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Embryonic Heart Failure in NFATc1ⴚ/ⴚ Mice
Novel Mechanistic Insights From In Utero Ultrasound Biomicroscopy
Colin K.L. Phoon, Rui Ping Ji, Orlando Aristizábal, Diane M. Worrad, Bin Zhou,
H. Scott Baldwin, Daniel H. Turnbull
Abstract—Gene targeting in the mouse has become a standard approach, yielding important new insights into the genetic factors
underlying cardiovascular development and disease. However, we still have very limited understanding of how mutations affect
developing cardiovascular function, and few studies have been performed to measure altered physiological parameters in mouse
mutant embryos. Indeed, although in utero lethality due to embryonic heart failure is one of the most common results of gene
targeting experiments in the mouse, the underlying physiological mechanisms responsible for embryonic demise remain elusive.
Using in utero ultrasound biomicroscopy (UBM), we studied embryonic day (E) 10.5 to 14.5 NFATc1⫺/⫺ embryos and control
littermates. NFATc1⫺/⫺ mice, which lack outflow valves, die at mid-late gestation from presumed defects in forward blood flow with
resultant heart failure. UBM showed increasing abnormal regurgitant flow in the aorta and extending into the embryonal–placental
circulation, which was evident after E12.5 when outflow valves normally first develop. Reduced NFATc1⫺/⫺ net volume flow and
diastolic dysfunction contributed to heart failure, but contractile function remained unexpectedly normal. Among 107 NFATc1⫺/⫺
embryos imaged, only 2 were observed to be in acute decline with progressive bradyarrhythmia, indicating that heart failure occurs
rapidly in individual NFATc1⫺/⫺ embryos. This study is among the first linking a specific physiological phenotype with a defined
genotype, and demonstrates that NFATc1⫺/⫺ embryonic heart failure is a complex phenomenon not simply attributable to contractile
dysfunction. (Circ Res. 2004;95:92-99.)
Key Words: cardiac development 䡲 embryonic circulation 䡲 heart failure 䡲 NFAT 䡲 ultrasound biomicroscopy
D
espite the proliferation of mouse models with aberrant
cardiovascular development and subsequent in utero demise, the inability to characterize embryonic circulatory physiology has severely limited our mechanistic understanding of
their targeted gene defects. In numerous such models, “heart
failure” is the presumed cause of embryonic lethality (see Copp1
and Rossant2), but mechanisms remain largely unknown. Characterization of hemodynamic changes would provide a powerful
link between a given genotype, its associated structural phenotype, and its physiological phenotype.
Null mutant mice lacking the nuclear factor of activated T
cells c1 gene (NFATc1⫺/⫺) do not form aortic and pulmonary
(outflow) valves and die in utero between embryonic days (E)
13.5 to 17.5.3,4 Notably, they demonstrate no morphological
outflow tract abnormalities before E12.5, the approximate
initial stage of outflow valve development.5,6 Moreover, there
are no associated problems in the formation of ventricular
myocardium (trabeculation or compaction), coronary vascularization, vascular endothelium, or extracellular matrix, nor
is there evidence of ventricular or atrial chamber dilatation or
unusual hypertrophy.3,4 The valvular problems in NFATc1⫺/⫺
mice presumably lead to severe defects in forward blood flow
with congestive heart failure.3,4 Therefore, it has been assumed that NFATc1⫺/⫺ mice, similar to other models of
cardiac disease and heart failure, die of progressive contractile failure as the heart fails to pump adequate blood to
support the embryo.
We hypothesized that NFATc1⫺/⫺ mouse embryos might
exhibit multiple mechanisms of heart failure, not appreciated on
postmortem histology. We sought to characterize blood flow in
this model before and after the time of valve formation, in order
to achieve a better mechanistic understanding of embryonic
heart failure, by using detailed multiparameter analyses of in
utero embryos with ultrasound biomicroscopy (UBM)-Doppler
(see review7). Our results demonstrate that embryonic heart
failure is a complex phenomenon, not simply due to contractile
failure, and that different mechanisms may underlie even contractile failure itself.
Materials and Methods
Animals
All animals were maintained according to protocols approved by the
Institutional Animal Care and Use Committee at New York Univer-
Original received February 12, 2004; revision received April 26, 2004; accepted May 18, 2004.
From the Pediatric Cardiology Program (C.K.L.P.), Skirball Institute of Biomolecular Medicine (C.K.L.P., R.P.J., O.A., D.H.T.), and Departments of
Radiology and Pathology (D.H.T.), New York University School of Medicine, New York, NY; Division of Cardiothoracic Surgery (D.M.W.), Children’s
Hospital of Philadelphia, Philadelphia, Pa; and Division of Pediatric Cardiology (B.Z., H.S.B.), Vanderbilt University School of Medicine, Nashville,
Tenn.
Correspondence to Colin K.L. Phoon, MPhil, MD, Pediatric Cardiology Program, 530 First Ave, FPT Suite 9U, New York, NY 10016. E-mail
[email protected]
© 2004 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000133681.99617.28
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sity School of Medicine, Children’s Hospital of Philadelphia, and
Vanderbilt University School of Medicine. The NFATc1 gene was
disrupted in embryonic stem cells (ES) of balbc mice as previously
described; the ES clone line (141) used in this study exhibits 100%
impaired aortic and pulmonary valve development.4 The mice were
rederived in the Transgenic Mouse/ES Cell Chimera Facility within
the Skirball Institute of Biomolecular Medicine at New York
University School of Medicine (New York). Embryos staged between E10.5 and 14.5 were obtained from heterozygous mutant
intercrosses (note: E0.5 was defined as noon of the day vaginal plugs
were detected after overnight mating).
Image Acquisition and Doppler Interrogation
The mouse preparation and imaging platform, and acquisition and
processing of UBM-Doppler data have been described previously,
modified for semiinvasive imaging to readily identify embryos for
genotype-phenotype correlation.8 –10 Our imaging technique allowed
embryos to remain in their normal in utero environment at physiological temperatures and yields physiological data.10 In order to
avoid observer bias, all measurements of Doppler parameters and
UBM dimensions (see next section) were made before any knowledge of the genotype data. Data from any obviously injured embryos
were excluded.
Imaging Protocols and Doppler Waveform and
UBM Image Analysis
We first sought to characterize middorsal aortic blood flow from
E10.5 to E13.5 and collected data from 383 embryos. As we
developed facility with imaging litters, we also undertook, in a
second protocol, to concurrently characterize dorsal aortic blood
flow, cardiac ventricular size and function, and umbilical arterial/
venous blood flow, in order to characterize changes in cardiac
function as well as distribution of blood flow. For the second
protocol, we obtained data from an additional 205 embryos from
E12.5 to E14.5, because our data from the first protocol indicated
that flows were essentially normal at E10.5 to E11.5. Thus, dorsal
aortic data were available from both protocols (n⫽588), spanning
stages E10.5 to E14.5.
For each embryo, three consecutive Doppler waveforms were
analyzed and the data averaged. Various flow parameters were
measured in the mid-dorsal aorta and umbilical vessels, as previously
described (see Phoon et al7,8). UBM images of the heart in a
four-chamber view allowed assessment of ventricular size and
function7,11 (see also Figure 3). Because the UBM maximal frame
rate is 8/second and embryonic heart rate is 150 to 250/minute,
consecutive cycles do not show the heart at maximal end-diastolic
and minimal end-systolic volumes. Therefore, to improve assessment of ventricular function by UBM, we recorded diastolic and
systolic ventricular dimensions for at least 10 consecutive cycles,
and used the maximal and minimal values to obtain the end-diastolic
area and end-systolic area, respectively, and to derive the fractional
area shortening (⫽[end-diastolic area⫺end-systolic area]/[end-diastolic area]). Because blood is highly echogenic at the ultrasound
frequencies used,11 endocardial borders cannot be delineated. Therefore, and because cardiac septation is not yet complete at the stages
studied, epicardial biventricular areas were used as surrogates for
ventricular volumes.
The limitations of small embryonic heart size (approximately 1 to
2 mm in any one dimension), low temporal resolution of UBM, and
blood echogenicity precluded certain refined functional analyses
often used in clinical cardiology, such as assessment of regional wall
motion abnormalities.
Dorsal aortic diameter (middorsal aorta) and umbilical artery/vein
diameters were measured from 3 separate frames, and cross-sectional
areas calculated from the average diameter. Blood volume flow was
calculated according to a recently developed model.9
Genotyping
Genotyping of the yolk sac or tail DNA of each embryo was
performed using standard polymerase chain reaction techniques.4
Embryonic Heart Failure in NFATc1ⴚ/ⴚ Mice
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Histology
Paraffin-embedded embryos were sectioned (6 ␮m thick) and stained
with hematoxylin and eosin (H&E) as previously described.4
Statistical Analysis
All data are expressed as mean⫾SD. Comparisons between two
groups were analyzed with the two-tailed Student’s t test, for
parametric distributions with equal variances; or Mann Whitney U
test, for nonparametric distributions with unequal variances. Because
we did not rely on any single statistical difference in our data
interpretation but rather looked for trends across stages, we did not
use techniques such as Bonferroni’s correction for large numbers of
comparisons, but instead set statistical significance at P⬍0.01.
Results
Wild-Type and Heterozygote Embryos Exhibit a
Normal Physiological Phenotype
Ratios of living wild-type (NFATc1⫹/⫹), heterozygote
(NFATc1⫹/⫺), and null mutant (NFATc1⫺/⫺) embryos at E10.5
to E14.5 were consistent with previous data indicating in
utero lethality occurring largely between E12.5 through
E14.5 (data not shown).3,4 We first compared UBM-Doppler
data between NFATc1⫹/⫹ (n⫽168) and NFATc1⫹/⫺ (n⫽313)
embryos. Because NFATc1⫹/⫺ embryos are viable and fertile,
and demonstrate no histological abnormalities,3,4 wild-type
and heterozygote mutant embryos should exhibit similar
hemodynamics, which was confirmed (data not shown).
Therefore, we increased the power of the study by pooling
NFATc1⫹/⫹and NFATc1⫹/⫺ data (control group, n⫽481), and
compared them with their NFATc1⫺/⫺ littermates (n⫽107).
Distinctive Abnormal Blood Flow Is Observed in
NFATc1ⴚ/ⴚ Embryos
To maintain efficient, unidirectional, forward blood flow to
the body, the aortic and pulmonary valves prevent regurgitation of flow back into the heart during diastole. Therefore,
their absence should result in distinctive, diastolic flow
reversal (regurgitation) evident on the aortic blood flow
Doppler waveform; this regurgitant flow would represent an
abnormal volume load to the embryonic heart. Indeed, this
hallmark finding (Figure 1A and 1B) was seen in ⬎80% of
E13.5 to E14.5 embryos but only 19% of E12.5 and 3% of
E10.5 to 11.5 embryos. Because true valve formation normally occurs at approximately E13,5,6 the outflow tract
endocardial cushions, which appose dynamically in a valvelike manner before true valve development, appear to function normally in NFATc1⫺/⫺, as expected from previous
histological data.4 After E12.5, NFATc1⫺/⫺ regurgitant flow
worsened with advancing gestation (Figure 1C and 1D).
Because the flow reversal waveform returns to baseline by
the end of diastole (that is, there is no flow between the aorta
and the ventricles), equalization of pressures has occurred by
the end of diastole, evidence of “free” (unrestricted) regurgitation. Aortic blood flow patterns correlated with the histological phenotype (Figure 1E through 1G).
Cardiac Contractile Function Is Preserved in
NFATc1ⴚ/ⴚ Embryos
Despite the abnormal flow in NFATc1⫺/⫺ embryos, UBMDoppler parameters of systolic function showed no consistent
differences between control and NFATc1⫺/⫺ mouse embryos,
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July 9, 2004
Figure 1. NFATc1⫺/⫺ embryos displayed a hallmark physiological phenotype, reversed diastolic aortic flow, that did not occur in
the presence of aortic and pulmonary valves. A, Normal dorsal aortic Doppler flow waveform in a wild-type E13.5 embryo contrasted with B, representative E13.5 NFATc1⫺/⫺ Doppler waveform showing characteristic regurgitant flow (asterisks). C, Abnormal
flow reversal commenced at approximately E13 in NFATc1⫺/⫺ embryos, the stage at which outflow valves should normally
develop. Regurgitant flow volume, as indicated by the reverse velocity-time integral (VTI) as a percentage of forward VTI, worsened with advancing gestation, as expected. D, Increasing peak regurgitant flow velocities (REV) in the dorsal aorta also indicated
worsening flow reversal with advancing gestation (*P⬍0.0001, NFATc1⫺/⫺, open circles, compared with control data, filled circles).
Notably, peak forward arterial flow velocity (FWD) increased normally in NFATc1⫺/⫺, without any increase in variances. E, Representative H&E sections in the sagittal plane show normal aortic valve development (arrow) in a wild-type E13.5 embryo, whereas
the littermate NFATc1⫺/⫺ embryo with retrograde aortic flow shows absence of valve formation (F). NFATc1⫺/⫺ embryos appear to
develop normal myocardium and, histologically, fail to exhibit either ventricular or atrial chamber dilatation. The occasional
NFATc1⫺/⫺ embryo that exhibited unexpectedly normal aortic flow (without retrograde flow) appeared slightly less mature, with the
outflow tract guarded still by endocardial cushions (arrow) (G); these embryos therefore behaved similarly to those at E12.5. a
indicates atria; v, ventricles. Scale bar in E⫽500 ␮m. Data are presented as mean⫾SD.
Phoon et al
Embryonic Heart Failure in NFATc1ⴚ/ⴚ Mice
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Figure 2. Multiparameter UBM-Doppler
analyses indicated preserved systolic
function and contractile ability, but demonstrated significantly lower indices of
cardiac output after accounting for
reversed flow. A, There were no significant differences between NFATc1⫺/⫺ and
normal embryos in the Doppler time
intervals cycle length (CL), ejection time
(ET), acceleration time (AT), or deceleration time (DT); therefore, NFATc1⫺/⫺
heart rate increased normally from
184⫾31 bpm at E10.5 to 254⫾47 bpm
at E14.5. Also normal were the following:
(B) biventricular diastolic areas, a surrogate index of ventricular volumes, and
fractional area shortening (FAS); (C) forward aortic velocity-time integral (AVTI);
and (D) forward stroke volume. Cardiac
output (⫽stroke volume⫻heart rate) also
did not differ between NFATc1⫺/⫺ and
normal embryos (data not shown). Variances of control and NFATc1⫺/⫺ data
appeared similar. However, when we
accounted for the regurgitant flow by subtracting reversed from forward flow volume
indices, significantly lower flow volume
indices and worsening net flow volumes
with increasing gestation at E13.5 to 14.5
became apparent (E, net AVTI; F, net
stroke volume; net cardiac output data not
shown). Filled circles are normal data;
open circles are NFATc1⫺/⫺ data, for all
panels. *P⬍0.01.
including heart rate, UBM-derived fractional area shortening,
and multiple Doppler parameters of contractility and systolic
stroke work, even at E13.5 and E14.5 (Figures 1D and 2C
through 2F).
In the mature human heart, the volume load imposed on the
left ventricle by aortic regurgitation often results first in
augmented systolic function, followed by progressive systolic
deterioration.12 Therefore, it is possible that systolic dysfunction in NFATc1⫺/⫺ embryos might be obscured by hyperdynamic systolic function during data averaging. Arguing
against this was the absence of increased variance in any of
our averaged data (Figures 1D and 2A through 2D).
Embryonic Blood Flow Is Reduced in NFATc1ⴚ/ⴚ
Embryos, Despite Preserved Contractile
Cardiac Function
Although forward systolic flow [as measured by velocitytime integral (VTI), stroke volume, or cardiac output] is an
indicator of contractile function, the embryo depends on the
total, or net, amount of blood flow distributed around its body
for nutrition and growth. We therefore characterized the net
blood flow by subtracting retrograde flow from forward flow
parameters, because reversed blood flow is of no apparent
physiological benefit to the embryo. Adjusted mean flow
indices were significantly lower in NFATc1⫺/⫺ embryos than
in controls from E11.5 through E14.5 (Figure 2E and 2F).
Diastolic Dysfunction Is Rarely Observed in
NFATc1ⴚ/ⴚ Embryos
Volume overload of the ventricles in the failing human heart
often leads to elevated ventricular diastolic pressures, which
are reflected in the atria and venous vascular bed. Because the
resultant hydrostatic forces typically lead to edema, we would
expect the NFATc1⫺/⫺ embryo with regurgitant flow to
exhibit pericardial effusions and generalized edema, such as
seen in human fetuses with absent aortic and pulmonary
valves.13 Pericardial effusions are frequently seen in
NFATc1⫺/⫺ mouse embryos after death4 (Figure 3), and such
effusions have been the basis for the presumption of heart
failure in this and many other mouse models.14 However, in
vivo UBM revealed that NFATc1⫺/⫺ ventricles were not
abnormally dilated (Figure 2), consistent with prior histological data.3,4 Interestingly, we observed abnormal pericardial
effusions in only 3 of 107 living NFATc1⫺/⫺ embryos in
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July 9, 2004
Figure 3. Pericardial effusions typically
described for embryonic heart failure
were found rarely in the living embryo. A
and B, UBM images of a dead
NFATc1⫺/⫺ embryo, with large pericardial
effusion (arrows). C, Wild-type embryo
with normal, small to minimal amount of
pericardial fluid. D, Typical living NFAT
embryo, with normal, minimal pericardial
fluid. E, Representative image of an
abnormal, moderate pericardial effusion in
a living NFATc1⫺/⫺ embryo. a indicates
atria; v, ventricles.
which we obtained Doppler data (Figure 3); a fourth exhibited a moderate pericardial effusion, but with extremely poor
contractions on UBM and no blood flow discernible by
Doppler (data not shown). That we observed so few embryos
with effusions suggests that they are late, not early, conditions. Thus, the presence of frequent pericardial effusions
postmortem may be secondary to cellular deterioration and
endothelial leakage after death; they do not truly reflect in
vivo hemodynamics. Our results suggest a noncompliant
myocardium (which does not dilate in the face of a volume
load) but with normal relaxation properties (that allows
normal or near-normal filling pressures). Taken together, our
data argue that, although diastolic dysfunction contributes to
embryonic demise, it is not as severe as postmortem histology
has suggested.
Embryonal–Placental Blood Flow Is Altered in
NFATc1ⴚ/ⴚ Embryos
We next investigated the hypothesis that alterations in dorsal
aortic flow in NFATc1⫺/⫺ embryos also alter umbilical flows
and the distribution of blood flow between embryo and
placenta. Alterations in embryonal–placental flow would
potentially influence embryonic oxygenation status and might
therefore contribute to embryonic lethality.
First, we observed NFATc1⫺/⫺ reversed diastolic flow
extending into the umbilical artery (Figure 4A). As in dorsal
aorta, Doppler time intervals in the umbilical artery appeared
normal in NFATc1⫺/⫺ (data not shown). In control embryos,
the proportion of blood in the dorsal aorta that flowed to the
placenta decreased from ⬇90% at E12.5 to ⬇70% at E14.5.
However, in NFATc1⫺/⫺ embryos, the proportion of aortic
flow to the placenta increased slightly from E12.5 through
E14.5 (Figure 4B).
In the midgestation mouse embryo, umbilical venous blood
flow exhibits continuous and undulating flow with a nonzero
diastolic velocity, because of a relatively high placental
impedance with low capacitance that allows partial transmission of the umbilical arterial pulse; the venous pulsatility
(magnitude of undulation) decreases with advancing gestation as impedance falls.8 As expected at E12.5, umbilical
venous pulsatility was normal, in the context of normal
umbilical arterial flow without retrograde diastolic flow.
However, in E13.5 to E14.5 NFATc1⫺/⫺ embryos, umbilical
venous pulsatility was higher than normal (Figure 4C), due to
a reduced diastolic velocity that would be predicted by the
diastolic flow reversal in the umbilical artery. Thus,
NFATc1⫺/⫺ embryos failed to exhibit the normal decrease in
umbilical venous pulsatility with advancing gestation.
Abnormal NFATc1ⴚ/ⴚ Hemodynamics Do Not
Appear Secondary to an Abnormal Peripheral
Vascular Bed
It is possible that an abnormally developed vascular bed
contributes to abnormal blood flow parameters and embryonic demise. However, the intraembryonic vascular system
appeared normally developed, as demonstrated by normal
dorsal aortic calibers in both control and NFATc1⫺/⫺ embryos
(data not shown) and by qualitative observations of all
embryos, both in vivo with UBM and ex vivo after dissection.
Similar analyses showed that the embryonal–placental vascular system, yolk sac formation, and placentation appeared
normally developed (data not shown). Our observations are
consistent with previous immunohistochemical analyses that
vascular endothelium appears unaffected in NFATc1⫺/⫺
embryos.3,4
NFATc1ⴚ/ⴚ Embryos Die Acutely Without Chronic
Progressive Deterioration
Four NFATc1⫺/⫺ embryos were excluded from the pooled
analysis, because they displayed rapidly progressive deterioration or arrhythmia that prevented a valid analysis, which
requires that embryos exhibit stable (unchanging) hemodynamics. Two E13.5 embryos displayed a rapidly progressive
bradycardia and irregular rhythm that were immediately
preceded by normal hemodynamics (Figure 5). The progressive bradyarrhythmia was not sudden in onset, but accompanied changes in the Doppler waveforms that indicated systolic failure; therefore, arrhythmia did not appear to be a
primary event. These were the only two that exhibited
arrhythmias. In two E14.5 embryos, UBM demonstrated very
poor contractions, with minimal or no blood flow by Doppler.
Neither exhibited obvious arrhythmias, and only one showed
Phoon et al
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97
a moderate pericardial effusion. That we observed so few
NFATc1⫺/⫺ embryos with poor hemodynamics also suggests
that in most, death is not due to chronic, but more likely to
acute heart failure.
No obvious problems were noted with the coronary arteries
or the myocardium, as previously reported.4
Discussion
This study definitively demonstrates that the lack of aortic
and pulmonary valves in the NFATc1⫺/⫺ mouse embryo
results in abnormal circulatory flow patterns, not primary
myocardial dysfunction. Using UBM-Doppler, we have now
specifically linked the physiological phenotype (regurgitant
diastolic arterial flow) with the structural phenotype (lack of
outflow valves) and genotype (NFATc1⫺/⫺) in the in utero
embryo. Although a few previous ultrasound studies have
correlated abnormal blood flow patterns with an abnormal
genotype, the limitations of poor spatial resolution in those
studies precluded a mechanistic understanding of embryonic
heart failure.15–17 In a recent study, diastolic dysfunction was
linked to abnormal ventricular myoarchitecture in explanted
FOG-2 mutant mouse embryos.18 Our data, for the first time,
implicate multiple possible mechanisms for embryonic heart
failure, even in a single model. Embryonic heart failure thus
appears to be a complex phenomenon not simply attributable
to contractile dysfunction or “pump failure.”
Cardiovascular Hemodynamics
In human children and adults, severe aortic or pulmonary
regurgitation frequently leads to chronic and progressive
ventricular dilation, with myocardial fibrosis and progressive
systolic failure.12,19 However, our multiparameter UBMDoppler analysis surprisingly failed to reveal any chronic,
progressive deterioration in contractile function, despite the
abnormal volume load imposed on the developing heart.
Reduced cardiac output and diastolic dysfunction appeared
to contribute to embryonic heart failure. It is notable that very
few living embryos displayed pericardial effusions, so that
the frequent effusions seen on histology are perhaps due to
cellular deterioration and leakage postmortem. Interestingly,
our data suggested that indices of net volume blood flow
supplied to the embryo demonstrated abnormalities as early
as E11.5 (Figure 2E and 2F), even though (lack of) proper
valve formation does not occur until ⬇E13. This timing
coincides with the stage at which a tissue boundary, normally
established between invading neural crest– derived mesenchyme and transformed endocardial cell– derived mesenFigure 4. Abnormal hemodynamics were also present in the
embryonal–placental circulation. A, NFATc1⫺/⫺ embryos exhibited diastolic flow reversal in the umbilical artery in a temporal
pattern very similar to that seen in the dorsal aorta. Similar to
dorsal aorta, time intervals in the umbilical artery showed no
differences between NFATc1⫺/⫺ and control embryos at any
stage (data not shown), thus further corroborating aortic data
indicating preserved systolic function in NFATc1⫺/⫺ embryos. B,
However, the percentage of forward dorsal aortic flow coursing
to the placenta was distinctly different in NFATc1⫺/⫺ embryos
(*P⬍0.01, at E12.5 and E13.5). Indeed, NFATc1⫺/⫺ embryos
failed to exhibit the normal decrease in the percentage of aortic
flow coursing to the placenta as gestation advanced. C, Umbilical venous pulsatility index (ratio of [peak systolic⫺trough dia-
stolic] to [peak systolic] flow velocities) was normal at E12.5, but
increased at E13.5 (*P⬍0.001) and E14.5 (**P⬍0.005), and
therefore lacked the normal progression toward a less pulsatile
waveform as observed in control embryos. Because umbilical
venous flow represents the net amount of flow from the umbilical artery that courses through the placental circulation, this
composite index of umbilical venous flow velocities provided
further evidence for abnormal placental blood flow in NFATc1⫺/⫺
mouse embryos. At E13.5, both peak systolic and trough diastolic velocities were lower, whereas at E14.5, peak systolic
velocity was higher than control with a lower trough diastolic
velocity (data not shown). Filled circles are normal data; open
circles are NFATc1⫺/⫺ data, for all panels.
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July 9, 2004
Figure 5. Representative data from one actively dying
NFATc1⫺/⫺ embryo show normal Doppler hemodynamics very
shortly before rapid deterioration at E13.5. The embryo, even in
the course of acute and rapid deterioration, failed to exhibit any
significant pericardial effusion (data not shown). A, Doppler
waveforms appeared normal just before rapid deterioration, a
finding arguing against chronic progressive systolic dysfunction
as an etiology of embryonic heart failure. UBM-Doppler parameters such as acceleration time (53 ms), ejection time (140 ms),
peak velocity (96 mm/second), aortic VTI (9.26 mm), stroke volume (0.37 mm3), and fractional area shortening (0.44) were all
within the normal range. B, During the rapid deterioration phase
approximately 5 to 10 minutes later, arrhythmia was evident, as
was a worsening of parameters of contractile function, such as
peak velocity, ejection time, and ejection time as a proportion of
the cycle length. C, Within a few minutes of the onset of arrhythmia, the heart has now severely failed. For all panels, the
velocity and time scales are identical.
chyme in the region of the semilunar valves, fails to develop
in NFATc1⫺/⫺ embryos (B. Zhou and H.S. Baldwin, unpublished data, 2004). Given evidence for subtle changes in
mesenchymal differentiation, we speculate that UBM-Doppler may be detecting subtle changes in cardiovascular function before gross histological changes.
Our data also support the possibility that alterations in
placental blood flow, and therefore oxygenation, contribute to
embryonic heart failure, although the exact physiological
effects of the observed altered flow are unclear; we speculate
that the abnormal umbilical venous flow patterns may alter
cardiac filling as well. The embryo is exquisitely sensitive to
even minor perturbations in cardiac output,20,21 and we
speculate that any combination of these mechanisms may
contribute to NFATc1⫺/⫺ embryonic lethality. We did not find
evidence of chronic arrhythmias as a cause of heart failure in
this model.
The seemingly brief window of heart failure and demise
limits the possibilities: these include acute electrical events
(arrhythmias) that compromise cardiac output, and “cata-
strophic” coronary insufficiency causing acute myocardial
failure. Because there were no indications of the former, we
speculate that acute coronary insufficiency may account for at
least some deaths. In the developing embryo, the difference
between diastolic aortic and left ventricular pressures, which
provides the necessary pressure gradient for coronary perfusion, is small,22–24 and as discussed, free outflow tract
regurgitation would abolish the necessary coronary arterial
perfusion pressure head, impairing coronary flow. It is
possible that embryos exhibit regional wall motion abnormalities caused by myocardial ischemia that are not discernible
by UBM before their acute demise. Why embryos should die
at different time points despite similar hemodynamics is not
known. Nevertheless, the timing suggested by our data is
consistent with that observed in mouse models of aberrant
epicardial and coronary arterial development,25–27 and may
help to define, for the first time, the onset of coronarydependent myocardial performance in utero. Although coronary vascular development begins with extracardiac investment of the epicardium derived from the proepicardial organ
at E9.5, the myocardial requirement for coronary-derived
circulation clearly occurs much later in development. These
data suggest that such a requirement occurs between E13 and
E14 in the mouse. However, further studies are needed to
define the exact time point and molecular components of this
critical transition during in utero cardiac development.
Insights Into Embryonic Myocardial Mechanics
The moderate degree of regurgitant flow and the lack of
ventricular dilatation suggest a noncompliant embryonic
myocardium. Other work in both mice and human fetuses has
also suggested a relatively stiff myocardium early in development, which becomes more compliant as it matures.15,18,28
The NFATc1⫺/⫺ embryonic heart appears to be remarkably
robust, with death occurring more as an acute event (occurring over minutes), rather than secondary to chronic deterioration (occurring over hours to days). It is not at all clear how
the embryonic heart is able to regulate blood flow in the
presence of abnormal hemodynamics.
Relevance to Human Disease Conditions
Absence of either or both outflow tract valves is rare and is
lethal.13,29 However, the condition is very likely more common than observed, because the observed incidence of
congenital cardiac malformations is skewed by intrauterine
death.13,30 It is not known at this time whether mutations in
NFATc1⫺/⫺ result in human cardiac disease. However, the
NFATc1⫺/⫺ model remains valuable because it presents the
embryonic mammalian heart with abnormal volume loads
that lead to heart failure, but does not appear to be complicated by coexistent multiple problems typical of other targeted genetic models.
Limitations
Extrapolation of these results to other mouse models of
cardiac maldevelopment with heart failure must be made with
caution. The NFATc1⫺/⫺ mouse nevertheless has revealed the
complexity of embryonic heart failure, a novel paradigm that
now may be explored in other models as well. More detailed
Phoon et al
data on the time course of death require serial, longitudinal
evaluation of embryonic hemodynamics, techniques which
are currently under development. Finally, anesthesia may
affect embryonic cardiac function. However, the anesthetic
choice does not likely influence our data interpretation,
because we have an appropriate control group (wild-type and
heterozygote littermates) and because UBM-Doppler data
derived under similar anesthetic conditions appear more
physiological than previous results obtained under invasive or
less-well controlled conditions (see Ji et al10). Although
UBM-Doppler has revealed some of the physiological mechanisms in play in this model of embryonic heart failure,
clearly, the underlying mechanisms at the cellular level will
require further elucidation.
Conclusions
UBM-Doppler is a powerful approach for characterizing
cardiovascular physiology in the in utero mouse embryo,
linking the physiological phenotype with genotype. Not only
have we linked abnormal cardiovascular flow dynamics with
the underlying genotype, but we have also moved toward a
mechanistic understanding of embryonic heart failure. Our
results indicate that, similar to human heart failure (see
Auslender31), embryonic heart failure is a complex syndrome
comprised of multiple mechanisms that require precise delineation, in different models, if optimal therapy is ultimately to
be achieved.
Acknowledgments
This work was supported by American Heart Association (Heritage
Affiliate) Scientist Development Grant 0030333T and NIH grants
K08 HL-04414 (to C.K.L.P.), R01 NS-038461 (to D.H.T.), and P50
HL-62177 (to H.S.B. and D.H.T.). We thank Dr Michael Artman for
his valuable input and Dr Glenn Fishman for his critical review of
this manuscript.
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