Ischemia/reperfusioninducedalterationsofenzymaticandsignalingfunctionsoftheratcardiacNa+/K+

ATPase:protectionbyouabainpreconditioning

Aude Belliard, Ph.D.1, *, Gaurav K. Gulati, Ph.D.1, *, #, Qiming Duan, Ph.D.1, $, Rosana Alves, Ph.D.1,
2 Shannon Brewer, B.Sc.1, Namrata Madan, Ph.D.2, Yoann Sottejeau, Ph.D. 1, , Xiaoliang Wang, M.D.2,

JenniferKalisz,B.Sc.1,andSandrineV.Pierre,Ph.D.2
1.DepartmentofBiochemistryandCancer
Biology,CollegeofMedicine,UniversityofToledo,Toledo,OH,USA.
2.MarshallInstituteforInterdisciplinaryResearch,Huntington,WV,USA.
*Authorshavecontributedequallytothework.
#Presentaffiliation:MaterialsScienceandEngineering
Department,UniversityofWashington,Seattle,WA,USA
$Presentaffiliation:GladstoneInstituteofCardiovascularDisease,SanFrancisco,CA,USA
Presentaffiliation:UniversitLille2,InsermU1011,Lille,France
Corresponding author: Sandrine V. Pierre, Marshall Institute for Interdisciplinary Research, Weisberg
EngineeringComplex,1628ThirdAvenue,Huntington,WV25703,USA.
Phonenumber:3046963549/3505
Faxnumber:3046963839
email:pierres@marshall.edu
RunningTitle:Na+ /K+ ATPaseActivityandSignalingintheIschemicHeart

Abstract


Cardiac glycosides (CG) are traditionally known as positive cardiac inotropes that inhibit Na+ /K+ ATPase
dependentiontransport.CGalsotriggerspecificsignalingpathwaysthroughthecardiacNa+ /K+ ATPase,with
beneficial effects in ischemia/reperfusion (I/R) injury (e.g. ouabain preconditioning, known as OPC) and
hypertrophy.OurcurrentunderstandingofhypersensitivitytoCGandsubsequenttoxicityintheischemicheart
ismostlybasedonspecificI/RinducedalterationsoftheNa+ /K+ ATPaseenzymaticfunctionandhasremained
incomplete.TheprimarygoalofthisstudywastoinvestigateandcomparetheimpactofI/RonNa+ /K+ ATPase
enzymaticandsignalingfunctions.Secondly,weassessedtheimpactofOPConbothfunctions. Langendorff
perfused rat hearts were exposed to 30 min of ischemia and 30 min of reperfusion. At the inotropic
concentration of 50M, ouabain increased ERK and Akt phosphorylation in control hearts. In I/R hearts, this
concentrationdidnotinducedpositiveinotropyandfailedtoinduceAktorERKphosphorylation.Theinotropic
responsetodobutamineaswellasinsulinsignalingpersisted,suggestingspecificalterationsofNa+ /K+ ATPase.
Indeed, Na+ /K+ ATPase protein expression was intact, but the enzyme activity was decreased by 60%
and the enzymatic function of the isoform with high affinity for ouabain was abolished following I/R.
Strikingly, OPCprevented all I/Rinduced alterations of the receptor. Further studies are needed to reveal the
respective roles of I/Rinduced modulations of Na+ /K+ ATPase enzymatic and signaling functions in
cardiomyocytedeath.

New&Noteworthy
CardiacischemiaandreperfusioninducesNa+ /K+ ATPasespecificalterations,whichresultinaspecificlossof
ouabaininduced inotropy and signaling in the postischemic heart. Transient exposure to a subinotropic
concentrationofouabainpreventsI/Rinducedalterations.

Introduction

Cardiacglycosides(CG)suchasouabainordigoxinarespecificinhibitorsofNa+ /K+ ATPaseactivity,which

results in subsequent increase in intracellular Na+ , increased Na+ /Ca2+ exchange, and cardiac positive
inotropy 1,2.OuabainbindingtothesubunitofNa+ /K+ ATPasealsoinitiatesspecificintracellularsignaling
pathways, most notably PI3K/Akt and cSrc/ERK in the cardiac tissue of various species 38. Based on
experimental evidence reported in recent years, the concept that these intracellular events occur at low (sub
inotropic) concentration and lead to additional cardiac actions of cardiac glycosides such as hypertrophic
growth, prevention of cardiac hypertrophy and failure, and protection against ischemiareperfusion injury has
emerged3,4,713.ThemostdocumentedexampleofprotectionagainstischemiareperfusioninjurybyaCGis
ouabainpreconditioning(OPC).OPCcanbetriggeredbyatransientexposuretoalowconcentrationofouabain
priortoasustainedischemia,andprovidesaprotectionagainstinfarctionandcontractiledefectscomparableto
thatobservedforischemicpreconditioning.KnownsignalingeventsdownstreamfromNa+ /K+ ATPasethatare
criticaltoOPCincludetheactivationofSrckinaseandPKC,openingofmitoKATPchannels,productionof
ROS,andactivationofPI3KIA.
The alteration of cardiac Na+ /K+ ATPase enzyme function during ischemia and reperfusion injury is well
documented12,1419,andisdetectedbeforecelldeathoccurs 20.InamodelofsimulatedI/Rinculturesofrat
neonatalcardiacmyocytes,wefoundthatOPCprotectedNa+ /K+ ATPasecatalyticproperties 12andprevented
I/Rinducedcelldeath.Inthiscellmodel,intrinsicNa+ /K+ ATPaseenzymaticactivitywasprotectedbyOPC,
but the cellular uptake of the K+ congener 86Rb+ remained reduced. As potential explanations for this
observation, we proposed the persistence of ouabain binding to the high affinity 3 isozyme expressed in rat
neonatal cardiac myocytes and/or the inhibition of catalytically competent Na+ /K+ ATPase by the labile
cytosolic compound described by Fuller et al. 21. As an important implication of thisfinding, the fact that
protectionofNa+ /K+ ATPasemediatediontransportwasnotcriticalforOPCmediatedcellsurvivalduringI/R
inthismodelledustoproposethatalterationofNa+ /K+ ATPasesignalingfunctionduringI/Randprotectionby
OPC could be involved. As a follow up, the present study was undertaken to thoroughly assess how I/R and
OPC impact Na+ /K+ ATPase enzymatic and signaling functions in the whole heart. The results obtained in
Langendorff rat heart preparations exposed to 30 min of global ischemia followed by 30 min of reperfusion
revealed marked functional changes of 2containing Na+ /K+ ATPase isoenzymes, with concomitant blunt of

both contractile and signaling responses to ouabain but not dobutamine or insulin. Preconditioning using a
subinotropicconcentrationofouabainpreventedischemiainducedalterationsofNa+ /K+ ATPaseenzymaticand
signalingproperties.

Methods
RatHeartLangendorffPreparation.ExperimentalprocedureswereconductedinaccordancewiththeGuidefor
theCareandUseofLaboratoryAnimals(NIHPublication,8thEdition,2011).Aratheartpreparationperfused
atconstantflowintheLangendorffmodewasusedaspreviouslydescribed11.MaleSpragueDawleyrats(320
350g,straincode001)wereanesthetizedbyintraperitonealinjectionofsodiumpentobarbital(80mg/kg).Hearts
were excised and rapidly placed into ice cold KrebsHenseleit (KH) buffer. Within 40 seconds, hearts were
perfusedintheLangendorffmodewithoxygenatedKHbuffercontaining(inmmol/L)NaCl(118.0),KCl(4.0),
CaCl2(1.8),KH2PO4(1.3),MgSO4(1.2),Ethyleneglycolbis(2aminoethylether)N,N,N,Ntetraaceticacid
(0.3), NaHCO3 (25), Dglucose (11). Isovolumic left ventricular developed pressure (LVDP) as well as end
diastolic pressure (EDP) were continuously monitored during the experiment through a waterfilled latex
balloon inserted into the left ventricle and analyzed subsequently using Lab Chart software (ADInstruments).
Heartswerepacedat4.5HzwithbipolarelectrodesattachedtotheleftventricleusingaGrassSD9stimulator,
andpacingwasmaintainedthroughoutallprotocols.EDPwasadjustedinitiallyat48mmHg.After20minutes
ofequilibration,oneofthetwoprotocolsdescribedinthenextsectionwasinitiated.
ExperimentalProtocols.ProtocolA.Todocumenttheeffectofischemiaandreperfusiononcardiacfunction,
tissue viability, and Na+ /K+ ATPase structure and enzyme function, hearts were subjected to the 80 minlong
protocol A shown in figure1. Control hearts (C) were perfused with KH buffer for 80
minutes. Ischemia/Reperfusion (I/R) hearts were perfused for 20 minutes with KH, then subjected to 30
minutes zeroflow (global) ischemia followed by 30 minutes of reperfusion. Ouabain Preconditioning (OPC)
heartswereperfusedwithKHfor8minutesfollowedbyouabain(10M)for4minutesandKHfor8minutes
before30minutesofglobalischemiaand30minutesofreperfusionaspreviouslydescribed11.Attheendof30
minutes of reperfusion, left ventricles were snap frozen in liquid nitrogen and stored at 80 C for Na+ /K+
ATPaseactivitymeasurementandwesternblotanalysisof1,2 and1isoformsofNa+ /K+ ATPase.Protocol

B.Toinvestigatetheeffectofischemiaandreperfusiononouabaininducedpositiveinotropyandsignaling,a15
minexposuretoKHor50Mouabainwasaddedtooneofthe80minprotocolsdescribedabove(i.e.C,I/R,or
OPC).Asubsetofheartswasexposedto15mindobutamine(75nM)orinsulin(0.3mU/ml)aftertheinitial80
minperiodasshowninfigure1,andusedascontrolsininotropyandsignalingstudies.
Lactatedehydrogenase(LDH)activitymeasurement.Coronaryeffluentswerecollectedfor30secondsattime
0,1,2,3,4,5,10,15,20and30minutesofreperfusion.LDHactivitywasdeterminedcolorimetricallyusinga
standardassaykit(Cytotoxicitydetectionkit(LDH),Version8.0,RocheDiagnostics,Indianapolis,IN,USA),
accordingtomanufacturerrecommendation.
Crude heart homogenates (KClenzyme preparations).Sample preparation was performed following a tissue
homogenizationprotocolmodifiedfrom 22.Briefly,200mgofleftventricularliquidnitrogenprocessedtissue
powderweredissolvedin10mlof1MKClsolutionandhomogenizedwithaPolytronPT10/STfor30seconds.
This method of homogenization coupled with a KCl treatment allows the disruption of the myofilament
contractileproteinspresentinthefraction.Thehomogenatewaspassedthroughadoublelayeredcheesecloth
andcentrifugedat1,000gfor10min.Thepelletwassuspendedin14mlof50mMKCland50mMTrisHCl
(pH 7.4) and centrifuged again. This washing procedure was repeated twice with 50 mM TrisHCl (pH
7.4).Thefinalpelletwassuspendedin1mlof1mMTrisEDTA(pH7.4)andthesuspensionwashomogenized
withaPotterElvehjenhomogenizer.Allprocedureswereconductedoniceandusingarefrigeratedcentrifugeat
4oC.
Na+/ K+ATPase activity. Na+ /K+ ATPase activity was measured by colorimetric determination of inorganic
phosphate released from ATP. To insure access of substrates and inhibitor to both the ATP and ouabain
binding sites of Na+ / K+ ATPase enzymes in closed membrane vesicles that may have formed during the
procedure,KClenzymepreparationswerepretreatedwithalamethicin(0.05mg/mgofprotein)for10minutes
atroomtemperatureasdescribedpreviously 23.Thereactionmixturefortheactivityassaycontained20 mM
TrisHCl(pH.7.2),3mMMgCl2,100mMNaCl,20mMKCl,1mMEGTATris,5mMNaN3,2mMATP,the
KCLenzyme preparation, and 1mM ouabain when indicated. After addition of Mg2+ /ATP, the enzymatic
reaction was allowed to run for 10 min. The reaction was terminated by addition of 1 ml cold 8%
tricholoroaceticacidandrapidplacementofeachtesttubeonice.Using an inorganic phosphate detection kit
(Biomol

Green,

Enzo

Life

Sciences,Farmingdale,

NY),

released

phosphate

was

quantified

spectrophotometrically at 620 nm. For each sample, the assay was done in the presence or absence of 1mM
ouabain to determine ouabaininsensitive and total Na+ /K+ ATPase activity, respectively. Ouabainsensitive
Na+ /K+ ATPase activity was then calculated as the difference between ouabaininsensitive and total Na+ /K+
ATPaseactivity.
Effect of various concentrations of ouabain on Na+/K+ATPase activity. Ouabain sensitive Na+ /K+ ATPase
activitywasdeterminedinKCLenzymepreparationinthepresenceof108to103Mouabaininthereaction
mixture. IC50 (50% inhibitory constant) and relative proportion of 1 and 2isoform of Na+ /K+ ATPase was
inferred from ouabain sensitive Na+ /K+ ATPase activity, as estimated from dose response curves on
permeabilizedcrudehearthomogenatepreparationsbyusingGraphPadPRISMSoftware(Version4.00).Curves
werefittoexperimentaldatabyanonlinearregressionmodelusingGraphPadPRISMSoftware.
TissuePreparation,SDSPAGEandWesternblots.Onehundredmgofpowderedleftventriclewereaddedto
anicecoldbuffercontaining1mMEDTA,1mMphenylmethylsulfonylfluoride,1mMsodiumorthovanadate,
1 mM NaF, 10 M okadaic acid, 10 g/ml aprotinin, 10 g/ml leupeptin and homogenized in a 30 ml
homogenizer by repeating 5 times a series of 8 upanddown strokes separated by 30 seconds intervals, and
rotatingfor90minutesat4 0C.Lysateswerethencentrifugedat16,000gfor15minutes,andsupernatants
were used. Equal amount of proteins were loaded and separated by 10% SDSPAGE, transferred onto
nitrocellulose membrane (Optitran BA S 83, 0.2 M, GE healthcare Life Sciences, UK), and immunoblotted
withantibodiesagainstNa+ /K+ ATPase1(6FDevelopmentalstudieshybridomabank,UniversityofIowa,
Iowa, USA), 2 (HERED primaryantibody a gift from Dr T. A. Pressley, Texas Tech University HSC,
Lubbock,TX)and1(05382,Upstate,CA)ofNa+ /K+ ATPasesubunits,phosphorylatedandtotalAkt(TAkt:
#9272,CellsignalingTech.,MAPAkt:S473,CellSignalingTech.,MA),ERK1/2(pERK1/2:Sc7383,Santa
Cruz, CA TERK1/2: Sc94, Santa Cruz, CA) and Src (CSrc: Sc8056, Santa Cruz, CA pSrc: 44660G,
Invitrogen,CA).GAPDH(Sc20357,SantaCruz,CA)wasprobedasaloadingcontrol.Rabbit(Sc2004,Santa
Cruz,CA),goat(Sc2020,SantaCruz, CA) and mouse (Sc2005,Santa Cruz, CA) secondary antibodies were
usedinthisstudy.Proteinbandsweredetectedusingchemiluminescenceandenhancedchemiluminescence,and
quantifiedusingImageJsoftware.

StatisticalAnalysis.AllvaluesareexpressedasmeanSEM.Comparisonsbetweengroupswereconducted
usingaonewayANOVAfollowedbyamultiplecomparisonposthoctest.Whentwogroupswerecompared,
unpairedbilateralStudentsttestwasused.P<0.05wasconsideredstatisticallysignificant.

Results
ImpactofI/RandOPCprotocolsoncontractilefunctionandtissueinjury
ThecontractileperformanceofcontrolheartsassessedbyrealtimerecordingofLVDPandEDPwasstableover
80 minutes of protocol A (Figure 1), as shown in figures 2A and 2B. In contrast, 30 minutes of ischemia
followed by 30 minutes of reperfusion resulted in a 68% decrease in LVDP at 80 minutes compared to pre
ischemicvalue(106 7 vs. 35 3 mmHg, P < 0.001), and a significant increase in EDP (3 2 vs. 71 5
mmHg,P<0.001).OPC resulted in a significantly better recovery of LVDP (76 5 mm Hgvs. 35 3 mm
HgP<0.001)andEDP(345mmHgvs.714mmHgP<0.001)at80mincomparedtoI/R.Asshownin
figure 2C, the protective effect of OPC was also associated with a 70% reduction of LDH release (0.08
0.03vs.0.180.03U/mlP<0.05).Takentogether,theabovefindingswereconsistentwithourearlyreportof
OPCinducedprotectioninthismodel11.

Na+/K+ATPasestructureandenzymefunction
AsshowninFigure3A,theouabainsensitiveactivitycorrespondingtoNa+ /K+ ATPaseinalamethicintreated
KClpreparationsfromcontrolheartswas7.40.8molPi/hr/mgofprotein.After30minofischemiaand30
minofreperfusion,Na+ /K+ ATPasewasdecreasedby 66% (2.50.6 mol Pi/hr/mg of protein P<0.001 vs.
control) and significantly protected by OPC (P<0.05 vs. I/R). Na+ /K+ ATPase in adult rat hearts typically
exhibit a biphasic doseresponse curve to ouabain, with a low affinity site attributed to 1containing
isoenzymes, and a high affinity component attributable to 2containing isoenzymes 24. As expected, the log
concentrationeffectcurveswerebiphasicforcontrolheartsinthepresentstudy,asshowninfigure3B(C).As
representedbythesolidline,doseresponsecurveswerebestmodelledassumingtwocomputedIC50(ouabain

concentration leading to 50% inhibition) of 5.9 X 108M and 9.1 X 105 M, with respective contributions of
33% and 67% (Table 1). In contrast, dose response curves from I/R hearts were best fitted assuming one
IC50ratherthantwo,resultinginamonophasiccurve(I/R).ThecomputedIC50oftheremainingsitewas1.8X
105 M, suggesting a loss of the high affinity site. OPC protected the activity of the site of high affinity, as
suggested by the biphasic curve. Contributions of the two isoenzymes were 64% for the site of low affinity
(1)and36%forthesiteofhighaffinity(2).Asshowninfigure4,I/RinducedalterationsofNa+ /K+ ATPase
activity and loss of high affinity enzymatic site or OPC protection were not accompanied by any detectable
changeintotalcardiacNa+ /K+ ATPase1,2or1proteincontentsasdeterminedbyWesternblotanalysis.

Effectofouabain50Moncontractilityandsignaling
We next evaluated the impact of I/R on positive inotropy and signaling induced by 50 M of ouabain. This
relativelyhighconcentrationwasselectedbecauseitisknowntotriggerarobustpositiveinotropiceffectwithout
signs of toxicity, as well as a clear activation of Na+ /K+ ATPase signaling in Langendorff preparations of rat
hearts5.Theeffectsincontrol,I/RandOPCheartswerecomparedonLVDPtomonitorchangesintheforceof
contraction,andERKandAktphosphorylationasmarkersofactivationofNa+ /K+ ATPasemediatedsignaling.
As expected, a 15 min exposure to ouabain 50 M according to protocol B (Figure 1) induced a robust and
significant increase ofLVDP in control hearts (956 mmHg to 1263 mmHg P<0.05 vs. C, Fig. 5). Also
expected,theI/RprotocolresultedinasignificantdecreaseinLVDPcomparedtocontrol.AfterI/R,exposureto
ouabain50Mfor15minfailedtoinducepositiveinotropy(figure5).Exposureto75nMofthe1adrenergic
agonist dobutamine, which produced a positive inotropic response comparable to ouabain 50 M in control
hearts (136 1 mmHg), still produced a significant increase of contractility in the I/R hearts (56 4
mmHgP<0.05vs.382mmHginI/R),suggestingthatthecontractileapparatusofmyocytesinI/Rheartswas
abletorespondtoinotropicstimuliotherthanouabain.
Asshowninfigure6A(leftpanel),ouabain50MresultedinarobustincreaseinAktSer473phosphorylation
in control hearts (2.44 0.29 in OUAvs. 1.04 0.11 in C P<0.01), but not in I/R hearts (6A, right panel).
Similarly,ouabain50MsignificantlyincreasedERK1/2phosphorylationincontrolhearts(1.000.07vs.2.77
0.17P<0.05),butnotinI/Rhearts.TotestwhetherthelackofAktresponsetoouabaininI/Rheartsresulted

from a general defect of Akt pathway after I/R, insulin 0.3 mU/ml was used according to Protocol B (figure
1).Asshowninfigure5(bluebars),thistreatmentdidnotsignificantlyaffectLVDPincontrolorI/Rhearts,
but induced a robust activation Akt Ser473 phosphorylation in both control and I/R hearts (Fig. 6C). OPC
preserved inotropic and signaling response to ouabain 50 M. Indeed, a significant increase of LVDP was
observed(647mmHgvs.10011P<0.05Figure5),whichwasaccompaniedbyasignificantincreasein
pAkt/AktandpERK1/2/ERK1/2ratios(Figure7).

Discussion
In Langendorffperfused rat heart preparations, we examined how I/R impacts Na+ /K+ ATPasemediated
signalinginresponseto50MoftheCGouabain.Na+ /K+ ATPasestructure,signalingandenzymefunction,as
well as cardiac contractile function were evaluated. The results suggest that I/R induces isoformspecific
Na+ /K+ ATPase alterations, which in turn modulate ouabain inotropic and signaling response in the ischemic
heart. They further revealed that OPC provides protection againstall I/Rinduced alterations of Na/KATPase
functions.

I/RinducedalterationofNa+/K+ATPaseisoenzymesandprotectionbyOPC
Numerous studies including ours have shown decreased sodium pump activity in cardiac tissue subjected to
I/R12,14,20,25.Inthepresentstudy,Na+ /K+ ATPaseincrudehomogenateswasreducedbyover60%,without
detectable changes in total protein expression of Na+ /K+ ATPase 1, 2 or 1 (Fig. 3A and 4). This is in
keeping with results from our recent study in rat neonatal cardiac myocytes 12, as is the complete protection
providedbyOPC.Intheadultratcardiactissue,itiswellestablishedthat1represents80%oftheNa+ /K+
ATPase catalyticsubunit expressed, and has a low affinity for ouabain. Na+ /K+ ATPase 2 represents the
remaining20%,with a higher affinity 26,27. Consistently, ouabain doseresponse curves were best fitted to a
modelwithtwophasesofinhibitionincontrolhearts,withamajorcontributionofthecomponentoflowaffinity

(Table1).Strikingly,ouabaininhibitioncurveswerenolongerbiphasicinpostischemichearts(Fig.3B).The
unique inhibitory constant was of low affinity, and the function of the high affinity component related to 2
containingisoenzymeswasnolongerdetected.Mechanistically,acombinedexposuretohighlevelsofreactive
oxygenspecies(ROS)andCa2+duringI/Rcouldexplaintheseisoenzymespecificalterationswithoutchangeof
total protein expression. Indeed, ROS preferentially alter 2mediated Na+ /K+ ATPase activity compared to
1 28,29,aselectivitythatismostlikelybasedonstructuralfeatures.Ithasalsobeenknownsincethe1980s
thatCa2+levelscriticallyaffectouabaindoseresponsesofcardiacNa+ /K+ ATPase.Specifically,thefunctionof
thecomponentofhighaffinityislostatsupraphysiologicalconcentrations 30,31.Whileallpreparationswere
perfused using a physiological concentration of 1.5 mM Ca2+ in the present study, a substantial I/Rinduced
increase in Ca2+ likely explains, at least in part, the monophasic ouabain doseresponse in I/R hearts.
Irrespectiveofthemechanisminvolved,thepresentstudyalsorevealedthatOPCpreventedtheseI/Rinduced
isoformspecificalterations.

ContractilityandsignalingresponsetoCGintheI/Rheart
AfterI/R,positiveinotropywasobservedinresponsetodobutaminebutnotouabain(Fig.5).Rather,ouabain50
MfurtherreducedLVDP,anadverseeffectsuggestiveoftoxicity.Indeed,althougharrythmogeniceffectsof
toxicconcentrationsofCGafterI/R32,33werenotobservedinourelectricallypacedpreparations,anincrease
of EDP was frequently observed upon addition of ouabain in I/R hearts, suggestive of Ca2+ overload (not
shown). Since LVDP was calculated as the difference between LVSP and EDP, this in turn contributed to a
decreaseinLVDPcomparedtoI/Ralone(Fig.5).AccordingtothemodelproposedbyMaixent&Lelievre17,
thelossofinotropicresponseandthetoxiceffectof50MouabainafterI/RaredirectconsequencesofI/R
inducedalterationsofNa+ /K+ ATPaseenzymaticproperties.Intheirmodel,thecomponentofhighaffinityfor
ouabain(2)isresponsiblefortheinotropicresponsewhereasinhibitionofthecomponentoflowaffinity(1)
mediatesthetoxiceffect.Consequently,theI/Rinducedlossoffunctionof2withincreasedcontributionof1
(see Table 1) led to the observed ouabainspecific loss of inotropic effect and toxicity 17, 34. In terms of
signaling,Aktphosphorylationpersistedinresponsetoinsulin(Fig.6C),butAktandERKphosphorylationno

longer occurred upon exposure to 50 M ouabain after I/R (Fig. 6B), suggesting that ouabain signaling was
specificallyaltered.OPCeffectivelyprotectedAktandERKresponsestoouabain(Fig.7).

Taken together, these findings indicate that both enzymatic andsignaling functions ofNa+ /K+ ATPase are
specificallyalteredintheischemicheart,andthatOPCpreservesallparameters.Twomainquestionsremainto
beaddressed.First,furtherstudiesareneededtoassesstheroleofNa+ /K+ ATPasenonenzymaticfunctionin
I/Rinduced cardiac myocyte cell death. Indeed, our previous study in rat neonatal cardiomyocytes provided
indirectevidencethatI/RinduceddisruptionofNa+ /K+ ATPasedriveniontransportmaynotbetheprimary
cause of I/Rinduced cell death 12 . This was based on the observation that OPC decreased I/Rinduced
cardiomyocyte death without observable protection of Na+ /K+ ATPasemediated ion transport (assessed by
ouabainsensitive86Rb+ uptake),andledustoproposethatanI/Rinducedeffectonthenonenzymaticfunction
of Na+ /K+ ATPase may occur. The present study provides the first evidence that at least some of Na+ /K+
ATPasemediated signaling pathways are indeed specifically disrupted by I/R and protected by OPC, which
warrantsfurtherstudiesofpossiblemechanisminvolved.

Acknowledgments
ThisworkwassupportedbyNationalHeart,Lung,andBloodInstituteGrantHL36573andbytheMarshall
InstituteforInterdisciplinaryResearch.

FigureLegends
Figure 1. Experimental protocols. Cardiac function was recorded in control (C), ischemiareperfusion (I/R)
and ouabain preconditioning (OPC) groups exposed to protocol A. The inotropic and signaling effects of
ouabain(OUA),dobutamine(DOB)andinsulin(INS)wereevaluatedusingprotocolB.KH:KrebsHenseleit
buffer.


Figure2.MyocardialFunction.Leftventriculardevelopedpressure(LVDP,2A)andenddiastolicpressure
(EDP,2B)werecontinuouslymeasuredincontrolhearts(C),heartsexposedto30minofischemiafollowedby
30minofreperfusion(I/R),orheartsexposedtoouabainpreconditioning(OPC)accordingtoprotocolA
(Figure1).Theamountoflactatedehydrogenase(LDH)wasmeasuredincoronaryeffluentduringreperfusion
(2C).ValuesaremeansSEMof610independentexperimentsforeachgroup.

Figure3.Na+/K+ATPaseenzymaticpropertiesincrudehearthomogenates.A.Na+/K+ATPaseinhearts
exposedtoprotocolA.ValuesaremeansSEMof8independentexperimentsforeachgroup.C:control,I/R:
ischemia/reperfusion,OPC:ouabainpreconditioning.**P<0.01vs.C, #P<0.05vs.I/R.B.Ouabaindose
dependentinhibitionofNa+/K+ATPase.ValuesaremeansSEM(n=6).Linesrepresentthetheoreticalcurves
afteranalysisusinganonlinearregressionmodel(seeMethods)bestfittedwithatwositesmodel(controland
OPC)oraonesitemodel(I/R).ComputedIC50andcontributionsofeachsitearereportedinTable1.

Figure4.Effectofischemia/reperfusionandOPConNa+/K+ATPasesubunits.Upper
Panels:RepresentativeimmunoblotsofNa+ /K+ ATPase1(A),2(B)andb1(C).Lowerpanels:Quantitative
analysisofimmunoblots.ValuesaremeansSEMof810independentsamplesforeachgroup,expressedas
subunit/GAPDHratios.TheseratiosarenormalizedtooneCsample/gel,whichwasassignedthevalueof1.C:
control,I/R:ischemia/reperfusion,OPC:ouabainpreconditioning.

Figure5.Effectofouabain50Moncontractilefunctionincontrol,I/RandOPChearts.Ouabain50M
(orange bars) or dobutamine 75nM (green bars) or insulin (0.3mU/ml) was applied for 15 min after aerobic
perfusionwithKrebsHenseleitbufferorfollowing30minofischemiaand30minreperfusionwithorwithout
OPC,accordingtoProtocolB(Figure1).ShownaremeansSEMfrom38independentexperimentsforeach
group.**P<0.001and***P<0.0001vs.C,#P<0.05vs.I/R,and$P<0.05vs.OPC.


Figure6.Effect of ouabain 50M on Akt and ERK phosphorylation in control and I/R hearts. Ouabain
50M(6A&6B)orinsulin0.3mU/ml(6C)wasaddedtotheperfusatefor15minafteraerobicperfusionwith
KrebsHenseleitbufferorfollowing30minofischemiaand30minreperfusion,accordingtoProtocolB(Figure
1). Crude homogenates were assayed for total and phosphorylated forms of Akt (A) and ERK (B). Upper
panels: representative immunoblots. Lower panels: activation quantified as ratio of phosphorylated to total
formoftheindicatedprotein.,normalizedtoC(leftpanels)orI/R(rightpanels).Theseratiosarenormalizedto
oneC(leftpanels)oroneI/R(leftpanel)sample/gel,whichwasassignedthevalueof1.Shownaremeans
SEMfrom36independentexperiments.*P<0.05and**P<0.01vs.correspondinguntreatedcondition.

Figure 7. Effect of ouabain 50M on Akt phosphorylation and ERK phosphorylation in hearts
preconditioned with ouabain.Total and phosphorylated forms of Akt (left) and ERK (right). Upper
panels:representativeimmunoblots.Lowerpanels:activationquantifiedasratioofphosphorylatedtototalform
oftheindicatedprotein.TheseratiosarenormalizedtooneOPCsample/gel,whichwasassignedthevalueof1.
ShownaremeansSEMfrom38independentexperiments.*P<0.05vsOPC.

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Figure1

Protocol
A

Protocol
B


min
95
50
0
80

Figure2

Figure3

Figure4

Na+/K+ATPase1

Figure5

Figure6

Akt

OUA
50M

OUA
50M

OUA
50M

OUA
50M

Figure6(continued)

INS

INS

I/R

Figure7

pAkt

Table1

Effect of ischemia and reperfusion on Na+/K+ATPase characteristics in crude heart

homogenates.

Control

Ischemia/Reperfusion

OuabainPreconditioning

(C)

(I/R)

(OPC)

(molPi/hr/mgofprotein)

7.40.8

2.50.6

6.40.7

Lowaffinitysite(1)

9.1x105

1.8x105

3.3x105

(67)

(100)

(64)

Na+/K+ATPaseactivity

IC50(M)
Contribution(%)
Highaffinitysite(2)
IC50(M)
Contribution(%)

5.9x108
(33)

6.9x108
(36)

ValuesaremeansSEM(n=6).