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Assessing the effect of hypoxia on cardiac metabolism using hyperpolarized 13C magnetic1
resonancespectroscopy2
3
(ShortTitle–LePageetal:Assessingtheresponseofcardiacmetabolismtohypoxiawith13CMRS)4
5
LydiaM.LePage1,2,3,Oliver J.Rider4,AndrewJ.Lewis4,VictoriaNoden1,MatthewKerr1,Lucia6
Giles1,LucyJ.A.Ambrose1,VickyBall1,LattMansor1,LisaC.Heather1*,andDamianJ.Tyler1,4*7
8
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
4
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
5
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
7
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
8
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
9
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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LePageetal:Assessingtheresponseofcardiacmetabolismtohypoxiawith13CMRS
11
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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LePageetal:Assessingtheresponseofcardiacmetabolismtohypoxiawith13CMRS
13
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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