caudally by a spiral ridge or cushion that separates the pulmonary artery and right ventricular outflow tract anteriorly from the aorta, and left ventricular outflow tract posteriorly (Romanoff, 1960; Seidl and Steding, 1981). The primordial aortic arches undergo an extensive remodeling from a symmetric pairwise system to an asymmetric system. This process may be regulated by cellular autonomous mechanisms. Other mechanisms involved in the aortic vessel morphogenesis are extracardiac mechanical forces, including traction by nerve plexi (Kirby, 1990) and torsion generated by the descended heart (Taber and Chabert, 2002). The hemodynamic forces generated by blood flow within the developing heart also have a molding influence on cardiovascular development. The left atrial ligation technique is well established as a model to generate cardiac outflow tract abnormalities (Rychter and Rychterova, 1981; Sedmera

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et al., 1999). We use this model to test the hypothesis that the redistributed intracardiac blood flow alters aortic arch development. Our integrated measurement of cardiovascular morphogenesis is essential to define the structure-function relationships in embryonic models of congenital cardiovascular anomalies.

MATERIAL AND METHODS Study Group Fertile white Leghorn chick eggs were incubated blunt-end up in a forced-draft 38.0 C incubator to stage 21 (31=2-day) (Hamburger and Hamilton, 1951). We positioned the egg under the stereomicroscope, and removed the egg shell and its membrane to expose the embryo. The embryo was gently turned to the left-side-up position, and an overhand knot 10-0 nylon suture was placed around the left atrium (Sedmera et al., 1999; Tobita and Keller, 2000). We constricted the left atrioventricular orifice by tightening the knot, thus decreasing the effective volume of the left atrium. The embryo was then gently returned to its original right-side-up position. Repositioning of the embryo did not alter heart rate or atrial function (Campbell et al., 1992). The opening in the egg was sealed with parafilm, and the eggs were returned to the incubator for hemodynamic measurements at stage 24 (4-day). We also incubated separate embryos for morphological analysis until stage 27 (5-day) or 34 (8-day). Sham-operated embryos had the suture tied adjacent to the left atrial wall. Normal embryos were not operated.

Morphometric Analysis At stage 24 (n  7), video images of the right atrium and ventricle were acquired using a photo-stereomicroscope (Leica MZ12, Bannockburn, IL), a video camera (Cohu 1300, San Diego CA), a video recorder (SONY SVO-9500MD, Norcross, GA), and a time-date generator (model VTG033 FOR.A, West Newton, MA) (Keller et al., 1991). After imaging, a 10-lm scale scribed-glass standard was recorded in the plane of each embryo. Starting from the onset of atrial diastole for each embryo, we selected video field of five complete and consecutive cardiac cycles to construct the atrial and ventricular volume. We used a planimeter (at 60 Hz) to measure sequential video fields for right atrial and ventricular cross-sectional areas using a video frame-grabbing board (Jandel Scientific, Corte Madera, CA), video analysis software (JAVA, Jandel Scientific, Corte Madera, CA). Cavity volume was calculated using a spherical atrial model (Vatrium ¼ 0.75 A3/2, where A is the atrial endocardial cross-sectional area), and an ellipsoidal ventricular model (Vventricle ¼ 1=3[4pab2]), where a is the semi-major axis, and b is the semi-minor axis of the ellipsoid). Ventricular stroke volumes were calculated from the difference of end-systolic and end-diastolic volumes of each cardiac cycle (Keller et al., 1994). Intra- and interobserver planimetric errors were small (P > 0.16 and P ¼ 0.12, respectively).

Hemodynamic Measurement We measured simultaneous dorsal aortic and atrioventricular blood velocities at stage 24 (n  10 in each parameter) with a 20-MHz ultrasound pulsed-Doppler

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flow meter (Clark and Hu, 1982; Hu and Clark, 1989). Dorsal aortic blood velocity was measured with a 750lm diameter piezoelectric crystal positioned over the dorsal aorta adjacent to the sinus venosus. Atrioventricular blood velocity was measured by positioning a second crystal at the apex of the ventricle, angled toward the atrioventricular orifice (Hu et al., 1991). Analog waveforms were sampled digitally at 500 Hz via an analog-todigital board (National Instruments AT-MIO16, Austin, TX), and viewed with custom analysis software (National Instruments Labview, Austin, TX). The converter offered 12 bits at an input range of 2.5 to 2.5 V and a real-time sampling rate of 70 KHz. A resolution of 12 bits or 4,096 levels yielded a signal resolution of 1.2207 mV or a velocity of 0.1351 mm/s with the Doppler. Left and right third and fourth aortic arch blood velocities (n  6) were measured with a custom-built fiberoptic laser-Doppler flow meter (Bioengineering Department, University of Utah) using a helium-neon laser operating at 633 nm wavelength. The sixth aortic arches were not measured due to the protrusion of the visceral arches. The optical beam from the fiber probe had a waist diameter of 6 lm and an effective detection distance of 20 lm. The size of the beam and detection distance enabled the probe to capture waveform from a single minute blood vessel without interference from the signals of neighboring circulations. A data acquisition card (National Instruments DAQCard-6062E, Austin, TX) with an analog-to-digital converter collected the Doppler spectrum at a sampling rate of 20 KHz from the photodetector, and transferred the data to the computer. The precision of the fiber-optic laser-Doppler flow meter was tested against a known velocity rotating disk that was covered with a white, reflective paper. The rotating paper acted as a random Gaussian scatterer that was statistically similar to the scattering from moving particles such as red blood cells. The laser-Doppler velocimeter yielded a regression analysis (y ¼ 1.0113x þ 0.0225, r2 ¼ 0.9998, SEE ¼ 0.0273) correlating linearly over the range of 0 to 7.5 mm/sec of the rotating disk. Reynolds number was less than 2.0 for both dorsal aortic and aortic arch blood flow indicating an orderly pattern of laminar flow (Hu et al., 1991). Cycle length was defined as the time between the upstrokes of the consecutive dorsal aortic blood velocity waveforms.

Analysis of Waveform Ventricular diastole of the atrioventricular blood velocity waveform was partitioned into a passive phase emerging from end-systole to onset of the a-wave, and an active phase from onset of a-wave to ventricular pressure upstroke (Hu et al., 1991). Dorsal aortic and aortic arch blood flow was calculated from the integral of flow velocity and the vessel cross-sectional area. The internal diameter of dorsal aorta was determined by video image planimetered against a 10-lm scribed glass standard at the sinus venosus level. The diameters of left and right in the third and fourth aortic arches also were calibrated with similar fashion at the mid-point of each arch. Passive and active atrioventricular stroke volumes were calculated as dorsal aortic stroke volume multiplied by the ratio of passive and active components. Aortic arch blood velocities were analyzed in MatLab (Natick, MA) using Fast Fourier Transformation to obtain the velocity pro-

file (Fig. 1). The pulse-Doppler frequency was directly measured by a built-in phase detector, whereas in laserDoppler velocimeter, the user did all the raw Doppler data postprocessing to obtain velocity information.

Blood Flow Pattern Intracardiac flow patterns (n  15 in each parameter) were recorded at 60 Hz using a video camera attached to the stereomicroscope and a videocassette recorder. 1 lL of India ink diluted (1:5) in normal saline was injected through the second bifurcation of the right vitelline vein. We recorded blood flow coursing through the beating atrium and primitive ventricle to aortic arches and dorsal aorta. Injection via right or left vitelline vein produced similar intracardiac flow patterns.

Micro-CT Scanning The cardiovascular system of stage 27 (5-day) embryos (n  10 in each parameter) was injected with radiopaque Microfil (Flow Tech, Carver, MA), and fixed in 70% ethanol. The tissue of the intact embryos was cleared with methyl salicylate after the embryos were dehydrated through an ascending ethanol series. The intact embryo was scanned by a l-CT system (Physiology Imaging Research Laboratory, Mayo Clinic, Rochester, MN) (Jorgensen et al., 1998). The resulting 3D images were displayed using Analyze image analysis software (AnalyzeDirect, Lenexa, KS). Computer-generated images display at different angles of view with 16-bit grayscale values of 6-lm voxels (on the side dimension). Individual image slices were edited in Adobe Photoshop (Adobe Systems, San Jose, CA) to segment the ventricle, bulbus, truncus, and aortic arches from the rest of the image.

Morphologic Analysis Stage 34 (8-day) embryos (n  10 in each parameter) were perfusion-fixed in diastole at high flow low pressure with 10% paraformaldehyde in an isotonic phosphate buffer containing 4  104 g/kg dose of diltiazem. We dissected the heart from the embryo to study the morphology of the atrium, ventricle, and vessels. The aortic arch artery derivatives were examined for presence and continuity. The right ventricular wall was removed to expose the ventricular septum, tricuspid valve, and right outflow tract.

Statistical Analysis Data are presented as mean  SEM. We used analysis of variance and Tukey’s significant difference test for statistical comparison. Significance level was defined as a P value less than 5% (P < 0.05).

RESULTS Hemodynamic Measurement (Stage 24) Cycle length and passive atrioventricular stroke volume were similar among normal, sham, and left atrial ligated embryos (P > 0.05) (Table 1). Active atrioventricular and dorsal aortic stroke volumes were decreased in ligated embryos (0.154  0.024 mm3, 0.304  0.025 mm3, respectively, P < 0.05) when compared with normal (0.316  0.023 mm3, 0.476  0.030 mm3, respectively) and sham

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Fig. 1. (a) A typical Doppler waveform obtained from the fourth left aortic arch in a stage-24 embryo using the laser Doppler velocity in time-domain. The frequency components in this signal are calculated with a Fast Fourier Transform routine using a window size of 512 samples. (b) A typical spectrum is obtained by FFT across a single win-

dow. (c) 3D plot of the spectra is presented for each window. (d) The mean Doppler frequency for each spectrum is calculated using the weighted mean algorithm showing the variation in the mean Doppler frequency as function of time. This variation in Doppler frequency is linearly related to blood velocity from the Doppler equation.

embryos (0.308  0.036 mm3, 0.448  0.032 mm3, respectively) (Fig. 2A). The right third and fourth aortic arch blood flow were decreased in ligated embryos (0.035  0.003 mm3/s, 0.037  0.002 mm3/s, respectively, P < 0.05) when compared with normal (0.048  0.003 mm3/s, 0.049  0.003 mm3/s, respectively) and sham embryos (0.047  0.002 mm3/s, 0.051  0.002 mm3/s, respectively) (Fig. 2B). Blood flow in the left third and fourth aortic arches also were decreased (P < 0.05) in the left atrial ligated embryo (Fig. 2B). Passive/active ratio increased in atrial ligated embryos (1.178  0.259) when compared with normal (0.550  0.089) and sham (0.532  0.043) controls (P < 0.05).

(Fig. 4) despite decreased dorsal aortic blood flow in the left atrial ligated embryo. Ventricular end-systolic anddiastolic volumes, in the left atrial ligated heart, were decreased by 46% and 45%, respectively (P < 0.05) when compared with normal and sham controls (Fig. 4). Passive atrioventricular blood flow occurred coincidentally with atrial filling.

Morphometric Analysis (Stage 24) The fourth aortic arch areas on the left and right displayed discordantly reduction in dimensions (Fig. 3). Right atrial and ventricular stroke volumes calculated from video images were similar in all embryos (P > 0.05)

Blood Flow Pattern (Stage 24) At least 60% of the left atrial ligated embryos showing the sequence of the blood flow pattern were altered in the right aortic arches. Blood flow in the third and sixth left aortic arches was obliterated. In normal embryos, blood flow emerged primarily in the fourth aortic arches, followed by the third, and then the sixth arches. In the left atrial ligated embryo, however, blood flowed dominantly through the sixth aortic arches rather than the fourth as in normal embryos (Fig. 5).

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TABLE 1. Cycle length and passive atrioventricular stroke volume of the normal, sham, and left atrial ligated embryos at stage 24 computed from the velocity waveforms

Cycle length (ms) Passive atrioventricular stroke volume (mm3)

Normal

Sham

Left atrial ligation

453.33  27.21 0.161  0.024

451.92  20.0 0.147  0.010

458.24  18.22 0.150  0.026

There is no statistical significance among the parameters. Data presented as mean  SEM.

Fig. 2. (a) Passive and active components of the atrioventricular stroke volumes, and dorsal aortic stroke volume of the normal, sham, and left atrial ligated embryos of stage-24 chick measured by pulseDoppler flow meter. The reduction of active atrioventricular filling attributes to the decrease in the dorsal aortic stroke volume. (b) Left

and right third and fourth aortic arches blood flow of stage-24 normal, sham, and left atrial ligated embryos using a laser-Doppler. Blood flow in all arches is decreased in left atrial ligated embryos. Data are presented as mean  SEM.

Micro-CT Scanning (Stage 27)

chamber with a pronounced effect on aortic arch malformations. One hundred percent of the left atrial ligated embryos surviving to stage 34 had hypoplastic left heart structures. More than seventy percent of these experimental embryos displayed some degree of malformations in the aortic arch arteries. The anomalies in the aortic arch arteries had a high variability including interrupted aortic arch, hypoplastic aortic arch, or persistence of some vessels that otherwise should have disappeared (Fig. 7).

More than 60% of the microfil perfused heart displayed some form of aortic arch abnormality in the left atrial ligated embryos. The type of anomalies varied from embryo to embryo. The major extracardiac disturbances were in the involution pattern or incomplete formation of the aortic arch derivatives. Conotruncus development digressed into a contorted appearance in comparison to the normal heart. The arrangement of the arches, particularly the left side, was noticeably asymmetric. The arches were askew and distorted in contrast to the arches of the normal embryo (Fig. 6). In some embryos, we observed that the left arches had conspicuously smaller diameters when compared with the right, and often displayed a twisted (off center) appearance. In others, the left fourth aortic arch was obliterated, and the continuity of the internal carotid arteries at the third aortic arches was interrupted. The left fourth arch (if present) and sixth arch exhibited a reduced diameter, and the sixth arch of the left side was more distorted than its counterpart. A partial absence of separation between the individual (left fourth and sixth) arches of the left side was prevalent.

Morphologic Analysis (Stage 34) Left atrial ligation in the chick embryo obstructed blood flow from the left atrium to the left ventricle, resulting in an acute increase in the right ventricular

DISCUSSION In the early embryonic heart, diverting blood flow from the left to the right side of the developing heart results in a decrease in active components of the atrioventricular and dorsal aortic blood flow, and associated aortic arch anomalies. The reduction of left atrial conduit and pump function alters ventricular diastolic filling. As a result, active atrial pump function is diminished, and dorsal aortic blood flow is decreased. Despite decreases in the left atrial conduit and pump function, we did not see a compensatory increase in the right atrial size. This suggests that atrial compensatory growth may be limited in early cardiac development. There cannot be adequate growth without adequate flow. Conversely, excessive flow causes increased growth when applied to the earlier stages of cardiovascular development (Clark, 1990). Ventricular ejection volume is not affected by the altered atrioventricular flow despite

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Fig. 3. Area of third and fourth left and right aortic arches among normal, sham, and left atrial ligated embryos. Data are presented as mean  SEM. All dimensions in the (third and fourth) aortic arch of the left atrial ligated show decrease in size. Yet, the fourth aortic arch in both left and right display statistical significance (P < 0.05) in the left atrial ligated embryos.

decreased ventricular end-diastolic and end-systolic volumes postligation. Decreased dorsal aortic blood flow with no change in ventricular ejection volume may reflect blood shunting away from the aortic arches due to altered intracardiac flow patterns. Ventricular contraction expels only a part of the blood contained in the chamber, and 30% to 60% of the end-diastolic volume remains in the ventricle at the end of systole (Milnor, 1989). The process of ligation alters the process of trabeculation and increases the intertrabecular spaces that compromise the trabecular struts to limit wall motion (Sedmera et al., 2000; Tobita and Keller, 2000). Consequently, ventricular ejection volume may have overestimated the total cardiac output. The limited trabeculated ventricle also may not be able to generate enough pressure to meet the increasing circulatory demands of the growing embryo. There are two separate streams of blood coursing through the left and right side of the heart (Rychter and Lemez, 1965; Hogers et al., 1997). The right blood stream is dominantly directed into the sixth aortic arches, while the left heart stream is directed into the third and fourth aortic arches. Antegrade blood flow through the atrioventricular cushions occurred during ventricular diastole, and no retrograde flow occurred during ventricular diastole or systole. The effects of abnormal communication between the circuits are maximized by obstructing one of the parallel circuits of blood flow from the atrium to the ventricles. Blood flows dominantly through the sixth aortic arches, whereas the third and fourth aortic arch blood flow is diminished in the left atrial ligated embryos. There is an increase in atrial contraction and ventricular compliance as the ventricular wall become structurally more complex (Lee and Downing, 1974; Mirsky and Krayenbuehl, 1981). By tying off the left atrium, blood volume required to fill the left atrial backs up into the right side of the common atrium. This causes a proportionally greater blood vol-

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Fig. 4. End systolic (ES) and end-diastolic (ED) volumes of right atrium and ventricle of stage-24 chick embryo acquired by video imaging. Although atrial volumes remain similar among the normal, sham, and left atrial ligated embryos, ventricular volumes are decreased in experimental embryos. Data are presented as mean  SEM.

ume to be forced through the right side of the common atrioventricular orifice to join and enlarge the right heart stream as it courses through the ventricle. Blood flow is distributed according to the hydrodynamic impedance to flow along each of the pathways in the aortic arches. Redistribution of myocardial blood flow occurs whenever there is a change in vascular resistance in one area within a flow-limited perfusion bed (Yoshigi et al., 1996). In a system of rigid tubes with low pressure, even with pulsatile flow, impedance is approximated by resistance. In measuring blood flow in the aortic arches of chick embryos, there is a dynamic reactance in the system that also needs to be considered. Arterial pressure, however, is preserved by a compensatory increase in impedance and a decrease in compliance. Although elastogenesis has not begun in the embryo at the aortic arch selection stage (Rosenquist et al., 1988), observations of the arches with high-resolution video show that they distend with systole. Furthermore, the aortic arches are part of a system which includes the entire embryo and vitelline vasculature. Even a small amount of vascular reactance to pulsatile flow will become significant when summed over such a large circulation (Lucitti et al., 2005). The dynamic component of the forces impeding blood flow must also be taken into account. Hemodynamic force plays a significant role in the arrangement of endothelial cells. Less blood flow decreases force and pressure against the walls, failing to properly mold the cells to a functional arrangement. Flow characteristics of the embryonic blood, such as velocity, viscosity and periodicity are taken into account to describe the responses of endothelial cells to shear stress as well as the sensors for this friction force (le Noble et al., 2005; Hierck et al., 2008). Icardo showed that venous clipping altered atrioventricular canal blood flow and endothelial alignment of the atrioventricular cushion, potentially due to change in surface shear stress (Icardo, 1989). Shear stress patterns may change due to abnormal flow. Endothelial cells may respond to altered shear stress by the regulation of cell growth by

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Fig. 5. Blood flow pattern of a normal (a–h) and left atrial ligated embryo (i–p) of stage-24 chick. The ventricle (V) is partially obscured by the allantois (A) in the normal embryo. Regurgitate flow occurs during atrial systole forcing the blood into the ductus cuvier (indicated by arrows; b–c). Blood flow emerges primarily in the fourth (IV) aortic

arches, followed by the third (III), and then the sixth (VI) arches (f–h). In contrast, in the left atrial ligated embryo, blood flows dominantly through the sixth aortic arches (denoted by arrowhead; o–p) rather than the fourth arch. (Atr, atrium; Ct, conotruncus; DA, dorsal aorta).

integrins, the expression of growth factor, and cytoskeletal rearrangement (Gittenberger-de Groot et al., 2006; Lehoux et al., 2006; Linask and Vanauker, 2007; Groenendijk et al., 2008). Hogers et al. confirmed that numerous unique streams of blood flow coursed between the embryonic heart and the extraembryonic vascular bed (Hogers et al., 1995). Peripheral venous clipping hampers intracardiac blood flow resulting in severe intracardiac and pharyngeal arch artery malformations (Rychter and Lemez 1965; Hogers et al., 1997, 1999). Venous clipping of the extraembryonic vitelline vein does not change the absolute circulating blood volume, but alters the left/right balance of venous inflow to the heart (Hogers et al., 1997, Ursem et al., 2004). In the left atrial ligated embryo, the proportion of left to right ventricle is markedly changed including decreased trabeculae in favor of a more compact myocardium and lumen despite the unaltered heart size (Sedmera et al., 1999). Some cardiac malformations can be attributed to a lack of proper looping of the heart (Bouman et al., 1995). Because of the abnormal blood flow, shear stress patterns might change. Altered blood flow likely affects the aortic

arches development. Studies have shown that subsequent to ablation of cranial neural crest, heart morphology and pharyngeal arch vessels (aortic arches) are altered before outflow tract septation normally occurred (Tomita et al., 1991). Alterations in the arch vessel apparatus were present by day three in chick embryos with neural crest ablation at stage 9–10 (Bockman et al., 1989). Mesenchyme was significantly reduced in quantity in the arches that also had lost the bilateral symmetry. There is a significant increase in the proportion of direct apposition of vessel endothelium with epithelium, without the intervening mesenchyme. Yet, studies have shown that functional changes in hemodynamics occur prior to any observable abnormal structural changes in the heart and aortic arch arteries with the cardiac neural crest ablation (Stewart et al., 1986). Thus, structure and function are integrally related in the cardiovascular system. The early chick heart, thought morphologically simple, adjusts to small changes in hemodynamic load. Any change in structure affects the response of the cardiovascular system to acute changes in hemodynamic load, and vice versa.

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Fig. 6. (a) Anterior right oblique view of the cardiovascular system from the l-CT-scan of a stage-27 normal embryo showing the third (III), fourth (IV), and sixth (VI) aortic arches. (b) Right side view of the same embryo with arrows indicating the bifurcation of conotruncal flow into third and fourth aortic arches (upper arrowhead), and sixth

aortic arches (lower arrowhead). (c) Anterior right oblique view of a left atrial ligated embryo. The right third and fourth aortic arches are missing. Insert shows a vessel disrupting the singularity of the left fourth and sixth aortic arches. (AA, allantoic artery; Atr, atrium; Ct, conotruncus; DA, dorsal aorta; Omp, fused omphalomesenteric veins).

Fig. 7. Frontal view of stage 34 (8-day) normal (a) and left atrial ligated chick hearts (b) and (c). Portion of the right ventricular wall (RV) is cut opened to show the interventricular septum (IVS). The aorta branches into right (B) and left (B0 ) brachiocephalic vessels. The right ventricle is dilated in the left atrial ligated hearts with a decrease in

the size of aortic arch (b, arrow); and missing the aortic arch with a supracristal ventricular septal defect (c, arrowhead). The right atrium is also distended, whereas the left atrium diminished in size when compared with the normal heart. (Ao, aortic arch; LA, left atrium; LV, left ventricle, PA, pulmonary artery; RA, right atrium. Scale bar ¼ 1 mm).

The combined analysis of blood flow and cardiac morphogenesis further refines our understanding of structure-function relationships in embryonic models of congenital cardiovascular anomalies. Our hypothesis does not explain the mechanism(s) causing the defects results. One significant factor is that an alteration in blood flow patterns occurs because of obstructions to flow at the sites affected. Result differences are contributed to the difference in severity, location, and timing of

the insult, along with the ability to withstand the insult. The ventricle retains its characteristic morphology despite the altered volume relationship. If flow volume had little effect on growth, we expect that any abnormality of the left atrium or mitral valve caused by injury at the time of surgery would be unaccompanied by any abnormalities in more distally located left heart structure. The altered flow does not appear to result in profound disturbance in basic early heart development. The major

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intracardiac disturbances are in the persistence and involution pattern of aortic arch derivatives. These defects provide further support for the hypothesis that altered growth caused by altered flow patterns results secondarily in abnormal aortic arch development.

ACKNOWLEDGMENT The authors sincerely appreciate Jackie Hinton’s help in editing the manuscript.

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