Ventricular diastolic filling characteristics in stage-24 chick embryos after extra-embryonic venous obstruction
1 Department of Obstetrics & Gynecology, Erasmus MC, Rotterdam, 3000 DR,
The Netherlands
2 Department of Anatomy and Embryology, Leiden University Medical Center,
Leiden, 2300 RC, The Netherlands
3 Department of Pediatrics, University of Utah, Salt Lake City, UT 84113,
USA
* Author for correspondence (e-mail: n.ursem{at}erasmusmc.nl)
Accepted 26 January 2004
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Summary |
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Key words: chick embryo, atrioventricular function, blood flow velocity, venous clip model
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Introduction |
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We recently demonstrated that venous clipping has major acute effects on
hemodynamics in the stage-17 chick embryo
(Stekelenburg-de Vos et al.,
2003). For the total study period of 5 h, dorsal aortic blood flow
remains lower than baseline values, whereas heart rate shows a recovery to
baseline within 2 h of clipping
(Stekelenburg-de Vos et al.,
2003
). This dramatic reduction in total circulating blood volume
after clipping could influence the functional characteristics of the embryonic
heart. Since the early developing cardiovascular system is not yet innervated
(Pappano, 1977
),
cardiovascular function is sensitive to mechanisms or interventions that alter
hemodynamic load, such as in the venous clip model. In contrast to the mature
cardiovascular system, the embryonic heart lacks the ability to acutely alter
heart rate to compensate for reduced ventricular preload
(Casillas et al., 1994
). We
therefore hypothesize that the decreased blood volume flow observed in the
venous clip embryo would affect ventricular diastolic function.
Embryonic diastolic function can be investigated using pulsed-Doppler
measurements of blood velocity and flow. The combination of simultaneous
atrioventricular and dorsal aortic blood flow profiles accurately defines
passive and active ventricular filling volumes
(Hu et al., 1991). We studied
embryonic cardiovascular performance in the stage-24 chick embryo during
normal growth and development and after venous clip intervention.
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Materials and methods |
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Dorsal aortic blood flow velocity and atrioventricular (AV) blood flow
velocity were recorded using a 20-MHz pulsed-Doppler velocity meter (model
545C-4; Iowa Doppler Products, Iowa City, IA, USA). Dorsal aortic blood
velocity was measured with a 750-µm piezoelectric crystal positioned at a
45° angle towards the dorsal aorta at the level of the developing wing
bud. Internal aortic diameter was calculated from a magnified video image
displaying the dorsal aorta using a custom-built analysis program (IMAQ
Vision; National Instruments, Austin, TX, USA)
(Ursem et al., 2001).
Atrioventricular blood flow velocity was measured with a second crystal
positioned at the apex of the heart towards the AV orifice. The Doppler audio
signals were digitized at 24 kHz and stored on hard disk. Using complex fast
Fourier transform analysis, the maximum velocity waveform was reconstructed. A
more detailed description of this method has been published previously
(Ursem et al., 2001
). Passive
filling (P) was defined in the AV flow velocity waveform from end-systole to
the onset of the A-wave, and active filling (A) from the onset of the A-wave
to the onset of systole (Fig.
1). Portions of passive and active filling overlapped each other
at faster heart rates. The demarcation between the passive and active
velocities was dependent on heart rate but was most conspicuous as heart rate
slowed. Therefore, heart rate was decreased to 100 beats
min1 by cooling of the embryo. Although environmental
temperature directly influences hemodynamics
(Wispé et al., 1983
), a
slowed heart rate of approximately 100 beats min1 was
necessary to discriminate between the passive and active filling phase and to
study both groups under similar conditions. Cycle length was defined as the
time between consecutive beats obtained from the dorsal aortic velocity
waveform. Dorsal aortic and both passive and active AV velocity profiles were
integrated over time. Dorsal aortic blood flow, an estimate of cardiac output,
was calculated as the product of the integrated velocity curve and the
cross-sectional area of the dorsal aorta. Passive ventricular filling volume
equaled dorsal aortic stroke volume multiplied by the fraction of passive
filling area, and active ventricular filling volume equaled dorsal aortic
stroke volume multiplied by the fraction of the active filling area
(Hu et al., 1991
).
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Fifteen clipped and 15 normal embryos were measured at stage 24. For each embryo, we analyzed five consecutive cycles. The data are presented as means ± S.E.M., and a statistical analysis was carried out using an unpaired t-test. When data were not normally distributed according to the ShapiroWilk test, a logarithmic transformation was performed prior to establishing difference between the two study groups. Statistical significance was reached at P<0.05. Calculations were performed with SPSS 10.1 software (SPSS, Inc., Chicago, IL, USA).
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Results |
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Dorsal aortic blood flow and stroke volume were similar in the clipped and normal embryos at stage 24 (Figs 2, 3). In the clipped embryos, mean passive ventricular filling volume decreased by 53%, while mean active ventricular filling volume increased 33%, when compared with normal controls (Fig. 3). The passive filling volume accounted for 15% of the stroke volume in the clipped embryos and 33% in the normal embryos. The ratio of passive to active ventricular filling volume was significantly decreased in the clipped embryos compared with normal embryos (0.19±0.02 vs 0.50±0.04) (P<0.001).
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Discussion |
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Rerouting of venous inflow by permanent obstruction of the right vitelline
vein in the early chick embryo results in cardiovascular malformations:
ventricular septal defects, valve anomalies and pharyngeal arch artery
malformations (Hogers et al.,
1997). Results of our hemodynamic study revealed that during the
first 5 h after clipping, dorsal aortic blood flow is markedly decreased and
is unable to return to baseline values
(Stekelenburg-de Vos et al.,
2003
). However, dorsal aortic blood flow measurements obtained 24
h (stage 21) after clipping demonstrated similar results in clipped and
control embryos (S. Stekelenburg-de Vos, unpublished data). We suggest that
the reduction in blood flow for at least 5 h may begin a cascade of events
that results in the cardiovascular malformations observed later during
development.
Proper functional loading is essential for normal cardiac morphogenesis, as
the structure and function of the developing heart are intimately linked.
Subjecting the chick embryonic heart to mechanically altered loading
conditions modifies the myocardial architecture
(Sedmera et al., 1999).
Decreased mechanical loading of the left ventricle by left atrial ligation
results in reduced levels of proliferation in the left ventricular compact
layer and trabeculae (Sedmera et al.,
2002
). During normal maturation of the heart, myofibrils increase
in number and alignment and this will probably affect the myocardial
properties of the ventricle (Clark et al.,
1986
). In the same left atrial ligation model, Tobita et al.
(2002
) demonstrated an
increase in passive stiffness of embryonic myocardium in response to reduced
mechanical load. Microtubules, important regulators of cellular organization
and fibrillogenesis, seem to be associated with the response of the embryonic
myocardium to altered load. An increase in microtubular density and an
acceleration of myofiber maturation were observed in the embryonic heart after
altered mechanical load and were related to the increased passive stiffness
noted after left atrial ligation (Schroder
et al., 2002
).
Venous clipping also reduces mechanical load and is likely to modify the
myocardial architecture, and this may subsequently result in increased passive
stiffness of the embryonic myocardium. This is supported by morphologic
examination of venous clipped embryos, demonstrating that, in addition to
delayed cardiac looping and impaired cushion formation, the compact layer of
ventricular myocardium was thinner and ventricular trabeculation was reduced
(Hogers et al., 1998). These
morphological changes in developing myocardium could also have an impact on
diastolic function and contribute to observed changes in ventricular filling
patterns.
By contrast, data of atrioventricular inflow patterns after left atrial
ligation showed a reduced contribution of atrial contraction to ventricular
filling and a decreased peak velocity from stage 21 to stage 27
(Tobita and Keller, 2000). In
the left atrial ligation model, the decrease in volume load is chronic,
whereas in the venous clip model the reduction in volume load is temporary. We
therefore suggest that the embryonic ventricle responds to temporarily reduced
loading conditions by a change in ventricular passive properties, resulting in
a reduction in passive filling that is compensated for by an increase of
atrial contraction to maintain constant cardiac output.
Experimental disruptions of the venous return by vitelline vein obstruction
or left atrial ligation cause cardiovascular malformations
(Harh et al., 1973;
Hogers et al., 1997
). These
observations indicate that responses of cardiac tissue to altered
biomechanical forces, including blood flow and shear stress, are critical
determinants of cardiac development. Also, a role for hemodynamics in
modulation of shape and arrangements of endocardial cells in the embryonic
chick has been reported (Icardo,
1989
). In addition, cultured vascular endothelial cells rearrange
their cytoskeletal architecture and change their gene expression profiles in
response to flow-induced forces (Davies and
Tripathi, 1993
; Galbraith et
al., 1998
; Topper and
Gimbrone, 1999
; Yoshigi et
al., 2003
). Thus, experimentally induced flow alterations that
translate fluid shear stress to changes in gene expression are likely
candidate regulating mechanisms for the response of the developing heart to
reduced loading conditions. The hypothesis that fluid shear stress plays an
important role in embryonic cardiogenesis was recently substantiated by a
study performed in zebrafish embryos. This study describes the quantitative
in vivo analysis of intracardiac blood flow and shear stress in
zebrafish embryos. These data strongly suggest that shear stress forces play
some role in the regulation of embryonic cardiogenesis
(Hove et al., 2003
).
In conclusion, early venous obstruction results in altered diastolic ventricular filling of the stage-24 chick embryo. Our study supports the paradigm that alteration in mechanical loading is a mechanism that can produce changes in cardiac function and structure.
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Acknowledgments |
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References |
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