Molecular Human Reproduction, Vol.20, No.7 pp.

690–700, 2014
Advanced Access publication on March 27, 2014 doi:10.1093/molehr/gau026

ORIGINAL RESEARCH

Metabolic gene profile in early human

fetal heart development
J.I. Iruretagoyena 1,*, W. Davis 2, C. Bird 1, J. Olsen 2, R. Radue 3,
A. Teo Broman 4, C. Kendziorski 4, S. Splinter BonDurant 2, T. Golos 5,
I. Bird 6, and D. Shah 1

Downloaded from https://academic.oup.com/molehr/article/20/7/690/2459849 by guest on 19 November 2020

1
MFM Division, OBGYN Department, University of Wisconsin, Madison, WI, USA 2Gene Expression Center, University of Wisconsin, Madison,
WI, USA 3University of Wisconsin School of Medicine and Public Health, Madison, WI, USA 4Department of Biostatistics and Medical Informatics,
University of Wisconsin, Madison, WI, USA 5National Primate Research Center, and Department of Comparative Biosciences, University of
Wisconsin, Madison, WI, USA 6Reproductive Sciences Division, OBGYN Department, University of Wisconsin, Madison, WI, USA

*Correspondence address. Division Maternal Fetal Medicine, OB/GYN Department, University of Wisconsin, 4th Floor McConnell Hall,
1010 Mound Street, Madison, WI 53715, USA. Tel: +1 (608) 417 6618; Fax: +1 (608) 257 1255; E-mail: iruretagoyen@wisc.edu

Submitted on January 31, 2014; resubmitted on February 28, 2014; accepted on March 20, 2014

abstract: The primitive cardiac tube starts beating 6–8 weeks post fertilization in the developing embryo. In order to describe normal
cardiac development during late first and early second trimester in human fetuses this study used microarray and pathways analysis and
created a corresponding ‘normal’ database. Fourteen fetal hearts from human fetuses between 10 and 18 weeks of gestational age (GA)
were prospectively collected at the time of elective termination of pregnancy. RNA from recovered tissues was used for transcriptome analysis
with Affymetrix 1.0 ST microarray chip. From the amassed data we investigated differences in cardiac development within the 10–18 GA period
dividing the sample by GA in three groups: 10 –12 (H1), 13 –15 (H2) and 16–18 (H3) weeks. A fold change of 2 or above adjusted for a false
discovery rate of 5% was used as initial cutoff to determine differential gene expression for individual genes. Test for enrichment to identify func-
tional groups was carried out using the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Array analysis cor-
rectly identified the cardiac specific genes, and transcripts reported to be differentially expressed were confirmed by qRT– PCR. Single transcript
and Ontology analysis showed first trimester heart expression of myosin-related genes to be up-regulated .5-fold compared with second tri-
mester heart. In contrast the second trimester hearts showed further gestation-related increases in many genes involved in energy production and
cardiac remodeling. In conclusion, fetal heart development during the first trimester was dominated by heart-specific genes coding for myocardial
development and differentiation. During the second trimester, transcripts related to energy generation and cardiomyocyte communication for
contractile coordination/proliferation were more dominant. Transcripts related to fatty acid metabolism can be seen as early as 10 weeks and
clearly increase as the heart matures. Retinol receptor and gamma-aminobutyric acid (GABA) receptor transcripts were detected, and have not
been described previously in human fetal heart during this period. For the first time global gene expression of heart has been described in human
samples to create a database of normal development to understand and compare with known abnormal fetal heart development.

Key words: fetal / human / microarray / renin-angiotensin / cardiac development

Introduction
Six to eight weeks after fertilization and toward the end of the embryonic
period, the primitive cardiac tube, with four chambers, starts beating. At
this time, the remaining cardiac stem cells became differentiated cardio-
myocytes. Through the remaining pregnancy period, the cardiomyocytes
then undergo differentiation and specialization into specific cardiac
tissues (Wheater et al., 1987; Moore, 1989; Gratacos et al., 2007).
Due to the obvious difficulty in obtaining human cardiac tissue, most of
the literature in early cardiac profiling is based on animal models, such
as mice and sheep. Studies looking for common pathways that may
explain cardiac failure have been performed in human fetal hearts
compared with failing adult heart profiles (Razeghi et al., 2001), but
such studies were limited to the 18- to 28-week period of gestation.
Previous cardiac development studies have elucidated, in part, the meta-
bolic profile of the early human heart. Fetal heart energy production has
been described as dependent on glucose metabolism during the whole
fetal life, which changes to fatty acid oxidation during the first weeks of
the neonatal period (Lopaschuk and Jaswal, 2010). The adult heart in
turn depends on fatty acid oxidation for energy generation. Previous experi-
ments using mouse models have also assessed the transition in energy
profile from embryonic stem cells to fetal cardiomyocyte during the first
stages of pregnancy. Cardiac stem cells appear to obtain energy from anaer-
obic metabolism that transforms into a mitochondria-mediated oxidative

& The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: journals.permissions@oup.com
Gene expression in early fetal human heart development
metabolism to meet increases in energy demand during cardiomyocyte
differentiation and development during fetal life (Chung et al., 2007;
Lopaschuk and Jaswal, 2010). No data are available to verify if this is
also true in humans.
The regulation of fetal cardiomyocyte growth is essential since the
number of cardiac cells at birth predetermines the number of cardiomyo-
cytes for life, which is a predictor for cardiac disease (Thornburg et al.,
2010). Studies in sheep have shown that proliferation of cardiomyocytes
is stimulated by angiotensin II, cortisol and insulin-like growth factor-1 in
the womb (Thornburg et al., 2010). Thus, in order to understand cardiac
disease in humans, it is imperative to study if these same endocrine
systems are present in early fetal life.
In the current study, the gene expression profile of human fetal heart
between 10 and 18 weeks’ gestation is presented. The working hypoth-
esis was that there is a developmental change in gene profile between
fetal hearts in the late first trimester (10 –12 weeks’ gestation) compared
with the early second trimester (16– 18 weeks’ gestation), which would
be detectable by transcript analysis and/or pathway analysis. To this end
we have analyzed fetal heart mRNA using Affymetrix arrays in an attempt
to both investigate changes in gene expression in human fetal heart and
provide a much needed reference database to guide future studies in
normal and abnormal human fetal development.
Methods
Tissue collection and RNA isolation
Human fetal cardiac tissue was collected after informed consent following in-
stitutional guidelines, from patients older than 18 years of age undergoing an
elective dilation and curettage for termination of pregnancy due to unwanted
pregnancy between 10 and 18 weeks of gestational age. Patients had already
undergone the surgical consent process with their health team before they
were approached to donate fetal tissue samples under an IRB approved
protocol. Those who expressed willingness for such research tissue donation
then underwent a research informed consent process. All pregnancies had a
dating ultrasound done within a week of the procedure. The suction was gen-
erated by a vacuum with a standard suction unit providing variable controlled
suction up to 550 mmHg, and a suction flow rate of 30 lpm.
Before the procedure, collection bottles were placed at 48C for 1 h. At the
time of the procedure, 100 ml (at 48C), RNAse-free water solution (BP561-1
Water, Sterile for RNA work, Fisher Scientific, USA) mixed with 10× PBS
buffer (Ambion, USA) at a final concentration of 10%, was added to the col-
lecting bottle.
The procedure was performed within 5 min of adding the cold water. After
the procedure, the heart was grasped with sterilized surgical instruments and
carefully isolated from the rest of the thoracic content to be placed in RNAse-
free plastic tubes. Fetal brain was also collected in the same fashion. No kar-
yotype was done to confirm normal chromosomes. The brain tissue was used
for comparison looking for major differences among the study group and the
brain group as an internal validation.
These tubes were immediately placed in liquid nitrogen for transport. The
tissue was then stored in a 2808C freezer.
Tissue RNA was extracted using RNA STAT-60 reagents, as recom-
mended by the manufacturer (Tel-Test, Inc., Friendswood, TX, USA). All
procedures were run on ice. A starting ratio of 1 ml reagent/100 mg tissue
was used to ensure efficient, intact RNA recovery. RNA concentration and
purity were initially determined using spectrophotometric A260 and A260/
280 ratios (NanoDrop Products, Thermo Fisher Scientific, Inc., Wilmington,
DE, USA). RNA quantification and quality control were performed using the
Agilent Bioanalyzer (Agilent Technology, Santa Clara, CA, USA) 260/280
691
ratios and standard RIN-values (RNA integrity number). A value of 1.7–2.1
was considered workable for further testing.
Array preparation
Fourteen hearts RNA samples were divided into three Affymetrix ST 1.0
arrays according to gestational age. Array #1 analyzed six hearts RNA
samples from 10 to 12 weeks’ gestation. Array #2 analyzed six hearts
RNA samples from 13 to 15 weeks’ gestation, and Array #3 analyzed two
hearts RNA samples from 16 to 18 weeks’ gestation. Ten brain samples
from the same fetuses were used as internal controls. cDNA was synthesized
from extracted RNA (300 ng) using the Ambion WT Expression Kit (Expres-
sion Kit Manual P/N 4425209 Rev. C). cDNA yield quantity and quality were
assessed using NanoDrop Spectrophotometer (NanoDrop Products,
Downloaded from https://academic.oup.com/molehr/article/20/7/690/2459849 by guest on 19 November 2020
Thermo Fisher Scientific, Inc., Wilmington, DE, USA) and Agilent Bioanalyzer
(Agilent Technology, Santa Clara, CA, USA). Extraction RNA samples with
an A260/280 ratio of 1.7 – 2.1 were further analyzed by gel electrophoresis
at the Biotech Center. Samples were considered acceptable for testing
when 28S and 18S rRNA bands resolved into two discrete bands that had
no significant smearing below each band. The 28S rRNA band had intensity
approximately twice that of the 18S rRNA band. These samples were then
further tested by cDNA generation for hybridization. cDNA was checked
for yield and size distribution using NanoDrop Spectrophotometer (Nano-
Drop Products, Thermo Fisher Scientific, Inc., Wilmington, DE, USA) and
Agilent profile (Agilent Bioanalyzer, Agilent Technology, Santa Clara, CA,
USA). Fragmentation check revealed a cDNA ,500 bp reliable for hybrid-
ization to Human Gene 1.0 ST arrays. All subsequent experiments were
then undertaken using RNA samples matching these requirements.
Fragmentation and end-terminus labeling of cDNAs were done at the Bio-
technology Center of the University of Wisconsin, Madison, using the Affy-
metrix WT Terminal Labeling Kit [see GeneChipw WT Terminal Labeling
and Hybridization User Manual (P/N 702808 Rev. 4)]. The samples were
then hybridized to Human Gene 1.0 ST arrays at 458C for 16 h O/N, follow-
ing all procedures outlined in the GeneChipw WT Terminal Labeling and
Hybridization User Manual (P/N 702808 Rev. 4) for Human Gene 1.0 ST
Arrays. GeneChips post-processed were done on the AFX Fluidics 450
Station, according to all AFX protocols and procedures defined for the
Human Gene 1.0 ST Array (FS450_0007), as outlined in the GeneChip
Expression Wash, Stain and Scan User Manual (P/N 702731 Rev. 3). Gene-
Chips were scanned on the GC3000 G7 Scanner. Data were extracted and
processed using Affymetrix Command Console version 3.1.1.1229.
Heart tissue was compared with brain tissue looking for organ-specific
genes. The heart’s top signals detected were Myosin light chain 7 (MYL7),
Myosin heavy chain 7 (MYH7), T-box 20 and Popeye domain which are
clearly appropriate to the heart. The brain’s top signals detected were Inter-
nexin, Septin, Doublecortin, Stathmin and NEL2 which are appropriate to the
heart.
Brain tissue was compared with heart tissue looking for organ-specific
genes. In the brain, the top signals detected are summarized in Fig. 1B.
Note the abundant expression of Internexin, Septin, Doublecortin, Stathmin
and NEL2, which are all clearly appropriate.
The array data can be found at GEO; the accession number is GSE56048.
Statistical methods
All analyses of GeneChip data were carried out in R (R Development Core
Team, 2007), a publicly available statistical analysis environment (available
at www.r-project.org). Specific software packages (affy, EBarrays, allez) are
available at Bioconductor (http://bioconductor.org/), an online suite of
tools for the analysis of genomic data (Huber and Gentleman, 2004). The
Affymetrix probe level data were processed using Robust Multi-Array
Analysis (RMA) as implemented in the affy package, in order to obtain
normalized summary expression scores for each probe set on each array
692 Iruretagoyena et al.

Downloaded from https://academic.oup.com/molehr/article/20/7/690/2459849 by guest on 19 November 2020

Figure 1 (A) Genes with the highest differential expression in the heart. This figure is based on pooled tissue samples from 14 fetal hearts from 10 to 18
weeks of gestational age for genes with 20-fold change or greater expression level. Human Gene 1.0 ST array (Affymetrix) was used. Genes were chosen
based on fold change compared with mRNA, after confirming a differential expression .95% with an false discover rate (FDR) of 5%. * ¼ genes verified by
qRT – PCR. (B) Genes with the highest differential expression in the brain. This figure is based on pooled tissue samples from 10 fetal brains from 10 to 18
weeks of gestational age for genes with 20-fold change or greater expression level. Human Gene 1.0 ST array (Affymetrix) was used. Genes were chosen
based on fold change compared with mRNA, after confirming a differential expression .95% with an FDR of 5%. * ¼ genes verified by qRT – PCR. (C)
Differentially expressed genes measured by fold change in mRNA levels between the hearts at different gestational ages. The late first trimester hearts
(H1) showed an increased number of differentially expressed genes (green bars) when compared with the early second trimester hearts (H3). Tissue
samples correspond to hearts from 10 – 12 weeks of gestational age (H1) and 16 – 18 weeks of gestational age (H3). Differences are measured by fold
change (.2), after adjusting for an FDR of 5% with a differential expression .95% using EBarrays (Newton et al., 2001; Kendziorski et al., 2003), to identify
differentially expressed genes in any two conditions. (D) Genes with the highest differential expression in the late first trimester hearts (H1). Tissue samples
correspond to hearts from 10 – 12 weeks of gestational age (H1) and 16 –18 weeks of gestational age (H3) pooled on two separate Human Gene 1.0 ST
array, according to gestational age. Differences are measured by fold change (.2), after adjusting for an FDR of 5% with a differential expression .95% using
EBarrays (Newton et al., 2001; Kendziorski et al., 2003), to identify differentially expressed genes in any two conditions. * ¼ gene verified by qRT – PCR.

(Irizarry et al., 2003). Cutoffs for sensitivity, ensuring a reliable positive detec-
tion result, were 0.95. Cutoffs for a significant change in expression were ini-
tially set at 2-fold.
Initial pools of the brain and heart derived from RNA were prepared for
validation using methods allowing for verification of the characteristics of
both the brain and heart. Results were those expected in the brain and
heart RNA.
In order to draw any conclusion, parameters had to first be established as to
how the output chips would be analyzed. In this presentation, the probe sets
are referred to as genes. RMA fits a linear model to the log probe intensities for
each probe set. The linear model includes a sample effect (the parameter of
interest), a probe effect and an error term. The standard errors are normalized
so that each probe set has the same median standard error across arrays.
EBarrays was used to identify differentially expressed (DE) genes (Newton
et al., 2001; Kendziorski et al., 2003). EBarrays is an empirical Bayes approach
which models the probability distribution of a set of expression measure-
ments. It accounts generally for differences in specific genes with regard to
their true underlying expression levels, measurement fluctuations and dis-
tinct expression patterns for a given gene among conditions (Kendziorski
et al., 2003). An expression pattern is an arrangement of the true underlying
intensities (m) in each condition. The number of patterns possible depends
on the number of conditions from which the expression measurements
were obtained. For example, when measurements are taken from two con-
ditions, two patterns of expression are possible; equivalent expression
(!m1 ¼ m2) and differential expression (!m1 = m2). Since there is no priori
stating which genes are in which patterns, the marginal distribution of the
Gene expression in early fetal human heart development
data is a mixture of all possible patterns with model parameters being deter-
mined by the full set of array data. This approach utilizes information across a
set of arrays optimizing model fit and is, therefore, more efficient than a
number of methods that make gene inferences one gene at a time (Kend-
ziorski et al., 2003). Fitted model parameters provide information on the
number of genes expected in each expression pattern. Furthermore, the
fitted model is used to assign probability distributions to every gene. Each
gene-specific distribution gives the posterior probability of that gene’s indi-
vidual expression pattern.
Thresholds were chosen to target an FDR of 5% when more than one
sample was available within the condition (e.g. heart versus brain), the log-
normal normal with moderated variance (LNNMV) model was used in EBar-
rays; otherwise, a lognormal normal model was used.
EBarrays then provides the posterior probability of differential expression
(DE) as well as equivalent expression (EE) for every gene (Table I). A gene
was defined as DE if its posterior probability of DE exceeded 0.95. This is
analogous to its posterior probability of EE being ≤0.05. This threshold
targets an overall false discovery rate (FDR) of 5%. In some cases (specified
in the text), a second fold-change based filter was applied to the list of DE
genes obtained from EBarrays.
Tests for enrichment of common function among sets of differentially
expressed genes were carried out using data from the Gene Ontology
(GO) annotations and the Kyoto Encyclopedia of Genes and Genomes
(KEGG). In GO, transcripts genes are categorized at varying levels of biologic-
al detail. The three broadest levels are molecular function, cellular compo-
nent and biological process. There are many subcategories within each
detail. The R package allez was used to perform tests of enrichment for
each GO category and KEGG pathway (Newton et al., 2001). In general,
the interpretation of P-values resulting from enrichment tests is not straight-
forward due to the many dependent hypotheses tested. Furthermore, the
enrichment test tends to result in small P-values when groups with few tran-
scripts are considered. The statistical methods underlying allez adjust for
these factors, allowing increased power and sensitivity for identifying sets
that are biologically meaningful (Tables II and III).
qRT –PCR
Seven selected genes (FABP4, MYH7B, APOA2, TNNI3, TMOD2, NRXN1,
RPLO) were further validated by quantitative real-time PCR (qRT– PCR) in
order to validate the array data. Human reference cDNA, oligo-dT primed
(Clontech) was used to create a standard dilution series for analysis in parallel
with samples on a Roche LightCycler480 using sequence-specific primers.
Each assay per sample was performed in triplicate on 96-well plates with
480 Sybr Green I Master (Roche), and 500 nM forward and reverse
primers. Cycling conditions were as follows: 10 min at 958C; 40 cycles at
958C for 10 s, 578C for 10 s and 728C for 5 s; followed by melt curve analysis.
Ethical approval
Sample collection was started after receiving Institutional Review Board
approval (H-2009-0071) over a 2-year period from 2009 to 2011.
Results
Confirmation of cardiac tissue
Pooled RNA from all cardiac tissue (10 –18 weeks) was compared with a
control tissue (fetal brain) to confirm specificity of the tissue. Genes like
natriuretic peptide (NPPA), troponin (TNNI3) and myosin heavy chain 7
(MYH7) cardiac muscle were readily detected (differential expression
.95%, after adjusting for an FDR of 5%) and correctly reported as
‘up-regulated’ relative to brain tissue (Fig. 1A). Subsequent qRT –PCR
693
confirmed expression of TNNI3 and MYH7. Brain tissue genes are
shown in Fig. 1B (Iruretagoyena et al., 2014).
In order to simplify the analysis of the data, samples were then sub-
grouped by gestational age within 2-week periods. H1 (10 –12 weeks),
H2 (13– 15 weeks) and H3 (16– 18 weeks). Given the magnitude of
the data set, statistical analysis was done comparing groups H1 versus
H3 (H2 group is shown in the figures to further illustrate the normal pro-
gression from first trimester to second trimester).
Changes according to gestational age on global
expression of genes
When fold changes of 2, 5 and above 20 were used to compared H1 to
Downloaded from https://academic.oup.com/molehr/article/20/7/690/2459849 by guest on 19 November 2020
H3 gene expression, there were 43, 13 and 5 individual gene transcripts
specifically up-regulated, respectively. Conversely, when H3 was com-
pared with H1 24, 4, 0 genes were up-regulated, respectively (Fig. 1C).
Heart contractile proteins
MYL1, MYH3, MYBPC1, SLN and ACTG2 genes coding for sarcoplas-
mic reticulum, actin and essential myosin contractile protein constitu-
ents of cardiac muscle were more highly expressed (differential
expression .95%, after adjusting for an FDR of 5%) in hearts of a
younger gestational age (Figs 1D and 2A). When gene ontology analysis
was applied, terms for structural constituents of muscle and myosin
filament were revealed, further supporting single transcript analysis
(Fig. 2B). qRT – PCR analysis was also confirmatory when MYH7 was
used as a representative target.
Energy generation metabolism in the heart
Individual transcript analysis showed that genes coding for FABP4,
FABP2, NRAP and ACE2, all part of either cardiac fatty acid metabolism
or structural remodeling, were among the highest expressed in H3
hearts. (Figs 2C and 3A). Gene ontology analysis showed ontology
terms in agreement with single transcript finding involving fatty acid me-
tabolism including chylomicron, positive regulation of cholesterol, lipo-
protein catabolic process, high and very low lipoprotein and
triglyceride-rich lipoprotein (Fig. 2B). qRT –PCR confirmed expression
of FABP4 and APOA2.
Complementing our findings on transcripts for proteins involved in
fatty acid metabolism as an energy source, single gene transcript analysis
for genes involved in glucose energy production including PPARa,
PPARb, PD, LD, LDH-A, HK-1 and HIF-1 also was present but not dif-
ferentially expressed between H1-H3 groups, after adjusting for an FDR
of 5% (Fig. 3B). Thus expression of the mRNA encoding these pathways
appears more constant with gestation.
Miscellaneous transcripts
GABRA4, GABRB1 and RBP7 genes were highly expressed in the H3
group, with a differential expression .95%, after adjusting for an FDR
of 5% (Fig. 2C). GABRA4 or GABRA1 were not found in the gene ontol-
ogy terms. Although RBP7 is not a direct participant in the ontology
terms described, it is indirectly involved in the Chylomicron and lipopro-
teins terms as part of fatty acid metabolism.
Retinol receptors (RAR, RXR), angiotensin receptors 1-2, IGF1-2,
IGFR1-2, IGFBP1 and glucocorticoid receptor NR3C1 were found to
be present but not differentially expressed (differential expression
,95%, after adjusting for an FDR of 5%) (Fig. 3C).
694 Iruretagoyena et al.

Table I Transcripts detected by array analysis with a fold change >5.

Entrez ID Gene Gene.Name PP.LNNMV. PP.LNNMV. FC

EE DE
.............................................................................................................................................................................................
253017 TECRL trans-2,3-enoyl-CoA reductase-like 0 1 63
58498 MYL7 Myosin, light chain 7, regulatory 0 1 56
64091 POPDC2 Popeye domain containing 2 0 1 46
57057 TBX20 T-box 20 0 1 46
4878 NPPA Natriuretic peptide precursor A 0 1 43
5350 PLN phospholamban 0 1 38
4625 MYH7 Myosin, heavy chain 7, cardiac muscle, beta 0 1 36

Downloaded from https://academic.oup.com/molehr/article/20/7/690/2459849 by guest on 19 November 2020

7137 TNNI3 Troponin I type 3 (cardiac) 0 1 30
115701 ALPK2 alpha-kinase 2 0 1 28
4888 NPY6R Neuropeptide Y receptor Y6 (pseudogene) 0 1 25
4633 MYL2 Myosin, light chain 2, regulatory, cardiac, slow 0 1 24
28965 SLC27A6 Solute carrier family 27 (fatty acid transporter), member 6 0 1 23
7134 TNNC1 Troponin C type 1 (slow) 0 1 21
4634 MYL3 Myosin, light chain 3, alkali; ventricular, skeletal, slow 0 1 21
137835 TMEM71 Transmembrane protein 71 0 1 20
51086 TNNI3K TNNI3 interacting kinase 0 1 19
10699 CORIN Corin, serine peptidase 0 1 19
4624 MYH6 Myosin, heavy chain 6, cardiac muscle, alpha 0 1 18
93649 MYOCD Myocardin 0 1 17
4607 MYBPC3 Myosin binding protein C, cardiac 0 1 17
27063 ANKRD1 Ankyrin repeat domain 1 (cardiac muscle) 0 1 16
27129 HSPB7 Heat shock 27 kDa protein family, member 7 (cardiovascular) 0 1 16
7139 TNNT2 Troponin T type 2 (cardiac) 0 1 16
150572 SMYD1 SET and MYND domain containing 1 0 1 15
4151 MB Myoglobin 0 1 14
51725 FBXO40 F-box protein 40 0 1 14
2170 FABP3 Fatty acid binding protein 3, muscle and heart (mammary-derived growth inhibitor) 0 1 13
376132 LRRC10 Leucine rich repeat containing 10 0 1 13
145781 GCOM1 GRINL1A complex locus 0 1 13
6523 SLC5A1 Solute carrier family 5 (sodium/glucose cotransporter), member 1 0 1 12
23676 SMPX Small muscle protein, X-linked 0 1 12
29119 CTNNA3 Catenin (cadherin-associated protein), alpha 3 0 1 12
8048 CSRP3 Cysteine and glycine-rich protein 3 (cardiac LIM protein) 0 1 12
8736 MYOM1 Myomesin 1, 185 kDa 0 1 11
5318 PKP2 Plakophilin 2 0 1 11
1160 CKMT2 Creatine kinase, mitochondrial 2 (sarcomeric) 0 1 11
11149 BVES Blood vessel epicardial substance 0 1 11
10529 NEBL nebulette 0 1 11
2274 FHL2 Four and a half LIM domains 2 0 1 10
23493 HEY2 Hairy/enhancer-of-split related with YRPW motif 2 0 1 10
285025 CCDC141 Coiled-coil domain containing 141 0 1 10
27302 BMP10 Bone morphogenetic protein 10 0 1 10
64208 POPDC3 Popeye domain containing 3 0 1 9
221662 RBM24 RNA binding motif protein 24 0 1 8
10611 PDLIM5 PDZ and LIM domain 5 0 1 8
146862 UNC45B Unc-45 homolog B (C. elegans) 0 1 8

Continued
Gene expression in early fetal human heart development 695

Table I Continued

Entrez ID Gene Gene.Name PP.LNNMV. PP.LNNMV. FC

EE DE
.............................................................................................................................................................................................
1410 CRYAB Crystallin, alpha B 0 1 8
6262 RYR2 Ryanodine receptor 2 (cardiac) 0 1 8
123722 FSD2 Fibronectin type III and SPRY domain containing 2 0 1 8
8988 HSPB3 Heat shock 27 kDa protein 3 0 1 8
7135 TNNI1 Troponin I type 1 (skeletal, slow) 0 1 7
23566 LPAR3 Lysophosphatidic acid receptor 3 0 1 7
6331 SCN5A Sodium channel, voltage-gated, type V, alpha subunit 0 1 7

Downloaded from https://academic.oup.com/molehr/article/20/7/690/2459849 by guest on 19 November 2020

27145 FILIP1 Filamin A interacting protein 1 0 1 7
167681 PRSS35 Protease, serine, 35 0 1 7
91624 NEXN Nexilin (F actin binding protein) 0 1 7
84803 AGPAT9 1-acylglycerol-3-phosphate O-acyltransferase 9 0 1 7
11155 LDB3 LIM domain binding 3 0 1 7
116496 FAM129A Family with sequence similarity 129, member A 0 1 7
1E+08 OCR1 Ovarian cancer-related protein 1 0 1 7
6450 SH3BGR SH3 domain binding glutamic acid-rich protein 0 1 7
59272 ACE2 Angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 0 1 7
91807 MYLK3 Myosin light chain kinase 3 0 1 7
2201 FBN2 Fibrillin 2 0 1 6
140456 ASB11 Ankyrin repeat and SOCS box-containing 11 0 1 6
6546 SLC8A1 Solute carrier family 8 (sodium/calcium exchanger), member 1 0 1 6
NA NA NA 0 1 6
NA NA NA 0 1 6
8470 SORBS2 Sorbin and SH3 domain containing 2 0 1 6
6910 TBX5 T-box 5 0 1 6
84675 TRIM55 Tripartite motif-containing 55 0 1 6
116362 RBP7 Retinol binding protein 7, cellular 0 1 6
84940 CORO6 Coronin 6 0 1 6
1573 CYP2J2 Cytochrome P450, family 2, subfamily J, polypeptide 2 0 1 6
144100 PLEKHA7 Pleckstrin homology domain containing, family A member 7 0 1 6
84676 TRIM63 Tripartite motif-containing 63 0 1 6
4286 MITF Microphthalmia-associated transcription factor 0 1 6
345557 PLCXD3 Phosphatidylinositol-specific phospholipase C, X domain containing 3 0 1 6
9200 PTPLA Protein tyrosine phosphatase-like (proline instead of catalytic arginine), member A 0 1 6
26548 ITGB1BP2 Integrin beta 1 binding protein (melusin) 2 0 1 6
88 ACTN2 Actinin, alpha 2 0 1 6
1832 DSP Desmoplakin 0 1 6
8490 RGS5 Regulator of G-protein signaling 5 0 1 6
NA NA NA 0 1 5
10398 MYL9 Myosin, light chain 9, regulatory 0 1 5
283358 B4GALNT3 beta-1,4-N-acetyl-galactosaminyl transferase 3 0 1 5
161247 FITM1 Fat storage-inducing transmembrane protein 1 0 1 5
387700 SLC16A12 Solute carrier family 16, member 12 (monocarboxylic acid transporter 12) 0 1 5
11030 RBPMS RNA binding protein with multiple splicing 0 1 5
90523 C6orf142 Chromosome 6 open reading frame 142 0 1 5
5322 PLA2G5 Phospholipase A2, group V 0 1 5
2702 GJA5 Gap junction protein, alpha 5, 40 kDa 0 1 5

Continued
696 Iruretagoyena et al.

Table I Continued

Entrez ID Gene Gene.Name PP.LNNMV. PP.LNNMV. FC

EE DE
.............................................................................................................................................................................................
51705 EMCN endomucin 0 1 5
8395 PIP5K1B Phosphatidylinositol-4-phosphate 5-kinase, type I, beta 0 1 5
606553 C8orf49 Chromosome 8 open reading frame 49 0 1 5
442721 LMOD2 Leiomodin 2 (cardiac) 0 1 5
358 AQP1 Aquaporin 1 (Colton blood group) 0 1 5

Downloaded from https://academic.oup.com/molehr/article/20/7/690/2459849 by guest on 19 November 2020

Table II Genontology classification.

GO GO.term n.genes z.score

.............................................................................................................................................................................................
GO:0042627 CC Chylomicron 12 12.01
GO:0010886 BP Positive regulation of cholesterol storage 6 11.26
GO:0008307 MF Structural constituent of muscle 43 10.29
GO:0032982 CC Myosin filament 18 10.08
GO:0042159 BP Lipoprotein catabolic process 6 9.96
GO:0033033 BP Negative regulation of myeloid cell apoptosis 6 9.92
GO:0008035 MF High-density lipoprotein binding 7 9.81
GO:0034361 CC Very-low-density lipoprotein particle 20 9.09
GO:0034385 CC Triglyceride-rich lipoprotein particle 20 9.09
GO:0031983 CC Vesicle lumen 38 8.64

Housekeeping genes
For validation purposes, mRNA expression of reference genes RPLO
and TBT (Stern-Straeter et al., 2009) did not show changes among the
different groups as expected (Fig. 3D).
Discussion
Our findings have shown that there are characteristic dynamic changes in
gene expression during early development in the human heart. While H1
heart gene expression profiles showed dominance by genes involved in
myocardium development and differentiation, H3 hearts showed rela-
tively increased expression of genes not only actively involved in
energy generation, but also advanced cellular functions like cardiomyo-
cyte communication and proliferation.
Heart contractile and remodeling proteins
A heart’s ultimate destiny is myocardial contraction and so it is clearly of
interest that various subtypes of myosin, the principal component of
muscle, are highly and differentially expressed in H1 hearts compared
with H3 hearts. Light and heavy chains of myosin and actin genes involved
in the cytoskeleton formation dominate this period (Tsuchimochi et al.,
1986; Yazaki et al., 1987). Genes like the Secreted Frizzled-Related
Protein 2 (SFRP2), which function to inhibit fibroblast growth (He
et al., 2010) and sarcolipin, which function to reduce the accumulation
of calcium in the sarcoplasmic reticulum (Babu et al., 2007) are differen-
tially expressed in the young hearts, potentially contributing to the right
proportion of muscle and collagen, as well as calcium dynamics based in
the findings of our study.
The renin-angiotensin system, specifically through ACE2 (angiotensin
converting enzyme), is one of the pathways hypothesized to play an
increased role in H3 hearts when compared with the H1 heart, based
on differential gene expression revealed in our studies. The H3 heart
expresses genes involved in advanced functions such as contractile co-
ordination, angiogenesis and utilization of energy. Studies in ovine
animal models have already shown angiotensin II acts in conjunction
with insulin-like growth factor (IGF) to control proliferation of cardio-
myocytes during the prenatal period through late gestation, when cardi-
omyocytes undergo terminal differentiation and proliferation diminishes
(Reini et al., 2009). Data from our study further suggest the initiation of
cardiomyocyte proliferation and remodeling in humans occurs around
weeks 16 –18 of gestation. Prior to this period, in the second trimester,
more specifically at the end of the first trimester (10–12 weeks’ gestation),
the major events occurring in the cardiomyocyte relate more to the early
differentiation of stem cells into active contractile cardiomyocytes.
Energy generation metabolism in the heart
Fetal cardiac metabolism has been characterized in animal models as de-
pendent on glucose metabolism for energy from the very early stages of
cardiac differentiation. This profile changes shortly after birth when the
heart energy metabolism depends on fatty acid oxidation in the mito-
chondria, and persists into adulthood (Kong et al., 2008). Our findings
using transcriptome analysis approaches in humans are entirely consist-
ent with Kong et al. findings.
Gene expression in early fetal human heart development 697

Table III Geontology classification by heart group.

H1 H3
.............................................................................................................................................................................................
GO:0008307 Structural constituent of muscle 43 GO:0042627 Chylomicron 12
GO:0032982 Myosin filament 18 GO:0010886 Positive regulation of cholesterol storage 6
GO:0033033 Negative regulation of myeloid cell apoptosis 6 GO:0042159 Lipoprotein catabolic process 6
GO:0008035 High-density lipoprotein binding 7
GO:0034361 Very-low-density lipoprotein particle 20
GO:0034385 Triglyceride-rich lipoprotein particle 20
GO:0031983 Vesicle lumen 38
Total genes 67 Total genes 109

Downloaded from https://academic.oup.com/molehr/article/20/7/690/2459849 by guest on 19 November 2020

H1: 10-12wks; H3: 16-18wks.

Figure 2 (A) mRNA gene expression of regulators of cardiomyocyte differentiation. All transcripts are increased in the late first trimester (H1) versus the
early second trimester (H3). Differences are measured by mRNA level on genes with a differential expression .95%, after adjusting for an FDR of 5% using
EBarrays (Newton et al., 2001; Kendziorski et al., 2003), to identify differentially expressed genes in any two conditions. SLN, Sarcolipin; MYL1, Myosin light
chain 1; MYH3, Myosin heavy chain 3; ACTG2, Actin gamma. * ¼ gene verified by qRT– PCR. (B) Bar chart showing terms from Gene Ontology in the heart,
classified according to biological process, cellular component and molecular function using test for enrichment of common functions among sets of differ-
entially expressed genes (Gene Ontology Annotations and the KEGG). Gene families involved in common functions for muscular development like myosin
filament and fatty acid metabolism including chylomicron, cholesterol, lipoprotein binding proteins and triglyceride lipoprotein are the highest ranked in the
cardiomyocytes from 10 to 18 weeks of gestational age (pooled data). (C) Genes with the highest differential expression in the early second trimester hearts
(H3). Tissue samples correspond to hearts from 10 – 12 weeks of gestational age (H1) and 16 – 18 weeks of gestational age (H3) pooled on two separate
Human Gene 1.0 ST array, according to gestational age. Differences are measured by fold change (.2), after adjusting for an FDR of 5% with a differential
expression .95% using EBarrays (Newton et al., 2001; Kendziorski et al., 2003), to identify differentially expressed genes in any two conditions. *¼ gene
verified by qRT –PCR.
698 Iruretagoyena et al.

Downloaded from https://academic.oup.com/molehr/article/20/7/690/2459849 by guest on 19 November 2020

Figure 3 (A) mRNA gene expression of regulators of the cardiac fatty acid metabolism and remodeling. All transcripts are increased in the early second
trimester as the heart matures, compared with the late first trimester. Differences are measured by mRNA level on genes with a differential expression
.95%, after adjusting for an FDR of 5% using EBarrays (Newton et al., 2001; Kendziorski et al., 2003), to identify differentially expressed genes in any
two conditions. *¼ genes verified by qRT– PCR. (B) mRNA expression of regulators of glucose energy generation by the cardiomyocytes. All transcripts
are high in both groups showing no significant differential expression (,0.005), after adjusting for an FDR of 5%. (C) mRNA gene expression of insulin
growth factor and angiotensin. Specific transcripts were examined due to the their suggested importance in previous studies (see introduction). mRNA
levels detected in each group are shown. Differential expression .95%, after adjusting for an FDR of 5% using EBarrays (Newton et al., 2001; Kendziorski
et al., 2003), was not achieved suggesting that these genes are not differentially controlled across early gestation. (D) Reference genes in the heart. The graph
shows mRNA expression of two candidate genes without significant change in expression, regardless of the experimental group (H1, H2 or H3). Reference
genes were validated by the method of Stern-Straeter et al. (2009). All H1, H2 and H3 groups of hearts were analyzed under exact conditions. Differential
expression was ,0.005 for both genes under the two conditions. RPLP0: Ribosomal protein, large; TBS: TATA box binding protein.

Single gene expression analysis showed H3 heart genes were differen-
tially expressed, favoring fatty acid oxidation metabolism (FABP 4, FABP
2 and RBP) (Figs 2C and 3A) during the H3 period otherwise not appar-
ent in the H1 hearts. Nonetheless this pathway was revealed when
grouped analysis was performed. This may be interpreted as further evi-
dence that an early sign of emerging fatty acid oxidation metabolism in the
H1 hearts that is not significant when transcripts are evaluated individu-
ally. At a later gestational age, when single gene expression is robust
enough to create a difference, such changes can be observed individually.
When gene ontology was further applied to a pooled sample of hearts
from 10 to 18 weeks of gestational age (Tables II and III), genes involved in
fatty acid metabolism either in cell components, molecular function or
biological processes were again identified as the major active pathways.
One remarkable finding was that genes driving metabolism of fatty acid
were highly differentially expressed in the H3 hearts, compared with
the H1 hearts. KEGG pathway analysis also showed pathways involved
in fatty acid metabolism and oxidative stress controls (ascorbate and
aldarate metabolism, retinol metabolism and PPAR signaling) are more
dominant in the H3 period.
In contrast to our findings on fatty acid metabolism, key genes coding
for HIF, HK1, LDH-A, and lactate dehydrogenase, as well as PPAR-alpha
and beta were more uniformly present and highly expressed in both H1
and H3 hearts. The apparent lack of differential gene expression for
genes involved in glucose metabolism is again consistent with previous
findings in animal studies of the heart as being dependent on glucose util-
ization for basal energy generation during the fetal period (Lopaschuk
and Jaswal, 2010). Combined, these observations may reflect the fact
that the fetal period is dominated by glucose energy production until
after birth, and no differential changes in the genes regulating this
process should be expected from 10 to 18 weeks of gestation.
Gene expression in early fetal human heart development 699

Interestingly, however, three genes involved in fatty acid oxidation
ranked among those with more than a 5-fold change in the H3 period
compared with H1. These genes are the FABP 4, FABP 2 and RBP.
Other specific transcripts
Beyond energy metabolism, transcript analysis also revealed many other
developmental changes of relevance. Retinol receptors are grouped in
two families; the RXRs and the RARs (Mangelsdorf et al., 1992). RARs
may function as receptors for thyroid hormones, vitamin D and peroxi-
some proliferators (PPAR) and co-operate with RXRs. Retinol and its
receptors may play an important role in cardiac development through
hormonal signaling and as cofactors in fatty acid oxidation for energy gen-
unchanged in both groups. While it was not clear in advance that this
would be the case for all housekeeping genes, it is reassuring that this
is the outcome, since it provides an internal quality control for this and
indeed future analysis. Previous studies validating reference genes in
myoblast development, utilizing different software, indicated that TBP
(TATA box binding protein) and RPLP0 (ribosomal protein large P0)
were the only two consistent genes that could be used to validate
changes in other genes (Stern-Straeter et al., 2009).
In conclusion, these experiments using fetal human hearts spanning
between 10 and 18 weeks of gestation confirmed previous findings in
metabolic profile showing, during this period, that energy derived from
glucose was the main source of fuel for the heart. Although the major

Downloaded from https://academic.oup.com/molehr/article/20/7/690/2459849 by guest on 19 November 2020

source of energy is glucose, it is not the only source. Fatty acid metabol-
eration (Sucov et al., 1994). Although RXR presence has been documen-
ism appears to also be a source of energy and transcripts related to that
ted in adult hearts, it has not been shown in the human fetal heart. In this
metabolic pathway can be found to be present as early as 10 weeks of
study, experiments found the presence mRNA coding for both RXR and
gestation. These genes involved in fatty acid metabolism thereafter
RAR in the human embryonic heart. There does not appear to be a dif-
show a stronger expression as the fetus develops, and this finding is con-
ference in mRNA expression when compared by gestational age.
sistent with our current knowledge showing a transition to a full utilization
However, when KEGG pathways are implemented, the retinol metabol-
of fatty acid oxidation as the energy source in the neonatal period and
ism and PPAR signaling pathway appear to be among the main pathways
adulthood. Many other individual transcript changes have also been iden-
activated in fetal hearts from 10 to 18 weeks. This finding may be linked,
tified that relate to the altered endocrine control of the heart and con-
since retinol is a cofactor in many peroxidation reactions where PPAR is
tractile proteins underlying its function. These findings not only serve
involved (Lopaschuk and Jaswal, 2010). Fatty acid metabolism and gluco-
as a database for comparison with older gestational ages and adults,
neogenesis are major processes regulated by these pathways.
but give a picture of the heart at a developmental stage not previously
A further intriguing finding in the H3 heart was the higher expression of
well characterized and yet critical to developmental success. It is tempt-
GABA receptors. These receptors have been extensively described in
ing to hypothesize that, between the gestational ages of 10 –18 weeks,
the brain and pre/post synaptic environment as neurotransmitters,
the heart fully differentiates and starts specializing, remodeling and
but have not been described in the heart. Kang et al. (2011) recently
adjusting to finally become the hemodynamic controller. Further
showed an association between neurotransmitters as GABA and sym-
studies will be necessary to both validate these findings at a functional
pathoexcitation of the heart in rats with heart failure. It would be reason-
level and establish physiologic correlates suggested by this data set.
able to associate the high expression of GABA receptors in the H3 heart
with sympathetic innervation control of the heart and advanced functions
such as heartbeat variability and blood pressure control. Supplementary data
Insulin growth factor-1 (IGF-1), cortisol receptor (NR3C1) and angio-
Supplementary data are available at http://molehr.oxfordjournals.org/.
tensin receptors 1 and 2 (AGT1R, AGT2R), previously described by
Thornburg et al. (2010) in sheep as important factors stimulating cardio-
myocyte proliferation, were found to be present in human fetal heart as Acknowledgements
early as 10 weeks of gestation. These genes were not differentially
We thank Mark Garthwaite for assistance with mRNA preparation.
expressed when comparison was made among groups based on gesta-
tional age. The mRNA expression though, showed that IGF-1 and
IGFBP1 were present in early gestational age (10 –12 weeks) as Authors’ roles
opposed to IGF-1 receptor that appeared to be mostly present at a
J.I.I.: substantial contributions to conception and design, acquisition of
later stage (16– 18 weeks).
data, analysis and interpretation of data, drafting the article and final
The angiotensin receptor 1 (AGT1R) mRNA expression appears to
approval of the version to be published. W.D.: substantial contributions
decrease with gestational age, the opposite of AGT2R which increases
to analysis and interpretation of data, drafting the article and final approv-
with gestational age. These changes may be related to the function of
al of the version to be published. C.B.: substantial contributions to acqui-
angiotensin II in cardiac remodeling during fetal life. AGT1R has been
sition of data, analysis and interpretation of data, drafting the article and
associated with vasoconstriction and hypertrophy while AGT2R has
final approval of the version to be published. J.O.: substantial contribu-
been associated with vasodilation counteracting AGT1R (Savoia and
tions to analysis and interpretation of data, drafting the article and final
Volpe, 2011). IGF and AGTR may be related. IGF has been previously
approval of the version to be published. R.R.: substantial contributions
described (Thornburg, 2011) to inhibit the expression of AGT2R
to acquisition of data, drafting the article and final approval of the
during hypoxic conditions in fetal rats. To our knowledge, none of
version to be published. A.T.B.: substantial contributions to analysis
these genes have been studied during early human fetal life.
and interpretation of data, drafting the article and final approval of the
version to be published. C.K.: substantial contributions to design, analysis
Reference genes and interpretation of data, drafting the article and final approval of the
In all array studies it is important to run negative controls. Interrogated version to be published. S.S.B.D.: substantial contributions to analysis
housekeeping genes, or reference genes, were indeed found to be and interpretation of data, drafting the article and final approval of the
700
version to be published. T.G.: substantial contributions to conception and
design and interpretation of data, drafting the article and final approval of
the version to be published. I.B.: substantial contributions to conception
and design, analysis and interpretation of data, drafting the article and
final approval of the version to be published. D.S.: substantial contributions
to conception and design, acquisition of data, analysis and interpretation of
data, drafting the article and final approval of the version to be published.
Funding
Maternal Fetal Medicine Research and Development Grant. Dept.
OBGYN, University of Wisconsin.
Conflict of interest
None declared.
References
Babu GJ, Bhupathy P, Carnes CA, Billman GE, Periasamy M. Differential
expression of sarcolipin protein during muscle development and cardiac
pathophysiology. J Mol Cell Cardiol 2007;43:215– 222.
Chung S, Dzeja PP, Faustino RS, Perez-Terzic C, Behfar A, Terzic A. Mitochondrial
oxidative metabolism is required for the cardiac differentiation of stem cells.
Nat Clin Pract Cardiovasc Med 2007;4(Suppl 1):S60–S67.
Gratacos E, Gomez R, Nicolaides K, Romero R, Cabero L. Medicina Fetal.
Spain: Editorial Medica Panamericana, 2007:822.
He W, Zhang L, Ni A, Zhang Z, Mirotsou M, Mao L, Pratt RE, Dzau VJ.
Exogenously administered secreted frizzled related protein 2 (Sfrp2)
reduces fibrosis and improves cardiac function in a rat model of
myocardial infarction. Proc Natl Acad Sci USA 2010;107:21110– 21115.
Huber W, Gentleman R. matchprobes: a Bioconductor package for the
sequence-matching of microarray probe elements. Bioinformatics 2004;
20:1651 – 1652.
Irizarry RA, Hobbs B, Collin F, Beazer-Barclay Y, Antonellis K, Scherf U,
Speed T. Exploration, normalization, and summaries of high density
oligonucleotide array probe level data. Biostatistics 2003;4:249– 264.
Iruretagoyena J.I, Davis W, Bird C, Olsen J, Radue R, Teo Broman A,
Kendziorski C, Splinter BonDurant S, Golos T, Bird I et al. Differential
changes in gene expression in human brain during late first trimester and
early second trimester of pregnancy. Prenat Diagn 2014. Jan 16; Epub
ahead of print.
Kang YM, Zhang AQ, Zhao XF, Cardinale JP, Elks C, Cao XM, Zhang ZW,
Francis J. Paraventricular nucleus corticotrophin releasing hormone
Iruretagoyena et al.
contributes to sympathoexcitation via interaction with neurotransmitters
in heart failure. Basic Res Cardiol 2011;106:473–483.
Kendziorski CM, Newton MA, Lan H, Gould MN. On parametric empirical
Bayes methods for comparing multiple groups using replicated gene
expression profiles. Stat Med 2003;22:3899 – 3914.
Kong B, Liu YL, Lu XD. Microarray-bioinformatics analysis of altered genomic
expression profiles between human fetal and infant myocardium. Chin Med
J (Engl) 2008;121:1257 – 1264.
Lopaschuk GD, Jaswal JS. Energy metabolic phenotype of the cardiomyocyte
during development, differentiation, and postnatal maturation. J Cardiovasc
Pharmacol 2010;56:130 – 140.
Mangelsdorf DJ, Borgmeyer U, Heyman RA, Zhou JY, Ong ES, Oro AE,
Kakizuka A, Evans RM. Characterization of three RXR genes that
Downloaded from https://academic.oup.com/molehr/article/20/7/690/2459849 by guest on 19 November 2020
mediate the action of 9-cis retinoic acid. Genes Dev 1992;6:329 – 344.
Moore K (ed). Embriologia Clinica, 4th edn. Mexico: Interamericana, 1989.
Newton MA, Kendziorski CM, Richmond CS, Blattner FR, Tsui KW. On
differential variability of expression ratios: improving statistical inference
about gene expression changes from microarray data. J Comput Biol
2001;8:37 – 52.
Razeghi P, Young ME, Alcorn JL, Moravec CS, Frazier OH, Taegtmeyer H.
Metabolic gene expression in fetal and failing human heart. Circulation
2001;104:2923 – 2931.
Reini SA, Wood CE, Keller-Wood M. The ontogeny of genes related to ovine
fetal cardiac growth. Gene Expr Patterns 2009;9:122 – 128.
Savoia C, Volpe M. Angiotensin receptor modulation and cardiovascular
remodeling. J Renin Angiotensin Aldosterone Syst 2011;12:381 – 384.
Stern-Straeter J, Bonaterra GA, Hormann K, Kinscherf R, Goessler UR.
Identification of valid reference genes during the differentiation of human
myoblasts. BMC Mol Biol 2009;10:66.
Sucov HM, Dyson E, Gumeringer CL, Price J, Chien KR, Evans RM. RXR alpha
mutant mice establish a genetic basis for vitamin A signaling in heart
morphogenesis. Genes Dev 1994;8:1007– 1018.
Thornburg KL. Foetal programming reveals the dark side of AT(2)R.
Cardiovasc Res 2011;89:260– 261.
Thornburg K, Jonker S, O’Tierney P, Chattergoon N, Louey S, Faber J,
Giraud G. Regulation of the cardiomyocyte population in the developing
heart. Prog Biophys Mol Biol 2010;106:289– 299.
Tsuchimochi H, Kuro-o M, Takaku F, Yoshida K, Kawana M, Kimata S,
Yazaki Y. Expression of myosin isozymes during the developmental stage
and their redistribution induced by pressure overload. Jpn Circ J 1986;
50:1044 – 1052.
Wheater PR, Burkitt HG, Daniels VG. Histologia Funcional, 2nd edn. Spain:
JIMS, 1987.
Yazaki Y, Tsuchimochi H, Kurabayashi M, Kawana M, Kimata S. Distribution
of cardiac myosin isozymes in cardiomyopathy: immunohistochemical and
gene analysis. Jpn Circ J 1987;51:676 – 681.