Download Microtubule Involvement in the Adaptation to Altered Mechanical

Microtubule Involvement in the Adaptation to Altered
Mechanical Load in Developing Chick Myocardium
Elizabeth A. Schroder, Kimimasa Tobita, Joseph P. Tinney, Jane K. Foldes, Bradley B. Keller
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Abstract—Mechanical load regulates ventricular growth, function, and structure from the earliest stages of cardiac
morphogenesis through senescence. Dramatic changes in cardiac form and function have been defined for developing
cardiovascular systems, and changes in mechanical loading conditions can produce structural malformations such as left
heart hypoplasia. To date, relatively little is known regarding the interactions between changes in mechanical load,
morphogenesis, and the material properties of the embryonic heart. We tested the hypothesis that passive material
properties in the embryonic heart change in response to altered mechanical load and that microtubules play an important
role in this adaptive response. We measured biaxial passive stress-strain relations in left ventricular (LV) myocardial
strips in chick embryos at Hamburger-Hamilton stage 27 following left atrial ligation (LAL) at stage 21 to reduce LV
volume load and create left heart hypoplasia. Following LAL, myocardial stresses at given strains and circumferential
stiffness increased versus control strips. Western blot analysis of LAL embryos showed an increase in both total and
polymerized ␤-tubulin and confocal microscopy confirmed an increase in microtubule density in the LV compact layer
versus control. Following colchicine treatment, LV stresses and stiffness normalized in LAL specimens and microtubule
density following colchicine was similar in LAL to control. In contrast, Taxol treatment increased myocardial stresses
and stiffness in control strips to levels beyond LAL specimens. Thus, the material properties of the developing
myocardium are regulated by mechanical load and microtubules play a role in this adaptive response during cardiac
morphogenesis. (Circ Res. 2002;91:353-359.)
Key Words: embryonic ventricle 䡲 cardiac morphogenesis 䡲 viscoelastic properties 䡲 ␤-tubulin 䡲 microtubules
T
syndrome (HLHS) in the chick embryo,10,11,14 whereas increased ventricular load produced by conotruncal (arterial)
banding accelerates myocardial growth via hyperplasia.9,10
Similar changes in mechanical load alter cardiac growth and
function in the fetal heart and young infant.15,16 Although the
relationship between changes in ventricular load, function,
and structure is well documented, we know very little
regarding the intracellular and extracellular mechanisms responsible for this adaptation in the developing myocardium.
Changes in microtubule content and distribution in response to altered mechanical load have been shown to
directly affect cardiomyocyte structure and function in feline,
canine, and human myocardium.17–20 Cardiomyocyte hypertrophy is also associated with increased density of the
intracellular microtubular network resulting in increased viscous damping.21 Drugs such as colchicine that depolymerize
microtubules have been shown to restore passive stiffness to
control values in some but not all models of myocardial
hypertrophy.22–25 Although microtubules may not be a major
determinant in the passive properties of normal adult
heart,21,23 microtubules have been shown to contribute to the
contractile dysfunction noted in hypertrophic and dilated
cardiomyopathy.20,21,26,27 Hatcher et al28 investigated the
he adaptation of ventricular function and structure to
altered mechanical load is a fundamental property of the
myocardium present from the earliest stages of cardiac
morphogenesis through senescence. Embryonic heart rate,
blood pressure, and cardiac output increase geometrically
during formation of the heart and blood vessels1–3 and the
developing heart undergoes a dramatic transformation in
3-dimensional structure coincident with rapid cardiomyocyte
clonal expansion and differentiation.1,2,4,5 Although there are
numerous examples of single-gene defects associated with
congenital cardiac malformations, most single-gene models
of congenital heart disease display a wide variation in
phenotype, suggesting an important role for modifying genes
as well as epigenetic or environmental factors.6 – 8
Experimental interventions that increase or decrease mechanical load during cardiac development result in both
changes in cardiac function and structure.9 –11 With a peak
systolic pressure of less than 10 mm Hg and an end-diastolic
pressure of less than 2 mm Hg during early cardiac morphogenesis, the developing myocardium responds rapidly to
changes in mechanical load to maintain cardiac output.12,13
Inflow occlusion of the developing left ventricle produced by
left-atrial ligation (LAL) produces hypoplastic left heart
Original received February 22, 2002; revision received June 21, 2002; accepted July 9, 2002.
From Cardiovascular Development Research Program, Department of Pediatrics, University of Kentucky, Lexington, Ky.
Correspondence to Bradley B. Keller, Dept of Pediatrics, University of Pittsburgh, 3705 Fifth Ave, Pittsburgh, PA 15213-2583. E-mail
[email protected]
© 2002 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000030179.78135.FA
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Circulation Research
August 23, 2002
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relationship between microtubules and the decrease in ventricular function noted in chick embryos after neural crest
ablation; however, no change in microtubule content was
found.
In the present study, we tested the hypothesis that microtubules are involved in the adaptive response of the developing myocardium to altered mechanical load. We measured
biaxial passive left ventricle (LV) stress-strain relations in
chick embryos after LAL known to reduce LV volume load
and produce HLHS.11 After LAL, myocardial stresses at
given strains and circumferential stiffness increased versus
control strips. Western blot analysis of LAL embryos showed
an increase in both total and polymerized ␤-tubulin and
confocal microscopy confirmed an increase in microtubule
density in LV compact myocardium versus control. After
colchicine treatment, LV stresses and stiffness normalized in
LAL specimens and microtubule density after colchicine was
similar in LAL to controls. Taxol treatment, which hyperpolymerizes microtubules, increased myocardial stresses and
stiffness in control strips to levels beyond LAL specimens.
Thus, the material properties of the developing myocardium
are regulated by mechanical load and microtubules play a role
in this adaptive response during cardiac morphogenesis.
Materials and Methods
Embryo Selection
Fertile White Leghorn chicken eggs (CBT Farms, Chestertown, Md)
were incubated in a forced-draft, constant-humidity incubator and
studied at Hamburger-Hamilton stage 27 (5 days) of a 46-stage (21
day) incubation period as previously described.29 At the selected
stage the embryonic ventricle has externally distinct RV and LV
chambers with common atrioventricular (AV) canal, large interventricular communication, and common outflow tract.30 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 US National Institutes of
Health (NIH Publication No. 85-23, revised 1985).
Left Atrial Ligation (LAL) to Reduce Left
Ventricular Preload
Embryos were initially incubated to stage 21. A 1-cm2 hole was
made in the shell and the inner shell and extraembryonic membranes
were removed to expose the developing embryo. The embryo was
then gently positioned left side up and microforceps were used to
make a slit-like opening in the thoracic wall above the primitive left
atrium. A loop of 10 to 0 nylon suture tied with an overhand knot
was then placed across the primitive left atrium and tightened,
decreasing the effective volume of the left atrium and thus redistributing blood from the left to the right ventricle.10,11,14 Each embryo
was then repositioned to its original right side up orientation, the
eggshell opening was sealed with parafilm, and reincubated until
stage 27.
Preparation of Myocardial Strips for
Mechanical Testing
Hamburger and Hamilton (1951) stage 27 embryos (normal and
LAL) were excised and placed in a Petri dish containing hypothermic
(25°C), hyperkalemic chick ringer solution (in mmol/L: 82 NaCl, 60
KCl, 2 CaCl2, 10 Trizma- HCl, and 10 Trizma-base) adjusted to pH
7.4 to arrest the heart during diastole.31 The ventricle was removed
from the embryo by cutting along the AV groove and across the
conotruncal cushions. Myocardial strips were dissected from the LV
free wall in both a longitudinal (LNG) and a circumferential (CIR)
orientation. We defined the LNG direction of the cardiac coordinate
system by an axis from the midpoint of the primitive AV valve to the
LV apex. Average cross-sectional area was not significantly different
between experimental groups cut in LNG (0.1790⫾0.0140 versus
0.1860⫾0.0162 mm2; normal versus LAL, respectively) or CIR
(0.1476⫾0.0107 versus 0.1362⫾0.0162 mm2; normal versus LAL,
respectively) orientations. After excision, strip length was measured
and then the specimens were mounted on our force workstation as
previously described.31 Microspheres (10 ␮m) were placed on the
epicardial surface of the ventricular strip in a triangular pattern such
that the centroid of the triangle aligned with the central axis of the
wires and was midway between their ends. Microsphere spacing
averaged 100 ␮m. A custom linear motor stretched the ventricular
segment in a triangular ramp pattern at a velocity of 0.2 mm/s at 30%
above the excised length. Myocardial strips were preconditioned for
1 minute at 30% stretch and then data were recorded for the range of
0 to 0.25 longitudinal strain. Myocardial strip dimensions and strain
patterns during stretch were monitored using a video microscopy
system and by internal length sensors in the displacement motor.
While the ventricle was being stretched, we recorded passive
force-strain measurements in isolated LV strips using a custom
analog-digital signal and image acquisition system.11 The data
acquisition system simultaneously acquired force and strain data at
100 Hz (LabVIEW, National Instruments) and 10 Hz (NIH image
1.62), respectively. Two-dimensional epicardial wall strain was
determined by tracking triangular arrays of microspheres from
acquired images.31
Embryonic LV geometry at the study stage is complex11,30 and the
ventricular wall is composed of an inner trabeculae layer with
varying intertrabecular spaces and a thin outer compact myocardium.
Because it is not technically feasible to acquire realistic
3-dimensional data of the trabeculated embryonic wall during
stretching, basic geometric assumptions are required in order to
estimate myocardial stresses. Therefore, for this study, we considered the embryonic LV myocardium to be a freely deforming solid
body composed of isotropic and incompressible elastic material.
Wall stresses at the midportion of the myocardial strip during stretch
were calculated as force divided by cross-sectional area, assuming a
cylindrical geometry. Passive stress-strain relations were fit by the
following equation32: ␴⫽a Exp(bE), where ␴, a, b, and E, are,
respectively, midwall stress, midwall stress at zero strain, myocardial
stiffness constant, and Lagrange strain. We chose “excision length”
as the reference state. We recognize that this passive reference state
is not a completely unloaded state due to residual stress in the
embryonic myocardium.33 To determine b from the stress-strain
relations in each embryo, we used a quasi-Newton algorithm to
minimize least square loss function. Convergence criterion was set to
0.0001. All calculations were performed using Sigma Plot (SPSS
Inc).
Normalized hysteresis loop area, a measure of viscous damping,
was calculated as the loop area between the stress-strain relationship
during an increase (loading) and decrease (unloading) in stress
divided by the total area under the loading portion of the curve.21,34,35
When stress is increased, the area under the stress strain relation is
equal to the potential energy gained by the muscle strip during
stretch. When stress is decreased, the energy returned to the system
is equal to the integral of the stress-strain relation. If damping occurs,
there will be a difference between the energy gained during stretch
and the energy returned during release. This loop area represents the
energy lost to heat and reflects the resistance to stretch associated
with viscosity.
Assessment of Microtubule Contribution to
Passive Properties
To examine the contribution of microtubule density on the passive
properties of the embryonic myocardium, excised specimens (CIR or
LNG strips) were placed in 50 ␮mol/L colchicine (Sigma)/chick
ringer solution on ice for 1 hour28 or in 25 ␮mol/L Taxol (Cytoskeleton Inc)/chick ringer solution at 37°C for 4 hours. Colchicine binds
tightly to the ␤-tubulin subunit, prevents polymerization, and results
in a rapid destabilization of microtubules. Taxol lowers the critical
concentration of ␣␤-tubulin heterodimers required to form microtu-
Schroder et al
Microtubules in Embryonic Heart
355
Figure 1. Average stress-strain relationships for stage 27 LV specimens. Black
circles indicate control specimens; white
circles, control specimens after colchicine treatment; gray circles, control
specimens after Taxol treatment; black
triangles, LAL specimens; white triangles,
LAL specimens after colchicine treatment. *P⬍0.05 vs stage 27 normal.
bules, thus increasing microtubule density via the hyperpolymerization of existing microtubules and the formation of aberrant, nonfunctional microtubules. Strips were then attached to the transducer and
motor and stretched following the aforementioned protocol.
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SDS-PAGE and Western Blot Analysis
of ␤-Tubulin
Whole cell lysates were prepared from LV specimens (control and
LAL groups, with and without colchicine treatment). Polymerized
␤-tubulin content was assessed following the protocol outlined by
Hatcher.28 SDS-PAGE (7% separating gel) and immunoblotting
were carried out following routine protocols. Mouse monoclonal
anti–␤-tubulin antibody (Sigma) was visualized with a horseradish
peroxidase-conjugated secondary antibody (Jackson Immunolaboratories) and chemiluminescence (Amersham). Each lane contained
protein prepared from 10 LV specimens (30 to 40 ␮g total protein).
Ventricular dimensions are comparable in control and LAL embryos
at this developmental stage.11 All Western blot experiments were
repeated at least 3 times to ensure that experimental observations
were reproducible. Known amounts of purified tubulin (Cytoskeleton Inc) were run on each gel to allow for the quantification of both
total and polymerized ␤-tubulin. Immunoblots were scanned on an
HP ScanJet 4c and quantified using densitometry (Scion Image,
Scion Corporation).
Confocal Microscopy and
Immunofluorescence Protocols
The embryonic heart was arrested by injection of 20 ␮L of 25°C
hyperkalemic chick Ringer solution containing (in mmol/L) 82
NaCl, 60 KCl, 2 CaCl2, 10 Trizma- HCl, and 10 Trizma-base, pH 7.4
via the sinus venosus. Each embryo was fixed in cold methanol with
1 mmol/L EGTA. After fixation, the heart was removed from the
embryo and then carefully placed in a LNG orientation within a 13%
polyacrylamide gel that was immediately polymerized with 2%
ammonium persulfate.36 Polyacrylamide-embedded hearts were sectioned at 100 ␮m with a standard vibrotome (VT1000S, Leica,
Germany). Sections representing LV myocardium were permeabilized with 0.1% Triton X-100 for 30 minutes and were stained for
f-actin with FITC-conjugated phalloidin (Molecular Probes) and for
␤-tubulin with mouse monoclonal anti–␤-tubulin primary antibody
(Sigma) and Alexa 568 (IgG1 specific, Molecular Probes) secondary
antibody. LV myocardium at the midventricular level was examined
using a Leica confocal microscopy system (Model TCS Laser
Scanning Confocal Microscope System, Leica, Germany). Laser
power, aperture, opening percentage, and gain of the confocal system
were selected to minimize background fluorescence and to optimize
the fluorescence intensity of the trabecular myocardium. Once this
protocol was established, similar settings were used for all specimens. Z-Serial optical sectioning was performed for a z-depth of 100
␮m at an interval of 1 ␮m using a 63⫻ water-immersion objective.
Stacks of 50 z-serial section images were captured and saved
digitally. The middle 20 consecutive sections from each stacked
image were projected using maximum intensity point projection for
subsequent image analysis.
Statistical Analysis
Data are presented as mean⫾SE. Two-factor repeated analysis of
variance (ANOVA) was performed to compare the mean values of
stress-strain relations between experimental groups. Two-factor
ANOVA was performed to compare mean values of wall stress,
strain components, and b between experimental groups. Individual
comparisons were performed using a Tukey test. Statistical significance was defined by a value of P⬍0.05. All calculations were
performed using SigmaStat (SPSS Inc).
Results
Passive Material Properties of Embryonic
LV Strips
LV myocardial strips from control and LAL embryos yielded
reproducible passive stress-strain curves in more than 90% of
experiments. Myocardial stresses at given strains and CIR
myocardial stiffness were significantly increased after LAL
(P⬍0.05 versus normal stage 27; Figure 1 and Table 1). This
suggests an increase in the stiffness of the passive elastic
component. After colchicine treatment, LV stress and stiffness were unchanged for control embryos; however, colchicine treatment reduced LAL values to control values
(P⬍0.05). Taxol treatment resulted in an increase in stresses
at given strains and myocardial stiffness in both the LNG and
CIR directions. Mean hysteresis loop energy, a measure of
viscous damping, was increased in LAL LV and Taxoltreated control specimens (both CIR and LNG) compared
with controls (Table 2).
Western Blot Analysis for ␤-Tubulin
Total and polymerized ␤-tubulin increased at stage 27 after
LAL compared with control (Figure 2). After colchicine
treatment, there was no significant difference in the amount
of polymerized ␤-tubulin between LAL and control groups.
Immunohistochemistry
Indirect immunofluorescence images of LV specimens
showed that microtubules form a reticular or randomly
arranged network pattern in the compact myocardium. In
normal developing LV, microtubular density in the compact
myocardium was lower than in the trabecular myocardium,
whereas the compact myocardial microtubular density increased after LAL (Figure 3). Colchicine treatment of LAL
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Circulation Research
TABLE 1.
August 23, 2002
Ventricular Curve Statistics
Left Ventricle
S 27 N
S 27 NColchicine
S 27 NTaxol
S 27 LAL
S 27 LALColchicine
Circumferential
n
8
9
4
6
7
a, mg/mm2
8.47⫾1.83
9.84⫾2.43
1.14⫾0.68*
10.56⫾1.28
8.32⫾2.97
Stiffness constant b
7.53⫾0.87
8.64⫾1.04
26.91⫾4.38*
11.37⫾1.04*
9.54⫾1.06
r
0.99⫾0.001
0.99⫾0.002
0.98⫾0.001
0.99⫾0.001
0.98⫾0.005
9
10
7
10
8
Longitudinal
n
2
a, mg/mm
5.14⫾0.93†
4.93⫾1.78†
4.47⫾2.8
12.93⫾1.57*
7.29⫾4.48
Stiffness constant b
7.79⫾0.50
9.01⫾1.01
19.72⫾1.68*
7.71⫾0.39†
10.09⫾1.09
r
0.99⫾0.007
0.98⫾0.004
0.97⫾0.017
0.99⫾0.001
0.99⫾0.002
S indicates Hamburger-Hamilton stage; N, normal; and LAL, left atrial ligation.
Mean⫾SE. *P⬍0.05 vs stage 27 normal; †P⬍0.05 vs circumferential.
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specimens reduced the microtubular density in both compact
and trabecular myocardium to the levels observed in control
embryos.
Discussion
Formation of the heart and blood vessels occurs within an
active biomechanical environment. During the period of
primary cardiac morphogenesis, the embryonic cardiovascular (CV) system operates at a peak systolic pressure of less
than 10 mm Hg and each of the parameters that influence
cardiac output have stage-specific optimized values.12 Despite relative structural immaturity, the embryonic heart
dynamically varies heart rate, preload, afterload, and viscoelastic properties influencing the final structure and function of the heart. Experimental models of congenital heart
disease in the chick embryo include left heart hypoplasia
produced by LAL,11,37 double outlet right ventricle produced
by altered venous return,38 and conotruncal defects produced
by neural crest ablation,39 and each has clinical correlates to
human anomalies. Previous studies have focused on the
changes in systolic properties that occur in these malformations,11,40 but our present study investigated changes in
passive material properties after LAL and identified an
increase in microtubules that may explain, in part, the
increased passive stiffness observed in the LAL LV.31 For the
low-pressure embryonic cardiovascular system, changes in
diastolic function may be a critical regulatory pathway during
morphogenesis of the heart.41
TABLE 2.
We assessed myocardial stiffness in the embryonic myocardium using uniaxial loading of specimens with biaxial
surface strain measurements and found that myocardial
stresses at given strains, circumferential myocardial stiffness,
and hysteresis loop area increased in response to reduced
mechanical load. This increase in passive stiffness of embryonic myocardium in response to reduced mechanical load is
in contrast to the response of the adult myocardium to
unloading.42,43 Rothen-Rutishauser et al44 observed distinct
differences in neonatal rat cardiomyocytes and adult rat
cardiomyocytes in their requirement for intact microtubules
in culture. They showed that adult cardiomyocytes lose a
certain degree of flexibility due to their longer adaptation to
specific situations in the heart, whereas neonatal cardiomyocytes can maintain and reassemble myofibrils even after
microtubules are destroyed. This adaptability to the environment may allow immature cardiomyocytes to remodel in
response to reduced mechanical load differently from the loss
of structural and functional integrity observed in unloaded
adult heart.42 Although LAL initially reduced LV loading, the
subsequent increase in passive stiffness after LAL more
closely resembled the changes in passive stiffness that occur
in adult myocardium in response to increased mechanical
load. We therefore investigated the role of microtubules in
our LAL model because increased microtubule content has
been associated with increased viscous damping in hypertrophied adult myocardium.21
Much work has been done defining the role of microtubules in pressure-overload hypertrophy models of the adult
Hysteresis Area Percent
Left Ventricle
S 27 N
S 27 NColchicine
S 27 NTaxol
S 27 LAL
S 27 LALColchicine
Circumferential
n
Hysteresis area percent
8
9
4
6
7
18.10⫾3.79
19.11⫾5.24
55.90⫾13.01*
37.30⫾9.20*
23.90⫾5.97†
Longitudinal
n
Hysteresis area percent
9
10
7
10
8
24.24⫾6.01
25.42⫾9.93
64.93⫾8.46*
42.83⫾4.96*
27.35⫾4.04†
S indicates Hamburger-Hamilton stage; N, normal; and LAL, left atrial ligation.
Mean⫾SE. *P⬍0.05 vs stage 27 normal; †P⬍0.05 vs untreated buffer.
Schroder et al
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Figure 2. Representative Western blot of total and polymerized
␤-tubulin before and after colchicine treatment for normal and
LAL left ventricular specimens. A 0.5-␮g standard of purified
tubulin is shown in the first Western lane. Corresponding summary data for total and polymerized ␤-tubulin before and after
colchicine treatment for normal (filled) and LAL (open) left ventricular specimens are directly beneath the matching Western
lane. *P⬍0.05 vs control.
heart.19,21,23–25,27,45 Increased microtubule content has been
suggested as one maladaptive mechanism that occurs in
pressure-overload hypertrophy,46 and data in the feline heart
support the view that microtubular stabilization is an important factor contributing to increased cytoskeletal stiffness and
contractile dysfunction.27 To date, however, very little has
been done to evaluate the role of microtubules in cardiac
morphogenesis and function. Microtubules have been shown
to be important regulators of cellular organization and myofibrillogenesis,47,48 and they mediate the transport of materi-
Figure 3. Representative photomicrographs of ␤-tubulin immunofluorescence in normal and LAL left ventricular specimens
before and after colchicine treatment. Note the increased density of microtubules in the compact myocardium after LAL. After
colchicine treatment, microtubule density appears similar to
control in both normal and LAL specimens. White double arrow
designates the compact myocardium. Scale bar⫽30 ␮m.
Microtubules in Embryonic Heart
357
als necessary for the production of new cellular structures.
Thus, it seemed reasonable to consider that microtubules may
play a role in mediating the adaptive response of the developing myocardium to altered mechanical load. Western blot
analysis and confocal images showed an increase in total
␤-tubulin content as well as an increase in polymerized
␤-tubulin present in the LV myocardium of LAL embryos
versus stage-matched controls. Colchicine treatment returned
protein levels to those observed in normal hearts. However,
the mechanisms that regulate tubulin synthesis and microtubule stabilization in response to altered mechanical stress
have not yet been defined.
We also noted a change in the viscous properties of the
embryonic myocardium after LAL, as assessed by hysteresis
loop area during stretch and release.21,34,35,49,50 This was
apparent as a decrease in force during release after stretch,
which resulted in an increase in loop area. This increase in
hysteresis loop area following LAL normalized after colchicine treatment and microtubule depolymerization. Taxol
treatment increased hysteresis loop area when compared with
control. Unlike changes that occur in vivo in response to
altered mechanical load (LAL), Taxol treatment in vitro
results in an unregulated hyperpolymerization of microtubules. These results are consistent with the association
between increased microtubule content and increased viscous
damping in adult myocytes from hypertrophied canine, feline,
and human heart failure specimens,20,21,27 although not all
laboratories concur with these results.23–25 The mechanism by
which increased microtubule density effects viscous properties is not well understood. In adult myocytes, microtubules
are concentrated in the perinuclear regions.47 When microtubular density increases, it occurs throughout all regions of the
cell, extending along the entire length of the sarcomere, thus
contributing to increased diastolic stiffness and viscous resistance. Our Western and confocal images show that microtubules form a reticular or randomly arranged network pattern
in the compact myocardium with an increase in both intensity
and density of microtubules in the LAL specimens. This
increase may contribute to both diastolic stiffness and viscous
resistance. Changes in the viscous properties of the developing myocardium could also impact diastolic function and
contribute to changes in ventricular filling patterns similar to
pathological states of diastolic dysfunction in the adult
heart.21,51–53
It is important to note that in the adult heart passive
elasticity is influenced by a wide range of intracellular,
cell-cell, and cell-matrix constituents including titin, desmin,
␣-actinin, collagen, microtubules as well as by myofilaments.21,23,54 Myofilament stiffness in the adult myocardium
is therefore determined by a large set of cytoskeletal components with a high degree of organization, which may minimize the effect of a change in one component, eg, microtubule content, on the measured stiffness of cellular
preparations from hypertrophied myocardium.21,35,51 However, the embryonic myocardium contains a significantly
lower content of organized collagen and a lower order of
cytoskeletal organization,1,55 perhaps explaining the increase
in passive stiffness associated with increased microtubule
content in LAL myocardium.56
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Circulation Research
August 23, 2002
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Cardiac morphogenesis occurs coincident with rapid cardiomyocyte clonal expansion.5,57 The compact myocardium
is a major source for dividing cardiomyocytes. Cell division
appears to cease as immature myocytes are recruited to form
mature trabeculae containing mature myocytes and Purkinje
cells.58 Confocal images showed that microtubules colocalized with f-actin in the compact layer of LAL embryos and
that the degree of myofilament organization appeared to be
accelerated in both the compact and trabecular layers in LAL
embryos versus controls. In addition to their critical role
regulating actin filament distribution during cell division,
microtubules have been shown to function as a temporary
scaffold to organize cellular components at early developmental stages.47 A lattice of actin filaments and microtubules
has been shown to permit the aggregation of myosin filaments in vitro.59 Sedmera et al57 recently showed that cell
proliferation was reduced in the LV after LAL. Further
experiments are needed to determine if the reduction in LV
mechanical load produced by LAL induces a reduction in cell
proliferation followed by increased microtubule synthesis and
accelerated cell differentiation, or vise versa.
Thus, this study is unique in that we have observed an
increase in microtubule density and ␤-tubulin protein after
reduced mechanical load in the embryonic heart produced by
a long-term reduction in mechanical load (LAL) associated
with decreased LV filling. Increased microtubule density was
associated with changes in both the viscous and elastic
components of LV passive properties in LAL chick embryonic myocardium and an acceleration of myofiber maturation.
Taken together, these data suggest a role for microtubules in
the response of the embryonic myocardium to altered mechanical load. Further research is needed to define the role
that microtubules play in translating regional changes in
ventricular mechanical load into changes in cardiac structure
and function.
Acknowledgments
This work was supported by an NIH individual NRSA No. F32
HL10200 (E. Schroder, PI) and NIH RO1 HL64626 (B. Keller, PI).
The authors would like to thank Jason B. Garrison for his technical
assistance.
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Microtubule Involvement in the Adaptation to Altered Mechanical Load in Developing
Chick Myocardium
Elizabeth A. Schroder, Kimimasa Tobita, Joseph P. Tinney, Jane K. Foldes and Bradley B.
Keller
Circ Res. 2002;91:353-359; originally published online July 18, 2002;
doi: 10.1161/01.RES.0000030179.78135.FA
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