Systematic Analysis of Gene Expression Differences between Left and Right Atria in Different Mouse Strains and in Human Atrial Tissue

Abstract

Normal development of the atria requires left-right differentiation during embryonic development. Reduced expression of Pitx2c (paired-like homeodomain transcription factor 2, isoform c), a key regulator of left-right asymmetry, has recently been linked to atrial fibrillation. We therefore systematically studied the molecular composition of left and right atrial tissue in adult murine and human atria.
We compared left and right atrial gene expression in healthy, adult mice of different strains and ages by employing whole genome array analyses on freshly frozen atrial tissue. Selected genes with enriched expression in either atrium were validated by RT-qPCR and Western blot in further animals and in shock-frozen left and right atrial appendages of patients undergoing open heart surgery.
We identified 77 genes with preferential expression in one atrium that were common in all strains and age groups analysed. Independent of strain and age, Pitx2c was the gene with the highest enrichment in left atrium, while Bmp10, a member of the TGFβ family, showed highest enrichment in right atrium. These differences were validated by RT-qPCR in murine and human tissue. Western blot showed a 2-fold left-right concentration gradient in PITX2 protein in adult human atria. Several of the genes and gene groups enriched in left atria have a known biological role for maintenance of healthy physiology, specifically the prevention of atrial pathologies involved in atrial fibrillation, including membrane electrophysiology, metabolic cellular function, and regulation of inflammatory processes. Comparison of the array datasets with published array analyses in heterozygous Pitx2c(+/-) atria suggested that approximately half of the genes with left-sided enrichment are regulated by Pitx2c.
Our study reveals systematic differences between left and right atrial gene expression and supports the hypothesis that Pitx2c has a functional role in maintaining "leftness" in the atrium in adult murine and human hearts.

Full text

Systematic Analysis of Gene Expression Differences
between Left and Right Atria in Different Mouse Strains
and in Human Atrial Tissue
Peter C. Kahr
1,2.
, Ilaria Piccini
1.
, Larissa Fabritz
1¤
, Boris Greber
3
, Hans Scho
¨ler
3
, Hans H. Scheld
4
,
Andreas Hoffmeier
4
, Nigel A. Brown
2
, Paulus Kirchhof
1
*
¤
1Department of Cardiology and Angiology, University Hospital Mu
¨nster, Mu
¨nster, Germany, 2Division of Biomedical Sciences, St. George’s University of London, London,
United Kingdom, 3Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Mu
¨nster, Germany, 4Department of Thoracic and
Cardiovascular Surgery, University Hospital Mu
¨nster, Mu
¨nster, Germany
Abstract
Background:
Normal development of the atria requires left-right differentiation during embryonic development. Reduced
expression of Pitx2c (paired-like homeodomain transcription factor 2, isoform c), a key regulator of left-right asymmetry, has
recently been linked to atrial fibrillation. We therefore systematically studied the molecular composition of left and right
atrial tissue in adult murine and human atria.
Methods:
We compared left and right atrial gene expression in healthy, adult mice of different strains and ages by
employing whole genome array analyses on freshly frozen atrial tissue. Selected genes with enriched expression in either
atrium were validated by RT-qPCR and Western blot in further animals and in shock-frozen left and right atrial appendages
of patients undergoing open heart surgery.
Results:
We identified 77 genes with preferential expression in one atrium that were common in all strains and age groups
analysed. Independent of strain and age, Pitx2c was the gene with the highest enrichment in left atrium, while Bmp10,a
member of the TGFbfamily, showed highest enrichment in right atrium. These differences were validated by RT-qPCR in
murine and human tissue. Western blot showed a 2-fold left-right concentration gradient in PITX2 protein in adult human
atria. Several of the genes and gene groups enriched in left atria have a known biological role for maintenance of healthy
physiology, specifically the prevention of atrial pathologies involved in atrial fibrillation, including membrane
electrophysiology, metabolic cellular function, and regulation of inflammatory processes. Comparison of the array datasets
with published array analyses in heterozygous Pitx2c
+/2
atria suggested that approximately half of the genes with left-sided
enrichment are regulated by Pitx2c.
Conclusions:
Our study reveals systematic differences between left and right atrial gene expression and supports the
hypothesis that Pitx2c has a functional role in maintaining ‘‘leftness’’ in the atrium in adult murine and human hearts.
Citation: Kahr PC, Piccini I, Fabritz L, Greber B, Scho
¨ler H, et al. (2011) Systematic Analysis of Gene Expression Differences between Left and Right Atria in Different
Mouse Strains and in Human Atrial Tissue. PLoS ONE 6(10): e26389. doi:10.1371/journal.pone.0026389
Editor: Leon J. de Windt, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands
Received June 21, 2011; Accepted September 26, 2011; Published October 19, 2011
Copyright: ß2011 Kahr et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Fondation Leducq (ENAFRA, http://www.fondationleducq.org, to PK) and by the European Union (E UT RAF ,
(http://www.eutraf.eu/, to PK and NB), as well as by Deutsche Forschungsgemeinschaft (DFG, Fa 413/3-1, h ttp:/ /gepr is. df g.d e/gep ris/O CTOPU S/;js ess io nid =
C247ACACA56F91B179F7F8456E22B886?module=gepris&task=showDetail&context=projekt&id=72005176, to LF, and Collaborative Research Center
(Sonderforschungsbereich) MoBil, SFB 656-A8, http://www.uni-muenster.de/Sfbmobil/projekte/targets/a8.html, to PK and LF). The funders had no ro le in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: p.kirchhof@bham.ac.uk
.These authors contributed equally to this work.
¤ Current address: University of Birmingham Centre for Cardiovascular Sciences, Birmingham, United Kingdom
Introduction
Atrial fibrillation is the most common sustained arrhythmia
[1,2,3,4]. Several forms of atrial damage, including electrical and
structural ‘‘remodelling’’, are the main cause of atrial fibrillation
[5,6]. It is generally accepted that atrial fibrillation is mainly a left
atrial disease. This old concept has been reinforced by the
development of ablation techniques to eliminate left atrial triggers
of atrial fibrillation [7], by recent analyses of ion channel expression
and function in left and right atria [8], and by the identification of
rare somatic mutations in left atrial myocardium associated with
atrial fibrillation [9].
In the last years, several genome-wide association studies in
European and Asian populations have confirmed an association
between atrial fibrillation and intergenic variations on chromo-
some 4q25, close to the Pitx2 transcription factor gene [10,11].
Heterozygous deletion of Pitx2c, the cardiac isoform of Pitx2,in
mice is sufficient to provoke increased inducibility of atrial fibrillation
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without obvious structural cardiac alterations [12,13], associated
with a shortening of the left atrial action potential duration [12].
Pitx2c has a number of developmental functions, particularly as the
final mediator in the left-right patterning pathway of mammalian
embryos [14], where it is expressed exclusively on the left, including
in the primitive left atrium [15]. Surprisingly, there is also a marked
chamber specificity of Pitx2c expression in the adult heart: mRNA
transcripts are expressed almost 100-fold higher in the left as
compared to the right adult human and murine atrium [12].
Taken together, these observations suggest that adult left and
right atria differ in their gene expression patterns, and that these
differences may generate specific molecular patterns with relevance
to atrial pathophysiology. The differences in gene expression
between right and left atria have, however, not yet been system-
atically studied using contemporary genomic techniques. We
therefore performed a genome-wide expression analysis in tissues
from left and right atria from mice of different genetic backgrounds
and age groups, and thereafter confirmed some of the main
identified differences in tissue from human atria. We identified
Pitx2c as the single most enriched gene in left atria of mice and
humans, and characterized 76 other atrial genes from different gene
groups that are differentially expressed in right and left atria. These
findings may help to better target research aimed at identifying new
molecular determinants of atrial fibrillation.
Results
Genome-wide differences in left and right atrial mRNA
expression
In the 3 microarray datasets (MF1_3, MF1_12, SA_12) a total
number of 624 gene probes were found to be significantly differentially
expressed in the mouse left and right atrium (Figure 1A, heat map in
Figure 1B, complete list in Supplementary Table S1), corresponding
to 576 transcripts of 534 genes. Of these, 118 gene probes overlapped
between two and 83 probes across all three datasets, corresponding to
77 genes (see Tables 1 and 2). All overlapping genes had consistently
higher expression values on the left or right atrial side across all
datasets. Overall, more gene probes had higher expression values on
the right atrial side (59%, 368 vs. 256). Different array probes targeting
the same genes usually gave highly consistent (.95% of cases) results
in terms of differential gene expression, suggesting overall reliable data
from a technical point of view.
Gene Set Enrichment Analysis
To investigate the potential relevance of differentially expressed
genes in left and right atrium without preformed hypotheses, we
performed gene set enrichment analyses (GSEA) in each of the
datasets independently. We identified a total of 119 differentially
expressed Gene Ontology (GO) terms, with the majority of these
enriched in right atrium (77 terms) and in the MF1_3 dataset (107
terms, Supplementary Table S2). Of those we found to be enriched
in left atrium, 10 were mitochondria-related GO terms (of which 3
were identified across datasets), ribosome-related terms, and protein
kinase regulation terms. Of those with right atrial predominance, we
found enriched GO terms relating to transforming growth factor-b
(TGFb) signalling, humoral processes, steroid biosynthesis, immune
response, muscle development, extracellular matrix composition,
and transmembrane receptor activity.
Single Gene Enrichment Analysis
To further evaluate how the preferential expression pattern in
the left atrium might affect left atrial function, we performed Gene
Figure 1. Results of the mRNA expression array experiments. AVenn diagram showing the overlap of transcripts (grey numbers) that are
significantly different between LA and RA (Fold-Change.1.5, P,0.05) in 3-month-old MF1 mice (MF1_3), 12-month-old MF1 mice (MF1-12), and 12-
month-old Swiss-Agouti mice (SA_12). The number of transcripts with increased level of expression in the left atrium (black) or right atrium (white)
are given in brackets. The overlapping 83 transcripts in the centre of the diagram refer to 77 single genes (see Tables 1 and 2). BTwo-dimensional
hierarchical clustering of relative change in gene expression using the 83 transcripts common to mice of all strains and age groups.
doi:10.1371/journal.pone.0026389.g001
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Ontology analysis using FatiGO. All 220 genes enriched in left
atrium by array analysis were recognized by the software that
could assign GO-biological process terms to 146 genes, GO-
cellular component terms to 115, and GO-molecular function to
155. Using the significantly enriched genes in left atrium as input
list, 9 GO-biological process terms (levels 3–9), 10 GO-cellular
component terms, and 8 GO-molecular function terms were
significantly enriched (P,0.01, Figure 2, for a complete list of GO
terms see Supplementary Table S3).
In both left and right atrium most of the specifically expressed
genes are coding for proteins expressed in the extracellular region
(extracellular matrix and space), as well as for contractile fibers
and myofibrils. The biological process analysis revealed in both
atria an over-representation of GO categories related to blood
vessels development and morphogenesis, response to external
stimuli, and cell adhesion. In the left atrium, we observed an
enrichment of genes involved in the regulation of muscle
contraction. In addition, we found groups of genes involved in
immune and inflammatory responses, cell differentiation and
proliferation, and regulation of transcription and apoptosis. In
both atria, molecular function terms specific for binding of growth
factors, receptors, carbohydrates, and calcium were enriched. In
the left atrium, chemokine activity and cytokine receptor binding
were also enriched.
Overall (see below), the pathway analyses confirm our main
observations of individual gene expression differences in murine
Table 1. Genes enriched in left atrium (P,0.05) identified in all 3 gene array datasets.
Symbol Gene Name
Fold change
(left vs. right atrium)
MF1_3 MF1_12 SA_12
Abcb4 ATP-binding cassette, sub-family B (MDR/TAP), member 4 1.56 1.88** 1.51
Ccl11 Small chemokine (C-C motif) ligand 11 1.80 2.37 3.18
Ccl21b Chemokine (C-C motif) ligand 21b 8.74* 4.22 2.72
Ccl21c Chemokine (C-C motif) ligand 21c (leucine) 5.16 3.08 3.04
Ckmt2 Creatine kinase, mitochondrial 2 1.55 1.54 2.30
Cxcl14 Chemokine (C-X-C motif) ligand 14 3.75 2.37 2.54
D630003M21Rik RIKEN cD D630003M21 gene 2.70 1.72 1.85
Ddit4l D-damage-inducible transcript 4-like 6.50 3.51 3.89
Entpd2 Ectonucleoside triphosphate diphosphohydrolase 2 1.87 1.80 2.23
F13a1 Coagulation factor XIII, A1 subunit 1.71 2.59 2.08
Fblim1 Filamin binding LIM protein 1 1.54 1.89 1.62
Fcna Ficolin A 1.79 2.64** 2.14
Gm1631 Gene model 1631, (NCBI) 2.16 1.80 1.54
Gucy1a3 Guanylate cyclase 1, soluble, alpha 3 2.29 2.36 1.86
LOC100041504 Similar to beta chemokine Exodus-2 (Ccl21c) 5.91 2.88 2.74
LOC100048554 Similar to monocyte chemoattractant protein-2 (MCP-2) 2.85 2.96 1.93
Mapk10 Mitogen-activated protein kise 10 2.75 1.92 1.50
Mfap4 Microfibrillar-associated protein 4 2.10 2.47 2.43
Mgl1 Macrophage galactose N-acetyl-galactosamine specific lectin 1 2.61 3.98 1.97
Phlda1 Pleckstrin homology-like domain, family A, member 1 4.57 3.82 5.09
Pi16 Peptidase inhibitor 16 3.20 3.19 2.85
Pitx2 Paired-like homeodomain transcription factor 2 15.43 8.07 10.27
Ppp1r1b Protein phosphatase 1, regulatory (inhibitor) subunit 1B 4.08 3.65 3.38
Psat1 Phosphoserine aminotransferase 1 1.82 2.69* 1.57
Reln Reelin 2.39 2.30 2.55
Scara5 Scavenger receptor class A, member 5 (putative) 3.81 4.57 4.13
Sh3gl2 SH3-domain GRB2-like 2 1.87 1.71 2.28
Slc41a3 Solute carrier family 41, member 3 1.74 2.01 1.71
Slco2b1 Solute carrier organic anion transporter family, member 2b1 2.86 2.40 2.17
Syn2 Sypsin II 1.90* 1.84 2.38
Timp4 Tissue inhibitor of metalloproteise 4 1.93 2.77* 2.33
Tnni2 Troponin I, skeletal, fast 2 3.78 2.60 2.33
Uts2d Urotensin 2 domain containing 2.65 4.99 2.30
Vtn Vitronectin 2.00 2.14 1.69
Asterisks indicate significant differences between gene expression levels in 3- and 12-months-old MF1 mice (1st column vs. 2nd column; * =P,0.05, ** = P,0.01).
doi:10.1371/journal.pone.0026389.t001
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Table 2. Genes enriched in right atrium (P,0.05) identified in all 3 gene array datasets.
Symbol Gene Name
Fold change
(left vs. right atrium)
MF1_3 MF1_12 SA_12
1500015O10Rik RIKEN cD 1500015O10 gene 2.06 2.62 2.06
Adm Adrenomedullin 7.84 7.66 4.02
Ahsg Alpha-2-HS-glycoprotein 2.18 3.36** 1.72
Aldh1l2 Aldehyde dehydrogese 1 family, member L2 6.23 2.64 1.74
Amigo2 Adhesion molecule with Ig like domain 2 1.98 2.36 2.00
Angptl7 Angiopoietin-like 7 3.13 2.93 1.87
Apoe Apolipoprotein E 1.51 2.07 1.76
Arc Activity regulated cytoskeletal-associated protein 1.69 1.70 1.63
Arpp21 Cyclic AMP-regulated phosphoprotein, 21 4.01 2.15 2.47
Asah3l N-acylsphingosine amidohydrolase 3-like 1.80 2.70* 2.00
BC022687 cD sequence BC022687 2.23 2.62 2.05
Bmp10 Bone morphogenetic protein 10 27.04* 4.23 12.55
Cd163 CD 163 antigen 1.52 1.66** 1.58
Cd207 CD 207 antigen 16.46 18.77** 10.72
Ckmt1 Creatine kinase, mitochondrial 1, ubiquitous 1.59 1.81 1.59
Cxcl13 Chemokine (C-X-C motif) ligand 13 13.98 14.52* 7.59
Cyp1b1 Cytochrome P450, family 1, subfamily b, polypeptide 1 1.61 1.85 1.54
Dbh Dopamine beta hydroxylase 4.04 5.39 2.86
Dok4 Docking protein 4 1.96 1.67 1.62
Ecm1 Extracellular matrix protein 1 1.78 1.86** 1.51
Emilin2 Elastin microfibril interfacer 2 2.82 2.74 2.24
Fxyd3 FXYD domain-containing ion transport regulator 3 4.97** 2.79 3.13
Gng13 Guanine nucleotide binding protein 13, gamma 3.41 3.14 2.60
Hamp Hepcidin antimicrobial peptide 10.78 17.15 7.00
Hamp2 Hepcidin antimicrobial peptide 2 9.05 12.89 9.21
Hdc Histidine decarboxylase 1.85 2.06 1.96
Hey1 Hairy/enhancer-of-split related with YRPW motif 1 2.50 2.55 2.24
Id1 Inhibitor of D binding 1 2.29* 1.83 1.80
Id2 Inhibitor of D binding 2 1.91 2.87 1.94
Id3 Inhibitor of D binding 3 1.54 1.84 1.69
Igfbp3 Insulin-like growth factor binding protein 3 2.60 2.15 3.81
Klk8 Kallikrein related-peptidase 8 1.59 1.60 1.65
LOC670044 Similar to Mothers against decapentaplegic homolog 6 2.61 2.99 2.67
Mrvi1 MRV integration site 1 1.57 1.70** 1.55
Msc Musculin 3.18 2.51 2.41
Pla2g5 Phospholipase A2, group V 1.98 1.65 1.65
Ptgds Prostaglandin D2 synthase 4.50 5.15 3.00
Ryr3 Rryanodine receptor 3 2.87 3.03** 2.72
Smarcd3 regulator of chromatin, subfamily d, member 3 1.92 2.08* 2.07
Tcf21 Transcription factor 21 1.87 1.95 1.97
Tmem108 Transmembrane protein 108 3.14 3.90 2.44
Vsig4 V-set and immunoglobulin domain containing 4 16.72 5.83 7.81
Vwf Von Willebrand factor homolog 1.79 2.03** 1.70
Asterisks indicate significant differences between gene expression levels in 3- and 12-months-old MF1 mice (1st column vs. 2nd column; * =P,0.05, ** = P,0.01).
doi:10.1371/journal.pone.0026389.t002
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atrial tissue, and are consistent with our validation experiments in
human atrial tissues.
Validation of array-based gene expression profiles by
RT-qPCR in mouse tissues
To validate the microarray data set, we selected left (n = 5) and
right (n = 4) marker candidates with different degrees of overlap
between the array datasets (2 genes identified in only 1 dataset, 1
gene in 2 datasets, and 6 genes identified in 3 datasets). We assessed
mRNA concentrations by RT-qPCR on additional samples of MF1
mice (n = 4, 3-month-old) and Swiss-Agouti (n = 4, 12-months-old),
and on a third wild-type mouse strain (CD1, n = 4, 3.5-months-old).
All genes tested by RT-qPCR were significantly differently
expressed between left and right atrial tissues in at least one of the
wild-type mouse strains (Figure 3A–B). For all genes, the atrium
showing enriched expression was the same by array and RT-qPCR.
Overall, RT-qPCR confirmed the microarray data, both in
enrichment in a given tissue (right versus left atrium) and across
different mouse strains (Figure 3A and 3B).
Age-dependent differences
Our array experiments were designed to identify differences in
gene expression that are independent of the strain and the age of
the mouse. The data set also allowed for an exploratory analysis of
age-dependent differences in gene expression in mouse atria of
MF_1 mice. This analysis identified 140 genes that were
upregulated by at least 2-fold (P,0.05) in the left atrium of 12-
months–old MF_1 mice, and 268 upregulated genes in left atrium
of 3-month–old. Of the 77 genes listed in Tables 1 and 2, four
genes showed enhanced expression in the left atrium of the older
mice and only 2 in left atrium of the younger mice. Ten genes
were enriched in the right atrium of the older mice and only 3 in
the right atrium of the younger mice. These genes are marked by
asterisks in Tables 1 and 2. Comparison of MF1 gene arrays in the
3- and 12-month–old mice revealed no significant difference in
Pitx2c expression. Expression of Irx3 was significantly downregu-
lated in the right atrium of the 12-month–old mice compared with
the 3-month–old mice, whereas expression of Scn4b was upregu-
lated with age (data not shown).
Validation of differential gene and protein levels in
human atrial tissues
To determine whether the differences identified in the left and
right atrial gene expression in the mouse are consistent in humans,
we performed RT-qPCR measurements on human left and right
atrial appendages (n = 5) harvested from patients undergoing open
Figure 2. Gene groups enriched in left atrium according to FatiGO gene ontology analysis. 220 genes significantly enriched in the left
atrium in any of the three microarray datasets (MF1_3; MF1_12; SA_12) were used as input list against the genome. 9 biological process, 10 cellular
component, and 8 molecular function enriched GO classes are highlighted in red (levels 3–9; P,0.01).
doi:10.1371/journal.pone.0026389.g002
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heart surgery. Clinical characteristics are given in Table 3. The
RT-qPCR measurements confirmed differential expression in the
left and right atrium in 5 of 9 tested human orthologues (Fig. 4A).
Adm,Irx3,Kcn4, and Bmp10 were confirmed to be expressed at
higher levels in right than in left atrium, whereas Pitx2c was
enriched in left atrium, as expected. The murine left atrium-
enriched genes Mapk10,Ppp1r1b, and Scn4b, and Ryr3, enriched in
right atrium by array analysis, were not confirmed as such in the
human tissue samples used for analysis.
Furthermore, we confirmed higher expression of three selected
genes at the protein level. Using Western blots of lysates prepared
from immediately shock-frozen human left and right atrial
appendages from the same human samples, we confirmed higher
PITX2 protein levels in left atrium and higher IRX3 and BMP10
protein levels in right atrium (n = 4, Fig. 4B and C).
Discussion
Main findings
This systematic analysis of gene expression profiles identified
major differences in gene expression between left and right adult
atrial tissue. These observations were not only consistent across
different strains of murine hearts, but also confirmed in human
atria. These data shed light onto potentially relevant physiological
differences between right and left atria: Our hypothesis-free
analysis identified Pitx2c, a gene that has recently been shown to be
involved in the pathophysiology of atrial fibrillation, as the single
most enriched gene in the left atrium, while Bmp10, a gene known
to be relevant for trabeculation of the right atrium and ventricle,
was the single most differentially expressed gene in the right
atrium. Other differentially expressed genes are involved in
membrane electrophysiology, metabolic cellular function, and
regulation of inflammatory cells.
Significant differences in left and right atrial gene
expression
We systematically characterized gene expression differences
between the right and left atria by gene arrays in healthy mice of
different strains and at different ages, and by employing different
commercially available gene array analyses in two different
laboratories. The degree of consistency in left-/rightness among
probes that were identified in murine atria from mice of different
Figure 3. RT-qPCR validation of the differential expression of candidate genes in left and right atria from wild-type mice of three
different strains. ARelative Log2 expression ratios between LA and RA in MF1 mice (blue, n =4), Swiss-Agouti mice (pink, n = 4), CD1 mice (yellow,
n = 4), and in wild-type mice (grey, n = 12). BCorresponding average mRNA expression in left (black) and right (white) atrial tissues of MF1, SA, and
CD1 mice (n = 4 each). Gapdh was used as control. Error bars indicate standard error of the mean (SEM). Statistically significant differences as assessed
by unpaired Student’s t-test are represented by asterisks (*P,0.05, **P,0.01).
doi:10.1371/journal.pone.0026389.g003
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strains and ages, as well as the number of transcripts that were
identified by multiple probes, highlight the robustness of the
observed differences. This finding is affirmed by the unbiased
detection of overlapping GO terms by GSEA and FatiGO in the
different datasets (Supplementary Tables S2 and S3). Although we
have highest confidence in those genes that were identified in all 3
datasets, we assume that some non-overlapping genes may
represent true biological differences. Finally, differences in
individual genes were validated by RT-qPCR in murine tissue.
Several differences were furthermore validated on human atrial
tissue at the RNA and protein levels.
What controls ‘‘sidedness’’?
Given that the constituent cells types of the left and right atria
do not differ markedly, the consistent differential gene expression
raises the question of how this is controlled. By extension of its role
Table 3. Experimental setup and samples.
Murine atria Human atria
MF1 MF1 Swiss Agouti CD1 P1 P2 P3 P4 P5
Age 1260w 5260w 5260w 1460 w 55 y 60 y 82 y 86 y 64 y
Sex 9f 4m 8f 2m+2f f m f m m
Operation - - - - AVR CABG AVR AVR CABG CABG
CHADS2 score#--- - 0223 2
Array xxx - -- -- -
RT-qPCR x- x x xxxx x
Western-Blot --- - xxxx -
Information on the types of samples used for the different experimental protocols. For human samples (P1–P5), clinical characteristics are given. All patients were in
permanent atrial fibrillation at the time of tissue harvesting during open heart procedures (AVR = Aortic Valve Replacement; CABG = Coronary Artery B ypass Grafting). All
sample pairs (left and right atrium) were preserved under standardized, pairwise identical conditions as described in the text.
#
The CHADS2 is a clinically used score that summarises comorbidities often found in atrial fibrillation patients.
It adds one point each for heart failure, hypertension, diabetes mellitus, age .75 years, and two points for a prior stroke.
doi:10.1371/journal.pone.0026389.t003
Figure 4. A) RT-qPCR measurements of orthologous transcripts in left atrial (black) and right atrial (white) human appendages
(n = 5). Actb was used as control. Error bars indicate standard error of the mean (SEM). Statistically significant differences as assessed by paired
Student’s t-test are represented by asterisks (*P,0.05). B) and C) PITX2, IRX3, and BMP10 protein expression level measured by Western blot of total
protein lysates from human left (black) and right (white) atrial appendages (n = 4). The data shown are averages of the results obtained from four
separate samples. Error bars indicate standard deviation (SD). Statistically significant differences as assessed by unpaired Student’s t-test are
represented by asterisks (*P,0.05).
doi:10.1371/journal.pone.0026389.g004
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in embryonic patterning, Pitx2c is one obvious candidate for a
controlling transcription factor. The embryonic right-side develop-
mental programme has been considered as the ‘‘default’’ pathway,
which will be followed unless signals are received instructing
otherwise. For heart development, Pitx2c is the key mediator for
such signalling, and in its complete absence, ‘‘leftness’’ is lost and
right isomerism results [16]. Our finding that Pitx2c is the most
highly differentially expressed gene in adult left atria is compatible
with a continuing control function. Only a few genes have been
identified as direct targets of Pitx2, including Cyclin D2 (Ccnd2) and
Lef-1, but none of the differentially expressed atrial genes we have
identified have been studied in this regard. Interestingly, our most
highly expressed right atrial gene, Bmp10, has been reported to be
regulated by Pitx2c, although a direct effect has not been tested [17].
In that case, as well as for some sinuatrial node genes [12,13], the
effect of Pitx2c is to repress expression in the left atrium. Bmp10 is
best known for its role in cardiomyocyte growth in trabecular and
compact myocardium [18,19]. As we found canonical target genes
of the BMP signaling pathway enriched in right atrium (Id1,Id2,Id3)
in our experiments and in a prior analysis of pooled murine atria
using self-constructed gene arrays [20], it may be speculated that
there is an ‘‘active’’ growth regulatory role for Bmp10 in the adult
right atrium. Alternatively, Bmp10 could have a reciprocal role to
Pitx2c in maintaining left-right atrial differences by suppressing
genes in the right atrium. In support of this notion, BMP signalling is
known to have a role in suppressing the nodal-pitx2 pathway in
early embryonic left-right patterning [21]. One approach to identify
genes that are regulated in the atria by Pitx2c would be correlation
analysis using microarrays, looking for genes that are co-regulated
with Pitx2c or with Bmp10 [22].
Pitx2c is the single most differentially expressed gene
between the left and right atrium
Pitx2c emerged as the single most differentially expressed gene
between left and right atrial tissue. This finding was consistent well
into advanced adulthood (12 months of age). Higher left than right
atrial expression of PITX2c was confirmed in human left atria. Of
the seven differentially expressed genes with a role in cardiac
development and morphogenesis, six are highly expressed in the
right atrium (Adm,Bmp10,Id1,Id2,Id3,Smarcd3) and only Pitx2c is
highly expressed in the left atrium. This differential expression is
conserved between mice and men, and results in a left-right
PITX2 protein gradient (Figure 4). This new observation
substantiates our prior assumption that Pitx2c has a functional
role in the adult left atrium [12], and that loss of Pitx2c function
may revert left atrial cardiomyocytes to right-sidedness.
Reduced Pitx2c expression reduces ‘‘leftness’’ of the
genomic signature in murine atria
To assess to what extent Pitx2c might control leftness in the
healthy murine atrium, we compared the 10 most highly dif-
ferentially expressed left and right atrial genes with a previously
published dataset of Pitx2c
+/2
mice, that were littermates to the
MF1_3 animals used in this present study [12]. Of the genes with
highest left-right fold-change (excluding Pitx2c), 5 of 9 were reduced
to 50–60% of wild-type levels in Pitx2c
+/2
left atria (Table 4; Ccl21b,
Ddit4l,LOC100041504,Ccl21c,andPpp1r1b). This suggests that
these genes are positively induced by Pitx2c; the remaining 4 genes
do not appear to be controlled by Pitx2c. Similarly, among the 10
genes most enriched genes in right atrium, 4 were increased by 140–
520% in the left atrium of Pitx2c
+/2
(Table 4; Bmp10, Vsig4, Cd207,
and Cxcl13), suggesting that these genes are repressed by Pitx2c.We
cannot say whether these apparent inductions and repressions are
direct or indirect. Nevertheless, this comparison suggests that about
half of the differences in gene expression seen in the left versus right
atria may be secondary to regulation by Pitx2c. Maintaining atrial
‘‘left-sidedness’’ through adequate Pitx2c expression could even be a
regulatory pathway that could help protect left atria against
development of atrial fibrillation [10,12,13].
Gene expression differences in ion channel composition
Several reports have consistently shown that left atrial action
potential duration (APD) is shorter than right atrial APD in mice
[23] and in canines [24]. The RNA for I
K,ACh
,Kcnc4, was
consistently expressed at higher levels in right atria in mice and
humans. Hence, a ‘‘loss of left-sidedness’’ would increase I
K,ACh
levels in the left atrium, which may contribute to atrial fibrillation
[25,26].
Cytokine 21 ligand genes are highly expressed in the left
atrium
Genes coding for cytokines and cytokines receptors are found in
both atria (left atrium: Ccl11,L0c100041504,Cxcl14,Ccl21c,Ccl21b,
Vtn; right atrium: Dbh,Ahsg,Cd163 Cxcl13). Of note, 3 transcripts
encoding the cytokine21 ligand (isoforms b and c) were robustly and
highly enriched in left atrium. It is tempting to speculate that this
cytokine receptor, which regulates chemotaxis and formation of
lymph nodes, may be involved in the production of myeloperox-
idase in the left atrium, which has recently been linked to
development of atrial fibrillation and atrial fibrosis [27]. Further
studies on the role for Ccl21 in left atrial function are warranted.
Consistent with this, we find other genes involved in inflammatory
processes with higher expression in the left atrium: Timp4 was
differentially expressed in all three data sets (Table 1), and Alox5,C3,
and Timp3 were differentially expressed in some, but not all tissues
examined (Supplementary Table S1). The role of local inflamma-
tion in the pathogenesis of atrial fibrillation has long been under
discussion [28,29], and anti-inflammatory agents can prevent post-
operative atrial fibrillation [3]. Recently, alterations in the
expression of Timp2 and Mmp2 have been shown to be associated
with atrial fibrillation in different respects [30,31], with the latter
enriched in left atrium in two out of three of our datasets
(Supplementary Table S1).
Table 4. Top 10 genes enriched in left atrium in MF1_3
dataset (excluding Pitx2c) compared to gene expression in left
atrium of 3-month–old Pitx2c
+/2
mice [12].
MF1_3
Pitx2c
+/2
vs. MF1_3
Gene FC (LA vs. RA) (LA)
Ccl21b 8.74 53.0%
Ddit4l 6.50 64.8%
LOC100041504 5.91 60.4%
Ccl21c 5.16 61.0%
Phlda1 4.57 101.7%
Ppp1r1b 4.08 58.5%
Scara5 3.81 114.5%
Tnni2 3.78 85.6%
Cxcl14 3.75 79.3%
doi:10.1371/journal.pone.0026389.t004
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PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e26389

Other genes enriched in the left atrium
Among the 77 genes enriched in the left or right atrium in all
three array experiments, 2 genes with a left-sided enrichment have
a role in the blood coagulation (F13a1,Entpd2). Four left-sided
genes (Fblim1,Reln,Vtn,Mfap4) and 3 right-sided genes (Emilin2,
Amigo2,Vwf) code for adhesion proteins. Tcf21,Hey1,Id1,Apoe,Dbh
(right atrium), and Pitx2c (left atrium) are involved in blood vessel
development. Left atrial clot formation is the basis for thrombem-
bolic stroke, one of the severe complications of atrial fibrillation. In
our dataset we also found genes related to coagulation, platelet
activity and thrombogenesis as being left-right differentially
expressed in the atria (left atrium: Entpd2,F13a1,Vtn; right
atrium: Vwf,Mrvi1,Cxcl14). Recently, Cxcl14, also known as Pf4
(platelet factor 4), was found to be slightly elevated in the left
atrium of patients with atrial fibrillation [32]. Ddit4l, another
highly differentially expressed gene, codes for a protein involved in
DNA damage and hypoxia-induced cell death. Ppp1r1b, another
gene with preferential left atrial expression, codes for a regulatory
subunit of protein phosphatase 1 (PP1). The catalytic subunit of
PP1 and its inhibitor are implicated in the development of heart
failure at the ventricular level [33,34,35]. The relevance of the
regulatory subunit encoded by Ppp1r1b in the atrial myocardium
has not been studied.
Implications
Our study identifies relevant differences in gene expression
between left and right atrium, suggesting that analysis of genes and
proteins should be separated for each of the two chambers when
functionally relevant differences are at stake. Our study also
confirms that Pitx2c is highly expressed in the left atrium,
suggesting a relevant role for this gene for left atrial function in
health and disease. Combined with the published information that
reduced Pitx2c expression is a predisposing factor for atrial
fibrillation [10,12,13], our data suggest that maintaining ‘‘left-
sidedness’’ in the left atrium may be important for normal left
atrial function in the adult heart.
Limitations
Here, we identify relevant differences in gene expression
between the right and left atria based on modern, relatively
comprehensive gene array technology. While we were able to
confirm some of our findings by RT-qPCR, including analyses of
diseased human tissues, our study will certainly miss further,
relevant biological differences between the right and left atria that
may be picked up by more sensitive, hypothesis-driven analyses.
This is a shortcoming generic to array analyses. The hypothesis-
free analysis of atrial tissue was confined to murine datasets in our
study. Our validation of the differences in the expression of some
murine genes in human atrial tissue harvested from patients with
heart disease requiring open heart surgery suggests that gene
expression differences may have biological relevance to human
atrial function. Further studies are warranted to study the effects of
heart disease on differential gene expression in the left and right
atrium. Furthermore, not all differences in mRNA concentrations
translate into differences of protein concentration [36].
Methods
Murine atrial tissues
We studied wild-type mice of three different outbred strains that
are commonly used in research laboratories (MF1, Swiss-Agouti,
and CD1). Animals were kept and sacrificed in one facility in
Mu¨ nster, Germany, following identical tissue extraction protocols.
The harvesting of animal tissues occurred within a research
program approved by our local authority (lanuv NRW). For array
analyses, we used 3-months-old (1260 weeks) and 12-months-old
(5260 weeks) MF1 mice and 3-months-old (1260 weeks) Swiss-
Agouti mice. For confirmatory RT-PCR experiments, we addition-
ally analyzed cardiac tissue from 3-month-old (1460 weeks) CD1
mice. Hearts were dissected in cold buffer solution and left and right
atria were stored separately. Tissues from 3-month-old MF1 mice
were placed into RNAlater (QIAGEN, Hilden, Germany) and
shipped to the second laboratory in London, United Kingdom, for
microarray experiments. All other samples were shock-frozen in
liquid nitrogen immediately after preparation and kept at 280uC
until further processing. Preparation time from the incision of the
murine thorax to preservation of the tissue was less than 3 minutes
(172621 seconds).
Human atrial tissue
Human samples from left and right atrial appendages were
collected during open heart surgery, shock-frozen and stored at
280uC. All patients gave written informed consent to the
preservation and analysis of their tissue, and the analysis was
approved by the ethics committee of A
¨rztekammer Westfalen-
Lippe and Medical Faculty of WWU Mu¨nster (AZ 2006-414-f-M).
Tissue homogenization and RNA isolation
All collected samples were handled independently and individ-
ually throughout the experiments, with no pooling of any samples,
at difference to prior reports using custom-made, non standardized
arrays [20]. Collected tissues were pulverised at 3000 rpm for
45 sec using a Mikro-Dismembrator S (Sartorius, Goettingen,
Germany). Half of the material was used for extraction of total
RNA, the other half for protein isolations. Total RNA was
extracted following standard techniques using RNeasy Fibrous
Tissue Mini Kit and Micro Kit (QIAGEN; including on-column
DNAse treatment) from human and mouse samples. Isolated RNA
was quantified by UV spectrophotometry and checked for
integrity by capillary electrophoresis using an Agilent 2100
Bioanalyzer (Agilent technologies, Boeblingen, Germany). RNA
Integrity Number .8 was accepted for further experiments.
Mircoarray hybridization
We performed 3 separate microarray experiments comparing
left and right atrial gene expression: ‘‘MF1_3’’ using 3-month-old
MF1 mice (n = 5, Illumina MouseWG-6 v2.0 Expression Bead-
Chip, 45,281 probes), ‘‘MF1_12’’ on 12-month-old MF1 mice
(n = 4, Illumina MouseRef-8 v2.0 Expression BeadChip, 25,697
probes) and ‘‘SA_12’’on 12-month-old Swiss-Agouti mice (n = 4,
Illumina MouseRef-8 v2.0 Expression BeadChip). For each
experiment, 100 ng of total RNA was labelled using the Illumina
Total Prep RNA Amplification Kit (Ambion, Austin, TX, USA)
and then hybridized to the chips. Scanning was performed on a
Bead Array Reader (Illumina, Eindhoven, The Netherlands) and
data was extracted from the images with Genome Studio (Version
2009.1, Illumina). All chips passed quality control to eliminate
scans with abnormal characteristics. Expression data was depos-
ited at the Gene Expression Omnibus (GEO accession numbers:
GSE22170, GSE29500).
Quantitative Real-Time Polymerase Chain Reaction
We used quantitative real-time polymerase chain reaction (RT-
qPCR) to validate the levels of expression from the microarrays.
Up to 1 mg of RNA per sample was reverse transcribed into cDNA
using oligo-dT
15
primers and M-MLV Reverse Transcriptase
(USB) following standard procedures. All primers were designed
Left-Right Gene Expression Differences in Atria
PLoS ONE | www.plosone.org 9 October 2011 | Volume 6 | Issue 10 | e26389

with Primer3 software [37] and validated with respect to efficiency
and specificity. Experiments were carried out using Power SYBR
Green PCR Master Mix (Applied Biosystems). All measurements
were performed in duplicate. Relative quantification of expression
was performed according to the ‘‘DDCt method’’ [38] and by
employing Glyceraldehyde-3-Phosphate Dehydrogenase (Gapdh)
or Beta-Actin (Actb) for normalization. (For full details on the
primer sequences and RT-qPCR settings, see Supplementary
Table S4.)
Protein biochemistry
Pulverized human left and right atrial samples were resuspensed
in lysis buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.5%
Triton X-100), supplemented with a protease inhibitor cocktail
tablet (Complete Mini, Roche diagnostics, Mannheim, Germany).
Homogenates were then centrifuged at 2500 rpm for 5 minutes.
50 mg of total protein lysate was loaded onto a 10% SDS-
polyacrylamide resolving gel (1-mm thick) and electrophoresed for
75 minutes at 180 V. Gels were blotted onto a nitrocellulose
membrane (Hybond ECL, GE Healthcare, Mu¨nchen, Germany)
by means of a wet transfer system for 60 minutes at 100 V. The
membranes were incubated overnight at 4uC with isoform-
unspecific rabbit-derived anti-PITX2 antibody (Capra Science,
#PA 1020-100 , 1:1,000 in blocking buffer), anti-IRX3 (Santa
Cruz Biotechnology, #sc-30157, 1:500 in blocking buffer),
mouse-derived anti-BMP10 (R&D Systems, #MAB2926, 1:500
in blocking buffer [39]) and mouse-derived anti-GAPDH (Am-
bion, #AM4300, 1:10,000 in blocking buffer). For better detection
of the mature, secreted form of BMP10 without its precursors,
membranes used for the human BMP10 blot were cut at 46 kD.
After washing, membranes were incubated with anti-rabbit or
anti-mouse IgG, HRP-Linked Whole Antibody (1:10,000; GE
Healthcare) for 2 hours at room temperature. Detection was
performed using ECL Western blot substrate (Thermo Fisher
Scientific, Waltham, MA, USA). Band densities were quantified
using Image J (National Institutes of Health, Bethesda, MD, USA).
Statistical analysis
All 3 microarray datasets (MF1_3, MF1_12, SA_12) were
analysed independently. In each dataset, samples were normalized
with Genespring (version GX11.0, Agilent technologies) by
applying a quantile algorithm and a baseline transformation to
the median of all samples. Gene probes with a fold-change .1.5
and a Benjamini-Hochberg corrected P-value,0.05 were accepted
as significantly different between left and right atria. Two-
dimensional hierarchical clustering was performed in Multi-
Experiment Viewer (TM4 Microarray Software Suite; [40]) on
gene probes that were identified as significantly different in all 3
datasets. Unpaired Student’s t-test was employed to test for
significant differences in relative RNA expression values, as
determined by RT-qPCR, and protein expression levels, from
Western blot experiments, between the left and right atrium. A
two-sided P-value,0.05 was accepted as significant.
Microarray functional analysis
Two independent and unbiased strategies were utilized to
identify enriched functional themes (GO terms) within the datasets.
Gene Set Enrichment Analysis (GSEA, Broad Institute; standard
settings, [41]) was performed on the unfiltered datasets. As
suggested for GSEA, a false discovery rate ,0.25 and a nominal
P-value,0.05 were accepted as significant. To assign biological
meaning to the group of genes significantly upregulated in the left or
right atrium in any of the three datasets (MF1_3; SA_12; MF1_12),
the FatiGO algorithm (Babelomics 4.2, corrected p-value,0.01)
was applied to compare these lists with the rest of the genome.
Additionally, we performed a common normalization for the
MF1_3 and MF1_12 datasets to determine whether there were any
age-dependent differences in this strain.
Supporting Information
Table S1 List of 624 gene probes that were identified in any of
the three independently prepared mRNA expression arrays
comparing left and right atrial gene expression in adult mice.
Gene probes that were not assigned in the ‘‘smaller’’ Illumina
MouseRef-8 v2 Expression BeadChip, which were used to
generate the MF1_12 and SA_12 datasets (compared with the
Mouse WG-6 v2 Expression BeadChip for the MF1_3 dataset) are
labelled as ‘‘na’’ in the table. If a gene probe did not exhibit a
significant difference between left and right atrium in one dataset,
the P-value column was left blank in this dataset.
(DOCX)
Table S2 Gene set enrichment analysis results.
(DOCX)
Table S3 FaTiGo results (Biological Process; Molecular Func-
tion, Cellular Compartment).
(DOCX)
Table S4 RT-qPCR primers for selected murine and ortholo-
gous human genes.
(DOCX)
Acknowledgments
The authors thank Janine Eisenmann, Nina Kreienkamp, Dana Kucerova,
Lisa Fortmueller, Sandra Laakmann, Meike Rolfing, Angela Schroeder,
and Ian Andrew for help with the study.
Author Contributions
Study design, experimental planning, experiments, paper writing and
review: PCK IP LF. Funding of experiments: LF. Study design,
experimental design, funding of study, review of manuscript: BG. Funding
of study, structural support, critical review of paper: HS. Collection of
human samples, critical review of paper: HHS. Collection of human
samples and clinical data, critical review of paper: AH. Design of study,
design of experiments, writing of paper, critical review of paper, funding of
study: NAB. Design of study, design and review of experiments, writing of
paper, funding of study: PK.
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