Cardiac effects of long-term active immunization with the second extracellular loop of human ß1- and/or ß3-adrenoceptors in Lewis rats

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Cardiac effects of long-term active immunization with

the second extracellular loop of human ß1- and/or

ß3-adrenoceptors in Lewis rats

E Montaudon, Laurence Dubreil, Valerie Lalanne, M. Vermot Des Roches,

Gilles Toumaniantz, Marion Fusellier, Jean-Claude Desfontis, Lionel

Martignat, Mohamed Yassine Mallem

To cite this version:

E Montaudon, Laurence Dubreil, Valerie Lalanne, M. Vermot Des Roches, Gilles Toumaniantz, et

al..

Cardiac effects of long-term active immunization with the second extracellular loop of

hu-man ß1- and/or ß3-adrenoceptors in Lewis rats. Pharmacological Research, 2015, 100, pp.210-219.

�10.1016/j.phrs.2015.08.006�. �hal-01208120�

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See discussions, stats, and author profiles for this publication at:

http://www.researchgate.net/publication/281082314

Cardiac effects of long-term active

immunization with the second extracellular

loop of human β1- and/or β3-adrenoceptors in

Lewis rats

ARTICLE

in

PHARMACOLOGICAL RESEARCH · AUGUST 2015

Impact Factor: 4.41 · DOI: 10.1016/j.phrs.2015.08.006 · Source: PubMed READS

13 9 AUTHORS

, INCLUDING:

Valerie Lalanne

École Nationale Vétérinaire, Agroalimentair…

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Marion Fusellier

École Nationale Vétérinaire, Agroalimentair…

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Desfontis Jean-Claude

École Nationale Vétérinaire, Agroalimentair…

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SEE PROFILE Available from: Elodie Montaudon Retrieved on: 01 October 2015

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ContentslistsavailableatScienceDirect

Pharmacological

Research

jo u r n al hom e p ag e :w w w . e l s e v i e r . c o m / lo c a t e / y p h r s

Cardiac

effects

of

long-term

active

immunization

with

the

second

extracellular

loop

of

human

!

1 -

and/or

!

3 -adrenoceptors

in

Lewis

rats

E. Montaudon

a

_,

_L.

_Dubreil

b

_,

_V.

_Lalanne

a

_,

_M.

_Vermot

_Des

_Roches

a

_,

_G.

_Toumaniantz

c

_,

M. Fusellier

d

_,

_J.-C.

_Desfontis

a

_,

_L.

_Martignat

e

_,

_M.Y.

_Mallem

a,∗

a_LUNAM_University,_Oniris,_UPSP₅₃₀₄_of_Animal_{Pathophysiology}_and_Functional_{Pharmacology,}_Atlanpôle_La_Chantrerie,_BP_40706,₄₄₃₀₇_Nantes,_France

b_LUNAM_University,_Oniris,_INRA_UMR_U703,_PanTHER,_Atlanpôle_La_Chantrerie,_BP_40706,₄₄₃₀₇_Nantes,_France

c_LUNAM_University,_INSERM,_UMR_1087/CNRS_6291,_Institut_du_Thorax,_Nantes_F44007_France

d_LUNAM_University,_Oniris,_INSERM_UMR_S_791,_LIOAD,_Atlanpôle_La_Chantrerie,_BP_40706,₄₄₃₀₇_Nantes,_France

e_LUNAM_University,_Oniris,_UPSP_SSBR,_Atlanpôle_La_Chantrerie,_BP_40706,₄₄₃₀₇_Nantes,_France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received6March2015

Receivedinrevisedform28June2015

Accepted7August2015

Availableonline11August2015

Keywords: Auto-antibodies !-Adrenoceptor Cardiaccontractility Dilatedcardiomyopathy Rats

a

b

s

t

r

a

c

t

!1-and!3-adrenoceptor(AR)auto-antibodiesweredetectedinpatientswithdilatedcardiomyopathy.

Manystudieshaveshownthat!1-ARauto-antibodieswithpartialagonist-likeeffectplayanimportant

roleinthepathogenesisofthisdisease.Moreover,arecentstudycarriedoutinourlaboratoryhasshown that!3-ARantibodies(!3-ABs),producedinrats,wereabletoreducecardiomyocytecontractilityvia

!3-ARactivation.Theaimsofthisstudywere(1)toinvestigate,inisolatedcardiomyocytesfromrabbit,

theroleofGiproteinsinthe!3-ABs-inducedcardiacnegativeinotropy,(2)todeterminewhether!3-ABs

mayexhibit!3-ARantagonisticpropertywhichischaracteristicofpartialagonists,and(3)todetermine

whetherlong-termactiveimmunizationproducingboth!1-ABsand/or!3-ABsleadstothedevelopment

ofcardiacdysfunctioninLewisrats.

Lewisratswereimmunizedfor6monthswithpeptidicsequencescorrespondingtothesecond extra-cellularloopofhuman!3-ARand/or!1-AR.Agonisticeffectof!3-ABswasevaluatedonelectrically

field-stimulatedisolatedcardiomyocytesfromadultrabbitbymeasuringthecellshortening. Echocar-diographyandexvivoisolatedperfusedheartstudieswereconductedonimmunizedrats.Finally,!-AR expressionwasquantifiedbyimmunofluorescenceandRT-qPCR.

SR58611A(10nM),apreferential!3-ARagonist,andpurified!3-ABs(25"g/ml)inducedadecrease

incellshortening(−39.71±4.9%(n=10)and−17.06±3.9%(n=10)respectively).Thiseffectwas signif-icantlyinhibitedwhenthecardiomyocyteswerepreincubatedwithpertussistoxin(0.3"g/ml),aGi

proteininhibitor(p<0.05).Inaddition,SR58611A-mediatednegativeinotropiceffectwasdecreased when cardiomyocytes were preincubated with !3-ABs (p<0.0001). Echocardiography revealed a

decreaseinthefractionalshorteningandejectionfractioninratsimmunizedagainst!1-ARandboth

!1-and!3-AR.However,thestudyonisolatedheartshowedadecreaseoftheisoproterenol-induced

lusitropicandinotropiceffectsinthe3groupsofimmunizedrats.Thesesystolicanddiastolicdysfunctions arecorrelatedwithadecreaseintheexpressionof!1-ARsandanincreaseof!3-ARsinratsimmunized

againstthe!1-ARandanincreaseofboth!3-ARand!1-ARinratsimmunizedagainstthe!3-AR.Forthe

firsttime,theseresultsshowedthat!3-ABshada!3-ARpartialagonist-likeactivitywhichmightplaya

roleinthepathogenesisofcardiacdysfunction.

Abbreviations: AB,antibody; AR,adrenoceptor;AAB, autoantibody;cAMP,

cyclicadenosinemonophosphate;DCM,dilatedcardiomyopathy;DP,developed

pressure;dP/dt,timederivativesofpressure;EDD,leftventricularend-diastolic

diameter; EDV, left ventricular end-diastolic volume; EF, ejection fraction;

ELISA, enzyme-linked immunosorbent assay; eNOS, endothelial nitric oxide

synthase;ESD,leftventricularend-systolicdiameter;ESV,leftventricular

end-systolic volume; FS, fractional shortening; IgG, immunoglobulin; MFI, mean

fluorescence intensity; NO, nitric oxide; OD, optical densities; PTX,

pertus-sistoxin;SR58611A,

[(RS)-N-[(25)-7-ethoxycarbonylmethoxy-1,2,3,4-tetrahydro-napht-2-yl]-(2R)-2(3-chlorophenyl)-2hydroethanaminehydrochloride].

∗ Correspondingauthor.

E-mailaddress:yassine.mallem@oniris-nantes.fr(M.Y.Mallem).

http://dx.doi.org/10.1016/j.phrs.2015.08.006

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E.Montaudonetal./PharmacologicalResearch100(2015)210–219

1. Introduction

Idiopathicdilatedcardiomyopathy(DCM) isoneof themain causeofsevereheartfailureinyoungadults.In60–70%ofcases,the etiologyremainspoorlyunderstoodandgrowingevidencessuggest thattheimmunitysystemmayplayakeyroleinthisdisease[1].

!1-Adrenoceptor (AR) auto-antibodies (AABs) were first

detected by ELISA (enzyme-linked immunosorbent) in 26 to 60% of patients with DCM [2,3]. These !1-AABs are directed

againstthesecondextracellularloopofhuman!1-AR[4].Invitro

and in vivo studies have shown that !1-AABs induce

posi-tive inotropicand chronotropic effects viathe!1-AR/adenylate

cyclase/cAMP-dependentproteinkinase-Apathway[5–8]. Accord-ingtoMagnussonetal.[7],these!1-AABssharesomepropertiesof

partialagonists.Theycanactasanagonistandactivateweaklythe !1-ARs.Onthecontrary,theycanactasanantagonistofthe!1-ARs

andblockthemwhenthecatecholaminelevelsarehigh. Further-more, rator rabbit immunizationwiththesecondextracellular loop of !1-ARhasbeenreported tobeable toinduce

myocar-dial dysfunctionthatmay leadtoventricledilatation similarto thatobservedinpatients[9,10].Thiseffectisconsideredtoresult fromlong-termoverstimulationofthe!1-ARsbythe!1-AABs.In

agreementwiththoseobservations,clinicalstudieshavealsofound that!1-AABssuppressionbyimmunoabsorptionimprovescardiac

functionofpatientswithDCM[11,12],strengtheningthe possibil-itythat!1-AABsplayanimportantpathophysiologicalroleinthe

developmentofthisdisease.

Morerecently,circulating!3-AABsdirectedagainstthesecond

extracellularloopofthe!3-AR,havealsobeendetectedin30%of

serafrompatientswithheartfailure[13].Arecentstudycarried outinourlaboratoryhasshownthat!3-ARantibodies(!3-ABs),

producedinrats,wereabletoreducecardiomyocytecontractility via!3-ARactivation[14].Nevertheless,veryfewstudieshavebeen

donetocharacterizetheseauto-antibodiesandtoevaluatetheir involvementinDCM.

In additionto!1-AR, !3-ARis alsoexpressedinthehuman

andanimalventriclemyocardium.Itsactivationwasdescribedto induceanegativeinotropiceffectthatinvolvedGiprotein/nitric

oxide(NO)pathway[15].Severalstudies,conductedinfailingor non-failing myocardiumhavereportedthat the!1-ARsand !3

-ARsarecross-regulatedbyinteractivecompensatorymechanisms [16–19].Theopposed changesinthe!1-ARand !3-AR-induced

myocardial contraction in response to adrenergic stimulation seemstoplayaroleintheworseningoftheheartcontractility. To the best of our knowledge, it is not known whether simi-lar regulationmay occurin response to !1-AABsand !3-AABs

that could possesssympathomimetic-like properties.Therefore, themainobjectiveofthisstudywastodeterminewhether long-termactiveimmunizationproducingboth!1-and/or!3-ABsleads

tothedevelopmentofcardiacdysfunctioninLewisrats.Moreover, consideringthe!3-ARs-mediatedpertussistoxin(PTX)-sensitive

effectintheheart[20]andthe!3-ARagonist-likeactivityofthe

!3-ABs[14],wefirstinvestigated,inisolatedcardiomyocytesfrom

rabbit,theroleofGiproteinsinthe!3-ABs-inducedcardiac

nega-tiveinotropyandwhether!3-ABsmayexhibit!3-ARantagonistic

property. 2. Methods 2.1. Animals

Whole experimental project was validated by local ethics committeeforanimalexperimentation(N◦_{CEEA.2012.76)}_and

con-ducted in accordance with“The guidefor the care and use of laboratoryanimals”publishedbytheNationalInstituteofHealth

(NIHpublication,eightedition,2010).Nineweek-oldmaleLewis rats from Janvier Labs (Le Genest Saint Isle, France) and New Zealand rabbits(2 kilograms) from Hypharm (Roussay, France) wereusedforthisstudy.Theywerehousedatconstant temper-ature(22±2◦_C)_and_subjected_to_a_cycle_of_dark/light_12:12_h,_with

standardchowanddrinkingwaterprovidedadlibitum. 2.2. Immunizationprotocol

Ratswereimmunizedbysubcutaneousinjectionsofan anti-gendissolvedin1mlofasolutioncontainingNa2CO3(0.1M)and

!-mercapto-ethanol1%,conjugatedwithFreund’sadjuvant(V/V) monthlyfor6months.Forthat,ratswererandomlydividedinto 4groups.Thefirstgroup(n=10)wasimmunizedwiththeantigen correspondingtothepeptidicsequenceofthesecondextracellular loop of human !1-AR (residues 197–222:

H-W-W-R-A-E-S-D-E-A-R-R-C-Y-N-D-P-K-C-C-D-F-V-T-N-R;1mg/ml)synthesizedby GeneCust (Dudelange, Luxembourg). The second group (n=10) was immunized with the antigen corresponding to the pep-tidicsequenceofthesecondextracellularloopofhuman!3-AR

(residues 176–200: R-V-G-A-D-A-E-A-Q-E-C-H-S-N-P-R-C-C-S-F-A-S-N-M-P; 2mg/ml) (GeneCust, Dudelange, Luxembourg). The thirdgroup(n=10)wasimmunizedwithboth !1-AR(1mg/ml)

and!3-AR(2mg/ml)peptidesandthelastgroup(n=10)

(adjuvant-treated)receivedonlysalineconjugatedwithFreund’sadjuvant (0.5ml).

2.3. ˇ1-andˇ3-adrenergicreceptorantibodiesdetectionand

purification

Serawerecollectedbeforethefirstimmunizationandafter2, 4and6monthsofimmunization.Theevolutionof!1-and!3-AB

titerswasfollowedbypeptide-basedELISA.

For that,!1-ARand !3-AR peptides(10"g/ml)used forthe

immunization were dissolved in BIC buffer (Na2CO3 0.1mol/l;

NaHCO30.1mol/l;indistilledwater;pH9.6)andcoatedona

96-wellmicroplate(polyNUNC,Denmark)overnightat4◦_C._Serum

dilutions(100"l)from1:400to1:51,200inPBS-Tween80-NaCl 0.5mol/l, wereusedtoreactwiththepeptidesfor 1hat37◦_C.

Afterwashing3timeswithPBS-Tween80,100"lofdonkey anti-ratimmunoglobulin(IgG)antibodyconjugatedwithhorseradish peroxidase (1:50,000 dilution in PBS-Tween 80-NaCl 0.5mol/l) (Jackson ImmunoResearch, USA) were added to the wells and incubated 1h at 37◦_C. _After ₃ _washings, ₁₀₀_"l _of _3,3$_, _5,5$

-tetramethylbenzidine(Sigma–Aldrich)wereincubatedat37◦_C_to

detecttheboundantibodies.Thereactionwasstoppedafter20min ofincubationbyadding50"lofsulphuricacid(0.1mol/l).Optical densities(OD)werereadat450nminamicroplatereader(TriStar, BertholdTechnologies,BadWildbad,Germany).Theantibodytiters weredefinedbytheODvalues.

IgGfractionswerepurifiedfromseracollectedafter6monthsof immunizationusingtheProteusProteinGkit(AbDSerotec,Colmar, France)incompliancewiththemanufacturer’sinstructions. Puri-fiedantibodyconcentrationsweredeterminedbythebicinchoninic acidproteinassay(Uptima,Interchim,Montluc¸on,France). 2.4. FunctionalcharacterizationofIgGscontainingˇ3-adrenergic

receptorantibodies

Thefunctionalityofpurified!3-ABswasevaluatedon

ventric-ularcardiomyocytesisolatedinhealthyrabbit,whichisknownto expressfunctional!3-ARs[21].Briefly,rabbitswereanesthetized

withpentobarbitone(54mg/kgIV) andheparinized (2500IU/kg IV).Cardiomyocyteswereisolatedbyperfusion(7ml/min)ofheart mountedona Langendorff apparatuswith1mg/ml collagenase typeII(Worthington,Lakewood,NI,USA)and0.04mg/mlprotease

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typeXIV(Sigma–Aldrich,France)(30"MCaCl2)for20min.After

gentlemanualstirring,cardiaccellswereprogressivelyexposed toincreasingCa2+_{concentrations}_in_100%_O₂_aerated_Tyrode

solu-tion(NaCl137mM;KCl5.4mM;MgCl21.2mM;Na2HPO41.2mM;

HEPES20mM;d-glucose10mM;pH7.4).Cardiomyocyteswere platedonpoly-l-lysine-coated35mmculturedishesandperfused ataflowrateof3–4ml/minwithTyrodesolution(1.8mMCaCl2)

at37◦_C._Cells_were_stimulated_by_an_electric_field₍₁_Hz)_at₉_V_and

cellshorteningwasrecordedusingavideo-imagingsystem (Coy-oteBayInstruments,Manchester,NH,USA).Imaginganalysiswas performedusingMatroxInspectorsoftware(CoyoteBay Instru-ments,Manchester,NH,USA).Myocytesselectedfordataanalysis presentedclearstriation,rod-shapedformanda stablediastolic lengthatbaseline.Tenconsecutiveheartcontractionswereused fortheanalysis.

2.5. Echocardiography

At6monthsofimmunization,ratswerelightlyanaesthetized with1–1.5%isofluraneuntiltheheartratestabilizedto350–400 beats per minute. Transthoracic echocardiographywas realized usingahighfrequencyultrasoundimagingsystem(MyLab70XVG, Esaote,Indianapolis,IN)withalinear18Mhztransducer.Thefocal length used was adjusted around 10mm. To identify whether the immunization leads to the development of functional and structuralcardiacdysfunctions,fractionshortening(FS),ejection fraction(EF),leftventricularend-systolicdiameter(ESD),left ven-tricularend-diastolicdiameter(EDD),leftventricularend-systolic volume (ESV) and left ventricular end-diastolic volume (EDV) weredeterminedusing M-Moderecordingsin theshort axisof the heart. ESV and EDV were calculated using the monoplane area-lengthmethod:V=8×A2_/3!L._FS_and_EF _were_respectively

calculated using theformulas FS=[(EDD−ESD)/EDD]×100and EF=[(EDV−ESV)/EDV]×100.Threeconsecutiveheartcycleswere measuredandtheaverageusedforanalysis.

2.6. Exvivocardiacfunction

At 6 months of immunization, half of immunized rats were anaesthetized by intraperitoneal injection of pentobarbi-tone (54mg/kg) and heparinized (2500IU/kg) (n=5, for each group). The heart was immediately removed and placed into ice-cold Krebs–Henseleit buffer (NaCl 118.3mM; KCl 4.7mM; MgSO4,7H2O 1.2mM; KH2PO4 1.2mM; NaHCO3 20mM; EDTA

0.016mM;Glucose 11.1mM;CaCl2 2.5mM; pH7.4). The aorta

wasrapidly cannulatedand theheart retrogradely perfused by a non-recirculating-Langendorfftechniqueat aconstant flowof 10ml/min in continuously gassed (95%O2/5%CO2) prewarmed

(37◦_C)_KH_buffer. _To_determine_left_ventricular_function,_a_latex

balloonwasinsertedintotheleftventriclethroughthemitralvalve andfilleduntiltheleftventricularend-diastolicpressurereached avalueof5mmHg.Anequilibrationperiodofatleast30minwas requiredtoensurethestabilityofrecordedparametersbeforeany additionofmolecule.Foreachimmunizedrat,systolic,diastolic, developedpressuresandtheheartratewererecordedinitiallyand aftertheadditionof(–)-isoproterenolhydrochloride(100nM).Left ventriculardevelopedpressure(DP)wasdeterminedasthe differ-encebetweenleftventricularsystolicpressureandleftventricular end-diastolicpressure.Timederivativesofpressurewere calcu-latedelectronicallyduringcontraction(dP/dTmax)andrelaxation (dP/dTmin)usinga Powerlab8/30Data Acquisitionsystemand analyzedbyLabChart®_Pro_software_(V7,_{ADInstruments,}_France).

2.7. RealtimequantitativeRT-PCR

Theotherhalfofimmunizedrats(n=5,foreachgroups)were usedtoperformrealtimeRT-qPCRtoevaluatetheeffectof immu-nizationsonthemRNAlevelsof!1-AR,!2-ARand!3-ARinthe

heart.Briefly,heartfragmentswererapidlyfrozeninliquid nitro-genandstoredat−80◦_C._Frozen_samples_were_dissolved_in_1.5_ml

ofTRIzol®_Reagent_and_ground_to_powder_in_a_potter,_according_to

manufacturer’sinstructions(LifeTechnologies,France).TotalRNA wereobtainedafterchloroformextractionandisopropanol precip-itationandresuspendedin50"lofRNase-freewater,heatedat 58◦_C_and_stored_at₋₂₀◦_C._The_total_RNA_integrity_was_checked_by

agarosegeleletrophoresis.TotalRNAconcentrations,260/280and 260/230ratiosweredeterminedbyspectrophotometry(Nanodrop 2000,ThermoScientific,France).

ToremoveanycontaminatinggenomicDNA,aDNasedigestion step(DNaseIrecomb,RocheDiagnosticsFrance,France)was car-riedoutaccordingtomanufacturer’sinstructions.DNA-freetotal RNAwererecoveredin20"lofRNase-freewater.Absenceof con-taminating DNA fragments was again verified with a sensitive GAPDHPCR(primersinTable1)andwithagarosegel electrophore-sis.DNA-freetotalRNAconcentrationsandDOratioswerethen determinedbyspectrophotometry.

Each sample underwent the following reverse transcription step:1"gofDNA-freetotalRNAwasusedin20"ltotalofmix solutionbasedontheSuperscriptIVReverse-Transcriptasekit(Life Technologies,France),withboth1"lofRandomPrimers(3"g/"l) and1"lofOligo(dT)20Primer(50"M)forRT.RetrotranscribedRNA

efficiencywasfurthervalidatedwithanewGAPDHPCRandwith agarosegelelectrophoresis.

RealtimePCRreactionsweresetin15"lwith5XHOTFIREPol®

EvaGreen® _qPCR _Mix _(high _ROX) _(Solis _BioDyne, _Riia, _Estonia),

includingeither5"lofdilutedcDNA(1:6)and1"lofeachprimers (10"M).Afteraninitialdenaturation/DNApolymeraseactivation step of 15min at 95◦_C, _the _PCR _forty _cycles _consisted _of ₁₀_s

at95◦_C_denaturation_step_and_of₁_min_at₆₀◦_C_(for_ADRB-1and

ADRB-2primers)or61◦_C_(for_ADRB-3_and_eNos_primers)

hybridiza-tion/elongationstep.Meltingcurveswereproducedtoconfirmthe presenceofasinglegenespecificpeakandtheabsenceofprimer dimers.Thequantificationofgeneexpressionwasbasedonthe 2−""Ct_method_[27]_._Briefly,_the_mean_Ct_value_of_the_control

house-keepinggene!-actinwassubtractedfrommeanthresholdcycle valuesofduplicatesforeachinterestmRNA.Thedifferenceinthe mean"Ctvaluesbetweenadjuvant-treatedratsand!-immunized ratsallowsthecalculationofrelativelevelsofinducedorrepressed expressionoftheinterestgene.

2.8. Immunofluorescenceandquantificationofˇ-adrenergic receptorexpression

RatheartusedfortheRT-qPCRwerealsousedtoquantify!-AR expressionin theheart (n=5,foreach groups).Forthat, imme-diately aftertheexcision,heartwastransversallysectionedand themiddlesectionwaspost-fixedbyincubationin4% phosphate-bufferedparaformaldehyde(pH7.4)for4hat4◦_C._After₃_washings

withPBSsolution,itwascryoprotectedbyovernightincubationin PBScontaining20%sucrose.Then,heartsectionwasembeddedin transversepositioninTissueTekOCTmediumandimmediately frozenbyimmersioninliquidisopentane.Frozensections(8"m thick) forimmunohistochemicalanalysiswerethawedand sub-mergedfor5mininacetoneat4◦_C_and_nonspecific_antigen_binding

wasblockedbyincubatingsectionsfor1hinaPBSsolution con-taining 0.3%Triton100Xand 2%bovineserumalbumin(Sigma) followingby1hat37◦_C_incubation_in_the_blocking_buffer_with_a

rabbitpolyclonalantibodyagainst!1-AR(1:50,SantaCruz

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Table1

Oligonucleotideprimersusedinthestudy.Oligonucleotidesequenceorientationsareindicatedasforward(Fw)andreverse(rv).

Primername Accession num-ber/publication Sequence(5$_–₃$₎ _Ampliconsize_(bp) ADRB-1-Fw ADRB-1-Rv NM012701 [22] CTGCTACAACGACCCCAAGTG AACACCCGGAGGTACACGAA 120 ADRB-2-Fw ADRB-2-Rv NM012492 Ourdesign CTCCTTAACTGGTTGGGCTATG TCCCATAGGTTTTCGAAGAAGA 127 ADRB-3-Fw ADRB-3-Rv NM013108 [23,24] GCCGAGACTACAGACCATAACCA CATTACGAGGAGTCCCACTACCA 79 eNos-Fw eNos-Rv NM 021838 [25] AGCTGGATGAAGCCCGGTGAC CCTCGTGGTAGCGTTGCTGA 60 B-Act-Fw B-Act-Rv NM007393 Ourdesign TTGCTGACAGGATGCAGAAG GTACTTGCGCTCAGGAGGAG 86 GAPDH-Fw GAPDH-Rv AF106860 [26] ACTGGCGTCTTCACCACCATGGAGAAGGCT CTCCTTGGAGGCCATGTAGGCCATGAGGTC 720

(1:100,SantaCruzBiotechnologyInc.,CA,USA)for1hat37◦_C._After

beingwashedinPBS,thesectionswereincubatedwiththe sec-ondaryantibody,AlexaFluor555-conjuguateddonkeyanti-rabbit (1:300,LifeTechnologies,SaintAubin,France)ordonkeyanti-goat (1:300,LifeTechnologies,SaintAubin,France)during1hatroom temperature.Afterbeingwashed,thesectionswerestainedwith nucleardyeDRAQ5(1/1000,BioStatus,Shepshed,UnitedKingtom) and mountedin Mowiol(Calbiochem,SanDiego,CA,USA). The immunolabeled sectionswerescannedseriallyusingthehelium neonlaser(543nm)toobserveAlexafluor555(!-AR immunola-bellings)andwithaheliumneonlaser(633nm)toobserveDRAQ5 signals(nuclei).Eachimagewasrecordedinaseparatedchannel (channelredforAlexafluor555andchannelblueforDRAQ5)and overlayed.Acquisitionswereperformedbyusingaconfocal micro-scope(Nikon,C1,Champigny-sur-Marne,France).Imageanalysis wereperformedtoevaluate!-ARexpressionlevelintheheart ven-tricleofimmunizedratsbyusingFijisoftware.MeanFluorescence Intensity(MFI)valuesfor!-ARimmunolabellingineachcondition wereacquiredfrom5differentfieldsofimmunolabeledventricule heartsectionsbysectionwith5ratsbyconditions.Finally, analy-seswereperformedatleaston550±100cardiacfibersbyanimal. Thesamethresholdwasusedtomeasurethesumintensity fluo-rescenceof!-ARimmunolabellingineachsectionandtheMFIwas reportedtototalareaofanalyzedsection.

2.9. Drugs

(–)-Isoproterenol hydrochloride, L-748337 hydrate, pertussis toxin from Bordetella pertussis were obtained from Sigma–Aldrich (France). SR58611A [(RS)-N-[(25)-7-ethoxycarbonylmethoxy-1,2,3,4-tetrahydro-napht-2-yl]-(2R)-2(3-chlorophenyl)-2hydroethanamine

hydrochloride] was agenerous giftfrom Sanofigroup (France). Alldrugswerepreparedindistilledwaterwiththeexceptionof SR58611A that wasdissolvedin ethanol. Sodiumpentobarbital solution was purchased from CEVA Santé Animale (Libourne, France)andheparinechoayfromSanofiAventis(Paris,France). 2.10. Statisticalanalysis

Resultswereexpressedasa mean±SEMwherenrepresents thenumberofratsorrabbitsusedforthestudy.Statistical differ-encesincomparisonwiththeadjuvant-treatedratswereevaluated usingunpairedStudent’sttestwithPrism®_software_V.5._The

con-ditionsforthet-testweretestedwithaKolmogorov–Smirnov’test fortheGaussiandistributionandwithanF-testforthe homogene-ityofvariances.Fortheexvivostudy,differencesweredetermined usingnonparametricMann-Whitneytestandforthe

immunoflu-orescenceanalysisusingaunivariateStudent’sttest(Rsoftware V3.0.1).Avalueforp<0.05wasconsideredstatisticallysignificant. 3. Results

3.1. ˇ–adrenoceptorantibodyproductionandcharacterization !1-ABsand!3-ABsweregeneratedbyimmunizingratsfor6

monthsandtheirrespectiveimmunoreactivitiesweredetermined byELISA.Twomonthsafterthefirstimmunization,weobserveda highproductionofspecific!1-ABsinratsimmunizedwith!1-ARor

!1-and!3-ARpeptides(Fig.1).Afterthe6monthsof

immuniza-tion,!1-ABtiterswerestillhighandstablecomparedtothelow

levelofproductioninantibodytitersin!3-AR-immunizedratsand

adjuvant-treatedrats(ODat6months:2.09±0.24and2.30±0.21 for!1-ARand!1-and!3-AR-immunizedratsrespectivelyversus

0.07±0.005and0.08±0.03foradjuvant-treatedratsand!3

-AR-immunizedratsrespectively).Inthesameway,weobservedahigh increaseof!3-ABtitersinboth!3-AR-immunizedratsand!1-and

!3-AR-immunizedratsafter6monthsofimmunizationcompared

withthe!1-AR-immunizedratsandadjuvant-treatedrats(ODat

6months:1.37±0.26and2.28±0.18for!3-ARand!1-and!3

-AR-immunizedratsrespectivelyversus0.07±0.006and0.14±0.02 foradjuvant-treatedratsand!1-AR-immunizedratsrespectively)

(Fig.1).Thedatahighlightedantibodyspecificityfortheirantigen andthelackofcross-reactivitybetweenthetwosubtypesof!-AR. 3.2. Negativeinotropiceffectofˇ3-adrenoceptorantibody

Todetermine whether!3-ABs exhibit an agonistic effect as

itwasalreadydescribed for!1-ABs [8],wemeasuredtheeffect

of!3-ABperfusiononelectricallyfield-stimulated cell

shorten-ing.!3-ABsinducedadecreaseofcellshortening(−17.06±3.9%,

n=10)andthis effectwassignificantly inhibitedinpresence of PTX(0.3"g/ml), a Gi proteininhibitor, and L-748337 (1"M), a

selective!3-ARantagonist(#<0.05)(Fig.2a).SR58611A(10nM),a

preferential!3-ARagonistproducedalsoasignificantdecreaseof

cellshorteningwhichwasstrongerthanthatinducedby!3-ABs

(−39.71±4.9%,n=10 and −3.88±3.4%,n=7, respectively).This negativeinotropiceffectwasnotobservedwhencardiomyocytes wereperfusedwiththeantibodiesfromadjuvant-treatedrats.

Then,assumingthat!3-ABsbehavedaspartial!3-ARagonists,

wetestedtheirability toexhibit !3-ARantagonisticproperties.

Incubationofrabbitcardiomyocytesfor2hwith!3-ABs(25"g/ml)

significantly reduced the SR58611A-induced negative inotropy (−7.22±3.8%,n=4)incomparisonwithantibodiesfrom ajduvant-treatedrats(−27.47±8.23%,n=7)(Fig.2b).

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Fig.1. Curvesshowingtheevolutionof!1-adrenoceptor(!1-AR)(a)and!3-adrenoceptor(!3-AR)(b)antibodytitersdeterminedbyELISAatdilution1/1600,inseraof

adjuvant-treatedrats(emptycircles,n=10),!1-AR-immunizedrats(emptysquares,n=10),!3-AR-immunizedrats(fullcircles,n=10)and!1-ARand!3-AR-immunized

rats(fullsquares,n=10).Antibodytitersaredefinedasmean±SEMofopticaldensityvalues,***p<0.0001versusadjuvant-treatedgroupdeterminedbyunpairedStudent’s

t-testatthedifferenttimes.

Fig.2. Effectsof!3-adrenoceptor(!3-AR)antibodiesonrabbitisolatedcardiomyocytecontractility.(a)Negativeinotropiceffectof!3-ARantibodies(25"g/ml)without

(blackbar,n=11)orwithpreincubation(2h)inthepresenceofpertussistoxin(0.3"g/ml)(n=4)orL-748337(1"M)(n=4),SR58611A(10nM)(whitebar,nn=10)and

adjuvantantibodies(25"g/ml)(greybar,n=7).(b)NegativeinotropiceffectofSR58611A(10nM)withpreincubation(2h)inthepresenceof!3-ARantibodies(25"g/ml)

(blackbar,n=6)oradjuvantantibodies(25"g/ml)(greybar,n=7).Valueswererepresentedasmean±SEM,*p<0.05,***p<0.0001versuscellsthatwerenotpreincubated,

determinedbyunpairedStudent’st-test.

3.3. Influenceofimmunizationsoncardiacfunction—invivo study

Toevaluatewhetherthedifferentimmunizationswereableto impactthecardiaccontractility,weperformedechocardiography. DataindicatedthatEFandFSweredecreasedinratsimmunized during 6 months with!1-AR (EF: −3.7% and FS: −5.1% versus

adjuvant-treated rats)or both !1/3-AR peptides (EF:−5.6% and

FS: −7.8% versus adjuvant-treated rats) compared to adjuvant-treatedrats(EF:77.50±0.47;FS:47.53±0.48)(Fig.3).TheEDDand ESDwerenotalteredbytheimmunizationin!1-AR-immunized

rats while in!1/3-AR-immunizedrats both EDDand ESD were

increased(EDD:+4.02%andESD:+11.13%versusadjuvant-treated rats)(Fig.3).Incontrast,immunizationwithonly!3-ARpeptide

didnotaltertheEFandFSbutincreasedtheEDD(+2.73%versus adjuvant-treatedrats)(Fig.3c).Moreover,systolicbloodpressure wasnotmodifiedduringthe6monthsofimmunizationinthe4 groups ofrats(datanotshown)suggestingthat theseobserved effectsoncardiaccontractilitywerenotduetochangesinarterial pressureanddidnotimpactbloodpressure.

3.4. Influenceofimmunizationsoncardiacfunction—exvivo study

The influence of chronic immunization on left ventricular contractilitywasalsoevaluatedexvivousinga non-recirculating-Langendorfftechnique.Foreachgroupofrats,systolic,diastolic, developed pressures and the heart rate were recorded under

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Fig.3. Influenceofimmunizationwithpeptidescorrespondingtothesecondextracellularloopof!1-adrenoceptoror!3-adrenoceptororboth!1-and!3-adrenoceptorand

withadjuvantoncardiacfunction.(a)Ejectionfraction(EF),(b)fractionalshortening(FS),(c)leftventricularend-diastolicdiameter(EDD),(d)leftventricularend-systolic

diameter(ESD).Valueswererepresentedasmean±SEM,*p<0.05,**p<0.001versusadjuvant-treatedrats,n=5ratsforeachgroups.

basalconditionsandaftertheadditionofisoproterenol(100nM). The data showed that isoproterenol increased the heart rate, the maximal and minimal first derivative of change in pres-sureovertime(dP/dt(max/min))andthedevelopedpressureof heartsfromadjuvant-treatedrats.Theseeffectsweredecreased inheartsfromratsimmunizedwith!1-ARor!3-ARor!1/3-AR

peptides (Fig.4).Given that heart contractilityat baseline was not altered by immunizations(data not shown),these findings revealedthatimmunizationswitha!-ARpeptide(!1-ARand/or

!3-AR)decreasedtheisoproterenol-inducedlusitropy(dP/dtmin)

andtheinotropyparameters(dP/dtmaxanddevelopedpressure). We showedthat!1-ARand !3-AR-immunizationshadasimilar

outcomeonheartfunction.

3.5. Influenceofimmunizationsonˇ-adrenoceptormRNAand expression

RT-qPCRwerecarriedouttodeterminewhetherchronic immu-nizations modifiedcardiac mRNAlevel of!-ARs.Thedata have shown that !1-AR-immunization induced a slight increase of

!1-AR transcripts compared to adjuvant-treated rats whereas

!2-AR, !3-AR and eNOS (endothelial NO synthase) transcripts

seemed tonot be changed. A similar transcription profile was alsoobservedin!1/!3-AR-immunized.The!3-AR-immunization

induced a slight increase of !1-AR, !3-AR transcripts but no

modification of the !2-AR and eNOS mRNA levels (Fig. 5). To

determine theexpression profileof the !1-ARand !3-AR

pro-teins on heart, immunofluorescence labeling were carried out. First, themembranelocalizationof!–ARs wascheckedby per-forming a co-labeling of !-AR/Dystrophin B to confirm the antibodyspecificity.Theco-labelingresultsshowedthat!1-ARs

and !3-ARs were expressed at the cell membrane in heart of

adjuvant-treatedrats(Fig.6).Thedataonexpression!1-ARand

!3-AR profile were generally in accordance with the

observa-tionsonmRNAlevels(Fig.7).The!1-AR-immunizationreduced

!1-AR expression (−11.89±9.74%; n=5) but increased that of

!3-ARs(+51.42±15.93%;n=5)whereasthe!3-AR-immunization

increasedboth !1-ARand !3-ARexpressions (+89.06±20.69%;

n=5and123.60±28.04%;n=5,respectively). 4. Discussion

Thepresentstudyclearlydemonstratedthat!3-ABs purified

from!3-AR-immunizedratsinduceda!3-ARpartialagonist-like

activityonisolatedrabbitcardiomyocytes.Inaddition,byexvivo approach,werevealedthatratimmunizationsfor6months pro-ducingfunctional!1-ABsand/or!3-ABsledtothedevelopmentof

bothsystolicanddiastolicdysfunctions.

Inthiswork,peptide-basedELISAmethodhasallowedtotest thespecificityofthe!3-ABsforthesecondextracellularloopofthe

!3-AR.Immunizedratshaveproducedhighconcentrationof!1-AR

and!3-ARIgG-likeABs.Inaddition,!3-ABsdidnotcross-reactwith

!1-ARsthatshareahighdegreeofstructuralandbiochemical

simi-laritieswith!3-ARs.AccordingtoBornholzetal.[2],!1-AABsfrom

patientswithDCMpreferentiallyrecognizeanative!1-AR

confor-mation.Therefore,thepeptide-basedELISAmethodseemsnottobe themostappropriatemethodtodetecthuman!1-AABs.However,

inthispresentwork,theuseofpeptidestodetectantibodies gen-eratedbyimmunizationinratswasnotanissuebecauseweused thesamepeptidesforboththeimmunizationandthedetectionof antibodies.

Wepreviouslyshowedthat!1-ABsproducedbyimmunization

ofratswith!1-ARpeptideinducedapositiveinotropiceffect[8].

Inordertotestwhether!3-ABspossessalsobiologicalactivity,

weperformedinotropic studyoncardiomyocytesisolatedfrom adulthealthyrabbit.The!3-ABs-inducedpertussistoxin-sensitive

effectstronglysuggeststhatthe!3-ABsareendowedwithnegative

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Fig.4.Cardiacresponsestoisoproterenol(100nM)inadjuvant-treatedrats(n=5)

andratsimmunizedwithpeptidescorrespondingtothesecondextracellularloopof

!1-adrenoceptor(AR)(n=5)or!3-AR(n=5)orboth!1-ARand!3-AR(n=5).Heart

rate(HR),timederivativesofpressureduringcontraction(dP/dtmax)and

relax-ation(dP/dtmin)anddevelopedpressure(DP)wererepresentedasmean±SEMof

percentageincreasefrombaselinewithsetat100%,*p<0.05versusadjuvant-treated

rats,determinedbynonparametricMann–Whitneytest.

Fig.5.ComparativeanalysisofthemRNAlevelsof!1-adrenoceptor(AR),!2-AR,!3

-ARandendothelialnitricoxidesynthase(eNOS)inheartfromadjuvant-treatedrats

(greybars),!1-AR-immunizedrats(blackbars),!3-AR-immunizedrats(whitebars)

andboth!1-and!3-AR-immunizedrats(strippedbars).Valueswereexpressedas

2−""Ct_using_!-actin_as_a_housekeeping_gene_and_were_represented_as_mean_±_SEM. pathway.Inaddition,thedecreaseofSR58611-inducednegative inotropyaftertheincubationofcardiomyocyteinpresenceof!3

-ABsindicatesthatthe!3-ABscanalsoantagonizethe!3-ARs.Taken

together,theseresultsareinfavorofapartialagonist-likeactivity ofthe!3-ABsonheart.

Theimmunizationprotocolusedtoproducespecificantibodies behaving likeAABs has beenalready applied tostudy the car-diostimulating effects of !1-AABs in rat and rabbit [8,28]. Our

findingsagreewitharecentstudyshowingthat!3-AABspurified

frompatientswithDCMinducednegativeinotropicandnegative chronotropiceffectsonratneonatalcardiomyocytes[29]. There-fore,itmightbearguedthat!3-ABsaswellas!1-ABswouldnot

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Fig.6. Immunofluorescentco-labelingof!1-adrenoceptors(ARs)or!3-ARsanddystrophinBinheartfromadjuvant-treatedrats.

Fig.7. Influenceofimmunizationwithpeptidescorrespondingtothesecondextracellularloopof!1-adrenoceptor(AR)(n=5)or!3-AR(n=5)on!1-AR(whitebars)and!3

-AR(blackbars)expressionsinheartcomparedtoadjuvant-treatedrats.Valueswererepresentedasmean±SEMofpercentageincreaseoffluorescencefromadjuvant-treated

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bejustabiomarker,asstatedbyMiaoetal.[30],butwouldhavea functionalrolewhichneedsmorethoroughinvestigation.

Toevaluatewhethercirculating!1-ABsand!3-ABshaving

ago-nisticpropertieswereabletoimpactthemyocardialfunction,rats wereimmunizedduring6monthswith!1-ARand/or!3-AR

pep-tides.

Asitwasalreadydescribed[31,32],ourresultsshowedaslight but a significantdecrease of fractional shortening and ejection fraction in rats immunized 6 months with the !1-AR peptide

without modifications of systolic arterialpressure, assessed by tailcuffmethod.Theseobservationswereconcordantwith pre-viousstudiesshowing thatleft ventricledysfunctions appeared after6monthsofimmunizationagainst!1-AR[9,10].However,

inourstudy,theinabilityof!1-ABstoinducethedevelopment

ofleftventricledilatation(i.e.,nomodificationofend-systolicand -diastolic diameters) is likely linked to thefact that 6 months ofimmunizationisnotsufficienttoobservenoticeablealteration inmyocardialcontractility.Inaccordancewithechocardiographic results,ourexvivofindingsalsorevealedbothsystolicand dias-tolic dysfunctions in rats immunized with the !1-AR or both

!1-and !3-ARpeptides.Thesedysfunctionswerecharacterized

by a reduction of isoproterenol-induced inotropy and lusitropy that likely involved an impairment of global !-AR reactivity (i.e., modification in !-AR expression or !-ARs-linked mecha-nisms). In linewiththis hypothesis, thequantification of !-AR expression by immunofluorescence revealed a downregulation of!1-ARsandanup-regulationof!3-ARsin!1-AR-immunized

rats when compared with theadjuvant-treated rats in the left ventricle. Thispatternof regulationwasalready reportedtobe involved in the alteration of myocardial contractility and was similar to that observed in patients with DCM [16,17]. Sur-prisingly, the !1-AR mRNA was not decreased and the !3-AR

mRNAwasnotsignificantlyincreasedin!1-AR-immunizedrats.

The reason of this discrepancy is not readily apparent in our study,butthechangeinthepost-transcriptionalorinthe post-translational mechanisms could explain the lack of correlation betweenmRNAandproteinexpressionswhichremainstobe elu-cidated.

Onepossibleexplanation toaccountforthecross-regulation of the !1-ARsand the!3-ARs in ourstudy is that !1-ABs, by

theirchronic!1-ARagonisticactionfollowingimmunization,could

desensitize !1-ARsand could upregulate!3-ARs asa

compen-satorymechanism.Somestudieshavedescribedthelossof!1-ARs

inresponseto!1-AABs[33]ortheexistenceofcross-regulation

between!1-ARsand!3-ARsintheheartwhen!1-ARsare

chron-ically stimulated[34].Theupregulationof!3-ARsisconsidered

usefulinthefirststageoftheheartfailurebecauseofitsprotective effectbyantagonizingthe!1-ARoverstimulation.

Similarlyto!1-AR-immunizedrats,exvivostudyhasrevealed

thedevelopmentofbothdiastolicandsystolicdysfunctionsin!3

-AR-immunizedrats,which werecharacterizedbya decreaseof isoproterenol-inducedinotropyandlusitropy.Thereby,weshowed in this study that theproduction of cardio-stimulatory !1-ABs

or cardio-inhibitory !3-ABs byimmunizing ratswith !1-ARor

!3-ARpeptidehad asimilaroutcomeonheartfunction.In rats

immunizedwith!3-AR peptide,theimmunofluorescenceassay

andRT–qPCRhavehighlightedanupregulationof!3-AR

expres-sionandmRNAintheleftventricle.Thus,itisnotunreasonable topostulatethatisoproterenol-mediatednegativeinotropiceffect via!3-AR activationcouldhave contributedtotheimpairment

ofcardiacfunctionin!3-AR-immunizedrats.Ideallytheeffetof

isoproterenol in thepresence of !3-AR antagonist shouldhave

beenperformedtoaccuratelyconfirmtheroleofthe!3-ARsin

theimpairmentofisoproterenol-inducedinotropy.Inourstudy, sinceeNOShasbeenshowntoplayaroleinthe!3-ARs-mediated

negativeinotropy[15],wetestedthehypothesisthatitsincreased

expressionthrougha largerproductionofNOcouldexplainthe depressedcontractilityobservedin!3-AR-immunizedrats.

Nev-ertheless,thelackofchangeofeNOSmRNAintheseratsargues against a roleof theGi protein/NO pathwaytoaccountfor the

reduced responsetoisoproterenolandsupportstheviewofthe existenceofalternativemechanisms(i.e.,reducedcAMPlevelvia Giproteins)thatneedfurtherinvestigation.

Althoughthepresentstudyhasnotspecificallyaddressedthe potential mechanism involved in the upregulation of ventricu-lar !3-ARs, sustainedactivation of!3-ARsby !3-ABsfollowing

long-term immunization could be considered as one possible explanationofthispatternofregulation.Inaccordancewiththis contention, recent studies have shown that stimulation of the !3-ARsinducedanincrease ofthe!3-ARexpression[35,36].In

addition,itwasshownthatanupregulationof!3-ARswasable

toaltercontractileresponsetoisoproterenolinmice[37]andin humanfailingmyocardium[16].Nevertheless,thosefindingsdo notagreewiththepreviousreport[29,38]showingtheprotective role of!3-AABsagainstcardiacdysfunction,anddonotsupport

the pointof viewthat !3-ARupregulation mayberegarded as

cardioprotectivemechanismthatmaybedevelopedtoprevent car-diomyocytedamage.

More interestingly,thedysfunctionsobservedexvivo inrats immunizedagainstthe!3-ARwerenotdetectedbyour

echocardio-graphicmeasurements.Weshowedthat6monthsofimmunization with!3-ARpeptidedidnotmodifythefractionalshorteningand

ejection fraction. The lack of correlation between ex vivo and echocardiographic parameters has already been reported [39]. In our study,thereason of this discrepancy is notclear, butit may be explained by theintervention of compensatory neuro-hormonalmechanismsthatmightoperateinvivothrough!1-ARs

to maintainmyocardial contractility.This emphasizesthe need totakeintoaccountthephysiologicalcontextintheinterpreting and theevaluationof leftventricularfunction.Moreover,based on our results, we cannot rule out the hypothesis that under invivoconditions,circulating!3-ABscouldhaveexhibiteda!3

-antagonisticpropertyinresponsetoendogenouscatecholamines that mightbe responsible for undetectablecardiac dysfunction implying!3-ARcomponent.However,whetherprolonged

immu-nizationperiodwith!3-ARpeptidecouldinduceimpairmentof

echocardiographic contractile parameters remains to be deter-mined.

Therearesomelimitationsofthisstudy.First,concerningthe functional study of !3-ABs, despite that rabbit cardiomyocytes

constitute a relevant approach to assess in vitro effects of !3

-ABs, translation toinvivoor totheclinicalsetting shouldonly madewithcaution.The!3-ABsproducedbyimmunizationinrats

couldhaveadifferentactionthanthatof!3-AABsfrompatients

withDCMwhich needsfurtherinvestigationinthefuture. Sec-ond,concerningtheinfluenceofimmunization,theimmunization time of6monthshasbeenselectedbecauseZuoetal.[9] have reportedthattheleftventriculardilationappearedfrom5months of immunization with!1-AR peptide.However, in viewof our

results,itseemsthattheputativeeffectof!3-ABsoncardiac

func-tionisamoreslowlyprocessandhence,theimmunizationtime would need tobeincreased in thefuture. Third,in contrastto the exvivo experiments,ourechocardiographicapproach failed tofindanyevidenceofcardiacdysfunctionin!3-AR-immunized

rats.Thissuggeststhatmeasurementsoffractionalshorteningand ejection fractionwould not bealways suitablefor the evaluat-ingofheartcontractilityunderbasalinvivoconditions.Therefore, future experiments using exogenous inotropic agents (i.e., iso-proterenol) willberequireedtoaccurately quantifythechange in the myocardial contractility that may occur in immunized rats.

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5. Conclusions

Theresultsofthepresentstudyshowedforthefirsttimethat !3-ABs induceda!3-ARpartialagonist-likeactivityinvolvinga

roleofGiproteinsinisolatedrabbitcardiomyocytes.Inaddition,

weshowthatimmunizationsfor6monthsproducingfunctional !1-ABsor!3-ABsledtothedevelopmentofsystolicanddiastolic

dysfunctionsinheartbyremodelingthe!1-AR/!3-ARratiointhe

leftventricle. Conflictsofinterest

None.

Acknowledgements

Theauthorswould liketothankChantalThorinforher help concerningthestatisticalanalysisandMireilleLedevinandSonia Becavinforthetechnicalassistance.

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