Norman Hu

Hemodynamics of the stage 12 to stage 29 chick embryo.

The heart is the first functioning organ in the embryo and provides blood flow during cardiac morphogenesis from a muscle-wrapped tube a few cells thick to the four-chambered pump. We described the hemodynamics of the chick embryo from stage 12 (50 hours of a 21-day incubation) to stage 29 (6 days), during which the embryo weight increased 120-fold. We measured ventricular, embryo and extraembryonic vascular bed wet weights, dorsal aortic blood flow with a directional pulsed-Doppler velocity meter, and ventricular and vitelline arterial blood pressures witfa a servo-null micropressure system. The data are reported as mean ± SEM. With rapid development and morphogenesis, dorsal aortic blood flow increased from 0.015 ± 0.004 to 2.40 ± 0.20 mm3/sec parallel to the geometric increase of wet embryo weight from 2.22 ± 0.10 to 267.5 ± 9.7 mg. Dorsal aortic blood flow normalized for embryo and extraembryonic weight remained relatively constant (Y=2.13+0.02X, r=0.23, SEE=0.03). Stroke volume increased from 0.01 ± 0.003 to 0.69 ± 0.03 mm3, and heart rate doubled from 103 ± 2 to 208 ± 5 beats/min. Systolic, diastolic, and mean vitelline arterial pressure increased linearly from 0∼32 ± 0.01, 0.23 ± 0.01, and 0.28 ± 0.01 mm Hg at stage 12 to 2.00 ± 0.06, 1.22 ± 0.03, and 1.51 ± 0.04 mm Hg, respectively, at stage 29. Ventricular peak systolic and end-diastolic pressure increased from 0.95 ± 0.04 and 0.24 ± 0.02 at stage 12 to 3.45 ± 0.10 and 0.82 ± 0.03 at stage 29, respectively. The hemodynamic waveforms were similar to those found in the four-chamber heart of the mature animal. These data are integral to understanding the interrelation of function and form during cardiac development.

Residual strain in the ventricle of the stage 16-24 chick embryo.

Residual stress and strain, i.e., the stress and strain remaining in a solid when all external loads are removed, may be produced in biological tissues by differential growth. During cardiac development, residual stress and strain may play a role in cardiac morphogenesis by affecting ventricular wall stress. After a transmural radial cut, a passive ventricular cross section opens into a sector, and the size of the opening angle provides a measure of the circumferential residual strain. Residual strains were characterized in this manner for the apical region of the diastolic embryonic chick heart for Hamburger-Hamilton stages 16, 18, 21, and 24 (approximately 2.5, 3.5, 4.0, and 4.5 days, respectively, of a 21-day incubation period). The average opening angle at these stages was 107 +/- 10 degrees, 79 +/- 10 degrees, 73 +/- 11 degrees, and 74 +/- 7 degrees, respectively (n > or = 5 for each stage). These measured angles were correlated with changes in ventricular morphology. Scanning electron micrographs of the apex revealed that the wall of the ventricle is smooth at stage 16. Then at stage 18, myocardial trabeculae develop, forming ridges with primarily a circumferential orientation. By stage 21, the trabeculae develop into a mesh, giving the ventricular wall a spongelike appearance, and the preferred orientation is lost by stage 24. The large decrease in opening angle between stages 16 and 18 corresponded to the onset of trabeculation, which is the greatest change in form during the studied stages. We speculate that residual strain is an important biomechanical factor during cardiac morphogenesis.

Diastolic Filling Characteristics in the Stage 12 to 27 Chick Embryo Ventricle

ABSTRACT: Cardiac output is affected by the diastolic filling characteristics of the ventricle. We hypothesized that the relative contributions of passive and active filling change as the ventricle develops from a smooth-walled tube to a trabeculated four-chamber heart. In stage 12 to 27 white Leghorn chick embryos, we simultaneously measured ventricular pressure with a servo-null micropressure system and dorsal aortic and atrioventricular velocities with a 20-MHz pulsed-Doppler velocity meter. The analog waveforms were sampled at 500 Hz and converted to digital format via an analog/digital board. We partitioned diastole into passive and active components. The passive phase began with the return of the pressure curve to baseline and extended to the onset of the a-wave. The active phase began with the upstroke of the atrial velocity curve and extended to the upstroke of the ventricular pressure curve at end-diastole. Data are presented as mean ± SEM (n ≥ 6 at each stage) and analyzed by analysis of variance and regression analysis. At similar cycle lengths ranging from 480 to 600 ms (p > 0.05), end-diastolic pressure increased from 0.24 ± 0.02 mm Hg at stage 12 to 0.55 ± 0.01 mm Hg at stage 27. Passive and active filling volumes were 92 (0.0038 ± 0.0005 mm3) and 8% (0.0004 ± 0.0002 mm3), respectively, at stage 12 and changed to 24 (0.23 ± 0.08 mm3) and 76% (0.62 ± 0.08 mm3), respectively, at stage 27. The ratio of passive to active filling volume decreased from 7.89 to 0.35. Thus, active ventricular filling became dominant as the trabeculae formed in the embryonic ventricle. These observations define the diastolic filling characteristics of the embryonic heart during primary cardiac morphogenesis.

Ventricular pressure-area loop characteristics in the stage 16 to 24 chick embryo.

The accurate description of embryonic cardiovascular function requires the adaption of standard measurement techniques to the small scale of the developing heart. In the mature heart, the analysis of ventricular pressure and volume accurately defines function. Because in vivo measures of volume are not feasible in the embryonic heart, we tested the hypothesis that ventricular pressure-area loops accurately define ventricular function in the stage 16 to stage 24 white Leghorn chick embryo. We simultaneously measured ventricular pressure with a servo-null pressure system and recorded video images at 60 Hz. The pressure waveform was superimposed onto the video image in real time. Video fields were planimetered for epicardial ventricular cross-sectional area and ventricular pressure. Pressure and area data were smoothed using a fast Fourier transform filter and plotted. Data are reported as mean +/- SEM, n greater than or equal to 4, and were tested by regression analysis and analysis of variance (p less than 0.05). Heart rate increased from 90 +/- 7 beats/min at stage 16 to 130 +/- 13 beats/min at stage 24. All pressure-area loops displayed diastolic filling, isometric contraction, ejection, and isometric relaxation, similar to pressure-volume loops of the mature heart. Isometric contraction time increased from 42 +/- 5 to 62 +/- 4 msec (p less than 0.05), while isometric relaxation time was 124 +/- 12 and 120 +/- 10 msec (p greater than 0.05) between stages 16 and 24, respectively. The maximum ratio of instantaneous ventricular pressure to area identified end systole better than peak ventricular pressure or minimum ventricular area. Thus, pressure-area relations define ventricular function in the embryonic chick heart.

Embryonic ventricular diastolic and systolic pressure-volume relations

The embryonic heart and vascular bed interact dynamically to support rapid growth of the embryo during cardiovascular development. Pressure-volume relations define ventricular function during alterations in loading conditions. We analyzed these relationships in the embryonic heart in order to define ventricular function and the response of the ventricle and vascular bed to acute changes in preload. We simultaneously measured ventricular pressure and recorded 60 video images per second in n≥6 stage 16, 18 and 21 white Leghorn chick embryos at baseline and during the infusion of 1–2 microliters of physiologic buffer into the venous sinus (sinus venosus). Ventricular tetany was then induced with the topical application of 2 Molar sodium chloride. Video fields were traced for ventricular pressure and epicardial cross-sectional area. Cross-sectional area was converted to volume assuming ellipsoid geometry, and cavity volume was calculated as total volume minus wall volume derived from the tetanized heart. We defined end-diastole at the onset of ventricular contraction and end-systole at maximum pressure/volume ratio. Stroke volume increased linearly with end-diastolic volume. End-diastolic pressure-volume relations were positive and linear, and end-systolic pressure-volume relations were curvilinear downward. Arterial elastance decreased with growth of the embryo and with volume infusion. Pressure-volume loop area, an index of consumption of energy, doubled between the embryonic stages. Thus, embryonic ventricular pressure-volume relations define diastolic and systolic function at rest and in response to altered preload.

Rate of coronary vascularization during embryonic chicken development is influenced by the rate of myocardial growth.

OBJECTIVE We tested the hypothesis that the degree of coronary microvessel formation in the embryonic heart is regulated by the magnitude of myocardial growth. METHODS The outflow tract of Hamburger-Hamilton stage 21 chicken hearts (prior to the onset of coronary vasculogenesis) was constricted in ovo with a loop of 10-0-nylon suture, and the hearts were studied at stages 29 and 36. RESULTS At stage 29 ventricular mass was 64% greater in the pressure-overloaded than in the hearts of sham-operated controls, but vascular volume density and numerical density, determined by electron microscopic morphometry, were identical. As demonstrated by histological morphometric evaluation, the compact region of the left ventricle at stage 29 was 43% thicker than the shams. However, by stage 36 heart mass, thickness of the compact region, and overall wall thickness (demonstrated by scanning electron microscopy) were significantly less than in the sham group of this stage, but vascular volume density was virtually identical in the two groups. Formation of the two main coronary arteries was clearly impeded in the banded hearts, i.e., the coronaries were stunted in their development or failed to completely form coronary ostia. CONCLUSIONS Vascular growth is proportional to myocardial growth in the embryonic, overloaded heart, but the persistence of the pressure overload results in a failure of or severe limitations in coronary artery development. These data support the hypothesis that vascular growth during this period of development is regulated, at least in part, by the rate and magnitude of myocardial growth.

Effect of Heart Rate Increase on Dorsal Aortic Flow before and after Volume Loading in the Stage 24 Chick Embryo

ABSTRACT: In the stage 24 chick embryo, a paced increase in heart rate reduces stroke volume, presumably by rate-dependent decrease in passive filling. We hypothesized that rate-dependent stroke volume reduction could be abolished by volume loading. Dorsal aortic blood velocity was measured with a 20 mHz puIsed-Doppler meter from a 0.75-mm piezoelectric crystal (eight embryos), and atrioventricular velocity was simultaneously measured from the ventricular apex (six embryos). Sinus venosus pacing (stimuli of 1 ms duration and <4 mA) was performed at intrinsic rate (P:I) and at 150% of intrinsic rate (P:150%I). Volume loading was performed during P:150%I by intravenous injection of 7.5 ixL of chick Ringers solution. Using atrioventricular velocity profile, stroke volume was divided into the proportion due to passive (E-phase) and active (Aphase) filling. Stroke volume was compared during P:I, P:150%I, immediately (P:150%I′) and 30 s after (P:150%I) volume loading. Data (mean ± SEM) were compared by ANOVA. During pacing, stroke volume (mm2/cycle) decreased but increased after volume loading (I, 0.43 ± 0.03; P:I, 0.37 ± 0.03; P:150%I, 0.19 ± 0.03; P:150%I′, 0.24 ± 0.05; P:150%I, 0.28 ± 0.04 (p < 0.005). During P:150%I, E-phase filling disappeared and was not restored by volume loading, whereas, A-phase filling diminished but was restored by volume loading. In stage 24 chick embryos, rate-dependent stroke volume decrease is reversed by volume loading that restores stroke volume due to an increase in active filling but not passive filling. Thus, even at rapid heart rate, the embryonic ventricle responds to volume loading, indicating that the Frank- Starling relationship functions during tachycardia in the embryonic heart.

Analysis of Dynamic Atrial Dimension and Function during Early Cardiac Development in the Chick Embryo

ABSTRACT: Although atrial morphologic changes are well documented, the description of early atrial function is limited. We used videomicroscopic methods to define the function of the contracting atrium in stage 16 to 24 white Leghorn chick embryos. We exposed the embryo in ovo (right side up) and imaged the ventricle, then repositioned the embryo (left side up) and imaged the atrium (n ≥ 8 per stage). We traced the atrial endocardial border and then measured atrial perimeter (mm) and cross-sectional area (mm2). A 20-MHz pulsed Doppler velocity meter was used to measure atrioventricular blood velocity during atrial imaging in an additional six stage 21 embryos. Data were tested by analysis of variance and regression analysis. Mean heart rate change after repositioning was −4 ± 1%. Atrial maximum and minimum area increased linearly versus embryo stage (y = 0.10x − 1.41, r = 0.89, p < 0.05 and y = 0.05x − 0.67, r = 0.82, p < 0.05, respectively). Shortening fraction (percentage of reduction) of atrial perimeter and area decreased from 32.3 ± 2.0% to 27.5 ± 1.8% (p < 0.05) and 56.2 ± 3.0% to 47.7 ± 2.0% (p < 0.05), respectively, from stage 16 to 24. During atrial contraction, the velocity of circumferential wall shortening increased linearly with stage (y = 0.22x − 2.08, r = 0.81, p < 0.01); however, the velocity of lengthening was similar between stages (p = 0.45). Simultaneous atrial imaging and pulsed Doppler velocity measurement showed that passive atrioventricular flow occurred late in atrial lengthening and active atrioventricular flow occurred during atrial contraction. Thus, atrial function increases in parallel with morphogenesis during early cardiac development, and measures of atrial function can now be incorporated into a physiologic model of the developing cardiovascular system.

Distribution of blood flow between embryo and vitelline bed in the stage 18, 21 and 24 chick embryo

OBJECTIVE We defined the distribution of blood flow between the embryo and the extraembryonic vascular bed as an initial step in understanding the control of flow distribution in the early developing heart. METHODS Dorsal aortic blood flow of stage 18, 21, and 24 chick embryo (n > or = 7 at each stage) was measured with a 20 MHz pulsed-Doppler velocity meter. Analog waveforms were digitally sampled at 500 Hz. 1-5 x 10(3) yellow microspheres in saline suspension were injected into the vitelline vein. The embryo and the extraembryonic vascular bed were harvested and separated from each other. The dye on the microspheres from each portion was extracted and extrapolated from the standard curve of the absorbance of dye concentrations per number of microspheres quantified by spectrophotometry. Blood flow was calculated from the integral of blood velocity and aortic cross-sectional area multiplied by the fraction distribution of microspheres in the embryo and extraembryonic vascular bed. Data were presented as mean +/- standard error of the mean. RESULTS The proportion distribution of microspheres between embryo and extraembryonic vascular bed shifted from 18.7 +/- 2.5 vs. 81.3 +/- 2.5% at stage 18, 25.1 +/- 3.0 vs. 74.9 +/- 3.0% at stage 21, and 34.2 +/- 2.4 vs. 65.8 +/- 2.4% at stage 24. Indices of blood flow normalized to wet weight (mean +/- 95% confidence interval) were similar between the embryo and the extraembryonic vascular bed, but increased throughout the stages. CONCLUSION During embryogenesis, blood flow per unit mass is evenly distributed between the metabolically active embryo and the extraembryonic vascular bed.

Effect of Atrial Natriuretic Peptide on Diastolic Filling in the Stage 21 Chick Embryo

ABSTRACT: Atrial natriuretic peptide (ANP) exerts hemodynamic effects by direct venodilation in the chick embryo. We hypothesized that ANP-induced venodilation affects ventricular diastolic filling resulting in reduced ventricular preload. Chick ANP (0.1 μg in 10 μL of normal saline) was suffused onto the vitelline vascular bed in stage 21 (3 1/2 d) chick embryos. Equivalent aliquots of normal saline were suffused as sham controls, and normal embryos received no suffusion. We measured simultaneously dorsal aortic blood velocity and atrioventricular blood velocity with a 20-MHz pulsed-Doppler velocity meter. Analog wave forms were digitally sampled at 500 Hz, and the dorsal aortic cross-sectional area was used to calculate dorsal aortic blood flow. Passive ventricular filling volume equaled dorsal aortic stroke volume multiplied by the fraction of passive area; active filling volume equaled dorsal aortic stroke volume multiplied by the fraction of active area. Data were summarized as mean ± SEM (n≥ 7 per group) and analyzed by analysis of variance. Cycle lengths were similar in ANP-suffused, sham control, and normal embryos. Dorsal aortic blood flow decreased from 0.49 ± 0.04 mm3/s at baseline to 0.27 ± 0.05 mm3/s at 4 min post-ANP suffusion (p< 0.05) and was unchanged in sham control and normal embryos (p> 0.05). Passive ventricular filling was reduced by ANP suffusion, whereas active filling was unaffected, resulting in a decreased passive/active filling ratio from 0.64 ± 0.07 at baseline to 0.32 ± 0.08 at 4 min in ANP-suffused embryos (p< 0.05). Passive/active ratio was unchanged in sham control and normal embryos. Thus, ANP-mediated vasodilation reduces cardiac output via decreased passive ventricular filling in the embryonic heart.