Download Pressure overload alters stress-strain properties of the developing

Am J Physiol Heart Circ Physiol 285: H1849–H1856, 2003.
First published July 10, 2003; 10.1152/ajpheart.00384.2002.
Pressure overload alters stress-strain properties
of the developing chick heart
Christine E. Miller,1 Chandra L. Wong,1 and David Sedmera2
1
Division of Pediatric Cardiology, University of Rochester School of Medicine and
Dentistry, Rochester, New York 14642; and 2Department of Cell Biology and
Anatomy, Medical University of South Carolina, Charleston, South Carolina 29425
Submitted 9 May 2002; accepted in final form 2 July 2003
cardiac development; trabeculae; left ventricle
respond to mechanical stimuli, producing biochemical signals that control gene
expression, in turn affecting tissue mass, shape, and/or
physical properties. It is well known that hemodynamic pressure overload in the mature left ventricle
(LV) results in increased mass with reversible or irreversible LV hypertrophy (1, 9, 18, 34). Whether physical properties change, in particular passive elastic
stress-strain relations, is questionable; similar (37, 41)
and significantly steeper (11, 36) diastolic stress-strain
relations have been observed after aortic banding-induced pressure overload in adult mammals.
Embryonic cardiac muscle also responds to increased
hemodynamic pressure with increased mass, but by
cardiomyocyte hyperplasia instead of hypertrophy (8).
Morphological and myoarchitectural changes occur, including chamber dilatation, double-outlet right ventri-
MANY CELL TYPES SENSE AND
Address for reprint requests and other correspondence: C. E.
Miller, Dept. of Mechanical Engr., Dana Bldg., Bucknell University,
Lewisburg, PA 17837 (E-mail: [email protected]).
http://www.ajpheart.org
cle (RV), persistent truncus arteriosus, ventricular septal defect, thickening of the compact myocardium and
trabeculae, and spiraling of the trabecular course (8,
40). Similar and reduced LV stress-strain relations
have been reported after ventricular pressure overload
(44).
Understanding the role of mechanical factors in
heart development is important because of the critical
function of the organ and the potential for congenital
defects. Several studies suggest that mechanical stress
and strain may play an important role. A cardiac model
incorporating end-diastolic sarcomere length and early-systolic stretch in feedback showed physiological
adaptations (2). Stress-related growth caused by enddiastolic pressure in a model of the chick ventricle at
Hamburger-Hamilton (HH) stages 21–29 correlated
well with experimental results (28). Cultured myocardial cells from HH31 chick were shown to proliferate in
response to mechanical strain in vitro (31). A fundamental question is whether a negative-feedback control mechanism exists, so that overall tissue response
reduces the difference between a mechanical quantity
sensed by the cell and a preset limit. The passive
mechanical structure of the heart, consisting of geometric and material properties, determines the magnitude of stress and strain for any given hemodynamic
pressure. Thus changes in passive mechanical structure are a likely effector in such a feedback scheme.
We hypothesized that the passive material properties of developing LV myocardium change in response
to chronically increased LV pressure. To test this hypothesis, LV pressure overload was created by
conotruncal banding (CTB) in the embryonic chick
heart. The chick heart parallels the human heart in
development, has been used extensively in studies of
embryonic cardiac function (6, 20, 24), and allows surgical interventions with reincubation to later stages.
CTB was applied at HH21 (19), and hearts were studied at HH27 (embryonic day 5), HH29 (embryonic day
6), and HH31 (embryonic day 7). These stages encompass the crucial period of chamber morphogenesis.
Stress-strain relations were constructed from uniaxial
cyclic loading with biaxial surface strain recording of
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0363-6135/03 $5.00 Copyright © 2003 the American Physiological Society
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Miller, Christine E., Chandra L. Wong, and David
Sedmera. Pressure overload alters stress-strain properties
of the developing chick heart. Am J Physiol Heart Circ
Physiol 285: H1849–H1856, 2003. First published July 10,
2003; 10.1152/ajpheart.00384.2002.—As a first step in investigating a control mechanism regulating stress and/or strain
in the embryonic heart, this study tests the hypothesis that
passive mechanical properties of left ventricular (LV) embryonic myocardium change with chronically increased pressure
during the chamber septation period. Conotruncal banding
(CTB) created ventricular pressure overload in chicks from
Hamburger-Hamilton (HH) stage 21 (HH21) to HH27, HH29,
or HH31. LV sections were cyclically stretched while biaxial
strains and force were measured. Wall architecture was
assessed with scanning electron microscopy. In controls, porosity-adjusted stress-strain relations decreased significantly
from HH27 to HH31. CTB at HH21 resulted in significantly
stiffer stress-strain relations by HH27, with larger increases
at HH29 and HH31, and nearly constant wall thickness.
Strain patterns, hysteresis, and loading-curve convergence
showed few differences after CTB. Trabecular extent decreased with age, but neither extent nor porosity changed
significantly after CTB. The stiffened stress-strain relations
and constant wall thickness suggest that mechanical load
may play a regulatory role in this response.
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PRESSURE OVERLOAD IN DEVELOPING CHICK HEART
excised, passive LV sections from normal and pressureoverloaded hearts. To eliminate possible bias from
loading direction, sections were excised and loaded in
two approximately perpendicular orientations. Wall
thickness; cyclic energy loss (hysteresis); normal,
transverse, and shear strains; and stress-strain curves
were constructed for each individual LV section. Scanning electron micrographs (SEM) from normal and
overloaded hearts were also examined to measure the
extent of the compact and trabecular myocardial regions and the percentage of intertrabecular spaces.
These results were used to adjust the measured stress
magnitudes for trabecular porosity.
MATERIALS AND METHODS
AJP-Heart Circ Physiol • VOL
Fig. 1. A: stereomicroscopic view of a chick heart at HamburgerHamilton (HH) stage 29 (HH29) showing approximate location of
excised sections. L, longitudinal section; T, transverse section; LV,
left ventricle; RV, right ventricle; LA, left atrium; RA, right atrium;
IVS, interventricular septum; CT, conotruncus. B: schematic diagram showing excised hexahedral LV section from HH29 chick heart.
Dimensions are means of all tested samples.
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Fertilized White Leghorn chicken eggs (Truslow Farms,
Chestertown, MD, and American Selected Products, Milton,
PA) were incubated blunt end upward at 38.5°C and 62%
humidity in a forced-downdraft incubator to the desired
stage. CTB was performed at HH21 (embryonic day 3.5),
which corresponds to embryonic day 10 in the mouse and
Streeter Horizon stage XIII in humans (42). The egg was
positioned under a dissecting microscope, and access to the
embryo was obtained by opening the shell and incising a
small region of the outer and inner shell membranes. A loop
of 10-0 nylon suture was tied in a secure but nonbinding
manner around the outflow track of the developing heart.
Embryos with visual signs of bleeding were discarded. The
opening in the shell was sealed with Parafilm, and the egg
was reincubated until sampling time. The shells of the
matched controls were left intact, and the matched controls
were not subjected to the banding procedure.
The CTB procedure increases LV pressure acutely and
chronically, with no changes in heart rate or cardiac output
(7, 25). At HH21, the interventricular septum (IVS) is not
formed; the ventral and dorsal endocardial cushions unite at
HH28, and the interventricular foramen closes at HH34
(embryonic day 8). Also, the conotruncus has not septated
into aorta and pulmonary artery, which occurs proximally at
HH34 (42). Thus the CTB procedure increases afterload to
the primitive RV and LV, although only the LV is studied
here.
Mechanical testing was performed on tissue from HH27,
HH29, and HH31 (embryonic days 5, 6, and 7, respectively)
hearts that had undergone CTB at HH21 and on matched
intact-shell controls. For all stages tested, the extraembryonic splanchnopleure and somatopleure were cut along the
border of the area opaca for removal of the embryos from the
yolk. The excised embryo was placed in a petri dish of
oxygenated 37°C Krebs-Henseleit buffer with 4 ⫻ 10⫺4
mmol/l verapamil, 30 mmol/l KCl, and 10 mmol/l EGTA.
These additives inhibit Ca2⫹ influx into myocardial cells
through slow Ca2⫹ channels, raise cellular resting potential
to inactivate Na⫹ channels, and chelate intracellular Ca2⫹,
respectively, with the total effect being to prevent Ca2⫹
release from the sarcoplasmic reticulum and, thus, prevent
activation of actin-myosin cross bridges. Myocardial cells are
thus maintained in a passive state. A major part of the
damage in cutting through myocardial cell membranes is due
to the destructive effects of the resulting contracture and
supercontracture. Cells interior to the cut can be affected,
because the extreme shortening causes adjacent cells to be
torn open; hence, the initial cutting injury at the surface is
propagated throughout the rest of the tissue by electrical and
mechanical propagation (35). Thus the verapamil, KCl, and
EGTA additives, which inhibit Ca2⫹ currents and prevent
cross-bridge formation, minimize the dissection injury.
Cutting through the atrioventricular and bulboventricular
grooves removed the LV and RV. The ventral and dorsal
halves of the ventricles were then separated. Hexahedral
sections were cut with microsurgical scissors from the ventral half of the LV (Fig. 1A). Longitudinal (L) sections were
oriented in an apicobasal direction, i.e., longitudinal from the
apex to the mitral orifice, approximately parallel to the IVS.
Section width was approximately equal to section thickness,
and total length varied from 400 ␮m (HH27) to 750 ␮m
(HH31). Transverse (T) sections were approximately perpendicular to L sections and parallel to the outer curvature of the
heart; width and length were as described for L sections (Fig.
1B). To HH29, the main trabecular sheets in the LV are
dorsoventrally oriented and perpendicular to the outer compact layer. In cross section, they appear parallel to the lateral
wall of the IVS, with very fine trabecular linkage to the
compact layer (39). The L sections are therefore parallel and
perpendicular to primary trabecular orientation at these
stages. At HH31, the predominant orientation of the main
LV trabecular sheets has changed from dorsoventral to apicobasal as they start the process of fusion to form the papillary muscles. Thus, at HH31, T sections are parallel and L
sections are perpendicular to primary trabecular orientation.
Images of all excised sections were recorded on videotape
before testing.
The excised sections were placed onto two 0.38-mm-diameter, stainless steel, bevel-tipped wires coated with ␣-cyanoacrylate adhesive and adjusted to a separation ⬃50% of the
length of the ventricular section. The wires extended into a
water bath filled with the warmed, oxygenated Krebs-Henseleit buffer and connected to an ultrasensitive force transducer and a linear actuator, as previously described (32, 33).
At least three 15-␮m spheres of styrene divinylbenzene were
placed on the anterior (myocardial) surface of the mounted
ventricular section midway between the ends to define a
central gage length. Custom LabVIEW software programs
(National Instruments, Houston, TX) with an analog-to-digital–digital-to-analog board (model AT-MIO-16XE, National
Instruments) controlled the motion of the linear actuator,
reversing direction at preset upper and lower limits, and
sampled the transducer signal at 500 Hz. The ventricular
section was stretched in a cyclic loading-and-unloading pattern at 0.5 Hz for 10 cycles to 15% longitudinal strain. The x-y
positions of the markers were determined during stretching
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The average longitudinal Lagrangian (2nd Piola-Kirchoff)
stress Tzz in the compact layer is then given by the following
equation: Tzz ⫽ F/[wt(1 ⫺ Po)]. Tzz was plotted vs. surface
longitudinal strain (Ezz) for each section. Stress-strain curves
were fit by various polynomial and exponential functions.
The strain energy (SE) density for the loading portion of each
curve (SE ⫽ 兰00.15TzzdEzz) was calculated as the area under
the stress-strain curves from 0 to 0.15 strain. Secant stiffness, the stress divided by the strain at each data point, was
calculated for all experimental data points from 0 to 0.15
strain for each tested section.
The six normal groups are designated by HH stage and
orientation (L or T) as 27L, 27T, 29L, 29T, 31L, and 31T. The
six banded groups are 27L-B, 27T-B, 29L-B, 29T-B, 31L-B,
and 31T-B. Mean ⫾ SD was calculated for each measurement
in the 16 groups. Student’s t-test gave the significance of
differences between the control and the banded group at each
stage and orientation and also between L and T orientations
at fixed stage and treatment (control and banded). ANOVA
with post hoc multiple-comparison Tukey’s test compared
differences between quantities across stages. Statistical significance level was P ⬍ 0.05.
RESULTS
Point-counting analysis of SEM sections from control
hearts showed that the percentage of cross-sectional
area occupied by the compact layer in control hearts
increased significantly with developmental stage from
HH27 (26%) to HH31 (48%; Table 1). Banding at all
stages produced no significant changes in the percentage of cross-sectional area occupied by the compact
layer. Trabecular layer porosity was similar in HH27
and HH31 (Table 1) but declined significantly from
HH29 (54%) to HH31 (48%, P ⬍ 0.05). Trabecular
porosity and overall porosity in corresponding banded
and control groups were similar (P ⫽ not significant).
In micrographs, LV dilatation and elongation with
the beginnings of trabecular spiraling and thickening
of the compact layer were observed in HH27 hearts
banded at HH21. At HH29, banding led to further LV
elongation, expansion of the trabecular-free lumen in
the apical part of the LV with concomitant reduction of
the trabecular extent, thickening of the compact layer,
Table 1. LV wall characteristics
ACL, %
PTL, %
Po, %
HH27
HH27-B
HH29
HH29-B
HH31
HH31-B
26 ⫾ 4
50 ⫾ 6
43
29 ⫾ 4
52 ⫾ 7
43
38 ⫾ 5*
54 ⫾ 7
42
40 ⫾ 5
49 ⫾ 7
37
48 ⫾ 7*†
48 ⫾ 4†
32
50 ⫾ 7
44 ⫾ 4
28
Values are means ⫾ SE. Geometric measurements were obtained
from the apical region of the left ventricular (LV) wall in embryonic
chick hearts. ACL, compact layer area, i.e., percentage of total crosssectional area of the myocardial wall occupied by the compact layer
as measured from point counting of scanning electron micrographs of
LV sections; PTL, trabecular layer porosity, i.e., percentage of crosssectional area of the trabecular region occupied by intertrabecular
spaces measured from scanning electron micrographs of LV sections
(43); Po, overall porosity, i.e., proportion of total cross-sectional area
(compact layer ⫹ trabecular layer) occupied by intertrabecular
spaces (see MATERIALS AND METHODS for calculation). Data from embryonic chick hearts at Hamburger-Hamilton (HH) stage 31 banded
at HH21 (HH31-B) are estimated from HH31 measurements and
HH29-B changes. * P ⬍ 0.001 vs. HH27; † P ⬍ 0.05 vs. HH29.
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as previously described (32) from digitized images from a
charge-coupled device video camera attached to the stereomicroscope (ComputerEyes/RT frame-grabber board, Digital Vision, Dedham, MA) and recorded to disk along with transducer output voltage and time. Strains were calculated from
the marker positions and the definition of the symmetrical
Green-Lagrangian strain tensor (Eij) (32). With z as the long
axis of the tissue section, x as the width, and y as the height,
the surface strains were Ezz, Exx, and Ezx. Principal strains
were also calculated. The calculated strain is homogeneous
within the triangle.
The remaining data analysis was done on recorded files of
time, voltage, and strain and on videotapes of tested sections.
All voltages were referred to the minimum at the end of the
third cycle, and force was calculated as transducer output
voltage multiplied by calibrated transducer sensitivity, 9.37
V/g. Width (w), length, and thickness (t) of each tested section
were measured from videotape (Scion Image program, Scion,
Frederick, MD) by averaging measurements at three locations. Thickness was the total distance from the epicardial to
the endocardial surface. Rectangularity was assessed by fitting lines through the epicardial and endocardial surfaces
from the side view and by measuring the linearity and
parallelity of the lines.
Normalized hysteresis area was calculated as the area
between the loading and unloading segments divided by the
total area under the loading segment in the fourth loading
cycle of the stress-strain plots. Hysteresis convergence was
the difference between the strain energy stored during loading in the first cycle and that stored during loading in the
fourth cycle.
SEM on a parallel sample population provided data on
typical cross-sectional characteristics. These data could not
be determined noninvasively on tested tissue sections. SEM
images of HH27, HH29, and HH31 normal hearts and hearts
banded at HH21 (40) were analyzed. Compact layer area
proportion (ACL), the percentage of the total cross-sectional
solid area occupied by the compact layer, was measured by an
unbiased point-counting technique on digitized SEM images
using the Analyze 7.1 image analysis system (CNSoftware,
London, UK) on a Hewlett-Packard UNIX platform. Trabecular layer area proportion (ATL) is then equal to 1 ⫺ ACL.
Trabecular gross porosity (PTL) was determined from the
point-counting technique as the percentage of the total area
of the trabecular layer (myocardial tissue ⫹ spaces) occupied
by the intertrabecular spaces. We calculated overall porosity
(Po), the percentage of the entire cross-sectional area occupied by intertrabecular spaces, as follows: Po ⫽ PTLATL/
ATL ⫹ (1 ⫺ PTL)ACL.
Until HH34, linear dimension shrinkage with SEM has
been shown to be 52% (39, 40). Independent experiments (39;
unpublished observations) were performed comparing trabecular morphology by SEM with confocal images of whole
mount phalloidin-stained hearts in the wet state by the
method of Germroth et al. (15) and uncut, optically sectioned
hearts stained with antimyosin antibody and cleared according to Kolker et al. (27). The trabecular morphology and
shape were in complete alignment, showing that critical
point drying in SEM results in homogeneous tissue shrinkage. Because of the uniform shrinkage, the dehydrated state
of specimens does not influence the values of porosity. Thus
ACL and PTL were not adjusted for tissue shrinkage.
To calculate stress, each tissue section was modeled as a
laminated bilayer bar of rectangular cross section with width
w and thickness t. Both layers contain identical material, one
closely packed and the other webbed. The ends are rigidly
held and stretched with force F. End effects are neglected.
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PRESSURE OVERLOAD IN DEVELOPING CHICK HEART
Fig. 2. Representative scanning electron microscope images of HH29 embryonic chick hearts sectioned in a transverse plane. A: normal heart, midventricular level. Arrow, trabecular sheet. B: midventricular level from heart banded at HH21 (2.5
days earlier); note absence of RV at this level. Arrows, trabecular sheets. C: normal heart, apical
level. D: banded heart, apical level; note thickened
compact layer, presence of trabeculae-free lumen,
and spiraling trabecular course.
Fig. 3. LV wall thickness of excised sections from chick embryos at
HH27, HH29, and HH31; suffix B denotes hearts subjected to pressure overload by conotruncal banding at HH21. Longitudinal and
transverse sections are pooled for each stage. Study population is as
follows: n ⫽ 17 for HH27, n ⫽ 16 for HH27-B and HH31, n ⫽ 19 for
HH29, n ⫽ 18 for HH29-B, and n ⫽ 20 for HH31-B. Statistical
comparisons were made between normal stages and within a stage
for normal vs. banded. For controls, significance is as follows: *P ⬍
0.001 vs. HH27. All other comparisons were not significant.
AJP-Heart Circ Physiol • VOL
ing possible bias in results due to greatly varying crosssectional shape.
During cyclic loading, peak longitudinal Green’s
strain (Ezz) on the epicardial surface of all 16 groups
was 0.16–0.19 and followed a cyclical pattern in phase
with force. The transverse epicardial strains (Exx) were
negative in all groups and were ⫺0.21 to ⫺0.37 times
longitudinal strain, except in 31L sections, where
transverse strain was only ⫺0.09 times longitudinal
strain. Exx of L and T orientations were similar, as was
Exx after banding, except at HH31. Exx in 31T sections
was 3.8 times that in 31L sections (P ⬍ 0.05), and Exx
in 31L-B sections was 2.8 times that in 31L sections
(P ⬍ 0.05).
The force-strain relations were well defined and repeatable. The loading and unloading branches were
separate, showing typical pseudoelastic behavior. In
all 12 groups, the loading branch was nonlinear, stiffening with increasing strain. Most curves showed inflection points between 0 and 0.07 strain. A preconditioning phenomenon was present, with the first loading
cycle at significantly higher force than the second and
subsequent loading cycles (P ⬍ 0.01 in all 12 groups).
The loading curves converged within 1% after three
cycles, resulting in reductions in strain energy of 33–
57%, with corresponding L and T orientations and
control and banded treatments similar, except at 29T.
The reduction was significantly more at 29T (57%)
than at 29L (33%, P ⫽ 0.02) or 29T-B (33%, P ⬍ 0.001).
The mean hysteresis, or energy loss, in a loadingunloading cycle ranged from 16% (27T) to 24% (29L).
Hysteresis was similar between L and T orientations
and between control and banded groups.
In control hearts, plots of porosity-adjusted mean
Lagrangian (2nd Piola-Kirchoff) stress vs. longitudinal
Lagrangian (Green’s) strain shifted significantly downward with developmental age from HH27 to HH29 for
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and spiraling of the trabeculae counterclockwise from
apex to base (Fig. 2).
The total wall thickness of sections from control hearts
increased significantly with age from HH27 to HH29 and
leveled off at HH31 (Fig. 3). Total wall thicknesses did
not increase significantly after CTB (Fig. 3). The mean
section widths (␮m) as cut for this study were 325 ⫾ 34
(HH27), 305 ⫾ 48 (HH27-B), 349 ⫾ 39 (HH29), 345 ⫾ 28
(HH29-B), 371 ⫾ 34 (HH31), and 388 ⫾ 64 (HH31-B).
These widths were approximately equal to the section
thickness; thus the excised sections had an almost square
cross section. Widths were also fairly uniform, eliminat-
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Fig. 4. Mean Lagrangian stress adjusted for trabecular porosity vs. longitudinal Green’s strain in excised LV
sections from control hearts. Numerical labels denote HH stage. A: longitudinal (L) sections. Study population is
as follows: n ⫽ 8 for 27L, 29L, and 31L.
B: transverse (T) sections. Study population is as follows: n ⫽ 9 for 27T, n ⫽
11 for 29T, and n ⫽ 8 for 31T. For
clarity, error bars (SD) are shown only
at 0.05, 0.10, and 0.15 strain. All differences between curves are significant
at P ⬍ 0.05 for strain energy. *P ⬍ 0.05
vs. HH27; ⫹P ⬍ 0.05 vs. HH29 for
secant stiffness.
29L and 29T were similar, but with 29L above 29T
(Fig. 5B; P ⫽ not significant). Curve shapes of 31L and
31T were also similar, but with 31T above 31L (Fig. 5C;
P ⬍ 0.05 above 0.10 strain).
Stress-strain curves from HH27, HH29, and HH31
hearts banded at HH21 shifted to larger stresses than
control hearts. The shifts were moderate at HH27,
with increases of 1.5–2.0 times in secant stiffness and
strain energy (P ⬍ 0.05 for T; Fig. 5A). At HH29,
banded stresses averaged 5.9 (L) to 7.4 (T) times larger
than control (P ⬍ 0.001 for L and T; Fig. 5B). The
difference was also significant at HH31: 5.0 (L) and
2.9(T) times larger than control (P ⬍ 0.001; Fig. 5C).
DISCUSSION
This study shows that pressure overload causes significant changes in the passive mechanical material
Fig. 5. Mean Lagrangian stress adjusted for trabecular porosity vs. longitudinal Green’s strain in control and
banded LV sections at HH27 (A),
HH29 (B), and HH31 (C). L, longitudinal orientation; T, transverse orientation; B, banded heart. Hearts were
banded at HH21. Study populations
are as follows: n ⫽ 8 for 27L, 27L-B,
27T-B, 29L, 29L-B, 31L, and 31T, n ⫽
9 for 27T, n ⫽ 10 for 29T-B, 31L-B, and
31T-B, and n ⫽ 11 for 29T. For clarity,
error bars are shown only at 0.05, 0.10,
and 0.15 strain. All differences between corresponding normal and
banded curves are significant at P ⬍
0.05 for strain energy. *P ⬍ 0.05 vs.
corresponding control group for secant
stiffness.
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L and T orientations (Fig. 4). Both stress-strain curves
had steeper curvature at HH29 than at HH31; 29L was
above 31L but 29T was below 31T. The curves were fit
well by a cubic polynomial (all R2 ⬎ 0.999), but not by
simple exponential equations. However, because emphasis could be too easily shifted between the linear,
quadratic, and cubic terms of the polynomial and because introduction of a mathematical description of the
curves having no unique physiological foundation was
deemed unadvisable, these fits were not used for statistical comparisons. Rather, ANOVA of total strain
energy showed P ⬍ 0.05 among all three curves at L
and T orientations. ANOVA of secant stiffness (Fig. 4)
also showed significant differences.
In control hearts, porosity-adjusted mean Lagrangian stress vs. longitudinal Lagrangian strain curves
were very similar in magnitude and shape for L and T
orientations at HH27 (Fig. 5A). The curve shapes of
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reported in the chick at HH24, HH27, and HH31 (43),
however; thus passive behavior may be different from
active behavior. The difference in stress-strain curves
and normalized transverse epicardial strains at HH31
suggests that the trabeculae, which have formed into
long, thick bundles (trabeculae carneae) attaching to
the ventricular wall or IVS along their length (38),
influence the mechanical behavior at this stage.
CTB initiated at HH21 induced significant changes
in the stress-strain relation by HH27, 1.5 days of overload, with passive stiffness almost doubling. Longer
exposure to overload resulted in much larger increases.
This period of overload encompasses HH24, when the
mitotic activity of myocardial cells is the highest in the
entire incubation period (17, 22). Because stress is
normalized to porosity-adjusted cross-sectional area,
these changes in passive stiffness are independent of
the increased growth observed after CTB at HH21, e.g.,
the increase in cell number by 43% at HH27 and 24%
at HH29, the increase in ventricular weight by 67% at
HH27 and 86% at HH29 (7), and the visible elongation
of the heart (40). Changes in the ECM may be responsible for the pressure overload-increased stiffness, because the characteristics and extent of the ECM are
important determinants of diastolic ventricular properties and function (16). Adult animal models showed
increased ventricular collagen (3, 11, 45) and constant
collagen concentration (26, 37) after pressure overload.
In contrast to the increase in stress-strain magnitude of 1.5–2.0 times (P ⬍ 0.05) at HH27 after CTB
found here, another study of myocardial material properties after CTB (44) did not measure any significant
changes in LV stress-strain relations at HH27 after
CTB at HH21 in pressure-strain and excised strip
tests. The reason for this difference is unclear but could
be due to a different amount of pressure overload, a
different location of strips, or larger strains used for
testing.
The present results support the possibility of a biological feedback control system in the developing heart,
with deformation or strain involved in the sensing and
regulatory process. LV wall stress due to internal pressure is directly proportional to pressure magnitude and
ventricular radius and inversely proportional to wall
thickness. An increase in wall stress due to pressure
overload with no change in tissue material properties
produces an increase in wall strain. Tissue remodeling
causing increased passive stiffness can decrease wall
strain, perhaps to a level that is “normal” for that
developmental stage. Although the effect is at the tissue level, the mechanisms for such stress- or strainregulated control likely reside at the cellular level and
may involve a number of cell-signaling mechanisms.
For example, increased tyrosine phosphorylation has
been observed in HH31 chick myocytes strained in
vitro on flexible membranes (31), and increased platelet-derived growth factor-like protein has been observed in banded embryonic chick ventricle (21).
The principal limitations of this study arise from the
small size and fragility of the embryonic heart. The
advantage of the in vitro direct loading method is
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properties of the early embryonic heart. These changes
depend on the duration of the overload. In addition,
testing of LV tissue from HH27 to HH31 control hearts
provides new information on changes in material properties with tissue maturation.
The geometric and material properties measured
here in control hearts are not unexpected for this
developmental range. Increases in wall thickness and
compact layer proportion from HH27 to HH31 are
indicative of the general growth and maturation of the
tissue. The nonlinear stress-strain curves (13, 16) and
preconditioning phenomenon (4, 12, 14, 23, 30, 32) are
typical of soft biological tissue. Less expected was the
finding that gross porosity of the trabecular layer decreased only slightly from HH27 to HH31, with most of
the decrease in overall porosity attributed to the decrease in the extent of the trabecular layer.
The significant decrease in the myocardial stressstrain relation from HH27 to HH29 and HH31 in
control hearts was unexpected. It cannot be explained
by changes in thickness of the compact and trabecular
layers and porosity, because these factors are accounted for in the area normalization factors. Although
the averaged overall porosities are means of a parallel
study group (total cross-sectional area is measured on
each tested specimen), they appear unlikely to bias the
results, because the range of porosity adjustments is
small relative to the decrease in stress with developmental stage. Further study is necessary to determine
the changes in intercellular, subcellular, and/or extracellular matrix (ECM) components and connections
responsible for this stress-strain decrease.
Much larger stress-strain magnitudes have been reported at HH27 from uniaxial loading of excised LV
strips (44). The data were modeled by the following
equation: stress ⫽ a[exp(b ⴱ strain)], where b for two
perpendicular directions is 7.5 and 7.9 and a is a
prestress included to achieve “excision length” and is
not explicitly given here. If a is in the range of 50
mg/mm2, as shown in pressure-strain graphs, then
Tobita et al. (44) report, e.g., 1.1 kPa unadjusted stress
at 0.10 strain, in contrast to the much lower stresses
measured here (0.20 kPa unadjusted stress at 0.10
strain). The differences between the present results
and those reported by Tobita et al. could be due to the
prestressing of the tissue, resulting in shifts to higher
stress-strain magnitudes, or to the larger strains used,
0.30 compared with 0.15, which may induce tissue
changes. Also, the tissue sections tested by Tobita et al.
may be from a different LV location, and morphological
studies (39) and measurement of intramyocardial pressure (5) suggest that material properties may vary
throughout the LV.
The similarity of stress-strain curves and transverse
strains in L and T orientations from HH27 to HH29
indicates that loading orientation does not bias the
results. It also implies that the relatively isotropic
compact layer, on which strains are measured, dominates the overall mechanical response of the LV myocardium in these tests. Anisotropic LV epicardial
strain patterns during active contraction have been
PRESSURE OVERLOAD IN DEVELOPING CHICK HEART
We thank Franco Ardizzoni for expert technical assistance with
SEM and Martina Sedmerova for assistance with point counting. We
acknowledge the support of Bucknell University, particularly
Weston Griffin and Dr. Keith Buffinton, for developing the linear
actuator.
DISCLOSURES
This research was supported by National Heart, Lung, and Blood
Institute Grant R01-HL-65908, American Heart Association, New
York State Affiliate, Grant-in-Aid 9951019T, and National Heart,
Lung, and Blood Institute Specialized Center of Research in Pediatric Cardiovascular Disease Grant HL-51498. Experimental work by
D. Sedmera was performed at the Institute of Physiology, University
of Lausanne (Lausanne, Switzerland).
AJP-Heart Circ Physiol • VOL
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