Aging Cell. 2020;19:e13213.  | 1 of 14https://doi.org/10.1111/acel.13213

wileyonlinelibrary.com/journal/acel

Received:20March2020  | Revised:16June2020  | Accepted:13July2020DOI: 10.1111/acel.13213

O R I G I N A L A R T I C L E

SS-31 and NMN: Two paths to improve metabolism and function in aged hearts

Jeremy A. Whitson1  | Alessandro Bitto1 | Huiliang Zhang1 | Mariya T. Sweetwyne1 | Rene Coig1 | Saakshi Bhayana2 | Eric G. Shankland2 | Lu Wang3 | Theo K. Bammler3 | Kathryn F. Mills4 | Shin-Ichiro Imai4 | Kevin E. Conley2 | David J. Marcinek2 | Peter S. Rabinovitch1

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,provided the original work is properly cited.©2020TheAuthors.Aging CellpublishedbyAnatomicalSocietyandJohnWiley&SonsLtd

1DepartmentofLaboratoryMedicineandPathology,UniversityofWashington,Seattle,Washington2DepartmentofRadiology,UniversityofWashington,Seattle,Washington3DepartmentofEnvironmental&OccupationalHealthSciences,UniversityofWashington,Seattle,Washington4DepartmentofDevelopmentalBiology,WashingtonUniversitySchoolofMedicine,St.Louis,Missouri

CorrespondencePeterS.Rabinovitch,1959NEPacificSt,HSBK-081,Seattle,WA98195.Email: petersr@u.washington.edu

Funding informationFunding for this research was provided bytheUWGeneticApproachestoAgingTrainingGrantT32AG000057-40,P01AG001751grant,andtheUWNathanShockCenter,andAFARGlennFoundationfor Medical Research Postdoctoral FellowshipforAgingResearchandAHA19CDA34660311toHZ.SS-31wasprovidedbyStealthBioTherapeutics(Newton,MA)freeofcharge.StealthBioTherapeutics did not play any role in thefunding,experimentaldesign,datacollection,orauthorshipofthisresearch.

AbstractThe effects of two different mitochondrial-targeted drugs, SS-31 and NMN, weretestedonOldmousehearts.Aftertreatmentwiththedrugs,individuallyorCombined,heartfunctionwasexaminedbyechocardiography.SS-31partiallyreversedanage-re-lateddeclineindiastolicfunctionwhileNMNfullyreversedanage-relateddeficiencyin systolic function at a higher workload. Metabolomic analysis revealed that both NMNandtheCombinedtreatmentincreasednicotinamideand1-methylnicotinamidelevels, indicatinggreaterNAD+ turnover,butonlytheCombinedtreatmentresultedinsignificantlygreatersteady-stateNAD(H)levels.Anovelmagneticresonancespec-troscopy approach was used to assess how metabolite levels responded to chang-ingcardiacworkload.PCr/ATPdecreased in response to increasedworkload inOldControl,butnotYoung,hearts,indicatinganage-relateddeclineinenergeticcapacity.BothdrugswereabletonormalizethePCr/ATPdynamics.SS-31andNMNtreatmentalso increasedmitochondrialNAD(P)Hproductionunderthehigherworkload,whileonlyNMN increasedNAD+ in response to increased work. These measures did not shift inheartsgiventheCombinedtreatment,whichmaybeowedtotheenhancedNAD(H)levelsintherestingstateafterthistreatment.Overall,theseresultsindicatethat both drugs are effective at restoring different aspects of mitochondrial and heart health and that combining them results in a synergistic effect that rejuvenates Old heartsandbestrecapitulatestheYoungstate.

K E Y W O R D Saging,heart,magneticresonancespectroscopy,metabolomics,NMN,SS-31

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1  |  INTRODUC TION

Cardiovascular disease remains the major cause of mortality in older adults (Heron&Anderson,2016;Tinettietal.,2012),eclipsingallformsofcancerbytheageof75(Rogeretal.,2011).Furthermore,age-relateddeclineinheartfunction,whichincludesdysfunctioninbothdiastoleandsystole(Dai,Chen,Johnson,Szeto,&Rabinovitch,2012), has been shown to be strongly associated with frailty(Leibowitz et al., 2016). Because of the immense burden of heartdysfunction on the elderly population, the development of treat-ments that can improve heart health in aged individuals remain a top priorityforextendinghumanhealthspan.

The majority of the heart's volume consists of cardiomyocytes (Zhou&Pu, 2016),whichmustmeet the incredible energetic de-mandofcontinuouscontractionand relaxation throughoutanen-tire lifespan.Tomeet this energeticneed,heart tissuehasoneofthehighestmitochondrialdensitiesfoundinthebody(Benardetal.,2006). As cells age, nicotinamide dinucleotide (NAD+) levels, ATPoutput, and mitochondrial biogenesis decrease, while oxidativestress, mtDNA damage, and mitochondrial structural alterationsincrease(Chistiakov,Sobenin,Revin,Orekhov,&Bobryshev,2014).Based on these physiological features, it has long been hypothe-sized that mitochondrial fitness is key to maintaining heart health (Herrmann&Decherd, 1939). According to this hypothesis, treat-ments that can restore mitochondrial parameters to more youthful states in aged cardiomyocytes have the potential to repair the func-tion of aged hearts.

Inthisstudy,wesetouttodeterminethefunctionalandmeta-boliceffectsonagedmouseheartsof twomitochondrial-targeteddrugsproposedtotreatage-relateddysfunction:SS-31andnicotin-amidemononucleotide(NMN).

SS-31,alsoknownaselamipretide, isasynthetictetrapeptideconsistingofD-arginine,2′,6′-dimethyl-l-tyrosine,lysine,andphe-nylalaninethatassociateswiththeheadgroupofcardiolipin,anes-sential phospholipid specific to the mitochondrial inner membrane (Szeto,2014).PrevioustestingofSS-31inmammalshasshownthatthedrugdecreasesoxidativestress(Houetal.,2016)andprotectsagainstischemia–reperfusioninjury(Caietal.,2018),althoughthespecific mechanisms by which the drug elicits these changes are still being actively investigated. There are indications that a combi-nationofpreventingcytochromecperoxidaseactivity,promotingcristae,andelectrontransportchaincomplexorganization,aswellas influencing othermitochondrial proteins, contributes to thesebeneficial effects (Birk, Chao, Bracken, Warren, & Szeto, 2014;Szeto,2014).WehaverecentlydemonstratedthatSS-31improvesdiastolicfunctionandreducesoxidativestressintheheartsofagedmicewhendeliveredfor8weeks(Chiaoetal.,2020).

NMNisanaturallyoccurringnucleotideprecursorofNAD+ that isgeneratedviatheNAD+salvagepathway(Yoshino,Baur,&Imai,2018).NMNanditsprecursornicotinamideriboside(NR)havebeenhypothesized to improve mitochondrial health by increasing cellular NAD+ availability and therefore improving energetic capacity and activating sirtuin deacetylase activity. Recent studies have provided

strongevidence for thishypothesis and shown thatNMNsupple-mentation bolstersNAD+ availability and improves many classical signsofaginginmice(Millsetal.,2016).NMNsupplementationhasalso been shown to partially restore heart function in a mouse model ofheartfailure(Leeetal.,2016).

AlthoughNMNandSS-31bothtargetmitochondria,theydosoviadifferentmechanismsofaction.Thus,wehypothesizedthatthedrugs would differ in effect on the heart and may have a synergistic effectwhenappliedtogether.Wesoughttocompareandcontrastthe mechanism and effects of these drugs in order to better under-standeachindividuallyandhowage-relateddeficits inheartfunc-tion can be addressed.

Because mice recapitulate many aspects of human aging (Vanhooren & Libert, 2013), including a general decline in heartfunction (Dai et al., 2012), they are an excellentmodel for deter-mining whether these drugs have the potential to rejuvenate aged hearts in humans and to determine the mechanisms of this im-provement. To test the effects of these drugs on the aged heart,miceaged24monthsatthestartoftreatment(Old)wererandomlysortedintoControl,SS-31,NMN,orCombined(SS-31+NMN)treat-mentgroupsforcomparisonwithmice5–6monthsofage(Young).TreatmentdosesofSS-31andNMNwereinalignmentwiththosethat have previously been shown to be effective in other healthspan measures(Chiaoetal.,2020;Millsetal.,2016).Invivomagneticres-onancespectroscopyistypicallyusedtoexamineATPandPCrlevelsinmuscletissue,thoughwhenappliedtobeatingheartsthespectramustbevoxel-localizedandtimedtocoincidewithheartandpulmo-narycycles.Inthisreport,wefurtherdevelopedandappliedforthefirsttimeanMRStechniquetodeterminehowadditionalheartme-tabolites,includingnicotinamidenucleotides,respondtoanincreasein cardiacworkload,definedbyanacceleratedheart rate, invivo.This analysis was coupled with measurements by echocardiography andliquidchromatographytandemmassspectrometry(LC-MS/MS)to obtain an integrated view of changes resulting from age and drug treatment from the metabolic to the functional level in the heart.

2  |  RESULTS

2.1  |  SS-31 and NMN treatments result in different functional improvements

Echocardiography was performed on all mice at baseline and again onOldmiceafter8weeksoftreatment.Diastolicfunctionwasas-sessed by determining the ratio of blood flow across the mitral valve inearlydiastole(Ea)totheflowinlatediastole(Aa).Ea/Aaisoneofthe primary markers used when assessing diastolic function in human patients(Mitter,Shah,&Thomas,2017)andistheparameterofdi-astolic function that has been used in composing a comprehensive frailtyindexofmouseaging(Kane,Keller,Heinze-Milne,Grandy,&Howlett,2019).OldControlmicehadasignificantly(p<0.05)lowerEa/Aa compared to Youngmice at their baseline, indicating a de-clineindiastolicfunctionwithage.Asfoundpreviously(Chiaoetal.,

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2020),OldmicetreatedwithSS-31showedasignificant(p<0.05)improvementinEa/Aa,restoringitapproximatelyhalfwaytothatofYoungEa/Aavalues,whereastheControlandNMN-treatedgroupsshowed no change in this parameter (Figure 1a). The Combinedtreatment group recapitulated the Ea/Aa change resulting fromSS-31alonebutdidnotshowanyfurtherimprovement.

Systolicfunctionwasassessedbymeasuringpercentfractionalshortening (i.e., the degree towhich the ventricular lumen closesduringsystole)fromechocardiograms.Sinceanesthetizedmicehavesubphysiologicalheartrates,alterationsinsystolicfunctionmaynotbe manifested under these low work conditions. Thus, fractionalshortening was also measured following injection of the ionotropic

agent dobutamine at a concentration that produced higher heart rateswithin thenormalphysiological range (Figure1b).Long-termassessment of heart rates showed that dobutamine administra-tion results in a similar significant (p<0.001)increaseinheartrateacrossallgroups(FigureS1).Therewerenosignificantdifferencesin fractional shortening between the groups at the low work state or within groups at treatment endpoints relative to their baselines (FigureS2).Underhigherworkloadconditions,however,OldControlmice manifested a significantly (p<0.0001)lowerpercentfractionalshorteningthanYoungmice, indicatingadecline inhighworksys-tolicfunctionwithage.SS-31treatmentdidnotsignificantlyaffectfractionalshortening,butNMNandtheCombinedtreatmentsboth

F I G U R E 1   EffectofSS-31andNMNonheartfunction.(a)Ea/Aaratioatbaseline(treatmentday0)andendpoint(8weeksoftreatment)and(b)percentfractionalshorteningatlowwork(-dobutamine)andhighwork(+dobutamine)atthe8weektreatmentendpoint,measuredby echocardiography. N=29Young,49OldControl,29OldSS-31,28OldNMN,10OldCombined.(c)Highwork(+dobutamine)percentfractionalshorteningmeasuredbyechocardiographyinYoungandOldmiceat0,1,6,10,20,30,42,and56daysofNMNtreatment.N=8Young,19Old.(d)Ratioofheartweight(g)totibialength(mm)assessedatendpoint(8weeksoftreatment)asameasureofcardiachypertrophy. N=21Young,21OldControl,16OldSS-31,15OldNMN,14OldCombined.

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significantly (p < 0.005) improved high work percent fractionalshorteninginOldmice,fullyrestoringittoYounglevels(Figure1b).

While our previous studies have shown that SS-31 induces apartial effect on diastolic function after 4 weeks of treatment and appearstoreachitspeakeffectat8weeks(Chiaoetal.,2020),itwasunknown on what timescale the cardiac functional improvements in-ducedbyNMNoccur.Totestthis,YoungandOldmiceweretreatedwith NMN and tested at regular intervals to determine changesin their highwork percent fractional shortening (Figure 1c). After10 days of treatment,Oldmice treatedwithNMN showed a sig-nificant (p<0.05)improvementinhighworkfractionalshortening,restoringittoYounglevels.Thisremainedconstant,withnofurtherenhancementfromprolongedtreatment.Youngfractionalshorten-ingwasunaffectedbyNMNtreatment.YoungmicewerealsogivenSS-31todeterminewhethertherewasanyeffectondiastolicfunc-tion, but the Ea/Aa ratiowas not significantly changed after 4 or8weeksofSS-31treatment(FigureS3).Thepersistenceofthefunc-tionalimprovementinducedbyNMNtreatmentwasalsoexamined.IncontrasttoSS-31,whichwehavepreviouslyshowntomaintainhalf its functional effect 4 weeks after cessation of treatment (Chiao etal.,2020),NMN'seffectonagedheartsismoretransient.AfterstoppingNMNtreatment,highworkfractionalshorteningremainedthesameonedaylaterbutthendeclinedatapproximatelytherateithadimprovedafterstartingtreatment,matchingthebaselineforOldheartsafter10dayswithouttreatment(FigureS4).

Relative toYoungmice,OldControlmiceshowedasignificant(p<0.05)increaseincardiachypertrophy,measuredastheratioofheartweight to tibia length (Figure 1d). NMN and the Combinedtreatments appeared to reduce this hypertrophy and partially re-storeheartweight/tibialengthtotheYoungstate,butSS-31alonedid not have a significant effect.

Mitochondrial function in isolated cardiomyocytes from Old ControlandNMN-treatedheartswasassessedbySeahorseassay(AgilentTechnologies,SantaClara,CA).WhilewehavepreviouslyshownbythismethodthatSS-31normalizedage-relatedincreasesinbasalrespirationandprotonleak(Chiaoetal.,2020),NMNtreat-mentdidnotappeartohaveanysucheffect,asmitochondrialoxy-gen consumption appeared unchanged from the Control group by all measures(FigureS5).

2.2  |  Metabolomic analyses of hearts reveal unique changes resulting from SS-31 and NMN treatments

Analysisofcardiacmetabolicdifferencesat theendpointof treat-mentwascompletedbytwoLC-MS/MSmethods:(a)afocusedanal-ysisofNAD-relatedmetabolitesand(b)ageneralscreeningof369common metabolites.

By the first method, the total NAD(H) pool of the heart ap-peared todeclinewithage (Figure2a),ashasbeenreportedelse-where(Braidyetal.,2011).NeitherSS-31norNMNtreatmentaloneresultedinanychangetototalNAD(H).However,whenNMNandSS-31weregivenincombination,therewasasignificant(p<0.05)increase in NAD(H) levels. The ratio of NAD+ to NADH did notchangesignificantlyinanyofthegroups.NADP+ also did not show anysignificantchangesbetweenthegroups(Figure2b),andNADPHwas undetectable by this assay. Of the assayed NAD precursors,onlynicotinamide(NAM)showedadifferencebetweengroups,witha significant (p<0.02)decrease in levelsbetweenYoungandOldControlhearts,andarestorationbybothNMNandCombinedtreat-ments (p<0.05)(Figure2c).NMN,nicotinamideriboside(NR),andnicotinicacid(NA)alsoshowedthesametrendofadecreasewithageandrestorationbyNMNandCombinedtreatmentsbutdidnotindividually reachsignificance (Figure2d–f).Nicotinamideadeninedinucleotide(NAAD)didnotfollowthistrendanddidnotshowanysignificantchanges(Figure2g).

In the general targeted metabolomics assay, which primarilyassessed metabolites involved in protein and carbohydrate energy metabolism,26metabolitesshowedsignificant(FDR<0.1)changeswhencomparingYoungtoOldControlmice(Table1).Noneofthetreatments appeared to have a significant effect on shifting the overallmetabolomeofOldheartsbacktowardtheYoungstate,andeachshowedonlyafewsignificant(FDR<0.1)changes.Mostnota-bly,theNMNandCombinedtreatmentsresultedinasharpincreasein 1-methylnicotinamide (MNA) (FDR< 0.0001) inOldmice com-paredtoYoung,OldControl,andOldSS-31mice(Figure3a).SS-31treatment resulted in modest increases in xanthine and choline(FDR<0.1)levels(Figure3b,c).Intriguingly,theCombinedtreatmentdidnotrecapitulatetheSS-31-inducedchangesandinsteadshowedsimilar levelsofbothcholineandxanthine to theOldControlandOldNMNgroups.The full setof comparisonsbetweenall groupscan be found in the Appendix S1. We investigated whether thechangeinMNAlevelscouldbeduetoadifferenceinnicotinamideN-methyltransferase(NNMT)levels;however,thisenzymewasnotdetectableinanymouseheartsbyWesternblot(FigureS6)orshot-gunproteomics(datanotshown).

Analysis of general trends in canonical pathways from thisdataset was performed based on all changes p<0.05 (unadjustedp-values) in Young relative to Old Control hearts using IngenuityPathwayAnalysis(Qiagen,Hilden,Germany).Thisrevealedthatthemajormetabolicpathwaysbeingaffectedbyageincludedglycolysis,citrulline biosynthesis, phosphatidylcholine biosynthesis, and oth-ers (Figure3d).Quantificationof relativechanges inthecanonicalpathways heatmap was split into metabolites that increased with age (Figure3e)andthosethatdecreasedwithage(Figure3f).Whilethereweresuggestivetrendstowardreversaloftheage-relatedchanges

F I G U R E 2   AnalysisofendpointNAD+-relatedmetabolitelevels.Endpoint(8weeksoftreatment)levelsof(a)NAD(H),(b)NADP+,(c)NAM,(d)NMN,(e)NR,(f)NA,and(g)NAADinmouseheartsasmeasuredbytargetedLC-MS/MS.Theheightofthestackedcolumnsin(a)indicatesthetotalNAD+NADHcontent.Thesignificancenotedisbasedonthesetotalvalues.N=11Young,10OldControl,11OldSS-31,11OldNMN,8OldCombined.

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to thesepathways, treatmenteffectsdidnot reachsignificance inthequantification.Meandifferenceplots foreachcomparisonareprovidedinthesupplementaldata(FigureS7).

2.3  |  Magnetic resonance spectroscopy probes dynamic metabolic shifts in vivo

31P-magnetic resonance spectroscopy (MRS) was applied at thetreatment endpoint in order to probe how metabolites in the heart respond to a dobutamine-induced increase in cardiacworkload invivo.Image-SelectedInVivoSpectroscopy(ISIS)wasusedtocollectdata localized to the heart with minimal inclusion of surrounding tis-sues.Arepresentativeheart-localizedvoxelandresultingMRSspec-trumfromthistechniqueareshowninFigure4a.

The principle difference in the energetic response to the in-creased heart rate was measured as the ratio of the peak areas of

phosphocreatine(PCr)toγ-ATP(givenasPCr/ATP;Figure4b).Thismeasurement provides an assessment of how effectively the heart can respond to increased work by determining the degree to which the high-energy phosphates stored in PCr are depleted from car-diomyocytesunderincreaseddemandforATP.WhileYoungmouseheartsshowednochangeinPCr/ATPinresponsetoincreasedwork-load,OldControlmouseheartshadasignificant(p<0.05)decreaseinPCr/ATPfollowingdobutamineinjection,indicatingadiminishedabilityof theheart tohandle suchaworkloadwithage.Althoughstill showinga slightdecrease inPCr/ATPat thehigherworkload,theSS-31,NMN,andCombinedtreatmentsallappearedtomitigatetheage-relatedchange,andPCr/ATPinthetreatedgroupswasnotsignificantlydifferentfromtheYoungstateortheirpredobutaminebaselines.

In order to analyze how less abundant metabolites in the heart respond to increased cardiac workload in these mice, peaks cor-responding to chemical standards of α-ATP, UDP-glucose, NAD+,

Metabolite

Fold change relative to old control

Young Old SS−31 Old NMNOld combined

5-Aminovalericacid 2.09(0.38) 1.79(0.38) 1.76(0.38) 1.81(0.41)

Carnosine 2.00(0.15) 0.78(0.16) 0.84(0.16) 0.94(0.17)

N-Formylmethionine 1.93(0.32) 1.60(0.32) 1.62(0.33) 1.79(0.35)

Hypotaurine 1.92(0.21) 1.16(0.22) 1.23(0.22) 1.35(0.23)

Beta-alanine 1.78(0.15) 0.95(0.15) 1.21(0.15) 1.12(0.16)

Anserine 1.64(0.10) 0.80(0.11) 0.89(0.11) 0.82(0.11)

Adenine 1.56(0.16) 1.09(0.17) 1.03(0.17) 1.21(0.18)

Cholesterol sulfate 1.46(0.19) 1.14(0.19) 1.08(0.19) 1.11(0.20)

Ethylmalonic acid 1.39(0.18) 1.00(0.18) 1.29(0.18) 1.06(0.19)

5′-Methylthioadenosine 1.35(0.14) 1.00(0.14) 0.96(0.14) 1.05(0.15)

ADP 1.32(0.08) 1.07(0.08) 1.02(0.08) 1.02(0.09)

Citrulline 1.29(0.10) 1.01(0.10) 1.04(0.10) 1.13(0.11)

L-Glutamicacid 1.20(0.10) 1.11(0.10) 0.98(0.10) 0.94(0.11)

Choline 1.14(0.06) 1.19(0.06) 0.99(0.06) 1.02(0.07)

L-Serine 0.81(0.10) 1.06(0.10) 0.90(0.10) 0.89(0.11)

Sedoheptulose7-phosphate 0.77(0.13) 1.20(0.13) 0.93(0.13) 1.04(0.14)

3-Methylhistidine 0.65(0.16) 0.89(0.16) 0.97(0.16) 0.87(0.18)

Pipecolic acid 0.63(0.20) 0.97(0.20) 0.86(0.20) 0.90(0.21)

Dihydroxyacetonephosphate

0.59(0.14) 0.72(0.14) 0.93(0.14) 0.81(0.16)

Lactose/trehalose 0.55(0.25) 0.81(0.26) 0.99(0.26) 1.14(0.28)

Glyceraldehyde-3-phosphate 0.54(0.16) 0.71(0.16) 0.91(0.16) 0.78(0.17)

Erythrose4-phosphate 0.50(0.28) 0.75(0.29) 0.91(0.29) 1.03(0.31)

Glucose-6-phosphate 0.49(0.29) 0.77(0.29) 0.93(0.29) 1.05(0.31)

Trimethyloxamine 0.47(0.31) 1.33(0.31) 1.14(0.31) 1.07(0.34)

Glucose-1,6-bisphosphate 0.40(0.32) 0.56(0.32) 0.90(0.32) 0.61(0.34)

L-Cystine 0.32(0.43) 0.85(0.43) 0.58(0.44) 0.62(0.47)

Values are untransformed fold changes with standard errors in parentheses. Bold numbers indicate significance(FDR<0.1).Orderedbyfoldchange.FullresultscanbefoundintheAppendixS1.

TA B L E 1   Significantage-relatedmetabolomics changes in mouse hearts

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NADH, NADP+, and NADPH were fit to the experimental spec-tra forquantificationusinga custommodel inOriginPro software(OriginLab, Northampton,MA). An additional peak correspondingtomitochondrialNAD(P)Hwasalsofittothedata,baseduponourpriorstudydemonstratingthepresenceofthispeakinvivo,whichhasanalteredchemicalshift relativetocytosolicNAD(P)Hduetothe unique environment of the mitochondrial matrix (Conley, Ali,Flores, Jubrias,&Shankland, 2016).A representative fit of exper-imental data using this model is shown in Figure 4c. To assess the performanceofthistechniqueforanalyzingthesemetabolites,MRSresults from Old Control mice prior to dobutamine injection were comparedwiththosefromthegeneralmetabolomicsscreen,whichalsoanalyzedNAD(P)(H)andUDP-glucose(Figure4d).Relativelev-els (normalized tomeanNAD+ for comparisonacrossmethods)ofNAD+, NADH, and UDP-glucose determined by both techniqueswere remarkably similar, but there was a large discrepancy inNADP+andNADPHlevels,whichweremuchhigherrelativelywhen

measuredbyMRS.Thisisunsurprisinggiventheknownissueswithextracting and preserving these metabolites from tissue samples(Lu,Wang,Chen,Hui,&Rabinowitz, 2018) and thusmay actuallydemonstrateastrengthoftheinvivoMRSmethod.MitochondrialNAD(P)Hcannotbedistinguishedfromthecytosol-localizedmole-cules by the metabolomics method and thus was not included in the comparison.Overall,thiscomparisonsupportstherelativequantifi-cationofthesemetabolitesfrominvivoheartMRSspectra.

TheMRSdatashowedatrendofNAD+ remaining stable or de-creasingslightlyinYoungheartswithhigherworkloadbutincreasingin Old Control hearts (p=0.066)(Figure4e).SS-31andtheCombinedtreatmentappearedtoreversethisage-relatedchangeinNAD+ dy-namics.Conversely,NMNtreatmentdidnotreversetheage-relatedchangeandtheincreaseinNAD+ at higher workload in these mice was significantly (p<0.05)differentfromtheYoungstateathigherworkload. Mitochondrial NAD(P)H also showed changing dynam-icswithdrug treatment (Figure4f).WhileYoungandOldControl

F I G U R E 3   Generalmetabolomicsanalysisofmousehearts.Endpoint(8weeksoftreatment)levelsof(a)1-methylnicotinamide(MNA),(b)xanthine,and(c)cholineasmeasuredbygeneraltargetedmetabolomics.N=12Young,10OldControl,12OldSS-31,11OldNMN,8OldCombined.Meansarecalculatedfrompostprocessedvaluesandarenotlog2-transformed.(d)Heatmapofmetabolicchangesinthetop20canonical pathways based on all changes with an unadjusted p<0.05whencomparingYoungtoOldControl.(eandf)Quantificationoftheheatmap,separatedintometabolitesthatdecreased(e)orincreased(f)withage.

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hearts had a trend of slightly decreased mitochondrial NAD(P)Hwith the increasedheart rate,SS-31andNMN-treatedhearts ap-peared to reverse this trend (p=0.074andp<0.05,respectively),asthesetreatmentsincreasedmitochondrialNAD(P)Hinresponsetoahigherworkload.Interestingly,theCombinedtreatmentdidnotrecapitulate the individual results of SS-31 andNMNand insteadshowedmitochondrialNAD(P)H levelsthatwereunchangedwhendobutamine was applied. Other metabolites did not show a signifi-cantdifferencebetweengroupswithincreasedworkload,andtheseresultsarepresentedinFigureS8.

3  |  DISCUSSION

3.1  |  SS-31 and NMN improve different aspects of heart health

SS-31andNMNtreatmentsdistinctlyimprovediastolicandhigherworkload systolic function, respectively. Since giving the drugs incombinationconferredbothdiastolicandsystolicimprovements,butneitherpreventednorfurtheraddedtotheindividualimprovements,

the data imply that the drugs use two independent mechanisms to achievethesefunctionalbenefits.Furthermore,sincethedrugsonlyshowedaneffectinOldhearts,thisappearstobeatruerejuvena-tion of heart function in both cases.

In thiscohortofmice,wefoundthatNMNandtheCombinedtreatmentwereeffectiveatreducingage-relatedhypertrophyoftheheartwhileSS-31alonewasnot.However,inothercohortsofmicewe have previously observed a modest but significant reduction in hypertrophy followingSS-31 treatment (Chiaoetal.,2020).Giventhe small difference in magnitude between these new results and thosealreadypublished,webelievethisdifferenceisprimarilybasedupon statistical variation and not the underlying biology.

NMNappears tohaveamuchmoreacuteeffecton theheartthan SS-31.While NMN achieves its peak functional effect afterjust10daysoftreatment,wehavepreviouslyobservedthatSS-31treatmenttakesapproximately8weekstoreachitspeakeffectontheheart (Chiaoetal.,2020).NMN'seffectwashalvedby6daysafter treatment cessation and completely gone after 10 days,whereasapartialSS-31effectpersistedforseveralweeksafterces-sationoftreatment(Chiaoetal.,2020).Thesedifferingtimelinesfitwith thehypothesis thatSS-31carriesout itseffectsby repairing

F I G U R E 4  Magneticresonancemeasurementsofdynamicmitochondrialmetabolism.(a)Representative1H-magneticresonanceimagingofmousechestcavity,heartvoxellocalizationforISIS,andresulting31Pspectrum.(b)ChangeinPCr/ATPratioafterinjectionofdobutamine.N=11Young,11OldControl,11OldSS-31,13OldNMN,10OldCombined.(c)Representativeimageoftheautomatedfittingmodel.(d)ComparisonofMRSandmetabolomicsOldControlresults.N=11MRS,10metabolomics.(eandf)Changein(e)NAD+and(f)mitochondrialNAD(P)Hafterinjectionofdobutamine.N=11Young,10OldControl,9OldSS-31,11OldNMN,10OldCombined.

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mitochondriaefficiencyandreducingoxidativestress,followedbymyocardialremodeling(Chiaoetal.,2020),whereasNMNprimarilyworksbyboostingNAD(H)biosynthesisand thusenhancingmyo-cardialenergeticsupplyviaoxidativephosphorylation.

Consistentwiththeseproposedmechanisms,wedidnotdetectanydifferenceinmitochondrialcoupling/efficiencyintheSeahorseassaywithunstimulatedcardiomyocytesisolatedfromNMN-treatedhearts. This is in clear contrast to SS-31, which improved protonleak and respiratory control ratio in the same assay (Zhang et al.,2020).WebelievethisisconsistentwithmitochondrialremodelingbySS-31thatdoesnotoccurfollowingNMNtreatment.However,thisdoesnotmeanthatNMNdoesnothaveanyeffectonthemito-chondria,asdiscussedbelow.

3.2  |  Metabolic crosstalk between SS-31 and NMN and the fate of NAD+

TheobservationthatNAMandMNAconcentrationsincreasedafterNMNtreatmentisunsurprising,giventhatbotharestepsalongthebreakdown pathway of NAD+ (Bender, 2003). Thus, the increasein bothmetabolites is an expected result of increasedNAD+ bio-synthesisandgreaterturnoverofNAD+ from its use as an enzyme cofactor, primarily for sirtuin deacetylases and poly ADP-ribosepolymerases(PARPs)(Pillai,Isbatan,Imai,&Gupta,2005).Thisen-hanced turnover ofNAD+may explainwhyNMN treatment doesnotsignificantlyincreaserestingNAD(H)levels,asnotedhereandinprevious study (Millset al.,2016).However, it is less clearwhyNMNandSS-31givenincombinationdidsignificantlyincreasetheNAD(H)poolwhenneitherdrugalonedid.Thiswouldappeartoin-dicateasynergisticeffectofthedrugs.OneplausibleexplanationisthatSS-31'sdemonstratedreductionofoxidativestress(Chiaoetal.,2020;Houetal.,2016)resultsinlessPARPactivationandthereforelessNAD+breakdown,allowingittoaccumulatetohigherlevelsinthe cell.

SinceNNMT, theenzymethatconvertsNAMtoMNAandre-moves it from reentering theNAD+ salvage pathway of synthesis (Bockwoldtetal.,2019),wasatundetectablelevelsintheheart, ifpresentatall,conversiontoMNAislikelyoccurringelsewhereinthemice,withtheMNAfoundinthehearthavingenteredfromthecir-culation.OneconcernwithincreasedMNAsynthesisisthepotentialfordepletionofmethyldonorsinthecell(Pissios,2017).NeithertheNMNorCombinedtreatmentgroupshowedasignificantdecreaseinS-adenosylmethionine(AppendixS1),themajormethyldonorofthecellandtheoneutilizedforMNAsynthesis,soitdoesnotappearthatmethyldonorsweredepleted to a significant level.However,enhancedusageofS-adenosylmethionine likelydoesexplainwhytheslightincreaseincholineinducedbySS-31treatmentwaspre-ventedbysimultaneoustreatmentwithNMN,sincecholineisusedtoproduceS-adenosylmethionine(Zeisel&Corbin,2012).

WhySS-31treatmentcausesanincreaseinxanthineandcholine,andwhy the addition ofNMNprevents the accumulation of xan-thine,islessclear.Inthecaseofcholine,theSS-31effectappears

tobearestorationofanage-relatedlossinthemetaboliteandcouldbedue to changes in phospholipidproduction inducedby SS-31'sremodelingof themitochondrial innermembrane.Conversely, theincrease in xanthine is a furthermovement away from theYoungstateandthereasonforthischangeisunclear.Adenineisonesourceof xanthine, by its deamination to hypoxanthine (Harrison, 2002),andmoreadenineisexpectedtobeusedforNAD+ synthesis with the addition ofNMN, providing one potential explanation for theNMNtreatmentpreventingtheSS-31-inducedincreaseinxanthine.Overall,thedifferencesinxanthineandcholineappeartobeminormetabolicconsequencesofthedrugsbutseeminglyunrelatedtothefunctional improvements in the heart.

Althoughawideselectionofmetaboliteswasscreened,therearemore that could be assessed to provide valuable information on the agingheartandtheeffectofthesedrugs.Lipidmetabolismisanareawe believe demands further investigation in future studies with this model system. Measurement of metabolic enzyme levels and activity couldalsoprovidevaluableinformationonthemechanismsofage-re-lated dysfunction and the restorative properties of these drugs.

3.3  |  SS-31 and NMN restore PCr/ATP dynamics in the heart

Inthisstudy,weapplied invivoMRStodemonstratethatthere isanage-relatedshift inthedynamicsofPCr/ATPinresponseto in-creased cardiac workload. Old Control hearts showed a drop in PCr/ATPwithahigherworkload,whereasthisparameterremainedsta-bleinYounghearts.Fromthis,wecanconcludethatmitochondriain Old hearts are less able to meet the energetic demand of a high workstateandPCrdecreasesasaconsequenceofitshigh-energyphosphatesbeingutilizedtomaintainATPstatusinmyofibrilsfasterthanoxidativephosphorylationcanrestoreit.Theattenuationoftheeffectofageon thePCr/ATPdynamicsbySS-31andNMNtreat-ment,bothindividuallyandincombination,suggeststhatbothdrugsimprove the ability of cardiac mitochondria to meet the metabolic demand of the increased workload.

3.4  |  Proposed model of dynamic mitochondrial metabolism changes

While limitedbysignal tonoiseandhighvariance, thenovelMRSmethod thatwedescribeherepresentsanexcitingnewmeans toanalyzemetabolicchangesinresponsetoastimulusinlivemice.Webelieve that the mitochondrial NAD(P)H peak we have describedconsistsprimarilyofNADHbasedonitsdynamicbehaviorhereandinourpriorstudyinwhichitwasfirstdescribed(Conleyetal.,2016),althoughweareunabletodefinitivelysaytowhatdegreeNADPHcontributes to the peak. Taking all of the above results into consid-eration,weproposethefollowingmodeltocontextualizethediffer-ences in metabolic shifts seen between the mouse groups with the increase in cardiac workload.

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Young hearts have a sufficient NAD(H) pool and undamagedmitochondria,which provide themwith adequateATPproductioncapacitytomeetahigherworkload.Asaresult,NAD+ and PCr do notshowanysignificantshiftsfromincreasingworkload,whilemi-tochondrialNAD(P)Htrendstowardaslightdecreaseduetogreaterutilization.OldControl heartshaveadiminishedNAD(H)pool, sohigherworkload places a greater strain on themitochondrialATPproductioncapacity,asevidencedbyaccumulatingNAD+,decreas-ingPCr,andreducedhighworksystolicfunction.

SS-31inOldheartsappearedtoimprovetheefficiencyofATPproductioninthemitochondria,as itnormalizedtheshifts inPCrandNAD+thatresultedfromincreasingthecardiacworkload.Area-sonforthismightbethatmitochondrialNAD(P)HshowedareverseinthedirectionofchangewithincreasingworkloadfollowingSS-31treatment, indicatingthatthesupply-demandmatchingofNADHproduction was improved, with supply now outpacing demandrather than lagging behind. This implies a potential improvement in theefficiencyofthetricarboxylicacid(TCA)cycle.AlthoughseveralTCAcycleintermediateswereevaluatedbythegeneralmetabolo-micsscreen,whichdidnotshowdifferencesresultingfromSS-31treatment (Appendix S1), this does not rule out the involvementof the TCA cycle since thesemeasurementswere only collectedfromtissuetakenatrest.WehavealsorecentlydemonstratedthatthereisadirectinteractionbetweenSS-31andTCAcycleenzymesinvolved in alpha-ketoglutaratemetabolism (Chavez et al., 2019).Other reasons why ATP production may be enhanced by SS-31lie in its direct effects on the mitochondrial inner membrane and inner membrane proteins. We have recently demonstrated thatSS-31 is highly effective at preventing an age-associated excessprotein leakacrossthemitochondrial innermembrane,whichap-pearstobemediatedbytheadeninenucleotidetranslocase(Zhangetal.,2020).Reducedproductionofreactiveoxygenspecies,whichotherwise cause damage to mitochondrial proteins and consume NADPH through antioxidant regeneration, alsooccurs and is an-other likelymechanism for the improved efficiency (Chiao et al.,2020).However,theseimprovementsareapparentlynotsufficientforSS-31torestorehigherworksystolicfunction.

NMNalsonormalizedPCr levelsunder thehigherworkload inOld hearts, but by a seemingly different mechanism than SS-31.NMN-treatedheartswere theonlygroup toshowasimultaneousincrease in both NAD+ andmitochondrial NAD(P)H content. Thiscould indicate that NMN-treated hearts responded to increasedworkload by rapidly increasing the synthesis ofNAD+ in order to raisemitochondrialNAD(H)levelsandprovideahighercapacityforwork in situ. This seemingly improved capacity allowed these hearts tomatchtheYoungstateinhighworksystolicfunction.

The Combined treatment in Old hearts appeared to best recapit-ulatetheYoungstate.ThisgroupdidnotshowanysignificantshiftsinPCr,NAD+,ormitochondrialNAD(P)Hfollowing the increase inworkload,whichwebelieve tobedue to thehigherenergetic ca-pacityaffordedbytherestorationofrestingNAD(H) levels.TheseheartsmatchedthehighworksystolicfunctionoftheYoungstate,

whilealsoexhibiting improveddiastolicfunction.Thus,thecombi-nationofSS-31andNMN,whichtogetherappearto increasefuelsupplyandcouplingefficiencywhilelimitingexcessoxidativestress,provides a synergistic improvement in energetic capacity so that supply can fully meet demand.

3.5  |  Mechanisms of diastolic improvement by SS-31

It is less clear whether the metabolic changes described above are directly responsible for the improvement in diastolic function result-ingfromSS-31treatment.Metabolicchangescertainlydoplayaroleindiastolicdysfunction,asitiswellestablishedthatADPlevelshavearegulatoryroleinsarcomerestiffnesswithhigherADPinhibitingrelaxationand increasingdiastolic stiffness (Sequeiraetal.,2015).ThepreviouslyreportedeffectsofageandSS-31onmitochondrialcouplingincardiomyocytes(Zhangetal.,2020)areconsistentwitharoleforalteredADPlevelsinthechangesindiastolicfunctionre-portedhere.However,wedidnotdetectanysignificantchangesinATPorADPatrestinOldheartstreatedwithSS-31inourmetabo-lomicsanalysis(AppendixS1).Furthermore,wefoundbyMRSthatPCr/ATPatrestwasunalteredbyageorSS-31(FigureS10),alsoin-dicatinga lackofdifference inrestingADP levels. Inaddition, theeffectsonSS-31mitochondrialcouplingwehavereportedappeartoberapidonset,whiletheimprovementsindiastolicdysfunctionrequirelongertermtreatment.Analternativemechanism,forwhichwehavepreviouslypublisheddirectevidence(Chiaoetal.,2020),istheeffectofSS-31onpost-translationalmodificationofmyofibrillarcontractileproteins.Inparticular,SS-31greatlyreducedage-relatedincreasesintheoxidationstateoftheproteome,includingcontrac-tileproteins,andrestoredthephosphorylationlevelsofthesarco-mericproteinmyosin-bindingproteinC,cardiac-type(cMyBP-C)totheyoungstate.Thesechangesareconsistentwithimprovedrelaxa-tionofthecardiacsarcomere,acriticalaspectofdiastolicfunction(Gilda&Gomes,2017;McNamara,Singh,&Sadayappan,2019).

3.6  |  Conclusions

BytestingSS-31andNMNtogetherinmice,wewereabletopro-vide new insight into the effects and mechanisms of each drug in the heart.Excitingly,thisworkshowsthatNMNandSS-31canbegivenin combination to rejuvenate both diastolic and systolic aspects of ventricularfunction,boostNAD(H)contentbetterthanNMNgivenalone,andstabilizemitochondrialNAD(H)dynamics.Theseresultsmay have strong clinical relevance and imply that synergistic treat-ment with the two drugs in combination may be more effective at treatingage-relatedheartdysfunctionthaneithergivenalone.Thisis also an illustration that complimentary use of appropriate drug combinations may become a more general model for achieving opti-malhealthspanextension.

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4  |  E XPERIMENTAL PROCEDURES

4.1  |  Animal use and care

AllmiceusedinthisstudyweremalesoftheC57BL/6strain.YoungandOldmicewereobtained from theNational InstituteonAgingCharlesRiver colony and further aged to5–6 and24months, re-spectively, before starting the study.Mice were kept under diur-nal conditions with ad libitum food and water. Mice were housed at20°CunderdiurnalconditionsinanAAALAC-accreditedfacilityunder Institutional Animal Care and Use Committee supervisionwith ad libitum access to food and water. Old mice were randomly assignedtoControl,SS-31,NMN,orCombinedtreatmentgroups.

4.2  |  Drug administration and treatment groups

SS-31wasprovidedbyStealthBioTherapeutics(Newton,MA)andadministered at a 3 mg/kg body weight/day dosage through osmotic minipumps (ALZET,Cupertino,CA) implanted surgicallyunder theskin on the left dorsal side of the mice. Pumps were modified to replace themetal stemwithPEEK tubing, according to themanu-facturer's instructions, for compatibility with magnetic resonancespectroscopy.After4weeks, theoriginalminipumpwassurgicallyremovedandanewminipumpwasimplantedtocontinuetheSS-31administration for another 4 weeks.

NMN was obtained from the Imai laboratory (WashingtonUniversity in St. Louis,MO) and administered through ad libitumdrinking water with a concentration based on each cage's measured waterconsumptionrateandmeanmousebodyweighttoapproxi-matea300mg/kgbodyweight/daydose.NMNwaterwastheonlysource of hydration given to these mice and was replaced every 3–7daysbaseduponpriortestsshowingthat itremainsstable inwateratroomtemperatureforatleast1week(Millsetal.,2016).

Combined treatment mice received SS-31 throughminipumpswhilesimultaneouslyreceivingNMNwaterastheirexclusivesourceof hydration as described above.

Initially,twodifferentcontrolsgroupswereused:micewithos-moticminipumpscontainingonlysalineasacontrolforSS-31andmice without minipumps receiving standard water as a control for NMN.However,ananalysisoffunctionalandMRSresultsrevealedthat there were no significant differences between these two con-trolgroups(FigureS9).Thus,thetwocontrolgroupswereintegratedinto a single control for all treatment conditions.

4.3  |  Echocardiography

Mice were anesthetized by 0.75%–2% isoflurane, and echocardi-ography was performed using a Siemens Acuson CV-70 (Munich,Germany)equippedwitha13MHzprobe.Heatsupportandheartrate monitoring were provided throughout the procedure with mouseheartratesbeingmaintainedintherangeof450–550bpmat

thelowworkstate.Analysisofsystolicfunctionincludedaninjec-tion of 3 µg/g body weight dobutamine in order to induce a higher cardiacworkload and analyzemaximal function.Highwork echo-cardiography was performed once the heart rate increased at least 100 bpm and remained stable.

4.4  |  Euthanasia and tissue handling

Mice were euthanized by live cervical dislocation. Hearts were im-mediatelyremoved,flushedwithPBStoremoveblood,andweighed.A2-mmsectionwasremovedfromtheventriclesforhistology,andthe remaining tissue was cut into ~2 mm3chunksandsnap-frozeninliquidN2 to store for further processing. Frozen tissue was mechani-callylysedintoafinepowderusingaTissuelyserII(Qiagen)priortopreparationforLC-MS/MS-basedmeasurements.

4.5  |  Targeted NAD+ metabolomics

LevelsofNAD+,NADH,NADP+,NADPH,NA,NAAD,NAM,NR,andNMNattreatmentendpointsweremeasuredbyUltraPerformanceLiquid Chromatography coupledwithMass Spectrometry as previ-ouslydescribed(Trammell&Brenner,2013),withsomemodifications.UnderCO2vapor,lysedtissuewasdissolvedinchilledbufferedmeth-anolsolution(75%methanol,25%10mMHEPESpH7.8)spikedwithstableisotope-labeledinternalstandardsofNAD+andNADHfornor-malization,andbroughttoafinalconcentrationof250mg/ml.Sampleswerethenspundownrepeatedlyat16,000g for 10 min and trans-ferred to new tubes until no pellet formed. Pellets were saved for pro-teinquantificationasanadditionalnormalizationstep.Supernatantswerefilteredusing4-mmand0.22-µMsyringefilters(MilliporeSigma,Burlington,MA).5µlof theextractwasseparatedonaBEHAmidecolumn(Waters,MilfordMA)usinganAcquityUPLC(Waters)andan-alyzedwithaXevoTQ(Waters)inmultiplereactionmonitoringmode(MRM).LCsolventswereA:H2O with 10 mM ammonium acetate and 0.4%NH4OH;andB:95:5acetonitrileH2O with 10 mM ammonium acetateand0.4%NH4OHforallmetabolites.Sampleswererunalong-sideanexternalstandardcurveforquantification.

4.6  |  General targeted metabolomics

Heart tissue samples were homogenized in water and methanol with spikedstableisotopesasinternalQCstandards.Sampleswerepuri-fiedbypelletingproteins,dryingofthesolutionviaSpeedvac(ThermoFisherScientific),andreconstitutinginHILICsolvent.ProteinpelletswereanalyzedbyBCA(ThermoFisherScientific)asanormalizationstep.LC-MS/MS-basedmetabolomicstargetingalistof369metabo-liteswasperformedonasystemconsistingofShimadzuNexeraXRLC-20ADpumps(Kyoto,Japan)coupledtoaSciex(Framingham,MA)6500+triplequadrupolespectrometeroperatinginscheduledMRMdetectionmodethroughtheSciexAnalyst1.6.3software,asdescribed

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elsewhere(NaganaGowda,Djukovic,Bettcher,Gu,&Raftery,2018).Thesystemincludesadual-columnsetupwithdedicatedcolumnsforpositive ionization mode and negative ionization mode. Metabolite concentrationswerequantifiedusingSciexMultiQuant3.0.2software.

AnalysisofthedatasetwasperformedusingR(version3.6.0).Inordertoremovethesystematicvariationbetweensamples,weper-formedaCyclicLOESSnormalization,whichhadgoodperformanceusingaMSbenchmarkdataset(Lietal.,2017).ThisnormalizationstepisimplementedusingthelimmaRpackage(Ritchieetal.,2015).Missing values are known to be a problematic issue for mass spec-trometry-baseddata.PreviousstudieshavesummarizedthethreemissingvaluemechanismsintermsofMS-baseddata(Lazar,Gatto,Ferro,Bruley,&Burger,2016;Weietal.,2018):MissingCompletelyAt Random (MCAR), which corresponds to the combination andpropagation of multiple minor errors or stochastic fluctuations. Eachmissingvalue cannotbedirectlyexplainedby thenatureofthefeatureorbyitsmeasuredintensity.MissingAtRandom(MAR)assumes the possibility of a variable being missing is determined byotherobservedvariables, for example, inaccuratepeakdetec-tion. Missing Not At Random (MNAR) corresponds to censoredmissing values caused by compound abundances that are below thelimitsofquantification(LOQ),a.k.a.left-censoreddata.Forthemetabolomicsdata,themissingvaluemechanismisbelievedtobeMNAR.Allthemetaboliteswith>40%missingnesswereexcluded,and a total of 191 metabolites were included in the imputation step (roughly0.9%ofthedataismissingvaluesafterfiltering).Weusedaquantileregressionapproachfortheimputationofleft-censoredmissingdata(QRILC),whichhasbeensuggestedasthefavoredim-putationmethod for left-censoredMNARdata (Weietal.,2018).Briefly, QRILC performs imputation by replacing missing valueswith random draws from a truncated distribution with parameters estimated using quantile regression. Thiswas implemented usingthe imputeLCMDRpackage. ImputationwasperformedafterthecyclicLOESSnormalization.Wefitalinearmodeltothenormalizedand imputed metabolomic data using the Bioconductor limma pack-age while adjusting the preparation batch and protein concentra-tion as covariates in our model. The limma package uses empirical Bayesmoderated statistics,which improvespowerby ‘borrowingstrength’ between metabolites in order to moderate the residual variance(Smyth,2004).Samplesizeforeachgroupafterremovingoutlierswas Young = 12,Old Control = 10,OldNMN= 11,OldSS-31=12,OldCombined=8.Falsediscoveryrate(FDR)waslim-itedto10%usingtheBenjamini–Hochbergmethod.

Heatmap generation was performed in R using the “heatmap” function tocreatea row-scaled imagebasedupon the top20sig-nificantlyalteredcanonicalpathways intheYoungvs.OldControlcomparisonasdeterminedbyIPA(Qiagen).

4.7  |  31P-magnetic resonance spectroscopy

Magnetic resonance spectroscopy was performed on live mice using a4.7Tesla instrumentoperatedby theTranslationalCenter

forMetabolicImagingattheUniversityofWashington.Micewereplaced into a custom apparatus with coils for both 1H and 31PNMRandanesthetizedminimallyvianoseconewith0.75%–1.5%isoflu-rane.Heartrate,respiratoryrate,andtemperatureweremonitoredusing a Model 1030 Monitoring & Gating System (Small AnimalInstruments, Inc., Stony Brook, NY). Anesthesia was adjusted asneeded to maintain respiratory rate at 100–130 bpm. Temperature was also adjusted as needed to keep mice at a body temperature of37°C.Micewereequippedwithanintraperitonealcathetercon-nected to a line of dobutamine to allow for remote injection without altering the position of the mouse.

Triggered, multi-slice, gradient echo, 1H-magnetic resonanceimageswereacquired(TR=160ms,TE=4ms,witha2mmslicethickness)toplaceavoxelisolatingtheheartfor31PISIS(Ordidge,Connelly,&Lohman,1986).31PISISspectra(TR=3s)werethere-sultof96averages(acquiringatotalof768acquisitions)using2mshyperbolicsecant180opulsesduringlocalization,butanominal90°square (50μs), non-selective pulse for readout. Typical voxel sizewas~0.8cm3 to sample the entire heart. Data were collected over a38-minacquisitionwithgatingbaseduponheart rateand respi-ratory rate to ensure the position of the heart was constant across measurements. Following the first acquisition,micewere injectedwith5µg/gdobutamine.Thesecondacquisitionwasstartedoncethe elevated heart rate became stable.

Manual phase adjustment of the spectra, alignment of PCr to−2.54ppm,andquantificationofPCrandγ-ATPintegralswereper-formed in TopSpin (Bruker, Billerica, MA). Baseline correction ofspectrawasperformedusingMnovasoftware(MestreLabResearch,S.L.,SantiagodeCompostela,Spain).

OriginPro software (OriginLab Corporation) was used tomodel the fitting of α-ATP,NAD+, cytosolicNADH,NADP+, cyto-solicNADPH,UDP-glucose, andmitochondrialNAD(P)Hpeaks tothe baseline-corrected experimental spectra for quantification ofthesemolecules.Lorentzianpeakswerepositionedandmodeledassinglets, doublets, or quartetsbaseduponanalysis chemical stan-dardsontheinstrumentunderphysiological(12mMMg2+,pH7.4)conditions (Figure S10). Peak width was variable across differentexperimental spectrabutwaskept consistent forNAD+, cytosolicNADH, NADP+, cytosolic NADPH, and UDP-glucose within eachfit.MitochondrialNAD(P)Hpeakwidthwasallowedtovarywithin1.5-tothreefoldthatoftheotherpeaks.Bestfitsweregeneratedautomaticallybasedonthismodel,withnomanualadjustmentused.Peakswereintegratedautomatically,andnon-ATPmetaboliteswerenormalized to α-ATP for relative quantification. OriginPromacrosareavailableuponrequest.

4.8  |  Statistical analysis

Statistical analysisof large-scalemetabolomicdata isdescribed inthe above section. Other statistical analyses were performed using Prism(GraphPadSoftware,SanDiego,CA).One-wayANOVAswereusedformeasurementswithasingletimepointorstatepergroup,

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andtwo-wayANOVAswereusedtocomparemeasurementswithmultiple timepoints or states for each group.One-sample t testswere used to determine whether delta means were significantly dif-ferentfrom0.Allresultsareplottedasmeans±SEM.

ACKNOWLEDG EMENTSAll authors listed contributed to the study design, experimentalproceedings,dataanalysis,and/ordraftingofthemanuscript.Wewould like to especially acknowledge the contributions in mag-neticresonancespectroscopymadebyDr.KevinE.Conley,whopassed away during the preparation of this paper. Although hewillnotbeable to see itspublication, thisworkwouldnothavebeen possible without Dr. Conley's expertise and support. Theauthors would also like to acknowledge the contributions of The NorthwestMetabolomicsResearchCenterregardingthegeneraltargeted metabolomics assay. Funding for this assay was provided bytheNathanShockCenterattheUniversityofWashington,andthe instrumentationgrantS10OD021562wasused topurchasethe mass spectrometer that analyzed the samples. OriginLabCorporation provided technical support for the programming of ourMRSfittingmodel.WethankNickBluthforcreatingtheheartimage used in the graphical abstract.

CONFLIC T OF INTERE S TThe authors declare that they have no conflict of interest.

DATA AVAIL ABILIT Y S TATEMENTSupplemental methods and results can be found in theSupplemental Data document. The full general targeted me-tabolomicsdataset canbe found in theAppendixS1. In accord-ance with Aging Cell'sdataavailabilitypolicy, thesedataarealsoavailableattheNationalMetabolomicsDataRepository(NMDR)(https://www.metabolomicsworkbench.org), which is supportedby NIH grant U2C-DK119886. It has been assigned Project IDPR000908andcanbeaccesseddirectlyviaitsProjecthttps://doi.org/10.21228/M8B11Q.

ORCIDJeremy A. Whitson https://orcid.org/0000-0002-5775-1142

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SUPPORTING INFORMATIONAdditional supporting information may be found online in theSupportingInformationsection.

How to cite this article: WhitsonJA,BittoA,ZhangH,etal.SS-31andNMN:Twopathstoimprovemetabolismandfunction in aged hearts. Aging Cell. 2020;19:e13213. https://doi.org/10.1111/acel.13213