Assessing the effect of hypoxia on cardiac metabolism using ...1 Assessing the effect of hypoxia on...

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

.CC-BY-NC-ND 4.0 International licenseavailable under awas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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

The copyright holder for this preprint (whichthis version posted December 13, 2018. ; https://doi.org/10.1101/495069doi: bioRxiv preprint

https://doi.org/10.1101/495069http://creativecommons.org/licenses/by-nc-nd/4.0/

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




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




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





5

rateinallgroups. Wefurthermeasuredbodyweightandhaematocrit inthelongerexposure106

groups(1weekand3weekshypoxia).Intheselattergroups,expressionlevelsofcardiacPDH107

regulatorspyruvatedehydrogenasekinase(PDK)1,2and4,andtheexpressionleveloflactate108

dehydrogenase(LDH),responsibleforconversionofpyruvatetolactate,werealsomeasuredin109

cardiactissue.110




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





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





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





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




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





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




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





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





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




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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