Alterations in cardiac DNA methylation in human dilated cardiomyopathy.

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by  Haas, Jan

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DNAmethylationinhumandilatedcardiomyopathy PROCESS ACCESS
Alterations in cardiac DNA methylation in
human dilated cardiomyopathy
Jan Haas1y, Karen S. Frese1y, Yoon Jung Park2,3y, Andreas Keller4, Britta Vogel1, Anders M. Lindroth2,
Dieter Weichenhan2, Jennifer Franke1, Simon Fischer1, Andrea Bauer5, Sabine Marquart1,
FarbodSedaghat-Hamedani1,ElhamKayvanpour1,Doreen K¨ohler1,NadineM.Wolf1,6,SarahHassel1,
Rouven Nietsch1, Thomas Wieland6,8, Philipp Ehlermann1, Jobst-Hendrik Schultz7, Andreas D¨osch1,
Derliz Mereles1, Stefan Hardt1, Johannes Backs1,9, J¨org D. Hoheisel5, Christoph Plass2,9,
Hugo A. Katus1,9, Benjamin Meder1,9*
Keywords: biomarker; dilated Dilated cardiomyopathies (DCM) show remarkable variability in their age of
cardiomyopathy; DNAmethylation; onset,phenotypicpresentation,andclinicalcourse.Hence,diseasemechanisms
epigenetics; heartfailure mustexistthatmodifytheoccurrenceandprogressionofDCM,eitherbygenetic
orepigeneticfactorsthatmayinteractwithenvironmentalstimuli.Inthepresent
study, we examined genome-wide cardiac DNA methylation in patients with
DOI10.1002/emmm.201201553
idiopathicDCMandcontrols.Wedetectedmethylationdifferencesinpathways
ReceivedMay08,2012 relatedtoheartdisease,butalsoingeneswithyetunknownfunctioninDCMor
RevisedNovember15,2012 heart failure, namely Lymphocyte antigen 75 (LY75), Tyrosine kinase-type cell
AcceptedNovember29,2012
surfacereceptorHER3(ERBB3),HomeoboxB13(HOXB13)andAdenosinereceptor
A2A(ADORA2A).Mass-spectrometricanalysisandbisulphite-sequencingenabled
confirmationoftheobservedDNAmethylationchangesinindependentcohorts.
Aberrant DNA methylation in DCM patients was associated with significant
changesinLY75andADORA2AmRNAexpression,butnotinERBB3andHOXB13.
In vivo studies of orthologous ly75 and adora2a in zebrafish demonstrate
a functional role of these genes in adaptive or maladaptive pathways in
heartfailure.
(1) DepartmentofInternalMedicineIII,UniversityofHeidelberg,Heidelberg, INTRODUCTION
Germany
(2) Division of Epigenomics and Cancer Risk Factors, German Cancer
Idiopathic dilated cardiomyopathy (DCM) is a frequent heart
ResearchCenter(DKFZ),Heidelberg,Germany
(3) Department of Nutritional Science and Food Management, Ewha muscle disease with an estimated prevalence of 1:2500
WomansUniversity,Seoul,SouthKorea (Karkkainen&Peuhkurinen, 2007). Theprogressivenatureof
(4) DepartmentofHumanGenetics,SaarlandUniversity,Germany the disorder is responsible for nearly 50,000 hospitalizations
(5) DivisionofFunctionalGenomeAnalysis,GermanCancerResearchCenter and10,000deathsperyearintheUSaloneandisthemaincause
(DKFZ),Heidelberg,Germany
forhearttransplantationinyoungadults(Dec&Fuster,1994).
(6) Medical Faculty Mannheim, Institute of Experimental and Clinical
Overall,theincidenceofthediseasehascontinuallyincreased
PharmacologyandToxicology,HeidelbergUniversity,Mannheim,Germany
over the past years and it was recognized that DCM has a
(7) DepartmentofGeneralInternalMedicineandPsychosomatics,University
HospitalHeidelberg,Heidelberg,Germany substantial genetic contribution (Grunig et al, 1998). It is
(8) DZHK (German Centre for Cardiovascular Research), partner site estimated that about 30–40% of all DCM cases show familial
Heidelberg/Mannheim,Mannheim,Germany aggregation and until now more than 40 different genes were
(9) DZHK (German Centre for Cardiovascular Research), partner site foundtocausegeneticDCM(Meder&Katus,2012).However,
Heidelberg/Mannheim,Heidelberg,Germany
sincethecourseofevenmonogeneticDCMishighlyvariable,
*Correspondingauthor:Tel:þ496221564835;Fax:þ496221564645;
genetic modifiers are thought to have an important influence
E-mail:[email protected]
on phenotypic characteristics and outcome (Friedrichs et al,
yTheseauthorscontributedequallytothiswork. 2009;Villardetal,2011).Accordingly,severalstudieshavenow
(cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO.Thisisanopenaccessarticleunder
thetermsoftheCreativeCommonsAttributionLicense(CCBY3.0),whichpermitsuse,distributionandreproduction
inanymedium,providedtheoriginalworkisproperlycited. EMBOMolMed(2013)5,413–429 413
ResearchArticle www.embomolmed.org
DNAmethylationinhumandilatedcardiomyopathy
identified common geneticpolymorphisms that areassociated Table1. Study sample characteristics of the screening and replication
withDCMorheartfailure(Friedrichsetal,2009;Villardetal, cohorts
2011).
Cohort N Female(%) Age(years) LV-EF(%)
Diseasemodificationthroughepigeneticalterationshasbeen
Screening
convincinglydemonstratedforanumberofdiseases(Feinberg
DCMcases 9 33 57(cid:1)5.4 27(cid:1)7.5
& Tycko, 2004; Jones & Baylin, 2002). In the cardiovascular
Controls 8 38 42(cid:1)14 60(cid:1)4.0
system, histone modifications and chromatin remodelling are Replication
thought to direct adaptive as well as maladaptive molecular DCMcases 30 37 58(cid:1)14 25.6(cid:1)8.4
pathwaysincardiachypertrophyandfailure(Montgomeryetal, Controls 28 46 57(cid:1)12 61.5(cid:1)5.3
2007),andDNAmethylationwasfoundtoberesponsibleforthe Forthecontrolpatients:Female,femaledonorstatusoftransplantedheart;
hypermutabilityofdistinctcardiacgenes(Meurs&Kuan,2011). LV-EF,leftventricularejectionfraction.
Furthermore,recentstudieshavehighlightedpotentialinterplay
between environmental factors and the disease phenotype
by epigenetic mechanisms (Herceg & Vaissiere, 2011; Jirtle We used the comprehensive screening datasets in a gene
& Skinner, 2007). However, the knowledge about the impact set enrichment analysis (GSEA) and identified within the
of epigenetic alterations on the disease phenotype in human top three (ranked by p values) enriched disease categories
patientsisstillverylimited. providedbytheNationalInstituteofAging(NIA)thedisease
Thepresentstudyinvestigatedforthefirsttimetheimpactof pathways ‘cardiovascular disease’, ‘metabolic disease’ and
genome-wide cardiac DNA methylation on human DCM in ‘pathological conditions’, all associated with DCM and
patients. We identified several candidate genes with altered with considerable overlaps between each other (Fig 1C). To
methylation status and replicated these findings in an detect patterns of genes with differential methylation in
independent cohort ofDCMpatientsandcontrols. Using gene the screening cohorts, we carried out a clustering approach
expressionanalysisandzebrafishasaninvivomodel,wecould on genes with known abundant expression in the human
furthermore show that appropriate mRNA levels of LY75 and heart (http://c-it.mpi-bn.mpg.de). From the annotated 2018
ADORA2Aareimportantforunconstrainedcardiacfunction. individual genes, 1858 (92.1%) were covered by the applied
Infinium assay. Since the degrees of methylation for the
genes were not normally distributed, we used two-tailed
RESULTS Wilcoxon rank-sum test to compute a significance value
foreachgene,whichresultedinatotalof90genessurpassing
DNAmethylationisalteredinpatientswithDCM statistical significance (p-value <0.05). While about one-
We performed two-staged, funnel-like DNA methylation map- third showed increased methylation in DCM patients,
ping in non-ischaemic, idiopathic DCM patients and controls approximately two-thirds were significantly less methylated.
(Table 1). In the screening stage, we assessed genome-wide Fig 1D gives a graphical representation of differential
DNA methylation levels of CpG islands (CGIs) using the methylation patterns by using hierarchical clustering on
Infinium HumanMethylation 27 platform. We first extracted the Euclidian distance. The heat map shows the patterns
1000ng of genomic DNA from LV biopsies from 10 DCM of higher- and lower methylated genes in DCM patients and
patientsand10controls.Aftermethylationprofiling,17datasets controls.
passed the stringent quality filter criteria, exemplarily shown Forthereplicationstageinanindependentcohort,methyla-
by reaching highly similar bead color signal intensities tionpatternsofsinglegeneswereusedtodefinecandidatesfor
(Fig 1A). Fig 1B shows a correlation plot of the 27,578 amassspectrometry-basedfine-mapping.Fig2AandBshows
individual methylation sites for all further analysed patients the five most hyper- and hypomethylated genes, while Fig 2C
and controls. While the degree of methylation for most CpG details the 20 most significantly dys-methylated genes of the
sites is highly correlated between the two groups, we screening stage. The final selection was based on unadjusted
detected several CGIs that are hypo- (green dots) or hyper- p values or absolute methylation difference, CGI localization,
methylated (red dots) in DCM compared to the controls capability to design specific assay probes, and known expres-
(unadjustedp-value<0.05). sionintheheart.
"
Figure1. DetectionofDNAmethylationpatterns.
A. Bargraphsshowingquantilenormalizedbeadcoloursignals(greenandred)frommethylationmeasurementsfromallpatientsandcontrolsofthescreening
stageincludedinthefurtheranalysis.SignalqualitywashighlysimilaroverallDCMandcontrolsamples.Errorbarsindicatestandarddeviation.
B. CorrelationplotshowingthepercentageofCpGmethylationincontrolsversusDCMpatients,resultinginanoverallveryhighcorrelation.Thecolouredsignals
thatarefurthestawayfromthebisectinglineshowsignificantlyhyper-(red)andhypo-methylated(green)CpGsinDCMpatients.
C. Gene-setenrichmentanalysisforNIAhumandiseasepathways.Thearea-proportionalVenndiagramshowsthatmethylationchangesincardiovascular
diseasegenesaresignificantlyenrichedtogetherwiththeoverlapoftheotherindicatedgenesets.
D. Clusteranalysisforgeneswithknownexpressioninthehumanheartandsignificantlyalteredmethylation.Thecolourcodeusedfortheheatmapisshownin
theupperleftcorner,valuesrangefrom1(samplewiththelowestmethylationfortheconsideredgenes)to17(samplewiththehighestmethylationforthe
consideredgenes).
414 (cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO. EMBOMolMed(2013)5,413–429
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JanHaasetal.
A
2) controls (n=8) DCM (n=9) controls (n=8) DCM (n=9)
g
o14
y (l12
sit
n10
e
nt 8
e i
c 6
n
ce 4
s
ore 2
flu 0
C
B Enriched human disease pathways
1.0
8) 60
s (n= 0.8 128 106
control 0.6 57 114 57
n
on i 0.4
ati 200
yl
eth 0.2
m Category Observed (n)
0.0 Cardiovascular Diseases 359
0.0 0.2 0.4 0.6 0.8 1.0 Nutritional and Metabolic Diseases 337
methylation in DCM (n=9) Pathological Conditions. Signs and Symptoms 428
D
DCM controls
Color Key
1 17
DCM controls
M
C
D
n
n i
w
o
d
M
C
D
n
p i
u
Figure1.
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DNAmethylationinhumandilatedcardiomyopathy
A B
controls (n=8)
controls (n=8)
DCM (n=9)
DCM (n=9)
1.0 1.0
0.8 0.8
n n
o o
ati ati
hyl 0.6 hyl 0.6
et et
m m
GI 0.4 GI 0.4
C C
0.2 0.2
0.0 0.0
C
1.0
controls (n=8)
DCM (n=9)
0.8
on 0.6
ati
yl
h
et
m
GI 0.4
C
0.2
0.0
Figure2. DifferentiallymethylatedgenesinDCMpatients.
A,B. BargraphsshowingthedegreeofmethylationofCGIsofthescreeningcohort(n¼9DCMpatients;n¼8controls).Thefivegeneswiththelargestincreasein
methylationinDCMpatientsareshownin(A)andgeneswiththelargestdecreaseinmethylationaredisplayedin(B).
C. The20geneswiththemostsignificantmethylationchangesinthescreeningphase.Errorbarsindicatestandarddeviation.
ValidationofaberrantDNAmethylationinDCM is displayed for LY75 and ERBB3 (Fig. 3), HOXB13 (Fig. 4)
As denoted above, we carried out an independent replication and ADORA2A (Supporting Information Fig 1). Interestingly,
andfinemappingoftheselectedgenesinalargercohortof30 ADORA2Ashowedsignificantlyalteredmethylationthroughout
idiopathicDCMpatientsand28controls.Allselectedcandidates all tested CpGs, while ERBB3 or LY75 showed methylation
were fine-mapped by using MassARRAY (Ehrich et al, 2005). alterations in a subset of the CpG nucleotides, possibly
For each gene, several CpGs were retrieved and their resulting in different functional consequences. Supporting
methylation status quantified. From 20 candidate genes, 12 InformationFig2givesthemeanmethylationoftheremaining
showedthesamedirectionofalteredmethylationbetweenthe investigated CGIs. Since epigenetic marks may be also
screening and the replication stage and four of them reached dependentonthegender,weadditionallymatchedthegender
statistical significance, namely LY75 (p¼0.000), ERBB3 ratioofcasesandcontrolsofthereplicationcohorttotheratioof
(p¼0.013), HOXB13 (p¼0.001), and ADORA2A (p¼0.011). thescreening cohort, leading tosignificance ofthesameCGIs
Figs 3 and 4 present the mean methylation changes of the shown above. This is also true when matching females and
replicatedgenes.Additionally,methylationofindividualCpGs males1:1.
416 (cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO. EMBOMolMed(2013)5,413–429
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JanHaasetal.
LY75
A
1000bp CGI 5000bp
p=0.000
controls DCM
0.5
CpGs
n 0.4 clones
o
ati
yl 0.3
h
et
m
GI 0.2
C
0.1
0.0
controls DCM
(n=28) (n=30)
1.0
controls (n=28) p=0.011
DCM (n=30)
0.8
on p=0.011 p=0.000
ati 0.6
yl
h
met 0.4 p=0.014
p=0.014
0.2 p=0.005
0.0
CpG1 CpG2 CpG3 CpG4 CpG5 CpG6 CpG7 CpG8 CpG9 CpG10
B ERBB3
1000bp CGI 4000bp
p=0.013
0.5 controls (n=28)
0.3 p=0.017 DCM (n=30)
0.4
p=0.018
methylation 0.2 methylation 00..23 p=0.003
GI 0.1
C
0.1 p=0.022
0.0 0.0
controls DCM
(n=28) (n=30)
Figure3. MassARRAY-basedvalidationofdifferentialmethylationinLY75andERBB3.DCMpatientsshowsignificantlyincreasedDNAmethylationinLY75(A),
whileERBB3issignificantlylowermethylated(B).TheschemasabovethemethylationgraphsrepresentthetestedCGI(redlines),inrelationtothepredicted
transcriptionstart-site(greenlines),theexons(blackbars)andalternativelysplicedexons(whiteboxes)ofthegenes.
A. Theboxandwhiskersplot(mintomax)ontheupperleftsiderepresentsthemeanCpGmethylationmeasuredbyMassARRAY,theindividualCpGmethylation
isshownatthebottom.ThepatternofCpGmethylationinmultipleclonesisshownontheupperrightinsert.
B. Theboxandwhiskersplot(mintomax)ontheleftsiderepresentsthemeanCpGmethylationmeasuredbyMassARRAY,theindividualCpGmethylationis
shownontheright.
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DNAmethylationinhumandilatedcardiomyopathy
HOXB13
A
0.15 0.3
controls (n=28)
p=0.001
DCM (n=30)
ation 0.10 ation 0.2 p=0.002
yl yl
h h
et et
m m
GI 0.05 G 0.1
C Cp
0.00 0.0
controls DCM CpG1 CpG2 CpG3 CpG4 CpG5 CpG6 CpG7 CpG8
(n=28) (n=30)
B ADORA2A
CGI-2 CGI-3 CGI-1
CTCF Max CTCF NFKB
Rad21 USF-1 TCF12 Oct-2
SMC3 TCF12 EBF PU.1
p=0.034 p=0.6233 p=0.011
0.3 1.0 1.0
methylation 0.2 methylation 00..68 methylation 00..68
GI-2 0.1 GI-3 0.4 GI-1 0.4
C C C
0.2 0.2
0.0 0.0 0.0
controls DCM controls DCM controls DCM
(n=25) (n=28) (n=23) (n=27) (n=28) (n=30)
controls DCM
CpGs
clones
Figure4. MassARRAY-basedvalidationofdifferentialmethylationinHOXB13andADORA2A.DCMpatientsshowsignificantlydecreasedDNAmethylationin
HOXB13(A)andADORA2A(B).
A. Theboxandwhiskersplot(mintomax)ontheleftsiderepresentsthemeanCpGmethylationmeasuredbyMassARRAY,theindividualCpGmethylationis
shownontheright.
B. TheschemeabovethegraphrepresentsthetestedCGI(redline),inrelationtothepredictedtranscriptionstartsite(greenline),theexons(blackboxes)and
alternativelysplicedexons(whiteboxes)ofadora2a.Dashedlinesindicatethe1500bpup-anddownstreamregionoftheCGIswhereinthelistedtranscription
factorbindingsitesarefound.BoxplotsbelowshowthemeanmethylationinDCMandcontrolsatthecorrespondingCGI.ThepatternofCpGmethylationas
assessedbybisulphite-sequencinginmultipleclonesisshownforCGI-1atthebottomofthefigure.
418 (cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO. EMBOMolMed(2013)5,413–429
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JanHaasetal.
InadditiontoMassARRAY,weappliedbisulphitesequencing methylationattheLY75,ERBB3,HOXB13,andADORA2Aloci
for LY75 and ADORA2A to fine-map and technically replicate on their gene expression by quantitative PCR (q-PCR) in
ourresults.Todoso,wegenerated14LY75and12ADORA2A controls, mild DCM (NYHA class II) and moderate to severe
clones from two DCM patients and two controls each, and DCM(NYHAclassIII–IV).
sequenced them before and after treatment with bisulphite For ADORA2A, we found a positive relationship of gene
(Fig 3A and B; black circles¼methylated CpG, white expression with methylation (relative expression 0.33;
circles¼unmethylatedCpG).Bisulphitesequencingconfirmed p¼0.002), while ERBB3 did not show significant alterations
thecorrespondingMassARRAYdataand,hence,demonstrated in cardiac expression levels (Fig 6B–D). HOXB13 transcripts
reliabilityofthelattertechnique. could not be PCR amplified in LV biopsies from patients and
Next,toruleoutapotentialeffectoftheimmunosuppressive controls. In case of LY75, the hypermethylated CpG island is
medication received by the control subjects (Table 2) on the relatively close to the transcriptional start site (distance
methylationoftheinvestigatedgenes,weanalysedthemethyla- 1395bp), whereas the distance between the CGIs in ERBB3
tionpatternsofgenomicDNAfromperipheralbloodof11subjects (7518bp) and ADORA2A (14,329bp) is markedly larger
drawnpre-andpost-hearttransplantation(HTX;meantimespan (Figs 3 and 4). Since we also observed a strong reduction in
after HTX¼37 months under medication). As shown in Fig 5 LY75expressioninmyocardialtissue(relativeexpression0.04;
we found highly comparable methylation levels (p¼n.s.) p¼0.001) in patients with DCM (Fig 6A and D), we asked if
of LY75, ADORA2A, ERBB3 and HOXB13 CGIs in pre- and increased promoter methylation is directly responsible for
post-HTX,indicatingthattheobservedmethylationdifferences decreased transcriptional activity. Hence, we performed a
in DCM are not due to methylation changes in the controls luciferase promoter assay with methylated and unmethylated
receivingimmunosuppressivemedication. LY75promoters.TheCpG-freecontrolreportervectorpCpGL-
CMV/EF1servedasanegativecontrol.AsshowninFig6E,we
Differentialgeneexpressionandfunctionalevaluationinvivo found a strong reduction in promoter activity after in vitro
suggestcontributionofLY75andADORA2AtoDCM methylation, supporting the functional relationship between
DNA methylation is often correlated with changes in the LY75hypermethylationandreducedexpressioninhumanDCM.
accessibility of DNA to transcriptional activators, enhancers, Since we found a rather unusual positive relationship of
or repressors. Hence, we first studied the impact of DNA ADORA2Ageneexpressionandmethylation,wefirstsoughtto
identify the predominant isoform of ADORA2A in the human
myocardium. By PCR, we found that the longer ADORA2A
Table2.Immunosuppressivemedicationofcontrolindividuals transcriptsENST00000337539andENST00000541988arehighly
expressed in the heart. However isoform ENST00000417596,
Medication N %
which is highly expressed in peripheral blood, is not
Tacrolimus 19 54
significantly expressed in the heart. We then investigated
Mycophenolat-mofetil 23 66
Ciclosporin 8 23 potential repressor regions in the close vicinity of the tested
Prednisolon 11 31 CGIs.Forthedetectionoftranscriptionfactorbindingsites,the
Everolimus 14 40 ENCODEtranscriptionfactorChIP-seqdatawasused.Wefound
Allcontrolindividualsreceivedoneormoreimmunosuppressives. for the aberrantly methylated CGI-1 two potential repressor
sites (CTCF and NFKB), for aberrantly methylated CGI-2 one
repressorsite(CTCF)andfortheunalteredCGI-3onerepressor
site(Max).Interestingly,bothhypomethylatedCGIs(1and2)
carry CTCF binding sites, which were recently identified as
pre-HTX (n=11)
epigenetickeyregulatorsinvariousotherdiseases.
post-HTX (n=11)
p=0.43 SinceLY75andADORA2Awerenotpreviouslyknowntobe
LY75 involvedinDCMorheartfailurepathogenesisandbothshowed
significantdownregulationinthemyocardiumofDCMpatients,
HOXB13 ERBB3 we investigated their functional roles by gene knockdown in
CGI methylation
p=0.97 p=0.20 zebrafish embryos (Dahme et al, 2009). We identified
10 20 30 orthologous sequences for human LY75 and ADORA2A by
0. 0. 0.
BLAST searches against the zebrafish Genbank database.
Protein sequence identity between the zebrafish and human
ADORA2A
version was 61% for adora2a (Fig 9A) and 37% for ly75
p=0.42
(SupportingInformationFig3).Similartothehumansituation,
we found adora2a and ly75 to be highly expressed in the
Figure5. CpGislandmethylationofcontrolsubjectsbeforeandafter
zebrafish heart using q-PCR and RNA antisense in situ
hearttransplantation.
hybridization (Figs 7–9). Additionally, as shown in cultured
ThelinediagramshowsthehighlysimilardegreeofmethylationofDNA
neonatal rat cells, both genes are higher expressed in
derivedfromperipheralbloodofcontrolsubjectspre-(lightblue)andpost-
(darkblue)hearttransplantation(n¼11)forLY75,ADORA2A,ERBB3and cardiomyocytesthanincardiofibroblasts(Fig7BandC).Hence,
HOXB13.Errorbarsindicatestandarddeviation. torecapitulatethedownregulationofbothgenesasobservedin
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DNAmethylationinhumandilatedcardiomyopathy
A B C
LY75 ERBB3 ADORA2A
1.5 1.5 1.5
relative expression 01..50 *** relative expression 01..50 relative expression 01..50 **
0 0 0
D
LY75 [ CT] ERBB3 [ CT] ADORA2A [ CT]
Controls (n=7) 8.51 ± 0.66 9.88 ± 0.80 8.65 ± 0.26
Moderate DCM (n=7) 9.11 ± 0.40 10.02 ± 0.63 8.90 ± 0.53
Severe DCM (n=5) 13.11 ± 0.78 9.90 ± 0.53 10.08 ± 0.20
1.0
E
firefly luciferase gene
CGI vity 0.8
cti
a
er 0.6
ot
m
LYp7ro5m coorteer- pCpGL-LY75 e pro 0.4
(5513bp) v
region ati
el
r 0.2
0.0
negative control pCpGL-LY75
Figure6. mRNAexpressionofgeneswithalteredmethylationstatusandgenepromoteranalysis.
A–C. BargraphsshowingrelativemRNAexpressionlevelsofLY75,ERBB3andADORA2Ainmildandmoderate-severeDCMincomparisontocontrols.(A)LY75
expressionisstronglyreduced(relativeexpression¼0.04),while(B)ERBB3mRNAlevelsarenotdifferentiallyregulated.(C)ADORA2Aisalsosignificantly
down-regulatedinDCM(relativeexpression¼0.37).
D. Givenarethemeandelta-CTvalues((cid:1)SEM)ofLY75,ERBB3,andADORA2Ainthedifferentgroups(controls,moderateDCM,severeDCM).Thereferenceis
basedonthemeanofthethreehousekeepergenes:GAPDH,RPL13,b-actin.
E. Left:SchemaofthevectorconstructtomeasureLY75promoteractivitywithoutandaftertreatmentwiththemethylaseSssI.Right:Relativepromoter
activity(meanofthreetechnicalreplicates(cid:1)SD)showingthestronglyreducedactivityinLY75aftermethylation,whereasthenegativecontroldoesnot
showsignificantdifferences.
thehumanheart,weinactivatedtheminzebrafishembryosby skipping of exon 8 (Fig 8D) and a consecutive frameshift
injection of Morpholino-modified antisense oligonucleotides thatpredictablyleadstoaprematurestopofproteintranslation.
directedagainstthesplicedonorsiteofly75orthetranslational As a result, ly75-morphants developed late-onset heart
start-siteofadora2a.Whilecontrol-injectedzebrafish(standard failure with dilation of the atrium (Fig 8C) and reduced
controlMorpholinoaswellasscrambledcontrolMorpholinos) ventricular contractility beginning at the 96h developmental
embryos did not show any obvious phenotype, splice site stage (FS¼46(cid:1)5% at 48hpf and 29(cid:1)4% at 96hpf; Fig 8E,
Morpholino-mediated knockdown of ly75 resulted in partial SupportingInformationMovie1).Additionally,ly75-morphants
420 (cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO. EMBOMolMed(2013)5,413–429
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JanHaasetal.
A whole fish heart showed a pronounced detachment and oedema of the skin
especiallynoticeableintheeyeandheadregion(Fig8C).MO-
4
adora2a-injected embryos also developed severe heart failure
n with progressively decreasing ventricular contractility as
o
si 3 measuredbyfractionalshortening(Fig9E,SupportingInforma-
s
pre tionMovie2).Indetail,theventricularcontractilityofadora2a-
ex morphants decreased from 47(cid:1)11% at 48hpf to 39(cid:1)8% at
e 2
v 72hpf. By 96hpf, both heart chambers became almost silent.
elati Occasionally, we also observed atrial fibrillation in adora2a-
r 1 morphants and embryos developed excessive pericardial
effusion and precardial blood congestion as a consequence of
0 the reduced cardiac function. For both, ly75- and adora2a-
ly75 adora2a
morphants, we saw no alterations in molecular chamber
cardiac fibroblasts cardiomyocytes specification and expression of atrial and ventricular myosin
heavy chain genes (Figs 8 and 9 and Supporting Information
B
Fig4).
3
DISCUSSION
n
o
si
s
pre 2 Epigenetic mechanisms are increasingly recognized as causes
x andmodulators ofhuman disease.Most studiesconducted so
e
e farhavefocusedoncancerandyetonlyfewhaveinvestigated
v
ati theroleofepigeneticmechanisms,suchasDNAmethylation,in
rel 1 cardiovascular disease (Movassagh et al, 2011). This is
surprisingsinceepigeneticmechanismsarethoughttocontrol
key adaptive and maladaptive processes such as cardiac
0 hypertrophy, fibrosis and failure (Backs et al, 2006, 2008).
LY75 Here,weinvestigatedDNAmethylationpatternsonagenome-
wide level in myocardium from patients with idiopathic DCM
cardiac fibroblasts cardiomyocytes
andfunctionallyunaffectedheartsofpatientswhohadreceived
C hearttransplantation. Wefoundandconfirmed aberrantDNA
50
methylation alterations in a number of CGIs, suggesting that
DNAmethylationisassociatedwithcardiacfunctionandmay
40 modulatephenotypiccharacteristicsofidiopathicDCM.
n Thereareseveralepigeneticmechanismsineukaryotesand
o
essi 30 somehavealreadybeenlinkedtoDCMorheartfailure.Firstly,
pr chromatin modifications through ATP-dependent enzymes
x
e from, e.g. the SWI/SNF genes enable the cell to regulate the
e
ativ 20 expressionofdistinctgeneprogramsinorgandevelopmentand
el adaptation(Ho&Crabtree,2010).Secondly,histonemodifiers
r
such as histone acetyltransferases (HATs) or histone deacety-
10
lases(HDACs)areknowntoplayakeyroleduringdevelopment
and in maintenance of cardiac function (Backs et al, 2011;
0 Montgomeryetal,2008;Zhouetal,2011).Cardiacspecificloss
ADORA2A
of HDAC1 and HDAC2 for example results in cardiac
arrhythmiasandheartfailure(Montgomeryetal,2007),while
Figure7. ly75andadora2atissueandcell-typeexpression. loss of HDAC3 function leads to cardiac hypertrophy (Mon-
A. Relativeexpressionlevelsofly75andadora2aquantifiedbyq-PCRin40 tgomery et al, 2008). Thirdly, short non-coding microRNAs
wholezebrafishembryos(greybars)and200isolatedzebrafish (miRNAs)broadlyinfluencegeneexpression(Humphreysetal,
embryonichearts(blackbars). 2005) and are increasingly recognized as valuable diagnostic
B,C. Expressionofly75(B)andadora2a(C)inneonatalratcardiomyocytes
(Keller et al, 2011; Meder et al, 2011a,b) and potential
andcardiofibroblasts(n¼3biologicalreplicates).Errorbarsindicate
therapeutic targets (Gambari et al, 2011). DCM caused by
standarddeviation.
deregulationofmiRNAssuchasmiRNA-133ahasbeenrecently
describedinanimalmodels(Chenetal,2008;Mederetal,2008;
van Rooij et al, 2007). The current study now adds DNA
EMBOMolMed(2013)5,413–429 (cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO. 421
ResearchArticle www.embomolmed.org
DNAmethylationinhumandilatedcardiomyopathy
A
ly75
llyy7755
VV
AAA
WT 72 hpf WWTT 72 hpf
B
MO3-control 72 hpf
C
MO-ly75 72 hpf
D E MO-ly75
MO1-control
60
%] 50
bp S [ 40
F
365 ar 30
ul
345 ntric 20
e 10
v
0
48 hpf 72 hpf 96 hpf
F
amhc vmhc MF20/S46
ol
r V
nt VVV V
o AA
c3- A A
O
M
7777777777777722222222222222222 hhhhhhhhhhhhhppppppppppppppppppfffffffffffffffffff 72 hpf MO3-control 72 hpf
amhc vmhc MF20/S46
VV
5 V
7
y V
lO- AAA A
M A
72 hpf 7777777222 hpf 72 hpf
Figure8.
422 (cid:1)2013TheAuthors.PublishedbyJohnWileyandSons,LtdonbehalfofEMBO. EMBOMolMed(2013)5,413–429