Research ArticleEchocardiographic Assessment ofEmbryonic and Fetal Mouse Heart DevelopmentA Focus on Haemodynamics and Morphology

Nathan D Hahurij12 Emmeline E Calkoen12 Monique R M Jongbloed23

Arno A W Roest1 Adriana C Gittenberger-de Groot23 Robert E Poelmann2

Marco C De Ruiter2 Conny J van Munsteren2 Paul Steendijk3 and Nico A Blom1

1 Department of Pediatric Cardiology Leiden University Medical Center (LUMC) Leiden PO Box 9600 2300 RCLeiden The Netherlands

2Department of Anatomy amp Embryology Leiden University Medical Center PO Box 9600 2300 RC Leiden The Netherlands3 Department of Cardiology Leiden University Medical Center PO Box 9600 2300 RC Leiden The Netherlands

Correspondence should be addressed to Nathan D Hahurij ndhahurijamcuvanl

Received 26 August 2013 Accepted 31 December 2013 Published 23 February 2014

Academic Editors H M Chamberlin and D M Garrity

Copyright copy 2014 Nathan D Hahurij et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Background Heart development is a complex process and abnormal development may result in congenital heart disease (CHD)Currently studies on animal models mainly focus on cardiac morphology and the availability of hemodynamic data especially ofthe right heart half is limited Here we aimed to assess the morphological and hemodynamic parameters of normal developingmouse embryosfetuses by using a high-frequency ultrasound system Methods A timed breeding program was initiated with aWTmouse line (Swiss129Sv background) All recordings were performed transabdominally in isoflurane sedated pregnant micein hearts of sequential developmental stages 125 145 and 175 days after conception (119899 = 105) Results Along development theheart rate increased significantly from 125plusmn 95 to 219plusmn 83 beats per minute Reliable flow measurements could be performedacross the developing mitral and tricuspid valves and outflow tract M-mode measurements could be obtained of all cardiaccompartments An overall increase of cardiac systolic and diastolic function with embryonicfetal development was observedConclusion High-frequency echocardiography is a promising and useful imaging modality for structural and hemodynamicanalysis of embryonicfetal mouse hearts

1 Introduction

Heart development is a complex process during which theheart will form from a single myocardial heart tube to a fullyseptated four-chambered heart with functional atrioven-tricular (AV) and ventriculoarterial valves and a separatedoutflow tract The primary heart tube is derived from thesplanchnic mesoderm in the embryonic plate and initiallyconsists mainly of a left ventricle (LV) and AV canal Duringfurther development significant contributions to the hearttube will continue to be made from the mesenchyme situatedbehind the heart the so-called second heart field (SHF) Thecells of the SHFwill form the right ventricle (RV) and outflow

tract at the arterial pole of the heart as well as myocardial andvascular structures at the venous pole of the heart [1]

Abnormal heart development results in congenital heartdisease (CHD) the most common birth defect with an esti-mated incidence of six per 1000 live born children [2] In thelast decade knowledge regarding genes growth and tran-scription factors important in heart development has dramat-ically increased which has led to the generation of specificgenetically mutated mouse models to study the etiology ofCHD [3] Up till now description of both normal and abnor-mal heart development in these mouse models mainlyfocused on the morphology of the heart whereas functionaldata like hemodynamics and in vivo imaging are limited

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 531324 11 pageshttpdxdoiorg1011552014531324

2 The Scientific World Journal

Furthermore functional parameters and their course at sub-sequent stages of normalmouse heart development have onlybeen described in a few studies [4ndash7] Specifically knowledgeregarding RV inflow andmyocardial function during embry-onic development is scarce

In clinical practice ultrasound is themost frequently usedminimally invasive imaging modality at pre- and postnatalstages of heart development Clinical ultrasound machineshave also been used for imaging of embryonic mouse heartshowever ultrasound frequencies (8ndash15MHz) are not suffi-cient for a detailed morphological and hemodynamic anal-ysis Recently developed high-frequency ultrasound systems(30ndash50MHz) are able to offer sufficient spatial-temporal res-olution for detailed and reliable imaging of embryonicmousehearts Thus far there are only limited studies describingthe hemodynamic and morphological changes at subsequentstages of heart development using such ultrasound systems[4 6 8]

Hemodynamics play an important role in normal heartdevelopment by the activation of shear-stress dependentsignaling pathways During development genes like Klf-2 [9]and periostin [10] are expressed in regions of the heart that areknown to be subjected to high mechanicalhemodynamicalstress It has also been demonstrated that artificial alterationsin hemodynamics throughout heart development result inaberrant activation of genes at the endocardial surface of theheart [11] which might lead to disturbed cardiogenesis thatis CHD A first step in the interpretation of hemodynamicparameters during development is the acquisition of a reliablereference series during normal heart development

In the current study we aim to provide an overview ofhemodynamic andmorphological changes during embryonicand fetal stages of normal mouse heart development usinga high-frequency ultrasound system Knowledge of thesefunctional parameters is highly relevant for the interpretationof results obtained in genetically mutated mouse models Weexpect that knowledge of how echocardiographic parametersshift over development will eventually contribute to the earlydetection of CHD during human pregnancy

2 Material and Methods

21 Animals Animal experiments were approved by the localanimal welfare committee (DEC number 10177) of the LeidenUniversity Medical Center A timed breeding program wasinitiated with a WT (Swiss129Sv background) mouse lineThe day after breeding was considered to be 05 days afterconception (dpc) All pregnant mice (119899 = 11) were subjectedto high-frequency ultrasound recordings at 3 subsequentstages of embryonicfetal development being 125 145 and175 dpc At 125 dpc the pregnant mother mouse was anaes-thetized using isoflurane (induction 5 maintenance duringassessment 15) the most commonly used anesthetic inmouse experiments [6 12] Subsequently the sedated mousewas placed in the experimental setup (VisualSonics Vevo770system Toronto Canada) which includes a heated table andrectal probe for maintenance and control of body temper-ature (365ndash375∘C) Electrodes were attached to the paws

formonitoring thematernal electrocardiogram andheart andbreathing rate during the experiments

Abdominal hair was removed using a commerciallyavailable chemical hair remover followed by application ofultrasound transmission gel (Aquasonic Parker LaboratoriesFairfield NY USA) The ultrasound recordings were per-formed with a high-frequency ultrasound system (30MHzVisualSonics Vevo 770 system Toronto Canada) with anaxial resolution of 55 120583m a focal length of 127mm and amaximal field of view of 20mm Scanning time was keptas short as possible according to the ALARA principle [13]Between the three subsequent developmental stages that wereassessed the pregnant mice were housed in cages in theanimal facility

22 Ultrasound Recording Protocol All ultrasound record-ings were performed by NDH and EEC During the ultra-sound recordings the two laterally located uterus horns werescanned for the presence and position of the embryosfetuses(119899 = 105) In individual embryosfetuses the left and rightside were established to identify the position of the heartincluding the individual cardiac compartments and struc-tures At 125 dpc these compartments included the left atrium(LA) right atrium (RA) LV RV the common AV canal(cAVC) encompassing the future mitral valve (MV) andtricuspid valve (TV) orifices common outflow tract (cOFT)developing interventricular septum (IVS) and the interven-tricular foramen that is the continuity between the LV andRV before completion of ventricular septation At later stages(145 and 175 dpc) the MV and TV orifices aorta (Ao) andpulmonary trunk (PT) were identified

23 Pulsed-Wave Doppler Recordings Themethods that wereused for pulsed-wave Doppler flow recordings are summa-rized in Table 1 For flow measurements automatic Dopplerangle corrections were accepted up to 45 degrees Flowrecordings across the cAVC (125 dpc future MV 119899 = 1future TV 119899 = 11) and developing MV and TV orifices(145 dpc MV 119899 = 17 TV 119899 = 20 175 dpc MV 119899 = 16 TV119899 = 14) were performed to assess the peak-E (earlypassiveventricular filling by ventricular relaxation in early diastole)and peak-A wave velocities (active ventricular filling due toatrial contraction) hence the EA ratios were calculated Therecordings across the MV orifice cAVC and developing werealso used to evaluate the embryonic heart rate (HR) in beatsper minute (BPM) cardiac cycle length (RR interval) dias-tolic ventricular filling time (DFT 125 dpc 119899 = 11 145 dpc119899 = 17 and 175 dpc 119899 = 16) ejection time (ET 125 dpc119899 = 11 145 dpc 119899 = 17 and 175 dpc 119899 = 16) isovolumetriccontraction time (IVCT 125 dpc 119899 = 7 145 dpc 119899 = 17 and175 dpc 119899 = 16) and isovolumetric relaxation time (IVRT125 dpc 119899 = 7 145 dpc 119899 = 17 and 175 dpc 119899 = 16) of theLV (Figure 1(a)) The myocardial performance index (MPI125 dpc 119899 = 5 145 dpc 119899 = 17 175 dpc 119899 = 16) also knownas Tei index evaluates the combined LV systolic and diastolicfunctionwas calculated using the following formula (IVRT+IVCT)ET [14]

Pulsed-wave Doppler recordings were also performed inthe cOFT at 125 dpc (119899 = 15) and Ao and PT at 145 dpc

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Table 1 The methods used for performing Doppler flow recordings per gestational age

Age (dpc)andalignment

Location Parameter (unit) View Doppler beam position

125Future MV

E-wave (ms)A-wave (ms)DFT (ms)IVCT (ms)IVRT (ms)ET (ms)

4 chambers cAVC parallel to flow direction at left side ofthe developing IVS

Future TV E-wave (ms)A-wave (ms) 4 chambers cAVC parallel to flow direction at the right

side of the developing IVS

cOFT future AoPT Peak flow (ms)VTI (mm) 5 chambersOFT Proximal part cOFT parallel to flow direction

145 and 175

MV

E-wave (ms)A-wave (ms)DFT (ms)IVCT (ms)IVRT (ms)ET (ms)

4 chambers LV below (developing) MV annulus parallel toflow direction at left side of the IVS

TV E-wave (ms)A-wave (ms) 4 chambers RV below (developing) TV annulus parallel to

flow direction at right side of IVS

Ao Peak flow (ms) 5 chambersLVOT Ao above (developing) valve annulus VTI(mm) parallel to flow direction

PT Peak flow (ms) RVOT PT above (developing) valve annulus parallelto flow direction

(119899 = 18 and 119899 = 12) and 175 dpc (119899 = 12 and 119899 = 4 Table 1)Furthermore the velocity time integral (VTI) that is the areaunder the Doppler velocity envelope for one heart beat wasdetermined (125 dpc 119899 = 15 145 dpc Ao 119899 = 18 and PT119899 = 12 175 dpc Ao 119899 = 12 and PA 119899 = 4) and the diametersof the cOFT (119899 = 2) and Ao (145 dpc 119899 = 3 and 175 dpc119899 = 2) were evaluated from B-mode echoloops in order tocalculate the mean stroke volume (SV) per developmentalstage according to the following formula mean SV = meanTVI times (mean diameter of Ao or cOFT2)2 times 120587 Hence themean cardiac output (CO) was calculated (mean CO =meanSV timesmean HR) for the three developmental stages

24 M-Mode Measurements For morphological assessmentM-mode measurements in short- or long-axis views of theventricles were performed to evaluate the end-systolic andend-diastolic ventricular inner diameter of the LV (LVID125 dpc 119899 = 9 145 dpc 119899 = 11 and 175 dpc 119899 = 14) andRV (RVID 125 dpc 119899 = 9 145 dpc 119899 = 10 and 175 dpc119899 = 12) and IVS diameter (125 dpc 119899 = 4 145 dpc 119899 = 6 and175 dpc 119899 = 9) Cardiac fractional shortening (FS) for theLV and RV was calculated using the formula (VIDdiastole minusVIDsystole)VIDdiastole times 100 Due to the size of thehearts at 125 dpc the resolution often limited the possibilityto discriminate the epicardial myocardial and endocardiallayer of the ventricular free wall Therefore at this stage thedistances from the epicardial layer to the IVS in end-systoleand end-diastole were used to calculate the FS

Although a complete hemodynamic and morphologi-cal assessment for each heart was aimed for (Table 2 for

overview) imaging possibilities were regularly limited by theposition of the embryofetus All echocardiographic record-ings were stored for offline data analysis by NDH and EECinterobserved analysis demonstrated a high level of consis-tency (119903 = 08ndash10 119875 lt 005) Flow parameters were calcu-lated by the average of pulsed-wave Doppler complexes ofthree consecutive beats similar methods were applied forM-mode recordings and diameter calculations on B-modeecholoops

25 Cardiac Morphology and Three-Dimensional Reconstruc-tions After the experiments the pregnant mouse was sac-rificed by cervical dislocation and embryosfetuses wereextractedWe refer to previous publications for details regard-ing embryonicfetal processing sectioning immunohisto-chemistry with MLC2a specific antibodies and preparationof the three-dimensional AMIRA reconstructions (AMIRAsoftware package Template Graphics Software San DiegoUSA) [15]

26 Statistics All statistics were performed with the statisti-cal package for the social sciences 150 (SPSS Inc Chicago ILUSA) andGraphPadPrism (GraphPad Software La Jolla CAUSA) A 119875 lt 005 (2-tailed) was considered to be significantall values are given as mean plusmn SEM All boxplots indicate theupper and lower quartile of the median which is indicatedby the horizontal bar The whiskers indicate maxmin rangeof the values

4 The Scientific World Journal

lowastns

ns80

60

40

20

125 145 175

ET (

)

(c)

40

20

0

minus20

minus40

Velo

city

(cm

s)

10

0

minus10

Freq

uenc

y (k

Hz)

7000 8000 Time (ms)

ECG 230mVResp 230mV

(a)

lowast

nsns80

60

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DFT

()

125 145 175

(b)

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15

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nsnsns

IVCT

()

125 145 175

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(d)

lowast

lowast20

15

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ns

125 145 175

Age (dpc)

IVRT

()

(e)

1

23

4

5

(1) Cycle length (RR)

(2) Diastolic filling time (DFT)

(3) Isovolumetric contraction time (IVCT)

(4) Ejection time (ET)

(5) Isovolumetric relaxation time (IVRT)

(a998400)

(a998400)

Figure 1 Development of LV inflow patterns (a) Shows an example of a pulsed-wave Doppler recording across the MV at 175 dpc (a1015840)Indicates the magnification of the boxed area in (a) in which the individual time intervals are indicated that is RR DFT IVCT ET andIVRT (b)Through (e) indicates the course of the DFT ET IVCT and IVRT respectively throughout embryonicfetal life All values in (bndashe)are expressed as percentage of the RR

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Table 2 Summary of the assessment of embryonic and fetal hearts

Method Parameter Unit Structure (abbreviation)

2D ultrasoundB-mode echoloops

Morphological assessmentposition and diameters ofcardiac compartments

mm

Right atrium (RA) left atrium (LA) right ventricle (RV) left ventrile (LV)common outflow tract (cOFT) pulmonary trunk (PT) aorta (Ao)inter ventricular septum (IVS) common atrioventricular canal (cAVC)mitral valve (MV) and tricuspid valve (TV)Definition Formula

Pulsed-waveDoppler

Peak E ms Early diastolic ventricular fillingPeak A ms Active diastolic ventricular fillingEA ratioRR interval ms Cycle lengthevaluation of heart rateDFT ms Diastolic filling timeIVRT ms Isovolumetric relaxation timeIVCT ms Isovolumetric contraction timeET ms Ejection timeMPITei index Myocardial performance index IVRT + IVCTETPeak velocities cOFT AoPT ms

VTI mm Velocity time integral

SV Stroke volume SV = TVI times (Ao orcOFT diameter2)2 times 120587

CO mLmin Cardiac output

M-mode

VID mm Ventricular inner diameterLVIDd mm End diastolic left ventricular inner diameterLVIDs mm End systolic left ventricular inner diameterRVIDd mm End diastolic right ventricular inner diameterRVIDs mm End systolic right ventricular inner diameterIVSd mm End diastolic interventricular septum diameterIVSs mm End systolic interventricular septum diameter

FS Fractional shortening (VIDdminusVIDs)VIDd times100

3 Results

31 Cardiac Morphology Reliable imaging of hearts as smallas 2mm was feasible in all stages examined Representativeexamples are shown in online Movie 1 (see SupplementaryMaterial available online at httpdxdoiorg1011552014531324)

At embryonic stages (125 dpc) ventricular septation hasbeen initiated but is not completed resulting in a primitiveinterventricular foramen that directly connects the lumen ofLV and RV M-mode recordings at these stages show a meansystolic IVS diameter of 020 plusmn 0045mm Figure 2(a) showsa reconstruction of a 125 dpc embryonic heart in which thelumen of the four cardiac chambers interventricular fora-men and theOFT can be identifiedThe atrial and ventricularchambers are connected via the cAVC that harbors large AVcushions which will contribute to formation of the MV andTV The AV canal is still partly positioned above the LV atthis stage The developing OFT is still positioned completelyabove the RV and contains cushion tissue that will form thesemilunar valves of the Ao and PTThey will further form thebasis for the muscular subpulmonary infundibulum and will

contribute to themembranous part of the IVS Consequentlythe blood from the LV runs via the primitive interventricularforamen into the cOFT that is future Ao and PT

At early fetal stages of 145 dpc the heart has increased insize (Figure 2(b)) and ventricular septation is now completeThe systolic diameter of the IVS has increased (024 plusmn0018mm) In the AVC the TV orifice has expanded and isnow situated entirely above the RV Also the MV orifice canbe identified as well as the developing AV valves The cardiacOFT now consists of a separate Ao and PT connecting to theLV and RV respectively The Ao and pulmonary valves stillshow immaturity and consist of cushion tissue

At late fetal stages (175 dpc) the size of the RV andLV and IVS (039 plusmn 0009mm) has increased significantly(Figure 2(c)) Mature valves are present at the levels of the in-and outflow parts of both ventricles

32 Diastolic and Systolic Function During embryonic andfetal development theHR increases significantly (119875 lt 00001)from 125 plusmn 95 up to 219 plusmn 83BPM Ventricular inflowpatterns across the developing MV and TV annulus werestudied by pulsed-wave Doppler imaging

6 The Scientific World Journal

(a) (b) (c)

lowast

lowastlowastlowast150

125

100

075

050

025

000

Dia

met

er lu

men

(mm

)

Left ventricle

Right ventricle

Diastole

Systole

125 145 175

Age (dpc)

ns

(d)

lowast

125 145 175

Age (dpc)

50

40

30

20

10

0

Shor

teni

ng fr

actio

n (

)

Left ventricle

Right ventricle

(e)

Figure 2 Cardiac morphology and FS calculation (a) Shows a reconstruction of the anterior view of an embryonic heart of 125 dpc Themyocardium is indicated in grey transparent The LA and RA are indicated in transparent dark grey The LV and RV lumen are indicated inred and blue respectively Note that the outflow tract lumen (purple) is positioned completely above the future RV which is surrounded bylarge outflow tract cushions (green transparent) At these stages development of the IVS has not yet completed leading to a direct connectionbetween the LV and RV via the interventricular foramen (arrow) (b) Anterior view of an early fetal heart of 145 dpc At this stage IVSdevelopment has been completed and four separate cardiac chambers can be identified The outflow tract consists of a separate Ao and PTincluding their valve apparatus which at these stages mainly consist of cushion tissue (green transparent) (c) Anterior view of a late fetalheart of 175 dpc At this stage the heart shows a mature morphological phenotype (d) Schematic representation of LV and RV diameters and(e) FS at the three consecutive stages of development

The DFT corrected for the RR interval (DFTRR) did notdiffer significantly at the three stages assessed (Figure 1(b))LV and RV filling were dominated by the peak-A waveBetween 125 and 175 dpc the peak-A wave of the developingMV annulus increased significantly (033 plusmn 0001ms versus042 plusmn 0014ms 119875 lt 00001) whereas that of the TV alsoshowed an increasing trend albeit not significant (037 plusmn0018ms versus 041 plusmn 0019ms 119875 = NS) The peak-Ewave also increased significantly for both LV (005plusmn0006msversus 018plusmn0005ms 119875 lt 00001) and RV (006plusmn0006msversus 018 plusmn 0012ms 119875 lt 00001) and showed a relativelygreater increase than the peak-A wave during development

As a result the EA ratio a measure for the ventricular dia-stolic function showed a significant increase for both LV(016 plusmn 0015 versus 044 plusmn 0013 119875 lt 00001) and RV(016 plusmn 0010 versus 044 plusmn 0014 119875 lt 00001) (Figures 3(a)ndash3(f))

The IVRT one of the measures for diastolic LV functioncorrected for the RR interval (IVRTRR) demonstrated asignificant (119875 = 0037) decrease between 125 (015 plusmn 0011)and 145 dpc (012 plusmn 0004) and remained stable at later fetalstages 175 dpc (012 plusmn 0003) (Figure 1(e)) indicating that LVmyocardial relaxation remains stable after completion of IVSdevelopment during fetal life

The Scientific World Journal 7

Mitral06

05

04

03

02

01

00

EA

ratio

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

125 145 175

(a)

06

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EA

ratio

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

125 145 175

Tricuspid

(b)

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125 145 175

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E (m

s)

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(c)

lowastlowastlowast

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Peak

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07

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(e)

125 145 175

Age (dpc)

Peak

A (m

s)

07

06

05

04

03

02

01

00

nsns

ns

(f)

Figure 3 Development of LV and RV inflow patternsThe graphs represent the course of the EA ratio (a b) peak-E wave (c d) and peak-Awave (e f) across the developing MV and TV at the three subsequent developmental stages

The systolic function of the developing ventricles wasassessed through studying the course of the FS IVCTRRand ETRR Throughout embryonic and fetal developmentthe FS remained constant for both ventricles and valuesranged between asymp30 and 40 Furthermore except for175 dpc (LV 406 RV 357 119875 = 0046) no significantdifferences were observed between the FS of the LV and RV

(Figures 2(d) and 2(e)) The mean IVCTRR remained stableduring all three stagesasymp009 the ETRR showed no changesbetween 125 dpc (042plusmn0015) and 145 dpc (043plusmn0010) butsignificantly decreased at 175 dpc (040 plusmn 0006 119875 = 0017)(Figures 1(c) and 1(d))

A combined assessment of LV systolic and diastolic func-tion was performed by calculating the MPI At embryonic

8 The Scientific World Journal

Table 3 The parameters used for calculation of the mean CO per gestational age

Parameter 125 dpc (cOFT) 145 dpc (Ao) 175 dpc (Ao)VTI (mm) 339 plusmn 137 423 plusmn 20 292 plusmn 205

Mean diameter (mm) 023 018 048 plusmn 003

Mean HR (bpm) 134 plusmn 112 148 plusmn 61 206 plusmn 98

Mean CO (mLmin) 019 016 096

lowastlowastlowast05

04

03

02

01

Peak

flow

(ms

)

Common OFT

Aorta

Pulmonary trunk

125 145 175

Age (dpc)

Figure 4 Pulsed-wave Doppler flow measurements in the cOFTAo and PT The graph demonstrates the significant increase of thepeak blood flowmeasured in the cOFT at 125 and Ao and PT at 145and 175 dpc

preseptated stages (125 dpc) the MPI was 064 plusmn 0012 anddecreased to 049 plusmn 0017 (119875 = 00002) after closure of theIVS at 145 dpc At late fetal stages (175 dpc) the MPI did notchange significantly (053 plusmn 0022 119875 = NS)

33 OutflowTract andCO Thepulsed-waveDoppler record-ings performed in the cOFT demonstrated a peak flow of027 plusmn 0021ms at preseptated stages As from 145 dpc twoindividual ventricular outflow tracts could be discriminatedespecially the high echodensity of the blood at these stagesenabled to discriminate the flow in the Ao (034 plusmn 0013ms)and PT (033 plusmn 0021ms) that are closely related to eachother As can be observed in Figure 4 the peak flow increasedsignificantly in the Ao (from 027 plusmn 0021ms to 036 plusmn0016ms 119875 = 0010) and PT (027 plusmn 0021ms to 044 plusmn0030ms 119875 = 0003) along fetal life

Table 3 summarizes theHRVTI and the diameters of thecOFT and Ao that were used to calculate the mean CO at thethree developmental stages Between 125 dpc and 145 dpc the

CO remained stable where after it increased to 096mLminat 175 dpc

4 Discussion

Heart development is a complex process which has beenextensively studied in both WT and genetically mutatedmouse models [3] It is only in the last decades that modernimaging techniques like ultrasound enable the assessmentof real-time hemodynamic and morphological changes thatoccur during mouse heart development [4ndash6 8 16ndash18]Knowledge regarding these functional parameters is essentialsince abnormal flow in the developing heart might lead toCHD through aberrant activation of shear-stress responsivegenes important in cardiogenesis [9 11] In early reportsdescribing the course of functional parameters during heartdevelopmentmajor variability exists inmeasured valuesThisis most likely related to the relatively low ultrasound frequen-cies of clinical ultrasound machines used to image the smallhearts of mouse embryosfetuses [16] and externalizationof uterus horns during ultrasound assessment [18] whichinfluences embryonicfetal physiology Low dose isofluraneanesthesia used in this study only has a minimal effect onhemodynamics and diastolic function in adult mice and theeffect on mouse embryos is supposed to be marginal [6]

In the current study we aimed to provide an overview ofhemodynamic parameters and changes that occur at subse-quent stages of heart development using a high-frequencyultrasound system

Compared to other reports in mouse using similar ultra-sound techniques we did not only focus on development ofthe future LV but also on that of the RV Key findings ofour study are (1) a reliable assessment of LV and RV in- andoutflow patterns can be performed from 125 dpc to term (2)a significant improvement occurs in the diastolic and systolicfunction of both LV and RV (3) over the developmentalperiod of 125 dpc to 175 dpc the ratio between RV and LVsize as well as the FS of both ventricles did not changesignificantly

Knowledge regarding the functional parameters of theRV is important since the RV is commonly affected in CHDDuring development the RV including the TV annulusdevelops subsequent to the LV by addition of SHF derivedcells to the developing heart [1] Many genes have beenidentified that have an important role in the SHF contributionto the developing heart andmutations in these genesmay leadto CHD [19]

At early embryonic stages endocardial cushion tissue ispositioned in the primitive AVC and OFT which later on

The Scientific World Journal 9

will contribute to formation of the TV and MV and Ao andPT semilunar valves Some clinical studies argued that AVvalve regurgitation is a commonphenomenon during the firsttrimester of pregnancy [20] Our study in accordance withseveral others in mouse [4 6] and human [13] did not showsigns of regurgitation at both early and late stages of heartdevelopment Pulsed-wave Doppler recordings across thecAVC at 125 dpc and MV and TV at 145 dpc demonstratedthat the AV cushionsmimic the function of normal AV valvesat 175 dpc In our opinion especially the flow in the cOFTat early embryonic stages which is completely positionedabove the RV (see Figure 2(a)) might wrongly be identifiedas regurgitation of the developing AV valvescushions Thisstresses the importance of detailed knowledge regarding therapid morphological changes that the heart undergoes atespecially the very early developmental stages

Regarding growth of the heart we demonstrated that therewas a 31 increase of LVID 34 of RIVD and 35 of IVSdiameter from 125 up to 175 dpcWe showed a trend towardsa smaller RVID as compared to the LVID at all stages whichwas not mentioned in other studies

41 Diastolic and Systolic Function during Heart DevelopmentImprovement of ventricular systolic and diastolic functionoccurs throughout the length of embryonic and fetal develop-mentWith respect to the diastolic function we demonstrateda significant increase of EA ratio across the developing MVand TV orifices which was related to an increase of the peak-E wave that is the fraction of diastolic ventricular filling thatis defined by ventricular relaxation These data are in accor-dance with the study of Zhou and colleagues who demon-strated that the EA ratio is increasing during developmentwhich seems to continue up to approximately 3 weeks afterbirth [6] Around day four after birth the peak-E wave mea-sured across theMV becomes more dominant than the peak-A wave indicating an improvement of cardiac complianceand that LV filling becomes dominated by passive ventricularrelaxation Remarkably these changes were not observed forthe RV inflow patterns where the peak-A wave remainsdominant [6] Human embryonic and fetal hearts show thesame maturation course of EA ratios across the developingMV and TV as was demonstrated in multiple studies [21ndash25]An abnormal course of EA ratio that is a decreasing[25 26] or an increasing trend [27] along development hasbeen related to the presence of CHD

With respect to systolic ventricular function the FSremained stable (asymp30ndash40) for both ventricles throughoutdevelopmentWe did find a significant difference favoring theLV FS as compared to the RV FS at 175 dpc Interestinglythe same phenomenon of FS difference between LV andRV has been reported in a study of human fetal heartsbetween 14 and 40weeks of pregnancy However as inmousedifferences between humanLVandRVFS is subtle [28] andwhether a decreased or decreasing FS is a predictor of CHDremains unknown Furthermore we did not find a decreasingtrend of both LV and RV FS as was recently demonstratedduring human fetal life [28 29] With respect to the CO wenoticed no change around the proces of ventricular septation(between 125 and 145 dpc) where after a major increase

occured to 096mLmin near term (175 dpc) The initial COdecreasemight be related to the fact that themeandiameter ofthe unseptated cOFT diameter was used at 125 dpc comparedto the isolated Ao diameter at 145 dpc The subsequent COincrease is mostly related to the dramatic increase of HRalong fetal life

The MPI or Tei index [14] is a measure for combinedsystolic and diastolic cardiac function and can be calculatedfor both ventricles In order to assess the MPI three differenttime intervals are needed the IVCT IVRT and ET For the LVthese parameters can be easily obtained since all the threeparameters can be assessed at once by placing the Dopplerultrasound beam across the developing MV orifice Notice-ably in literature there is less data regarding RV MPI andits course along development which might be related to thefact that for the RV not all the three time intervals can beassessed in one view as was also suggested in a recent reviewby Godfrey et al [14] The current study demonstrated asignificant decrease of LV MPI between 125 and 145 dpcwhich might be related to the rapid morphological changesthe heart goes through including completion of IVS leadingto altered hemodynamic conditions in the developing heart Itis however also important to notice that at preseptation stagesthe IVCT and IVRT cannot always be identified separately sothat calculation of the MPI is frequently not possible at veryearly stages of embryonic development Between 145 and175 dpcwenoticed a slight increase of theMPI suggesting thatcardiac function improves at subsequent fetal stages similarresults have also been demonstrated inmouse [4] and human[30] However discussion remains regarding the exact courseof theMPI IVCT IVRT andETandwhether isolated changesof one of the factors is a predictor for fetal demise and CHDInterestinglywith respect to IVRTweobserved a decreasingtrend near completion of the IVS (between 125 and 145)whereafter it more or less remained stable throughout fetallife A similar course was observed in a recent ultrasoundstudy in embryonicfetal C57Bl6 mice [4] Human embryosalso showed a decreasing course of the IVRT [13 21] and anelevated IVRT has been implicated as a predictor of CHD[13]

5 Conclusion

We demonstrated the course of several morphologic andhemodynamic parameters along embryonic and fetal lifeOur data show that a reliable assessment can be made notonly of the left but also of right ventricular function duringembryonicfetal stages We demonstrate an overall improve-ment of cardiac diastolic and systolic function of both LV andRV along heart development The implementation of high-frequency ultrasound in embryonicfetal mouse heart devel-opment is a promising and useful tool The data presented inthis studymight be useful as reference values in future studiesin genetic mutated mouse models of CHD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

10 The Scientific World Journal

Authorsrsquo Contribution

Nathan D Hahurij and Emmeline E Calkoen contributedequally to this work and shared first authorship

Acknowledgment

This work is supported by the DutchHeart Foundation Grantno 2009T070 (Nathan D Hahurij)

References

[1] R G Kelly ldquoMolecular inroads into the anterior heart fieldrdquoTrends inCardiovascularMedicine vol 15 no 2 pp 51ndash56 2005

[2] J I E Hoffman and S Kaplan ldquoThe incidence of congenitalheart diseaserdquo Journal of the AmericanCollege of Cardiology vol39 no 12 pp 1890ndash1900 2002

[3] D Srivastava ldquoGenetic assembly of the heart implications forcongenital heart diseaserdquo Annual Review of Physiology vol 63pp 451ndash469 2001

[4] N Corrigan D P Brazil and F M Auliffe ldquoHigh-frequencyultrasound assessment of the murine heart from embryothrough to juvenilerdquoReproductive Sciences vol 17 no 2 pp 147ndash157 2010

[5] C F Spurney CW Lo and L Leatherbury ldquoFetal mouse imag-ing using echocardiography a review of current technologyrdquoEchocardiography vol 23 no 10 pp 891ndash899 2006

[6] Y-Q Zhou F S Foster R Parkes and S L Adamson ldquoDevel-opmental changes in left and right ventricular diastolic fillingpatterns in micerdquo American Journal of Physiology vol 285 no4 pp H1563ndashH1575 2003

[7] Q Yu L Leatherbury X Tian and C W Lo ldquoCardiovascularassessment of fetal mice by in utero echocardiographyrdquo Ultra-sound in Medicine and Biology vol 34 no 5 pp 741ndash752 2008

[8] C K L Phoon ldquoImaging tools for the developmental biologistultrasound biomicroscopy of mouse embryonic developmentrdquoPediatric Research vol 60 no 1 pp 14ndash21 2006

[9] J S Lee Q Yu J T Shin et al ldquoKlf2 is an essential regulator ofvascular hemodynamic forces in vivordquo Developmental Cell vol11 no 6 pp 845ndash857 2006

[10] R A Morris B Damon V Mironov et al ldquoPeriostin regulatescollagen fibrillogenesis and the biomechanical properties ofconnective tissuesrdquo Journal of Cellular Biochemistry vol 101 no3 pp 695ndash711 2007

[11] B C W Groenendijk B P Hierck J Vrolijk et al ldquoChanges inshear stress-related gene expression after experimentally alteredvenous return in the chicken embryordquoCirculation Research vol96 no 12 pp 1291ndash1298 2005

[12] C J Zuurbier V M Emons and C Ince ldquoHemodynamics ofanesthetized ventilated mouse models aspects of anestheticsfluid support and strainrdquo American Journal of Physiology vol282 no 6 pp H2099ndashH2105 2002

[13] A Włoch W Rozmus-Warcholinska B Czuba et al ldquoDopplerstudy of the embryonic heart in normal pregnant womenrdquoJournal of Maternal-Fetal and Neonatal Medicine vol 20 no 7pp 533ndash539 2007

[14] M E Godfrey B Messing S M Cohen D V Valsky andS Yagel ldquoFunctional assessment of the fetal heart a reviewrdquo

Ultrasound in Obstetrics and Gynecology vol 39 no 2 pp 131ndash144 2012

[15] M R M Jongbloed M C E F Wijffels M J Schalij et alldquoDevelopment of the right ventricular inflow tract and moder-ator band a possible morphological and functional explanationforMahaim tachycardiardquoCirculation Research vol 96 no 7 pp776ndash783 2005

[16] Y-HGui KK Linask PKhowsathit and J CHuhta ldquoDopplerechocardiography of normal and abnormal embryonic mouseheartrdquo Pediatric Research vol 40 no 4 pp 633ndash642 1996

[17] C F Spurney L Leatherbury and C W Lo ldquoHigh-frequencyultrasound database profiling growth development and car-diovascular function in C57BL6J mouse fetusesrdquo Journal of theAmerican Society of Echocardiography vol 17 no 8 pp 893ndash900 2004

[18] R P Ji C K L Phoon O Aristizabal K E McGrath JPalis and D H Turnbull ldquoOnset of cardiac function duringearly mouse embryogenesis coincides with entry of primitiveerythroblasts into the embryo properrdquoCirculation Research vol92 no 2 pp 133ndash135 2003

[19] M R Jongbloed S R Vicente N D Hahurij et al ldquoNormaland abnormal development of the cardiac conduction systemimplications for conduction and rhythm disorders in the childand adultrdquo Differentiation vol 84 no 1 pp 131ndash148 2012

[20] K Makikallio P Jouppila and J Rasanen ldquoHuman fetal cardiacfunction during the first trimester of pregnancyrdquo Heart vol 91no 3 pp 334ndash338 2005

[21] W Rozmus-Warcholinska A Wloch G Acharya et al ldquoRef-erence values for variables of fetal cardiocirculatory dynamicsat 11-14 weeks of gestationrdquo Ultrasound in Obstetrics andGynecology vol 35 no 5 pp 540ndash547 2010

[22] G Tulzer P Khowsathit S Gudmundsson et al ldquoDiastolicfunction of the fetal heart during second and third trimestera prospective longitudinal Doppler-echocardiographic studyrdquoEuropean Journal of Pediatrics vol 153 no 3 pp 151ndash154 1994

[23] MC Leiva J E Tolosa CN Binotto et al ldquoFetal cardiac devel-opment and hemodynamics in the first trimesterrdquoUltrasound inObstetrics and Gynecology vol 14 no 3 pp 169ndash174 1999

[24] I P Van Splunder and J W Wladimiroff ldquoCardiac functionalchanges in the human fetus in the late first and early secondtrimestersrdquo Ultrasound in Obstetrics and Gynecology vol 7 no6 pp 411ndash415 1996

[25] S A Clur R K Oude B W Mol J Ottenkamp and CM Bilardo ldquoFetal cardiac function between 11 and 35 weeksrsquogestation and nuchal translucency thicknessrdquo Ultrasound inObstetrics amp Gynecology vol 37 no 1 pp 48ndash56 2011

[26] G Rizzo A Muscatello E Angelini and A Capponi ldquoAbnor-mal cardiac function in fetuses with increased nuchal translu-cencyrdquo Ultrasound in Obstetrics and Gynecology vol 21 no 6pp 539ndash542 2003

[27] F Crispi E Hernandez-Andrade M M A L Pelsers et alldquoCardiac dysfunction and cell damage across clinical stagesof severity in growth-restricted fetusesrdquo American Journal ofObstetrics and Gynecology vol 199 no 3 pp 254ndash258 2008

[28] S Luewan Y Yanase F Tongprasert K Srisupundit and TTongsong ldquoFetal cardiac dimensions at 14-40 weeksrsquo gestationobtained using cardio-STIC-Mrdquo Ultrasound in Obstetrics andGynecology vol 37 no 4 pp 416ndash422 2011

The Scientific World Journal 11

[29] T Tongsong C Wanapirak W Piyamongkol et al ldquoFetalventricular shortening fraction in hydrops fetalisrdquo Obstetricsand Gynecology vol 117 no 1 pp 84ndash91 2011

[30] E Hernandez-Andrade H Figueroa-Diesel C Kottman etal ldquoGestational-age-adjusted reference values for the modifiedmyocardial performance index for evaluation of fetal left car-diac functionrdquoUltrasound in Obstetrics and Gynecology vol 29no 3 pp 321ndash325 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Signal TransductionJournal of

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Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 The Scientific World Journal

Furthermore functional parameters and their course at sub-sequent stages of normalmouse heart development have onlybeen described in a few studies [4ndash7] Specifically knowledgeregarding RV inflow andmyocardial function during embry-onic development is scarce

In clinical practice ultrasound is themost frequently usedminimally invasive imaging modality at pre- and postnatalstages of heart development Clinical ultrasound machineshave also been used for imaging of embryonic mouse heartshowever ultrasound frequencies (8ndash15MHz) are not suffi-cient for a detailed morphological and hemodynamic anal-ysis Recently developed high-frequency ultrasound systems(30ndash50MHz) are able to offer sufficient spatial-temporal res-olution for detailed and reliable imaging of embryonicmousehearts Thus far there are only limited studies describingthe hemodynamic and morphological changes at subsequentstages of heart development using such ultrasound systems[4 6 8]

Hemodynamics play an important role in normal heartdevelopment by the activation of shear-stress dependentsignaling pathways During development genes like Klf-2 [9]and periostin [10] are expressed in regions of the heart that areknown to be subjected to high mechanicalhemodynamicalstress It has also been demonstrated that artificial alterationsin hemodynamics throughout heart development result inaberrant activation of genes at the endocardial surface of theheart [11] which might lead to disturbed cardiogenesis thatis CHD A first step in the interpretation of hemodynamicparameters during development is the acquisition of a reliablereference series during normal heart development

In the current study we aim to provide an overview ofhemodynamic andmorphological changes during embryonicand fetal stages of normal mouse heart development usinga high-frequency ultrasound system Knowledge of thesefunctional parameters is highly relevant for the interpretationof results obtained in genetically mutated mouse models Weexpect that knowledge of how echocardiographic parametersshift over development will eventually contribute to the earlydetection of CHD during human pregnancy

2 Material and Methods

21 Animals Animal experiments were approved by the localanimal welfare committee (DEC number 10177) of the LeidenUniversity Medical Center A timed breeding program wasinitiated with a WT (Swiss129Sv background) mouse lineThe day after breeding was considered to be 05 days afterconception (dpc) All pregnant mice (119899 = 11) were subjectedto high-frequency ultrasound recordings at 3 subsequentstages of embryonicfetal development being 125 145 and175 dpc At 125 dpc the pregnant mother mouse was anaes-thetized using isoflurane (induction 5 maintenance duringassessment 15) the most commonly used anesthetic inmouse experiments [6 12] Subsequently the sedated mousewas placed in the experimental setup (VisualSonics Vevo770system Toronto Canada) which includes a heated table andrectal probe for maintenance and control of body temper-ature (365ndash375∘C) Electrodes were attached to the paws

formonitoring thematernal electrocardiogram andheart andbreathing rate during the experiments

Abdominal hair was removed using a commerciallyavailable chemical hair remover followed by application ofultrasound transmission gel (Aquasonic Parker LaboratoriesFairfield NY USA) The ultrasound recordings were per-formed with a high-frequency ultrasound system (30MHzVisualSonics Vevo 770 system Toronto Canada) with anaxial resolution of 55 120583m a focal length of 127mm and amaximal field of view of 20mm Scanning time was keptas short as possible according to the ALARA principle [13]Between the three subsequent developmental stages that wereassessed the pregnant mice were housed in cages in theanimal facility

22 Ultrasound Recording Protocol All ultrasound record-ings were performed by NDH and EEC During the ultra-sound recordings the two laterally located uterus horns werescanned for the presence and position of the embryosfetuses(119899 = 105) In individual embryosfetuses the left and rightside were established to identify the position of the heartincluding the individual cardiac compartments and struc-tures At 125 dpc these compartments included the left atrium(LA) right atrium (RA) LV RV the common AV canal(cAVC) encompassing the future mitral valve (MV) andtricuspid valve (TV) orifices common outflow tract (cOFT)developing interventricular septum (IVS) and the interven-tricular foramen that is the continuity between the LV andRV before completion of ventricular septation At later stages(145 and 175 dpc) the MV and TV orifices aorta (Ao) andpulmonary trunk (PT) were identified

23 Pulsed-Wave Doppler Recordings Themethods that wereused for pulsed-wave Doppler flow recordings are summa-rized in Table 1 For flow measurements automatic Dopplerangle corrections were accepted up to 45 degrees Flowrecordings across the cAVC (125 dpc future MV 119899 = 1future TV 119899 = 11) and developing MV and TV orifices(145 dpc MV 119899 = 17 TV 119899 = 20 175 dpc MV 119899 = 16 TV119899 = 14) were performed to assess the peak-E (earlypassiveventricular filling by ventricular relaxation in early diastole)and peak-A wave velocities (active ventricular filling due toatrial contraction) hence the EA ratios were calculated Therecordings across the MV orifice cAVC and developing werealso used to evaluate the embryonic heart rate (HR) in beatsper minute (BPM) cardiac cycle length (RR interval) dias-tolic ventricular filling time (DFT 125 dpc 119899 = 11 145 dpc119899 = 17 and 175 dpc 119899 = 16) ejection time (ET 125 dpc119899 = 11 145 dpc 119899 = 17 and 175 dpc 119899 = 16) isovolumetriccontraction time (IVCT 125 dpc 119899 = 7 145 dpc 119899 = 17 and175 dpc 119899 = 16) and isovolumetric relaxation time (IVRT125 dpc 119899 = 7 145 dpc 119899 = 17 and 175 dpc 119899 = 16) of theLV (Figure 1(a)) The myocardial performance index (MPI125 dpc 119899 = 5 145 dpc 119899 = 17 175 dpc 119899 = 16) also knownas Tei index evaluates the combined LV systolic and diastolicfunctionwas calculated using the following formula (IVRT+IVCT)ET [14]

Pulsed-wave Doppler recordings were also performed inthe cOFT at 125 dpc (119899 = 15) and Ao and PT at 145 dpc

The Scientific World Journal 3

Table 1 The methods used for performing Doppler flow recordings per gestational age

Age (dpc)andalignment

Location Parameter (unit) View Doppler beam position

125Future MV

E-wave (ms)A-wave (ms)DFT (ms)IVCT (ms)IVRT (ms)ET (ms)

4 chambers cAVC parallel to flow direction at left side ofthe developing IVS

Future TV E-wave (ms)A-wave (ms) 4 chambers cAVC parallel to flow direction at the right

side of the developing IVS

cOFT future AoPT Peak flow (ms)VTI (mm) 5 chambersOFT Proximal part cOFT parallel to flow direction

145 and 175

MV

E-wave (ms)A-wave (ms)DFT (ms)IVCT (ms)IVRT (ms)ET (ms)

4 chambers LV below (developing) MV annulus parallel toflow direction at left side of the IVS

TV E-wave (ms)A-wave (ms) 4 chambers RV below (developing) TV annulus parallel to

flow direction at right side of IVS

Ao Peak flow (ms) 5 chambersLVOT Ao above (developing) valve annulus VTI(mm) parallel to flow direction

PT Peak flow (ms) RVOT PT above (developing) valve annulus parallelto flow direction

(119899 = 18 and 119899 = 12) and 175 dpc (119899 = 12 and 119899 = 4 Table 1)Furthermore the velocity time integral (VTI) that is the areaunder the Doppler velocity envelope for one heart beat wasdetermined (125 dpc 119899 = 15 145 dpc Ao 119899 = 18 and PT119899 = 12 175 dpc Ao 119899 = 12 and PA 119899 = 4) and the diametersof the cOFT (119899 = 2) and Ao (145 dpc 119899 = 3 and 175 dpc119899 = 2) were evaluated from B-mode echoloops in order tocalculate the mean stroke volume (SV) per developmentalstage according to the following formula mean SV = meanTVI times (mean diameter of Ao or cOFT2)2 times 120587 Hence themean cardiac output (CO) was calculated (mean CO =meanSV timesmean HR) for the three developmental stages

24 M-Mode Measurements For morphological assessmentM-mode measurements in short- or long-axis views of theventricles were performed to evaluate the end-systolic andend-diastolic ventricular inner diameter of the LV (LVID125 dpc 119899 = 9 145 dpc 119899 = 11 and 175 dpc 119899 = 14) andRV (RVID 125 dpc 119899 = 9 145 dpc 119899 = 10 and 175 dpc119899 = 12) and IVS diameter (125 dpc 119899 = 4 145 dpc 119899 = 6 and175 dpc 119899 = 9) Cardiac fractional shortening (FS) for theLV and RV was calculated using the formula (VIDdiastole minusVIDsystole)VIDdiastole times 100 Due to the size of thehearts at 125 dpc the resolution often limited the possibilityto discriminate the epicardial myocardial and endocardiallayer of the ventricular free wall Therefore at this stage thedistances from the epicardial layer to the IVS in end-systoleand end-diastole were used to calculate the FS

Although a complete hemodynamic and morphologi-cal assessment for each heart was aimed for (Table 2 for

overview) imaging possibilities were regularly limited by theposition of the embryofetus All echocardiographic record-ings were stored for offline data analysis by NDH and EECinterobserved analysis demonstrated a high level of consis-tency (119903 = 08ndash10 119875 lt 005) Flow parameters were calcu-lated by the average of pulsed-wave Doppler complexes ofthree consecutive beats similar methods were applied forM-mode recordings and diameter calculations on B-modeecholoops

25 Cardiac Morphology and Three-Dimensional Reconstruc-tions After the experiments the pregnant mouse was sac-rificed by cervical dislocation and embryosfetuses wereextractedWe refer to previous publications for details regard-ing embryonicfetal processing sectioning immunohisto-chemistry with MLC2a specific antibodies and preparationof the three-dimensional AMIRA reconstructions (AMIRAsoftware package Template Graphics Software San DiegoUSA) [15]

26 Statistics All statistics were performed with the statisti-cal package for the social sciences 150 (SPSS Inc Chicago ILUSA) andGraphPadPrism (GraphPad Software La Jolla CAUSA) A 119875 lt 005 (2-tailed) was considered to be significantall values are given as mean plusmn SEM All boxplots indicate theupper and lower quartile of the median which is indicatedby the horizontal bar The whiskers indicate maxmin rangeof the values

4 The Scientific World Journal

lowastns

ns80

60

40

20

125 145 175

ET (

)

(c)

40

20

0

minus20

minus40

Velo

city

(cm

s)

10

0

minus10

Freq

uenc

y (k

Hz)

7000 8000 Time (ms)

ECG 230mVResp 230mV

(a)

lowast

nsns80

60

40

20

DFT

()

125 145 175

(b)

20

15

10

5

nsnsns

IVCT

()

125 145 175

Age (dpc)

(d)

lowast

lowast20

15

10

5

ns

125 145 175

Age (dpc)

IVRT

()

(e)

1

23

4

5

(1) Cycle length (RR)

(2) Diastolic filling time (DFT)

(3) Isovolumetric contraction time (IVCT)

(4) Ejection time (ET)

(5) Isovolumetric relaxation time (IVRT)

(a998400)

(a998400)

Figure 1 Development of LV inflow patterns (a) Shows an example of a pulsed-wave Doppler recording across the MV at 175 dpc (a1015840)Indicates the magnification of the boxed area in (a) in which the individual time intervals are indicated that is RR DFT IVCT ET andIVRT (b)Through (e) indicates the course of the DFT ET IVCT and IVRT respectively throughout embryonicfetal life All values in (bndashe)are expressed as percentage of the RR

The Scientific World Journal 5

Table 2 Summary of the assessment of embryonic and fetal hearts

Method Parameter Unit Structure (abbreviation)

2D ultrasoundB-mode echoloops

Morphological assessmentposition and diameters ofcardiac compartments

mm

Right atrium (RA) left atrium (LA) right ventricle (RV) left ventrile (LV)common outflow tract (cOFT) pulmonary trunk (PT) aorta (Ao)inter ventricular septum (IVS) common atrioventricular canal (cAVC)mitral valve (MV) and tricuspid valve (TV)Definition Formula

Pulsed-waveDoppler

Peak E ms Early diastolic ventricular fillingPeak A ms Active diastolic ventricular fillingEA ratioRR interval ms Cycle lengthevaluation of heart rateDFT ms Diastolic filling timeIVRT ms Isovolumetric relaxation timeIVCT ms Isovolumetric contraction timeET ms Ejection timeMPITei index Myocardial performance index IVRT + IVCTETPeak velocities cOFT AoPT ms

VTI mm Velocity time integral

SV Stroke volume SV = TVI times (Ao orcOFT diameter2)2 times 120587

CO mLmin Cardiac output

M-mode

VID mm Ventricular inner diameterLVIDd mm End diastolic left ventricular inner diameterLVIDs mm End systolic left ventricular inner diameterRVIDd mm End diastolic right ventricular inner diameterRVIDs mm End systolic right ventricular inner diameterIVSd mm End diastolic interventricular septum diameterIVSs mm End systolic interventricular septum diameter

FS Fractional shortening (VIDdminusVIDs)VIDd times100

3 Results

31 Cardiac Morphology Reliable imaging of hearts as smallas 2mm was feasible in all stages examined Representativeexamples are shown in online Movie 1 (see SupplementaryMaterial available online at httpdxdoiorg1011552014531324)

At embryonic stages (125 dpc) ventricular septation hasbeen initiated but is not completed resulting in a primitiveinterventricular foramen that directly connects the lumen ofLV and RV M-mode recordings at these stages show a meansystolic IVS diameter of 020 plusmn 0045mm Figure 2(a) showsa reconstruction of a 125 dpc embryonic heart in which thelumen of the four cardiac chambers interventricular fora-men and theOFT can be identifiedThe atrial and ventricularchambers are connected via the cAVC that harbors large AVcushions which will contribute to formation of the MV andTV The AV canal is still partly positioned above the LV atthis stage The developing OFT is still positioned completelyabove the RV and contains cushion tissue that will form thesemilunar valves of the Ao and PTThey will further form thebasis for the muscular subpulmonary infundibulum and will

contribute to themembranous part of the IVS Consequentlythe blood from the LV runs via the primitive interventricularforamen into the cOFT that is future Ao and PT

At early fetal stages of 145 dpc the heart has increased insize (Figure 2(b)) and ventricular septation is now completeThe systolic diameter of the IVS has increased (024 plusmn0018mm) In the AVC the TV orifice has expanded and isnow situated entirely above the RV Also the MV orifice canbe identified as well as the developing AV valves The cardiacOFT now consists of a separate Ao and PT connecting to theLV and RV respectively The Ao and pulmonary valves stillshow immaturity and consist of cushion tissue

At late fetal stages (175 dpc) the size of the RV andLV and IVS (039 plusmn 0009mm) has increased significantly(Figure 2(c)) Mature valves are present at the levels of the in-and outflow parts of both ventricles

32 Diastolic and Systolic Function During embryonic andfetal development theHR increases significantly (119875 lt 00001)from 125 plusmn 95 up to 219 plusmn 83BPM Ventricular inflowpatterns across the developing MV and TV annulus werestudied by pulsed-wave Doppler imaging

6 The Scientific World Journal

(a) (b) (c)

lowast

lowastlowastlowast150

125

100

075

050

025

000

Dia

met

er lu

men

(mm

)

Left ventricle

Right ventricle

Diastole

Systole

125 145 175

Age (dpc)

ns

(d)

lowast

125 145 175

Age (dpc)

50

40

30

20

10

0

Shor

teni

ng fr

actio

n (

)

Left ventricle

Right ventricle

(e)

Figure 2 Cardiac morphology and FS calculation (a) Shows a reconstruction of the anterior view of an embryonic heart of 125 dpc Themyocardium is indicated in grey transparent The LA and RA are indicated in transparent dark grey The LV and RV lumen are indicated inred and blue respectively Note that the outflow tract lumen (purple) is positioned completely above the future RV which is surrounded bylarge outflow tract cushions (green transparent) At these stages development of the IVS has not yet completed leading to a direct connectionbetween the LV and RV via the interventricular foramen (arrow) (b) Anterior view of an early fetal heart of 145 dpc At this stage IVSdevelopment has been completed and four separate cardiac chambers can be identified The outflow tract consists of a separate Ao and PTincluding their valve apparatus which at these stages mainly consist of cushion tissue (green transparent) (c) Anterior view of a late fetalheart of 175 dpc At this stage the heart shows a mature morphological phenotype (d) Schematic representation of LV and RV diameters and(e) FS at the three consecutive stages of development

The DFT corrected for the RR interval (DFTRR) did notdiffer significantly at the three stages assessed (Figure 1(b))LV and RV filling were dominated by the peak-A waveBetween 125 and 175 dpc the peak-A wave of the developingMV annulus increased significantly (033 plusmn 0001ms versus042 plusmn 0014ms 119875 lt 00001) whereas that of the TV alsoshowed an increasing trend albeit not significant (037 plusmn0018ms versus 041 plusmn 0019ms 119875 = NS) The peak-Ewave also increased significantly for both LV (005plusmn0006msversus 018plusmn0005ms 119875 lt 00001) and RV (006plusmn0006msversus 018 plusmn 0012ms 119875 lt 00001) and showed a relativelygreater increase than the peak-A wave during development

As a result the EA ratio a measure for the ventricular dia-stolic function showed a significant increase for both LV(016 plusmn 0015 versus 044 plusmn 0013 119875 lt 00001) and RV(016 plusmn 0010 versus 044 plusmn 0014 119875 lt 00001) (Figures 3(a)ndash3(f))

The IVRT one of the measures for diastolic LV functioncorrected for the RR interval (IVRTRR) demonstrated asignificant (119875 = 0037) decrease between 125 (015 plusmn 0011)and 145 dpc (012 plusmn 0004) and remained stable at later fetalstages 175 dpc (012 plusmn 0003) (Figure 1(e)) indicating that LVmyocardial relaxation remains stable after completion of IVSdevelopment during fetal life

The Scientific World Journal 7

Mitral06

05

04

03

02

01

00

EA

ratio

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

125 145 175

(a)

06

05

04

03

02

01

00

EA

ratio

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

125 145 175

Tricuspid

(b)

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

125 145 175

Peak

E (m

s)

04

03

02

01

00

(c)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

125 145 175

Peak

E (m

s)

04

03

02

01

00

(d)

lowastlowastlowast

125 145 175

Age (dpc)

Peak

A (m

s)

07

06

05

04

03

02

01

00

nslowastlowast

(e)

125 145 175

Age (dpc)

Peak

A (m

s)

07

06

05

04

03

02

01

00

nsns

ns

(f)

Figure 3 Development of LV and RV inflow patternsThe graphs represent the course of the EA ratio (a b) peak-E wave (c d) and peak-Awave (e f) across the developing MV and TV at the three subsequent developmental stages

The systolic function of the developing ventricles wasassessed through studying the course of the FS IVCTRRand ETRR Throughout embryonic and fetal developmentthe FS remained constant for both ventricles and valuesranged between asymp30 and 40 Furthermore except for175 dpc (LV 406 RV 357 119875 = 0046) no significantdifferences were observed between the FS of the LV and RV

(Figures 2(d) and 2(e)) The mean IVCTRR remained stableduring all three stagesasymp009 the ETRR showed no changesbetween 125 dpc (042plusmn0015) and 145 dpc (043plusmn0010) butsignificantly decreased at 175 dpc (040 plusmn 0006 119875 = 0017)(Figures 1(c) and 1(d))

A combined assessment of LV systolic and diastolic func-tion was performed by calculating the MPI At embryonic

8 The Scientific World Journal

Table 3 The parameters used for calculation of the mean CO per gestational age

Parameter 125 dpc (cOFT) 145 dpc (Ao) 175 dpc (Ao)VTI (mm) 339 plusmn 137 423 plusmn 20 292 plusmn 205

Mean diameter (mm) 023 018 048 plusmn 003

Mean HR (bpm) 134 plusmn 112 148 plusmn 61 206 plusmn 98

Mean CO (mLmin) 019 016 096

lowastlowastlowast05

04

03

02

01

Peak

flow

(ms

)

Common OFT

Aorta

Pulmonary trunk

125 145 175

Age (dpc)

Figure 4 Pulsed-wave Doppler flow measurements in the cOFTAo and PT The graph demonstrates the significant increase of thepeak blood flowmeasured in the cOFT at 125 and Ao and PT at 145and 175 dpc

preseptated stages (125 dpc) the MPI was 064 plusmn 0012 anddecreased to 049 plusmn 0017 (119875 = 00002) after closure of theIVS at 145 dpc At late fetal stages (175 dpc) the MPI did notchange significantly (053 plusmn 0022 119875 = NS)

33 OutflowTract andCO Thepulsed-waveDoppler record-ings performed in the cOFT demonstrated a peak flow of027 plusmn 0021ms at preseptated stages As from 145 dpc twoindividual ventricular outflow tracts could be discriminatedespecially the high echodensity of the blood at these stagesenabled to discriminate the flow in the Ao (034 plusmn 0013ms)and PT (033 plusmn 0021ms) that are closely related to eachother As can be observed in Figure 4 the peak flow increasedsignificantly in the Ao (from 027 plusmn 0021ms to 036 plusmn0016ms 119875 = 0010) and PT (027 plusmn 0021ms to 044 plusmn0030ms 119875 = 0003) along fetal life

Table 3 summarizes theHRVTI and the diameters of thecOFT and Ao that were used to calculate the mean CO at thethree developmental stages Between 125 dpc and 145 dpc the

CO remained stable where after it increased to 096mLminat 175 dpc

4 Discussion

Heart development is a complex process which has beenextensively studied in both WT and genetically mutatedmouse models [3] It is only in the last decades that modernimaging techniques like ultrasound enable the assessmentof real-time hemodynamic and morphological changes thatoccur during mouse heart development [4ndash6 8 16ndash18]Knowledge regarding these functional parameters is essentialsince abnormal flow in the developing heart might lead toCHD through aberrant activation of shear-stress responsivegenes important in cardiogenesis [9 11] In early reportsdescribing the course of functional parameters during heartdevelopmentmajor variability exists inmeasured valuesThisis most likely related to the relatively low ultrasound frequen-cies of clinical ultrasound machines used to image the smallhearts of mouse embryosfetuses [16] and externalizationof uterus horns during ultrasound assessment [18] whichinfluences embryonicfetal physiology Low dose isofluraneanesthesia used in this study only has a minimal effect onhemodynamics and diastolic function in adult mice and theeffect on mouse embryos is supposed to be marginal [6]

In the current study we aimed to provide an overview ofhemodynamic parameters and changes that occur at subse-quent stages of heart development using a high-frequencyultrasound system

Compared to other reports in mouse using similar ultra-sound techniques we did not only focus on development ofthe future LV but also on that of the RV Key findings ofour study are (1) a reliable assessment of LV and RV in- andoutflow patterns can be performed from 125 dpc to term (2)a significant improvement occurs in the diastolic and systolicfunction of both LV and RV (3) over the developmentalperiod of 125 dpc to 175 dpc the ratio between RV and LVsize as well as the FS of both ventricles did not changesignificantly

Knowledge regarding the functional parameters of theRV is important since the RV is commonly affected in CHDDuring development the RV including the TV annulusdevelops subsequent to the LV by addition of SHF derivedcells to the developing heart [1] Many genes have beenidentified that have an important role in the SHF contributionto the developing heart andmutations in these genesmay leadto CHD [19]

At early embryonic stages endocardial cushion tissue ispositioned in the primitive AVC and OFT which later on

The Scientific World Journal 9

will contribute to formation of the TV and MV and Ao andPT semilunar valves Some clinical studies argued that AVvalve regurgitation is a commonphenomenon during the firsttrimester of pregnancy [20] Our study in accordance withseveral others in mouse [4 6] and human [13] did not showsigns of regurgitation at both early and late stages of heartdevelopment Pulsed-wave Doppler recordings across thecAVC at 125 dpc and MV and TV at 145 dpc demonstratedthat the AV cushionsmimic the function of normal AV valvesat 175 dpc In our opinion especially the flow in the cOFTat early embryonic stages which is completely positionedabove the RV (see Figure 2(a)) might wrongly be identifiedas regurgitation of the developing AV valvescushions Thisstresses the importance of detailed knowledge regarding therapid morphological changes that the heart undergoes atespecially the very early developmental stages

Regarding growth of the heart we demonstrated that therewas a 31 increase of LVID 34 of RIVD and 35 of IVSdiameter from 125 up to 175 dpcWe showed a trend towardsa smaller RVID as compared to the LVID at all stages whichwas not mentioned in other studies

41 Diastolic and Systolic Function during Heart DevelopmentImprovement of ventricular systolic and diastolic functionoccurs throughout the length of embryonic and fetal develop-mentWith respect to the diastolic function we demonstrateda significant increase of EA ratio across the developing MVand TV orifices which was related to an increase of the peak-E wave that is the fraction of diastolic ventricular filling thatis defined by ventricular relaxation These data are in accor-dance with the study of Zhou and colleagues who demon-strated that the EA ratio is increasing during developmentwhich seems to continue up to approximately 3 weeks afterbirth [6] Around day four after birth the peak-E wave mea-sured across theMV becomes more dominant than the peak-A wave indicating an improvement of cardiac complianceand that LV filling becomes dominated by passive ventricularrelaxation Remarkably these changes were not observed forthe RV inflow patterns where the peak-A wave remainsdominant [6] Human embryonic and fetal hearts show thesame maturation course of EA ratios across the developingMV and TV as was demonstrated in multiple studies [21ndash25]An abnormal course of EA ratio that is a decreasing[25 26] or an increasing trend [27] along development hasbeen related to the presence of CHD

With respect to systolic ventricular function the FSremained stable (asymp30ndash40) for both ventricles throughoutdevelopmentWe did find a significant difference favoring theLV FS as compared to the RV FS at 175 dpc Interestinglythe same phenomenon of FS difference between LV andRV has been reported in a study of human fetal heartsbetween 14 and 40weeks of pregnancy However as inmousedifferences between humanLVandRVFS is subtle [28] andwhether a decreased or decreasing FS is a predictor of CHDremains unknown Furthermore we did not find a decreasingtrend of both LV and RV FS as was recently demonstratedduring human fetal life [28 29] With respect to the CO wenoticed no change around the proces of ventricular septation(between 125 and 145 dpc) where after a major increase

occured to 096mLmin near term (175 dpc) The initial COdecreasemight be related to the fact that themeandiameter ofthe unseptated cOFT diameter was used at 125 dpc comparedto the isolated Ao diameter at 145 dpc The subsequent COincrease is mostly related to the dramatic increase of HRalong fetal life

The MPI or Tei index [14] is a measure for combinedsystolic and diastolic cardiac function and can be calculatedfor both ventricles In order to assess the MPI three differenttime intervals are needed the IVCT IVRT and ET For the LVthese parameters can be easily obtained since all the threeparameters can be assessed at once by placing the Dopplerultrasound beam across the developing MV orifice Notice-ably in literature there is less data regarding RV MPI andits course along development which might be related to thefact that for the RV not all the three time intervals can beassessed in one view as was also suggested in a recent reviewby Godfrey et al [14] The current study demonstrated asignificant decrease of LV MPI between 125 and 145 dpcwhich might be related to the rapid morphological changesthe heart goes through including completion of IVS leadingto altered hemodynamic conditions in the developing heart Itis however also important to notice that at preseptation stagesthe IVCT and IVRT cannot always be identified separately sothat calculation of the MPI is frequently not possible at veryearly stages of embryonic development Between 145 and175 dpcwenoticed a slight increase of theMPI suggesting thatcardiac function improves at subsequent fetal stages similarresults have also been demonstrated inmouse [4] and human[30] However discussion remains regarding the exact courseof theMPI IVCT IVRT andETandwhether isolated changesof one of the factors is a predictor for fetal demise and CHDInterestinglywith respect to IVRTweobserved a decreasingtrend near completion of the IVS (between 125 and 145)whereafter it more or less remained stable throughout fetallife A similar course was observed in a recent ultrasoundstudy in embryonicfetal C57Bl6 mice [4] Human embryosalso showed a decreasing course of the IVRT [13 21] and anelevated IVRT has been implicated as a predictor of CHD[13]

5 Conclusion

We demonstrated the course of several morphologic andhemodynamic parameters along embryonic and fetal lifeOur data show that a reliable assessment can be made notonly of the left but also of right ventricular function duringembryonicfetal stages We demonstrate an overall improve-ment of cardiac diastolic and systolic function of both LV andRV along heart development The implementation of high-frequency ultrasound in embryonicfetal mouse heart devel-opment is a promising and useful tool The data presented inthis studymight be useful as reference values in future studiesin genetic mutated mouse models of CHD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

10 The Scientific World Journal

Authorsrsquo Contribution

Nathan D Hahurij and Emmeline E Calkoen contributedequally to this work and shared first authorship

Acknowledgment

This work is supported by the DutchHeart Foundation Grantno 2009T070 (Nathan D Hahurij)

References

[1] R G Kelly ldquoMolecular inroads into the anterior heart fieldrdquoTrends inCardiovascularMedicine vol 15 no 2 pp 51ndash56 2005

[2] J I E Hoffman and S Kaplan ldquoThe incidence of congenitalheart diseaserdquo Journal of the AmericanCollege of Cardiology vol39 no 12 pp 1890ndash1900 2002

[3] D Srivastava ldquoGenetic assembly of the heart implications forcongenital heart diseaserdquo Annual Review of Physiology vol 63pp 451ndash469 2001

[4] N Corrigan D P Brazil and F M Auliffe ldquoHigh-frequencyultrasound assessment of the murine heart from embryothrough to juvenilerdquoReproductive Sciences vol 17 no 2 pp 147ndash157 2010

[5] C F Spurney CW Lo and L Leatherbury ldquoFetal mouse imag-ing using echocardiography a review of current technologyrdquoEchocardiography vol 23 no 10 pp 891ndash899 2006

[6] Y-Q Zhou F S Foster R Parkes and S L Adamson ldquoDevel-opmental changes in left and right ventricular diastolic fillingpatterns in micerdquo American Journal of Physiology vol 285 no4 pp H1563ndashH1575 2003

[7] Q Yu L Leatherbury X Tian and C W Lo ldquoCardiovascularassessment of fetal mice by in utero echocardiographyrdquo Ultra-sound in Medicine and Biology vol 34 no 5 pp 741ndash752 2008

[8] C K L Phoon ldquoImaging tools for the developmental biologistultrasound biomicroscopy of mouse embryonic developmentrdquoPediatric Research vol 60 no 1 pp 14ndash21 2006

[9] J S Lee Q Yu J T Shin et al ldquoKlf2 is an essential regulator ofvascular hemodynamic forces in vivordquo Developmental Cell vol11 no 6 pp 845ndash857 2006

[10] R A Morris B Damon V Mironov et al ldquoPeriostin regulatescollagen fibrillogenesis and the biomechanical properties ofconnective tissuesrdquo Journal of Cellular Biochemistry vol 101 no3 pp 695ndash711 2007

[11] B C W Groenendijk B P Hierck J Vrolijk et al ldquoChanges inshear stress-related gene expression after experimentally alteredvenous return in the chicken embryordquoCirculation Research vol96 no 12 pp 1291ndash1298 2005

[12] C J Zuurbier V M Emons and C Ince ldquoHemodynamics ofanesthetized ventilated mouse models aspects of anestheticsfluid support and strainrdquo American Journal of Physiology vol282 no 6 pp H2099ndashH2105 2002

[13] A Włoch W Rozmus-Warcholinska B Czuba et al ldquoDopplerstudy of the embryonic heart in normal pregnant womenrdquoJournal of Maternal-Fetal and Neonatal Medicine vol 20 no 7pp 533ndash539 2007

[14] M E Godfrey B Messing S M Cohen D V Valsky andS Yagel ldquoFunctional assessment of the fetal heart a reviewrdquo

Ultrasound in Obstetrics and Gynecology vol 39 no 2 pp 131ndash144 2012

[15] M R M Jongbloed M C E F Wijffels M J Schalij et alldquoDevelopment of the right ventricular inflow tract and moder-ator band a possible morphological and functional explanationforMahaim tachycardiardquoCirculation Research vol 96 no 7 pp776ndash783 2005

[16] Y-HGui KK Linask PKhowsathit and J CHuhta ldquoDopplerechocardiography of normal and abnormal embryonic mouseheartrdquo Pediatric Research vol 40 no 4 pp 633ndash642 1996

[17] C F Spurney L Leatherbury and C W Lo ldquoHigh-frequencyultrasound database profiling growth development and car-diovascular function in C57BL6J mouse fetusesrdquo Journal of theAmerican Society of Echocardiography vol 17 no 8 pp 893ndash900 2004

[18] R P Ji C K L Phoon O Aristizabal K E McGrath JPalis and D H Turnbull ldquoOnset of cardiac function duringearly mouse embryogenesis coincides with entry of primitiveerythroblasts into the embryo properrdquoCirculation Research vol92 no 2 pp 133ndash135 2003

[19] M R Jongbloed S R Vicente N D Hahurij et al ldquoNormaland abnormal development of the cardiac conduction systemimplications for conduction and rhythm disorders in the childand adultrdquo Differentiation vol 84 no 1 pp 131ndash148 2012

[20] K Makikallio P Jouppila and J Rasanen ldquoHuman fetal cardiacfunction during the first trimester of pregnancyrdquo Heart vol 91no 3 pp 334ndash338 2005

[21] W Rozmus-Warcholinska A Wloch G Acharya et al ldquoRef-erence values for variables of fetal cardiocirculatory dynamicsat 11-14 weeks of gestationrdquo Ultrasound in Obstetrics andGynecology vol 35 no 5 pp 540ndash547 2010

[22] G Tulzer P Khowsathit S Gudmundsson et al ldquoDiastolicfunction of the fetal heart during second and third trimestera prospective longitudinal Doppler-echocardiographic studyrdquoEuropean Journal of Pediatrics vol 153 no 3 pp 151ndash154 1994

[23] MC Leiva J E Tolosa CN Binotto et al ldquoFetal cardiac devel-opment and hemodynamics in the first trimesterrdquoUltrasound inObstetrics and Gynecology vol 14 no 3 pp 169ndash174 1999

[24] I P Van Splunder and J W Wladimiroff ldquoCardiac functionalchanges in the human fetus in the late first and early secondtrimestersrdquo Ultrasound in Obstetrics and Gynecology vol 7 no6 pp 411ndash415 1996

[25] S A Clur R K Oude B W Mol J Ottenkamp and CM Bilardo ldquoFetal cardiac function between 11 and 35 weeksrsquogestation and nuchal translucency thicknessrdquo Ultrasound inObstetrics amp Gynecology vol 37 no 1 pp 48ndash56 2011

[26] G Rizzo A Muscatello E Angelini and A Capponi ldquoAbnor-mal cardiac function in fetuses with increased nuchal translu-cencyrdquo Ultrasound in Obstetrics and Gynecology vol 21 no 6pp 539ndash542 2003

[27] F Crispi E Hernandez-Andrade M M A L Pelsers et alldquoCardiac dysfunction and cell damage across clinical stagesof severity in growth-restricted fetusesrdquo American Journal ofObstetrics and Gynecology vol 199 no 3 pp 254ndash258 2008

[28] S Luewan Y Yanase F Tongprasert K Srisupundit and TTongsong ldquoFetal cardiac dimensions at 14-40 weeksrsquo gestationobtained using cardio-STIC-Mrdquo Ultrasound in Obstetrics andGynecology vol 37 no 4 pp 416ndash422 2011

The Scientific World Journal 11

[29] T Tongsong C Wanapirak W Piyamongkol et al ldquoFetalventricular shortening fraction in hydrops fetalisrdquo Obstetricsand Gynecology vol 117 no 1 pp 84ndash91 2011

[30] E Hernandez-Andrade H Figueroa-Diesel C Kottman etal ldquoGestational-age-adjusted reference values for the modifiedmyocardial performance index for evaluation of fetal left car-diac functionrdquoUltrasound in Obstetrics and Gynecology vol 29no 3 pp 321ndash325 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

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International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Signal TransductionJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World Journal 3

Table 1 The methods used for performing Doppler flow recordings per gestational age

Age (dpc)andalignment

Location Parameter (unit) View Doppler beam position

125Future MV

E-wave (ms)A-wave (ms)DFT (ms)IVCT (ms)IVRT (ms)ET (ms)

4 chambers cAVC parallel to flow direction at left side ofthe developing IVS

Future TV E-wave (ms)A-wave (ms) 4 chambers cAVC parallel to flow direction at the right

side of the developing IVS

cOFT future AoPT Peak flow (ms)VTI (mm) 5 chambersOFT Proximal part cOFT parallel to flow direction

145 and 175

MV

E-wave (ms)A-wave (ms)DFT (ms)IVCT (ms)IVRT (ms)ET (ms)

4 chambers LV below (developing) MV annulus parallel toflow direction at left side of the IVS

TV E-wave (ms)A-wave (ms) 4 chambers RV below (developing) TV annulus parallel to

flow direction at right side of IVS

Ao Peak flow (ms) 5 chambersLVOT Ao above (developing) valve annulus VTI(mm) parallel to flow direction

PT Peak flow (ms) RVOT PT above (developing) valve annulus parallelto flow direction

(119899 = 18 and 119899 = 12) and 175 dpc (119899 = 12 and 119899 = 4 Table 1)Furthermore the velocity time integral (VTI) that is the areaunder the Doppler velocity envelope for one heart beat wasdetermined (125 dpc 119899 = 15 145 dpc Ao 119899 = 18 and PT119899 = 12 175 dpc Ao 119899 = 12 and PA 119899 = 4) and the diametersof the cOFT (119899 = 2) and Ao (145 dpc 119899 = 3 and 175 dpc119899 = 2) were evaluated from B-mode echoloops in order tocalculate the mean stroke volume (SV) per developmentalstage according to the following formula mean SV = meanTVI times (mean diameter of Ao or cOFT2)2 times 120587 Hence themean cardiac output (CO) was calculated (mean CO =meanSV timesmean HR) for the three developmental stages

24 M-Mode Measurements For morphological assessmentM-mode measurements in short- or long-axis views of theventricles were performed to evaluate the end-systolic andend-diastolic ventricular inner diameter of the LV (LVID125 dpc 119899 = 9 145 dpc 119899 = 11 and 175 dpc 119899 = 14) andRV (RVID 125 dpc 119899 = 9 145 dpc 119899 = 10 and 175 dpc119899 = 12) and IVS diameter (125 dpc 119899 = 4 145 dpc 119899 = 6 and175 dpc 119899 = 9) Cardiac fractional shortening (FS) for theLV and RV was calculated using the formula (VIDdiastole minusVIDsystole)VIDdiastole times 100 Due to the size of thehearts at 125 dpc the resolution often limited the possibilityto discriminate the epicardial myocardial and endocardiallayer of the ventricular free wall Therefore at this stage thedistances from the epicardial layer to the IVS in end-systoleand end-diastole were used to calculate the FS

Although a complete hemodynamic and morphologi-cal assessment for each heart was aimed for (Table 2 for

overview) imaging possibilities were regularly limited by theposition of the embryofetus All echocardiographic record-ings were stored for offline data analysis by NDH and EECinterobserved analysis demonstrated a high level of consis-tency (119903 = 08ndash10 119875 lt 005) Flow parameters were calcu-lated by the average of pulsed-wave Doppler complexes ofthree consecutive beats similar methods were applied forM-mode recordings and diameter calculations on B-modeecholoops

25 Cardiac Morphology and Three-Dimensional Reconstruc-tions After the experiments the pregnant mouse was sac-rificed by cervical dislocation and embryosfetuses wereextractedWe refer to previous publications for details regard-ing embryonicfetal processing sectioning immunohisto-chemistry with MLC2a specific antibodies and preparationof the three-dimensional AMIRA reconstructions (AMIRAsoftware package Template Graphics Software San DiegoUSA) [15]

26 Statistics All statistics were performed with the statisti-cal package for the social sciences 150 (SPSS Inc Chicago ILUSA) andGraphPadPrism (GraphPad Software La Jolla CAUSA) A 119875 lt 005 (2-tailed) was considered to be significantall values are given as mean plusmn SEM All boxplots indicate theupper and lower quartile of the median which is indicatedby the horizontal bar The whiskers indicate maxmin rangeof the values

4 The Scientific World Journal

lowastns

ns80

60

40

20

125 145 175

ET (

)

(c)

40

20

0

minus20

minus40

Velo

city

(cm

s)

10

0

minus10

Freq

uenc

y (k

Hz)

7000 8000 Time (ms)

ECG 230mVResp 230mV

(a)

lowast

nsns80

60

40

20

DFT

()

125 145 175

(b)

20

15

10

5

nsnsns

IVCT

()

125 145 175

Age (dpc)

(d)

lowast

lowast20

15

10

5

ns

125 145 175

Age (dpc)

IVRT

()

(e)

1

23

4

5

(1) Cycle length (RR)

(2) Diastolic filling time (DFT)

(3) Isovolumetric contraction time (IVCT)

(4) Ejection time (ET)

(5) Isovolumetric relaxation time (IVRT)

(a998400)

(a998400)

Figure 1 Development of LV inflow patterns (a) Shows an example of a pulsed-wave Doppler recording across the MV at 175 dpc (a1015840)Indicates the magnification of the boxed area in (a) in which the individual time intervals are indicated that is RR DFT IVCT ET andIVRT (b)Through (e) indicates the course of the DFT ET IVCT and IVRT respectively throughout embryonicfetal life All values in (bndashe)are expressed as percentage of the RR

The Scientific World Journal 5

Table 2 Summary of the assessment of embryonic and fetal hearts

Method Parameter Unit Structure (abbreviation)

2D ultrasoundB-mode echoloops

Morphological assessmentposition and diameters ofcardiac compartments

mm

Right atrium (RA) left atrium (LA) right ventricle (RV) left ventrile (LV)common outflow tract (cOFT) pulmonary trunk (PT) aorta (Ao)inter ventricular septum (IVS) common atrioventricular canal (cAVC)mitral valve (MV) and tricuspid valve (TV)Definition Formula

Pulsed-waveDoppler

Peak E ms Early diastolic ventricular fillingPeak A ms Active diastolic ventricular fillingEA ratioRR interval ms Cycle lengthevaluation of heart rateDFT ms Diastolic filling timeIVRT ms Isovolumetric relaxation timeIVCT ms Isovolumetric contraction timeET ms Ejection timeMPITei index Myocardial performance index IVRT + IVCTETPeak velocities cOFT AoPT ms

VTI mm Velocity time integral

SV Stroke volume SV = TVI times (Ao orcOFT diameter2)2 times 120587

CO mLmin Cardiac output

M-mode

VID mm Ventricular inner diameterLVIDd mm End diastolic left ventricular inner diameterLVIDs mm End systolic left ventricular inner diameterRVIDd mm End diastolic right ventricular inner diameterRVIDs mm End systolic right ventricular inner diameterIVSd mm End diastolic interventricular septum diameterIVSs mm End systolic interventricular septum diameter

FS Fractional shortening (VIDdminusVIDs)VIDd times100

3 Results

31 Cardiac Morphology Reliable imaging of hearts as smallas 2mm was feasible in all stages examined Representativeexamples are shown in online Movie 1 (see SupplementaryMaterial available online at httpdxdoiorg1011552014531324)

At embryonic stages (125 dpc) ventricular septation hasbeen initiated but is not completed resulting in a primitiveinterventricular foramen that directly connects the lumen ofLV and RV M-mode recordings at these stages show a meansystolic IVS diameter of 020 plusmn 0045mm Figure 2(a) showsa reconstruction of a 125 dpc embryonic heart in which thelumen of the four cardiac chambers interventricular fora-men and theOFT can be identifiedThe atrial and ventricularchambers are connected via the cAVC that harbors large AVcushions which will contribute to formation of the MV andTV The AV canal is still partly positioned above the LV atthis stage The developing OFT is still positioned completelyabove the RV and contains cushion tissue that will form thesemilunar valves of the Ao and PTThey will further form thebasis for the muscular subpulmonary infundibulum and will

contribute to themembranous part of the IVS Consequentlythe blood from the LV runs via the primitive interventricularforamen into the cOFT that is future Ao and PT

At early fetal stages of 145 dpc the heart has increased insize (Figure 2(b)) and ventricular septation is now completeThe systolic diameter of the IVS has increased (024 plusmn0018mm) In the AVC the TV orifice has expanded and isnow situated entirely above the RV Also the MV orifice canbe identified as well as the developing AV valves The cardiacOFT now consists of a separate Ao and PT connecting to theLV and RV respectively The Ao and pulmonary valves stillshow immaturity and consist of cushion tissue

At late fetal stages (175 dpc) the size of the RV andLV and IVS (039 plusmn 0009mm) has increased significantly(Figure 2(c)) Mature valves are present at the levels of the in-and outflow parts of both ventricles

32 Diastolic and Systolic Function During embryonic andfetal development theHR increases significantly (119875 lt 00001)from 125 plusmn 95 up to 219 plusmn 83BPM Ventricular inflowpatterns across the developing MV and TV annulus werestudied by pulsed-wave Doppler imaging

6 The Scientific World Journal

(a) (b) (c)

lowast

lowastlowastlowast150

125

100

075

050

025

000

Dia

met

er lu

men

(mm

)

Left ventricle

Right ventricle

Diastole

Systole

125 145 175

Age (dpc)

ns

(d)

lowast

125 145 175

Age (dpc)

50

40

30

20

10

0

Shor

teni

ng fr

actio

n (

)

Left ventricle

Right ventricle

(e)

Figure 2 Cardiac morphology and FS calculation (a) Shows a reconstruction of the anterior view of an embryonic heart of 125 dpc Themyocardium is indicated in grey transparent The LA and RA are indicated in transparent dark grey The LV and RV lumen are indicated inred and blue respectively Note that the outflow tract lumen (purple) is positioned completely above the future RV which is surrounded bylarge outflow tract cushions (green transparent) At these stages development of the IVS has not yet completed leading to a direct connectionbetween the LV and RV via the interventricular foramen (arrow) (b) Anterior view of an early fetal heart of 145 dpc At this stage IVSdevelopment has been completed and four separate cardiac chambers can be identified The outflow tract consists of a separate Ao and PTincluding their valve apparatus which at these stages mainly consist of cushion tissue (green transparent) (c) Anterior view of a late fetalheart of 175 dpc At this stage the heart shows a mature morphological phenotype (d) Schematic representation of LV and RV diameters and(e) FS at the three consecutive stages of development

The DFT corrected for the RR interval (DFTRR) did notdiffer significantly at the three stages assessed (Figure 1(b))LV and RV filling were dominated by the peak-A waveBetween 125 and 175 dpc the peak-A wave of the developingMV annulus increased significantly (033 plusmn 0001ms versus042 plusmn 0014ms 119875 lt 00001) whereas that of the TV alsoshowed an increasing trend albeit not significant (037 plusmn0018ms versus 041 plusmn 0019ms 119875 = NS) The peak-Ewave also increased significantly for both LV (005plusmn0006msversus 018plusmn0005ms 119875 lt 00001) and RV (006plusmn0006msversus 018 plusmn 0012ms 119875 lt 00001) and showed a relativelygreater increase than the peak-A wave during development

As a result the EA ratio a measure for the ventricular dia-stolic function showed a significant increase for both LV(016 plusmn 0015 versus 044 plusmn 0013 119875 lt 00001) and RV(016 plusmn 0010 versus 044 plusmn 0014 119875 lt 00001) (Figures 3(a)ndash3(f))

The IVRT one of the measures for diastolic LV functioncorrected for the RR interval (IVRTRR) demonstrated asignificant (119875 = 0037) decrease between 125 (015 plusmn 0011)and 145 dpc (012 plusmn 0004) and remained stable at later fetalstages 175 dpc (012 plusmn 0003) (Figure 1(e)) indicating that LVmyocardial relaxation remains stable after completion of IVSdevelopment during fetal life

The Scientific World Journal 7

Mitral06

05

04

03

02

01

00

EA

ratio

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

125 145 175

(a)

06

05

04

03

02

01

00

EA

ratio

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

125 145 175

Tricuspid

(b)

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

125 145 175

Peak

E (m

s)

04

03

02

01

00

(c)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

125 145 175

Peak

E (m

s)

04

03

02

01

00

(d)

lowastlowastlowast

125 145 175

Age (dpc)

Peak

A (m

s)

07

06

05

04

03

02

01

00

nslowastlowast

(e)

125 145 175

Age (dpc)

Peak

A (m

s)

07

06

05

04

03

02

01

00

nsns

ns

(f)

Figure 3 Development of LV and RV inflow patternsThe graphs represent the course of the EA ratio (a b) peak-E wave (c d) and peak-Awave (e f) across the developing MV and TV at the three subsequent developmental stages

The systolic function of the developing ventricles wasassessed through studying the course of the FS IVCTRRand ETRR Throughout embryonic and fetal developmentthe FS remained constant for both ventricles and valuesranged between asymp30 and 40 Furthermore except for175 dpc (LV 406 RV 357 119875 = 0046) no significantdifferences were observed between the FS of the LV and RV

(Figures 2(d) and 2(e)) The mean IVCTRR remained stableduring all three stagesasymp009 the ETRR showed no changesbetween 125 dpc (042plusmn0015) and 145 dpc (043plusmn0010) butsignificantly decreased at 175 dpc (040 plusmn 0006 119875 = 0017)(Figures 1(c) and 1(d))

A combined assessment of LV systolic and diastolic func-tion was performed by calculating the MPI At embryonic

8 The Scientific World Journal

Table 3 The parameters used for calculation of the mean CO per gestational age

Parameter 125 dpc (cOFT) 145 dpc (Ao) 175 dpc (Ao)VTI (mm) 339 plusmn 137 423 plusmn 20 292 plusmn 205

Mean diameter (mm) 023 018 048 plusmn 003

Mean HR (bpm) 134 plusmn 112 148 plusmn 61 206 plusmn 98

Mean CO (mLmin) 019 016 096

lowastlowastlowast05

04

03

02

01

Peak

flow

(ms

)

Common OFT

Aorta

Pulmonary trunk

125 145 175

Age (dpc)

Figure 4 Pulsed-wave Doppler flow measurements in the cOFTAo and PT The graph demonstrates the significant increase of thepeak blood flowmeasured in the cOFT at 125 and Ao and PT at 145and 175 dpc

preseptated stages (125 dpc) the MPI was 064 plusmn 0012 anddecreased to 049 plusmn 0017 (119875 = 00002) after closure of theIVS at 145 dpc At late fetal stages (175 dpc) the MPI did notchange significantly (053 plusmn 0022 119875 = NS)

33 OutflowTract andCO Thepulsed-waveDoppler record-ings performed in the cOFT demonstrated a peak flow of027 plusmn 0021ms at preseptated stages As from 145 dpc twoindividual ventricular outflow tracts could be discriminatedespecially the high echodensity of the blood at these stagesenabled to discriminate the flow in the Ao (034 plusmn 0013ms)and PT (033 plusmn 0021ms) that are closely related to eachother As can be observed in Figure 4 the peak flow increasedsignificantly in the Ao (from 027 plusmn 0021ms to 036 plusmn0016ms 119875 = 0010) and PT (027 plusmn 0021ms to 044 plusmn0030ms 119875 = 0003) along fetal life

Table 3 summarizes theHRVTI and the diameters of thecOFT and Ao that were used to calculate the mean CO at thethree developmental stages Between 125 dpc and 145 dpc the

CO remained stable where after it increased to 096mLminat 175 dpc

4 Discussion

Heart development is a complex process which has beenextensively studied in both WT and genetically mutatedmouse models [3] It is only in the last decades that modernimaging techniques like ultrasound enable the assessmentof real-time hemodynamic and morphological changes thatoccur during mouse heart development [4ndash6 8 16ndash18]Knowledge regarding these functional parameters is essentialsince abnormal flow in the developing heart might lead toCHD through aberrant activation of shear-stress responsivegenes important in cardiogenesis [9 11] In early reportsdescribing the course of functional parameters during heartdevelopmentmajor variability exists inmeasured valuesThisis most likely related to the relatively low ultrasound frequen-cies of clinical ultrasound machines used to image the smallhearts of mouse embryosfetuses [16] and externalizationof uterus horns during ultrasound assessment [18] whichinfluences embryonicfetal physiology Low dose isofluraneanesthesia used in this study only has a minimal effect onhemodynamics and diastolic function in adult mice and theeffect on mouse embryos is supposed to be marginal [6]

In the current study we aimed to provide an overview ofhemodynamic parameters and changes that occur at subse-quent stages of heart development using a high-frequencyultrasound system

Compared to other reports in mouse using similar ultra-sound techniques we did not only focus on development ofthe future LV but also on that of the RV Key findings ofour study are (1) a reliable assessment of LV and RV in- andoutflow patterns can be performed from 125 dpc to term (2)a significant improvement occurs in the diastolic and systolicfunction of both LV and RV (3) over the developmentalperiod of 125 dpc to 175 dpc the ratio between RV and LVsize as well as the FS of both ventricles did not changesignificantly

Knowledge regarding the functional parameters of theRV is important since the RV is commonly affected in CHDDuring development the RV including the TV annulusdevelops subsequent to the LV by addition of SHF derivedcells to the developing heart [1] Many genes have beenidentified that have an important role in the SHF contributionto the developing heart andmutations in these genesmay leadto CHD [19]

At early embryonic stages endocardial cushion tissue ispositioned in the primitive AVC and OFT which later on

The Scientific World Journal 9

will contribute to formation of the TV and MV and Ao andPT semilunar valves Some clinical studies argued that AVvalve regurgitation is a commonphenomenon during the firsttrimester of pregnancy [20] Our study in accordance withseveral others in mouse [4 6] and human [13] did not showsigns of regurgitation at both early and late stages of heartdevelopment Pulsed-wave Doppler recordings across thecAVC at 125 dpc and MV and TV at 145 dpc demonstratedthat the AV cushionsmimic the function of normal AV valvesat 175 dpc In our opinion especially the flow in the cOFTat early embryonic stages which is completely positionedabove the RV (see Figure 2(a)) might wrongly be identifiedas regurgitation of the developing AV valvescushions Thisstresses the importance of detailed knowledge regarding therapid morphological changes that the heart undergoes atespecially the very early developmental stages

Regarding growth of the heart we demonstrated that therewas a 31 increase of LVID 34 of RIVD and 35 of IVSdiameter from 125 up to 175 dpcWe showed a trend towardsa smaller RVID as compared to the LVID at all stages whichwas not mentioned in other studies

41 Diastolic and Systolic Function during Heart DevelopmentImprovement of ventricular systolic and diastolic functionoccurs throughout the length of embryonic and fetal develop-mentWith respect to the diastolic function we demonstrateda significant increase of EA ratio across the developing MVand TV orifices which was related to an increase of the peak-E wave that is the fraction of diastolic ventricular filling thatis defined by ventricular relaxation These data are in accor-dance with the study of Zhou and colleagues who demon-strated that the EA ratio is increasing during developmentwhich seems to continue up to approximately 3 weeks afterbirth [6] Around day four after birth the peak-E wave mea-sured across theMV becomes more dominant than the peak-A wave indicating an improvement of cardiac complianceand that LV filling becomes dominated by passive ventricularrelaxation Remarkably these changes were not observed forthe RV inflow patterns where the peak-A wave remainsdominant [6] Human embryonic and fetal hearts show thesame maturation course of EA ratios across the developingMV and TV as was demonstrated in multiple studies [21ndash25]An abnormal course of EA ratio that is a decreasing[25 26] or an increasing trend [27] along development hasbeen related to the presence of CHD

With respect to systolic ventricular function the FSremained stable (asymp30ndash40) for both ventricles throughoutdevelopmentWe did find a significant difference favoring theLV FS as compared to the RV FS at 175 dpc Interestinglythe same phenomenon of FS difference between LV andRV has been reported in a study of human fetal heartsbetween 14 and 40weeks of pregnancy However as inmousedifferences between humanLVandRVFS is subtle [28] andwhether a decreased or decreasing FS is a predictor of CHDremains unknown Furthermore we did not find a decreasingtrend of both LV and RV FS as was recently demonstratedduring human fetal life [28 29] With respect to the CO wenoticed no change around the proces of ventricular septation(between 125 and 145 dpc) where after a major increase

occured to 096mLmin near term (175 dpc) The initial COdecreasemight be related to the fact that themeandiameter ofthe unseptated cOFT diameter was used at 125 dpc comparedto the isolated Ao diameter at 145 dpc The subsequent COincrease is mostly related to the dramatic increase of HRalong fetal life

The MPI or Tei index [14] is a measure for combinedsystolic and diastolic cardiac function and can be calculatedfor both ventricles In order to assess the MPI three differenttime intervals are needed the IVCT IVRT and ET For the LVthese parameters can be easily obtained since all the threeparameters can be assessed at once by placing the Dopplerultrasound beam across the developing MV orifice Notice-ably in literature there is less data regarding RV MPI andits course along development which might be related to thefact that for the RV not all the three time intervals can beassessed in one view as was also suggested in a recent reviewby Godfrey et al [14] The current study demonstrated asignificant decrease of LV MPI between 125 and 145 dpcwhich might be related to the rapid morphological changesthe heart goes through including completion of IVS leadingto altered hemodynamic conditions in the developing heart Itis however also important to notice that at preseptation stagesthe IVCT and IVRT cannot always be identified separately sothat calculation of the MPI is frequently not possible at veryearly stages of embryonic development Between 145 and175 dpcwenoticed a slight increase of theMPI suggesting thatcardiac function improves at subsequent fetal stages similarresults have also been demonstrated inmouse [4] and human[30] However discussion remains regarding the exact courseof theMPI IVCT IVRT andETandwhether isolated changesof one of the factors is a predictor for fetal demise and CHDInterestinglywith respect to IVRTweobserved a decreasingtrend near completion of the IVS (between 125 and 145)whereafter it more or less remained stable throughout fetallife A similar course was observed in a recent ultrasoundstudy in embryonicfetal C57Bl6 mice [4] Human embryosalso showed a decreasing course of the IVRT [13 21] and anelevated IVRT has been implicated as a predictor of CHD[13]

5 Conclusion

We demonstrated the course of several morphologic andhemodynamic parameters along embryonic and fetal lifeOur data show that a reliable assessment can be made notonly of the left but also of right ventricular function duringembryonicfetal stages We demonstrate an overall improve-ment of cardiac diastolic and systolic function of both LV andRV along heart development The implementation of high-frequency ultrasound in embryonicfetal mouse heart devel-opment is a promising and useful tool The data presented inthis studymight be useful as reference values in future studiesin genetic mutated mouse models of CHD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

10 The Scientific World Journal

Authorsrsquo Contribution

Nathan D Hahurij and Emmeline E Calkoen contributedequally to this work and shared first authorship

Acknowledgment

This work is supported by the DutchHeart Foundation Grantno 2009T070 (Nathan D Hahurij)

References

[1] R G Kelly ldquoMolecular inroads into the anterior heart fieldrdquoTrends inCardiovascularMedicine vol 15 no 2 pp 51ndash56 2005

[2] J I E Hoffman and S Kaplan ldquoThe incidence of congenitalheart diseaserdquo Journal of the AmericanCollege of Cardiology vol39 no 12 pp 1890ndash1900 2002

[3] D Srivastava ldquoGenetic assembly of the heart implications forcongenital heart diseaserdquo Annual Review of Physiology vol 63pp 451ndash469 2001

[4] N Corrigan D P Brazil and F M Auliffe ldquoHigh-frequencyultrasound assessment of the murine heart from embryothrough to juvenilerdquoReproductive Sciences vol 17 no 2 pp 147ndash157 2010

[5] C F Spurney CW Lo and L Leatherbury ldquoFetal mouse imag-ing using echocardiography a review of current technologyrdquoEchocardiography vol 23 no 10 pp 891ndash899 2006

[6] Y-Q Zhou F S Foster R Parkes and S L Adamson ldquoDevel-opmental changes in left and right ventricular diastolic fillingpatterns in micerdquo American Journal of Physiology vol 285 no4 pp H1563ndashH1575 2003

[7] Q Yu L Leatherbury X Tian and C W Lo ldquoCardiovascularassessment of fetal mice by in utero echocardiographyrdquo Ultra-sound in Medicine and Biology vol 34 no 5 pp 741ndash752 2008

[8] C K L Phoon ldquoImaging tools for the developmental biologistultrasound biomicroscopy of mouse embryonic developmentrdquoPediatric Research vol 60 no 1 pp 14ndash21 2006

[9] J S Lee Q Yu J T Shin et al ldquoKlf2 is an essential regulator ofvascular hemodynamic forces in vivordquo Developmental Cell vol11 no 6 pp 845ndash857 2006

[10] R A Morris B Damon V Mironov et al ldquoPeriostin regulatescollagen fibrillogenesis and the biomechanical properties ofconnective tissuesrdquo Journal of Cellular Biochemistry vol 101 no3 pp 695ndash711 2007

[11] B C W Groenendijk B P Hierck J Vrolijk et al ldquoChanges inshear stress-related gene expression after experimentally alteredvenous return in the chicken embryordquoCirculation Research vol96 no 12 pp 1291ndash1298 2005

[12] C J Zuurbier V M Emons and C Ince ldquoHemodynamics ofanesthetized ventilated mouse models aspects of anestheticsfluid support and strainrdquo American Journal of Physiology vol282 no 6 pp H2099ndashH2105 2002

[13] A Włoch W Rozmus-Warcholinska B Czuba et al ldquoDopplerstudy of the embryonic heart in normal pregnant womenrdquoJournal of Maternal-Fetal and Neonatal Medicine vol 20 no 7pp 533ndash539 2007

[14] M E Godfrey B Messing S M Cohen D V Valsky andS Yagel ldquoFunctional assessment of the fetal heart a reviewrdquo

Ultrasound in Obstetrics and Gynecology vol 39 no 2 pp 131ndash144 2012

[15] M R M Jongbloed M C E F Wijffels M J Schalij et alldquoDevelopment of the right ventricular inflow tract and moder-ator band a possible morphological and functional explanationforMahaim tachycardiardquoCirculation Research vol 96 no 7 pp776ndash783 2005

[16] Y-HGui KK Linask PKhowsathit and J CHuhta ldquoDopplerechocardiography of normal and abnormal embryonic mouseheartrdquo Pediatric Research vol 40 no 4 pp 633ndash642 1996

[17] C F Spurney L Leatherbury and C W Lo ldquoHigh-frequencyultrasound database profiling growth development and car-diovascular function in C57BL6J mouse fetusesrdquo Journal of theAmerican Society of Echocardiography vol 17 no 8 pp 893ndash900 2004

[18] R P Ji C K L Phoon O Aristizabal K E McGrath JPalis and D H Turnbull ldquoOnset of cardiac function duringearly mouse embryogenesis coincides with entry of primitiveerythroblasts into the embryo properrdquoCirculation Research vol92 no 2 pp 133ndash135 2003

[19] M R Jongbloed S R Vicente N D Hahurij et al ldquoNormaland abnormal development of the cardiac conduction systemimplications for conduction and rhythm disorders in the childand adultrdquo Differentiation vol 84 no 1 pp 131ndash148 2012

[20] K Makikallio P Jouppila and J Rasanen ldquoHuman fetal cardiacfunction during the first trimester of pregnancyrdquo Heart vol 91no 3 pp 334ndash338 2005

[21] W Rozmus-Warcholinska A Wloch G Acharya et al ldquoRef-erence values for variables of fetal cardiocirculatory dynamicsat 11-14 weeks of gestationrdquo Ultrasound in Obstetrics andGynecology vol 35 no 5 pp 540ndash547 2010

[22] G Tulzer P Khowsathit S Gudmundsson et al ldquoDiastolicfunction of the fetal heart during second and third trimestera prospective longitudinal Doppler-echocardiographic studyrdquoEuropean Journal of Pediatrics vol 153 no 3 pp 151ndash154 1994

[23] MC Leiva J E Tolosa CN Binotto et al ldquoFetal cardiac devel-opment and hemodynamics in the first trimesterrdquoUltrasound inObstetrics and Gynecology vol 14 no 3 pp 169ndash174 1999

[24] I P Van Splunder and J W Wladimiroff ldquoCardiac functionalchanges in the human fetus in the late first and early secondtrimestersrdquo Ultrasound in Obstetrics and Gynecology vol 7 no6 pp 411ndash415 1996

[25] S A Clur R K Oude B W Mol J Ottenkamp and CM Bilardo ldquoFetal cardiac function between 11 and 35 weeksrsquogestation and nuchal translucency thicknessrdquo Ultrasound inObstetrics amp Gynecology vol 37 no 1 pp 48ndash56 2011

[26] G Rizzo A Muscatello E Angelini and A Capponi ldquoAbnor-mal cardiac function in fetuses with increased nuchal translu-cencyrdquo Ultrasound in Obstetrics and Gynecology vol 21 no 6pp 539ndash542 2003

[27] F Crispi E Hernandez-Andrade M M A L Pelsers et alldquoCardiac dysfunction and cell damage across clinical stagesof severity in growth-restricted fetusesrdquo American Journal ofObstetrics and Gynecology vol 199 no 3 pp 254ndash258 2008

[28] S Luewan Y Yanase F Tongprasert K Srisupundit and TTongsong ldquoFetal cardiac dimensions at 14-40 weeksrsquo gestationobtained using cardio-STIC-Mrdquo Ultrasound in Obstetrics andGynecology vol 37 no 4 pp 416ndash422 2011

The Scientific World Journal 11

[29] T Tongsong C Wanapirak W Piyamongkol et al ldquoFetalventricular shortening fraction in hydrops fetalisrdquo Obstetricsand Gynecology vol 117 no 1 pp 84ndash91 2011

[30] E Hernandez-Andrade H Figueroa-Diesel C Kottman etal ldquoGestational-age-adjusted reference values for the modifiedmyocardial performance index for evaluation of fetal left car-diac functionrdquoUltrasound in Obstetrics and Gynecology vol 29no 3 pp 321ndash325 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 The Scientific World Journal

lowastns

ns80

60

40

20

125 145 175

ET (

)

(c)

40

20

0

minus20

minus40

Velo

city

(cm

s)

10

0

minus10

Freq

uenc

y (k

Hz)

7000 8000 Time (ms)

ECG 230mVResp 230mV

(a)

lowast

nsns80

60

40

20

DFT

()

125 145 175

(b)

20

15

10

5

nsnsns

IVCT

()

125 145 175

Age (dpc)

(d)

lowast

lowast20

15

10

5

ns

125 145 175

Age (dpc)

IVRT

()

(e)

1

23

4

5

(1) Cycle length (RR)

(2) Diastolic filling time (DFT)

(3) Isovolumetric contraction time (IVCT)

(4) Ejection time (ET)

(5) Isovolumetric relaxation time (IVRT)

(a998400)

(a998400)

Figure 1 Development of LV inflow patterns (a) Shows an example of a pulsed-wave Doppler recording across the MV at 175 dpc (a1015840)Indicates the magnification of the boxed area in (a) in which the individual time intervals are indicated that is RR DFT IVCT ET andIVRT (b)Through (e) indicates the course of the DFT ET IVCT and IVRT respectively throughout embryonicfetal life All values in (bndashe)are expressed as percentage of the RR

The Scientific World Journal 5

Table 2 Summary of the assessment of embryonic and fetal hearts

Method Parameter Unit Structure (abbreviation)

2D ultrasoundB-mode echoloops

Morphological assessmentposition and diameters ofcardiac compartments

mm

Right atrium (RA) left atrium (LA) right ventricle (RV) left ventrile (LV)common outflow tract (cOFT) pulmonary trunk (PT) aorta (Ao)inter ventricular septum (IVS) common atrioventricular canal (cAVC)mitral valve (MV) and tricuspid valve (TV)Definition Formula

Pulsed-waveDoppler

Peak E ms Early diastolic ventricular fillingPeak A ms Active diastolic ventricular fillingEA ratioRR interval ms Cycle lengthevaluation of heart rateDFT ms Diastolic filling timeIVRT ms Isovolumetric relaxation timeIVCT ms Isovolumetric contraction timeET ms Ejection timeMPITei index Myocardial performance index IVRT + IVCTETPeak velocities cOFT AoPT ms

VTI mm Velocity time integral

SV Stroke volume SV = TVI times (Ao orcOFT diameter2)2 times 120587

CO mLmin Cardiac output

M-mode

VID mm Ventricular inner diameterLVIDd mm End diastolic left ventricular inner diameterLVIDs mm End systolic left ventricular inner diameterRVIDd mm End diastolic right ventricular inner diameterRVIDs mm End systolic right ventricular inner diameterIVSd mm End diastolic interventricular septum diameterIVSs mm End systolic interventricular septum diameter

FS Fractional shortening (VIDdminusVIDs)VIDd times100

3 Results

31 Cardiac Morphology Reliable imaging of hearts as smallas 2mm was feasible in all stages examined Representativeexamples are shown in online Movie 1 (see SupplementaryMaterial available online at httpdxdoiorg1011552014531324)

At embryonic stages (125 dpc) ventricular septation hasbeen initiated but is not completed resulting in a primitiveinterventricular foramen that directly connects the lumen ofLV and RV M-mode recordings at these stages show a meansystolic IVS diameter of 020 plusmn 0045mm Figure 2(a) showsa reconstruction of a 125 dpc embryonic heart in which thelumen of the four cardiac chambers interventricular fora-men and theOFT can be identifiedThe atrial and ventricularchambers are connected via the cAVC that harbors large AVcushions which will contribute to formation of the MV andTV The AV canal is still partly positioned above the LV atthis stage The developing OFT is still positioned completelyabove the RV and contains cushion tissue that will form thesemilunar valves of the Ao and PTThey will further form thebasis for the muscular subpulmonary infundibulum and will

contribute to themembranous part of the IVS Consequentlythe blood from the LV runs via the primitive interventricularforamen into the cOFT that is future Ao and PT

At early fetal stages of 145 dpc the heart has increased insize (Figure 2(b)) and ventricular septation is now completeThe systolic diameter of the IVS has increased (024 plusmn0018mm) In the AVC the TV orifice has expanded and isnow situated entirely above the RV Also the MV orifice canbe identified as well as the developing AV valves The cardiacOFT now consists of a separate Ao and PT connecting to theLV and RV respectively The Ao and pulmonary valves stillshow immaturity and consist of cushion tissue

At late fetal stages (175 dpc) the size of the RV andLV and IVS (039 plusmn 0009mm) has increased significantly(Figure 2(c)) Mature valves are present at the levels of the in-and outflow parts of both ventricles

32 Diastolic and Systolic Function During embryonic andfetal development theHR increases significantly (119875 lt 00001)from 125 plusmn 95 up to 219 plusmn 83BPM Ventricular inflowpatterns across the developing MV and TV annulus werestudied by pulsed-wave Doppler imaging

6 The Scientific World Journal

(a) (b) (c)

lowast

lowastlowastlowast150

125

100

075

050

025

000

Dia

met

er lu

men

(mm

)

Left ventricle

Right ventricle

Diastole

Systole

125 145 175

Age (dpc)

ns

(d)

lowast

125 145 175

Age (dpc)

50

40

30

20

10

0

Shor

teni

ng fr

actio

n (

)

Left ventricle

Right ventricle

(e)

Figure 2 Cardiac morphology and FS calculation (a) Shows a reconstruction of the anterior view of an embryonic heart of 125 dpc Themyocardium is indicated in grey transparent The LA and RA are indicated in transparent dark grey The LV and RV lumen are indicated inred and blue respectively Note that the outflow tract lumen (purple) is positioned completely above the future RV which is surrounded bylarge outflow tract cushions (green transparent) At these stages development of the IVS has not yet completed leading to a direct connectionbetween the LV and RV via the interventricular foramen (arrow) (b) Anterior view of an early fetal heart of 145 dpc At this stage IVSdevelopment has been completed and four separate cardiac chambers can be identified The outflow tract consists of a separate Ao and PTincluding their valve apparatus which at these stages mainly consist of cushion tissue (green transparent) (c) Anterior view of a late fetalheart of 175 dpc At this stage the heart shows a mature morphological phenotype (d) Schematic representation of LV and RV diameters and(e) FS at the three consecutive stages of development

The DFT corrected for the RR interval (DFTRR) did notdiffer significantly at the three stages assessed (Figure 1(b))LV and RV filling were dominated by the peak-A waveBetween 125 and 175 dpc the peak-A wave of the developingMV annulus increased significantly (033 plusmn 0001ms versus042 plusmn 0014ms 119875 lt 00001) whereas that of the TV alsoshowed an increasing trend albeit not significant (037 plusmn0018ms versus 041 plusmn 0019ms 119875 = NS) The peak-Ewave also increased significantly for both LV (005plusmn0006msversus 018plusmn0005ms 119875 lt 00001) and RV (006plusmn0006msversus 018 plusmn 0012ms 119875 lt 00001) and showed a relativelygreater increase than the peak-A wave during development

As a result the EA ratio a measure for the ventricular dia-stolic function showed a significant increase for both LV(016 plusmn 0015 versus 044 plusmn 0013 119875 lt 00001) and RV(016 plusmn 0010 versus 044 plusmn 0014 119875 lt 00001) (Figures 3(a)ndash3(f))

The IVRT one of the measures for diastolic LV functioncorrected for the RR interval (IVRTRR) demonstrated asignificant (119875 = 0037) decrease between 125 (015 plusmn 0011)and 145 dpc (012 plusmn 0004) and remained stable at later fetalstages 175 dpc (012 plusmn 0003) (Figure 1(e)) indicating that LVmyocardial relaxation remains stable after completion of IVSdevelopment during fetal life

The Scientific World Journal 7

Mitral06

05

04

03

02

01

00

EA

ratio

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

125 145 175

(a)

06

05

04

03

02

01

00

EA

ratio

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

125 145 175

Tricuspid

(b)

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

125 145 175

Peak

E (m

s)

04

03

02

01

00

(c)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

125 145 175

Peak

E (m

s)

04

03

02

01

00

(d)

lowastlowastlowast

125 145 175

Age (dpc)

Peak

A (m

s)

07

06

05

04

03

02

01

00

nslowastlowast

(e)

125 145 175

Age (dpc)

Peak

A (m

s)

07

06

05

04

03

02

01

00

nsns

ns

(f)

Figure 3 Development of LV and RV inflow patternsThe graphs represent the course of the EA ratio (a b) peak-E wave (c d) and peak-Awave (e f) across the developing MV and TV at the three subsequent developmental stages

The systolic function of the developing ventricles wasassessed through studying the course of the FS IVCTRRand ETRR Throughout embryonic and fetal developmentthe FS remained constant for both ventricles and valuesranged between asymp30 and 40 Furthermore except for175 dpc (LV 406 RV 357 119875 = 0046) no significantdifferences were observed between the FS of the LV and RV

(Figures 2(d) and 2(e)) The mean IVCTRR remained stableduring all three stagesasymp009 the ETRR showed no changesbetween 125 dpc (042plusmn0015) and 145 dpc (043plusmn0010) butsignificantly decreased at 175 dpc (040 plusmn 0006 119875 = 0017)(Figures 1(c) and 1(d))

A combined assessment of LV systolic and diastolic func-tion was performed by calculating the MPI At embryonic

8 The Scientific World Journal

Table 3 The parameters used for calculation of the mean CO per gestational age

Parameter 125 dpc (cOFT) 145 dpc (Ao) 175 dpc (Ao)VTI (mm) 339 plusmn 137 423 plusmn 20 292 plusmn 205

Mean diameter (mm) 023 018 048 plusmn 003

Mean HR (bpm) 134 plusmn 112 148 plusmn 61 206 plusmn 98

Mean CO (mLmin) 019 016 096

lowastlowastlowast05

04

03

02

01

Peak

flow

(ms

)

Common OFT

Aorta

Pulmonary trunk

125 145 175

Age (dpc)

Figure 4 Pulsed-wave Doppler flow measurements in the cOFTAo and PT The graph demonstrates the significant increase of thepeak blood flowmeasured in the cOFT at 125 and Ao and PT at 145and 175 dpc

preseptated stages (125 dpc) the MPI was 064 plusmn 0012 anddecreased to 049 plusmn 0017 (119875 = 00002) after closure of theIVS at 145 dpc At late fetal stages (175 dpc) the MPI did notchange significantly (053 plusmn 0022 119875 = NS)

33 OutflowTract andCO Thepulsed-waveDoppler record-ings performed in the cOFT demonstrated a peak flow of027 plusmn 0021ms at preseptated stages As from 145 dpc twoindividual ventricular outflow tracts could be discriminatedespecially the high echodensity of the blood at these stagesenabled to discriminate the flow in the Ao (034 plusmn 0013ms)and PT (033 plusmn 0021ms) that are closely related to eachother As can be observed in Figure 4 the peak flow increasedsignificantly in the Ao (from 027 plusmn 0021ms to 036 plusmn0016ms 119875 = 0010) and PT (027 plusmn 0021ms to 044 plusmn0030ms 119875 = 0003) along fetal life

Table 3 summarizes theHRVTI and the diameters of thecOFT and Ao that were used to calculate the mean CO at thethree developmental stages Between 125 dpc and 145 dpc the

CO remained stable where after it increased to 096mLminat 175 dpc

4 Discussion

Heart development is a complex process which has beenextensively studied in both WT and genetically mutatedmouse models [3] It is only in the last decades that modernimaging techniques like ultrasound enable the assessmentof real-time hemodynamic and morphological changes thatoccur during mouse heart development [4ndash6 8 16ndash18]Knowledge regarding these functional parameters is essentialsince abnormal flow in the developing heart might lead toCHD through aberrant activation of shear-stress responsivegenes important in cardiogenesis [9 11] In early reportsdescribing the course of functional parameters during heartdevelopmentmajor variability exists inmeasured valuesThisis most likely related to the relatively low ultrasound frequen-cies of clinical ultrasound machines used to image the smallhearts of mouse embryosfetuses [16] and externalizationof uterus horns during ultrasound assessment [18] whichinfluences embryonicfetal physiology Low dose isofluraneanesthesia used in this study only has a minimal effect onhemodynamics and diastolic function in adult mice and theeffect on mouse embryos is supposed to be marginal [6]

In the current study we aimed to provide an overview ofhemodynamic parameters and changes that occur at subse-quent stages of heart development using a high-frequencyultrasound system

Compared to other reports in mouse using similar ultra-sound techniques we did not only focus on development ofthe future LV but also on that of the RV Key findings ofour study are (1) a reliable assessment of LV and RV in- andoutflow patterns can be performed from 125 dpc to term (2)a significant improvement occurs in the diastolic and systolicfunction of both LV and RV (3) over the developmentalperiod of 125 dpc to 175 dpc the ratio between RV and LVsize as well as the FS of both ventricles did not changesignificantly

Knowledge regarding the functional parameters of theRV is important since the RV is commonly affected in CHDDuring development the RV including the TV annulusdevelops subsequent to the LV by addition of SHF derivedcells to the developing heart [1] Many genes have beenidentified that have an important role in the SHF contributionto the developing heart andmutations in these genesmay leadto CHD [19]

At early embryonic stages endocardial cushion tissue ispositioned in the primitive AVC and OFT which later on

The Scientific World Journal 9

will contribute to formation of the TV and MV and Ao andPT semilunar valves Some clinical studies argued that AVvalve regurgitation is a commonphenomenon during the firsttrimester of pregnancy [20] Our study in accordance withseveral others in mouse [4 6] and human [13] did not showsigns of regurgitation at both early and late stages of heartdevelopment Pulsed-wave Doppler recordings across thecAVC at 125 dpc and MV and TV at 145 dpc demonstratedthat the AV cushionsmimic the function of normal AV valvesat 175 dpc In our opinion especially the flow in the cOFTat early embryonic stages which is completely positionedabove the RV (see Figure 2(a)) might wrongly be identifiedas regurgitation of the developing AV valvescushions Thisstresses the importance of detailed knowledge regarding therapid morphological changes that the heart undergoes atespecially the very early developmental stages

Regarding growth of the heart we demonstrated that therewas a 31 increase of LVID 34 of RIVD and 35 of IVSdiameter from 125 up to 175 dpcWe showed a trend towardsa smaller RVID as compared to the LVID at all stages whichwas not mentioned in other studies

41 Diastolic and Systolic Function during Heart DevelopmentImprovement of ventricular systolic and diastolic functionoccurs throughout the length of embryonic and fetal develop-mentWith respect to the diastolic function we demonstrateda significant increase of EA ratio across the developing MVand TV orifices which was related to an increase of the peak-E wave that is the fraction of diastolic ventricular filling thatis defined by ventricular relaxation These data are in accor-dance with the study of Zhou and colleagues who demon-strated that the EA ratio is increasing during developmentwhich seems to continue up to approximately 3 weeks afterbirth [6] Around day four after birth the peak-E wave mea-sured across theMV becomes more dominant than the peak-A wave indicating an improvement of cardiac complianceand that LV filling becomes dominated by passive ventricularrelaxation Remarkably these changes were not observed forthe RV inflow patterns where the peak-A wave remainsdominant [6] Human embryonic and fetal hearts show thesame maturation course of EA ratios across the developingMV and TV as was demonstrated in multiple studies [21ndash25]An abnormal course of EA ratio that is a decreasing[25 26] or an increasing trend [27] along development hasbeen related to the presence of CHD

With respect to systolic ventricular function the FSremained stable (asymp30ndash40) for both ventricles throughoutdevelopmentWe did find a significant difference favoring theLV FS as compared to the RV FS at 175 dpc Interestinglythe same phenomenon of FS difference between LV andRV has been reported in a study of human fetal heartsbetween 14 and 40weeks of pregnancy However as inmousedifferences between humanLVandRVFS is subtle [28] andwhether a decreased or decreasing FS is a predictor of CHDremains unknown Furthermore we did not find a decreasingtrend of both LV and RV FS as was recently demonstratedduring human fetal life [28 29] With respect to the CO wenoticed no change around the proces of ventricular septation(between 125 and 145 dpc) where after a major increase

occured to 096mLmin near term (175 dpc) The initial COdecreasemight be related to the fact that themeandiameter ofthe unseptated cOFT diameter was used at 125 dpc comparedto the isolated Ao diameter at 145 dpc The subsequent COincrease is mostly related to the dramatic increase of HRalong fetal life

The MPI or Tei index [14] is a measure for combinedsystolic and diastolic cardiac function and can be calculatedfor both ventricles In order to assess the MPI three differenttime intervals are needed the IVCT IVRT and ET For the LVthese parameters can be easily obtained since all the threeparameters can be assessed at once by placing the Dopplerultrasound beam across the developing MV orifice Notice-ably in literature there is less data regarding RV MPI andits course along development which might be related to thefact that for the RV not all the three time intervals can beassessed in one view as was also suggested in a recent reviewby Godfrey et al [14] The current study demonstrated asignificant decrease of LV MPI between 125 and 145 dpcwhich might be related to the rapid morphological changesthe heart goes through including completion of IVS leadingto altered hemodynamic conditions in the developing heart Itis however also important to notice that at preseptation stagesthe IVCT and IVRT cannot always be identified separately sothat calculation of the MPI is frequently not possible at veryearly stages of embryonic development Between 145 and175 dpcwenoticed a slight increase of theMPI suggesting thatcardiac function improves at subsequent fetal stages similarresults have also been demonstrated inmouse [4] and human[30] However discussion remains regarding the exact courseof theMPI IVCT IVRT andETandwhether isolated changesof one of the factors is a predictor for fetal demise and CHDInterestinglywith respect to IVRTweobserved a decreasingtrend near completion of the IVS (between 125 and 145)whereafter it more or less remained stable throughout fetallife A similar course was observed in a recent ultrasoundstudy in embryonicfetal C57Bl6 mice [4] Human embryosalso showed a decreasing course of the IVRT [13 21] and anelevated IVRT has been implicated as a predictor of CHD[13]

5 Conclusion

We demonstrated the course of several morphologic andhemodynamic parameters along embryonic and fetal lifeOur data show that a reliable assessment can be made notonly of the left but also of right ventricular function duringembryonicfetal stages We demonstrate an overall improve-ment of cardiac diastolic and systolic function of both LV andRV along heart development The implementation of high-frequency ultrasound in embryonicfetal mouse heart devel-opment is a promising and useful tool The data presented inthis studymight be useful as reference values in future studiesin genetic mutated mouse models of CHD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

10 The Scientific World Journal

Authorsrsquo Contribution

Nathan D Hahurij and Emmeline E Calkoen contributedequally to this work and shared first authorship

Acknowledgment

This work is supported by the DutchHeart Foundation Grantno 2009T070 (Nathan D Hahurij)

References

[1] R G Kelly ldquoMolecular inroads into the anterior heart fieldrdquoTrends inCardiovascularMedicine vol 15 no 2 pp 51ndash56 2005

[2] J I E Hoffman and S Kaplan ldquoThe incidence of congenitalheart diseaserdquo Journal of the AmericanCollege of Cardiology vol39 no 12 pp 1890ndash1900 2002

[3] D Srivastava ldquoGenetic assembly of the heart implications forcongenital heart diseaserdquo Annual Review of Physiology vol 63pp 451ndash469 2001

[4] N Corrigan D P Brazil and F M Auliffe ldquoHigh-frequencyultrasound assessment of the murine heart from embryothrough to juvenilerdquoReproductive Sciences vol 17 no 2 pp 147ndash157 2010

[5] C F Spurney CW Lo and L Leatherbury ldquoFetal mouse imag-ing using echocardiography a review of current technologyrdquoEchocardiography vol 23 no 10 pp 891ndash899 2006

[6] Y-Q Zhou F S Foster R Parkes and S L Adamson ldquoDevel-opmental changes in left and right ventricular diastolic fillingpatterns in micerdquo American Journal of Physiology vol 285 no4 pp H1563ndashH1575 2003

[7] Q Yu L Leatherbury X Tian and C W Lo ldquoCardiovascularassessment of fetal mice by in utero echocardiographyrdquo Ultra-sound in Medicine and Biology vol 34 no 5 pp 741ndash752 2008

[8] C K L Phoon ldquoImaging tools for the developmental biologistultrasound biomicroscopy of mouse embryonic developmentrdquoPediatric Research vol 60 no 1 pp 14ndash21 2006

[9] J S Lee Q Yu J T Shin et al ldquoKlf2 is an essential regulator ofvascular hemodynamic forces in vivordquo Developmental Cell vol11 no 6 pp 845ndash857 2006

[10] R A Morris B Damon V Mironov et al ldquoPeriostin regulatescollagen fibrillogenesis and the biomechanical properties ofconnective tissuesrdquo Journal of Cellular Biochemistry vol 101 no3 pp 695ndash711 2007

[11] B C W Groenendijk B P Hierck J Vrolijk et al ldquoChanges inshear stress-related gene expression after experimentally alteredvenous return in the chicken embryordquoCirculation Research vol96 no 12 pp 1291ndash1298 2005

[12] C J Zuurbier V M Emons and C Ince ldquoHemodynamics ofanesthetized ventilated mouse models aspects of anestheticsfluid support and strainrdquo American Journal of Physiology vol282 no 6 pp H2099ndashH2105 2002

[13] A Włoch W Rozmus-Warcholinska B Czuba et al ldquoDopplerstudy of the embryonic heart in normal pregnant womenrdquoJournal of Maternal-Fetal and Neonatal Medicine vol 20 no 7pp 533ndash539 2007

[14] M E Godfrey B Messing S M Cohen D V Valsky andS Yagel ldquoFunctional assessment of the fetal heart a reviewrdquo

Ultrasound in Obstetrics and Gynecology vol 39 no 2 pp 131ndash144 2012

[15] M R M Jongbloed M C E F Wijffels M J Schalij et alldquoDevelopment of the right ventricular inflow tract and moder-ator band a possible morphological and functional explanationforMahaim tachycardiardquoCirculation Research vol 96 no 7 pp776ndash783 2005

[16] Y-HGui KK Linask PKhowsathit and J CHuhta ldquoDopplerechocardiography of normal and abnormal embryonic mouseheartrdquo Pediatric Research vol 40 no 4 pp 633ndash642 1996

[17] C F Spurney L Leatherbury and C W Lo ldquoHigh-frequencyultrasound database profiling growth development and car-diovascular function in C57BL6J mouse fetusesrdquo Journal of theAmerican Society of Echocardiography vol 17 no 8 pp 893ndash900 2004

[18] R P Ji C K L Phoon O Aristizabal K E McGrath JPalis and D H Turnbull ldquoOnset of cardiac function duringearly mouse embryogenesis coincides with entry of primitiveerythroblasts into the embryo properrdquoCirculation Research vol92 no 2 pp 133ndash135 2003

[19] M R Jongbloed S R Vicente N D Hahurij et al ldquoNormaland abnormal development of the cardiac conduction systemimplications for conduction and rhythm disorders in the childand adultrdquo Differentiation vol 84 no 1 pp 131ndash148 2012

[20] K Makikallio P Jouppila and J Rasanen ldquoHuman fetal cardiacfunction during the first trimester of pregnancyrdquo Heart vol 91no 3 pp 334ndash338 2005

[21] W Rozmus-Warcholinska A Wloch G Acharya et al ldquoRef-erence values for variables of fetal cardiocirculatory dynamicsat 11-14 weeks of gestationrdquo Ultrasound in Obstetrics andGynecology vol 35 no 5 pp 540ndash547 2010

[22] G Tulzer P Khowsathit S Gudmundsson et al ldquoDiastolicfunction of the fetal heart during second and third trimestera prospective longitudinal Doppler-echocardiographic studyrdquoEuropean Journal of Pediatrics vol 153 no 3 pp 151ndash154 1994

[23] MC Leiva J E Tolosa CN Binotto et al ldquoFetal cardiac devel-opment and hemodynamics in the first trimesterrdquoUltrasound inObstetrics and Gynecology vol 14 no 3 pp 169ndash174 1999

[24] I P Van Splunder and J W Wladimiroff ldquoCardiac functionalchanges in the human fetus in the late first and early secondtrimestersrdquo Ultrasound in Obstetrics and Gynecology vol 7 no6 pp 411ndash415 1996

[25] S A Clur R K Oude B W Mol J Ottenkamp and CM Bilardo ldquoFetal cardiac function between 11 and 35 weeksrsquogestation and nuchal translucency thicknessrdquo Ultrasound inObstetrics amp Gynecology vol 37 no 1 pp 48ndash56 2011

[26] G Rizzo A Muscatello E Angelini and A Capponi ldquoAbnor-mal cardiac function in fetuses with increased nuchal translu-cencyrdquo Ultrasound in Obstetrics and Gynecology vol 21 no 6pp 539ndash542 2003

[27] F Crispi E Hernandez-Andrade M M A L Pelsers et alldquoCardiac dysfunction and cell damage across clinical stagesof severity in growth-restricted fetusesrdquo American Journal ofObstetrics and Gynecology vol 199 no 3 pp 254ndash258 2008

[28] S Luewan Y Yanase F Tongprasert K Srisupundit and TTongsong ldquoFetal cardiac dimensions at 14-40 weeksrsquo gestationobtained using cardio-STIC-Mrdquo Ultrasound in Obstetrics andGynecology vol 37 no 4 pp 416ndash422 2011

The Scientific World Journal 11

[29] T Tongsong C Wanapirak W Piyamongkol et al ldquoFetalventricular shortening fraction in hydrops fetalisrdquo Obstetricsand Gynecology vol 117 no 1 pp 84ndash91 2011

[30] E Hernandez-Andrade H Figueroa-Diesel C Kottman etal ldquoGestational-age-adjusted reference values for the modifiedmyocardial performance index for evaluation of fetal left car-diac functionrdquoUltrasound in Obstetrics and Gynecology vol 29no 3 pp 321ndash325 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Signal TransductionJournal of

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Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World Journal 5

Table 2 Summary of the assessment of embryonic and fetal hearts

Method Parameter Unit Structure (abbreviation)

2D ultrasoundB-mode echoloops

Morphological assessmentposition and diameters ofcardiac compartments

mm

Right atrium (RA) left atrium (LA) right ventricle (RV) left ventrile (LV)common outflow tract (cOFT) pulmonary trunk (PT) aorta (Ao)inter ventricular septum (IVS) common atrioventricular canal (cAVC)mitral valve (MV) and tricuspid valve (TV)Definition Formula

Pulsed-waveDoppler

Peak E ms Early diastolic ventricular fillingPeak A ms Active diastolic ventricular fillingEA ratioRR interval ms Cycle lengthevaluation of heart rateDFT ms Diastolic filling timeIVRT ms Isovolumetric relaxation timeIVCT ms Isovolumetric contraction timeET ms Ejection timeMPITei index Myocardial performance index IVRT + IVCTETPeak velocities cOFT AoPT ms

VTI mm Velocity time integral

SV Stroke volume SV = TVI times (Ao orcOFT diameter2)2 times 120587

CO mLmin Cardiac output

M-mode

VID mm Ventricular inner diameterLVIDd mm End diastolic left ventricular inner diameterLVIDs mm End systolic left ventricular inner diameterRVIDd mm End diastolic right ventricular inner diameterRVIDs mm End systolic right ventricular inner diameterIVSd mm End diastolic interventricular septum diameterIVSs mm End systolic interventricular septum diameter

FS Fractional shortening (VIDdminusVIDs)VIDd times100

3 Results

31 Cardiac Morphology Reliable imaging of hearts as smallas 2mm was feasible in all stages examined Representativeexamples are shown in online Movie 1 (see SupplementaryMaterial available online at httpdxdoiorg1011552014531324)

At embryonic stages (125 dpc) ventricular septation hasbeen initiated but is not completed resulting in a primitiveinterventricular foramen that directly connects the lumen ofLV and RV M-mode recordings at these stages show a meansystolic IVS diameter of 020 plusmn 0045mm Figure 2(a) showsa reconstruction of a 125 dpc embryonic heart in which thelumen of the four cardiac chambers interventricular fora-men and theOFT can be identifiedThe atrial and ventricularchambers are connected via the cAVC that harbors large AVcushions which will contribute to formation of the MV andTV The AV canal is still partly positioned above the LV atthis stage The developing OFT is still positioned completelyabove the RV and contains cushion tissue that will form thesemilunar valves of the Ao and PTThey will further form thebasis for the muscular subpulmonary infundibulum and will

contribute to themembranous part of the IVS Consequentlythe blood from the LV runs via the primitive interventricularforamen into the cOFT that is future Ao and PT

At early fetal stages of 145 dpc the heart has increased insize (Figure 2(b)) and ventricular septation is now completeThe systolic diameter of the IVS has increased (024 plusmn0018mm) In the AVC the TV orifice has expanded and isnow situated entirely above the RV Also the MV orifice canbe identified as well as the developing AV valves The cardiacOFT now consists of a separate Ao and PT connecting to theLV and RV respectively The Ao and pulmonary valves stillshow immaturity and consist of cushion tissue

At late fetal stages (175 dpc) the size of the RV andLV and IVS (039 plusmn 0009mm) has increased significantly(Figure 2(c)) Mature valves are present at the levels of the in-and outflow parts of both ventricles

32 Diastolic and Systolic Function During embryonic andfetal development theHR increases significantly (119875 lt 00001)from 125 plusmn 95 up to 219 plusmn 83BPM Ventricular inflowpatterns across the developing MV and TV annulus werestudied by pulsed-wave Doppler imaging

6 The Scientific World Journal

(a) (b) (c)

lowast

lowastlowastlowast150

125

100

075

050

025

000

Dia

met

er lu

men

(mm

)

Left ventricle

Right ventricle

Diastole

Systole

125 145 175

Age (dpc)

ns

(d)

lowast

125 145 175

Age (dpc)

50

40

30

20

10

0

Shor

teni

ng fr

actio

n (

)

Left ventricle

Right ventricle

(e)

Figure 2 Cardiac morphology and FS calculation (a) Shows a reconstruction of the anterior view of an embryonic heart of 125 dpc Themyocardium is indicated in grey transparent The LA and RA are indicated in transparent dark grey The LV and RV lumen are indicated inred and blue respectively Note that the outflow tract lumen (purple) is positioned completely above the future RV which is surrounded bylarge outflow tract cushions (green transparent) At these stages development of the IVS has not yet completed leading to a direct connectionbetween the LV and RV via the interventricular foramen (arrow) (b) Anterior view of an early fetal heart of 145 dpc At this stage IVSdevelopment has been completed and four separate cardiac chambers can be identified The outflow tract consists of a separate Ao and PTincluding their valve apparatus which at these stages mainly consist of cushion tissue (green transparent) (c) Anterior view of a late fetalheart of 175 dpc At this stage the heart shows a mature morphological phenotype (d) Schematic representation of LV and RV diameters and(e) FS at the three consecutive stages of development

The DFT corrected for the RR interval (DFTRR) did notdiffer significantly at the three stages assessed (Figure 1(b))LV and RV filling were dominated by the peak-A waveBetween 125 and 175 dpc the peak-A wave of the developingMV annulus increased significantly (033 plusmn 0001ms versus042 plusmn 0014ms 119875 lt 00001) whereas that of the TV alsoshowed an increasing trend albeit not significant (037 plusmn0018ms versus 041 plusmn 0019ms 119875 = NS) The peak-Ewave also increased significantly for both LV (005plusmn0006msversus 018plusmn0005ms 119875 lt 00001) and RV (006plusmn0006msversus 018 plusmn 0012ms 119875 lt 00001) and showed a relativelygreater increase than the peak-A wave during development

As a result the EA ratio a measure for the ventricular dia-stolic function showed a significant increase for both LV(016 plusmn 0015 versus 044 plusmn 0013 119875 lt 00001) and RV(016 plusmn 0010 versus 044 plusmn 0014 119875 lt 00001) (Figures 3(a)ndash3(f))

The IVRT one of the measures for diastolic LV functioncorrected for the RR interval (IVRTRR) demonstrated asignificant (119875 = 0037) decrease between 125 (015 plusmn 0011)and 145 dpc (012 plusmn 0004) and remained stable at later fetalstages 175 dpc (012 plusmn 0003) (Figure 1(e)) indicating that LVmyocardial relaxation remains stable after completion of IVSdevelopment during fetal life

The Scientific World Journal 7

Mitral06

05

04

03

02

01

00

EA

ratio

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

125 145 175

(a)

06

05

04

03

02

01

00

EA

ratio

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

125 145 175

Tricuspid

(b)

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

125 145 175

Peak

E (m

s)

04

03

02

01

00

(c)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

125 145 175

Peak

E (m

s)

04

03

02

01

00

(d)

lowastlowastlowast

125 145 175

Age (dpc)

Peak

A (m

s)

07

06

05

04

03

02

01

00

nslowastlowast

(e)

125 145 175

Age (dpc)

Peak

A (m

s)

07

06

05

04

03

02

01

00

nsns

ns

(f)

Figure 3 Development of LV and RV inflow patternsThe graphs represent the course of the EA ratio (a b) peak-E wave (c d) and peak-Awave (e f) across the developing MV and TV at the three subsequent developmental stages

The systolic function of the developing ventricles wasassessed through studying the course of the FS IVCTRRand ETRR Throughout embryonic and fetal developmentthe FS remained constant for both ventricles and valuesranged between asymp30 and 40 Furthermore except for175 dpc (LV 406 RV 357 119875 = 0046) no significantdifferences were observed between the FS of the LV and RV

(Figures 2(d) and 2(e)) The mean IVCTRR remained stableduring all three stagesasymp009 the ETRR showed no changesbetween 125 dpc (042plusmn0015) and 145 dpc (043plusmn0010) butsignificantly decreased at 175 dpc (040 plusmn 0006 119875 = 0017)(Figures 1(c) and 1(d))

A combined assessment of LV systolic and diastolic func-tion was performed by calculating the MPI At embryonic

8 The Scientific World Journal

Table 3 The parameters used for calculation of the mean CO per gestational age

Parameter 125 dpc (cOFT) 145 dpc (Ao) 175 dpc (Ao)VTI (mm) 339 plusmn 137 423 plusmn 20 292 plusmn 205

Mean diameter (mm) 023 018 048 plusmn 003

Mean HR (bpm) 134 plusmn 112 148 plusmn 61 206 plusmn 98

Mean CO (mLmin) 019 016 096

lowastlowastlowast05

04

03

02

01

Peak

flow

(ms

)

Common OFT

Aorta

Pulmonary trunk

125 145 175

Age (dpc)

Figure 4 Pulsed-wave Doppler flow measurements in the cOFTAo and PT The graph demonstrates the significant increase of thepeak blood flowmeasured in the cOFT at 125 and Ao and PT at 145and 175 dpc

preseptated stages (125 dpc) the MPI was 064 plusmn 0012 anddecreased to 049 plusmn 0017 (119875 = 00002) after closure of theIVS at 145 dpc At late fetal stages (175 dpc) the MPI did notchange significantly (053 plusmn 0022 119875 = NS)

33 OutflowTract andCO Thepulsed-waveDoppler record-ings performed in the cOFT demonstrated a peak flow of027 plusmn 0021ms at preseptated stages As from 145 dpc twoindividual ventricular outflow tracts could be discriminatedespecially the high echodensity of the blood at these stagesenabled to discriminate the flow in the Ao (034 plusmn 0013ms)and PT (033 plusmn 0021ms) that are closely related to eachother As can be observed in Figure 4 the peak flow increasedsignificantly in the Ao (from 027 plusmn 0021ms to 036 plusmn0016ms 119875 = 0010) and PT (027 plusmn 0021ms to 044 plusmn0030ms 119875 = 0003) along fetal life

Table 3 summarizes theHRVTI and the diameters of thecOFT and Ao that were used to calculate the mean CO at thethree developmental stages Between 125 dpc and 145 dpc the

CO remained stable where after it increased to 096mLminat 175 dpc

4 Discussion

Heart development is a complex process which has beenextensively studied in both WT and genetically mutatedmouse models [3] It is only in the last decades that modernimaging techniques like ultrasound enable the assessmentof real-time hemodynamic and morphological changes thatoccur during mouse heart development [4ndash6 8 16ndash18]Knowledge regarding these functional parameters is essentialsince abnormal flow in the developing heart might lead toCHD through aberrant activation of shear-stress responsivegenes important in cardiogenesis [9 11] In early reportsdescribing the course of functional parameters during heartdevelopmentmajor variability exists inmeasured valuesThisis most likely related to the relatively low ultrasound frequen-cies of clinical ultrasound machines used to image the smallhearts of mouse embryosfetuses [16] and externalizationof uterus horns during ultrasound assessment [18] whichinfluences embryonicfetal physiology Low dose isofluraneanesthesia used in this study only has a minimal effect onhemodynamics and diastolic function in adult mice and theeffect on mouse embryos is supposed to be marginal [6]

In the current study we aimed to provide an overview ofhemodynamic parameters and changes that occur at subse-quent stages of heart development using a high-frequencyultrasound system

Compared to other reports in mouse using similar ultra-sound techniques we did not only focus on development ofthe future LV but also on that of the RV Key findings ofour study are (1) a reliable assessment of LV and RV in- andoutflow patterns can be performed from 125 dpc to term (2)a significant improvement occurs in the diastolic and systolicfunction of both LV and RV (3) over the developmentalperiod of 125 dpc to 175 dpc the ratio between RV and LVsize as well as the FS of both ventricles did not changesignificantly

Knowledge regarding the functional parameters of theRV is important since the RV is commonly affected in CHDDuring development the RV including the TV annulusdevelops subsequent to the LV by addition of SHF derivedcells to the developing heart [1] Many genes have beenidentified that have an important role in the SHF contributionto the developing heart andmutations in these genesmay leadto CHD [19]

At early embryonic stages endocardial cushion tissue ispositioned in the primitive AVC and OFT which later on

The Scientific World Journal 9

will contribute to formation of the TV and MV and Ao andPT semilunar valves Some clinical studies argued that AVvalve regurgitation is a commonphenomenon during the firsttrimester of pregnancy [20] Our study in accordance withseveral others in mouse [4 6] and human [13] did not showsigns of regurgitation at both early and late stages of heartdevelopment Pulsed-wave Doppler recordings across thecAVC at 125 dpc and MV and TV at 145 dpc demonstratedthat the AV cushionsmimic the function of normal AV valvesat 175 dpc In our opinion especially the flow in the cOFTat early embryonic stages which is completely positionedabove the RV (see Figure 2(a)) might wrongly be identifiedas regurgitation of the developing AV valvescushions Thisstresses the importance of detailed knowledge regarding therapid morphological changes that the heart undergoes atespecially the very early developmental stages

Regarding growth of the heart we demonstrated that therewas a 31 increase of LVID 34 of RIVD and 35 of IVSdiameter from 125 up to 175 dpcWe showed a trend towardsa smaller RVID as compared to the LVID at all stages whichwas not mentioned in other studies

41 Diastolic and Systolic Function during Heart DevelopmentImprovement of ventricular systolic and diastolic functionoccurs throughout the length of embryonic and fetal develop-mentWith respect to the diastolic function we demonstrateda significant increase of EA ratio across the developing MVand TV orifices which was related to an increase of the peak-E wave that is the fraction of diastolic ventricular filling thatis defined by ventricular relaxation These data are in accor-dance with the study of Zhou and colleagues who demon-strated that the EA ratio is increasing during developmentwhich seems to continue up to approximately 3 weeks afterbirth [6] Around day four after birth the peak-E wave mea-sured across theMV becomes more dominant than the peak-A wave indicating an improvement of cardiac complianceand that LV filling becomes dominated by passive ventricularrelaxation Remarkably these changes were not observed forthe RV inflow patterns where the peak-A wave remainsdominant [6] Human embryonic and fetal hearts show thesame maturation course of EA ratios across the developingMV and TV as was demonstrated in multiple studies [21ndash25]An abnormal course of EA ratio that is a decreasing[25 26] or an increasing trend [27] along development hasbeen related to the presence of CHD

With respect to systolic ventricular function the FSremained stable (asymp30ndash40) for both ventricles throughoutdevelopmentWe did find a significant difference favoring theLV FS as compared to the RV FS at 175 dpc Interestinglythe same phenomenon of FS difference between LV andRV has been reported in a study of human fetal heartsbetween 14 and 40weeks of pregnancy However as inmousedifferences between humanLVandRVFS is subtle [28] andwhether a decreased or decreasing FS is a predictor of CHDremains unknown Furthermore we did not find a decreasingtrend of both LV and RV FS as was recently demonstratedduring human fetal life [28 29] With respect to the CO wenoticed no change around the proces of ventricular septation(between 125 and 145 dpc) where after a major increase

occured to 096mLmin near term (175 dpc) The initial COdecreasemight be related to the fact that themeandiameter ofthe unseptated cOFT diameter was used at 125 dpc comparedto the isolated Ao diameter at 145 dpc The subsequent COincrease is mostly related to the dramatic increase of HRalong fetal life

The MPI or Tei index [14] is a measure for combinedsystolic and diastolic cardiac function and can be calculatedfor both ventricles In order to assess the MPI three differenttime intervals are needed the IVCT IVRT and ET For the LVthese parameters can be easily obtained since all the threeparameters can be assessed at once by placing the Dopplerultrasound beam across the developing MV orifice Notice-ably in literature there is less data regarding RV MPI andits course along development which might be related to thefact that for the RV not all the three time intervals can beassessed in one view as was also suggested in a recent reviewby Godfrey et al [14] The current study demonstrated asignificant decrease of LV MPI between 125 and 145 dpcwhich might be related to the rapid morphological changesthe heart goes through including completion of IVS leadingto altered hemodynamic conditions in the developing heart Itis however also important to notice that at preseptation stagesthe IVCT and IVRT cannot always be identified separately sothat calculation of the MPI is frequently not possible at veryearly stages of embryonic development Between 145 and175 dpcwenoticed a slight increase of theMPI suggesting thatcardiac function improves at subsequent fetal stages similarresults have also been demonstrated inmouse [4] and human[30] However discussion remains regarding the exact courseof theMPI IVCT IVRT andETandwhether isolated changesof one of the factors is a predictor for fetal demise and CHDInterestinglywith respect to IVRTweobserved a decreasingtrend near completion of the IVS (between 125 and 145)whereafter it more or less remained stable throughout fetallife A similar course was observed in a recent ultrasoundstudy in embryonicfetal C57Bl6 mice [4] Human embryosalso showed a decreasing course of the IVRT [13 21] and anelevated IVRT has been implicated as a predictor of CHD[13]

5 Conclusion

We demonstrated the course of several morphologic andhemodynamic parameters along embryonic and fetal lifeOur data show that a reliable assessment can be made notonly of the left but also of right ventricular function duringembryonicfetal stages We demonstrate an overall improve-ment of cardiac diastolic and systolic function of both LV andRV along heart development The implementation of high-frequency ultrasound in embryonicfetal mouse heart devel-opment is a promising and useful tool The data presented inthis studymight be useful as reference values in future studiesin genetic mutated mouse models of CHD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

10 The Scientific World Journal

Authorsrsquo Contribution

Nathan D Hahurij and Emmeline E Calkoen contributedequally to this work and shared first authorship

Acknowledgment

This work is supported by the DutchHeart Foundation Grantno 2009T070 (Nathan D Hahurij)

References

[1] R G Kelly ldquoMolecular inroads into the anterior heart fieldrdquoTrends inCardiovascularMedicine vol 15 no 2 pp 51ndash56 2005

[2] J I E Hoffman and S Kaplan ldquoThe incidence of congenitalheart diseaserdquo Journal of the AmericanCollege of Cardiology vol39 no 12 pp 1890ndash1900 2002

[3] D Srivastava ldquoGenetic assembly of the heart implications forcongenital heart diseaserdquo Annual Review of Physiology vol 63pp 451ndash469 2001

[4] N Corrigan D P Brazil and F M Auliffe ldquoHigh-frequencyultrasound assessment of the murine heart from embryothrough to juvenilerdquoReproductive Sciences vol 17 no 2 pp 147ndash157 2010

[5] C F Spurney CW Lo and L Leatherbury ldquoFetal mouse imag-ing using echocardiography a review of current technologyrdquoEchocardiography vol 23 no 10 pp 891ndash899 2006

[6] Y-Q Zhou F S Foster R Parkes and S L Adamson ldquoDevel-opmental changes in left and right ventricular diastolic fillingpatterns in micerdquo American Journal of Physiology vol 285 no4 pp H1563ndashH1575 2003

[7] Q Yu L Leatherbury X Tian and C W Lo ldquoCardiovascularassessment of fetal mice by in utero echocardiographyrdquo Ultra-sound in Medicine and Biology vol 34 no 5 pp 741ndash752 2008

[8] C K L Phoon ldquoImaging tools for the developmental biologistultrasound biomicroscopy of mouse embryonic developmentrdquoPediatric Research vol 60 no 1 pp 14ndash21 2006

[9] J S Lee Q Yu J T Shin et al ldquoKlf2 is an essential regulator ofvascular hemodynamic forces in vivordquo Developmental Cell vol11 no 6 pp 845ndash857 2006

[10] R A Morris B Damon V Mironov et al ldquoPeriostin regulatescollagen fibrillogenesis and the biomechanical properties ofconnective tissuesrdquo Journal of Cellular Biochemistry vol 101 no3 pp 695ndash711 2007

[11] B C W Groenendijk B P Hierck J Vrolijk et al ldquoChanges inshear stress-related gene expression after experimentally alteredvenous return in the chicken embryordquoCirculation Research vol96 no 12 pp 1291ndash1298 2005

[12] C J Zuurbier V M Emons and C Ince ldquoHemodynamics ofanesthetized ventilated mouse models aspects of anestheticsfluid support and strainrdquo American Journal of Physiology vol282 no 6 pp H2099ndashH2105 2002

[13] A Włoch W Rozmus-Warcholinska B Czuba et al ldquoDopplerstudy of the embryonic heart in normal pregnant womenrdquoJournal of Maternal-Fetal and Neonatal Medicine vol 20 no 7pp 533ndash539 2007

[14] M E Godfrey B Messing S M Cohen D V Valsky andS Yagel ldquoFunctional assessment of the fetal heart a reviewrdquo

Ultrasound in Obstetrics and Gynecology vol 39 no 2 pp 131ndash144 2012

[15] M R M Jongbloed M C E F Wijffels M J Schalij et alldquoDevelopment of the right ventricular inflow tract and moder-ator band a possible morphological and functional explanationforMahaim tachycardiardquoCirculation Research vol 96 no 7 pp776ndash783 2005

[16] Y-HGui KK Linask PKhowsathit and J CHuhta ldquoDopplerechocardiography of normal and abnormal embryonic mouseheartrdquo Pediatric Research vol 40 no 4 pp 633ndash642 1996

[17] C F Spurney L Leatherbury and C W Lo ldquoHigh-frequencyultrasound database profiling growth development and car-diovascular function in C57BL6J mouse fetusesrdquo Journal of theAmerican Society of Echocardiography vol 17 no 8 pp 893ndash900 2004

[18] R P Ji C K L Phoon O Aristizabal K E McGrath JPalis and D H Turnbull ldquoOnset of cardiac function duringearly mouse embryogenesis coincides with entry of primitiveerythroblasts into the embryo properrdquoCirculation Research vol92 no 2 pp 133ndash135 2003

[19] M R Jongbloed S R Vicente N D Hahurij et al ldquoNormaland abnormal development of the cardiac conduction systemimplications for conduction and rhythm disorders in the childand adultrdquo Differentiation vol 84 no 1 pp 131ndash148 2012

[20] K Makikallio P Jouppila and J Rasanen ldquoHuman fetal cardiacfunction during the first trimester of pregnancyrdquo Heart vol 91no 3 pp 334ndash338 2005

[21] W Rozmus-Warcholinska A Wloch G Acharya et al ldquoRef-erence values for variables of fetal cardiocirculatory dynamicsat 11-14 weeks of gestationrdquo Ultrasound in Obstetrics andGynecology vol 35 no 5 pp 540ndash547 2010

[22] G Tulzer P Khowsathit S Gudmundsson et al ldquoDiastolicfunction of the fetal heart during second and third trimestera prospective longitudinal Doppler-echocardiographic studyrdquoEuropean Journal of Pediatrics vol 153 no 3 pp 151ndash154 1994

[23] MC Leiva J E Tolosa CN Binotto et al ldquoFetal cardiac devel-opment and hemodynamics in the first trimesterrdquoUltrasound inObstetrics and Gynecology vol 14 no 3 pp 169ndash174 1999

[24] I P Van Splunder and J W Wladimiroff ldquoCardiac functionalchanges in the human fetus in the late first and early secondtrimestersrdquo Ultrasound in Obstetrics and Gynecology vol 7 no6 pp 411ndash415 1996

[25] S A Clur R K Oude B W Mol J Ottenkamp and CM Bilardo ldquoFetal cardiac function between 11 and 35 weeksrsquogestation and nuchal translucency thicknessrdquo Ultrasound inObstetrics amp Gynecology vol 37 no 1 pp 48ndash56 2011

[26] G Rizzo A Muscatello E Angelini and A Capponi ldquoAbnor-mal cardiac function in fetuses with increased nuchal translu-cencyrdquo Ultrasound in Obstetrics and Gynecology vol 21 no 6pp 539ndash542 2003

[27] F Crispi E Hernandez-Andrade M M A L Pelsers et alldquoCardiac dysfunction and cell damage across clinical stagesof severity in growth-restricted fetusesrdquo American Journal ofObstetrics and Gynecology vol 199 no 3 pp 254ndash258 2008

[28] S Luewan Y Yanase F Tongprasert K Srisupundit and TTongsong ldquoFetal cardiac dimensions at 14-40 weeksrsquo gestationobtained using cardio-STIC-Mrdquo Ultrasound in Obstetrics andGynecology vol 37 no 4 pp 416ndash422 2011

The Scientific World Journal 11

[29] T Tongsong C Wanapirak W Piyamongkol et al ldquoFetalventricular shortening fraction in hydrops fetalisrdquo Obstetricsand Gynecology vol 117 no 1 pp 84ndash91 2011

[30] E Hernandez-Andrade H Figueroa-Diesel C Kottman etal ldquoGestational-age-adjusted reference values for the modifiedmyocardial performance index for evaluation of fetal left car-diac functionrdquoUltrasound in Obstetrics and Gynecology vol 29no 3 pp 321ndash325 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 The Scientific World Journal

(a) (b) (c)

lowast

lowastlowastlowast150

125

100

075

050

025

000

Dia

met

er lu

men

(mm

)

Left ventricle

Right ventricle

Diastole

Systole

125 145 175

Age (dpc)

ns

(d)

lowast

125 145 175

Age (dpc)

50

40

30

20

10

0

Shor

teni

ng fr

actio

n (

)

Left ventricle

Right ventricle

(e)

Figure 2 Cardiac morphology and FS calculation (a) Shows a reconstruction of the anterior view of an embryonic heart of 125 dpc Themyocardium is indicated in grey transparent The LA and RA are indicated in transparent dark grey The LV and RV lumen are indicated inred and blue respectively Note that the outflow tract lumen (purple) is positioned completely above the future RV which is surrounded bylarge outflow tract cushions (green transparent) At these stages development of the IVS has not yet completed leading to a direct connectionbetween the LV and RV via the interventricular foramen (arrow) (b) Anterior view of an early fetal heart of 145 dpc At this stage IVSdevelopment has been completed and four separate cardiac chambers can be identified The outflow tract consists of a separate Ao and PTincluding their valve apparatus which at these stages mainly consist of cushion tissue (green transparent) (c) Anterior view of a late fetalheart of 175 dpc At this stage the heart shows a mature morphological phenotype (d) Schematic representation of LV and RV diameters and(e) FS at the three consecutive stages of development

The DFT corrected for the RR interval (DFTRR) did notdiffer significantly at the three stages assessed (Figure 1(b))LV and RV filling were dominated by the peak-A waveBetween 125 and 175 dpc the peak-A wave of the developingMV annulus increased significantly (033 plusmn 0001ms versus042 plusmn 0014ms 119875 lt 00001) whereas that of the TV alsoshowed an increasing trend albeit not significant (037 plusmn0018ms versus 041 plusmn 0019ms 119875 = NS) The peak-Ewave also increased significantly for both LV (005plusmn0006msversus 018plusmn0005ms 119875 lt 00001) and RV (006plusmn0006msversus 018 plusmn 0012ms 119875 lt 00001) and showed a relativelygreater increase than the peak-A wave during development

As a result the EA ratio a measure for the ventricular dia-stolic function showed a significant increase for both LV(016 plusmn 0015 versus 044 plusmn 0013 119875 lt 00001) and RV(016 plusmn 0010 versus 044 plusmn 0014 119875 lt 00001) (Figures 3(a)ndash3(f))

The IVRT one of the measures for diastolic LV functioncorrected for the RR interval (IVRTRR) demonstrated asignificant (119875 = 0037) decrease between 125 (015 plusmn 0011)and 145 dpc (012 plusmn 0004) and remained stable at later fetalstages 175 dpc (012 plusmn 0003) (Figure 1(e)) indicating that LVmyocardial relaxation remains stable after completion of IVSdevelopment during fetal life

The Scientific World Journal 7

Mitral06

05

04

03

02

01

00

EA

ratio

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

125 145 175

(a)

06

05

04

03

02

01

00

EA

ratio

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

125 145 175

Tricuspid

(b)

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

125 145 175

Peak

E (m

s)

04

03

02

01

00

(c)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

125 145 175

Peak

E (m

s)

04

03

02

01

00

(d)

lowastlowastlowast

125 145 175

Age (dpc)

Peak

A (m

s)

07

06

05

04

03

02

01

00

nslowastlowast

(e)

125 145 175

Age (dpc)

Peak

A (m

s)

07

06

05

04

03

02

01

00

nsns

ns

(f)

Figure 3 Development of LV and RV inflow patternsThe graphs represent the course of the EA ratio (a b) peak-E wave (c d) and peak-Awave (e f) across the developing MV and TV at the three subsequent developmental stages

The systolic function of the developing ventricles wasassessed through studying the course of the FS IVCTRRand ETRR Throughout embryonic and fetal developmentthe FS remained constant for both ventricles and valuesranged between asymp30 and 40 Furthermore except for175 dpc (LV 406 RV 357 119875 = 0046) no significantdifferences were observed between the FS of the LV and RV

(Figures 2(d) and 2(e)) The mean IVCTRR remained stableduring all three stagesasymp009 the ETRR showed no changesbetween 125 dpc (042plusmn0015) and 145 dpc (043plusmn0010) butsignificantly decreased at 175 dpc (040 plusmn 0006 119875 = 0017)(Figures 1(c) and 1(d))

A combined assessment of LV systolic and diastolic func-tion was performed by calculating the MPI At embryonic

8 The Scientific World Journal

Table 3 The parameters used for calculation of the mean CO per gestational age

Parameter 125 dpc (cOFT) 145 dpc (Ao) 175 dpc (Ao)VTI (mm) 339 plusmn 137 423 plusmn 20 292 plusmn 205

Mean diameter (mm) 023 018 048 plusmn 003

Mean HR (bpm) 134 plusmn 112 148 plusmn 61 206 plusmn 98

Mean CO (mLmin) 019 016 096

lowastlowastlowast05

04

03

02

01

Peak

flow

(ms

)

Common OFT

Aorta

Pulmonary trunk

125 145 175

Age (dpc)

Figure 4 Pulsed-wave Doppler flow measurements in the cOFTAo and PT The graph demonstrates the significant increase of thepeak blood flowmeasured in the cOFT at 125 and Ao and PT at 145and 175 dpc

preseptated stages (125 dpc) the MPI was 064 plusmn 0012 anddecreased to 049 plusmn 0017 (119875 = 00002) after closure of theIVS at 145 dpc At late fetal stages (175 dpc) the MPI did notchange significantly (053 plusmn 0022 119875 = NS)

33 OutflowTract andCO Thepulsed-waveDoppler record-ings performed in the cOFT demonstrated a peak flow of027 plusmn 0021ms at preseptated stages As from 145 dpc twoindividual ventricular outflow tracts could be discriminatedespecially the high echodensity of the blood at these stagesenabled to discriminate the flow in the Ao (034 plusmn 0013ms)and PT (033 plusmn 0021ms) that are closely related to eachother As can be observed in Figure 4 the peak flow increasedsignificantly in the Ao (from 027 plusmn 0021ms to 036 plusmn0016ms 119875 = 0010) and PT (027 plusmn 0021ms to 044 plusmn0030ms 119875 = 0003) along fetal life

Table 3 summarizes theHRVTI and the diameters of thecOFT and Ao that were used to calculate the mean CO at thethree developmental stages Between 125 dpc and 145 dpc the

CO remained stable where after it increased to 096mLminat 175 dpc

4 Discussion

Heart development is a complex process which has beenextensively studied in both WT and genetically mutatedmouse models [3] It is only in the last decades that modernimaging techniques like ultrasound enable the assessmentof real-time hemodynamic and morphological changes thatoccur during mouse heart development [4ndash6 8 16ndash18]Knowledge regarding these functional parameters is essentialsince abnormal flow in the developing heart might lead toCHD through aberrant activation of shear-stress responsivegenes important in cardiogenesis [9 11] In early reportsdescribing the course of functional parameters during heartdevelopmentmajor variability exists inmeasured valuesThisis most likely related to the relatively low ultrasound frequen-cies of clinical ultrasound machines used to image the smallhearts of mouse embryosfetuses [16] and externalizationof uterus horns during ultrasound assessment [18] whichinfluences embryonicfetal physiology Low dose isofluraneanesthesia used in this study only has a minimal effect onhemodynamics and diastolic function in adult mice and theeffect on mouse embryos is supposed to be marginal [6]

In the current study we aimed to provide an overview ofhemodynamic parameters and changes that occur at subse-quent stages of heart development using a high-frequencyultrasound system

Compared to other reports in mouse using similar ultra-sound techniques we did not only focus on development ofthe future LV but also on that of the RV Key findings ofour study are (1) a reliable assessment of LV and RV in- andoutflow patterns can be performed from 125 dpc to term (2)a significant improvement occurs in the diastolic and systolicfunction of both LV and RV (3) over the developmentalperiod of 125 dpc to 175 dpc the ratio between RV and LVsize as well as the FS of both ventricles did not changesignificantly

Knowledge regarding the functional parameters of theRV is important since the RV is commonly affected in CHDDuring development the RV including the TV annulusdevelops subsequent to the LV by addition of SHF derivedcells to the developing heart [1] Many genes have beenidentified that have an important role in the SHF contributionto the developing heart andmutations in these genesmay leadto CHD [19]

At early embryonic stages endocardial cushion tissue ispositioned in the primitive AVC and OFT which later on

The Scientific World Journal 9

will contribute to formation of the TV and MV and Ao andPT semilunar valves Some clinical studies argued that AVvalve regurgitation is a commonphenomenon during the firsttrimester of pregnancy [20] Our study in accordance withseveral others in mouse [4 6] and human [13] did not showsigns of regurgitation at both early and late stages of heartdevelopment Pulsed-wave Doppler recordings across thecAVC at 125 dpc and MV and TV at 145 dpc demonstratedthat the AV cushionsmimic the function of normal AV valvesat 175 dpc In our opinion especially the flow in the cOFTat early embryonic stages which is completely positionedabove the RV (see Figure 2(a)) might wrongly be identifiedas regurgitation of the developing AV valvescushions Thisstresses the importance of detailed knowledge regarding therapid morphological changes that the heart undergoes atespecially the very early developmental stages

Regarding growth of the heart we demonstrated that therewas a 31 increase of LVID 34 of RIVD and 35 of IVSdiameter from 125 up to 175 dpcWe showed a trend towardsa smaller RVID as compared to the LVID at all stages whichwas not mentioned in other studies

41 Diastolic and Systolic Function during Heart DevelopmentImprovement of ventricular systolic and diastolic functionoccurs throughout the length of embryonic and fetal develop-mentWith respect to the diastolic function we demonstrateda significant increase of EA ratio across the developing MVand TV orifices which was related to an increase of the peak-E wave that is the fraction of diastolic ventricular filling thatis defined by ventricular relaxation These data are in accor-dance with the study of Zhou and colleagues who demon-strated that the EA ratio is increasing during developmentwhich seems to continue up to approximately 3 weeks afterbirth [6] Around day four after birth the peak-E wave mea-sured across theMV becomes more dominant than the peak-A wave indicating an improvement of cardiac complianceand that LV filling becomes dominated by passive ventricularrelaxation Remarkably these changes were not observed forthe RV inflow patterns where the peak-A wave remainsdominant [6] Human embryonic and fetal hearts show thesame maturation course of EA ratios across the developingMV and TV as was demonstrated in multiple studies [21ndash25]An abnormal course of EA ratio that is a decreasing[25 26] or an increasing trend [27] along development hasbeen related to the presence of CHD

With respect to systolic ventricular function the FSremained stable (asymp30ndash40) for both ventricles throughoutdevelopmentWe did find a significant difference favoring theLV FS as compared to the RV FS at 175 dpc Interestinglythe same phenomenon of FS difference between LV andRV has been reported in a study of human fetal heartsbetween 14 and 40weeks of pregnancy However as inmousedifferences between humanLVandRVFS is subtle [28] andwhether a decreased or decreasing FS is a predictor of CHDremains unknown Furthermore we did not find a decreasingtrend of both LV and RV FS as was recently demonstratedduring human fetal life [28 29] With respect to the CO wenoticed no change around the proces of ventricular septation(between 125 and 145 dpc) where after a major increase

occured to 096mLmin near term (175 dpc) The initial COdecreasemight be related to the fact that themeandiameter ofthe unseptated cOFT diameter was used at 125 dpc comparedto the isolated Ao diameter at 145 dpc The subsequent COincrease is mostly related to the dramatic increase of HRalong fetal life

The MPI or Tei index [14] is a measure for combinedsystolic and diastolic cardiac function and can be calculatedfor both ventricles In order to assess the MPI three differenttime intervals are needed the IVCT IVRT and ET For the LVthese parameters can be easily obtained since all the threeparameters can be assessed at once by placing the Dopplerultrasound beam across the developing MV orifice Notice-ably in literature there is less data regarding RV MPI andits course along development which might be related to thefact that for the RV not all the three time intervals can beassessed in one view as was also suggested in a recent reviewby Godfrey et al [14] The current study demonstrated asignificant decrease of LV MPI between 125 and 145 dpcwhich might be related to the rapid morphological changesthe heart goes through including completion of IVS leadingto altered hemodynamic conditions in the developing heart Itis however also important to notice that at preseptation stagesthe IVCT and IVRT cannot always be identified separately sothat calculation of the MPI is frequently not possible at veryearly stages of embryonic development Between 145 and175 dpcwenoticed a slight increase of theMPI suggesting thatcardiac function improves at subsequent fetal stages similarresults have also been demonstrated inmouse [4] and human[30] However discussion remains regarding the exact courseof theMPI IVCT IVRT andETandwhether isolated changesof one of the factors is a predictor for fetal demise and CHDInterestinglywith respect to IVRTweobserved a decreasingtrend near completion of the IVS (between 125 and 145)whereafter it more or less remained stable throughout fetallife A similar course was observed in a recent ultrasoundstudy in embryonicfetal C57Bl6 mice [4] Human embryosalso showed a decreasing course of the IVRT [13 21] and anelevated IVRT has been implicated as a predictor of CHD[13]

5 Conclusion

We demonstrated the course of several morphologic andhemodynamic parameters along embryonic and fetal lifeOur data show that a reliable assessment can be made notonly of the left but also of right ventricular function duringembryonicfetal stages We demonstrate an overall improve-ment of cardiac diastolic and systolic function of both LV andRV along heart development The implementation of high-frequency ultrasound in embryonicfetal mouse heart devel-opment is a promising and useful tool The data presented inthis studymight be useful as reference values in future studiesin genetic mutated mouse models of CHD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

10 The Scientific World Journal

Authorsrsquo Contribution

Nathan D Hahurij and Emmeline E Calkoen contributedequally to this work and shared first authorship

Acknowledgment

This work is supported by the DutchHeart Foundation Grantno 2009T070 (Nathan D Hahurij)

References

[1] R G Kelly ldquoMolecular inroads into the anterior heart fieldrdquoTrends inCardiovascularMedicine vol 15 no 2 pp 51ndash56 2005

[2] J I E Hoffman and S Kaplan ldquoThe incidence of congenitalheart diseaserdquo Journal of the AmericanCollege of Cardiology vol39 no 12 pp 1890ndash1900 2002

[3] D Srivastava ldquoGenetic assembly of the heart implications forcongenital heart diseaserdquo Annual Review of Physiology vol 63pp 451ndash469 2001

[4] N Corrigan D P Brazil and F M Auliffe ldquoHigh-frequencyultrasound assessment of the murine heart from embryothrough to juvenilerdquoReproductive Sciences vol 17 no 2 pp 147ndash157 2010

[5] C F Spurney CW Lo and L Leatherbury ldquoFetal mouse imag-ing using echocardiography a review of current technologyrdquoEchocardiography vol 23 no 10 pp 891ndash899 2006

[6] Y-Q Zhou F S Foster R Parkes and S L Adamson ldquoDevel-opmental changes in left and right ventricular diastolic fillingpatterns in micerdquo American Journal of Physiology vol 285 no4 pp H1563ndashH1575 2003

[7] Q Yu L Leatherbury X Tian and C W Lo ldquoCardiovascularassessment of fetal mice by in utero echocardiographyrdquo Ultra-sound in Medicine and Biology vol 34 no 5 pp 741ndash752 2008

[8] C K L Phoon ldquoImaging tools for the developmental biologistultrasound biomicroscopy of mouse embryonic developmentrdquoPediatric Research vol 60 no 1 pp 14ndash21 2006

[9] J S Lee Q Yu J T Shin et al ldquoKlf2 is an essential regulator ofvascular hemodynamic forces in vivordquo Developmental Cell vol11 no 6 pp 845ndash857 2006

[10] R A Morris B Damon V Mironov et al ldquoPeriostin regulatescollagen fibrillogenesis and the biomechanical properties ofconnective tissuesrdquo Journal of Cellular Biochemistry vol 101 no3 pp 695ndash711 2007

[11] B C W Groenendijk B P Hierck J Vrolijk et al ldquoChanges inshear stress-related gene expression after experimentally alteredvenous return in the chicken embryordquoCirculation Research vol96 no 12 pp 1291ndash1298 2005

[12] C J Zuurbier V M Emons and C Ince ldquoHemodynamics ofanesthetized ventilated mouse models aspects of anestheticsfluid support and strainrdquo American Journal of Physiology vol282 no 6 pp H2099ndashH2105 2002

[13] A Włoch W Rozmus-Warcholinska B Czuba et al ldquoDopplerstudy of the embryonic heart in normal pregnant womenrdquoJournal of Maternal-Fetal and Neonatal Medicine vol 20 no 7pp 533ndash539 2007

[14] M E Godfrey B Messing S M Cohen D V Valsky andS Yagel ldquoFunctional assessment of the fetal heart a reviewrdquo

Ultrasound in Obstetrics and Gynecology vol 39 no 2 pp 131ndash144 2012

[15] M R M Jongbloed M C E F Wijffels M J Schalij et alldquoDevelopment of the right ventricular inflow tract and moder-ator band a possible morphological and functional explanationforMahaim tachycardiardquoCirculation Research vol 96 no 7 pp776ndash783 2005

[16] Y-HGui KK Linask PKhowsathit and J CHuhta ldquoDopplerechocardiography of normal and abnormal embryonic mouseheartrdquo Pediatric Research vol 40 no 4 pp 633ndash642 1996

[17] C F Spurney L Leatherbury and C W Lo ldquoHigh-frequencyultrasound database profiling growth development and car-diovascular function in C57BL6J mouse fetusesrdquo Journal of theAmerican Society of Echocardiography vol 17 no 8 pp 893ndash900 2004

[18] R P Ji C K L Phoon O Aristizabal K E McGrath JPalis and D H Turnbull ldquoOnset of cardiac function duringearly mouse embryogenesis coincides with entry of primitiveerythroblasts into the embryo properrdquoCirculation Research vol92 no 2 pp 133ndash135 2003

[19] M R Jongbloed S R Vicente N D Hahurij et al ldquoNormaland abnormal development of the cardiac conduction systemimplications for conduction and rhythm disorders in the childand adultrdquo Differentiation vol 84 no 1 pp 131ndash148 2012

[20] K Makikallio P Jouppila and J Rasanen ldquoHuman fetal cardiacfunction during the first trimester of pregnancyrdquo Heart vol 91no 3 pp 334ndash338 2005

[21] W Rozmus-Warcholinska A Wloch G Acharya et al ldquoRef-erence values for variables of fetal cardiocirculatory dynamicsat 11-14 weeks of gestationrdquo Ultrasound in Obstetrics andGynecology vol 35 no 5 pp 540ndash547 2010

[22] G Tulzer P Khowsathit S Gudmundsson et al ldquoDiastolicfunction of the fetal heart during second and third trimestera prospective longitudinal Doppler-echocardiographic studyrdquoEuropean Journal of Pediatrics vol 153 no 3 pp 151ndash154 1994

[23] MC Leiva J E Tolosa CN Binotto et al ldquoFetal cardiac devel-opment and hemodynamics in the first trimesterrdquoUltrasound inObstetrics and Gynecology vol 14 no 3 pp 169ndash174 1999

[24] I P Van Splunder and J W Wladimiroff ldquoCardiac functionalchanges in the human fetus in the late first and early secondtrimestersrdquo Ultrasound in Obstetrics and Gynecology vol 7 no6 pp 411ndash415 1996

[25] S A Clur R K Oude B W Mol J Ottenkamp and CM Bilardo ldquoFetal cardiac function between 11 and 35 weeksrsquogestation and nuchal translucency thicknessrdquo Ultrasound inObstetrics amp Gynecology vol 37 no 1 pp 48ndash56 2011

[26] G Rizzo A Muscatello E Angelini and A Capponi ldquoAbnor-mal cardiac function in fetuses with increased nuchal translu-cencyrdquo Ultrasound in Obstetrics and Gynecology vol 21 no 6pp 539ndash542 2003

[27] F Crispi E Hernandez-Andrade M M A L Pelsers et alldquoCardiac dysfunction and cell damage across clinical stagesof severity in growth-restricted fetusesrdquo American Journal ofObstetrics and Gynecology vol 199 no 3 pp 254ndash258 2008

[28] S Luewan Y Yanase F Tongprasert K Srisupundit and TTongsong ldquoFetal cardiac dimensions at 14-40 weeksrsquo gestationobtained using cardio-STIC-Mrdquo Ultrasound in Obstetrics andGynecology vol 37 no 4 pp 416ndash422 2011

The Scientific World Journal 11

[29] T Tongsong C Wanapirak W Piyamongkol et al ldquoFetalventricular shortening fraction in hydrops fetalisrdquo Obstetricsand Gynecology vol 117 no 1 pp 84ndash91 2011

[30] E Hernandez-Andrade H Figueroa-Diesel C Kottman etal ldquoGestational-age-adjusted reference values for the modifiedmyocardial performance index for evaluation of fetal left car-diac functionrdquoUltrasound in Obstetrics and Gynecology vol 29no 3 pp 321ndash325 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World Journal 7

Mitral06

05

04

03

02

01

00

EA

ratio

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

125 145 175

(a)

06

05

04

03

02

01

00

EA

ratio

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

125 145 175

Tricuspid

(b)

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

125 145 175

Peak

E (m

s)

04

03

02

01

00

(c)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

125 145 175

Peak

E (m

s)

04

03

02

01

00

(d)

lowastlowastlowast

125 145 175

Age (dpc)

Peak

A (m

s)

07

06

05

04

03

02

01

00

nslowastlowast

(e)

125 145 175

Age (dpc)

Peak

A (m

s)

07

06

05

04

03

02

01

00

nsns

ns

(f)

Figure 3 Development of LV and RV inflow patternsThe graphs represent the course of the EA ratio (a b) peak-E wave (c d) and peak-Awave (e f) across the developing MV and TV at the three subsequent developmental stages

The systolic function of the developing ventricles wasassessed through studying the course of the FS IVCTRRand ETRR Throughout embryonic and fetal developmentthe FS remained constant for both ventricles and valuesranged between asymp30 and 40 Furthermore except for175 dpc (LV 406 RV 357 119875 = 0046) no significantdifferences were observed between the FS of the LV and RV

(Figures 2(d) and 2(e)) The mean IVCTRR remained stableduring all three stagesasymp009 the ETRR showed no changesbetween 125 dpc (042plusmn0015) and 145 dpc (043plusmn0010) butsignificantly decreased at 175 dpc (040 plusmn 0006 119875 = 0017)(Figures 1(c) and 1(d))

A combined assessment of LV systolic and diastolic func-tion was performed by calculating the MPI At embryonic

8 The Scientific World Journal

Table 3 The parameters used for calculation of the mean CO per gestational age

Parameter 125 dpc (cOFT) 145 dpc (Ao) 175 dpc (Ao)VTI (mm) 339 plusmn 137 423 plusmn 20 292 plusmn 205

Mean diameter (mm) 023 018 048 plusmn 003

Mean HR (bpm) 134 plusmn 112 148 plusmn 61 206 plusmn 98

Mean CO (mLmin) 019 016 096

lowastlowastlowast05

04

03

02

01

Peak

flow

(ms

)

Common OFT

Aorta

Pulmonary trunk

125 145 175

Age (dpc)

Figure 4 Pulsed-wave Doppler flow measurements in the cOFTAo and PT The graph demonstrates the significant increase of thepeak blood flowmeasured in the cOFT at 125 and Ao and PT at 145and 175 dpc

preseptated stages (125 dpc) the MPI was 064 plusmn 0012 anddecreased to 049 plusmn 0017 (119875 = 00002) after closure of theIVS at 145 dpc At late fetal stages (175 dpc) the MPI did notchange significantly (053 plusmn 0022 119875 = NS)

33 OutflowTract andCO Thepulsed-waveDoppler record-ings performed in the cOFT demonstrated a peak flow of027 plusmn 0021ms at preseptated stages As from 145 dpc twoindividual ventricular outflow tracts could be discriminatedespecially the high echodensity of the blood at these stagesenabled to discriminate the flow in the Ao (034 plusmn 0013ms)and PT (033 plusmn 0021ms) that are closely related to eachother As can be observed in Figure 4 the peak flow increasedsignificantly in the Ao (from 027 plusmn 0021ms to 036 plusmn0016ms 119875 = 0010) and PT (027 plusmn 0021ms to 044 plusmn0030ms 119875 = 0003) along fetal life

Table 3 summarizes theHRVTI and the diameters of thecOFT and Ao that were used to calculate the mean CO at thethree developmental stages Between 125 dpc and 145 dpc the

CO remained stable where after it increased to 096mLminat 175 dpc

4 Discussion

Heart development is a complex process which has beenextensively studied in both WT and genetically mutatedmouse models [3] It is only in the last decades that modernimaging techniques like ultrasound enable the assessmentof real-time hemodynamic and morphological changes thatoccur during mouse heart development [4ndash6 8 16ndash18]Knowledge regarding these functional parameters is essentialsince abnormal flow in the developing heart might lead toCHD through aberrant activation of shear-stress responsivegenes important in cardiogenesis [9 11] In early reportsdescribing the course of functional parameters during heartdevelopmentmajor variability exists inmeasured valuesThisis most likely related to the relatively low ultrasound frequen-cies of clinical ultrasound machines used to image the smallhearts of mouse embryosfetuses [16] and externalizationof uterus horns during ultrasound assessment [18] whichinfluences embryonicfetal physiology Low dose isofluraneanesthesia used in this study only has a minimal effect onhemodynamics and diastolic function in adult mice and theeffect on mouse embryos is supposed to be marginal [6]

In the current study we aimed to provide an overview ofhemodynamic parameters and changes that occur at subse-quent stages of heart development using a high-frequencyultrasound system

Compared to other reports in mouse using similar ultra-sound techniques we did not only focus on development ofthe future LV but also on that of the RV Key findings ofour study are (1) a reliable assessment of LV and RV in- andoutflow patterns can be performed from 125 dpc to term (2)a significant improvement occurs in the diastolic and systolicfunction of both LV and RV (3) over the developmentalperiod of 125 dpc to 175 dpc the ratio between RV and LVsize as well as the FS of both ventricles did not changesignificantly

Knowledge regarding the functional parameters of theRV is important since the RV is commonly affected in CHDDuring development the RV including the TV annulusdevelops subsequent to the LV by addition of SHF derivedcells to the developing heart [1] Many genes have beenidentified that have an important role in the SHF contributionto the developing heart andmutations in these genesmay leadto CHD [19]

At early embryonic stages endocardial cushion tissue ispositioned in the primitive AVC and OFT which later on

The Scientific World Journal 9

will contribute to formation of the TV and MV and Ao andPT semilunar valves Some clinical studies argued that AVvalve regurgitation is a commonphenomenon during the firsttrimester of pregnancy [20] Our study in accordance withseveral others in mouse [4 6] and human [13] did not showsigns of regurgitation at both early and late stages of heartdevelopment Pulsed-wave Doppler recordings across thecAVC at 125 dpc and MV and TV at 145 dpc demonstratedthat the AV cushionsmimic the function of normal AV valvesat 175 dpc In our opinion especially the flow in the cOFTat early embryonic stages which is completely positionedabove the RV (see Figure 2(a)) might wrongly be identifiedas regurgitation of the developing AV valvescushions Thisstresses the importance of detailed knowledge regarding therapid morphological changes that the heart undergoes atespecially the very early developmental stages

Regarding growth of the heart we demonstrated that therewas a 31 increase of LVID 34 of RIVD and 35 of IVSdiameter from 125 up to 175 dpcWe showed a trend towardsa smaller RVID as compared to the LVID at all stages whichwas not mentioned in other studies

41 Diastolic and Systolic Function during Heart DevelopmentImprovement of ventricular systolic and diastolic functionoccurs throughout the length of embryonic and fetal develop-mentWith respect to the diastolic function we demonstrateda significant increase of EA ratio across the developing MVand TV orifices which was related to an increase of the peak-E wave that is the fraction of diastolic ventricular filling thatis defined by ventricular relaxation These data are in accor-dance with the study of Zhou and colleagues who demon-strated that the EA ratio is increasing during developmentwhich seems to continue up to approximately 3 weeks afterbirth [6] Around day four after birth the peak-E wave mea-sured across theMV becomes more dominant than the peak-A wave indicating an improvement of cardiac complianceand that LV filling becomes dominated by passive ventricularrelaxation Remarkably these changes were not observed forthe RV inflow patterns where the peak-A wave remainsdominant [6] Human embryonic and fetal hearts show thesame maturation course of EA ratios across the developingMV and TV as was demonstrated in multiple studies [21ndash25]An abnormal course of EA ratio that is a decreasing[25 26] or an increasing trend [27] along development hasbeen related to the presence of CHD

With respect to systolic ventricular function the FSremained stable (asymp30ndash40) for both ventricles throughoutdevelopmentWe did find a significant difference favoring theLV FS as compared to the RV FS at 175 dpc Interestinglythe same phenomenon of FS difference between LV andRV has been reported in a study of human fetal heartsbetween 14 and 40weeks of pregnancy However as inmousedifferences between humanLVandRVFS is subtle [28] andwhether a decreased or decreasing FS is a predictor of CHDremains unknown Furthermore we did not find a decreasingtrend of both LV and RV FS as was recently demonstratedduring human fetal life [28 29] With respect to the CO wenoticed no change around the proces of ventricular septation(between 125 and 145 dpc) where after a major increase

occured to 096mLmin near term (175 dpc) The initial COdecreasemight be related to the fact that themeandiameter ofthe unseptated cOFT diameter was used at 125 dpc comparedto the isolated Ao diameter at 145 dpc The subsequent COincrease is mostly related to the dramatic increase of HRalong fetal life

The MPI or Tei index [14] is a measure for combinedsystolic and diastolic cardiac function and can be calculatedfor both ventricles In order to assess the MPI three differenttime intervals are needed the IVCT IVRT and ET For the LVthese parameters can be easily obtained since all the threeparameters can be assessed at once by placing the Dopplerultrasound beam across the developing MV orifice Notice-ably in literature there is less data regarding RV MPI andits course along development which might be related to thefact that for the RV not all the three time intervals can beassessed in one view as was also suggested in a recent reviewby Godfrey et al [14] The current study demonstrated asignificant decrease of LV MPI between 125 and 145 dpcwhich might be related to the rapid morphological changesthe heart goes through including completion of IVS leadingto altered hemodynamic conditions in the developing heart Itis however also important to notice that at preseptation stagesthe IVCT and IVRT cannot always be identified separately sothat calculation of the MPI is frequently not possible at veryearly stages of embryonic development Between 145 and175 dpcwenoticed a slight increase of theMPI suggesting thatcardiac function improves at subsequent fetal stages similarresults have also been demonstrated inmouse [4] and human[30] However discussion remains regarding the exact courseof theMPI IVCT IVRT andETandwhether isolated changesof one of the factors is a predictor for fetal demise and CHDInterestinglywith respect to IVRTweobserved a decreasingtrend near completion of the IVS (between 125 and 145)whereafter it more or less remained stable throughout fetallife A similar course was observed in a recent ultrasoundstudy in embryonicfetal C57Bl6 mice [4] Human embryosalso showed a decreasing course of the IVRT [13 21] and anelevated IVRT has been implicated as a predictor of CHD[13]

5 Conclusion

We demonstrated the course of several morphologic andhemodynamic parameters along embryonic and fetal lifeOur data show that a reliable assessment can be made notonly of the left but also of right ventricular function duringembryonicfetal stages We demonstrate an overall improve-ment of cardiac diastolic and systolic function of both LV andRV along heart development The implementation of high-frequency ultrasound in embryonicfetal mouse heart devel-opment is a promising and useful tool The data presented inthis studymight be useful as reference values in future studiesin genetic mutated mouse models of CHD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

10 The Scientific World Journal

Authorsrsquo Contribution

Nathan D Hahurij and Emmeline E Calkoen contributedequally to this work and shared first authorship

Acknowledgment

This work is supported by the DutchHeart Foundation Grantno 2009T070 (Nathan D Hahurij)

References

[1] R G Kelly ldquoMolecular inroads into the anterior heart fieldrdquoTrends inCardiovascularMedicine vol 15 no 2 pp 51ndash56 2005

[2] J I E Hoffman and S Kaplan ldquoThe incidence of congenitalheart diseaserdquo Journal of the AmericanCollege of Cardiology vol39 no 12 pp 1890ndash1900 2002

[3] D Srivastava ldquoGenetic assembly of the heart implications forcongenital heart diseaserdquo Annual Review of Physiology vol 63pp 451ndash469 2001

[4] N Corrigan D P Brazil and F M Auliffe ldquoHigh-frequencyultrasound assessment of the murine heart from embryothrough to juvenilerdquoReproductive Sciences vol 17 no 2 pp 147ndash157 2010

[5] C F Spurney CW Lo and L Leatherbury ldquoFetal mouse imag-ing using echocardiography a review of current technologyrdquoEchocardiography vol 23 no 10 pp 891ndash899 2006

[6] Y-Q Zhou F S Foster R Parkes and S L Adamson ldquoDevel-opmental changes in left and right ventricular diastolic fillingpatterns in micerdquo American Journal of Physiology vol 285 no4 pp H1563ndashH1575 2003

[7] Q Yu L Leatherbury X Tian and C W Lo ldquoCardiovascularassessment of fetal mice by in utero echocardiographyrdquo Ultra-sound in Medicine and Biology vol 34 no 5 pp 741ndash752 2008

[8] C K L Phoon ldquoImaging tools for the developmental biologistultrasound biomicroscopy of mouse embryonic developmentrdquoPediatric Research vol 60 no 1 pp 14ndash21 2006

[9] J S Lee Q Yu J T Shin et al ldquoKlf2 is an essential regulator ofvascular hemodynamic forces in vivordquo Developmental Cell vol11 no 6 pp 845ndash857 2006

[10] R A Morris B Damon V Mironov et al ldquoPeriostin regulatescollagen fibrillogenesis and the biomechanical properties ofconnective tissuesrdquo Journal of Cellular Biochemistry vol 101 no3 pp 695ndash711 2007

[11] B C W Groenendijk B P Hierck J Vrolijk et al ldquoChanges inshear stress-related gene expression after experimentally alteredvenous return in the chicken embryordquoCirculation Research vol96 no 12 pp 1291ndash1298 2005

[12] C J Zuurbier V M Emons and C Ince ldquoHemodynamics ofanesthetized ventilated mouse models aspects of anestheticsfluid support and strainrdquo American Journal of Physiology vol282 no 6 pp H2099ndashH2105 2002

[13] A Włoch W Rozmus-Warcholinska B Czuba et al ldquoDopplerstudy of the embryonic heart in normal pregnant womenrdquoJournal of Maternal-Fetal and Neonatal Medicine vol 20 no 7pp 533ndash539 2007

[14] M E Godfrey B Messing S M Cohen D V Valsky andS Yagel ldquoFunctional assessment of the fetal heart a reviewrdquo

Ultrasound in Obstetrics and Gynecology vol 39 no 2 pp 131ndash144 2012

[15] M R M Jongbloed M C E F Wijffels M J Schalij et alldquoDevelopment of the right ventricular inflow tract and moder-ator band a possible morphological and functional explanationforMahaim tachycardiardquoCirculation Research vol 96 no 7 pp776ndash783 2005

[16] Y-HGui KK Linask PKhowsathit and J CHuhta ldquoDopplerechocardiography of normal and abnormal embryonic mouseheartrdquo Pediatric Research vol 40 no 4 pp 633ndash642 1996

[17] C F Spurney L Leatherbury and C W Lo ldquoHigh-frequencyultrasound database profiling growth development and car-diovascular function in C57BL6J mouse fetusesrdquo Journal of theAmerican Society of Echocardiography vol 17 no 8 pp 893ndash900 2004

[18] R P Ji C K L Phoon O Aristizabal K E McGrath JPalis and D H Turnbull ldquoOnset of cardiac function duringearly mouse embryogenesis coincides with entry of primitiveerythroblasts into the embryo properrdquoCirculation Research vol92 no 2 pp 133ndash135 2003

[19] M R Jongbloed S R Vicente N D Hahurij et al ldquoNormaland abnormal development of the cardiac conduction systemimplications for conduction and rhythm disorders in the childand adultrdquo Differentiation vol 84 no 1 pp 131ndash148 2012

[20] K Makikallio P Jouppila and J Rasanen ldquoHuman fetal cardiacfunction during the first trimester of pregnancyrdquo Heart vol 91no 3 pp 334ndash338 2005

[21] W Rozmus-Warcholinska A Wloch G Acharya et al ldquoRef-erence values for variables of fetal cardiocirculatory dynamicsat 11-14 weeks of gestationrdquo Ultrasound in Obstetrics andGynecology vol 35 no 5 pp 540ndash547 2010

[22] G Tulzer P Khowsathit S Gudmundsson et al ldquoDiastolicfunction of the fetal heart during second and third trimestera prospective longitudinal Doppler-echocardiographic studyrdquoEuropean Journal of Pediatrics vol 153 no 3 pp 151ndash154 1994

[23] MC Leiva J E Tolosa CN Binotto et al ldquoFetal cardiac devel-opment and hemodynamics in the first trimesterrdquoUltrasound inObstetrics and Gynecology vol 14 no 3 pp 169ndash174 1999

[24] I P Van Splunder and J W Wladimiroff ldquoCardiac functionalchanges in the human fetus in the late first and early secondtrimestersrdquo Ultrasound in Obstetrics and Gynecology vol 7 no6 pp 411ndash415 1996

[25] S A Clur R K Oude B W Mol J Ottenkamp and CM Bilardo ldquoFetal cardiac function between 11 and 35 weeksrsquogestation and nuchal translucency thicknessrdquo Ultrasound inObstetrics amp Gynecology vol 37 no 1 pp 48ndash56 2011

[26] G Rizzo A Muscatello E Angelini and A Capponi ldquoAbnor-mal cardiac function in fetuses with increased nuchal translu-cencyrdquo Ultrasound in Obstetrics and Gynecology vol 21 no 6pp 539ndash542 2003

[27] F Crispi E Hernandez-Andrade M M A L Pelsers et alldquoCardiac dysfunction and cell damage across clinical stagesof severity in growth-restricted fetusesrdquo American Journal ofObstetrics and Gynecology vol 199 no 3 pp 254ndash258 2008

[28] S Luewan Y Yanase F Tongprasert K Srisupundit and TTongsong ldquoFetal cardiac dimensions at 14-40 weeksrsquo gestationobtained using cardio-STIC-Mrdquo Ultrasound in Obstetrics andGynecology vol 37 no 4 pp 416ndash422 2011

The Scientific World Journal 11

[29] T Tongsong C Wanapirak W Piyamongkol et al ldquoFetalventricular shortening fraction in hydrops fetalisrdquo Obstetricsand Gynecology vol 117 no 1 pp 84ndash91 2011

[30] E Hernandez-Andrade H Figueroa-Diesel C Kottman etal ldquoGestational-age-adjusted reference values for the modifiedmyocardial performance index for evaluation of fetal left car-diac functionrdquoUltrasound in Obstetrics and Gynecology vol 29no 3 pp 321ndash325 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 The Scientific World Journal

Table 3 The parameters used for calculation of the mean CO per gestational age

Parameter 125 dpc (cOFT) 145 dpc (Ao) 175 dpc (Ao)VTI (mm) 339 plusmn 137 423 plusmn 20 292 plusmn 205

Mean diameter (mm) 023 018 048 plusmn 003

Mean HR (bpm) 134 plusmn 112 148 plusmn 61 206 plusmn 98

Mean CO (mLmin) 019 016 096

lowastlowastlowast05

04

03

02

01

Peak

flow

(ms

)

Common OFT

Aorta

Pulmonary trunk

125 145 175

Age (dpc)

Figure 4 Pulsed-wave Doppler flow measurements in the cOFTAo and PT The graph demonstrates the significant increase of thepeak blood flowmeasured in the cOFT at 125 and Ao and PT at 145and 175 dpc

preseptated stages (125 dpc) the MPI was 064 plusmn 0012 anddecreased to 049 plusmn 0017 (119875 = 00002) after closure of theIVS at 145 dpc At late fetal stages (175 dpc) the MPI did notchange significantly (053 plusmn 0022 119875 = NS)

33 OutflowTract andCO Thepulsed-waveDoppler record-ings performed in the cOFT demonstrated a peak flow of027 plusmn 0021ms at preseptated stages As from 145 dpc twoindividual ventricular outflow tracts could be discriminatedespecially the high echodensity of the blood at these stagesenabled to discriminate the flow in the Ao (034 plusmn 0013ms)and PT (033 plusmn 0021ms) that are closely related to eachother As can be observed in Figure 4 the peak flow increasedsignificantly in the Ao (from 027 plusmn 0021ms to 036 plusmn0016ms 119875 = 0010) and PT (027 plusmn 0021ms to 044 plusmn0030ms 119875 = 0003) along fetal life

Table 3 summarizes theHRVTI and the diameters of thecOFT and Ao that were used to calculate the mean CO at thethree developmental stages Between 125 dpc and 145 dpc the

CO remained stable where after it increased to 096mLminat 175 dpc

4 Discussion

Heart development is a complex process which has beenextensively studied in both WT and genetically mutatedmouse models [3] It is only in the last decades that modernimaging techniques like ultrasound enable the assessmentof real-time hemodynamic and morphological changes thatoccur during mouse heart development [4ndash6 8 16ndash18]Knowledge regarding these functional parameters is essentialsince abnormal flow in the developing heart might lead toCHD through aberrant activation of shear-stress responsivegenes important in cardiogenesis [9 11] In early reportsdescribing the course of functional parameters during heartdevelopmentmajor variability exists inmeasured valuesThisis most likely related to the relatively low ultrasound frequen-cies of clinical ultrasound machines used to image the smallhearts of mouse embryosfetuses [16] and externalizationof uterus horns during ultrasound assessment [18] whichinfluences embryonicfetal physiology Low dose isofluraneanesthesia used in this study only has a minimal effect onhemodynamics and diastolic function in adult mice and theeffect on mouse embryos is supposed to be marginal [6]

In the current study we aimed to provide an overview ofhemodynamic parameters and changes that occur at subse-quent stages of heart development using a high-frequencyultrasound system

Compared to other reports in mouse using similar ultra-sound techniques we did not only focus on development ofthe future LV but also on that of the RV Key findings ofour study are (1) a reliable assessment of LV and RV in- andoutflow patterns can be performed from 125 dpc to term (2)a significant improvement occurs in the diastolic and systolicfunction of both LV and RV (3) over the developmentalperiod of 125 dpc to 175 dpc the ratio between RV and LVsize as well as the FS of both ventricles did not changesignificantly

Knowledge regarding the functional parameters of theRV is important since the RV is commonly affected in CHDDuring development the RV including the TV annulusdevelops subsequent to the LV by addition of SHF derivedcells to the developing heart [1] Many genes have beenidentified that have an important role in the SHF contributionto the developing heart andmutations in these genesmay leadto CHD [19]

At early embryonic stages endocardial cushion tissue ispositioned in the primitive AVC and OFT which later on

The Scientific World Journal 9

will contribute to formation of the TV and MV and Ao andPT semilunar valves Some clinical studies argued that AVvalve regurgitation is a commonphenomenon during the firsttrimester of pregnancy [20] Our study in accordance withseveral others in mouse [4 6] and human [13] did not showsigns of regurgitation at both early and late stages of heartdevelopment Pulsed-wave Doppler recordings across thecAVC at 125 dpc and MV and TV at 145 dpc demonstratedthat the AV cushionsmimic the function of normal AV valvesat 175 dpc In our opinion especially the flow in the cOFTat early embryonic stages which is completely positionedabove the RV (see Figure 2(a)) might wrongly be identifiedas regurgitation of the developing AV valvescushions Thisstresses the importance of detailed knowledge regarding therapid morphological changes that the heart undergoes atespecially the very early developmental stages

Regarding growth of the heart we demonstrated that therewas a 31 increase of LVID 34 of RIVD and 35 of IVSdiameter from 125 up to 175 dpcWe showed a trend towardsa smaller RVID as compared to the LVID at all stages whichwas not mentioned in other studies

41 Diastolic and Systolic Function during Heart DevelopmentImprovement of ventricular systolic and diastolic functionoccurs throughout the length of embryonic and fetal develop-mentWith respect to the diastolic function we demonstrateda significant increase of EA ratio across the developing MVand TV orifices which was related to an increase of the peak-E wave that is the fraction of diastolic ventricular filling thatis defined by ventricular relaxation These data are in accor-dance with the study of Zhou and colleagues who demon-strated that the EA ratio is increasing during developmentwhich seems to continue up to approximately 3 weeks afterbirth [6] Around day four after birth the peak-E wave mea-sured across theMV becomes more dominant than the peak-A wave indicating an improvement of cardiac complianceand that LV filling becomes dominated by passive ventricularrelaxation Remarkably these changes were not observed forthe RV inflow patterns where the peak-A wave remainsdominant [6] Human embryonic and fetal hearts show thesame maturation course of EA ratios across the developingMV and TV as was demonstrated in multiple studies [21ndash25]An abnormal course of EA ratio that is a decreasing[25 26] or an increasing trend [27] along development hasbeen related to the presence of CHD

With respect to systolic ventricular function the FSremained stable (asymp30ndash40) for both ventricles throughoutdevelopmentWe did find a significant difference favoring theLV FS as compared to the RV FS at 175 dpc Interestinglythe same phenomenon of FS difference between LV andRV has been reported in a study of human fetal heartsbetween 14 and 40weeks of pregnancy However as inmousedifferences between humanLVandRVFS is subtle [28] andwhether a decreased or decreasing FS is a predictor of CHDremains unknown Furthermore we did not find a decreasingtrend of both LV and RV FS as was recently demonstratedduring human fetal life [28 29] With respect to the CO wenoticed no change around the proces of ventricular septation(between 125 and 145 dpc) where after a major increase

occured to 096mLmin near term (175 dpc) The initial COdecreasemight be related to the fact that themeandiameter ofthe unseptated cOFT diameter was used at 125 dpc comparedto the isolated Ao diameter at 145 dpc The subsequent COincrease is mostly related to the dramatic increase of HRalong fetal life

The MPI or Tei index [14] is a measure for combinedsystolic and diastolic cardiac function and can be calculatedfor both ventricles In order to assess the MPI three differenttime intervals are needed the IVCT IVRT and ET For the LVthese parameters can be easily obtained since all the threeparameters can be assessed at once by placing the Dopplerultrasound beam across the developing MV orifice Notice-ably in literature there is less data regarding RV MPI andits course along development which might be related to thefact that for the RV not all the three time intervals can beassessed in one view as was also suggested in a recent reviewby Godfrey et al [14] The current study demonstrated asignificant decrease of LV MPI between 125 and 145 dpcwhich might be related to the rapid morphological changesthe heart goes through including completion of IVS leadingto altered hemodynamic conditions in the developing heart Itis however also important to notice that at preseptation stagesthe IVCT and IVRT cannot always be identified separately sothat calculation of the MPI is frequently not possible at veryearly stages of embryonic development Between 145 and175 dpcwenoticed a slight increase of theMPI suggesting thatcardiac function improves at subsequent fetal stages similarresults have also been demonstrated inmouse [4] and human[30] However discussion remains regarding the exact courseof theMPI IVCT IVRT andETandwhether isolated changesof one of the factors is a predictor for fetal demise and CHDInterestinglywith respect to IVRTweobserved a decreasingtrend near completion of the IVS (between 125 and 145)whereafter it more or less remained stable throughout fetallife A similar course was observed in a recent ultrasoundstudy in embryonicfetal C57Bl6 mice [4] Human embryosalso showed a decreasing course of the IVRT [13 21] and anelevated IVRT has been implicated as a predictor of CHD[13]

5 Conclusion

We demonstrated the course of several morphologic andhemodynamic parameters along embryonic and fetal lifeOur data show that a reliable assessment can be made notonly of the left but also of right ventricular function duringembryonicfetal stages We demonstrate an overall improve-ment of cardiac diastolic and systolic function of both LV andRV along heart development The implementation of high-frequency ultrasound in embryonicfetal mouse heart devel-opment is a promising and useful tool The data presented inthis studymight be useful as reference values in future studiesin genetic mutated mouse models of CHD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

10 The Scientific World Journal

Authorsrsquo Contribution

Nathan D Hahurij and Emmeline E Calkoen contributedequally to this work and shared first authorship

Acknowledgment

This work is supported by the DutchHeart Foundation Grantno 2009T070 (Nathan D Hahurij)

References

[1] R G Kelly ldquoMolecular inroads into the anterior heart fieldrdquoTrends inCardiovascularMedicine vol 15 no 2 pp 51ndash56 2005

[2] J I E Hoffman and S Kaplan ldquoThe incidence of congenitalheart diseaserdquo Journal of the AmericanCollege of Cardiology vol39 no 12 pp 1890ndash1900 2002

[3] D Srivastava ldquoGenetic assembly of the heart implications forcongenital heart diseaserdquo Annual Review of Physiology vol 63pp 451ndash469 2001

[4] N Corrigan D P Brazil and F M Auliffe ldquoHigh-frequencyultrasound assessment of the murine heart from embryothrough to juvenilerdquoReproductive Sciences vol 17 no 2 pp 147ndash157 2010

[5] C F Spurney CW Lo and L Leatherbury ldquoFetal mouse imag-ing using echocardiography a review of current technologyrdquoEchocardiography vol 23 no 10 pp 891ndash899 2006

[6] Y-Q Zhou F S Foster R Parkes and S L Adamson ldquoDevel-opmental changes in left and right ventricular diastolic fillingpatterns in micerdquo American Journal of Physiology vol 285 no4 pp H1563ndashH1575 2003

[7] Q Yu L Leatherbury X Tian and C W Lo ldquoCardiovascularassessment of fetal mice by in utero echocardiographyrdquo Ultra-sound in Medicine and Biology vol 34 no 5 pp 741ndash752 2008

[8] C K L Phoon ldquoImaging tools for the developmental biologistultrasound biomicroscopy of mouse embryonic developmentrdquoPediatric Research vol 60 no 1 pp 14ndash21 2006

[9] J S Lee Q Yu J T Shin et al ldquoKlf2 is an essential regulator ofvascular hemodynamic forces in vivordquo Developmental Cell vol11 no 6 pp 845ndash857 2006

[10] R A Morris B Damon V Mironov et al ldquoPeriostin regulatescollagen fibrillogenesis and the biomechanical properties ofconnective tissuesrdquo Journal of Cellular Biochemistry vol 101 no3 pp 695ndash711 2007

[11] B C W Groenendijk B P Hierck J Vrolijk et al ldquoChanges inshear stress-related gene expression after experimentally alteredvenous return in the chicken embryordquoCirculation Research vol96 no 12 pp 1291ndash1298 2005

[12] C J Zuurbier V M Emons and C Ince ldquoHemodynamics ofanesthetized ventilated mouse models aspects of anestheticsfluid support and strainrdquo American Journal of Physiology vol282 no 6 pp H2099ndashH2105 2002

[13] A Włoch W Rozmus-Warcholinska B Czuba et al ldquoDopplerstudy of the embryonic heart in normal pregnant womenrdquoJournal of Maternal-Fetal and Neonatal Medicine vol 20 no 7pp 533ndash539 2007

[14] M E Godfrey B Messing S M Cohen D V Valsky andS Yagel ldquoFunctional assessment of the fetal heart a reviewrdquo

Ultrasound in Obstetrics and Gynecology vol 39 no 2 pp 131ndash144 2012

[15] M R M Jongbloed M C E F Wijffels M J Schalij et alldquoDevelopment of the right ventricular inflow tract and moder-ator band a possible morphological and functional explanationforMahaim tachycardiardquoCirculation Research vol 96 no 7 pp776ndash783 2005

[16] Y-HGui KK Linask PKhowsathit and J CHuhta ldquoDopplerechocardiography of normal and abnormal embryonic mouseheartrdquo Pediatric Research vol 40 no 4 pp 633ndash642 1996

[17] C F Spurney L Leatherbury and C W Lo ldquoHigh-frequencyultrasound database profiling growth development and car-diovascular function in C57BL6J mouse fetusesrdquo Journal of theAmerican Society of Echocardiography vol 17 no 8 pp 893ndash900 2004

[18] R P Ji C K L Phoon O Aristizabal K E McGrath JPalis and D H Turnbull ldquoOnset of cardiac function duringearly mouse embryogenesis coincides with entry of primitiveerythroblasts into the embryo properrdquoCirculation Research vol92 no 2 pp 133ndash135 2003

[19] M R Jongbloed S R Vicente N D Hahurij et al ldquoNormaland abnormal development of the cardiac conduction systemimplications for conduction and rhythm disorders in the childand adultrdquo Differentiation vol 84 no 1 pp 131ndash148 2012

[20] K Makikallio P Jouppila and J Rasanen ldquoHuman fetal cardiacfunction during the first trimester of pregnancyrdquo Heart vol 91no 3 pp 334ndash338 2005

[21] W Rozmus-Warcholinska A Wloch G Acharya et al ldquoRef-erence values for variables of fetal cardiocirculatory dynamicsat 11-14 weeks of gestationrdquo Ultrasound in Obstetrics andGynecology vol 35 no 5 pp 540ndash547 2010

[22] G Tulzer P Khowsathit S Gudmundsson et al ldquoDiastolicfunction of the fetal heart during second and third trimestera prospective longitudinal Doppler-echocardiographic studyrdquoEuropean Journal of Pediatrics vol 153 no 3 pp 151ndash154 1994

[23] MC Leiva J E Tolosa CN Binotto et al ldquoFetal cardiac devel-opment and hemodynamics in the first trimesterrdquoUltrasound inObstetrics and Gynecology vol 14 no 3 pp 169ndash174 1999

[24] I P Van Splunder and J W Wladimiroff ldquoCardiac functionalchanges in the human fetus in the late first and early secondtrimestersrdquo Ultrasound in Obstetrics and Gynecology vol 7 no6 pp 411ndash415 1996

[25] S A Clur R K Oude B W Mol J Ottenkamp and CM Bilardo ldquoFetal cardiac function between 11 and 35 weeksrsquogestation and nuchal translucency thicknessrdquo Ultrasound inObstetrics amp Gynecology vol 37 no 1 pp 48ndash56 2011

[26] G Rizzo A Muscatello E Angelini and A Capponi ldquoAbnor-mal cardiac function in fetuses with increased nuchal translu-cencyrdquo Ultrasound in Obstetrics and Gynecology vol 21 no 6pp 539ndash542 2003

[27] F Crispi E Hernandez-Andrade M M A L Pelsers et alldquoCardiac dysfunction and cell damage across clinical stagesof severity in growth-restricted fetusesrdquo American Journal ofObstetrics and Gynecology vol 199 no 3 pp 254ndash258 2008

[28] S Luewan Y Yanase F Tongprasert K Srisupundit and TTongsong ldquoFetal cardiac dimensions at 14-40 weeksrsquo gestationobtained using cardio-STIC-Mrdquo Ultrasound in Obstetrics andGynecology vol 37 no 4 pp 416ndash422 2011

The Scientific World Journal 11

[29] T Tongsong C Wanapirak W Piyamongkol et al ldquoFetalventricular shortening fraction in hydrops fetalisrdquo Obstetricsand Gynecology vol 117 no 1 pp 84ndash91 2011

[30] E Hernandez-Andrade H Figueroa-Diesel C Kottman etal ldquoGestational-age-adjusted reference values for the modifiedmyocardial performance index for evaluation of fetal left car-diac functionrdquoUltrasound in Obstetrics and Gynecology vol 29no 3 pp 321ndash325 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World Journal 9

will contribute to formation of the TV and MV and Ao andPT semilunar valves Some clinical studies argued that AVvalve regurgitation is a commonphenomenon during the firsttrimester of pregnancy [20] Our study in accordance withseveral others in mouse [4 6] and human [13] did not showsigns of regurgitation at both early and late stages of heartdevelopment Pulsed-wave Doppler recordings across thecAVC at 125 dpc and MV and TV at 145 dpc demonstratedthat the AV cushionsmimic the function of normal AV valvesat 175 dpc In our opinion especially the flow in the cOFTat early embryonic stages which is completely positionedabove the RV (see Figure 2(a)) might wrongly be identifiedas regurgitation of the developing AV valvescushions Thisstresses the importance of detailed knowledge regarding therapid morphological changes that the heart undergoes atespecially the very early developmental stages

Regarding growth of the heart we demonstrated that therewas a 31 increase of LVID 34 of RIVD and 35 of IVSdiameter from 125 up to 175 dpcWe showed a trend towardsa smaller RVID as compared to the LVID at all stages whichwas not mentioned in other studies

41 Diastolic and Systolic Function during Heart DevelopmentImprovement of ventricular systolic and diastolic functionoccurs throughout the length of embryonic and fetal develop-mentWith respect to the diastolic function we demonstrateda significant increase of EA ratio across the developing MVand TV orifices which was related to an increase of the peak-E wave that is the fraction of diastolic ventricular filling thatis defined by ventricular relaxation These data are in accor-dance with the study of Zhou and colleagues who demon-strated that the EA ratio is increasing during developmentwhich seems to continue up to approximately 3 weeks afterbirth [6] Around day four after birth the peak-E wave mea-sured across theMV becomes more dominant than the peak-A wave indicating an improvement of cardiac complianceand that LV filling becomes dominated by passive ventricularrelaxation Remarkably these changes were not observed forthe RV inflow patterns where the peak-A wave remainsdominant [6] Human embryonic and fetal hearts show thesame maturation course of EA ratios across the developingMV and TV as was demonstrated in multiple studies [21ndash25]An abnormal course of EA ratio that is a decreasing[25 26] or an increasing trend [27] along development hasbeen related to the presence of CHD

With respect to systolic ventricular function the FSremained stable (asymp30ndash40) for both ventricles throughoutdevelopmentWe did find a significant difference favoring theLV FS as compared to the RV FS at 175 dpc Interestinglythe same phenomenon of FS difference between LV andRV has been reported in a study of human fetal heartsbetween 14 and 40weeks of pregnancy However as inmousedifferences between humanLVandRVFS is subtle [28] andwhether a decreased or decreasing FS is a predictor of CHDremains unknown Furthermore we did not find a decreasingtrend of both LV and RV FS as was recently demonstratedduring human fetal life [28 29] With respect to the CO wenoticed no change around the proces of ventricular septation(between 125 and 145 dpc) where after a major increase

occured to 096mLmin near term (175 dpc) The initial COdecreasemight be related to the fact that themeandiameter ofthe unseptated cOFT diameter was used at 125 dpc comparedto the isolated Ao diameter at 145 dpc The subsequent COincrease is mostly related to the dramatic increase of HRalong fetal life

The MPI or Tei index [14] is a measure for combinedsystolic and diastolic cardiac function and can be calculatedfor both ventricles In order to assess the MPI three differenttime intervals are needed the IVCT IVRT and ET For the LVthese parameters can be easily obtained since all the threeparameters can be assessed at once by placing the Dopplerultrasound beam across the developing MV orifice Notice-ably in literature there is less data regarding RV MPI andits course along development which might be related to thefact that for the RV not all the three time intervals can beassessed in one view as was also suggested in a recent reviewby Godfrey et al [14] The current study demonstrated asignificant decrease of LV MPI between 125 and 145 dpcwhich might be related to the rapid morphological changesthe heart goes through including completion of IVS leadingto altered hemodynamic conditions in the developing heart Itis however also important to notice that at preseptation stagesthe IVCT and IVRT cannot always be identified separately sothat calculation of the MPI is frequently not possible at veryearly stages of embryonic development Between 145 and175 dpcwenoticed a slight increase of theMPI suggesting thatcardiac function improves at subsequent fetal stages similarresults have also been demonstrated inmouse [4] and human[30] However discussion remains regarding the exact courseof theMPI IVCT IVRT andETandwhether isolated changesof one of the factors is a predictor for fetal demise and CHDInterestinglywith respect to IVRTweobserved a decreasingtrend near completion of the IVS (between 125 and 145)whereafter it more or less remained stable throughout fetallife A similar course was observed in a recent ultrasoundstudy in embryonicfetal C57Bl6 mice [4] Human embryosalso showed a decreasing course of the IVRT [13 21] and anelevated IVRT has been implicated as a predictor of CHD[13]

5 Conclusion

We demonstrated the course of several morphologic andhemodynamic parameters along embryonic and fetal lifeOur data show that a reliable assessment can be made notonly of the left but also of right ventricular function duringembryonicfetal stages We demonstrate an overall improve-ment of cardiac diastolic and systolic function of both LV andRV along heart development The implementation of high-frequency ultrasound in embryonicfetal mouse heart devel-opment is a promising and useful tool The data presented inthis studymight be useful as reference values in future studiesin genetic mutated mouse models of CHD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

10 The Scientific World Journal

Authorsrsquo Contribution

Nathan D Hahurij and Emmeline E Calkoen contributedequally to this work and shared first authorship

Acknowledgment

This work is supported by the DutchHeart Foundation Grantno 2009T070 (Nathan D Hahurij)

References

[1] R G Kelly ldquoMolecular inroads into the anterior heart fieldrdquoTrends inCardiovascularMedicine vol 15 no 2 pp 51ndash56 2005

[2] J I E Hoffman and S Kaplan ldquoThe incidence of congenitalheart diseaserdquo Journal of the AmericanCollege of Cardiology vol39 no 12 pp 1890ndash1900 2002

[3] D Srivastava ldquoGenetic assembly of the heart implications forcongenital heart diseaserdquo Annual Review of Physiology vol 63pp 451ndash469 2001

[4] N Corrigan D P Brazil and F M Auliffe ldquoHigh-frequencyultrasound assessment of the murine heart from embryothrough to juvenilerdquoReproductive Sciences vol 17 no 2 pp 147ndash157 2010

[5] C F Spurney CW Lo and L Leatherbury ldquoFetal mouse imag-ing using echocardiography a review of current technologyrdquoEchocardiography vol 23 no 10 pp 891ndash899 2006

[6] Y-Q Zhou F S Foster R Parkes and S L Adamson ldquoDevel-opmental changes in left and right ventricular diastolic fillingpatterns in micerdquo American Journal of Physiology vol 285 no4 pp H1563ndashH1575 2003

[7] Q Yu L Leatherbury X Tian and C W Lo ldquoCardiovascularassessment of fetal mice by in utero echocardiographyrdquo Ultra-sound in Medicine and Biology vol 34 no 5 pp 741ndash752 2008

[8] C K L Phoon ldquoImaging tools for the developmental biologistultrasound biomicroscopy of mouse embryonic developmentrdquoPediatric Research vol 60 no 1 pp 14ndash21 2006

[9] J S Lee Q Yu J T Shin et al ldquoKlf2 is an essential regulator ofvascular hemodynamic forces in vivordquo Developmental Cell vol11 no 6 pp 845ndash857 2006

[10] R A Morris B Damon V Mironov et al ldquoPeriostin regulatescollagen fibrillogenesis and the biomechanical properties ofconnective tissuesrdquo Journal of Cellular Biochemistry vol 101 no3 pp 695ndash711 2007

[11] B C W Groenendijk B P Hierck J Vrolijk et al ldquoChanges inshear stress-related gene expression after experimentally alteredvenous return in the chicken embryordquoCirculation Research vol96 no 12 pp 1291ndash1298 2005

[12] C J Zuurbier V M Emons and C Ince ldquoHemodynamics ofanesthetized ventilated mouse models aspects of anestheticsfluid support and strainrdquo American Journal of Physiology vol282 no 6 pp H2099ndashH2105 2002

[13] A Włoch W Rozmus-Warcholinska B Czuba et al ldquoDopplerstudy of the embryonic heart in normal pregnant womenrdquoJournal of Maternal-Fetal and Neonatal Medicine vol 20 no 7pp 533ndash539 2007

[14] M E Godfrey B Messing S M Cohen D V Valsky andS Yagel ldquoFunctional assessment of the fetal heart a reviewrdquo

Ultrasound in Obstetrics and Gynecology vol 39 no 2 pp 131ndash144 2012

[15] M R M Jongbloed M C E F Wijffels M J Schalij et alldquoDevelopment of the right ventricular inflow tract and moder-ator band a possible morphological and functional explanationforMahaim tachycardiardquoCirculation Research vol 96 no 7 pp776ndash783 2005

[16] Y-HGui KK Linask PKhowsathit and J CHuhta ldquoDopplerechocardiography of normal and abnormal embryonic mouseheartrdquo Pediatric Research vol 40 no 4 pp 633ndash642 1996

[17] C F Spurney L Leatherbury and C W Lo ldquoHigh-frequencyultrasound database profiling growth development and car-diovascular function in C57BL6J mouse fetusesrdquo Journal of theAmerican Society of Echocardiography vol 17 no 8 pp 893ndash900 2004

[18] R P Ji C K L Phoon O Aristizabal K E McGrath JPalis and D H Turnbull ldquoOnset of cardiac function duringearly mouse embryogenesis coincides with entry of primitiveerythroblasts into the embryo properrdquoCirculation Research vol92 no 2 pp 133ndash135 2003

[19] M R Jongbloed S R Vicente N D Hahurij et al ldquoNormaland abnormal development of the cardiac conduction systemimplications for conduction and rhythm disorders in the childand adultrdquo Differentiation vol 84 no 1 pp 131ndash148 2012

[20] K Makikallio P Jouppila and J Rasanen ldquoHuman fetal cardiacfunction during the first trimester of pregnancyrdquo Heart vol 91no 3 pp 334ndash338 2005

[21] W Rozmus-Warcholinska A Wloch G Acharya et al ldquoRef-erence values for variables of fetal cardiocirculatory dynamicsat 11-14 weeks of gestationrdquo Ultrasound in Obstetrics andGynecology vol 35 no 5 pp 540ndash547 2010

[22] G Tulzer P Khowsathit S Gudmundsson et al ldquoDiastolicfunction of the fetal heart during second and third trimestera prospective longitudinal Doppler-echocardiographic studyrdquoEuropean Journal of Pediatrics vol 153 no 3 pp 151ndash154 1994

[23] MC Leiva J E Tolosa CN Binotto et al ldquoFetal cardiac devel-opment and hemodynamics in the first trimesterrdquoUltrasound inObstetrics and Gynecology vol 14 no 3 pp 169ndash174 1999

[24] I P Van Splunder and J W Wladimiroff ldquoCardiac functionalchanges in the human fetus in the late first and early secondtrimestersrdquo Ultrasound in Obstetrics and Gynecology vol 7 no6 pp 411ndash415 1996

[25] S A Clur R K Oude B W Mol J Ottenkamp and CM Bilardo ldquoFetal cardiac function between 11 and 35 weeksrsquogestation and nuchal translucency thicknessrdquo Ultrasound inObstetrics amp Gynecology vol 37 no 1 pp 48ndash56 2011

[26] G Rizzo A Muscatello E Angelini and A Capponi ldquoAbnor-mal cardiac function in fetuses with increased nuchal translu-cencyrdquo Ultrasound in Obstetrics and Gynecology vol 21 no 6pp 539ndash542 2003

[27] F Crispi E Hernandez-Andrade M M A L Pelsers et alldquoCardiac dysfunction and cell damage across clinical stagesof severity in growth-restricted fetusesrdquo American Journal ofObstetrics and Gynecology vol 199 no 3 pp 254ndash258 2008

[28] S Luewan Y Yanase F Tongprasert K Srisupundit and TTongsong ldquoFetal cardiac dimensions at 14-40 weeksrsquo gestationobtained using cardio-STIC-Mrdquo Ultrasound in Obstetrics andGynecology vol 37 no 4 pp 416ndash422 2011

The Scientific World Journal 11

[29] T Tongsong C Wanapirak W Piyamongkol et al ldquoFetalventricular shortening fraction in hydrops fetalisrdquo Obstetricsand Gynecology vol 117 no 1 pp 84ndash91 2011

[30] E Hernandez-Andrade H Figueroa-Diesel C Kottman etal ldquoGestational-age-adjusted reference values for the modifiedmyocardial performance index for evaluation of fetal left car-diac functionrdquoUltrasound in Obstetrics and Gynecology vol 29no 3 pp 321ndash325 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

10 The Scientific World Journal

Authorsrsquo Contribution

Nathan D Hahurij and Emmeline E Calkoen contributedequally to this work and shared first authorship

Acknowledgment

This work is supported by the DutchHeart Foundation Grantno 2009T070 (Nathan D Hahurij)

References

[1] R G Kelly ldquoMolecular inroads into the anterior heart fieldrdquoTrends inCardiovascularMedicine vol 15 no 2 pp 51ndash56 2005

[2] J I E Hoffman and S Kaplan ldquoThe incidence of congenitalheart diseaserdquo Journal of the AmericanCollege of Cardiology vol39 no 12 pp 1890ndash1900 2002

[3] D Srivastava ldquoGenetic assembly of the heart implications forcongenital heart diseaserdquo Annual Review of Physiology vol 63pp 451ndash469 2001

[4] N Corrigan D P Brazil and F M Auliffe ldquoHigh-frequencyultrasound assessment of the murine heart from embryothrough to juvenilerdquoReproductive Sciences vol 17 no 2 pp 147ndash157 2010

[5] C F Spurney CW Lo and L Leatherbury ldquoFetal mouse imag-ing using echocardiography a review of current technologyrdquoEchocardiography vol 23 no 10 pp 891ndash899 2006

[6] Y-Q Zhou F S Foster R Parkes and S L Adamson ldquoDevel-opmental changes in left and right ventricular diastolic fillingpatterns in micerdquo American Journal of Physiology vol 285 no4 pp H1563ndashH1575 2003

[7] Q Yu L Leatherbury X Tian and C W Lo ldquoCardiovascularassessment of fetal mice by in utero echocardiographyrdquo Ultra-sound in Medicine and Biology vol 34 no 5 pp 741ndash752 2008

[8] C K L Phoon ldquoImaging tools for the developmental biologistultrasound biomicroscopy of mouse embryonic developmentrdquoPediatric Research vol 60 no 1 pp 14ndash21 2006

[9] J S Lee Q Yu J T Shin et al ldquoKlf2 is an essential regulator ofvascular hemodynamic forces in vivordquo Developmental Cell vol11 no 6 pp 845ndash857 2006

[10] R A Morris B Damon V Mironov et al ldquoPeriostin regulatescollagen fibrillogenesis and the biomechanical properties ofconnective tissuesrdquo Journal of Cellular Biochemistry vol 101 no3 pp 695ndash711 2007

[11] B C W Groenendijk B P Hierck J Vrolijk et al ldquoChanges inshear stress-related gene expression after experimentally alteredvenous return in the chicken embryordquoCirculation Research vol96 no 12 pp 1291ndash1298 2005

[12] C J Zuurbier V M Emons and C Ince ldquoHemodynamics ofanesthetized ventilated mouse models aspects of anestheticsfluid support and strainrdquo American Journal of Physiology vol282 no 6 pp H2099ndashH2105 2002

[13] A Włoch W Rozmus-Warcholinska B Czuba et al ldquoDopplerstudy of the embryonic heart in normal pregnant womenrdquoJournal of Maternal-Fetal and Neonatal Medicine vol 20 no 7pp 533ndash539 2007

[14] M E Godfrey B Messing S M Cohen D V Valsky andS Yagel ldquoFunctional assessment of the fetal heart a reviewrdquo

Ultrasound in Obstetrics and Gynecology vol 39 no 2 pp 131ndash144 2012

[15] M R M Jongbloed M C E F Wijffels M J Schalij et alldquoDevelopment of the right ventricular inflow tract and moder-ator band a possible morphological and functional explanationforMahaim tachycardiardquoCirculation Research vol 96 no 7 pp776ndash783 2005

[16] Y-HGui KK Linask PKhowsathit and J CHuhta ldquoDopplerechocardiography of normal and abnormal embryonic mouseheartrdquo Pediatric Research vol 40 no 4 pp 633ndash642 1996

[17] C F Spurney L Leatherbury and C W Lo ldquoHigh-frequencyultrasound database profiling growth development and car-diovascular function in C57BL6J mouse fetusesrdquo Journal of theAmerican Society of Echocardiography vol 17 no 8 pp 893ndash900 2004

[18] R P Ji C K L Phoon O Aristizabal K E McGrath JPalis and D H Turnbull ldquoOnset of cardiac function duringearly mouse embryogenesis coincides with entry of primitiveerythroblasts into the embryo properrdquoCirculation Research vol92 no 2 pp 133ndash135 2003

[19] M R Jongbloed S R Vicente N D Hahurij et al ldquoNormaland abnormal development of the cardiac conduction systemimplications for conduction and rhythm disorders in the childand adultrdquo Differentiation vol 84 no 1 pp 131ndash148 2012

[20] K Makikallio P Jouppila and J Rasanen ldquoHuman fetal cardiacfunction during the first trimester of pregnancyrdquo Heart vol 91no 3 pp 334ndash338 2005

[21] W Rozmus-Warcholinska A Wloch G Acharya et al ldquoRef-erence values for variables of fetal cardiocirculatory dynamicsat 11-14 weeks of gestationrdquo Ultrasound in Obstetrics andGynecology vol 35 no 5 pp 540ndash547 2010

[22] G Tulzer P Khowsathit S Gudmundsson et al ldquoDiastolicfunction of the fetal heart during second and third trimestera prospective longitudinal Doppler-echocardiographic studyrdquoEuropean Journal of Pediatrics vol 153 no 3 pp 151ndash154 1994

[23] MC Leiva J E Tolosa CN Binotto et al ldquoFetal cardiac devel-opment and hemodynamics in the first trimesterrdquoUltrasound inObstetrics and Gynecology vol 14 no 3 pp 169ndash174 1999

[24] I P Van Splunder and J W Wladimiroff ldquoCardiac functionalchanges in the human fetus in the late first and early secondtrimestersrdquo Ultrasound in Obstetrics and Gynecology vol 7 no6 pp 411ndash415 1996

[25] S A Clur R K Oude B W Mol J Ottenkamp and CM Bilardo ldquoFetal cardiac function between 11 and 35 weeksrsquogestation and nuchal translucency thicknessrdquo Ultrasound inObstetrics amp Gynecology vol 37 no 1 pp 48ndash56 2011

[26] G Rizzo A Muscatello E Angelini and A Capponi ldquoAbnor-mal cardiac function in fetuses with increased nuchal translu-cencyrdquo Ultrasound in Obstetrics and Gynecology vol 21 no 6pp 539ndash542 2003

[27] F Crispi E Hernandez-Andrade M M A L Pelsers et alldquoCardiac dysfunction and cell damage across clinical stagesof severity in growth-restricted fetusesrdquo American Journal ofObstetrics and Gynecology vol 199 no 3 pp 254ndash258 2008

[28] S Luewan Y Yanase F Tongprasert K Srisupundit and TTongsong ldquoFetal cardiac dimensions at 14-40 weeksrsquo gestationobtained using cardio-STIC-Mrdquo Ultrasound in Obstetrics andGynecology vol 37 no 4 pp 416ndash422 2011

The Scientific World Journal 11

[29] T Tongsong C Wanapirak W Piyamongkol et al ldquoFetalventricular shortening fraction in hydrops fetalisrdquo Obstetricsand Gynecology vol 117 no 1 pp 84ndash91 2011

[30] E Hernandez-Andrade H Figueroa-Diesel C Kottman etal ldquoGestational-age-adjusted reference values for the modifiedmyocardial performance index for evaluation of fetal left car-diac functionrdquoUltrasound in Obstetrics and Gynecology vol 29no 3 pp 321ndash325 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World Journal 11

[29] T Tongsong C Wanapirak W Piyamongkol et al ldquoFetalventricular shortening fraction in hydrops fetalisrdquo Obstetricsand Gynecology vol 117 no 1 pp 84ndash91 2011

[30] E Hernandez-Andrade H Figueroa-Diesel C Kottman etal ldquoGestational-age-adjusted reference values for the modifiedmyocardial performance index for evaluation of fetal left car-diac functionrdquoUltrasound in Obstetrics and Gynecology vol 29no 3 pp 321ndash325 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology