Assessing the effect of hypoxia on cardiac metabolism using ...1 Assessing the effect of hypoxia on...
Transcript of Assessing the effect of hypoxia on cardiac metabolism using ...1 Assessing the effect of hypoxia on...
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Assessing the effect of hypoxia on cardiac metabolism using hyperpolarized 13C magnetic1
resonancespectroscopy2
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(ShortTitle–LePageetal:Assessingtheresponseofcardiacmetabolismtohypoxiawith13CMRS)4
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LydiaM.LePage1,2,3,Oliver J.Rider4,AndrewJ.Lewis4,VictoriaNoden1,MatthewKerr1,Lucia6
Giles1,LucyJ.A.Ambrose1,VickyBall1,LattMansor1,LisaC.Heather1*,andDamianJ.Tyler1,4*7
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1DepartmentofPhysiology,AnatomyandGenetics,UniversityofOxford,Oxford,UK9
2Department of Physical Therapy and Rehabilitation Science, University of California, San10
Francisco,SanFrancisco,USA11
3DepartmentofRadiologyandBiomedicalImaging,UniversityofCalifornia,SanFrancisco,San12
Francisco,USA13
4OxfordCentreforClinicalMagneticResonanceResearch,DivisionofCardiovascularMedicine,14
UniversityofOxford,Oxford,UK15
*jointlastauthor16
17Correspondingauthor: 18
DrLydiaLePage,DepartmentsofPhysicalTherapyandRehabilitationScience,andRadiology19
andBiomedicalImaging,UniversityofCalifornia,SanFrancisco,SanFrancisco,94158,CA,USA.20
Email:[email protected]
22
WordCount:3,15223
Keywords:hyperpolarized13C,cardiacmetabolism,hypoxia,magneticresonancespectroscopy24
Abbreviations:HP:hyperpolarized;PDH:pyruvatedehydrogenase;LDH:lactatedehydrogenase;25
BOLD: blood-oxygen-level dependent; PET: positron emission tomography; PDK: pyruvate26
dehydrogenasekinase;HIF:hypoxia-induciblefactor27
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Abstractsummary28
Hypoxiaplaysaroleinmanydiseasesandcanhaveawiderangeofeffectsoncardiacmetabolism29
dependingontheextentofthehypoxicinsult.Non-invasiveimagingmethodscouldshedvaluable30
lightonthemetaboliceffectsofhypoxiaontheheartinvivo.Hyperpolarizedcarbon-13magnetic31
resonance spectroscopy (HP 13C MRS) in particular is an exciting technique for imaging32
metabolismthatcouldprovidesuchinformation.33
Theaimofourworkwas,therefore,toestablishwhetherhyperpolarized13CMRScanbeusedto34
assesstheinvivoresponseofcardiacmetabolismtosystemicacuteandchronichypoxicexposure.35
GroupsofhealthymaleWistarratswereexposedtoeitheracute(30minutes),oneweekorthree36
weeks of hypoxia. In vivoMRS of hyperpolarized [1-13C]pyruvatewas carried out alongwith37
assessmentsofphysiologicalparametersandejectionfraction.Nosignificantchangesinheart38
rate, respiration rate, or ejection fraction were observed at any timepoint. Haematocrit was39
elevatedafteroneweekandthreeweeksofhypoxia.40
Thirtyminutesofhypoxiaresultedinasignificantreductioninpyruvatedehydrogenase(PDH)41
flux,whereasoneor threeweeksof hypoxia resulted inaPDH flux thatwasnotdifferent to42
normoxicanimals.Conversionofhyperpolarized[1-13C]pyruvateinto[1-13C]lactatewaselevated43
followingacutehypoxia,suggestiveofenhancedanaerobicglycolysis.ElevatedHPpyruvateto44
lactateconversionwasalsoseenattheone-weektimepoint,inconcertwithanincreaseinlactate45
dehydrogenase (LDH) expression. Following three weeks of hypoxic exposure, cardiac46
metabolismwascomparabletothatobservedinnormoxia.47
Wehavesuccessfullyvisualizedoftheeffectsofsystemichypoxiaoncardiacmetabolismusing48
hyperpolarized13CMRS,withdifferencesobservedfollowing30minutesand1weekofhypoxia.49
This demonstrates the potential of in vivo hyperpolarized 13C MRS data for assessing the50
cardiometaboliceffectsofhypoxiaindisease. 51
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Introduction52
Oxygenation of tissue is key to survival andmaintenance of organ health. The heart has the53
potential tobe exposed to a spectrumofhypoxic insults, ranging frommildandtransient, to54
prolongedandsevere.Themetaboliceffectsofacutehypoxiaarewelldocumented,andnotably55
involveincreasedglycolyticfluxandtransientlactateacidosis1,2.Prolongedandseverehypoxia56
requires reprogramming of cardiac metabolism; the heart downregulates oxygen-consuming57
processesandupregulatesglycolysisinanattempttomaximizeATPproductionunderoxygen58
restrictedconditions3–5.Theeffectsofchronichypoxiaareobservedinresponsetohighaltitude6,59
orasafactorinmanypathologicalconditions;examplesincludechronicobstructivepulmonary60
disease7, complications in pregnancy8, sleep apnoea9, myocardial infarction (the peri-infarct61
region)10andheartfailure11.62
However,muchofthisexistingliteraturereliesonexvivoassessmentofthemetabolicchanges63
thatoccur.Assuch,non-invasiveinvivomeasuresoftheeffectofoxygenlevelsoncardiactissue64
wouldbevaluable,especiallyasthehypoxicresponsecanbeverytransient12.Imagingtechniques65
havebeguntoprobeinvivooxygenlevels,andcurrentprominentmethodsincludeblood-oxygen-66
leveldependent(BOLD)MRIandpositronemissiontomography(PET)imaging,althoughneither67
isstandardclinicalpracticeasyet.BOLDMRIenablesassessmentofvascularoxygenation,using68
theparamagneticnatureofdeoxyhaemoglobintocreateimagecontrast13;thistechniquehasnot69
yetreachedtheclinicduetoacombinationofmanychallengesincludinglowsignal-to-noiseand70
aneed forrobustanalysis14,whichstudieshavebeguntoaddress15.There isalsoanongoing71
searchforPETprobestoassesshypoxia,themostpromisingofwhichiscurrently11C-acetate.72
Clearanceofthistracerisdependentonoxidativemetabolism,andsoaccumulationindicateslow73
oxygenpresence16.Itdoes,however,haveashorthalf-life17andsousagedependsonanearby74
cyclotron,andaswithallPEToptions,patientswillbeexposedtoionizingradiationwhichmay75
prohibitrepeatedmeasurements.76
Spectroscopic imaging holds potential for providing non-invasive, non-radioactive metabolic77
data.Imagingofcarbon-13(13C)inparticularcanbeveryinformativegiventheabundanceof78
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LePageetal:Assessingtheresponseofcardiacmetabolismtohypoxiawith13CMRS
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carbonpresentinmetabolites,includingthoseinpathwaysaffectedbyoxygenlevel.Although13C79
spectroscopy suffers from inherently low sensitivity in vivo, the adventof hyperpolarized 13C80
magneticresonancespectroscopy(HP13CMRS)offerstheuniqueabilitytomeasuretherateof81
enzymefluxinvivo18.Itprovidesanenhancementofthe13Csignalof>10,000fold,andassuch,82
enablesanon-invasivemeasurementofenzymaticfluxinrealtime.Intheheart,theglycolytic83
pathwayiscentraltothemetabolicchangesthatoccurasoxygenlevelsfall.Themostestablished84
hyperpolarized13C-labelledprobe,[1-13C]pyruvate,isrelevanttothispathway,asitallowsusto85
visualise the fateofpyruvateeither throughmitochondrialpyruvatedehydrogenase(PDH) to86
bicarbonate,orthroughcytosoliclactatedehydrogenase(LDH)intolactate19.Apreviousstudyby87
Laustsenetal.20showedthevalueofhyperpolarizedpyruvateintheinvestigationofhypoxiain88
thediabeticratkidney–demonstratinganabilitytomeasureincreasedlactateproductionafter89
fifteenminutesofhypoxicanaesthesia.Hypoxiaisalsooneofmanypathologicalfactorsoftumor90
development21,fluctuatingovertimeandinregionsofthetumor22,andassuchIvesonetal.used91
HP13CMRSinamousetumormodel,showingthatinspirationofahypoxicatmospherecaused92
increasedlactateproductionintumors23 .Oxidativestresshasbeeninvestigatedinafewnon-93
cardiac studies, using HP dehydroascorbate24,25, but the toxicity of this compoundmay limit94
translationtoclinicalstudies26.Indeed,thechallengesandfutureofhyperpolarizedprobesfor95
assessingrenalandcardiacoxygenmetabolismhavebeendiscussedinareviewbySchroeder96
andLaustsen27.Thusfar,nostudieshaveinvestigatedtheuseofHP13CMRStoassesstheeffect97
ofhypoxiaonglucosemetabolismintheinvivoheart.98
Inthisstudywehavethereforeassessedtheeffectofthreelengthsofhypoxicexposure–thirty99
minutes, one week, and three weeks - on the in vivo rat heart, using hyperpolarized [1-13C]100
pyruvate.WehavemeasuredtheconversionofHPpyruvatetobicarbonate,lactateandalanine101
(Figure1A shows thebiochemical pathways involved).The level of oxygen saturation in the102
bloodwasmatchedacrossgroups,andestablishedfollowingmeasurementinanimalshousedat103
11%oxygenfrompreviousrodentstudiesinourlaboratory3,4.Alongsidecardiacmetabolismby104
MRS,weassessedejectionfractionbyCINEMRIimaging,andmeasuredheartrateandrespiration105
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LePageetal:Assessingtheresponseofcardiacmetabolismtohypoxiawith13CMRS
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rateinallgroups. Wefurthermeasuredbodyweightandhaematocrit inthelongerexposure106
groups(1weekand3weekshypoxia).Intheselattergroups,expressionlevelsofcardiacPDH107
regulatorspyruvatedehydrogenasekinase(PDK)1,2and4,andtheexpressionleveloflactate108
dehydrogenase(LDH),responsibleforconversionofpyruvatetolactate,werealsomeasuredin109
cardiactissue.110
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Methods111
Animalhandling:MaleWistarrats(initialbodyweight~200g,Harlan,UK)werehousedona112
12:12-hlight/darkcycleinanimalfacilitiesattheUniversityofOxford.Allimagingstudieswere113
performedbetween6amand1pmwithanimalsinthefedstate.Allproceduresconformedtothe114
HomeOfficeGuidanceontheOperationoftheAnimals(ScientificProcedures)Actof1986andto115
UniversityofOxfordinstitutionalguidelines.116
117
Hypoxicexposure118
Agroupofhypoxically-housedanimals(n=6)andagroupofanimalshousedinnormoxia(n=4)119
wereusedtoassessbloodoxygensaturation.Saturationwasmeasuredtobe74±2%(Figure1B)120
in hypoxia, using a pulse oximeter on their hind paw (MouseOx, Starr Life Sciences). This121
concentrationwassubsequentlymatchedforallhypoxicexposures.122
123
Experimentalgroupsforinvivoimaging124
Three groups of animalswere exposed to three different lengths of hypoxia. Control groups125
experiencednormoxiaonly.ThegroupsaresummarizedinFigure1C.126
127
Thirtyminutes (acute)hypoxia:Animals (n=9)were anaesthetisedusing isoflurane (2%) in128
100%O2(2L/min).Metabolicandfunctionaldatawereacquiredinnormoxiaasdescribedinthe129
imaging protocol below. Animals were then slowly introduced to hypoxia by increasing130
replacementofoxygenwithnitrogenoverthirtyminutes,untilabloodoxygensaturationwhich131
matchedthatoftheanimalshousedinthehypoxicchamberwasachieved(describedabove).A132
second injection of hyperpolarized [1-13C]pyruvate was administered and a second data set133
acquired.Acutehypoxiaelicitedsomerapidphysiologicalresponsessuchasincreasedventilation134
andheartrate28,whichsettledpriortodataacquisition,allowingacquisitionofdatainastable135
hypoxicstate.136
137
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LePageetal:Assessingtheresponseofcardiacmetabolismtohypoxiawith13CMRS
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Oneweekofhypoxia:Animals (n=8)werehoused inanormobarichypoxic chamber forone138
week,duringwhichtimetheoxygenconcentrationwasreduceddailyby1-2%untilatthefinal139
daytheconcentrationwas11%.Animalswereweigheddaily,whichresultedinbriefexposureto140
normoxia(nolongerthan5minutes).Animalsweresubsequentlyanaesthetisedunderhypoxia141
(O2/N2mix)outsidethechamber,beforebeingplacedinthemagnetandtheimagingprotocol.142
executed. A control group (n=6)was housed outside the hypoxic chamber in room air (21%143
oxygen)foroneweekfromwhichnormoxicdatawereacquired.144
145
Threeweeksofhypoxia:Animals(n=8)wereintroducedtothenormobarichypoxicchamberas146
fortheone-weekexperiments,butremainedinthechamberforafurther14daysat11%oxygen.147
Animalswerethenanaesthetisedunderhypoxiaoutsidethechamber(O2/N2mix)andunderwent148
theMRprotocolasfortheone-weekanimals,toobtaininvivocardiacmetabolicdata.Acontrol149
group(n=8)washousedoutsidethehypoxicchamberinroomair(21%oxygen)forthreeweeks150
fromwhichnormoxicdatawereacquired.151
152
Magnetic resonance (MR) protocol: Animals were anaesthetised with isoflurane (3.5%153
induction and 2%maintenance). Ratswere positioned in a 7 T horizontal boreMR scanner154
interfaced toaDirectDrive console (VarianMedicalSystems,Yarnton,UK),andahome-built155
1H/13Cbutterflycoil(loopdiameter,2cm)wasplacedoverthechest.Correctpositioningwas156
confirmed by the acquisition of an axial proton fast low-angle shot (FLASH) image (TE/TR,157
1.17/2.33ms;matrixsize,64x64;FOV,60x60mm;slicethickness,2.5mm;excitationflipangle,158
15°).AnECG-gatedaxialCINEimagewasobtained(slicethickness:1.6mm,matrixsize:128×128,159
TE/TR:1.67/4.6ms, flip angle:15°) at the level of the papillarymuscles for ejection fraction160
calculation. An ECG-gated shim was used to reduce the proton linewidth to ~120 Hz.161
Hyperpolarized[1-13C]pyruvate(Sigma-Aldrich,Gillingham,UK)waspreparedby40minutesof162
hyperpolarization at ~1K as described by Ardenkjaer-Larsen et al.18, before being rapidly163
dissolved in a pressurised and heated alkaline solution. This produced a solution of 80mM164
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LePageetal:Assessingtheresponseofcardiacmetabolismtohypoxiawith13CMRS
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hyperpolarizedsodium[1-13C]pyruvateatphysiologicaltemperatureandpH,withapolarization165
of~30%.Onemillilitreofthissolutionwasinjectedovertensecondsviaatailveincannula(dose166
of ~0.32 mmol/kg). Sixty individual ECG-gated 13C MR slice selective, pulse-acquire cardiac167
spectrawereacquiredover60safterinjection(TR,1s;excitationflipangle,5°;slicethickness168
10mm,sweepwidth13,593Hz;acquiredpoints2,048;frequencycentredontheC1pyruvate169
resonance)29.170
171
Tissue collection: All animals were sacrificed with an overdose of isoflurane following172
completionof theMRprotocol. The heartwas rapidly removed,washedbriefly inphosphate173
bufferedsaline,andsnap-frozeninliquidnitrogen.174
175
Blood analyses: Samples of blood were collected from the chest cavity on sacrificing, and176
centrifugedat8,000rpmfor10minutes.Haematocritwasmeasuredusingamicrohaematocrit177
reader(Hawksley,UK).178
179
Tissueanalysis:ForWesternblottingofcardiactissuefromoneweekandthreeweekgroups,180
frozentissuewascrushedandlysisbufferaddedbeforetissuewashomogenised;aproteinassay181
establishedtheproteinconcentrationofeachlysate.Thesameconcentrationofproteinfromeach182
sample was loaded on to 12.5% SDS-PAGE gels and separated by electrophoresis30. Primary183
antibodiesforPDK1and2werepurchasedfromNewEnglandBiolabsandAbgent,respectively;184
anantibody forPDK4waskindlydonatedbyProf.MarySugden(QueenMary’s,Universityof185
London,UK).AprimaryantibodyforLDHwaspurchasedfromAbcam(ab52488).Evenprotein186
loadingandtransferwereconfirmedbyPonceaustaining(0.1%w/vin5%v/vaceticacid,Sigma-187
Aldrich),andinternalstandardswereusedtoensurehomogeneitybetweensamplesandgels.188
BandswerequantifiedusingUN-SCAN-ITgelsoftware(SilkScientific,USA)andallsampleswere189
runinduplicateonseparategelstoconfirmresults.190
191
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LePageetal:Assessingtheresponseofcardiacmetabolismtohypoxiawith13CMRS
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Magnetic resonancedataanalysis:All cardiac13C spectrawere analysedusing theAMARES192
algorithminthejMRUIsoftwarepackage31.Figure1Dshowsexamplespectrasummedover30193
seconds of acquisition in normoxic animals, acutely hypoxic animals and animals housed in194
hypoxia for one and three weeks, showing cardiometabolic conversion of the injected195
hyperpolarizedpyruvateintothedownstreamproductslactate,alanineandbicarbonate.Spectra196
were DC offset-corrected based on the last half of acquired points. The peak areas of [1-197
13C]pyruvate, [1-13C]lactate, [1-13C]alanine and[13C]bicarbonate at each time point were198
quantifiedandusedasinputdataforakineticmodelbasedonthatdevelopedbyZierhutetal.32199
andAthertonetal.33.PDHfluxwasquantifiedastherateof13Clabeltransferfrompyruvateto200
bicarbonate.Therateof 13C labeltransfer frompyruvate to lactateandalaninewasusedasa201
marker of lactate dehydrogenase activity and alanine aminotransferase activity respectively.202
CINE images were analyzed using cmr42 software (Circle Cardiovascular Imaging, Calgary,203
Canada)byanexperiencedanalystblindedtoexperimentalgroup.204
205
Statistical analyses: No significant differences were observed between the three normoxic206
groups(acute,oneweekandthreeweeks)foranyparameter,thereforeallnormoxicvalueswere207
combined for subsequent analysis. Values are reported as the mean ± standard deviation.208
Differences between groups were assessed using a one-way ANOVA followed by a Tukey’s209
multiplecomparisonstest.ThiswasperformedusingGraphPadPrismversion6.0gforMacOSX210
(GraphPadSoftware, La JollaCaliforniaUSA,www.graphpad.com). Statistical significancewas211
consideredifp≤0.05.212
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Results213
Oxygensaturationwassuccessfullyreducedinallhypoxicgroupscomparedwithnormoxicdata214
(Figure2A).215
216
Physiological Effects of Hypoxia: Hypoxia did not significantly affect in vivo heart rate,217
respirationrateorleftventricularejectionfractioninanygroup(Figure2B,C,D).However,the218
ANOVAforheartrategaveapvalueof0.051,andsoacomparisonbetweenthe30minuteand1-219
weekhypoxiadatashouldbenoted(p=0.04).Oneweekofhypoxiacausedasignificantincrease220
inhaematocritcomparedtonormoxia(49.3±0.6%and43±2%respectively),andhaematocritin221
three-weekhypoxicanimalswassignificantly increasedcompared toone-weekandnormoxic222
values(58±2%)(Figure2E);thisdemonstratessystemicadaptationtohypoxiaovertime.223
Animalshousedinhypoxiaforoneweekshowedsignificantlylowerbodyweightsthannormoxic224
animals. Following three weeks of hypoxia however, body weights were no different from225
controls.226
227
MetabolicEffectsofHypoxia:228
Invivodata:Following30minutesofhypoxia,animalsdemonstratedasignificantreductionin229
PDH flux (50%) compared to normoxic animals (0.009±0.003 s-1 and 0.017±0.007 s-1230
respectively;Figure 3A). In contrast, both 1 and3weeks of hypoxic exposure didnot show231
significantlyalteredPDH flux,with valuesnot significantlydifferent fromcontrols (one-week232
hypoxia:0.013±0.007s-1;three-weekshypoxia:0.017±0.011s-1;normoxia:0.017±0.007s-1).233
234
A significant (58%) increase inHP 13C label transfer to lactate (Figure3B),wasobserved in235
comparing30minuteshypoxicexposuretonormoxicdata(0.032±0.008s-1and0.020±0.006s-1236
respectively),indicativeofashort-termmetabolicshifttowardsanaerobicmetabolism.Afterone237
weekof hypoxia, theunchangedPDH fluxwasaccompaniedby an increased rateof 13C label238
transfertolactate(by40%)comparedtonormoxicanimals(0.028±0.008s-1and0.020±0.006s-239
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1respectively).Nodifferenceinfluxto13Clactatewasobservedfollowingthreeweeksofhypoxia240
comparedtonormoxicdata(0.023±0.002s-1and0.020±0.001s-1respectively).Nochangeinthe241
rateof13Clabeltransfertoalaninewasseenatanytimepoint(Figure3C).242
243
Biochemicalanalyses:244
Cardiac tissue from the one-week and three-week hypoxic groups was assessed ex vivo. In245
agreementwiththeunchangedPDHfluxatboththesetimepoints,nosignificantdifferencesin246
theproteinexpressionlevelsoftheregulatorycardiacPDKisoforms(1,2and4)wereobserved247
(Figure4).AsignificantlyhigherexpressionofLDHwasobservedinthe1-weekhypoxictissue,248
inlinewiththeincreasedHPpyruvatetolactateconversionseeninvivo.249
250
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Discussion251
Inhypoxia,metabolicchangeshavetooccurinorderforcardiacfunctiontobemaintainedunder252
theseoxygen-restrictedconditions.Firstlyconsideringtheresponsetoacutehypoxia,theheart253
mustrapidlyshiftmetabolismtowardsamoreanaerobicphenotype,whichischaracterisedby254
increased glycolysis, increased lactate efflux34 and decreased oxidative mitochondrial255
metabolism.Indeed,intheanimalsexposedto30minutesofhypoxia,cardiacpyruvatetolactate256
conversioninvivowassignificantlyincreased,andPDHfluxsignificantlydecreased.Therapid257
response that we observed, in line with the expected metabolic signature of anaerobic258
respiration, is likely mediated by changes in the NAD+/NADH ratio as a direct result of the259
decreased oxygen availability35. The reduced oxygen results in decreased mitochondrial260
respiration4, increasing NADH, inhibiting NAD-dependent dehydrogenases such as PDH and261
promotingNADH-dependentdehydrogenasessuchasLDH.262
Afteroneweekofhypoxicexposure,weobservedasignificantlyincreasedhaematocritlevel,as263
theanimalsunderwentadaptationtotheincreasinglevelofhypoxia.Thispotentiallyindicatesa264
partialadaptationtothehypoxicenvironment,aparticularlyviablesuggestionwhenconsidered265
alongside the three-weekhaematocritdata,which showsan additional significant increase in266
haematocrit.Thishypothesisof‘interim’adaptationissupportedbyatrendtoincreasedheart267
rate as a compensatory mechanism to ensure sufficient systemic oxygen delivery, and a268
significantlyreducedbodyweight.Similarparametershavebeenobservedinhumansadapting269
to altitude showing increasedheart rate36 anda lower calorie intake37, the latter of hasbeen270
suggestedtobeduetoincreasedleptinlevels38.271
Theincreasedhaematocritleveldemonstratedbyourone-weekandthree-weekhypoxicanimals272
is a hallmark of systemic adaptation to physiological hypoxia, driven by HIF-2α-stimulated273
productionof erythropoietin39,40.Glycolytic changeshavebeen reported tobepredominantly274
HIF-1α-regulated41suchasthatoflactatedehydrogenase42,theenzymeresponsiblefortheHP275
conversionwemeasured invivo.Glycolyticallyderived lactatewas increased in theone-week276
hypoxic animals, as assessed by HP pyruvate to lactate conversion, in line with significantly277
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increasedLDHexpressionincomparisontonormoxicdata.PDHfluxwasnotdecreased,which278
was supported by our assessment of expression levels of its PDK regulators, perhaps279
unexpectedlyduetopreviousstudiesdiscussingthehypoxia-induciblenatureofPDK143,44.Our280
three-weekhypoxicexposurealsoresultedinnometabolicdifferences(intheconversionofHP281
pyruvatetolactateorbicarbonate)incomparisonwithnormoxicdata,assupportedbymeasures282
ofPDKandLDHexpression.283
MuchresearchhashoweverfocussedontheeffectofhypoxiaonPDKexpressionincellculture.284
Kimetal.43andPapandreouetal.44showedupregulationofPDK1,inmouseembryonicfibroblasts285
following24-72hin0.5%hypoxia.Geneticover-activationofHIF1αincreasesPDK1and4protein286
levelsinmuscle45.Ithasgenerallybeenassumedthatthistranslatestotheheart,intheinvivo287
setting.Equally,measuredchangesintheseregulatorykinaseshavebeenextrapolatedtomeana288
changeinPDHactivity.However,ourdatasuggeststhatthismaynotenablecommentonlong-289
terminvivocardiachypoxia.Indeed,astudybyLeMoineetal.demonstratednoelevationofPDK1290
expressioninskeletalmusclefollowingoneweekofhypoxicexposure46.Previousstudiesfrom291
our group have shown that this three-week protocol of chronic hypoxia at 11% oxygen is292
sufficienttometabolicallyreprogramtheheartspecificallytobecomemoreoxygenefficient5in293
waysnotassessedinthisstudy.Further,studiesinanimalmodelsofhypertrophyhaverevealed294
unchangedPDHactivity47,48andnodifferences inPDK isoforms,whichappearedatoddswith295
cellularstudiesonhypoxia.Ourdatacontributestotheseobservationsandmayinfuturehelp296
explainthesituationindisease.297
298
Limitations299
This study did not measure ex vivo PDH activity, which could contribute to the in vivo HP300
measures,andcouldbealteredinspiteofunchangedPDKexpression.HowevertheworkbyLe301
Moine et al. demonstrated that ex vivo skeletal PDH activity inmice exposed to oneweek of302
hypoxiawasunchangedcomparedtonormoxicanimals49.Concomitantly,workbyAthertonetal.303
.CC-BY-NC-ND 4.0 International licenseavailable under awas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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-
LePageetal:Assessingtheresponseofcardiacmetabolismtohypoxiawith13CMRS
14
demonstratedasignificantcorrelationbetweeninvivodataacquiredusingHP[1-13C]pyruvate304
andPDHactivityassessedfromexvivotissue50,strengtheningthevalidityofourinvivoHPdata.305
Apulse-acquiresequencewasusedinthisstudy,anddataacquiredusingasurfacecoil.Future306
work could involve implementing a more elegant acquisition protocol51 to provide more307
informationonregionalhypoxiawithintheheart.308
Finally,normoxicanimalswereimagedusing100%oxygen,which,althoughcommonprocedure309
inpreclinicalanimalstudies,mayexacerbatethedifferenceswehaveseenhere.Futurestudies310
couldincludeanaesthesiaataloweroxygenpercentage.311
312
Conclusion313
Inconclusion,wehavedemonstratedtheabilityofHP[1-13C]pyruvatetonon-invasivelyassess314
metabolicchangesinthehealthyheartinresponsetothreelengthsofexposuretohypoxia.This315
couldthereforebeaviabletechniqueforassessinghypoxiainawiderangeofdiseasesandin316
responsetotherapy.317
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-
Funding318
This study was funded by grants from the British Heart Foundation (FS/10/002/28078,319
FS/14/17/30634)andDiabetesUK(11/0004175)andequipmentsupportwasprovidedbyGE320
Healthcare.321
322
Acknowledgements323
TheauthorswouldliketothankDr.LouiseUptonandProf.MarySugdenforthekindprovision324
ofthepulseoximeterandaprimaryantibodyforPDK4respectively.L.L.Pwouldalsoliketothank325
RichardandJocelynLePagefortechnicalassistanceinpreparingthemanuscript,andAsst.Prof.326
MyriamChaumeilforvaluablediscussions.327
328
Conflictsofinterest329
Lydia Le Page was supported in the form of a partial contribution to her D.Phil studies by330
AstraZenecaPLC,London,UK.331
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