Carbamylated erythropoietin enhances mice ventilatory responses to changes in O2 but not CO2 levels

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Carbamylated erythropoietin enhances mice ventilatory responses to changes in O2 but not CO2 levels

Hanan Khemiri, Marc Maresca, Christian Gestreau

To cite this version:

Hanan Khemiri, Marc Maresca, Christian Gestreau. Carbamylated erythropoietin enhances mice

ventilatory responses to changes in O2 but not CO2 levels. Respiratory Physiology and Neurobiology,

Elsevier, 2016, 232, pp.1 - 12. �10.1016/j.resp.2016.06.002�. �hal-01475546�

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Contents lists available atScienceDirect

Respiratory Physiology & Neurobiology

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / r e s p h y s i o l

Carbamylated erythropoietin enhances mice ventilatory responses to changes in O 2 but not CO 2 levels

Hanan Khemiri

^a

, Marc Maresca

^b

, Christian Gestreau

^a,∗

aAixMarseilleUniv,CNRS,CRN2M,Marseille,France

bAixMarseilleUniv,CNRS,CentraleMarseille,iSm2,Marseille,France

a r t i c l e i n f o

Articlehistory:

Received26May2016

Receivedinrevisedform14June2016 Accepted15June2016

Availableonline16June2016

Keywords:

Erythropoietin Carbamylatedderivative Breathing

Chemoreﬂexes Plethysmography

a b s t r a c t

Erythropoietin(EPO)hasbeneﬁcialtissue-protectiveeffectsinseveraldiseasesbuterythrocytosismay causedeleteriouseffectsinEPO-treatedpatients.Thuscarbamylated-EPO(C-EPO)andotherderivatives retainingtissue-protectivebutlackingbonemarrow-stimulatingactionshavebeendeveloped.Although EPOmodulatesventilatoryresponses,theeffectsofC-EPOonventilationhavenotbeeninvestigated.

Here,basalbreathingandrespiratorychemoreflexesweremeasuredbyplethysmographyafteracute andchronictreatmentswithrecombinanthumanC-EPO(rhC-EPO;15,000IU/kgduring5days)orsaline (controlgroup).Hematocrit,plasmaandbrainstemrhC-EPOlevelswerealsoquantified.ChronicrhC- EPOsignificantlyelevatedtissuerhC-EPOlevelsbutnothematocrit.Noneofthedrugregimenaltered basalventilation(normoxia).ChronicbutnotacuterhC-EPOenhancedhyperoxicventilatorydepression, andsustainedthehypoxicventilatoryresponsemainlyviaareductionoftheroll-offphase.Bycontrast, rhC-EPOdidnotblunttheventilatoryresponsetohypercapnia.Thus,chronicC-EPOmaybeapromis- ingtherapytoimprovebreathingduringhypoxiawhileminimizingadverseeffectsoncardiovascular function.

1. Introduction

Erythropoietin(EPO)playsamajorroleinoxygen(O₂)homeo- stasisin mammals.Thishormone ismainlysynthetizedin fetal liverandadultkidneys,and itsbasal levelisupregulated when thearterialPO₂decreasesbelownormallevels(Becketal.,1991;

Haase,2013;Jelkmann,1992).HigherplasmaEPOlevelsstimulate redbloodcellsproduction,therebyincreasingthebloodO2 car- ryingcapacity.Accordingly,theuseof recombinanthumanEPO (rhEPO)inpatientswithsevereanemiaand/orchronickidneydis- easeshoweda reduced requirementfor bloodtransfusions and improved quality-of-life assessments (Canadian Erythropoietin Study Group, 1990; Eschbach et al., 1989; Kato, 2010; Pfeffer et al., 2009). Besides its erythropoietic effect, EPO has tissue- protective properties in a broadrange of pathologies including hypoxiaandischemia-inducedbraindamages(Chateauvieuxetal., 2011;Jelkmann,2007;Kumraletal.,2011;Sanchis-Gomaretal.,

∗ Corresponding author at: Aix−Marseille Université and CNRS, Centre de RechercheenNeurobiologieetNeurophysiologiedeMarseille(CRN2M),UMR7286, FacultédeMédecineSecteurNord,51BoulevardPierreDramard,13344Marseille cedex15,France.

E-mailaddress:[email protected](C.Gestreau).

2013;Sirenetal.,2009).Theseneuroprotectiveeffectsaremediated byendogenousEPO(i.e.producedbyneuronsandastrocytes)or exogenousrhEPOactingonEPO-receptors(EPO-R)thatareexten- sivelypresentinbrain(Martietal.,1996).However,thedosesof rhEPOrequiredtoobtainsuchtissue-protectiveeffectsstimulate erythropoieisisand increasethehematocrit,leadingtoasubse- quent procoagulant status and elevatedblood pressure (Marti, 2004; Velly et al., 2010).Serious side effects of chronic rhEPO includehypertension,polycythemia,myocardialinfarction,stroke andseizures,plateletactivationandincreasedthromboembolicrisk (Banachetal., 2010; Macdougallet al.,2012;Ponikowskietal., 2007;Shinetal.,2012;vanVeldhuisenetal.,2007).Thisprompted for thedevelopmentofnewgeneration ofEPO-Ragonistdrugs topreservetissue-protectivepropertiesandavoiderythropoiesis (Sirenetal.,2009).CarbamylatedEPO(C-EPO)isoneofthepromis- ingderivativesthatmayprovidebetteroutcomeinclinicaltrials (Choietal.,2014;Leconteetal.,2011;Leistetal.,2004).

Inadditiontoitserythropoieticandtissue-protectiveeffects, EPOisthoughttointeractwithperipheralandcentralchemore- ceptorstoincreaseventilationinresponsetohypoxia(Gassmann andSoliz,2009;Khemirietal.,2012;Soliz,2013;Solizetal.,2005).

EPOmayplayaroleinthecentralregulationofbreathingasEPO injectionsintheadultratbrainincreaseminuteventilation(Yalcin etal.,2007).Also,thehypoxicventilatoryresponseisenhancedin http://dx.doi.org/10.1016/j.resp.2016.06.002

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transgenicmicewithchroniccerebralrhEPOoverexpressioncom- pared to wildtype animals (Caravagna et al., 2015). Similarly, bath application of rhEPOprior to recording of fictive breath- ingpreventedhypoxicdepressioninbrainstempreparationsfrom miceneonates (Khemiri et al.,2012).Therefore, chronicrhEPO therapywassuggestedasareliableapproachfortreating respi- ratorydisorders (Caravagnaet al.,2015; Soliz,2013).However, we have recently shown that polycythemia alone or increased rhEPOlevelsinplasmaandbrainbluntsthehypercapnicventila- toryresponse(Menuetet al.,2016).Thispotentially deleterious effect on breathing may represent another limitation for pre- scriptionofchronicrhEPOtreatmentinpatients.Thus,idealdrug therapy to compensate for the lack of O₂ at multiple levels shouldinvolvemoleculesretainingbeneficialrespiratory-related andtissue-protectiveeffectsindependentoferythrocyteproduc- tion. Although the effects of C-EPO on the neural control of breathinghavenotbeeninvestigated,this moleculecouldfulfill these criteria. Indeed, in 2004,Leist and collaborators demon- stratedthatcarbamylationofEPO,whichtransformslysineresidues intohomocitrullinesand alterstheproteinconformation,main- tained the tissue-protective effects of EPO and eliminated its hematopoieticeffects(Leistetal.,2004).Inthelastyears,several studiesconfirmedtheseobservationsand provedtheneuropro- tectiveeffects ofC-EPOin numerousCNS diseases(Chenet al., 2015).Therefore,thisstudywasdesignedtomeasureventilationby plethysmographyafteracuteandchronictreatmentswithrhC-EPO, andtoevaluatedrugeffectsinnormoxia,hyperoxia,hypoxiaand hypercapnia.WealsocomparedplasmaandbrainrhC-EPOlevels aswellashematocritinsalineorrhC-EPOtreatedmice.

2. Materialandmethods 2.1. Ethicsstatements

AdultC57BL6 malemice(n=20;Janvier, France)from 90to 120postnataldayswereusedinthisstudy.Animalswerehoused underpathogen-freeconditions,hadunlimitedaccesstofoodand water,andweremaintainedonareversed12:12hlight/darkcycle inatemperature-controlledroom(25^◦C±2^◦C)for1weekprior toexperimentalmanipulation.Theexperimentalprocedureswere carriedoutinaccordancewithFrenchnationallegislation(JO87- 848,EuropeanCommunitiesCouncilDirective2010/63/EU,74)and localethicscommittee“DirectionDépartementaledelaProtection desPopulations”,withpermitnumber13-227deliveredtoCG.

2.2. PreparationandidentiﬁcationofrhC-EPO

RhC-EPO was prepared from rhEPO (Eprex, Janssen Cilag, France) aspreviously described (Leistet al., 2004).Brieﬂy, one volumeofrhEPO(1mg/ml) wasmixedwithonevolumeof1M Na-borate(pH8.8)andrecrystallizedKOCNwasaddedtoaﬁnal concentrationof1M.Themixturewasincubatedat37^◦Cfor24h.

SampleswereimmediatelydialyzedagainstMill-Qwaterandsub- sequentlyagainst20mMSodiumcitratein0.1MNaCl,pH6.0.After dialysis,thesampleswereconcentratedusingCentriconwithpore sizeof3kDa(Amicon).Theproteincontentwasdeterminedwitha Bradforddosage.EfficientcarbamylationofrhEPOwasconfirmed bytestingtheresistanceofnativeandcarbamylatedrhEPOtopro- teolysisbyLys-Cproteinase(Leistetal.,2004).Briefly,10␮gof rhEPOorrhC-EPOwereincubatedwith0.4␮gofLys-Cproteinase at37^◦Cfor20h.Laemmlibufferwasthenaddedandsampleswere boiledfor5min.Thedigestionproductswerefinallyseparatedby SDS-PAGEelectrophoresison15%acrylamidegelandproteinswere visualizedbysilverstaining.

2.3. Druginjections

Miceweredividedinto2groups(10animalspergroup).Mice wereinjectedi.pwithsterileNaCl(0.9%)orrhC-EPOat15,000IU/kg dilutedinﬁnalvolume100␮lofNaCl.ThisdoseofrhC-EPOhasbeen chosenbasedonbothpreviousstudiesonneuroprotection(Banks etal.,2004;Ehrenreichetal.,2004;Xenocostasetal.,2005)and ourpreliminaryplethysmographyexperimentssuggestingcentral effects ofEPO.Injectionsweremadein theeveningduringﬁve consecutivedays.

2.4. Hematocritmeasurements

Afterrecordingsofventilationondays1and5,bloodsamples (100␮l)wereobtainedafterasmallincisionwasmadeatthetipof thetail.Thesampleswerecollectedinheparinizedplasticcapillar- iesandhematocrit(Htc)wasmeasuredbycentrifugationmethod (5000gfor5minat4^◦C).

2.5. Determinationofventilationbyplethysmography

Ventilation of conscious animals was recorded using non- invasivewhole-bodybarometric plethysmography(EMKATech- nologies, Paris,France), as previously described (Menuet et al., 2016).Twodaysbeforerecordings,themicewerehabituatedtothe plethysmographchambertoreducestress.Onthedayoftheexper- iment,micewereweightedandrectaltemperaturewasmeasured beforeand after ventilatorymeasurement. We used a constant ﬂow plethysmograph (EMKA Technologies, Paris, France) with 200-mlanimalchamberventilatedwithair(600ml/min).Thevar- iousgasmixturescorrespondingtonormoxia,hyperoxia,hypoxia andhypercapniaweredeliveredusingaprogrammablegasmixer (GSM3,CWE,USA).Spirogramswerestoredandanalyzed(Spike2;

CambridgeElectronicDesign,Cambridge,UK)tocalculatethemean respiratoryfrequency(Rf,cycles/min),tidalvolumenormalizedby thebodyweight(V_T,ml/100g)andminuteventilationnormalized bythebodyweight(VE,ml/min/100g,withVE=Rf×VT).Volume calibrationswereperformedbyinjectingsmallamount(100␮l)of airintherecordingchambers,andthesemeasurementswereused tocalculateVT(DrorbaughandFenn,1955;Menuetetal.,2016).

Micewereinitiallyexposedtonormoxia(21%O2balanceN2) andbasalventilation(V_E,RfandV_T)wasrecordedforatleastthirty minutes.Ondays1and5,animalswereexposedtohypercapnia (21%O2,5%CO2 balanceN2),hyperoxia(100%O2),andhypoxia (10%O₂ balanceN₂).Ventilationwasrecordedfor5minduring thehypercapnic(HCVR)andhyperoxic(HxVR)challenges,andfor 20mininhypoxiatostudytheearlyphase(from0to5min)andthe latephase(from15to20min)ofthehypoxicventilatoryresponse (HVR)(MaxovaandVizek,2001;Powelletal.,1998).Eachchallenge wasseparatedbyarecoveryperiodofatleast20mininnormoxia, duringwhichbasalventilationwasreassessedfor5minbeforethe nextchallenge.Onlyperiodsofbreathingwithoutbodymovements werestudied.Insomecases,animalsweretooagitatedduringa particularrecordingperiodandcorrespondingdatawereexcluded fromtheanalysis.

2.6. ELISAassay

Onthelastexperimentalday(Day5),brainstemandplasma werecollectedtodeterminerhC-EPOconcentrations.Afterdeep anesthesiaofthemicewithisoﬂurane(5%inair),bloodsamples wereobtainedbycardiacpunctionandcollectedinheparinized tubes. They were immediately centrifuged (760g for 15min at 4^◦C)and100␮lofplasmawerecollected.Aftertranscardiacperfu- sionwithheparinizedPBS,brainstemwasdissectedandremoved.

Plasmaandbrainstem sampleswereimmediatelyfrozeninliq-

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uidnitrogen,andstoredat−80^◦Cuntilfurtheruse.Theanalysis ofrhC-EPOlevelsinbrainstemrequiredthehomogenizationofthe tissues.Brieﬂy,foreachgroup,3sampleswerepreparedbypooling togetherbrainstemfrom3miceinFastPrep@LysingmatrixDtubes (fromMP),thetubesbeingmaintainedonice.Ice-coldlysisbuffer (150␮l,0.9% NaCl with protease inhibitor cocktail from Sigma Aldrich)wasthenaddedtothetubesandtissueswerehomoge- nizedusinglysishomogenizer(40shomogenization).Tubeswere centrifugedtoremovecelldebris(12,000gfor20minat4^◦C)and clearsupernatantswerecollectedandanalyzed.Concentrationsof rhC-Epoinplasmaandtissuehomogenatesweremeasuredusing ELISAkit(fromR&Dsystems,Minneapolis,MN)accordingtoman- ufacturer’sinstructions(Menuetetal.,2016).Thesensitivityofthe assaywas∼1.0mIU/mL.ResultsareexpressedasmIUpergramof tissue(mIU/g)forthebrainstem,andasmIU/mLfortheplasma.

2.7. Statisticalanalysis

All statistical tests were performed using GraphpadPrism 6 (Graphpadsoftware,LaJolla,California,USA),andvaluesexpressed asmean (SD).Resultsof theELISA assaywerecompared using unpairedttests.Hematocrit,bodyweight,rectaltemperature,basal ventilationandvaluesofdeltaVEwerecomparedusingatwo-way ANOVAwithtreatmentandtimeeffectsasmainfactors.RawHxVR, HVRandHCVRwerecomparedusingtwo-wayrepeatedmeasures ANOVAwithtreatmentandFiO2orFiCO2effectsasmainfactors.

Whenappropriate,post-hocprotectedleastsigniﬁcantdifference ofFisher(PLSD)testswereused.Differenceswereconsideredsig- niﬁcantatP<0.05.

3. Results

3.1. ValidationofrhEPOcarbamylation

Afterchemical modificationofrhEPOtorhC-EPO,rhEPOand rhC-EPOweredigestedbyLys-CandanalyzedbySDS-PAGE.After digestionbyLys-C,rhEpowasentirelydegradedsothatnobandsof rhEPOcouldbeobserved.Bycontrast,rhC-EPOresistedproteolysis sincenodegradationpeptidescouldbedetected,confirmingthe efficientcarbamylationofrhEpo(Fig.1).

3.2. AcuteorchronicrhC-EPOdidnotincreasehematocrit

Resultsofhematocrit(Htc)measurementsinmiceafteracute (Day1)andchronic(Day5)treatmentswithNaClorrhC-EPOare showninTable1.Noneofthestatisticalcomparisonsrevealedsig- niﬁcantdifferences.Thus,controlandrhC-EPOgroupshadsimilar hematocritafteracuteandchronicinjections.

Fig.1. IdentificationofrhC-EPO.TheefficiencyofcarbamylationofrhEPOintorhC- EPOwasconfirmedbySDS-PAGEafterdigestionwiththeendoproteinaseLys-C andvisualizationbysilverstaining.Lane1:molecular-weight-markers(MW).Lane 2:rhEPOwithoutLys-Cdigestion.Lane3:rhEpowithLys-Cdigestion.Lane4:rhC- EPOwithoutLys-Cdigestion.Lane5:rhC-EPOwithLys-Cdigestion.Notethelackof degradationofrhC-EPOafterincubationwithLys-C.

Table1

Comparisonsofhematocritlevelswith2-wayrepeatedmeasuresANOVAanalysis.

Dataareexpressedasmean(SD).

Time Groups Hematocrit Timeeffect Treatmenteffect Interaction Day

1

Control(n=7) 41.14(3.01) F=0.727 F=3.310 F=0.040 rhC-EPO(n=7) 43.41(1.66) P=0.411 P=0.094 P=0.845 Day

5

Control(n=7) 39.89(2.93) rhC-EPO(n=7) 42.64(5.07)

Table2

ComparisonsofplasmaandbrainstemrhC-EPOconcentrationsonday5with unpairedt-test.

Parameter Groups Mean(SD) t-Values

Plasmaconcentration Control(n=5) 29.55(31.46) −8.266 rhC-EPO(n=5) 1009^∗∗∗(263.1)

Brainstemconcentration Control(n=3) 0.008(0.002) −3.166 rhC-EPO(n=3) 0.019^∗(0.005)

3.3. ChronicinjectionsincreasedrhC-EPOlevelsinplasmaand brainstem

Tissuelevelsof rhC-EPOweremeasuredincontrol andrhC- EPOgroupsofmiceonday5.Resultsindicatedthatchronicdrug injectionssigniﬁcantlyelevatedrhC-EPOconcentrationsinplasma and brainstemcompared tocontrolvalues (Table2).Therefore, peripherallyadministratedrhC-EPOpartially crossedtheblood- brainbarrierandreachedthebrainstem.

Table3

Comparisonsofbodyweightandrectaltemperaturewith2-wayrepeatedmeasuresANOVAanalysis.Dataareexpressedasmean(SD).

Time Groups Weight(g) Temperature(^◦C) Timeeffect Treatmenteffect Interaction

Day 0

Control(n=8) 23.7(1.08) 37.6(0.86) Statisticsforbodyweight:

rhC-EPO(n=9) 23.5(1.74) 37.7(0.88) F=19.50 F=0.056 F=0.054

p<0.001 p=0.816 p=0.947

Day 1

Control(n=8) 24.0(1.23) 37.5(0.74) rhC-EPO(n=9) 23.8(1.86) 37.9(0.66)

Statisticsfortemperature:

Day 5

Control(n=8) 24.6(1.10) 37.1(0.66) F=2.371 F=0.702 F=0.795

rhC-EPO(n=9) 24.4(1.91) 37.3(0.87) p=0.110 p=0.415 p=0.461

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Fig.2.Measurementsofrespiratoryparametersinnormoxia(basalventilation) beforetreatment(Day0),afteracute(Day1)andchronic(Day5)druginjections.A:

Minuteventilation(VE).B:Tidalvolume(VT).C:Respiratoryfrequency(Rf).Values areexpressedasmean(SD).Basalventilationdidnotchangesigniﬁcantlybetween thetwogroupsofmiceovertheexperimentalprotocol.

3.4. rhC-EPOdidnotalterbodyweightandrectaltemperature

Bodyweightandrectaltemperatureweremeasuredondays0, 1and5.Statisticalresultsshowedasigniﬁcantmaintimeeffectfor bodyweightonly(Table3).Post-hoccomparisons,however,didnot revealsigniﬁcantwithin-groupdifferences(P>0.05forallcompar- isons).Thus,thetwogroupsofmicehadsimilarbodyweightand rectaltemperatureovertheexperimentalprotocol.

3.5. BasalventilationwasnotaffectedbyrhC-EPO

Basalventilatoryparameters(V_E,V_TandRfinnormoxia)were measuredbeforetreatment(Day0),aswellasafteracute(Day 1)andchronic(Day5)druginjectionsincontrol(n=8)andrhC- EPOtreated(n=9)mice(Fig.2).Allstatisticalgroupcomparisons yieldednon-signiﬁcantresults(datanotshown).Therefore,control

andrhC-EPOgroupsofmicehadsimilarbasalventilationoverthe experimentalprotocol.

3.6. ChronicbutnotacuterhC-EPOincreasedthehyperoxic ventilatorydepression

Ventilatoryresponsestohyperoxia(HxVR,100%O₂for5min) weremeasuredincontrolandrhC-EPOgroupsofmiceafteracute andchronicdruginjections.Originaltracesfromplethysmography areshowninFigs.3a–cand4a–c.Inbothgroups,hyperoxiasignif- icantlydecreasedVE,thisventilatorydepressionbeingmainlydue toadecreaseinRf(Fig.3d).However,therewasnotreatmentxFiO2

interactionforV_E,V_TandRfafteracuteinjection(Day1;Table4).

Thus,acuterhC-EPOtreatmenthadnoeffectonHxVR.

After chronic drug injections (Day 5), significant treatment xFiO₂ interactions were foundfor V_E and V_T denoting greater decreasesinbothparametersduringhyperoxiainmicetreatedwith rhC-EPO(Fig.4d,Table4).Themagnitudeofventilatorydepression (orDeltaV_E)wasalsocomparedtofurtherexaminetheeffectsof treatmentsovertime(Fig.5).Thisconfirmedalackofsignificant effectonday1(P=0.332),andasignificantdecrease(P<0.01)in DeltaV_E inrhC-EPOtreatedmicecomparedtocontrolgroupon day5.Thus,thehyperoxicventilatorydepressionincreasedafter chronictreatmentwithrhC-EPO.

3.7. ChronicbutnotacuterhC-EPOenhancedthehypoxic ventilatoryresponse

The effects of rhC-EPO on thehypoxic ventilatory response (HVR,10%O₂for20min)weremeasuredafteracute(Day1,Fig.6) andchronic(Day5,Fig.7)treatments.Representativetracesfrom plethysmographyareshowninFigs.6a-dand7a-d,respectively.

DatacomparisonswithANOVAanalysesaredetailedinTable5.

Onday1,bothgroupshadsignificantlyelevatedVEintheearly phaseofHVRcomparedtobasalventilation(significantmainFiO₂ effect).ThelatephaseofHVRwascharacterizedbyasignificant ventilatorydepression in thetwo groupsof mice(i.e.a signifi- cantdecreaseinVEcomparedtotheearlyphaseofHVR),mainly explainedbydecreasesinRfand/orV_T(Fig.6e,Table5).Notreat- mentxFiO₂interactionwasfound(Table5).Thus,acutetreatment withrhC-EPOhadnoeffectontheHVR.Onday5,however,asignif- icanttreatmentxFiO₂interactionwasfoundforV_E(Table5).The twogroupshadanelevatedV_Einboththeearlyandlatephases ofHVRcomparedtobasalventilation,duetoelevatedVTandRf.

However,onlycontrolmicehadaclearventilatorydepressionin thelatephaseofHVRwhereasnodifferenceinrespiratoryparam- eterscouldbedetectedbetweentheearlyandlatephasesofHVR inrhC-EPOtreatedmice(Fig.7e,Table5).Tofurtherexaminethe effectsoftreatmentsovertime,wealsocomparedthemagnitude oftheresponsesasDelta VE ondays1 and5(Fig.8).Thiscon- firmedalackofsignificanteffectsonday1forthemagnitudeof theearly,lateandroll-offphasesofHVR(P>0.05forallcompar- isons),andsignificantincreases(P<0.05)inDeltaVEforthelate androll-offphasesofHVRinrhC-EPOcomparedtocontrolmiceon day5.Thus,rhC-EPOenhancedthelatephaseofHVRandreduced hypoxicventilatorydepression.

3.8. rhC-EPOdidnotalterventilatoryresponsetohypercapnia

Ventilatoryparametersrecordedduringhypercapniaafteracute andchronicrhC-EPOinjectionsareillustratedinFigs.9 and10, respectively. Inthetwo groups of mice,statisticalcomparisons revealedsigniﬁcantlyelevatedVE,VTandRfinhypercapniacom- paredtobasalcondition(mainFiCO₂effect)afterbothacuteand chronictreatments.However,inbothcases,therewasnotreatment effectandnotreatmentxFiCO2 interaction(Table6).Therefore,

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Fig.3. Changesinventilationinresponsetohyperoxiaafteracutedruginjections(Day1).A:Originaltracingsfromplethysmography.B-C:Enlargementsofthespirogram atperiodsindicatedbyarrowsinA.B:basalventilation(basal)undernormoxia(N).C:Ventilatoryresponsetohyperoxia(100%O2).Verticalscalebarscorrespondtoone arbitraryunit.D:Measurementsofminuteventilation(VE),tidalvolume(VT),andrespiratoryfrequency(Rf).Valuesareexpressedasmean(SD).*,**,***:P<0.05,P<0.01 andP<0.001post-hocdifferencesinhyperoxiavsbasalventilation.Notethesimilarventilatorydepressioninresponsetohyperoxiainthetwogroupsofmice,asevidenced byANOVAanalysesdetailedinTable4.

Table4

Comparisonsofventilatoryparametersinhyperoxiaafteracute(Day1)andchronic(Day5)treatmentswith2-wayrepeatedmeasuresANOVAanalysis.

Time Parameter Groups FiO2effect Treatmenteffect Interaction

Day1 VE(Basal,Hyperoxia) Control(n=7) rhC-EPO(n=8)

F=26.53 F=0.203 F=1.015

P<0.001 P=0.660 P=0.332

VT(Basal,Hyperoxia) F=3.921 F=2.027 F=0.402

P=0.069 P=0.178 P=0.537

Rf(Basal,Hyperoxia) F=36.91 F=2.793 F=0.854

P<0.001 P=0.119 P=0.372

Day5 VE(Basal,Hyperoxia) Control(n=8) rhC-EPO(n=9)

F=95.27 F=0.068 F=9.001

P<0.001 P=0.797 P=0.009

VT(Basal,Hyperoxia) F=46.05 F=0.479 F=6.452

P<0.001 P=0.500 P=0.026

Rf(Basal,Hyperoxia) F=18.98 F=0.955 F=0.874

P<0.001 P=0.344 P=0.365

Table5

Comparisonsofventilatoryparametersinhypoxiaafteracute(Day1)andchronic(Day5)treatmentswith2-wayrepeatedmeasuresANOVAanalysis.

Time Parameter Groups FiO2effect Treatmenteffect Interaction

Day1 VE(Basal,EarlyandLatehypoxia) Control(n=8) rhC-EPO(n=9)

F=46.64 F=0.268 F=0.787

P<0.001 P=0.613 P=0.464

VT(Basal,EarlyandLatehypoxia) F=41.18 F=1.641 F=1.515

P<0.001 P=0.220 P=0.236

Rf(Basal,EarlyandLatehypoxia) F=55.14 F=1.046 F=0.294

P<0.001 P=0.323 P=0.747

Day5 VE(Basal,EarlyandLatehypoxia) Control(n=7) rhC-EPO(n=7)

F=30.23 F=2.818 F=5.353

P<0.001 P=0.119 P=0.012

VT(Basal,EarlyandLatehypoxia) F=30.69 F=6.935 F=3.827

P<0.001 P=0.022 P=0.036

Rf(Basal,EarlyandLatehypoxia) F=44.30 F=0.424 F=4.045

P<0.001 P=0.527 P=0.031

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Fig.4.Changesinventilationinresponsetohyperoxiaafterchronicdruginjections(Day5).A:Originaltracingsfromplethysmography.B-C:Enlargementsofthespirogram atperiodsindicatedbyarrowsinA.B:basalventilation(basal)undernormoxia(N).C:Ventilatoryresponsetohyperoxia(100%O2).Verticalscalebarscorrespondtoone arbitraryunit.D:Measurementsofminuteventilation(VE),tidalvolume(VT),andrespiratoryfrequency(Rf).Valuesareexpressedasmean(SD).*,**,***:P<0.05,P<0.01 andP<0.001post-hocdifferencesinhyperoxiavsbasalventilation.VentilatorydepressioninresponsetohyperoxiawasmorepronouncedinrhC-EPOmice,asevidenced byANOVAanalysesdetailedinTable4.

Table6

Comparisonsofventilatoryparametersinhypercapniaafteracute(Day1)andchronic(Day5)treatmentswith2-wayrepeatedmeasuresANOVAanalysis.

Time Parameter Groups FiCO2effect Treatmenteffect Interaction

Day1 VE(Basal,Hypercapnia) Control(n=8) rhC-EPO(n=9)

F=57.39 F=0.199 F=0.088

P<0.001 P=0.662 P=0.771

VT(Basal,Hypercapnia) F=73.02 F=0.060 F=0.471

P<0.001 P=0.810 P=0.503

Rf(Basal,Hypercapnia) F=84.57 F=1.638 F=1.375

P<0.001 P=0.220 P=0.259

Day5 VE(Basal,Hypercapnia) Control(n=7) rhC-EPO(n=7)

F=96.51 F=0.034 F=0.297

P<0.001 P=0.855 P=0.593

VT(Basal,Hypercapnia) F=125.0 F=0.002 F=0.188

P<0.001 P=0.967 P=0.670

Rf(Basal,Hypercapnia) F=123.6 F=0.106 F=1.266

P<0.001 P=0.749 P=0.277

controlandrhC-EPOtreatedmicehadsimilarpatternsofventila- tioninhypercapniaovertheentireexperimentalprotocol.

4. Discussion

ThisstudyexaminedtheeffectsofrhC-EPOonmiceventilation andotherphysiologicalparameters.Asexpected,chronicrhC-EPO injectionsdidnotstimulateerythropoiesis despiteanincreased plasmarhC-EPOconcentration.Resultsdemonstratedthat1/acute or chronic drug treatment did not change basal breathing in normoxiccondition,2/chronic butnotacuterhC-EPOtreatment enhancedthehyperoxicventilatoryresponse,andsustainedventi- lationinresponsetohypoxiabyreducingthehypoxicventilatory depression(theroll-offphaseoftheHVR),and3/noneofthedrug

regimenalteredventilatoryresponsetohypercapnia.Wediscuss therelevanceoftheseﬁndingswithavailableliterature,including theeffectsofEPOonneuralcontrolofbreathing.

4.1. Methodologicalconsiderations

Drug wasinjected at 15,000IU/kg usingan intra-peritoneal route.In humans, sucha doseand route of rhEPOadministra- tionallowthedrugtocrosstheblood-brainbarrierandreachthe cerebralparenchyma(Rasmussenetal.,1985;Xenocostasetal., 2005).ThisisinagreementwithourELISAmeasurementsshowing increasedlevelsofrhC-EPOinmicebrainstemtissueafterchronic treatment.Besides,druglevelswerehigherinplasmathaninbrain- stem,consistentwithpreviousobservationsdemonstrating that

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Fig.5.Differencesinminuteventilation(DeltaVE)betweennormoxiaandhyper- oxiaafteracute(Day1)andchronic(Day5)druginjections.Valuesareexpressed asmean(SD).*:P<0.05vscontrolgroup.ChronicbutnotacuterhC-EPOinjections enhancedtheventilatorydepressionseeninhyperoxia.

onlyafractionofcirculatingrhEPOcouldreachthecerebrospinal ﬂuid(BrinesandCerami,2005).Leistandcollaboratorssuggested thatrhEPOandrhC-EPOsharesimilarpharmacokinetics(Leistetal.,

2004).SincerhC-EPOtissueconcentrationwasmeasuredatonly onetimepoint(Day5)inthepresentstudy,ourdatacannotcon- ﬁrmthissuggestion.Lowdruglevelswerealsodetectedintissues fromcontrolanimals.Theselevelslikelycorrespondtoendogenous mouseEPOasahighdegreeofsequencehomologyexistsbetween humanandmurineEPO(Wenetal.,1993).Thelackofsigniﬁcant effectsofchronicrhC-EPOtreatmentonbodyweightandcorebody temperatureinourstudyisconsistentwithanotherobservationin mice(Fantaccietal.,2006).Thisisparticularlyimportantforval- idationofourventilatorymeasurementswithplethysmography, aschangesinbodytemperature(e.g.whenmiceareexposedto hypoxia)mayconfoundinterpretationofresults.

4.2. LackofacuterhC-EPOtreatmentonventilation

AcuterhC-EPOhasnoeffectonventilationinnormoxia,hyperoxia,hypoxiaorhypercapnia.Thisisinagreementwithaprevious reportshowingasimilarlackofdrugeffectonbothbasalventila- tionandinresponsetomoderatehypoxia(10%O2)2hafteracute injectionof2000IU/kgrhEPO(Solizetal.,2005).Althoughdrug concentrationintissueswasnotmeasuredonday1,plasmalevels ofrhEPOandrhC-EPOremainedelevatedfor24hafterasingledrug

Fig.6.Changesinventilationinresponsetohypoxia(10%O2)afteracutedruginjections(Day1).A:Originaltracingsfromplethysmography.B-D:Enlargementsofthe spirogramatperiodsindicatedbyarrowsinA.B:basalventilation(basal)undernormoxia(N).C-D:Earlyandlatephasesofthehypoxicventilatoryresponse,respectively.

Verticalscalebarscorrespondtoonearbitraryunit.E:Measurementsofminuteventilation(VE),tidalvolume(VT),andrespiratoryfrequency(Rf).Valuesareexpressedas mean(SD).*,**,***:P<0.05,P<0.01andP<0.001post-hocdifferencesinhypoxiavsbasalventilation.$,$$,$$$:P<0.05,P<0.01andP<0.001post-hocdifferencesinlate vsearlyhypoxia.Bothgroupshadsimilarincreasesinventilationintheearlyphaseofhypoxiafollowedbyasigniﬁcantdecline(orventilatorydepression)inthelatephase ofhypoxia(seealsoTable5).

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Fig.7. Changesinventilationinresponsetohypoxia(10%O2)afterchronicdruginjections(Day1).A:Originaltracingsfromplethysmography.B-D:Enlargementsofthe spirogramatperiodsindicatedbyarrowsinA.B:basalventilation(basal)undernormoxia(N).C-D:Earlyandlatephasesofthehypoxicventilatoryresponse,respectively.

Verticalscalebarscorrespondtoonearbitraryunit.E:Measurementsofminuteventilation(VE),tidalvolume(VT),andrespiratoryfrequency(Rf).Valuesareexpressedas mean(SD).*,**,***:P<0.05,P<0.01andP<0.001post-hocdifferencesinhypoxiavsbasalventilation.$,$$,$$$:P<0.05,P<0.01andP<0.001post-hocdifferencesinlate vsearlyhypoxia.NotethelackofventilationdepressioninthelatephaseofhypoxiainrhC-EPOmice(seealsoTable5).

injectionatsimilardosesinrats(Leistetal.,2004).Also,acuteinjec- tionwithahigherdoseofrhEPO(77,000IU/kg)inmiceresultedin elevatedrhEPOlevelsinbothplasmaandbrainupto48hpost- injection(Schuleretal.,2012).Thus,ourresultsreinforcetheidea thatstimulationofEPOreceptorsafteracuteinjectionofrhC-EPO doesnotexertsigniﬁcanteffectsonbasalventilation,noronrespi- ratorychemoreﬂexes,despiteelevateddruglevelsintissues.

4.3. LackofchroniceffectsofrhC-EPOonbasalventilation

Chronic treatmentwith rhC-EPO didnot change ventilatory parameters measured in normoxia. Similar ﬁndings have been reportedintransgenicmicewithchronicoverexpressionofrhEPO inbothplasmaandbrain(Tg6mice)orinbrainonly(Tg21mice), aswellasinwildtypemiceafterchronicrhEPOinjections(Menuet etal.,2016;Solizetal.,2007;Solizetal.,2005).Thepresentresults inadultmicealsocorroboratedpreviousobservationsinnewborns, demonstratingnochangeinﬁctiverespiratoryactivityafterprein- cubationofisolatedbrainstemwithrhEPO(Khemirietal.,2012).

Thusourdatasuggestthat,similarlytorhEPO,chronictreatment withrhC-EPOdoesnotalterneuronalmechanismsinvolvedinbasal breathing.

4.4. rhC-EPOenhancedventilatoryresponsestochangesinO₂ levels

It iswidely acceptedthat changesin ventilation afterexpo- sureofanimalsto100%O₂,theso-calledDejour’stest(Dejours, 1962),isduetothesilencingofcarotidbodies.Thus,thistestcan beusedasanindirectmeasureofcarotidbodies’sensitivitytoO2

(Ramanantsoaetal.,2006;Solizetal.,2007).Ourresultsobtained inmiceinjectedwithrhC-EPOoverﬁveconsecutivedaysshowed greaterventilatorydepressioninhyperoxiathancontrolmice.A similarﬁndingwasreportedinTg6micewithanoverexpressionof rhEPOinbothplasmaandbrain(Solizetal.,2007).Thissuggests thatchronictreatmentwithoneoranotherdrugincreasedcarotid bodies’ O₂ sensitivity. Interestingly, since a normal hematocrit wasobservedinrhC-EPOtreatedmicewhereaschronicrh-EPOis knowntoinducepolycythemia(Menuetetal.,2016),itisunlikely thatdistinctbloodcarryingO2capacitiescontributetochangesin hypoxicbreathingpatterns.

ChronicrhC-EPOtreatmentdidnotchangetheearlyphase(ﬁrst 5min)oftheHVR.Atﬁrstglance,theseresultsaresurprisingsince 1/theinitialincreaseinventilationinresponsetohypoxiaismainly attributedto carotid bodies (Powellet al., 1998; Teppema and Dahan,2010),and2/enhancementoftheHxVRafterchronicrhC- EPOsuggestedanincrease incarotid bodies’O₂ sensitivity(see

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Fig.8. Differencesinminuteventilation(DeltaVE)betweennormoxiaandhypoxiaincontrolandrhC-EPOmiceondays1and5.A:Magnitudesoftheearlyandlatephases ofhypoxia.B:Magnitudeoftheroll-offphase.Valuesareexpressedasmean(SD).*:P<0.05vscontrolgrouponday5.$$:P<0.01withingroupdifferencesinlatevsearly hypoxia.#:P<0.05betweengroupsdifferenceinlatehypoxia.N.S.:notstatisticallysigniﬁcant.ChronicbutnotacuterhC-EPOinjectionsdecreasedthehypoxicventilatory depression.

above).Interestingly,rhEPOalsoenhancescarotidbodies’O2sen- sitivityinmalemicebutdoesnotaltertheearlyphaseofHVRin responsetomoderatehypoxia(10%O₂)(Solizetal.,2005).Inthelat- terstudy,however,changesinpatternsofventilationattributable to a role of EPO on carotid body cells could be demonstrated onlyafteraseverehypoxicchallenge(6%O₂)inbothTg21mice withchronicrhEPOoverexpressionandwildtypemicereceiving anacuteinjectionofrhEPO(2000IU/kg,2hbeforehypoxia).There- fore,itispossiblethattheearlyphaseofHVRdidnotincreasein ourstudyduetoinsufficientchangesinarterialPO₂betweennor- moxia(21%O2)andmoderatehypoxia(10%O2).Bycontrast,drastic changesin arterialPO₂ occurredbetweennormoxiaand hyperoxia(100%O₂)sothattheresultingfiringalterationofperipheral chemosensitivefibersmaybesufficienttoenhancetheHxVR.

AnotherstrikingsimilaritybetweentheeffectsofrhC-EPOand rhEPOisthereductionofventilatorydepressioninthelatephase (last5min) oftheHVR(MaxovaandVizek,2001;Powelletal., 1998),therebyenhancingventilationinresponsetolowO₂ levels.Hypoxicventilatorydepression,alsocalledtheroll-offphase, isverylikelyattributabletoacentralinhibitoryeffectofhypoxia althoughtheprecisemechanismsarestilldebated(Gestreauetal., 2010;MaxovaandVizek,2001).TransgenicTg21micewithacon-

stitutivecerebraloverexpressionofrhEPOalsohavea sustained ventilationinthelatephaseofHVRatasimilarlevelofhypoxia(10%

O₂)(Solizetal.,2005).Also,Tg21micedisplayasustainedhypoxic responseafterbilateraltransectionofcarotidsinusnervewhich iscorrelatedwithalterationsincatecholaminesynthesisinboth medullaandpons(Solizetal.,2007;Solizetal.,2005).Thus,asimple interpretationofourresultsisthatrhC-EPOmayplayamodulatory roleoncentralstructuresinvolvedinthecontrolofbreathingdur- inghypoxia.TheelevatedbrainstemrhC-EPOconcentrationfound afterchronicdruginjection(presentresults),andthewideexpres- sionofEPO-Rinbrainstemnucleiinvolvedinrespiratorycontrol (Solizetal.,2005)areinlinewiththishypothesis.

To furtherstudy centralchemoreception, we also measured changes inventilation inresponse tohypercapniaafter chronic rhC-EPOorsalineinjections.Resultsrevealedsimilarincreasein ventilatoryparametersinbothmicegroups,suggestingalackof effectofcarbamylatedEPOoncentralchemoreceptorsdetecting changesinCO₂/pH,suchasneuronsoftheretrotrapezoidnucleus (GuyenetandBayliss,2015;Guyenetetal.,2010;Wangetal.,2013).

Thus,ourstudypointedtoamajordifferencebetweentheventila- toryeffectsofrhEPOandrhC-EPOsincechronicrhEPOtreatment bluntstheHCVR(Menuetetal.,2016).Consequently,itisunlikely

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Fig.9.Changesinventilationinresponsetohypercapniaafteracutedruginjections(Day1).A:Originaltracingsfromplethysmography.B-C:Enlargementsofthespirogram atperiodsindicatedbyarrowsinA.B:basalventilation(basal)undernormoxia(N).C:Ventilatoryresponsetohypercapnia(5%CO2).Verticalscalebarscorrespondtoone arbitraryunit.D:Measurementsofminuteventilation(VE),tidalvolume(VT),andrespiratoryfrequency(Rf).Valuesareexpressedasmean(SD).***:P<0.001post-hoc differencesinhypercapniavsbasalventilation.Bothgroupsofmicehadsimilarincreasesinventilationinresponsetohypercapnia(seealsoTable6).

thattheenhancedHVRreportedhereinafterchronicrhC-EPOtreat- mentcouldbeexplainedbychangesincentralCO₂sensitivityin thesemice.

4.5. AretheheterodimericEPOreceptorsresponsibleforthe effectsonventilation?

EPOisthoughttomodulaterespiratoryactivitybyactingonclas- sicalhomodimeric(EPO-R/EPO-R)EPOreceptors(Bunn,2013).This viewissupportedbyexpressionofEPOreceptorsincarotidbodies andmanybrainstemrespiratoryareas(Solizetal.,2005).However, thespeciﬁcityofmostantibodiesdirectedagainstEPO-Rhasbeen recentlychallenged(Elliottetal.,2006;Elliottetal.,2014;Kirkeby etal.,2007).Here,weshowthatrhC-EPO,likerhEPO,alteredboth hyperoxicandhypoxicventilatoryresponsesalthoughthisdrug lackserythropoieticeffectandbindsheterodimeric(EPO-R/␤c-R) receptors(Brinesetal.,2004;Bunn,2013;Chamorroetal.,2013;

Chateauvieuxetal.,2011).Therefore,modulationofbreathingin responsetochangesinO₂levelsmaybemediatedbyinteractionof bothdrugswithEPO-R/␤cRreceptors.The␤cRsubunitiscommon forseveralcytokinereceptors(D’AndreaandGonda,2000).Itwas detectedinvariousstructuresofthecentralnervoussystem(Brines etal.,2004;Nadametal.,2007;Sanchezetal.,2009),butnoinfor- mationisavailableonitslocalizationinthebrainstem,inparticular inrespiratoryareas,noratthelevelofthecarotidbodies.Future studiesshouldidentifytheprecisemechanismsofEPO-mediated

respiratoryeffects, includingthetargetcells andtheidentityof theirEPOreceptors.

4.6. Conclusions

Chronic treatmentwithrhC-EPO altered bothhyperoxic and hypoxic ventilatory responses in the absence of polycythemia.

These effects are very likely mediated by neuromodulation of peripheralandcentralstructurescontributingtoventilatoryadap- tationtochangesinO₂ levels.Interestingly,rhC-EPOissafeand welltoleratedinHumans(Boeschetal.,2014).Thus,heterodimeric EPO-R/␤c-RagonistssuchasC-EPOcouldrepresentgoodcandi- datesforthetreatmentofdiseaseslinkedtorespiratorynetwork dysfunctionwhenerythropoiesis-stimulatingeffectisunwanted.

SincechronicrhEPO,butnotrhC-EPO,bluntsthehypercapnicven- tilatoryresponseandmaybedeleteriousforbreathing(Menuet etal.,2016),clinicaluseofrhC-EPOmayprovidegreaterefﬁcacy thanrhEPO.

Conﬂictofinterest

Theauthors declarethat theresearchwasconducted inthe absenceofanycommercialorﬁnancialrelationshipsthatcouldbe construedasapotentialconﬂictofinterest.