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Cardiovascular Development Research Program, Department of Pediatrics, University of Kentucky, Lexington, Kentucky 40536-0298
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Mechanical load influences embryonic ventricular growth, morphogenesis, and function. However, little is known about changes in regional passive ventricular properties during the development of altered mechanical loading conditions in the embryo. We tested the hypothesis that regional mechanical loads are a critical determinant of embryonic ventricular passive properties. We measured biaxial passive right and left ventricular (RV and LV, respectively) stress-strain relations in chick embryos at Hamburger-Hamilton stages 21 and 27 after conotruncal banding (CTB) to increase biventricular pressure load or left atrial ligation (LAL) to reduce LV volume load and increase RV volume load. In the RV, wall strains at end-diastolic (ED) pressure normalized whereas ED stresses increased after either CTB or LAL during development. In the left ventricle, both ED strain and stress normalized after CTB, whereas both remained reduced with significantly increased myocardial stiffness after LAL. These results suggest that the embryonic ventricle adapts to chronically altered mechanical loading conditions by changing specific RV and LV passive properties. Thus regional mechanical load has a critical role during cardiogenesis.
ventricle; cardiac morphogenesis; mechanical load
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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REGIONAL MECHANICAL LOAD INFLUENCES embryonic ventricular growth, morphogenesis, and function. After the onset of the embryonic heartbeat, the heart initially functions in series with common atrial, ventricular, and outflow tract segments (12). Embryonic heart rate, blood pressure, and cardiac output increase geometrically (1, 13, 21). One of the unique aspects of embryonic circulation is that the heart undergoes a dramatic transformation in three-dimensional structure coincident with rapid cardiomyocyte clonal expansion and differentiation and a geometric increase in function (1, 12, 13, 18).
With a maximum operating pressure of <10 mmHg during early cardiac morphogenesis, embryonic heart function is dramatically affected by small changes in mechanical loading conditions (2, 26, 30). The embryonic ventricle acutely adapts contractile function in response to altered mechanical loads (14). Chronic alterations in ventricular loads influence both ventricular growth and morphogenesis, which results in cardiovascular malformations (2, 10, 11, 26, 30). These studies support a function-structure paradigm during cardiac morphogenesis.
Several studies in mature cardiac tissue have shown that mechanical factors such as myocardial stress and strain regulate ventricular growth and remodeling (23). Presumably, ventricular geometry and/or myocardial properties are changed so as to reduce initial increases in diastolic wall stresses or strains. In contrast to the mature myocardium, little is known about how these mechanical factors regulate embryonic ventricular growth or morphogenesis during normal and altered mechanical loads. Previous work in our laboratory (30) demonstrated that regional ventricular contraction patterns of the embryonic ventricle change from globally uniform to ventricular-specific patterns during normal development, and that these contraction patterns are influenced by altered mechanical loads that result in abnormal ventricular morphogenesis.
In the present study, we tested the hypothesis that regional mechanical loading conditions are a critical determinant of the normal maturation of embryonic ventricular passive properties during ventricular morphogenesis. To test our hypothesis, we measured biaxial passive right and left ventricular (RV and LV, respectively) stress-strain relations after chronically increased ventricular pressure load that was produced by conotruncal banding (CTB), or chronically reduced LV volume load and compensatory increased RV volume load that was produced by left atrial ligation (LAL) in white Leghorn chick embryos. We found that in response to increased mechanical loads, the right ventricle normalized end-diastolic (ED) strain rather than ED stress, whereas the left ventricle normalized both ED strain and stress. In response to reduced LV volume load, both ED strain and stress were not normalized, and myocardial stiffness was significantly increased. The embryonic ventricle adapted to the chronically altered mechanical loading conditions by changing specific RV and LV passive properties. Thus regional mechanical load has a critical role during cardiogenesis.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Embryo selection.
Fertile white Leghorn chicken eggs were incubated in a forced-draft,
constant-humidity incubator and studied at Hamburger-Hamilton stages 21 (3.5 days) and 27 (5 days) of a
46-stage (21-day) incubation period as previously described
(9). The selected stages represent the period during which
the embryonic ventricle transforms from a single pulsatile chamber to
form externally distinct RV and LV chambers with a common
atrioventricular (AV) canal, large interventricular communication, and
common outflow tract (Fig.
1, Ref.
27). Normal embryos were studied acutely at each stage.
Embryos that were dysmorphic or exhibited overt bleeding were excluded.
Our research protocols conform to the Guide for the Care and Use
of Laboratory Animals published by the National Institutes of
Health (NIH Publication No. 85-23, Revised 1985).
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Chronic CTB to increase RV and LV pressure load. Embryos were initially incubated to stage 21. A 1-cm2 hole was made in the shell and the inner shell membranes were removed. A monofilament 10-0 nylon suture was passed underneath the midportion of the conotruncus and then tied snugly in an overhand knot until ventricular dimensions were obviously increased without blood flow cessation or ventricular arrhythmia. The eggshell opening was sealed with Parafilm and reincubated until stage 27 (2).
Chronic LAL to reduce LV preload. Embryos were initially incubated to stage 21. A 1-cm2 hole was made in the shell and the inner shell and extra-embryonic membranes were removed to expose the developing embryo. The embryo was then gently positioned with the left side up, and microforceps were used to make a slitlike opening in the thoracic wall above the primitive left atrium. A loop of 10-0 nylon suture was placed across the primitive left atrium and tightened, which decreased the effective volume of the left atrium (10, 26, 30). Each embryo was then repositioned to its original right-side-up orientation. The eggshell opening was sealed with Parafilm and reincubated until stage 27.
Intraventricular pressure in ovo.
We measured intraventricular pressure in ovo with a servo-null pressure
system (model 900A, World Precision Instruments; Sarasota, FL) as
previously described (1). Briefly, a glass micropipette with a 7-to-10-µm-diameter tip was filled with 2 M NaCl and
positioned with the use of a micromanipulator (Leitz; Wezlar, Germany)
to puncture the ventricle. The servo-null system measures the
resistance of the 2 M NaCl-filled pipette tip and then prevents changes
in resistance by generating an opposing pressure to the pressure present at the tip. We set the pipette resistance at 0.3-0.4 M
(damping ratio 
0.01; Ref. 31). The servo-null system
was linear (y = 0.995x 
0.23, r = 0.99, SE = 0.11 mmHg) to a standing water column over the range of 0-10 mmHg (1, 14).
Intraventricular pressure was calculated as the difference between the
measured pressure and the pressure recorded when the tip was placed in extra-embryonic fluid adjacent to the midventricular level. ED ventricular pressure was defined as the point at which the first derivative of the pressure waveform rose sharply from the baseline (1).
Pressure-strain relations.
RV and LV pressure-strain relations in isolated embryonic ventricles
were obtained using a custom pressure system and epicardial surface-strain measurement while the ventricle was passively inflated with a chick Ringer solution. The embryonic heart was arrested by
injection via the sinus venosus of 20 µl of 25°C hyperkalemic chick
Ringer solution containing (in mM) 82 NaCl, 60 KCl, 2 CaCl2, 10 Trizma-HCl, and 10 Trizma base, pH 7.4. The heart
was rapidly excised from the embryo and placed in a petri dish
containing hyperkalemic chick Ringer solution at 37°C and pH 7.4. The
conotruncus was occluded using a 10-0 nylon suture. A 50-µm-diameter
tip glass micropipette connected to the custom-made pressure system was inserted into the ventricle through the AV canal from the atrium. The
AV canal was then occluded using a 10-0 nylon suture tied around the AV
groove (Fig. 2).
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3x, r = 0.99, SE = 9.0 × 104 mV) to a standing water column over the
range of 0-10 mmHg. We first validated the sensitivity of the
pressure data obtained from the custom pressure system by
simultaneously measuring intraventricular pressure using a combined
servo-null pressure system and a custom pressure system in
stage-matched embryonic ventricles (n = 16). During
inflation, the micromanometer pressure rose sharply within the range of
0-0.2 mmHg recorded by the servo-null system. Above 0.2 mmHg, the
pressure of the two systems changed in parallel during inflation (Fig.
3A). Therefore, we calibrated
the custom pressure system starting at the pressure recorded when the
servo-null system was 0.2 mmHg. There was an excellent linear
correlation in a range of intraventricular pressure from 0.2 to 2.0 mmHg of the servo-null system (y = 1.00x 0.20; x: servo-null system, y: micromanometer, r = 0.99, SE = 0.05 mmHg; Fig. 3B). In each experiment, we first recorded the
pressure and strain data using the custom pressure system. We then
recorded the intraventricular pressure simultaneously using the two
systems in the same ventricle and we estimated the intraventricular
pressure.
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S0) and after deformation (s) in the following way:
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(1) |
a1 and a2 are the
circumferential and longitudinal cardiac coordinate components of
S0 (CIR and LNG, respectively), which are defined by
choosing a2 to be the line between the
midpoint of the conotruncal valve and the ventricular apex in the right
ventricle and the midpoint of the primitive AV valve and the
ventricular apex in the left ventricle. Therefore, E11 is circumferential,
E22 is longitudinal, and
E12 = E21 are shear-strain components, respectively.
We monitored the intraventricular pressure and epicardial strain
patterns during inflation in each experiment. We disregarded the data
that fell into either of the following two categories: 1)
intraventricular pressure did not reach 2 mmHg at the end of inflation,
which indicated pressure leakage during inflation; or 2) one
or more microspheres moved out of focus during inflation, which
occurred when the epicardial surface did not remain approximately planar. After obtaining simultaneous pressure-strain data, the data
were interpolated using rational interpolation (LabVIEW) in each
ventricle. From each pressure-strain curve, strain was measured in the
range of 0.2-1 mmHg with 0.2-mmHg increments and was then averaged
for each experimental group.
Passive wall stress-strain relations and myocardial properties. Embryonic ventricular geometry at the stages studied is complex (see Fig. 1). The ventricular wall is composed of an inner trabeculae layer of varying intertrabecular spaces and a thin outer compact myocardium. It is not technically feasible to use a realistic three-dimensional geometric model of the complex trabeculated wall to estimate material properties especially during inflation. Therefore, in this study we focused on the outer compact myocardium because the compact myocardium is the future myocardial wall and has highly proliferative activity generating new cells for the trabecular layer during study stages (18, 28). We modeled the embryonic RV or LV free wall as a locally thin-walled axisymmetric ellipsoid of revolution with two equal minor radii and with equal wall thickness. The compact myocardium was assumed as a freely deforming solid body composed of isotropic and incompressible elastic material. We also neglected inertial and torsion effects. We defined the major axis of the stage 27 RV ellipsoid as the CIR and that of the stage 21 right ventricle and stages 21 and 27 left ventricle as the LNG according to external ventricular shape (see Figs. 1 and 4).
Right and left midventricular compact myocardium thickness was measured from 7-µm-thick ventricular transverse cross sections (Fig. 5). The embryonic heart was arrested by the method described (see Pressure-strain relations) and fixed with 4% paraformaldehyde at 4°C for 12 h. The heart was embedded in optimum cutting temperature compound (Tissue-Tek, Sakura; Torrance, CA) in transverse orientation. Serial sections were cut and stained for
-myosin heavy chain with MF-20 monoclonal antibody
(Developmental Studies Hybridoma Bank, University of Iowa, Iowa City,
IA). Alexa 488 (IgG2b specific, Molecular Probes; Eugene, OR) was used
as a secondary antibody. Images were captured digitally on a Nikon E600
microscope with an epifluorescence attachment (Nikon; Tokyo, Japan) and
a SPOT RT slider camera (Diagnostic Instruments, Sterling Heights, MI). Compact myocardium thickness was measured on the midportion slice that
contained both ventricles at the intercepts of the lines perpendicular
to the interventricular septum at 0°, +5°, and 5° in each
ventricular wall.
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(2) |
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= l/l0) is expressed as
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(3) |
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(4) |
epi and
R
epi are, respectively, the epicardial major
and minor principal radii of curvature of the ellipsoid at equator
level. The reference length l0 (zero pressure
length) was defined as Repi0 = Depi0 in the CIR direction and
Repi0 = L
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(5) |
![<IT>+</IT>(10<IT>/</IT>3)<IT>D</IT><SUB>in</SUB><IT>h</IT><SUP>2</SUP><IT>+h</IT><SUP>3</SUP>]<IT>, h></IT>0](/content/vol282/issue6/fulltext/H2386/img011.gif)
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![&sfgr;<SUB>&thgr;</SUB>=(PD<SUB>m</SUB><IT>/h</IT>)[1<IT>−</IT>(<IT>D</IT><SUP>2</SUP><SUB>m</SUB><IT>/</IT>2<IT>L</IT><SUP>2</SUP><SUB>m</SUB>)]](/content/vol282/issue6/fulltext/H2386/img012.gif)
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(6) |
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, and
are intraventricular pressure, CIR, and LNG midwall
stresses, respectively.
The estimation of RV and LV wall stress-strain relations from the
inflation test are influenced by the interaction between RV and LV. To
address whether estimated wall stress-strain relations from the
pressure-strain relations represent regional ventricular material
properties, we measured stress-strain relations from myocardial strips
of RV and LV free wall in the Hamburger-Hamilton stage 27 embryonic ventricle in each experimental group.
The embryonic heart was arrested and excised from the embryo and placed
in cold chick Ringer solution by the method described. The myocardial
strip was then dissected from both the right and left ventricles in
either a CIR or LNG cardiac coordinate orientation. After excision,
strip length was measured parallel to the cardiac coordinate from which
it was cut. The myocardial strip was then transferred in a small-volume
Plexiglas chamber that was perfused with 37°C oxygenated buffer
solution at a constant flow rate of 5 ml/min and were attached between
two stainless steel bevel-tipped wires using an ultrapure
low-viscosity, fast cure, 
-cyanoacrylate ester (model 262, Permabond
International; Englewood, NJ; Ref. 19). The cyanoacrylate
adhesive does not produce tissue injury beyond the glue joints
(19). We also separately tested the tissue viability of
the excised myocardial strip via electrical pacing in each experimental
group (n > 5 in each group). We could obtain reproducible active contraction force after pacing for up to 3 h.
One wire was connected to a force transducer (model 403A, Aurora Scientific; Ontario, Canada) and the other to a custom linear displacement motor mounted on a micromanipulator. The strip was then
adjusted to its "excision length." The 10-µm microspheres were
positioned with the centriod of the triangle aligned with the central
axis of the wires and midway between the tips. A custom linear motor
displaced the wire and stretched the myocardial strip up to 30% of the
excision length in a triangular wave pattern at a velocity of 0.2 mm/s.
We monitored changes in myocardial strip length using both the imaging
system and internal length sensors in the displacement motor.
Simultaneous force and strain data were acquired digitally at a rate of
100 Hz (LabVIEW) and 10 Hz (NIH Image 1.62), respectively.
Cross-sectional area was determined using an eyepiece retical assuming
cylindrical geometry. We reviewed surface-strain patterns and
displacement values in each ventricle and disregarded the specimens for
a variety of strain-measurement problems, which fell into two
categories (19): 1) one or more microsphere
markers moved out of focus during stretch, indicating that the
myocardial surface of the mounted segment surrounding the microspheres
did not remain approximately planar; or 2) values of
longitudinal strain (parallel to stretch direction) <25%, large shear
strain >2%, or positive circumferential strain. Small value of
longitudinal strain occurred when the sample did not adhere to the
wires properly. Large shear or positive circumferential strains
occurred when the mounted myocardial strip stretched with obvious
torsion or with asymmetric nonuniaxial motion. The myocardial strips
were preconditioned with three loading cycles of 30% stretch. After
preconditioning, data were obtained at a range of 0-0.25 strain.
Passive CIR or LNG stress-strain relations were fitted by the following
exponential equation (15)
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(7) |
, a, b, and E are
myocardial stress, intercept at zero strain, myocardial stiffness
constant (dimensionless), and Lagrange strain, respectively. We chose
the zero-pressure state as the reference state for the pressure-strain
relations and the excision length for the myocardial strip. Note that
these reference states are not the unloaded state due to the presence
of residual stress. To determine myocardial constant b from
the stress-strain relations, we used a quasi-Newton algorithm to
minimize the least-square loss function. Convergence criterion was set
to 0.0001. All calculations were performed using STATISTICA (Statsoft;
Tulsa, OK).
It is technically difficult to measure all of the parameters of
intraventricular pressure in ovo, compact myocardium thickness, RV or
LV passive-pressure strain, and stress-strain relations of a myocardial
strip from the same 1-mm diameter embryonic heart. Thus we measured
each parameter from different embryos. Intraventricular pressure
measurement was performed in the following groups: normal embryos,
immediately after the procedure of CTB or LAL at stage 21,
and stage 27 normal, CTB, and LAL groups. Ventricular
dimensions and passive pressure-strain relations were measured from the
same embryo in stage 21, stage 27 normal, CTB,
and LAL groups. Compact myocardium thickness was measured in
stage 21, stage 27 normal, CTB, and LAL groups.
Stretch tests of myocardial strips were performed in stage
27 normal, CTB, and LAL embryos.
Statistical analysis. Data are presented as means ± SE. Two-factor repeated ANOVA was performed to compare the mean values of pressure-strain relations between experimental groups. Two-factor ANOVA was performed to compare the mean values of intraventricular pressure, ventricular dimensions, compact myocardium thickness, ED strain components, and myocardial stiffness constant between experimental groups. Individual comparisons were performed using Tukey's test. Statistical significance was defined by a value of P < 0.05. All calculations were performed using SigmaStat (SPSS; Chicago, IL).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Intraventricular pressure in ovo.
Peak and ED pressure increased in parallel with development in all
experimental groups (P < 0.05; Table
1). At stage 21, peak and ED
pressure increased after CTB and decreased after LAL consistent with
the acute effects of increased ventricular pressure load or reduced
volume load (P < 0.05 vs. normal stage 21).
At stage 27, peak and ED pressure in CTB remained higher
than normal, whereas those values in LAL were lower (P < 0.05 vs. normal stage 27).
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Ventricular dimensions at zero pressure.
Epicardial major and minor axis diameters at zero pressure increased
during development in both the right and left ventricles (Table
2). At stage 27, both RV and
LV diameters increased after CTB (P = 0.10 in LV major
axis, others, P < 0.05 vs. normal stage 27). Compact myocardium thickness increased in both right and left
ventricles during development. After CTB, both RV and LV compact
myocardium thickness increased significantly by stage 27 (P < 0.05 vs. stage 27 normal). After LAL,
RV compact myocardium thickness was decreased (P < 0.05), whereas LV compact myocardium thickness was similar to normal
(Table 2 and Fig. 5).
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Pressure-strain relations.
Figure 6 shows RV and LV pressure-strain
relations during inflation tests for each group. With increasing
ventricular pressure, both CIR and LNG strains became more positive,
which indicates that ventricles underwent in-plane stretch in all
groups. Stage 27 LAL LV strains at a range of 0.6-1.0
mmHg pressure were significantly smaller than normal (P < 0.05 vs. stage 27 normal) in both CIR and LNG directions.
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Wall stress-strain relations and myocardial stiffness.
Table 3 summarizes ED strain in all
groups. During normal development, ED strain changed from globally
uniform to right- and left ventricle-specific patterns. At stage
27, RV ED strains were normalized in both CTB and LAL groups. In
the left ventricle, ED strains were normalized after CTB whereas ED
strains remained reduced after LAL (P < 0.05 vs.
normal stage 27). Figure 7
shows average RV and LV stress-strain curves estimated from the
pressure-strain relations. The stress-strain relations appeared
exponential in all groups (Table 4). At
stage 21, ED stress-strain points shifted to the right after
CTB, whereas they shifted to the left after LAL in both right
and left ventricles. At stage 27, RV ED stresses increased
after CTB and LAL. In the LV, ED stresses were normalized after CTB,
whereas ED stresses remained reduced after LAL in both CIR and LNG
directions. After LAL, LV CIR myocardial stiffness was significantly
larger than normal in both inflation tests and myocardial strips
(P < 0.05 vs. normal myocardial strip, Table 4).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The present study is the first to examine selective embryonic passive biaxial RV and LV myocardial stress-strain relations during normal and experimentally altered mechanical loading conditions. We demonstrated that the embryonic ventricle adapts to altered mechanical loading conditions by changing specific RV or LV passive properties. In response to increased pressure- and volume-loading conditions, the right ventricle normalized ED strain rather than ED stress, whereas the left ventricle normalized both ED strain and stress. In response to reduced LV volume load, both ED strain and stress were not normalized. In addition, regional myocardial stiffness was significantly increased.
Chamber-specific deformation patterns reflect the integration of mechanical loading conditions with both active and passive myocardial properties (30). Our previous study of systolic wall strains showed that embryonic ventricular contraction patterns are changed from being globally uniform at stage 21 to being RV- or LV-specific patterns at stage 27 in which the right ventricle contracts preferentially in the CIR direction whereas the left ventricle contracts in the LNG direction. Developmental changes of RV- or LV-contraction patterns likely reflect changes in the principal trabecular arrangement (30). Regional ventricular contraction patterns are mainly related to the active properties of the myocardium (muscle shortening); however, the regional passive properties of the embryonic myocardium are not well understood. In this study, we showed that passive ED strains increased and changed from uniform to RV- or LV-specific patterns during development. In addition, regional compact-layer ED stress increased >50% in RV LNG and LV CIR and LNG directions by stage 27 (Fig. 7). These results suggest that changes in the passive material properties may relate to changes in myocardial architecture during normal development. In the present study we estimated myocardial stress at the compact myocardium because it is difficult to use a feasible geometric model of the complex three-dimensional trabeculated, ventricular myocardium. Further studies are needed to discern whether changes in ED stress-strain relations reflect passive myocardial properties throughout the ventricular wall during normal development.
The embryonic myocardium responds to increased mechanical loads by clonal cardiomyocyte expansion (hyperplasia) rather than myocardial hypertrophy (2). Myocardial architecture is also changed in response to increased loading conditions. Increased pressure load after CTB induces ventricular dilatation, which is compensated by cardiomyocyte proliferation, trabecular and compact myocardium thickening, and precocious spiraling and compaction. In some instances, structural abnormalities such as double-outlet right ventricle or persistent truncus arteriosus are present. Increased volume load to the right ventricle after LAL induces increased spacing and thinning of RV trabecular sheets with delayed compaction (26).
Studies of the hypertrophic response of mature myocardium show that mechanical factors have an important role in regulating ventricular growth and remodeling in both pressure and volume overload (23). Florenzano and Glantz (6) showed that ED stress regulates volume overload hypertrophy. Nguyen et al. (22) showed that the ED stress also regulates pressure overload hypertrophy. Emery and Omens (5) recently showed that ED stress and strain are increased acutely in response to volume overload, and during remodeling, ED strain is normalized; however, ED stress remained elevated with increased myocardial stiffness. They concluded that ED strain rather than stress regulates ventricular growth and remodeling in volume-overload hypertrophy. In the present study, the embryonic myocardium responds to the increased mechanical loads with RV- and LV-specific patterns. In the right ventricle, ED strain rather than stress was normalized in response to either pressure- or volume-overloading conditions. In the left ventricle, both ED strain and stress were normalized in response to pressure overload. Regional myocardial stiffness was not changed significantly.
In contrast to the hypertrophic response, reduced mechanical load induces myocardial atrophy and/or fibrosis in the mature myocardium (24). A recent study of mechanical circulatory support to the failing human heart showed that reduced mechanical load changed LV structure (reverse remodeling) including a reduction of LV dimensions and muscle mass and an improvement in passive and contractile properties (4). In addition, surgical intervention for congenital cardiovascular malformations such as total anomalous pulmonary venous return proves that correction of abnormal intracardiac blood flow normalizes ventricular structure and function (17). In the present study, the embryonic LV myocardial response to reduced volume load differed markedly from the response observed with increased loads. Harh et al. (10) initially produced a model of hypoplastic left heart syndrome in the chick embryo. They speculated that after interference with blood flow between the left atrium and left ventricle, RV-inflow volume would increase and LV-inflow volume would decrease, thereby resulting in RV hyperplasia and LV hypoplasia. Our previous study (30) showed that reduced volume load to the left ventricle affected LV contraction patterns and induced LV hypoplasia. The present study showed that changes in stage 27 LV dimensions after LAL were relatively mild. However, both ED strain and stress remained decreased during development with significant increases in CIR myocardial stiffness. Changes in passive myocardial properties also precede morphological change after LAL. These results suggest that the chamber-specific response to altered mechanical loads in the embryonic ventricle affects both normal myocardial growth and chamber morphogenesis resulting in structural malformations.
In contrast to the mature myocardium, little is known about how mechanical factors directly influence embryonic ventricular growth and remodeling during cardiogenesis. Lin and Taber (16) described a wall-stress-dependent linear growth law using a mathematical model in chick embryonic ventricles from stage 21 to 29. They showed that ED stress increases during normal development and the normal increases of wall stress drive cardiac growth. In response to pressure overload, the embryonic ventricle normalizes ED stress by changing ventricular geometry. However, their theoretical model was a highly idealized cylindrical tube with constant passive material properties during development. The wall-stress-dependent growth law excludes strain as a growth stimulus because of difficulty in defining a reference length. Cowin (3) described the strain-dependent growth law by extending the theory of the stress-dependent growth law that Lin and Taber used in their study. He showed the possibility that strain is a mechanical factor that stimulates growth and/or remodeling. In addition, he pointed out that the reference configuration in either the strain- or stress-dependent growth law must be updated on a time scale larger than the time for short-term loading events and shorter than the time that it takes for significant growth or remodeling changes. Our results showed that there are likely RV- and LV-chamber-specific responses to altered loading conditions. However, we could not define which mechanical factor predominates in regulating ventricular growth and/or morphogenesis in the embryonic heart. Further studies are needed to understand the mechanism regulating ventricular growth, morphogenesis, and/or remodeling during cardiac morphogenesis.
Changes in the geometry and material properties of the embryonic myocardium are also reflected in changes at the tissue and cellular level. Mikawa et al. (18) showed that embryonic cardiomyocytes clonally divide and extend from the epicardial to the endocardial direction during normal development. Clark et al. (1, 2) showed that myofibril volume density in a cardiomyocyte increases during normal development but does not change after CTB (ventricular hyperplasia). At the tissue and cellular levels, either myocytes or the extracellular matrix or both are likely candidates to sense mechanical stimuli and initiate protein production in the cardiomyocyte via complex biochemical pathways (8, 25). However, at the present time the mechanisms that translate regional mechanical loading conditions into cardiomyocyte division, differentiation, and remodeling during cardiac morphogenesis are not well understood.
The results of the present study are certainly subject to limitations. Estimated regional wall stress-strain relations from the inflation test were likely influenced by interaction between right and left ventricles. We measured biaxial passive stress-strain relations of a thin-walled latex ellipsoidal balloon during passive inflation. When one-half of the balloon surface is glued to restrict deformation (no deformation) during inflation, the stress-strain relations of the deformable side of the balloon are changed. On the other hand, the stress-strain relations from a myocardial strip were independent of this interaction. Myocardial stiffness in these two experimental settings showed similar trends. It is better to normalize myocardial stiffness between two experimental settings to minimize the error of ventricular interaction (7). However, the cause of this error was not only due to the influence of RV and LV interaction, but also to reference-point definition. Figure 7 shows that the y-intercepts (constant a) of the stress-strain curves were different in each experimental group, which suggests that the residual stress may vary between the groups. Residual stress also influences passive material properties (16). Owing to a lack of precise structural information of the embryonic myocardium (myofiber alignment, etc.), it is difficult to define the unloaded state in the embryonic ventricle even in isolated myocardial strips. Finally, geometric assumptions have great influence on the estimation of wall stress in either the chamber or the myocardial strip. Further studies are needed.
In conclusion, our study supports the paradigm that regional alterations in mechanical loading conditions can produce important changes in cardiac structure and function during cardiac morphogenesis. There may be a threshold of mechanical load required to stimulate "normal" cardiomyocyte proliferation and differentiation during cardiac morphogenesis.
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ACKNOWLEDGEMENTS |
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This research was supported by National Institutes of Health Grant RO1 H-64626-01 (to B. B. Keller) and National Research Service Award Grant F32 HL-10200-01 (to E. A. Schroder).
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FOOTNOTES |
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Address for reprint requests and other correspondence: K. Tobita, Cardiovascular Development Research Program, Dept. of Pediatrics, Univ. of Kentucky, 800 Rose St., Rm. MN475, Lexington, KY 40536-0298 (E-mail: ktobi0{at}.uky.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 21, 2002;10.1152/ajpheart.00879.2001
Received 10 October 2001; accepted in final form 18 February 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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)
after CTB (
) and shift leftward after LAL
(
) in both CIR and LNG directions. In stage
27 right ventricle, CIR ED stress after CTB (dashed line and
) increases, and both CIR and LNG ED stresses increase
after LAL (dotted line and 
) compared with normal
(solid line and 
). In the stage 27 left
ventricle, ED stress-strain points after CTB are similar to normal,
whereas both CIR and LNG ED stress-strain points are smaller than
normal after LAL.