rc_AI232374_g_at103838.com

1029HypertensResVol.
29(2006)No.
12p.
1029-1045OriginalArticleComparisonofGeneExpressionProlinginPressureandVolumeOverload–InducedMyocardialHypertrophiesinRatsHiroshiMIYAZAKI1),NaokiOKA1),AkimasaKOGA1),HaruyaOHMURA1),TamenobuUEDA1),andTsutomuIMAIZUMI1),2)Geneexpressionprofilinghasbeenconductedinratheartssubjectedtopressureoverload(PO).
However,pressureandvolumeoverloadproducemorphologicallyandfunctionallydistinctformsofcardiachypertro-phy.
Surprisingly,geneexpressionprofilinghasnotbeenreportedforinananimalmodelofvolumeover-load(VO).
WethereforecomparedthegeneexpressionprofilesinthehypertrophiedmyocardiumofratssubjectedtoPOandVOusingDNAchiptechnology(AffymetrixU34A).
ConstrictionoftheabdominalaortaandabdominalaortocavalshuntingwereusedtoinducePOandVO,respectively.
Thegeneexpressionpro-filesoftheleftventricle(LV)4weeksaftertheprocedurewereanalyzedbyDNAchips.
Therewerecompa-rableincreasesintheleftventricularweight/bodyweightratioinratssubjectedtoPOandVO.
EchocardiographyrevealedconcentrichypertrophyinthePOanimals,buteccentrichypertrophyintheratssubjectedtoVO.
TheexpressionsofmanygeneswerealteredinVO,PO,orboth.
Amongthegenesthatwereupregulatedinbothformsofhypertrophy,greatlyincreasedexpressionsofB-typenatriureticpeptide,lysyloxidase–likeprotein1andmetallothionein-1(MT)wereconfirmedbyreal-timereversetranscription–polymerasechainreaction(RT-PCR).
Becausefreeradicalsareincreasedinthehypertrophiedheartandmaycontributetoapoptosis,weexaminedtheroleofMT,afreeradicalscavenger,inapoptosis.
Theover-expressionofMTinH9c2cellsinhibitednorepinephrine-inducedapoptosis,suggestingthatMTmayactasananti-apoptoticmoleculeincardiachypertrophy.
Inconclusion,wefoundthatmanygeneswereregulatedinVO,PO,orboth.
Inaddition,anovelroleofMTinthehypertrophiedmyocardiumwassuggested.
(Hyper-tensRes2006;29:1029–1045)KeyWords:geneexpression,hypertrophy,DNAchip,metallothionein-1,apoptosisIntroductionPressureandvolumeoverload(POandVO,respectively)producemorphologicallyandfunctionallydistinctformsofcardiachypertrophy(1–4);POproducesconcentrichypertro-phywhereasVOproduceseccentrichypertrophy.
Atthecel-lularlevel,cardiomyocytesgrowverticallyinPOandlongitudinallyinVO.
Themolecularmechanismsofthesedifferenceshavebeenunknown.
ChangesofsomespecificgeneshavebeenreportedintheheartsofPOandVO,i.
e.
,theexpressionsofcollagenisoforms(5,6)andmatrixmetallo-Fromthe1)DepartmentofMedicine,DivisionofCardio-VascularMedicineand2)CardiovascularResearchInstitute,KurumeUniversitySchoolofMed-icine,Kurume,Japan.
ThisstudywassupportedinpartbyagrantfromtheKimuraMemorialHeartFoundation/PfizerGrantforResearchonAutonomicNervousSystemandHypertension(toN.
O.
),andagrantforScienceFrontierResearchPromotionCentersandaGrant-in-AidfortheEncouragementofYoungScientists(toH.
M.
andN.
O.
)fromtheMinistryofEducation,Culture,Sports,ScienceandTechnology,Japan.
AddressforReprints:NaokiOka,M.
D.
,Ph.
D.
,DepartmentofMedicine,DivisionofCardio-VascularMedicine,KurumeUniversitySchoolofMedi-cine,67Asahi-machi,Kurume830–0011,Japan.
E-mail:noka@med.
kurume-u.
ac.
jpReceivedFebruary27,2006;AcceptedinrevisedformSeptember4,2006.
1030HypertensResVol.
29,No.
12(2006)proteinases(7),theexpressionofβmyosinheavychain(8),thetranscriptionalregulationofadrenomedullin(9),andtheexpressionsofgrowthfactors(10,11)andβ-tubulin(12).
Basedonthesepreviousreports,itisanticipatedthatsomespecificgenesareregulatedsimilarlyordifferentiallyinPOandVO,buttheregulationofgenesotherthanthoselistedabovehasnotbeenclarified.
Microarrayanalysisisausefulmethodtoanalyzethebehaviorofmanygenes.
Withthismethod,geneprofilinghasbeenconductedinanimalheartssubjectedtoPO(13,14).
Surprisingly,however,geneexpres-sionprofilinghasnotbeenconductedinheartssubjectedtoVO.
Accordingly,wecomparedthegeneexpressionprofilesinthehypertrophiedmyocardiumofratssubjectedtoPOandVOusingtheDNAchiptechnology.
MethodsAnimalModelsMaleWistarratswereusedfortheexperiments.
POwaspro-ducedinratsbyabdominalaorticbanding(15),andVObyaortocavalshunting(16).
Theratsweresacrificed28daysaftertheprocedure.
AllanimalprocedureswereconductedaccordingtotheguidelinesprovidedbytheKurumeUniver-sityInstitutionalAnimalCareandUseCommitteeunderanapprovedprotocol.
EchocardiographyAtthedaybeforesacrifice,ratswerelightlyanesthetizedwithpentobarbital,thenexaminedbyechocardiographywithanSSD5500anda7.
5MHzprobe(Aloka,Tokyo,Japan).
DNAChipAnalysisDNAchips(GeneChipRatGenomeU34Aarrays)werepur-chasedfromAffymetrix(SantaClara,USA).
Poly(A)RNAwasextractedfromtheleftventricle(LV)byusingaFASTtrackRNApurificationkit(Invitrogen,Carlsbad,USA).
Wemixedpoly(A)RNAspreparedfromtheLVoffiveratsandthesampleswereanalyzedbythreeDNAchipsineachgroup.
DNAchipanalysiswasperformedaccordingtothemanufac-turer'srecommendedprotocol.
Eachofthegroupswascom-paredwitheachoftheothergroups,resultingin27comparisontests.
AverageintensityvaluesforeachprobesetwereobtainedfromtheAffymetrixMicroArraySuite4.
0.
Apvaluelessthan0.
01wasarbitrarilyassignedasthelevelofstatisticalsignificance.
Weconsideredgenesthatwereele-vatedby>2foldcomparedwithcontrolsas"upregulated,"genesthatwerereducedto<0.
5as"downregulated,"andgenesthatwerealteredby0.
8–1.
2foldasshowing"nochange.
"Thegenesthatwereexpressedatverylowlevels(thosewithasignalintensitylessthan100)orgenesthatwereconsiderednottobepresentbasedonlowsignalintensitybytheAffymetrixsoftwareunderallconditionswereomitted.
Forclusteringanalysis,theCELfileswereconvertedintoDCPfilesusingdCHIPanalysissoftware(www.
biostat.
harvard.
edu/compalab/cdhip/),asdescribedpreviouslybyLiandWong(17).
Genechipswerenormalized,andmodel-basedexpressionvaluesweregenerated.
Weusedhierarchicalclus-Fig.
1.
Echocardiographicmeasurementsofthehypertrophiedratheart.
Echocardiographywasperformed28daysafterhemo-dynamicoverloadasdescribedinMethods.
VO,volumeoverloadgroup;PO,pressureoverloadgroup;LVDd,end-diastolicdiameteroftheleftventricle;IVST,interventricularseptalwallthickness;%FS,percentfractionalshortening;LAD,leftatrialdiameter.
Valuesarethemean±SD.
**p<0.
01vs.
theshamgroup.
LVDd(mm)0102468OPOVmahS)mm(TSVIOPOVmahS5.
05.
15.
2012********)mm(DALOPmahSOV0123456****OPOVmahS%FractionalShortening0010203040506BADCMiyazakietal:GeneExpressionProfilinginCardiacHypertrophy1031teringwiththeaveragelinkagemethod.
Thefunctionsofthesealteredgeneswereclassifiedbyclustersoforthologousgroupsanalysis.
Real-TimeReverseTranscription–PolymeraseChainReactionTotalRNAwaspreparedwithanRNeasyMidiKit(QIAGEN,Tokyo,Japan)fromLV.
Real-timereversetran-scription–polymerasechainreaction(RT-PCR)wasper-formedbyaGeneAmp5700andaTaqManOne-StepRT-PCRMasterMixReagentsKit(AppliedBiosystems,FosterCity,USA)accordingtothemanufacturer'sinstruction.
Expressionvaluesforeachgenewerenormalizedto18Sribo-somalRNA(TaqManribosomalRNAcontrolreagents;AppliedBiosystems).
TheprimersandTaqManprobesusedintheseexperimentswereasfollows:guanosinemonophos-phate(GMP)reductaseTaqManprobe,CTCTGACGGAAGCTGCACATGTCCA;GMPreductasesense-primer,GGCCTCAAGGGACACATCA;GMPreductaseantisense-primer,AAAGGCTTTGGCGACATCTC;metallothionein-1(MT)TaqManprobe,TGCAAAGGTGCCTCGGACAAGTGC;MTsense-primer,GTGGGCTGCTCCAAATGTG;MTantisense-primer,GGTCCGGAAATTATTTACACCTGA;B-typenatriureticpeptide(BNP)TaqManProbe,CGGCGCAGTCAGTCGCTTGG;BNPsense-primer,TGGGCAGAAGATAGACCGGA;BNPantisense-primer,ACAACCTCAGCCCGTCACAG;lysyloxidase–likeprotein1Fig.
2.
Leftventricularweightandheartweight28daysafterhemodynamicoverload.
A:LVW/BW.
B:HW/BW.
VO,volumeoverloadgroup;PO,pressureoverloadgroup;LVW/BW,leftventricularweightnormalizedtobodyweight;HW/BW,heartweightnormalizedtobodyweight.
Valuesarethemean±SD,*p<0.
05,**p<0.
01vs.
theshamgroup.
Fig.
3.
Comparisonsofdifferentiallyregulatedgenes.
Venndiagramswereproducedtocomparethenumberofgenesalteredinthevolumeoverload(VO),pressureoverload(PO),andshamgroups.
A:GenesdifferentiallyupregulatedbetweentheshamgroupandtheVOgroup,POgroup,orboth.
B:GenesdifferentiallydownregulatedbetweentheshamgroupandtheVOgroup,POgroup,orboth.
C:GenesunalteredbetweentheshamgroupandtheVOgroup,POgroup,orboth.
D:GenesupregulatedinthePOgroup,downregulatedintheVOgroup,orboth.
E:GenesupregulatedintheVOgroup,downregulatedinthePOgroup,orboth.
0123401234567OPOVmahSOPOVmahSHW/BW(mg/g)LVW/BW(mg/g)******BAABCDEdetalugernwodseneGdetalugerpuseneGderetlanuseneGOPOVOPOVOPOVOPOV8864251433111327508011164038OPOV77811yllaitnereffiddevahebseneGyllaitnereffiddevahebseneG1032HypertensResVol.
29,No.
12(2006)Table1.
GenesThatRespondtoPressureorVolumeOverloadAccessionProbesetIDGenenameVOPOMolecularfunctionReferenceVO↑andPO↑AA799773rc_AA799773_atFilaminC,γ2.
422.
03actinbinding19AA892378rc_AA892378_atSimilartotetratricopeptiderepeatdomain11(LOC288584)2.
272.
05apoptosisS69383S69383_at12-Lipoxygenase2.
612.
02arachidonate12-lipoxygenaseactivity,ironionbinding20L18948L18948_atS100calcium-bindingproteinA9(calgranulinB)3.
253.
22calciumionbinding19U17919U17919_s_atAllograftinflammatoryfactor-12.
052.
00calciumionbindingAI102562rc_AI102562_atMetallothionein-1(MT-1)4.
053.
82copperionbinding,metalionbinding,zincionbinding14,19,21L16532L16532_at2′,3′-cyclicnucleotide3′-phosphodiesterase(CnpII)3.
282.
94cyclicnucleotidecatabolismU17254U17254_g_atImmediateearlygenetranscriptionfactorNGFI-B2.
142.
06DNAbinding,ligand-dependentnuclearreceptoractivityU75397U75397UTR#1_s_atEarlygrowthresponse1(Egr1)2.
372.
03DNAbinding,nucleicacidbinding,transcriptionfactoractivity21L22761L22761_atGATAbindingprotein4(Gata4)2.
423.
06DNAbinding,transcriptionfactoractivity13AA859805rc_AA859805_atLysyloxidaselikeprotein(LOXL)3.
952.
48elastogenesis19AA800517rc_AA800517_s_atVesicleassociatedprotein(VAP1)2.
072.
02endosomeproteinAI231472rc_AI231472_s_atCollagenα1typeI3.
673.
90extracellularmatrixprotein13,14M17526M17526_atGTP(guaninenucleotideprotein)-bindingprotein(Gαo)2.
092.
49GDPbinding19AA800784rc_AA800784_atCysteine-richprotein61(CYR61)2.
182.
18growthfactorbinding,heparinbinding14AA799498rc_AA799498_atNatriureticpeptideprecursortypeB4.
452.
62hormoneactivity13,19,22E00775E00775cds_s_atNatriureticpeptideprecursortypeA4.
122.
56hormoneactivity13,14,19,20,22M32062M32062_atFc-γreceptor(IgG)2.
882.
31IgGbinding,IgGreceptoractivity,receptoractivityAI231213rc_AI231213_atMetastasissuppressorhomolog(KAI1)2.
032.
01integraltomembraneX04979X04979_atApolipoproteinE2.
162.
00lipidtransporteractivity,lipidbinding,heparinbindingJ00692J00692_atSkeletalmuscleα-actingene2.
472.
02motoractivity,structuralconstituentofcytoskeleton14AA799678rc_AA799678_s_atEGLninehomolog32.
332.
00oxidoreductaseactivity19X71127X71127_atComplementproteinC1qβ-chain2.
232.
00phosphatetransport,immuneresponse,complementactivationAI069982rc_AI069982_s_atMyotonicdystrophykinase–relatedCdc42-bindingkinase(MRCK)2.
092.
02proteinserine/threoninekinaseactivityAA875405rc_AA875405_atForkhead-like182.
212.
07transcription,regulationoftranscription,developmentAI639185rc_AI639185_s_atPotassiumchannel,subfamilyK,member33.
203.
26transport,iontransport,potassiumiontransportM91235M91235_f_atVL30element2.
602.
15—AA892847rc_AA892847_atSimilartoα-N-acetyl-galactosaminidase(LOC315165)2.
952.
71—AA891828rc_AA891828_g_atSimilartoUVexcisionrepairproteinRAD23homologA2.
032.
07—AA799992rc_AA799992_atSimilartoC11orf17protein(LOC361624)2.
572.
18—AA800678rc_AA800678_atSimilartoAW046014protein(LOC363328)2.
032.
00—AA875523rc_AA875523_s_atSimilarto17kDamyosinlightchain(LOC362816)2.
042.
00—AA800803rc_AA800803_g_atEST190300(LungderivedcDNA)2.
052.
11—VO↑andPO→AI105348rc_AI105348_i_atCofilin1(Cfl1)2.
201.
13actinbindingM58404M58404_atThymosinβ-10(testis-specific)gene2.
441.
04actinbinding,actinmonomerbindingMiyazakietal:GeneExpressionProfilinginCardiacHypertrophy1033Table1.
(Continued)AccessionProbesetIDGenenameVOPOMolecularfunctionReferenceAI180288rc_AI180288_s_atCaldesmon1(Cald1)3.
791.
14actinbinding,calmodulinbinding,myosinbindingAA875132rc_AA875132_atTropomyosin1,α2.
360.
95actinbinding,structuralconstituentofcytoskeletonAA893280rc_AA893280_atAdiposedifferentiation–relatedprotein2.
010.
82adipocytedifferentiationM23601M23601_atMonoamineoxidaseB(Maobf3)3.
711.
13amineoxidaseactivity,oxidoreductaseactivityAA859829rc_AA859829_g_atMacrophageerythroblastattacher2.
080.
96apoptosis,celladhesion,developmentS54008S54008_i_atFibroblastgrowthfactorreceptor1β-isoform2.
221.
12ATPbinding,fibroblastgrowthfactorreceptoractivityAA875002rc_AA875002_atLeucine-richrepeat-containing8(LOC311846)2.
520.
84BcelldevelopmentM83143M83143_g_atβ-Galactoside-α2,6-sialyltransferase2.
060.
81β-galactosideα-2,6-sialyltransferaseactivityU90121U90121_atThrombomodulin3.
221.
11bloodcoagulationAF027984AF027984_atLowvoltage-activated,T-typecalciumchannelαsubunit2.
131.
11calciumchannelactivity,calciumionbindingAA891204rc_AA891204_s_atSecretedacidiccysteinerichglycoprotein(Sparc)orosteonectin2.
461.
13calciumionbinding22X06916X06916_atProteinp9Kahomologoustocalcium-bindingprotein2.
140.
85calciumionbindingAA849769rc_AA849769_atFollistatin-relatedproteinprecursororFollistatin-like(Fstl)3.
221.
19calciumionbinding,heparinbinding21X51529X51529_atPlateletphospholipaseA3.
790.
85calciumionbinding,phospholipaseA2activityX89963X89963_atThrombospondin4(Thbs4)4.
410.
93calciumionbinding,structuralmoleculeactivityAI145680rc_AI145680_s_atMonocarboxylatetransporterorSolutecarrierfamily16,member12.
171.
14carrieractivity,monocarboxylateporteractivityAB000778AB000778_s_atPhospholipaseD2.
460.
85catalyticactivity,hydrolaseactivity,phospholipaseDactivityX82396X82396_atCathepsinB2.
121.
12cathepsinBactivity,cysteine-typeendopeptidaseactivityL03201L03201_atCathepsinS2.
380.
90cathepsinSactivity,cysteine-typeendopeptidaseactivityAF020618AF020618_g_atMyeloiddifferentiationprimaryresponsegene1162.
240.
86celldifferentiationAA874848rc_AA874848_s_atThy-1geneforcell-surfaceglycoprotein2.
421.
20cell-cellinteractionAA799803rc_AA799803_atComplementcomponent1,rsubcomponent2.
360.
96chymotrypsinactivity,trypsinactivityM14656M14656_atOsteopontin3.
431.
03cytokineactivity,growthfactoractivity,integrinbindingX59864X59864mRNA_g_atASM15gene2.
981.
15cytoplasmicproteinU16025U16025_atClassIbRT1orM3protein(M3gene)2.
821.
18defenseresponse,immuneresponseAI232374rc_AI232374_g_atH1histonefamily,member0(H1f0)2.
071.
19DNAbindingM31322M31322_atSpermmembraneprotein(YWK-II)2.
201.
19DNAbinding,endopeptidaseinhibitoractivityAA800613rc_AA800613_atGeneforTIS11orZincfingerprotein36(Zfp36)2.
830.
94DNAbinding,transcriptionalactivatoractivityM65149M65149_atCCAAT/enhancerbinding,protein(C/EBP)δ(Cebpd)2.
300.
83DNAbinding,proteinbindingX60769X60769mRNA_atInterleukin-6-dependentbindingprotein2.
571.
05DNAbinding,proteinbinding,transcriptionalactivatoractivityAA891041rc_AA891041_atJun-Boncogene3.
210.
93DNAbinding,transcriptionfactoractivityAB012231AB012231_s_atNF1-B22.
111.
11DNAbinding,transcriptionfactoractivityAA891717rc_AA891717_atUpstreamtranscriptionfactor12.
321.
19DNAbinding,transcriptionfactoractivityAA892520rc_AA892520_g_atVesicleaminetransportprotein1homolog2.
270.
99DNAbinding,zincionbinding,oxidoreductaseactivityAF041066AF041066_atRibonuclease42.
091.
17endonucleaseactivity,hydrolaseactivity,nucleaseactivityU42719U42719_atComplementcomponent4a2.
000.
82endopeptidaseinhibitoractivityJ04035J04035_atTropoelastin2.
541.
17extracellularmatrixconstituentconferringelasticity1034HypertensResVol.
29,No.
12(2006)Table1.
(Continued)AccessionProbesetIDGenenameVOPOMolecularfunctionReferenceU17834U17834_atBiglycan2.
661.
16extracellularmatrixproteinX84039X84039_atLumican2.
101.
03extracellularmatrixproteinX17053X17053mRNA_s_atImmediate-earlyserum-responsiveJEgene2.
150.
81G-protein–coupledreceptorbinding,chemokineactivityX05834X05834_atFibronectingene3′end2.
460.
82heparinbinding,mercuryionbinding,oxidoreductaseactivityM36317M36317_s_atThyrotropin-releasinghormone(TRH)precursor3.
340.
86hormoneactivity,thyrotropin-releasinghormoneactivityH31839rc_H31839_atBCL2-antagonist/killer1(Bak1)2.
170.
94inductionofapoptosisAI233219rc_AI233219_atEndothelialcell-specificmolecule14.
781.
19insulin-likegrowthfactorbindingAA894029rc_AA894029_atEndothelialtypegp91-phoxgene3.
031.
09ionchannelactivity,voltage-gatedionchannelactivityX16554X16554_atPhosphoribosylpyrophosphatesynthetasesubunitI2.
561.
19kinaseactivity,lipoate-proteinligaseBactivityU02553U02553cds_s_atProteintyrosinephosphatase2.
741.
19MAPkinasephosphataseactivityS74351S74351_s_atProteintyrosinephosphatase2.
301.
11MAPkinasephosphataseactivityS81478S81478_s_atOxidativestress–inducibleproteintyrosinephosphatase2.
200.
84MAPkinasephosphataseactivityAI169327rc_AI169327_atTissueinhibitorofmetalloproteinase-1(TIMP1)2.
321.
19metalloendopeptidaseinhibitoractivityX06801X06801cds_i_atVascularα-actin3.
091.
15motoractivity,structuralconstituentofcytoskeleton22L00088L00088expanded_cds#2Fastmyosinalkalilightchains2.
900.
81motoractivity,structuralconstituentofmuscleAA866276rc_AA866276_atMyeloid-associateddifferentiationmarker2.
430.
94myeloidbloodcelldifferentiationAA945737rc_AA945737_atChemokinereceptorLCR12.
350.
95neuronalcellrecognitionU90610U90610_atCXCchemokinereceptor(CXCR4)2.
181.
11neuronalcellrecognitionAA874999rc_AA874999_atSec61βsubunit2.
111.
14proteintargeting,proteintransportU42627U42627_atDual-specificityproteintyrosinephosphatase(rVH6)2.
581.
18proteintyrosine/serine/threoninephosphataseactivityAA874889rc_AA874889_g_atUnc-5homologB2.
290.
95receptoractivity,netrinreceptoractivity,proteinbindingX07636X07636_atHepaticlectin2.
351.
14receptoractivity,sugarbindingM91590M91590_atβ-Arrestin22.
651.
08regulationofG-proteincoupledreceptorproteinsignalingAI008423rc_AI008423_atUnc-50relatedprotein(UNCL)2.
011.
19RNAbindingAA860043rc_AA860043_atGuaninenucleotidebindingproteinγsubunit11(Gng11)2.
261.
03signaltransduceractivityAA894345rc_AA894345_atPhosphoproteinenrichedinastrocytes152.
170.
85sugarporteractivity,proteinbindingAA893235rc_AA893235_atSimilartoG0S2-likeprotein2.
600.
96—X52815X52815cds_f_atSimilartoγactin–likeprotein2.
011.
17—AA892391rc_AA892391_atSimilartohypotheticalproteinFLJ20531(LOC303164)3.
851.
16—AA893267rc_AA893267_atSimilartoproline-serine-threoninephosphatase–interactingprotein12.
060.
84—AA799571rc_AA799571_atSimilartoRIKENcDNA1110001M20(LOC298308)2.
011.
19—AA892578rc_AA892578_atSimilartoRIKENcDNA6330406I15(LOC360757)2.
831.
00—AA891940rc_AA891940_atSimilartotransformingproteinRhoC(H9)2.
360.
91—AA892333rc_AA892333_atSimilartotubulinα-62.
130.
81—20AA891690rc_AA891690_g_atSimilartotumornecrosisfactorligandsuperfamilymember132.
371.
07—AA894292rc_AA894292_atEST198095(SpleenderivedcDNA)2.
310.
87—AA894092rc_AA894092_atEST197895(SpleenderivedcDNA)4.
981.
14—AA891677rc_AA891677_atEST195480(kidneyderivedcDNA)2.
071.
14—Miyazakietal:GeneExpressionProfilinginCardiacHypertrophy1035Table1.
(Continued)AccessionProbesetIDGenenameVOPOMolecularfunctionReferenceVO↑andPO↓J02780J02780_atTropomyosin4(Tpm4)2.
700.
48actinbinding19M34043M34043_atThymosinβ-4(pTB4G)2.
320.
49actinbinding19M83107M83107_atTransgelin(Smoothmuscle22protein)(Tagln)2.
280.
44actinfilamentbinding,proteinbinding,bridgingL12380L12380_atADP-ribosylationfactor1(Arf1)2.
950.
40GTPbinding,GTPaseactivity,proteintransporteractivityX74402X74402_atGuanosinediphosphatedissociationinhibitor1(Gdi1)2.
450.
47GTPaseactivatoractivity,RABGDP-dissociationinhibitoractivityD30740D30740_at14-3-3proteinmRNAformitochondrialimportstimulationfactorS12.
360.
44monooxygenaseactivity,proteindomainspecificbindingAA799520rc_AA799520_atIntegralmembraneprotein2B2.
000.
43neurogenesis21X76489X76489cds_g_atCD9mRNAforcellsurfaceglycoprotein2.
370.
33proteinbindingAA875033rc_AA875033_atFibulin5(Fbln5)2.
160.
49regulationofcellgrowth19AA850734rc_AA850734_atVascularendothelialgrowthfactorA(Vegfa)2.
120.
49vascularendothelialgrowthfactorreceptorbindingAA875537rc_AA875537_atSimilartosplicingfactorarginine/serinerich2(SC-38)2.
550.
32—VO→andPO↑M60655M60655_atα-1Badrenergicreceptor1.
032.
05α1-adrenergicreceptoractivityAI639058rc_AI639058_s_atTransmembrane,prostateandrogeninducedRNA1.
172.
19androgenreceptorsignalingpathwayAF014009AF014009_atAcidiccalcium-independentphospholipaseA20.
802.
02antioxidantactivity,catalyticactivity,hydrolaseactivity21M22631M22631_atα-Propionyl-CoAcarboxylase1.
142.
02ATPbinding,biotinbinding,propionyl-CoAcarboxylaseactiv-ityU10357U10357_atPyruvatedehydrogenasekinase2subunitp451.
192.
30ATPbinding,pyruvatedehydrogenasekinaseactivityX75253X75253_atPhosphatidylethanolaminebindingprotein(Pbp)0.
832.
00ATPbinding,serine-typeendopeptidaseinhibitoractivityD86039D86039_atPotassiuminwardlyrectifyingchannel,subfamilyJ,member110.
982.
18ATP-activatedinwardrectifierpotassiumchannelactivityAI169417rc_AI169417_s_atPhosphoglyceratemutasetypeBsubunit1.
123.
61bisphosphoglyceratephosphataseactivity,catalyticactivityM86621M86621_atDihydropyridine-sesitiveL-typecalciumchannelα-2subunit1.
022.
18calciumchannelactivity,ionchannelactivityAA892511rc_AA892511_atTescalcin0.
922.
07calciumionbindingAA892146rc_AA892146_f_atSimilartocarboxypeptidaseBgene1.
132.
02carboxypeptidaseactivity,metallopeptidaseactivityX66494X66494_atCholinetransporter(CHOT1)0.
912.
00cholinetransporteractivity,creatinetransporteractivityAB012234AB012234_g_atNuclearfactorI/X(Nfix)1.
112.
01DNAbinding,transcriptionfactoractivityU17837U17837UTR#1_g_atZincfingerproteinRIZ0.
862.
06DNAbindingtranscriptionfactor19AA899106rc_AA899106_atG1/S-specificcyclinD2(VIN-1proto-oncogene)(LOC297611)1.
142.
19G1/Stransitionofmitoticcellcycle,cellgrowthAA799788rc_AA799788_s_atCelldivisioncycle340.
992.
10G2checkpointAA799570rc_AA799570_atDnaJ(Hsp40)homolog,subfamilyA,member41.
112.
02heatshockproteinbinding,unfoldedproteinbinding19M33962M33962_g_atProtein-tyrosine-phospatase0.
932.
02hydrolaseactivity,phosphoproteinphosphataseactivityAJ006971AJ006971_g_atDeath-associatedlikekinase(Dapk)0.
802.
08inductionofapoptosisAI043968rc_AI043968_atCaveolin-31.
032.
00integraltomembraneAA859981rc_AA859981_atSimilartomyo-inositolmonophosphatase2(Impa2)0.
822.
02magnesiumionbinding,inositol-1(or4)-monophosphataseactivity1036HypertensResVol.
29,No.
12(2006)Table1.
(Continued)AccessionProbesetIDGenenameVOPOMolecularfunctionReferenceX53054X53054_atRT1.
Dβchain0.
992.
16MHCclassIIproteinAI227608rc_AI227608_s_atMicrotubule-associatedproteintau(Mapt)1.
162.
02microtubule-basedprocessU59126U59126_atTR4-NSorphanreceptor(TR4)gene1.
135.
34nuclearhormonereceptorAF077354AF077354_atIschemiaresponsive94kDaprotein(irp94)1.
002.
20responsetoheatM29293M29293_atSmallnuclearribonucleoparticle-associatedprotein(snRNP)1.
122.
03RNAbinding,pre-mRNAsplicingfactoractivityU82224U82224_atCardiactitinN2Bisoform(Ttn)1.
153.
79sarcomereorganizationX78985X78985cds_s_atCD5orHocyteantigenCD50.
814.
28scavengerreceptoractivityAA892843rc_AA892843_atMitochondrialribosomalproteinL241.
062.
04structuralconstituentofribosomeX56228X56228_g_atRhodaneseorthiosulfatesulfurtransferase(Tst)0.
802.
31thiosulfatesulfurtransferaseactivity,transferaseactivityAA858586rc_AA858586_atSuppressorofTy5homolog(S.
cerevisiae)(Supt5h)1.
142.
16transcriptionelongationfactoractivityM62752M62752_atStatin-relatedprotein(S1)gene0.
872.
10translationelongationfactoractivity,GTPbindingAA892507rc_AA892507_atImmaturecoloncarcinomatranscript11.
122.
00translationreleasefactoractivityAA799691rc_AA799691_atK-ClcotransporterKCC41.
012.
00transport,iontransport,potassiumiontransportAA799475rc_AA799475_atAnkyrinrepeatdomain240.
882.
05—AA875261rc_AA875261_atCSX-associatedLIM1.
172.
08—AA893821rc_AA893821_atP34protein1.
192.
12—AA892005rc_AA892005_atSCIRP10-relatedprotein1.
022.
08—AA891802rc_AA891802_atSimilartocysteineandhistidinerich11.
162.
04—AA800039rc_AA800039_s_atSimilartohypotheticalproteinMGC6696(LOC293719)1.
022.
12—AA799766rc_AA799766_atSimilartoJtv1-pendingprotein0.
922.
00—AA892768rc_AA892768_atSimilartoputativebreastadenocarcinomamarker(32kDa)1.
192.
30—AA799654rc_AA799654_g_atSimilartoWDrepeat-containingF-boxproteinFBW51.
022.
12—AA866454rc_AA866454_atSimilartoα-2(I)promoter1.
162.
00—AA892773rc_AA892773_atEST196576(kidneyderivedcDNA)1.
022.
02—AA891631rc_AA891631_atEST195434(kidneyderivedcDNA)0.
932.
02—AA799932rc_AA799932_atEST189429(heartderivedcDNA)1.
092.
09—VO→andPO↓U93306U93306_atVEGFreceptor-2/FLK-11.
010.
49ATPbinding,kinaseactivity,proteinkinaseactivityX53363X53363cds_s_atCalreticulin(Calr)0.
820.
36calciumionbinding,calciumionstorageactivityAF047707AF047707_atUDP-glucose:ceramideglycosyltransferase1.
130.
45ceramideglucosyltransferaseactivityU95727U95727_atDnaJ(Hsp40)homolog,subfamilyA,member2(Dnaja2)0.
890.
45chaperoneactivity19X16043X16043cds_atPhosphatase2Acatalyticsubunitisotypeα0.
920.
47CTDphosphataseactivity,calcium-dependentphosphataseactivityD17711D17711cds_s_atHeterogeneousnuclearribonucleoproteinK(Hnrpk)1.
130.
32DNAbinding,RNAbinding,nucleicacidbindingAA799582rc_AA799582_atHeterogeneousnuclearribonucleoproteinK(Hnrpk)1.
170.
48DNAbinding,RNAbinding,nucleicacidbindingU53922U53922_atDnaJ-likeprotein(RDJ1)0.
820.
47DNAdamageresponse,perceptionofDNAdamageD21800D21800_atProteasomesubunitRC10-II0.
830.
43endopeptidaseactivity,hydrolaseactivity,peptidaseactivityMiyazakietal:GeneExpressionProfilinginCardiacHypertrophy1037Table1.
(Continued)AccessionProbesetIDGenenameVOPOMolecularfunctionReferenceAF072411AF072411_g_atFattyacidtranslocase/CD361.
130.
35fattyacidbinding,receptoractivityS81353S81353_s_atSulfatedglycoprotein-1orProsaposin(Psap)0.
890.
25glycolipidtransport,sphingolipidmetabolismM83676M83676_atSimilartoRAB12,memberRASoncogenefamily(Rab12)0.
920.
49GTPbinding,proteintransporteractivityD84477D84477_atPlysiaras-relatedhomologA2(Arha2)1.
190.
25GTPaseactivity,GTPbindingX68101X68101_atDedicatorofcytokinesis91.
160.
46guanyl-nucleotideexchangefactoractivity,GTPbindingAF000942AF000942_atInhibitorofDNAbinding31.
110.
43inhibitoroftranscriptionM19533M19533mRNA_i_atCyclophilinorpeptidylprolylisomeraseA(Ppia)0.
880.
46isomeraseactivity,peptidyl-prolylcis-transisomeraseactivityAI102620rc_AI102620_atMitogenactivatedproteinkinasekinasekinase10.
950.
47magnesiumionbinding,proteinkinaseactivityAI011706rc_AI011706_atSplicingfactor,arginine/serine-rich3(SRp20)0.
990.
49nucleicacidbinding,RNAbindingD84346D84346_s_atNCK-associatedprotein1(Nap1)proteinorHem-20.
880.
21proteinbindingD78303D78303_atYT521forRNAsplicing-relatedprotein1.
090.
49proteinbindingAA891035rc_AA891035_atBeclin1(coiled-coil,myosin-likeBCL2-interacting)1.
110.
45proteinbindingS59892S59892_f_atLa=autoantigenSS-B/La(3′region,cloneK4)0.
890.
43RNAbinding,nucleicacidbindingAF030091AF030091UTR#1_g_atCyclinania-6a1.
100.
49RNAprocessingAB022209AB022209_s_atRibonucleoproteinF1.
180.
46TATA-bindingproteinbinding,pre-mRNAsplicingfactoractiv-ityAF048687AF048687_s_atUDP-Gal:glucosylceramideβ-1,4-galactosyltransferase1.
180.
45UDP-galactose-glucosylceramidegalactosyltransferaseactivityAF034237AF034237_s_atDD6A4-11.
130.
49—U64705U64705cds_i_atEukaryotictranslationinitiationfactor4A20.
890.
49—AA891535rc_AA891535_atHippocampusabundantgenetranscript11.
180.
49—AA892376rc_AA892376_atProteinassociatedwithPRK11.
190.
44—AA892918rc_AA892918_atSimilartotightjunctionprotein1(Tjp1)1.
190.
47—VO↓andPO↑L22294L22294_atPyruvatedehydrogenasekinase-1(Pdk1)0.
442.
02ATPbinding,pyruvatedehydrogenasekinaseactivity19D10754D10754_atProteasomesubunitR-DELTA0.
482.
19endopeptidaseactivity,hydrolaseactivity,peptidaseactivityX62660X62660mRNA_atGlutathionetransferasesubunit80.
462.
06—AA891037rc_AA891037_g_atSimilarto60SribosomalproteinL3-like0.
472.
05—AA799888rc_AA799888_atSimilartomitochondrialribosomalproteinL400.
492.
00—VO↓andPO→D16478D16478_atHydroxyacyl-CoAdehydrogenase/3-ketoacyl-CoAhiolase/enoyl-CoAhydrataseαsubunit0.
490.
923-hydroxyacyl-CoAdehydrogenaseactivityD00512D00512_atMitochondrialacetoacetyl-CoAthiolase0.
430.
98acetyl-CoAC-acetyltransferaseactivity,acyltransferaseactivityJ02752J02752_atAcyl-coAoxidase0.
480.
83acyl-CoAoxidaseactivityM60322M60322_g_atAldehydereductase1(lowKmaldosereductase)(Aldr1)0.
491.
08aldehydereductaseactivity,oxidoreductaseactivityM21060M21060_s_atCopper-zinccontainingsuperoxidedismutase0.
461.
05antioxidantactivity,copper,zincsuperoxidedismutaseactivityAI104924rc_AI104924_f_atα-Cardiacmyosinheavychain0.
481.
06ATPbinding,actinbinding,calmodulinbinding,motoractivityAA800190rc_AA800190_g_atBrainglycogenphosphorylase(Pygb)0.
430.
89catalyticactivity,glycogenphosphorylaseactivity1038HypertensResVol.
29,No.
12(2006)Table1.
(Continued)AccessionProbesetIDGenenameVOPOMolecularfunctionReferenceY00497Y00497_s_atSuperoxidedismutase20.
410.
84copper,zincsuperoxidedismutaseactivityAI229620rc_AI229620_s_atCytochromecoxidasesubunitVb0.
460.
95cytochrome-coxidaseactivity,oxidoreductaseactivityX15030X15030_atCoxVaformitochondrialcytochromecoxidasesubunitVa0.
480.
97cytochrome-coxidaseactivity,oxidoreductaseactivityU95001U95001UTR#1_s_atDevelopmentally-regulatedcardiacfactor(DRCF-5)0.
440.
88diphosphoinositol-polyphosphatediphosphataseactivityK00750K00750exon#2-3_g_atCytochromecgene,nucleargeneformitochondrialproduct0.
470.
88electrontransporteractivity,lipidbindingAI176422rc_AI176422_g_atElectron-transferring–flavoproteindehydrogenase0.
470.
90electron-transferring–flavoproteindehydrogenaseactivityM83680M83680_atSprague-Dawley(cloneLRB13)RAB14orGTPaseRab140.
441.
02GTPbinding,proteintransporteractivityAA799474rc_AA799474_atSimilartocytochromec-1(LOC300047)0.
370.
93hemebinding,electrontransporterD13120D13120_s_atATPsynthasesubunitd0.
440.
81hydrogeniontransporteractivity22AA875444rc_AA875444_atDihydropyrimidinase-like2(Dpysl2)0.
330.
85hydrolaseactivityAI235358rc_AI235358_atUbiquinol-cytochromecreductasecoreproteinII0.
430.
89metalloendopeptidaseactivityM20131M20131cds_s_atcytochromeP450,family2,subfamilye,polypeptide10.
481.
16monooxygenaseactivity,oxidoreductaseactivityAI013297rc_AI013297_g_atNADHdehydrogenase(ubiquinone)Fe-Sprotein40.
470.
83NADHdehydrogenase(ubiquinone)activityAA819547rc_AA819547_atNADHdehydrogenase(ubiquinone)1αsubcomplex0.
470.
85NADHdehydrogenase(ubiquinone)activityAA851403rc_AA851403_atNADHdehydrogenase(ubiquinone)1βsubcomplex80.
491.
04NADHdehydrogenase(ubiquinone)activityAA891651rc_AA891651_g_atNADHdehydrogenase(ubiquinone)1,α/βsubcomplex,10.
450.
83NADHdehydrogenase(ubiquinone)activity,acylcarrieractiv-ityAF095927AF095927_g_atProteinphosphatase2C0.
480.
89negativeregulationofcellcycleD12769D12769_g_atBasictranscriptionelementbindingprotein10.
380.
91nucleicacidbinding,DNAbinding,zincionbindingAA875327rc_AA875327_g_atEukaryotictranslationinitiationfactor4H0.
440.
81nucleotidebinding,translationinitiationfactoractivityAI136977rc_AI136977_atFK506bindingprotein40.
370.
87peptidyl-prolylcis-transisomeraseactivityU90261U90261UTR#1_atHypertension-regulatedvascularfactor-1(HRVF-1)0.
450.
99proteinbindingD32209D32209_atLANPforleucine-richacidicnuclearprotein0.
480.
94proteinbindingAA892544rc_AA892544_s_atNeuralprecursorcellexpressed,developmentallydown-regulatedgene80.
490.
95proteinbindingAI103838rc_AI103838_g_atHeatshock20-kDaprotein(Loc192245)0.
450.
99regulationofmusclecontractionAF090867AF090867_atGuanosinemonophosphatereductase(GMP-reductase)0.
311.
12responsetocoldD84480D84480_s_atPMSG-inducedovarianmRNA,3′sequence,N20.
350.
84RNAbinding,structuralconstituentofribosomeAA900476rc_AA900476_g_atTranscriptionfactorMRG10.
440.
96transcriptionfactoractivity,transcriptioncofactoractivityAA892554rc_AA892554_g_atAA891476BACcloneRP24-216M6from50.
490.
82—D16554D16554_atPolyubiquitin(fourrepetitiveubiquitinsintandem)0.
490.
92—AA891476rc_AA891476_atSimilaritytoproteinpdb:1LBG(E.
coli)BChainB0.
480.
91—AA946040rc_AA946040_atSimilartoUroplakinIa(UPIa)(UPKa)(LOC365227)0.
490.
79—AI177256rc_AI177256_atEST220870(ovaryderivedcDNA)0.
490.
95—AI011556rc_AI011556_s_atEST206007(ovaryderivedcDNA)0.
481.
05—AI010292rc_AI010292_s_atEST204743(SimilartomitochondrialcytochromeoxidasesubunitII)0.
440.
89—Miyazakietal:GeneExpressionProfilinginCardiacHypertrophy1039Table1.
(Continued)AccessionProbesetIDGenenameVOPOMolecularfunctionReferenceVO↓andPO↓J03481J03481mRNA_g_atDihydropteridinereductase0.
490.
496,7-dihydropteridinereductaseactivity,oxidoreductaseactivityJ02791J02791_atAcetylcoenzymeAdehydrogenasemediumchain0.
470.
42acyl-CoAdehydrogenaseactivity,oxidoreductaseactivityD00688D00688_s_atMonoamineoxidase0.
440.
43amineoxidaseactivity,oxidoreductaseactivityL19998L19998_atMinoxidilsulfotransferase0.
490.
47arylsulfotransferaseactivity,sulfotransferaseactivityAA946532rc_AA946532_atSimilartoATP-bindingcassette,sub-familyD(ALD)0.
270.
38ATPbinding,ATPaseactivityD49785D49785_atProteinkinase(MUK)norvegicusmitogenactivatedproteinkinase0.
330.
21ATPbinding,cAMP-dependentproteinkinaseactivityAI007820rc_AI007820_s_atHeatshock90kDaprotein1,β0.
480.
49ATPbinding,nitric-oxidesynthaseregulatoractivityAI176546rc_AI176546_atHeatshockprotein860.
440.
49ATPbinding,unfoldedproteinbindingAA858640rc_AA858640_s_atHeatshockprotein10.
460.
46ATPbinding,unfoldedproteinbindingAI235707rc_AI235707_g_atCalnexin0.
480.
48calciumionbinding,sugarbinding,unfoldedproteinbindingM26125M26125_atEpoxidehydrolase10.
480.
47catalyticactivity,aminopeptidaseactivity19X60328X60328_atCytosolicepoxidehydrolase0.
360.
38catalyticactivity,epoxidehydrolaseactivity,hydrolaseactivityAA818226rc_AA818226_s_atMitochondrialcytochromecoxidasesubunit0.
450.
49cytochrome-coxidaseactivity,oxidoreductaseactivityAI136891rc_AI136891_atZincfingerprotein360.
450.
45DNAbinding,nucleicacidbindingAI171355rc_AI171355_s_atcytochromeb,mitochondrial0.
380.
46electrontransporteractivity19AA945054rc_AA945054_s_atCytochromeb50.
390.
41electrontransporteractivity19AI103396rc_AI103396_g_atMitochondrialgeneforcytochromeb0.
490.
49electrontransporteractivity19AI234604rc_AI234604_s_atHeatshockcognate71kDaprotein0.
460.
49heatshockproteinactivityAA818858rc_AA818858_s_atPeptidylprolylisomeraseA(Ppia)0.
470.
47isomeraseactivity,peptidyl-prolylcis-transisomeraseactivityAI228674rc_AI228674_s_atPeptidyl-prolylcis-transisomeraseA(PPIase)0.
490.
49isomeraseactivity,peptidyl-prolylcis-transisomeraseactivityAI103874rc_AI103874_atFK506bindingprotein30.
450.
49peptidyl-prolylcis-transisomeraseactivity,receptoractivityAI230406rc_AI230406_atRAB10,memberRASoncogenefamily0.
420.
45proteinbinding,GTPbinding,proteintransporteractivityAI007614rc_AI007614_atMitogen-activatedproteinkinasekinasekinasekinase40.
430.
36proteinkinaseactivity,smallGTPaseregulatoractivityAB016532AB016532_atrPER2,periodhomolog2(Per2)0.
410.
44signaltransduceractivity19X62951X62951mRNA_s_atEndogenousretrovirus0.
410.
37—AB013454AB013454_atSimilartoAc2-2100.
340.
32—AI009132rc_AI009132_atSimilartochromosome13openreadingframe120.
470.
47—AA892056rc_AA892056_atSimilartodeoxycytidyltransferase0.
430.
46—AI104035rc_AI104035_s_atEST213324(heartderivedcDNA)0.
430.
47—AA945152rc_AA945152_s_atEST20065(liverderivedcDNA)0.
320.
49—AI009141rc_AI009141_atEST203592(embryoderivedcDNA)0.
370.
42—GeneBankaccessionnumbers,probesetID,genename,andtheirfunctionsarelistedforeachgene.
TheholdchangeingeneexpressioninPOandVOvs.
shamsamplesisgiven.
"Refer-ence"indicatesthattheresultsweresimilartotheindicatedreferences.
1040HypertensResVol.
29,No.
12(2006)(LOXL)TaqManProbe,AGGCAGCCTCCCCCAAGAAGCA;LOXLsense-primer,TTGAAAAGCAGGACCTGCTTC;LOXLantisense-primer,CTCCGGCTAGGCGGCT;pyruvatedehydrogenasekinases-1(PDK-1)TaqManProbe,CCGTCGCCACTCTCCATGAAGCA;PDK-1sense-primer,GGACTTCTATGCGCGCTTCT;PDK-1antisense-primer,ACTGACCCGAAGTCCAGGAA;transgelinTaqManProbe,CCGCCCTCCATGGTCTTCAAGCA;transgelinsense-primer,GCCAGTGAAGGTGCCTGAGA;transgelinantisense-primer,AGAATTGAGCCACCTGTTCCA.
SubcloningofRatMTRatMTcDNAwasclonedfromratheartbyRT-PCR.
Thefollowingprimerswereusedforthisexperiment:senseprimer,5′-ACTGCCTTCTTGTCGCTTAC-3′;antisenseprimer,5′-AGGGCAGCAGCACTGTTCGT-3′.
ThisPCRproductwassubclonedintopCR2.
1andthentransferredtopcDNA3.
1(Invitrogen).
CellCultureandTransfectionH9c2cells(acardiacmyoblastcellline)wereobtainedfromtheAmericanTypeCultureCollection(Rockville,USA).
CellsweretransfectedwithratMT-pcDNA3.
1orpcDNA3.
1(emptyvector)byLipofectamine2000(AmershamBio-sciences,Piscataway,USA).
At24hafterthetransfection,cellswereexposedtonorepinephrine(NE;104mol/l)andculturedforanadditional24h.
Theefficiencyofthetransfec-tionwasassessedbytheco-transfectionofpQBI50,whichencodesbluefluorescentprotein(Takara,Tokyo,Japan).
AssessmentofApoptosisandCaspase3AssayApoptosiswasassessedbyTUNELstaining(InSituCellDeathDetectionkit,RocheDiagnostics,Mannheim,Ger-many).
Caspase-3activitywasmeasuredbyaCaspACEassaysystem(Promega,Madison,USA).
StatisticalAnalysisDataarepresentedasthemean±SEM.
Comparisonsofmeanvalueswereperformedbyone-wayANOVAfollowedbyScheffe'stest.
Valuesofp<0.
05wereconsideredtoindicatestatisticallysignificantdifferences.
ResultsFigure1showstheechocardiographicfindings4weeksafterthesurgery.
Leftventriculardiastolicdiameter(LVDd)wassignificantlyhigherintheratssubjectedtoVOcomparedwiththosesubjectedtoPOandthoseundergoingshamoper-ation(hereaftertheVO,PO,andshamgroups,respectively)(PO:96±8%ofthesham-groupvalue;VO:119±9%ofthesham-groupvalue).
Whileinterventricularseptalwallthick-ness(IVST)wasincreasedinbothtypesofhypertrophy(PO,126±12%ofthesham-groupvalue;VO,120±11%ofthesham-groupvalue),theIVSTofthePOgroupwasgreaterthanthatoftheVOgroup.
Thus,POproducedconcentrichypertrophyandVOproducedeccentrichypertrophy.
TherewasnodifferenceamongthePO,VOandshamgroupsinper-centfractionalshortening(%FS).
Theleftatrialdiameter(LAD)ofbothhypertrophygroupswaslargerthanthatoftheshamgroup.
AsshowninFig.
2,theleftventricularweight/bodyweightratioandheartweight/bodyweightratioweresignificantlyincreasedinbothhypertrophygroups,comparedwiththeshamgroup.
TheseratioswereslightlylargerintheVOthanthePOgroup(PO:139±11%ofthesham-groupvalue;VO:157±16%ofthesham-groupvalue).
Figure3andTable1showresultsofDNAchipanalysis.
Atotalof33geneswereupregulatedinbothformsofhypertro-phy,whereas31weredownregulated.
ThegenesthatwereupregulatedinbothformsincludedBNP,atrialnatriureticpeptide(ANP),andskeletalα-actin,whichareknowntobeupregulatedinthehypertrophiedmyocardium.
InthePOgroup,52geneswereselectivelyupregulatedand41genesweredownregulated.
IntheVOgroup,88geneswereselec-tivelyupregulatedand46genesweredownregulated.
Subsequently,weperformedhierarchicalclusteringwiththeaveragelinkagemethod.
Figure4showstheclusterofupregulatedgenes(A)anddownregulatedgenes(B)inbothhypertrophygroups.
ThedifferentiallyexpressedgenesweregroupedbyfunctionalcategoryclassificationaccordingtotheGeneOntology(http://www.
geneontology.
org/)termsbasedontheNCBILocusLink(http://www.
ncbi.
nlm.
nih.
gov/LocusLink/)database.
Thegeneclusterthatshowedthemostmarkedincreasewasthenatriureticpeptidefamily.
Thehydrogen-transportingtwo-sectorATPasegroup,thecyto-skeletalorganizationandbiogenesisgroup,andthemitochon-drialsubstratecarriergroupwereupregulatedinbothhypertrophiedhearts.
Ontheotherhand,thecalciumion–bindinggroupandthebasic-leucinezippertranscriptionfac-torgroupweredownregulatedinbothhypertrophygroups.
WeobservedselectiveupregulationofthegenesthatbindtoactinintheVOgroup(e.
g.
,tropomyosin4,thymosinβ-4,andtransgelin).
Next,weperformedreal-timeRT-PCRanalysistoconfirmtheresultsoftheDNAchipanalysis(Fig.
5).
Real-timeRT-PCRrevealedthatBNP,MTandLOXLmRNAswereallupregulatedinboththeVOandPOgroups,comparedwiththeshamgroup.
MTmRNAexpressionintheVOgroupwassignificantlyhigherthanthatinthePOgroup.
ThelevelsofBNPandLOXLmRNAsintheVOgroupwerealsohigherthanthoseinthePOgroup,butthesedifferencesdidnotreachthelevelofstatisticalsignificance.
DNAchipanalysisshowedthatGMPreductasemRNAwasselectivelydownreg-ulatedintheVOgroup.
Thiswasconfirmedbyreal-timeRT-PCR.
WethenexaminedtheexpressionsofsomeofthegenesthatweredifferentiallyalteredinVOandPOhearts.
DNAMiyazakietal:GeneExpressionProfilinginCardiacHypertrophy1041chipanalysisrevealedthattransgelinwasincreasedintheVOgroupbutdownregulatedinthePOgroup,andPDK1wasdecreasedinVObutupregulatedinPO.
Real-timeRT-PCRconfirmedthesechanges(Fig.
5E,F).
InourDNAchipanalysis,BNPandANPshowedthemostremarkableupregulationsinhypertrophiedLVs.
MTshowedthenexthighestlevelofupregulation.
Accordingly,weexam-inedtheroleofMTinthedevelopmentofcardiachypertro-phy(Fig.
6).
MTisafreeradicalscavengerandissuggestedtoplayaprotectiveroleinmanycelltypes.
Therefore,wehypothesizedthatMTplaysananti-apoptoticroleincardiachypertrophyandperformedthefollowingexperiments.
MTcDNAorcontrolvector(pcDNA3.
1)wasoverexpressedinH9c2cells.
Thecellswerethenexposedtoahighconcentra-tionofNEfor24h,andTUNELstainingwasperformed.
Incellsoverexpressingthecontrolvector,NEincreasedTUNEL-positivecells.
OverexpressionofMTinhibitedtheincreaseofTUNEL-positivecells(Fig.
6).
Toconfirmtheanti-apoptoticeffectsofMT,wenextexaminedthelevelofcaspase-3activity,whichiscriticallyinvolvedinapoptosis,inthesecells(Fig.
7).
Caspase-3activitywasincreasedincellstreatedwithNE.
TheoverexpressionofMTreducedtheaug-mentationofcaspase-3activityby42±9.
9%comparedwiththatinthecellstransfectedwiththecontrolvector(pCDNA3.
1;Fig.
7).
DiscussionWeexaminedthesimilarityanddifferenceofthegeneexpressionprofilingbetweenthePOandVOhearts.
Wefoundmanygenesthatareregulatedsimilarlyordifferentiallybetweenthetwogroups.
Thealterationsofsomespecificgeneswereconfirmedbyreal-timeRT-PCR.
Amongthegenesthatwereupregulatedinbothformsofhypertrophy,weexaminedtheroleofMTinapoptosis,andfoundthatthisgeneplayedanovelroleinanti-apoptosis.
Fig.
4.
Hierarchicalclusteranalysisofexpressionprofiles.
Regulatedgenesaredepictedusingacolorscale(red,upregulatedgenes;blue,downregulatedgenes).
A:Theoneoftheclusterconsistingofgenesthatwereincreasedinbothformsofhypertro-phy.
B:Theoneoftheclusterconsistingofgenesthatweredecreasedinbothformsofhypertrophy.
1042HypertensResVol.
29,No.
12(2006)Fig.
5.
Real-timeRT-PCR.
A:B-typenatriureticpeptide(BNP).
B:Metallothionein-1(MT).
C:Lysyloxidase–likeprotein1(LOXL).
D:GMPreductase.
E:Transgelin.
F:Pyruvatedehydrogenasekinases-1(PDK-1).
Valuesarethemean±SD.
*p<0.
05,**p<0.
01.
Fig.
6.
EffectsofoverexpressionofMTonthepercentageofTUNEL-positivecells.
A:TUNELstaining.
B:PercentageofTUNEL-positivecells.
H9c2cellsweretransfectedwithMT-pcDNA3orpcDNA3alone.
At24hafterthetransfection,cellswereexposedtonorepinephrine(104mol/l)andculturedforanadditional24h.
TUNELstainingwasperformedasdescribedinMethods.
Valuesarethemean±SD.
**p<0.
01.
1-nienoihtollateMPNB01234567OPOVmahS0010203OPOVmahSFoldIncreaseofShamFoldIncreaseofSham*********BALXOL0501OPOVmahSFoldIncreaseofShamesatcudeRPMG05.
00.
15.
1OPOVmahSFoldIncreaseofSham********DCE05.
015.
125.
2OPOVmahSFoldIncreaseofShamnilegsnarT***F1-KDP05.
015.
125.
2OPOVmahS***FoldIncreaseofSham%ofTUNELPositiveCells3ANDcp3ANDcp/1-TM001020304**BAelciheVenirhpeniperoN3ANDcp/1-TM3ANDcpenirhpeniperoNelciheVnoitcefsnartoNnoitcefsnartoNMiyazakietal:GeneExpressionProfilinginCardiacHypertrophy1043MethodologicalConsiderationsInthisstudy,weusedtwodifferentprocedurestoinducecar-diachypertrophy:abdominalconstrictionforpressureover-loadandaortocavalshuntforvolumeoverload.
Thesetwoproceduresproducedthesamemagnitudeofhypertrophywithdistinctmorphologicaldifferencesasshownbyechocar-diography.
Thusthesimilaritiesingeneprofilingmaybeattributabletohypertrophyperseandthedissimilaritytothedifferentmorphologiesoftheheart,i.
e.
,concentrichypertro-phyoreccentrichypertrophy.
Inbothformsofhypertrophy,cardiacfunctionassessedbyfractionalshorteningwasnor-mal,indicatingthatchangesingeneprofilingwerenotattrib-utabletothoseofthemyocardiumatthefailingstage.
Thesemechanicalstressesactivatemanymoleculesintheheart.
Therearetwopeaksintheactivationsofbiochemicalsignalsafteranexposuretohemodynamicload(18).
Acuteresponsesoccurwithinafewminutes,andhypertrophicresponsesfollowoverthenextseveraldaysorweeks.
Asapilotstudy,wemeasuredthetime-dependentchangeoftheexpressionofBNPmRNAinratPOhearts.
Theleftventricu-larweight/bodyweightratioincreasedgraduallyuntilday28aftertheinitiationofPOandcontinuedataplateaulevelthereafter.
BNPmRNAlevelsshowedabiphasicresponse,i.
e.
,aninitialincreaseandalatergradualincrease.
Thefirstpeakoftheexpressionwasat24handthesubsequentpeakwasatday28(datanotshown).
Thus,atday28acutegeneresponseswerenolongerobservedandhypertrophicgeneresponsesweremaximalandstable.
Thisisthetimewhenhypertrophywasmorphologicallyestablished.
Inordertoinvestigategeneresponseswithhypertrophybutwithoutheartfailure,wechoseday28.
Weexaminedgeneprofilingatthestageofestablishedcar-diachypertrophybutdidnotexamineitchronologically.
Thusthegeneprofilinginourstudymaynotrepresentthatofthedevelopmentalstageofhypertrophy,butmerelythatofthecompensationofhypertrophy.
Moreover,weexaminedgeneprofilingofthewholeheart.
Thusourresultsdonotnecessar-ilyindicategeneprofilingofcardiomyocytes,butmayalsoincludeprofilingfortheinterstitialtissues.
GeneProfilinginPOandVOThepresentstudyrevealedgeneexpressionprofilesthatmaybeassociatedwithhypertrophyperseordifferenttypesofremodeling.
Inagreementwithpreviousreports(13,14,19,20),wefoundchangesingeneprofilinginPO.
AsshowninFig.
3,mostgeneswereunalteredinthehypertrophicmyocar-diuminducedbyPO.
Only85geneswereupregulated.
Thus,thenumberofgenesupregulatedinthehypertrophiedmyo-cardiuminducedbyPOwaslessthanthatofseveralpreviousreports.
Thisdiscrepancymaybeascribabletothecriteriausedforthegeneselectionandtheuseofdifferentkindsofchips.
Thepresentstudyisthefirsttodescribegeneexpres-sionsinVO.
AsshowninFig.
3andTable1,thelevelsofmostofthegeneswereunaltered,justasinPO.
Only121geneswereupregulated.
ItmaybeinterestingtonotethatsomegeneswereregulateddifferentiallybetweenVOandPO.
Amongthosegenes,extracellularmatrix–relatedpro-teins,e.
g.
,osteopontin,secretedacidiccysteinerichglyco-protein,andtropoelastin,werepredominantlyupregulatedinVO.
TheupregulationofthesegenesmayberelatedtothespecificremodelinginVO,butnottohypertrophyperse.
Inaddition,theselectiveupregulationofgenesinVOthatbindtoactin(e.
g.
,tropomyosin4,thymosinβ-4,andtransgelin)mayexplainthedifferenceinstructuralventricularremodel-ingbetweenVOandPO.
Severalgeneprofileshavebeenreportedinothermodelsofhypertrophy(21,22).
Thesereportshaveshownthatthecommongeneticeventsinhyper-trophyincludeupregulationsofANP,BNP,andskeletalα-actin.
Thesegeneswereincreasedinbothformsofhypertro-phyinthepresentstudyaswell.
GMPreductasewaspredominantlydownregulatedinVO.
BecauseGMPreductaseincreasestheadeninenucleotidepoolthroughsynthesisofinosinemonophosphate(23),thedecreaseofGMPreductasemayhavesomedeteriorativeeffectsonmyocardialmetabolism.
WealsofoundthattheexpressionsofPDK-1andtransgelinweredifferentiallyreg-ulatedinVOandPOhearts(Fig.
5E,F).
Transgelin(alsoknownasSM22α)isa22-kDaproteinthatisassociatedwithcytoskeletalactinfilamentbundlesincontractilesmoothmusclecell(SMC)(24,25).
Itisexpressedincardiac,smooth,andskeletalmusclecellsduringearlyembryogenesisbutbecomesrestrictedtovisceralandvascularSMCatlateembryonicstagesandthroughoutadulthood.
TransgelinwasupregulatedinVOanddownregulatedinPOheartsintheFig.
7.
EffectsofoverexpressionofMToncaspase-3activ-ity.
H9c2cellsweretransfectedwithMT-pcDNA3orpcDNA3alone.
At24hafterthetransfection,cellswereexposedtonorepinephrine(104mol/l)andculturedforanadditional24h.
Thecaspase-3assaywasperformedasdescribedinMethods.
Dataarefromfourindependentexper-iments.
Valuesarethemean±SD.
**p<0.
01.
Caspase-3activity(foldincrease)3ANDcp3ANDcp/1-TMelciheVenirhpeniperoNnoitcefsnartoN05.
015.
125.
2****1044HypertensResVol.
29,No.
12(2006)presentstudy(Fig.
5E),butthesignificanceofthesechangesisunknown.
Transgelinmayregulateseveralgenesthatareassociatedwithvasculardevelopment,remodeling,andalter-ations.
Thedifferentialexpressionofthisgenemayreflectthedifferenceofvascularremodelingbetweenconcentricandeccentriccardiachypertrophy.
PDK-1wasdownregulatedinVObutupregulatedinPO(Fig.
5F).
PDKsphosphorylatethemitochondrialpyruvatedehydrogenasecomplex,whichcata-lyzestheoxidativedecarboxylationofpyruvate,andtherebyinactivateit(26).
Althoughthesignificanceofalterationsofthisenzymeinthehypertrophiedheartsisunclear,thesechangesmaybeassociatedwiththedifferenceofthemyocar-dialenergyproductioninbothformsofhypertrophy.
UpregulatedGenesinBothFormsofHypertro-phyOurDNAchipanalysisshowedupregulationof33genesinbothformshypertrophy(Table1).
Thesegenesmaybeasso-ciatedwithhypertrophyperse.
Amongthesegenes,wecon-firmedtheupregulationsofBNP,MTandLOXLmRNA.
Wangetal.
(13)andWeinbergetal.
(14)havereportedthatMTisupregulatedsignificantlyinaorticbandingmice.
Inthepresentstudy,MTwasoneofgenesshowingthehighestlevelofupregulationinbothformsofhypertrophiedheart.
Accord-ingly,weexaminedtheroleofMTincardiachypertrophy.
MTisacysteine-richproteinwithastrongaffinityforZn2+(reviewedinPalmitter(27),andKang(28)).
MTisknowntodetoxifytheheavymetals.
RecentstudiesrevealedthatMTbindstoraremetalsinvivoandregulatesmetalproteinandmetal-dependenttranscriptionalfactors.
Moreover,MTisapotentfreeradicalscavenger.
Andintheheartsofmicewithadriamycin-inducedcardiomyopathy,MTexpressionhasbeenshowntobeincreased(29).
Moreover,ithasbeenreportedthatoverexpressionofMTintheheartsoftransgenicmicesuppressesdoxorubicincardiotoxicityandischemia-reperfusioninjury(30,31).
ThusMTplayscytoprotectiverolesinmanycelltypes.
Inthisstudy,MTsuppressedtheNE-inducedincreaseofTUNEL-positivecells.
Thisinhibitionwasassociatedwiththesuppressionofcaspase-3activity,suggestingthatMTinhibitstheapoptosisofcardiomyocytes.
Ithasbeenreportedthatthemyocardiumofbothratmodelsofhypertrophyshowsapoptoticcardiomyocytedeath(32,33).
Therefore,weconsiderthatMTmayexertanti-apoptoticeffectsunderphysiologicalconditions.
Aronowandcowork-ersshowedthatMTwasuniversallyincreasedinhypertro-phiedheartsproducedbyfourdifferentmechanisms,suggestingthattheupregulationofMTisahighlyconservedgeneticeventinhypertrophy(21).
Interestingly,theexpres-sionofMTwasgreaterinthehypertrophymodelthanintheheartfailuremodelintheirstudy(21).
Thissuggeststhatapo-ptosisofthehypertrophiedmyocardium,whichleadstoheartfailure,ispreventedbytheexpressionofMT.
Takentogether,thesefindingsindicatethatMTmayhaveaprotectiveeffectontheheartthroughitsanti-apoptoticeffects.
LOXLwasalsosignificantlyupregulatedinbothformsofhypertrophy.
LOXLisamemberofthelysyloxidasegenefamily,whichisanextracellular,copper-dependentenzymefamilythatinitiatesthecross-linkingofcollagensandelastin(34,35).
ItmaybepossiblethatLOXLisassociatedwiththeremodelingoftheextracellularmatrixinthehypertrophiedmyocardium.
Amongtheupregulatedgenes,GATA-4isknowntobeessentialfortheinductionofseveralcardiac-spe-cificgenes(36).
TheupregulationofGATA-4supportstheideathatthisproteinmaybethecommonregulatorofhyper-trophicresponses.
Inconclusion,wefoundmanygenesthataresimilarlyordissimilarlyregulatedintheheartsofVOandPO.
ThealteredexpressionsofBNP,MT,GMPreductase,LOXL,transgelin,andPDK-1wereconfirmedbyreal-timeRT-PCR.
Genepro-filingbyDNAchiptechnologyrepresentsausefultoolforidentificationofgenesthatmaycontributetomorphologicalandfunctionaldifferencesbetweenPOandVO.
Moreover,theoverexpressionofMTinH9c2cellsattenuatedthenorepi-nephrine-inducedapoptosis.
MTmayplayanovelprotectiveroleinthedevelopmentofcardiachypertrophy.
Althoughwefoundthatmanygeneswerealteredinthehypertrophiedhearts,itremainsuncertainwhetherthesechangesplayedaroleinorweresecondarytothedevelopmentofhypertrophy.
Toclarifythesequestions,functionalstudiesshouldbeper-formedforeachmolecule.
AcknowledgementsWethankYumikaTamotoforvaluabletechnicalassistance.
References1.
LorellBH,CarabelloBA:Leftventricularhypertrophy:pathogenesis,detection,andprognosis.
Circulation2000;102:470–479.
2.
HunterJJ,ChienKR:Signalingpathwaysforcardiachyper-trophyandfailure.
NEnglJMed1999;341:1276–1283.
3.
HomcyCJ:Signalinghypertrophy:howmanyswitches,howmanywires.
Circulation1998;97:1890–1892.
4.
DiamondJA,PhillipsRA:Hypertensiveheartdisease.
HypertensRes2005;28:191–202.
5.
ChapmanD,WeberKT,EghbaliM:RegulationoffibrillarcollagentypesIandIIIandbasementmembranetypeIVcollagengeneexpressioninpressureoverloadedratmyo-cardium.
CircRes1990;67:787–794.
6.
NambaT,TsutsuiH,TagawaH,etal:Regulationoffibril-larcollagengeneexpressionandproteinaccumulationinvolume-overloadedcardiachypertrophy.
Circulation1997;95:2448–2454.
7.
NagatomoY,CarabelloBA,CokerML,etal:DifferentialeffectsofpressureorvolumeoverloadonmyocardialMMPlevelsandinhibitorycontrol.
AmJPhysiolHeartCircPhysiol2000;278:H151–H161.
8.
ImamuraT,McDermottPJ,KentRL,NagatsuM,CooperG4th,CarabelloBA:Acutechangesinmyosinheavychainsynthesisrateinpressureversusvolumeoverload.
CircResMiyazakietal:GeneExpressionProfilinginCardiacHypertrophy10451994;75:418–425.
9.
YoshiharaF,NishikimiT,HorioT,etal:Ventricularadrenomedullinconcentrationisasensitivebiochemicalmarkerforvolumeandpressureoverloadinrats.
AmJPhysiolHeartCircPhysiol2000;278:H633–H642.
10.
CalderoneA,TakahashiN,IzzoNJJr,ThaikCM,ColucciWS:Pressure-andvolume-inducedleftventricularhyper-trophiesareassociatedwithdistinctmyocytephenotypesanddifferentialinductionofpeptidegrowthfactormRNAs.
Circulation1995;92:2385–2390.
11.
ModestiPA,VanniS,BertolozziI,etal:Earlysequenceofcardiacadaptationsandgrowthfactorformationinpres-sure-andvolume-overloadhypertrophy.
AmJPhysiolHeartCircPhysiol2000;279:H976–H985.
12.
TsutsuiH,TagawaH,KentRL,etal:Roleofmicrotubulesincontractiledysfunctionofhypertrophiedcardiocytes.
Circulation1994;90:533–555.
13.
WangD,OparilS,FengJA,etal:Effectsofpressureover-loadonextracellularmatrixexpressionintheheartoftheatrialnatriureticpeptide-nullmouse.
Hypertension2003;42:88–95.
14.
WeinbergEO,MirotsouM,GannonJ,DzauVJ,LeeRT,PrattRE:Sexdependenceandtemporaldependenceoftheleftventriculargenomicresponsetopressureoverload.
PhysiolGenomics2003;12:113–127.
15.
LimasCJ:Increasednumberofbeta-adrenergicreceptorsinthehypertrophiedmyocardium.
BiochimBiophysActa1979;588:174–178.
16.
GarciaR,DieboldS:Simple,rapid,andeffectivemethodofproducingaortocavalshuntsintherat.
CardiovascRes1990;24:430–432.
17.
LiC,WongWH:Model-basedanalysisofoligonucleotidearrays:expressionindexcomputationandoutlierdetection.
ProcNatlAcadSciUSA2001;98:31–36.
18.
HoshijimaM,ChienKR:Mixedsignalsinheartfailure:cancerrules.
JClinInvest2002;109:849–855.
19.
KongSW,BodyakN,YueP,etal:Geneticexpressionpro-filesduringphysiologicalandpathologicalcardiachyper-trophyandheartfailureinrats.
PhysiolGenomics2005;21:34–42.
20.
UenoS,OhkiR,HashimotoT,etal:DNAmicroarrayanal-ysisofinvivoprogressionmechanismofheartfailure.
Bio-chemBiophysResCommun2003;307:771–777.
21.
AronowBJ,ToyokawaT,CanningA,etal:Divergenttran-scriptionalresponsestoindependentgeneticcausesofcar-diachypertrophy.
PhysiolGenomics2001;6:19–28.
22.
FriddleCJ,KogaT,RubinEM,BristowJ:Expressionpro-filingrevealsdistinctsetsofgenesalteredduringinductionandregressionofcardiachypertrophy.
ProcNatlAcadSciUSA2000;97:6745–6750.
23.
Zoref-ShaniE,ShirinC,SidiY,BrombergY,SperlingO:Metabolismofguanineandguaninenucleotidesinprimaryratcardiomyocytecultures.
BiochemMolMed1995;55:149–155.
24.
FeilS,HofmannF,FeilR:SM22alphamodulatesvascularsmoothmusclecellphenotypeduringatherogenesis.
CircRes2004;94:863–865.
25.
MorganKG,GangopadhyaySS:Invitedreview:cross-bridgeregulationbythinfilamentassociatedproteins.
JApplPhysiol2001;91:953–962.
26.
SugdenMC,HolnessMJ:RecentadvancesinmechanismsregulatingglucoseoxidationatthelevelofthepyruvatedehydrogenasecomplexbyPDKs.
AmJPhysiolEndocrinolMetab2003;284:E855–E862.
27.
PalmiterRD:Theelusivefunctionofmetallothioneins.
ProcNatlAcadSciUSA1998;95:8428–8430.
28.
KangYJ:Theantioxidantfunctionofmetallothioneinintheheart.
ProcSocExpBiolMed1999;222:263–273.
29.
YinX,WuH,ChenY,KangYJ:Inductionofantioxidantsbyadriamycininmouseheart.
BiochemPharmacol1998;56:87–93.
30.
KangYJ,ChenY,YuA,Voss-McCowanM,EpsteinPN:Overexpressionofmetallothioneinintheheartoftransgenicmicesuppressesdoxorubicincardiotoxicity.
JClinInvest1997;100:1501–1506.
31.
KangYJ,LiY,SunX:Antiapoptoticeffectandinhibitionofischemia/reperfusion-inducedmyocardialinjuryinmet-allothionein-overexpressingtransgenicmice.
AmJPathol2003;163:1579–1586.
32.
TeigerE,ThanVD,RichardL,etal:Apoptosisinpressureoverloadinducedhearthypertrophyintherat.
JClinInvest1996;97:2891–2897.
33.
CoxMJ,SoodHS,HuntMJ,etal:Apoptosisintheleftven-tricleofchronicvolumeoverloadcausesendocardialendo-thelialdysfunctioninrats.
AmJPhysiolHeartCircPhysiol2002;282:H1197–H1205.
34.
KaganHM,LiW:Lysyloxidase:properties,specificity,andbiologicalrolesinsideandoutsideofthecell.
JCellBiochem2003;88:660–672.
35.
BankRA,vanHinsberghVW:Lysyloxidase:newlooksonLOX.
ArteriosclerThrombVascBiol2002;22:1365–1366.
36.
MolkentinJD:Thezincfinger-containingtranscriptionfac-torsGATA-4,-5,and-6.
Ubiquitouslyexpressedregulatorsoftissue-specificgeneexpression.
JBiolChem2000;275:38949–38952.