EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02055 © EMBO 1 p53’s choice ofmyocardialdeath orsurvival:Oxygen protects infarctmyocardium by recruiting p53 on NOS3 promoterthrough regulation ofp53-Lys118 acetylation RajanGogna,EshaMadan,MahmoodKhan,UttamPatiandPeriannanKuppusamy Correspondingauthor:PeriannanKuppusamy, DartmouthCollege Review timeline: Submissiondate: 06 August2013 EditorialDecision: 20 November2013 Revisionreceived: 24 January 2013 EditorialDecision: 13 February 2013 Revisionreceived: 10 July 2013 EditorialDecision: 18 July 2013 Revisionreceived: 26 July 2013 EditorialDecision: 30 July 2013 Revisionreceived: 06 August2013 Accepted: 09 August2013 Transaction Report: (Note:With the exception ofthe correction oftypographicalor spelling errors thatcould be a source ofambiguity, letters and reports are not edited. The originalformatting oflettersand referee reportsmaynotbe reflected in this compilation.) Editor: RobertoBuccione 1st EditorialDecision 20 November2013 Thankyouforthe submission ofyourmanuscript" p53's choice ofmyocardialdeath orsurvival: Oxygenprotectsinfarctmyocardium byrecruitingp53onNOS3promoterthroughregulationof p53-Lys118acetylation". Weareverysorryforthedelayingettingbacktoyouwith theReviewers'evaluation on yourwork. Unfortunately,inthiscaseweexperiencedunusualdifficultiesinsecuringthreeappropriate reviewers in a timely manner.We have now heard back from the three referees whom we asked to evaluate yourmanuscript. YouwillseethatalthoughReviewers2and3underlinetheconsiderablepotentialinterestofyour work,togetherwithReviewer1,theyalsoraisesignificantconcernsthatpreventusfrom considering publication atthistime. Reviewer1iscriticalofthe experimental approaches, choice of cell model and the interpretations/conclusions drawn from your data. S/he complains the lack of histology data to supportp53/eNOS localisation during myocardialremodelling and challenges the use ofH9c2 cells to explorep53 function in cardiomyocytes. Reviewer2issupportiveofyourstudy,butmentionsanumberofspecificissuesthatweakenyourEMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02055 © EMBO 2 currentclaims.Forinstance,s/he pointsto the need ofqPCR to validate RT-PCR data,ELISA to quantify theeffectsof oxygen on p53 in hearts and the generallack of controls in immunoprecipitation, co-immunoprecipitation and other experiments. Reviewer3,whilealsobeinggenerallysupportive,suggestssomeimprovementstoincreaseflow, readability and discussion ofthe medical implications of your study. Whileitisclearthatpublicationofthepapercannotbeconsideredatthisstage,Iamopentothe submission ofa substantially revised manuscript,provided,however,thatthe Reviewers'concerns are fully addressed with additionalexperimentaldata where appropriate. PleasenotethatitisEMBO MolecularMedicinepolicytoallow asingleroundofrevisiononlyand that, therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next,finalversion of the manuscript. Asyouknow,EMBO MolecularMedicinehasa"scoopingprotection"policy,wherebysimilar findings thatare published by others during review orrevision are nota criterion forrejection. I understand thattheamountofwork thatwould berequired to submitarevised version ofyour manuscriptissignificantandIwouldunderstandifyoudecidedtosubmityourworkelsewhere.If you should decideto submitarevised version,Ido ask you to let us know and then to get in touch withusafterthreemonthsifyouhavenotcompletedit,toupdateusonthestatus.Pleasealso contactusassoon aspossible ifsimilarwork ispublished elsewhere. ***** Reviewer'scomments***** Referee#1(CommentsonNovelty/ModelSystem): NOS3functionischaracterizedinendothelialcells.It'sfunctioninprimarycardiomyocytesis unclear.P53 functionsin acelltypespecificway.Wholeorgan lysatesarethewrong way to go. Referee#1(GeneralRemarks): Oxygendependentregulatoryfunction ofp53 related to acetylation statusisvery interesting. Howevertheauthorsdonotprovideanyinvivorelevantdata.NOS3functionsarewell characterized in endothelialcells.It'sfunction in primary cardiomyocytesisunclear.P53 functions in a celltype specific way.Whole organ lysatesare the wrong way to go. Therearenohistologydatashowincolocalizationofp53/eNOSduringmyocardialremodeling.In vitro studieswereperformed in H9c2 which isnotan adequatesystem to explorep53 function in cardiomyocytes.Mostexperimentsare missing importantcontrols.Data are overinterpretated. Referee#2(CommentsonNovelty/ModelSystem): Novelty:Theconceptofachangeinacetylationofasinglep53lysinealteringp53preferencefor one promoteroranotherwith significantfunctionaleffectsisparticularly noveland intriguing. Technicalquality:Technicalstrengthsofthestudyincludetheuseofavarietyofapproachestotest this hypothesis and the high quality of the data presented in the manuscript. Medicalrelevance:Thephysiologicalandmedicalrelevanceoftheworkishigh,sincetheauthors show the role ofa simple biologicalmolecule such as oxygen in regulation oftranscription,post- translational modifications and, mostimportantly,pro-survivalresponses in infarctmyocardium. I have no major problems with the adequacy of the modelsystem. EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02055 © EMBO 3 Referee#2(GeneralRemarks): ThemanuscriptbyGognaetaldescribesaninterestingresearchworkabouthow oxygencanswitch p53 signaling from apro-death to pro-survivalprogram by preventing acetylation ofLys118 residue. Myocardialinfarctioncausesoxygendeficiencyintissueandinducesp53-mediatedcell-death through the BAX apoptosis pathway while oxygenation of the infarctheartcan preventcell-death and tissue damage.The conceptofa change in acetylation ofa single p53 lysine altering p53 preferenceforonepromoteroranotherwith significantfunctionaleffectsisintriguing.Thestudy shows thatoxygenation canshiftp53 binding from BAX-p53RE to NOS3-p53RE,which upregulatesNOS3 expression to promotecellsurvival.Low p53 Lys118 acetylation by theoxygen- mediatedreductionofTIP60acetylaseisshowntoplayarolebyswitchingthebindingofp53from BAX-p53REtoNOS3-p53RE.Thispreventstheexpression ofvariouscell-death proteinswhile inducing expression of multiple anti-apoptotic genes.The research work iswellorganized and strengths ofthe study include the use ofa variety ofapproaches to testthishypothesisand thehigh quality ofthedatapresented in themanuscript.Theresultsobtained supporttheconclusionsand the physiologicalrelevanceofthework ishigh,sincetheauthorsshow theroleofasimplebiological moleculesuchasoxygeninregulation oftranscription,post-translational modifications and pro- survivalresponses in infarctmyocardium.Although the manuscriptis wellorganized authors have missedsomeimportantexperimentsandadditionofthefollowingexperimentswillfurtherimprove the quality of the data. 1)In Fig 1atheresultsoftheRT-PCR mustbecountervalidatedandquantifiedusingrealtime PCR. 2)In Fig 1b authorsmustincludetechniqueslikeELISA to quantify theeffectofoxygen on p53 expression in infarctand treated hearts. 3)In Fig 1ctheIP experimentsmusthaveanegativecontrolsuch asIP withoutany antibody and withanunrelatedantibody. 4)In Fig1d theauthorsmustprovidethecontrolsin thesupplementary dataforobtaining thetrue nuclearand cytoplasmic fractions from the tissue samples. 5)Fig 1ftheresultsofco-IP mustbe repeated with both p53 and p300 antibodies,currently the authorshave shown the IP data with only p53 Ab. 6)In Fig 2b theblotmustincludeanegativecontrolsuch asNOS3siRNA. 7)In Fig 2b and 2cthesignificanceofthedifferencesin theluciferasereading mustbeshown. 8)In Fig 2d theauthorsmush show thesuper-shiftofthe p53-DNA complexandaddp21p53REas a positive control. 9)In Fig 3d authorsshould representthe binding of p53 to both bax-p53RE and NOS3-p53 RE in oneEMSA experiment. 10)In Fig 4atheauthorsmustpresentwestern blotforindividualposttranslationalmodifications. TheIPdatamustbequantifiedwithproteinquantificationtechniquessuch as ELISA. 11)In Fig 5cand 5d thesignificanceoftheresultsmustberepresented. Referee#3(CommentsonNovelty/ModelSystem): Muchmoreconsiderationsholdbegiventothemedicalconsequencesofthiswork.Thisistheweak partofthispaper Referee#3(GeneralRemarks): Thispaperisinterestingbecauseittacklesaproblem offundamentalbasicbiologywhichisof interest i.e. the role of p53 in cardioprotection under conditions of enhanced oxygenation in the myocardium. A strongpoint is the molecular methodology, which in my view is impeccable. Theweakpointsdealmainlywiththebio-MEDICALimplicationsofthisstudy MajorpointsIfind: 1.- Itis well-known thatnecrosisplaysaMAJOR rolein celldeath in myocardialinfarction (seefor instance: "The injurious impact of myocardial ischaemia comes from a mixture of pro-apoptotic and necrosis-promoting signalCardiovascRes(2000)45 (3):630-641".Theword (ortheconcept) "necrosis" does notappearin the paper. EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02055 © EMBO 4 2.- Theintroduction isfartoo long.Onesometimesismisled by somethoughtsthatarenotrequired forthe understanding of this paper 3.- Pleasemakeanexplanatoryfigureofhow p53SENSES oxygen.Thisisastrongstatementinthe paperand needsclarification (see also point5)To thisreviewer'sknowledge,the mechanismsby whichHIFsensesoxygenaremuchclearer. 4.- Pleasecommentonthelackofeffectivenessofpreconditioninginmyocardialinfarction treatment ( clinical trials have so far failed to report positiveeffectsin humans) 5.- Pleasemakethepapereasiertoread.(Forinstance,toomanyabbreviations) 1stRevision - authors'response Reviewer#1 R1.1 NOS3functionischaracterizedinendothelialcells.Itsfunctioninprimarycardiomyocytes is unclear. WeagreewiththereviewerthatNOS3iswell-characterized in endothelialcells.However, cardiomyocytesalso expressNOS3 enzyme.Studieshave demonstrated thatNO production from cardiomyocyte-residentNOS3 is necessary for postnatalcardiomyocyteproliferationandmaturation (Lepicetal,2006). Both, iNOS and NOS3 (eNOS) isoforms are prominently expressed during early stages ofcardiomyogenesis (Blochetal, 1999). Studies have also shown that deficiency in NOS3 resultsin congenitalatrialand ventricularseptaldefects(Fengetal,2002). R1.2 p53 functionsin a celltypespecificway.Wholeorgan lysatesarethewrong wayto go. It’s true thatp53 functions in a cell-type specific way but study of p53 expression in the whole organswhich consistofcellsofavariety ofdifferentlineagessuch asbrain,liver,prostrate,lungs, colon,testis,ovariesare conducted in past(Chrestaetal,1996; Iggo etal,1990; Zhenetal,1999). Theprimereasonforthisisthatthebiomedicalorphysiologicalimportanceoftheeffectofp53on wholeorgansholdsacriticalimportanceandfurtheritisveryhardandvirtuallynon-practicalto isolate cells of different origin from a tissue for all research studies. Thus the major physiological and biomedicalrelevance ofthe study liesin the observation,which showsthe effectofoxygenation and p53-survivalpathway in the heartas an organ,rather than individualcells of some lineage.In this study we have also used serum-deprived H9c2 cardiomyoblasts(Bonavitaetal,2003) to study the function of p53 in myocytes. We have observed similar results with respect p53 activation and function suggesting thatp53’s functions are notdifferentin the tissue lysate containing cardiomyocytesand othercells. R1.3 Oxygendependentregulatoryfunctionofp53relatedtoacetylationstatusisvery interesting. However the authors do not provide any in vivo relevant data. NOS3 functions are well characterized in endothelialcells.Itsfunction in primary cardiomyocytesis unclear. P53 functions in a cell type specific way. Whole organ lysates are the wrong way to go. PleaseseeourresponsetoR1.1& R1.2,above. R1.4 Therearenohistologydatashowingco-localization of p53/eNOS during myocardial remodeling. Asthereviewersuggested wehaveprovided histology datashowing co-localization of p53 and eNOS (NOS3)during myocardialremodeling.The new data ispresented in SupplementalFigure 6 in the revised version. R1.5 In vitro studies were performed in H9c2 which is notan adequatesystem to explorep53 function in cardiomyocytes. H9c2isapermanentcelllinederivedfrom embryonicrathearttissue.H9c2cellsshow electrophysiologicaland biochemicalpropertiesofboth skeletaland cardiac tissues. TheH9c2cells haveemerged asan excellentin vitro alternativeto primary cardiacmyocytes. Thecellscanbe engineered to expressforeign genesatcontrollable levels,making them a suitable system to study molecularresponsestooxidativedamage.Watkinsetal(Watkinsetal,2011) showed the importance of H9c2 cells as a model for in vitro studies of cardiac hypertrophy and similarity with human cardiomyocytecelllinesforprospectivemolecularstudiesin heart development and disease. H9c2cellsshowedalmostidenticalhypertrophicresponsestothoseobservedinprimaryEMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02055 © EMBO 5 cardiomyocytes.Although the H9c2 cellslack the elaborate contractile apparatusofbona fide cardiac myocytes,they cellselicitrobusthypertrophy-associated signature offetalgene expression. Additionally,similartowhatoccursintheintactheart,pathologicalhypertrophyofH9c2cardiac myocytescouldbeattenuatedbypan-HDACinhibitors. R1.6 Mostexperimentsaremissingimportantcontrols.Data areoverinterpreted. Wehavethoroughlyrevisedourmanuscript,andaddedappropriatecontrolsassuggestedbythis,as wellasbyotherreviewers.Further,wehavesimplifieddatainterpretationusinganadditionalmodel in the discussion section (Figure 8). Reviewer#2 ThemanuscriptbyGognaetaldescribesaninterestingresearchworkabouthow oxygencanswitch p53 signaling from a pro-death to pro-survivalprogram by preventing acetylation ofLys118 residue.Although the manuscriptis well organized authors have missed some important experimentsand addition ofthe following experimentswillfurtherimprove the quality ofthe data. R2.1 In Fig 1a the results ofthe RT-PCR mustbecountervalidatedandquantifiedusingreal time PCR. Asthe reviewer suggested we have added qPCR data in Figure 1 (see Fig 1b in the revised manuscript). R2.2 In Fig 1b authors mustinclude techniques like ELISA to quantify the effectofoxygen on p53 expression in infarctand treated hearts. Assuggested,wehaveaddedin vivo ELISA datainFigure1(seeFig1dintherevisedmanuscript). R2.3 In Fig 1c the IP experiments musthave a negative controlsuch as IP withoutany antibody and with an unrelated antibody. Asthereviewersuggested,wehaverepeated theIP experimentwith negativecontrols,IP without any antibody,IP with anti-tubulin antibody. The new data is presented in Fig 1e in the revised manuscript. R2.4 In Fig 1d the authors mustprovide the controls in the supplementary data for obtainingthe true nuclear and cytoplasmic fractions from the tissue samples. Asthereviewersuggested,wehaveprovidedanextrafigure(FigureS1intherevisedmanuscript) showing controls forobtaining true nuclear(NF)and cytoplasmic (CF)fractions. R2.5 In Fig 1ftheresultsofco-IP mustbe repeated with both p53 and p300 antibodies, currently the authorshave shown the IP data with only p53 Ab. Asthereviewersuggested,wehaverepeatedtheanalysisandaddedresultsshowingco-IP with both p53 and p300 antibodies(seeFig 1h in therevised manuscript). R2.6 In Fig 2b the blotmustinclude a negative controlsuch as NOS3 siRNA. Asthereviewersuggested,wehaveaddedNOS3siRNA asanegativecontrolinFigure2b R2.7 In Fig 2b and 2c the significance ofthe differences in the luciferase reading mustbe shown. Wehavenowmentionedthesignificance(Pvalue)ofresultsinfigure2b/2clegends. R2.8 In Fig 2d the authors mush show the super-shiftofthe p53-DNAcomplexand add p21 p53RE asa positivecontrol. Asthereviewersuggested,werepeatedthisexperimentandaddedthelaneshowingsuper-shiftof the p53-DNA complexandalsowehaveaddedp21p53RE-p53 interaction laneaspositivecontrolin this experiment. The new dataisshown in Fig 2d in therevised manuscript. R2.9 In Fig 3d authors should representthe binding ofp53 to both bax-p53RE and NOS3-p53 RE inoneEMSA experiment.EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02055 © EMBO 6 Asthereviewersuggested,wehaverepeatedthisexperimentandwehavenow shownthe binding of Bax-p53RE and NOS3-p53 RE in oneEMSA experiment(Fig 3d). R2.10 In Fig 4a the authors mustpresentwestern blotfor individualpost-translational modifications.TheIPdatamustbequantifiedwithproteinquantificationtechniquessuchasELISA. Asthereviewersuggested,wehaveaddedadditionalcontrolsintheimmunoprecipitationdata. Further,wehaverepeatedtheexperimentusingin vivo ELISA andthenew resultsarepresentedin FigureS10. R2.11 In Fig 5c and 5d the significance ofthe results must be represented. WehavenowmentionedthesignificanceandPvaluesinfigure5cand5dandinfigurelegends Reviewer#3 R3.0 Muchmoreconsiderationshouldbegiventothemedicalconsequencesofthiswork.Thisis the weak part of this paper. Wehavediscussedthebio-medicalimplicationsofthisresearchworkasaseparatesectioninthe discussion.Thefollowing isareproduction ofthenew textadded attheend ofDiscussion: “Thepresentstudy providesanovelmechanisticinsightand therapeutic strategy to targetthe infarction-induced myocyte apoptosis in the heart. Theresultshaveimportantbiomedicaland physiologicalrelevancein thetreatmentofmyocardialinfarction.Oxygen therapy isexpected to improve the oxygenation of the ischemic myocardium, reduce infarct size, and consequently morbidityandmortality.Although, the use of supplemental oxygen in the treatment of acute MI has been in practiceforover100 years,thereisno conclusivedataon itsbeneficialeffect(Beasleyetal, 2007; Wijesingheetal,2009). Controversies continue to emerge regarding the applicability and efficacy ofoxygen therapy forMIpatients(Kones,2011). One-time administration of hyperoxygenation,intended asapre-conditioning treatmentbefore induction ofmyocardialinjury, hasbeen shown to bebeneficial(Cabigasetal,2006; Yogaratnam etal,2008; Yogaratnam etal, 2010). However, these studies lacked the clinical relevance for treating post-MIpatients.Onthe otherhand,clinicalprotocolsroutinely useinhalation ofhigh-flow oxygen in the first24 hours after acute MI.These clinicalstudiesprovided conflicting results, even detrimental effects, largely attributed to vasoconstrictive effectofoxygen (Kones,2011; Wijesingheetal,2009). Our study providesapost-MIapproachwithdailycyclesofbriefperiodsofoxygenation,whichismore practicaland clinically relevant.Furthermore,thepresentstudy also provides the underlying molecularmechanism bywhichperiodicadministrationofsupplementaloxygenationresultsinpro- survivalresponses in the infarctheart.” Thispaperisinterestingbecauseittacklesaproblem offundamentalbasicbiologywhichis of interest i.e. the role of p53 in cardioprotection under conditions of enhanced oxygenation in the myocardium.Astrongpointisthemolecularmethodology,whichinmyviewisimpeccable. Wethankthereviewerfortherecognition. R3.1 Theweakpointsdealmainlywiththebio-MEDICALimplicationsofthisstudy.Major pointsIfind:Itiswell-known thatnecrosisplaysa MAJOR role in celldeath in myocardial infarction (see for instance: "The injurious impact of myocardial ischaemia comes from a mixture of pro-apoptoticand necrosis-promoting signalCardiovascRes(2000)45 (3):630-641".Theword (or the concept) "necrosis" does not appear in the paper. Wehavediscussedthebiomedicalimplicationsofourworkintherevisedpaper.Please see our response to R3.0 above. Bothnecrosisandapoptosishavebeenimplicatedintheloss(death)ofcardiomyocytesinthe reperfused heart(Kajsturaetal,1996; Yaoitaetal,2000). Studies using animal models of MI indicate that myocyte death due to necrosis is an early event that beginswith prolonged ischemia, furtherexacerbated with the onsetof reperfusion,and may lastup to 24 hours (Eeftingetal,2004; Oerlemansetal,2012). On the other hand, apoptotic cell death is largely initiated during reperfusion and continuesto occurin the MIheartforlongerduration (Gottliebetal,1994; Zhaoetal,2000). Weusedoxygenationtreatment48 hoursafterinduction ofMIby ischemia-reperfusion.The reason EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02055 © EMBO 7 forthe 48-hourdelay wasto avoid introduction ofhyperoxygenation during theonsetofreactive oxidantproduction during theearly phaseofreperfusion,and subsequentinflammation. WehaveaddedthisinformationinthebeginningofIntroductionintherevisedmanuscript. R3.2 Theintroductionisfartoolong.Onesometimesismisledbysomethoughtsthatarenot required for the understanding ofthis paper. Asthereviewersuggestedwehavenow shortenedtheintroductionto835 wordsfrom 1248 words in the previous version. We have also made the Introduction more streamlined and reader friendly. R3.3 Pleasemakeanexplanatoryfigureofhow p53SENSESoxygen. This is a strong statement in the paper and needs clarification (see also point 5). To this reviewer's knowledge, the mechanismsbywhichHIFsensesoxygenaremuchclearer. Wehavepresentedanextramodel(Figure8)inthediscussionsectionwhichtriesto explain a putativemechanism ofoxygen sensing viaap53-TIP60pathway. R3.4 Pleasecommentonthelackofeffectivenessofpreconditioninginmyocardialinfarction treatment (clinical trials have so far failed to report positive effects in humans). Asthereviewersuggestedwehavementionedtheimportantissuewhichdealswiththelackof effectivenessofpreconditioning in myocardialinfarction treatmentin the discussion section. R3.5 Pleasemakethepapereasiertoread.(Forinstance,toomany abbreviations) Wehaveremovedunnecessaryabbreviations,simplifiedandshortenedthepaper. ------------------- REFERENCES BeasleyR,AldingtonS,WeatherallM,RobinsonG,McHaffieD (2007)Oxygentherapyin myocardialinfarction:anhistoricalperspective.J R Soc Med 100:130-133 BlochW,FleischmannBK,LorkeDE,AndressenC,HopsB,HeschelerJ,AddicksK (1999)Nitric oxide synthase expression and role during cardiomyogenesis. CardiovascRes43:675-684 BonavitaF,StefanelliC,GiordanoE,ColumbaroM,FacchiniA,BonafeF,CaldareraCM, GuarnieriC(2003)H9c2cardiacmyoblastsundergoapoptosisinamodelofischemiaconsisting ofserum deprivation and hypoxia:inhibition by PMA.FEBSletters536:85-91 CabigasBP,SuJ,HutchinsW,ShiY,SchaeferRB,RecinosRF,NilakantanV,KindwallE, NiezgodaJA,BakerJE(2006)Hyperoxicandhyperbaric-induced cardioprotection: roleofnitric oxidesynthase3.CardiovascRes72:143-151 ChrestaCM,MastersJR,HickmanJA (1996)Hypersensitivityofhumantesticulartumorsto etoposide-induced apoptosis is associated with functional p53 and a high Bax:Bcl-2 ratio.Cancer research56: 1834-1841 EeftingF,RensingB,WigmanJ,PannekoekWJ,LiuWM,CramerMJ,LipsDJ,DoevendansPA (2004) Role of apoptosis in reperfusion injury.CardiovascRes61:414-426 FengQ,SongW,LuX,HamiltonJA,LeiM,PengT,YeeSP (2002)Developmentofheartfailure and congenitalseptaldefectsin mice lacking endothelialnitric oxide synthase.Circulation 106: 873-879 GottliebRA,BurlesonKO,KlonerRA,BabiorBM,EnglerRL(1994)Reperfusioninjuryinduces apoptosisin rabbitcardiomyocytes.TheJournal of clinical investigation 94:1621-1628 Iggo R,Gatter K,Bartek J,Lane D,Harris AL (1990) Increased expression of mutantforms of p53 oncogenein primary lung cancer.Lancet335:675-679 KajsturaJ,ChengW,ReissK,ClarkWA,SonnenblickEH,Krajewski S, Reed JC, Olivetti G, AnversaP(1996)Apoptoticandnecroticmyocytecelldeathsareindependentcontributing variablesofinfarctsizein rats.LabInvest74:86-107 EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02055 © EMBO 8 KonesR(2011)Oxygentherapyforacutemyocardialinfarction-then and now. A century of uncertainty.Am JMed124:1000-1005 LepicE,BurgerD,LuX,SongW,FengQ (2006)Lackofendothelialnitricoxidesynthase decreasescardiomyocyteproliferation and delayscardiacmaturation.Am JPhysiolCellPhysiol 291:C1240-1246 OerlemansMI,KoudstaalS,ChamuleauSA,deKleijnDP,DoevendansPA,SluijterJP(2012) Targetingcelldeathinthereperfusedheart:Pharmacologicalapproachesforcardioprotection.Int J Cardiol WatkinsSJ,BorthwickGM,ArthurHM(2011)TheH9C2cellline and primary neonatal cardiomyocyte cellsshow similarhypertrophic responsesin vitro.In Vitro CellDev BiolAnim 47:125-131 WijesingheM,PerrinK,RanchordA,SimmondsM,WeatherallM,BeasleyR(2009)Routineuse ofoxygen in thetreatmentofmyocardialinfarction:systematic review.Heart95:198-202 YaoitaH,OgawaK,MaeharaK,MaruyamaY (2000)Apoptosisinrelevantclinicalsituations: contribution ofapoptosisin myocardialinfarction.CardiovascRes45:630-641 Yogaratnam JZ,LadenG,Guvendik L, Cowen M, Cale A, Griffin S (2008) Pharmacological preconditioning with hyperbaricoxygen:can thistherapy attenuatemyocardialischemic reperfusion injury and induce myocardialprotection via nitric oxide? J Surg Res149:155-164 Yogaratnam JZ,Laden G,Guvendik L,Cowen M,CaleA,Griffin S (2010)Hyperbaricoxygen preconditioning improvesmyocardialfunction,reduceslength ofintensivecarestay,and limits complicationspostcoronary artery bypassgraftsurgery.CardiovascRevascMed 11:8-19 Zhao ZQ,NakamuraM,Wang NP,Wilcox JN,ShearerS,Ronson RS,Guyton RA,Vinten- Johansen J (2000)Reperfusion induces myocardialapoptotic celldeath.CardiovascRes45:651- 660 ZhenHN,ZhangX,BuXY,ZhangZW,HuangWJ,ZhangP,LiangJW,WangXL (1999) Expression ofthesimian virus40 largetumorantigen (Tag)and formation ofTag-p53 and Tag- pRb complexesin human brain tumors.Cancer86:2124-2132 2ndEditorialDecision 13 February 2013 ThankyouforthesubmissionofyourrevisedmanuscripttoEMBOMolecularMedicine. WehavenowheardbackfromthetwoReviewerswhomweaskedtoevaluateyourmanuscript.You willseethat,whileReviewer2issatisfiedwiththerevision,Reviewer1notesthathis/herrequests havenotbeen addressed and thusdoesnotsupportpublication. Reviewer1remainsconcernedthattheclonallyderivedmyoblastcelllineH9c2isnotsufficientto draw conclusionson p53 function in cardiomyocytes.S/hecorrectly notesthatneonatalrat cardiomyocytesare available and thusmustbeused to confirm key findings.Reviewer1 had made this point clear in his/her first evaluation and furthermore, in my decision letter, I had asked you to comply with the Reviewers'requests.Ithusconcurwith Reviewer1'scurrentassessment. Asyouknow,wewouldnormallynotallow asecondrevision.Iam preparedinthiscase,however, to give you another opportunity to improve your manuscript, with the understanding that acceptance orrejection ofthemanuscriptwilldepend thesatisfactory validation ofthekey findingsin cardiomyocytesin the next,finalversion ofthe manuscript. Asyouknow,EMBO MolecularMedicinehasa"scoopingprotection"policy,wherebysimilar findings thatare published by others during review orrevision are notacriterion forrejection. However,Idoaskyoutogetintouchwithusafterthreemonthsifyouhavenotcompletedyour revision,to update us on the status.Please also contactus as soon as possible if similar work is published elsewhere. EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02055 © EMBO 9 I look forward to seeing a revised form of yourmanuscriptas soon as possible. Shouldyoufindthattherequestedrevisionsarenotfeasiblewithintheconstraintsoutlinedhereand choose,therefore,to submityourpaperelsewhere,we would welcome a message to this effect. ***** Reviewer'scomments***** Referee#1(GeneralRemarks): TherevisedversionofthemanuscriptbyGognaetaladdressedseveralconcernsbutthemain concernswere notaddressed adequately. TheauthorsimplythatNOS3isanimportantmodulatorofp53incardiomyocytesandthatthis pathway isan importantprosurvivalmechanism forcardiomyocytes.My concernsarethatthe findings from whole organ lysates do notreflectsignaling changes in cardiomyocytes during cardiac remodeling.In fact,stainings for NOS3 and p53 supportthese concerns.There is no expression of NOS3andp53innormalcardiomyocytesasshown.Afterinjurythereisdramaticupregulationof NOS3andp53incellpopulationsthatdo notappearto be related to cardiomyocytes.Any changes in the expression of these genes in whole organ lysates are probably related to signaling changes in non cardiomyocytesand through changesin cellpopulations(infiltration ofinflammatory cells, massivedeathofothercells,proliferationoflocalnoncardiomyocytesect)duringtheremodeling process. ThereisevidenceintheliteraturethatNOS3isnotexpressedincardiomyocytes(Tambasciaetal Hypertension2001;37:1427-1428).Theauthorsmustshowthatp53andNOS3areco-expressed during theremodeling process. TheauthorschoosetouseH9c2cellswhichisaclonallyderivedmyoblastlinesthatwasgenerated over40 yearsago.Wemustassumethatthesecellsbypassnormalsenescencethrough transformation affecting genetic stability. P53 is the gate keeper for genetic integrity. It is likely that p53 function isdefectivein H9s2 and thereforethislineislesssuitableto study p53 function. Neonatalratcardiomyocytesareavailableandmustbeusedatleasttoconfirm keyfindings. Referee#2(CommentsonNovelty/ModelSystem): Technicalquality:Themolecularbiologyandbiochemistryexperimentaldesigns,thetechnical correctnessand data presentation isofthe highestquality.Alltheimportantmolecularbiologyand biochemistry toolhavebeen used to reach theconclusions. Novelty:Theresearchbringsaboutthenovelroleofoxygeninfunctioningasacardioprotectorby altering the transcriptionalability ofp53. MedicalImpact:The use ofoxygen to treatmyocardialinfraction willhave huge bio-medical implications. Adequacyofmodelsystem:Theauthorshaveusedappropriatein-vivo modelsystemsto conduct experimentsand to reach the conclusions. Referee#2(GeneralRemarks): Therevisedversionofthemanuscripttitled"p53'schoiceofmyocardialdeathorsurvival:Oxygen protectsinfarctmyocardium by recruiting p53 on NOS3 promoterthrough regulation ofp53-Lys118 acetylation" now clearly establishesthe role ofoxygen asamodulatorofp53'stranscriptionalability to achieve cardioprotection via p53-NOS3activationpathwaycontrarytothep53-BAX activation pathway which existsin themyocardialinfarcthearts.Theauthorshaveaddressed allmy concerns related to addition ofappropriate controlsin the experimentsand improving the overallquality of the manuscript. The paper now is suitable for publication in EMBO Molecular Medicine. 2nd Revision - authors'response 10 July 2013 EMM-2012-02055-Gogna et al. P53’schoiceofmyocardial death or survival …” 1 Response to reviewer’s comments Editor E: Reviewer1remainsconcerned thattheclonally derivedmyoblastcellline H9c2isnot sufficientto draw conclusions on p53 function in cardiomyocytes. S/he correctly notes that neonatal rat cardiomyocytes are available andthus must be used to confirm key findings. Reviewer 1 hadmade this point clear inhis/herfirstevaluationandfurthermore,inmydecisionletter,I hadaskedyou to complywith the Reviewers'requests.IthusconcurwithReviewer1'scurrentassessment.As youknow, we wouldnormally not allow asecondrevision. I am prepared inthis case, however,to give you another opportunity to improve your manuscript, with the understanding that acceptance or rejection of the manuscript will depend the satisfactory validation of the key findings incardiomyocytesinthe next, final versionof the manuscript. We thankthe editor for givingus anopportunity to revisethe manuscript.As suggested by the editor/reviewer 1, we have now performed the key experiments using rat neonatal cardiomyocytes. Accordingly, we have revised our manuscript and added the following new figures: Figure 6 (panel A-F), Figure 7 (panel B), Figure 9 (panel A-G). We haveelucidatedthe whole molecular pathway in rat neonatal cardiomyocytes in addition to the H9C2 cells. The results observed with rat neonatal cardiomyocytes are similar to those inH9C2 cells andthus showsthe criticalroleofoxygen in theregulationof p53 transcriptional activity. Reviewer #1 R1.1 The authors implythat NOS3isanimportantmodulator ofp53incardiomyocytes andthat this pathwayis animportant prosurvival mechanism for cardiomyocytes. My concerns are that the findings from whole organ lysates do not reflect signaling changes in cardiomyocytes during cardiac remodeling. In fact, stainings for NOS3 and p53 support these concerns. There is no expressionof NOS3 andp53 innormal cardiomyocytes as shown. Afterinjury there isdramatic upregulation of NOS3 and p53 in cell populations that do not appear to be related to cardiomyocytes. Any changes inthe expressionofthesegenes inwhole organlysates areprobably related to signaling changes in non cardiomyocytes and through changes in cell populations (infiltration of inflammatory cells, massive death of other cells, proliferation of local non cardiomyocytesect)duringthe remodelingprocess.Thereis evidence intheliteraturethat NOS3 is not expressed in cardiomyocytes (Tambascia et al Hypertension 2001; 37:1427-1428). The authors must show that p53 and NOS3 are co-expressed during the remodeling process. The authors chooseto use H9c2 cellswhichare aclonally derivedmyoblast lines thatwas generated over 40 years ago. We must assume that these cells bypass normal senescence through transformationaffectinggeneticstability.p53isthegate keeper for geneticintegrity.Itislikely that p53functionis defective inH9c2 andtherefore this line is less suitable to study p53 function. Neonatal rat cardiomyocytes areavailable andmust be used at least to confirm key findings. We acknowledge the questions raised by the reviewer. As mentioned above, we have carefully addressed the concerns of the reviewer by repeating key experiments using rat neonatal cardiomyocytes(RNC).Thenew results confirm the results from H9C2cells. Inour assessment themajor concern of therevieweroriginated from the uncertaintyregarding the validityoftheproposedstudy usingH9C2cells, whichare clonallyderived myoblastcells and not necessarilycardiomyocytes.Thereviewerraised two majorpoints:(1)Existence of eNOSor NOS3 as asignalingmolecule incardiomyocytes. (2) Suitability of H9C2 cells asbasis of our in- vitrostudymodel. We thank thereviewerforraisingthesequestionsandsuggestingustouse rat neonatal cardiomyocytes. Inthis revised versionwe have included the new results obtained usingRNCs andcomparedwith that obtained using H9C2 cells. We have validated the entire pathway in rat neonatalEMM-2012-02055-Gogna et al. P53’schoiceofmyocardial death or survival …” 2 cardiomyocytes. Our results with RNC support the previous findings with H9C2 cells where oxygenationinhibits theexpressionofTIP60acetylase, resultinginthe lack ofp53 acetylationat Lys118 residue whichpromotes p53-NOS3 survival pathway. This phenomenonhas now been observed by us ininfarct hearts, H9C2 cells, andrat neonatal cardiomyocytes. We have added Figure 6a,6b,6c, 6d, 6e,6f, 7b,8a,8b,8c, 8d, 8e,8f and8ginsupportofourfindings. We now sincerelyhopethatthe reviewerwill findthat we havesuccessfully addressed theconcerns. Inaddition, wewouldlike toaddresstheother concernofthereviewerthateNOS orNOS3 do not playany signalingrole inthe cardiomyocytes. In2007,Barbara Casadei’s researchlaboratory publishedtherole ofeNOSincellularsignalingincardiomyocytes(Seddonetal., 2007).Usingthe model (shown below) the authors clearly showed the role of eNOS in signaling both in the coronary vasculature andincardiomyocytes. In2012,AllenSamarel andgroupfromUniversity of Chicagoshowedthatallthree isoformsare expressed incardiomyocytes (Chuet al., 2012). They further showed that while eNOS and nNOS are constitutively expressed, iNOS is up- regulated under pathological conditions. These reports confirm our findings where we have discovered a very important role of molecular oxygen in inducing a p53-dependent survival pathway via NOS3 upregulationin the infarcthearts. Cardiomyocytesaseffectorsof nitricoxide signaling. MikeSeddon et al, CardiovascularResearch75(2007)315–326 References Chu, M., Koshman, Y., Iyengar, R., Kim, T., Russell, B., and Samarel, A.M. (2012). Contractile Activity Regulates Inducible Nitric Oxide Synthase ExpressionandNO(i) ProductioninCardiomyocytes via a FAK- DependentSignalingPathway. Journal ofsignal transduction 2012, 473410. Seddon, M., Shah, A.M., andCasadei, B. (2007). Cardiomyocytes as effectors of nitric oxide signalling. Cardiovascular Research75, 315-326.EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02055 © EMBO 10 3rdEditorialDecision 18 July 2013 ThankyouforthesubmissionofyourrevisedmanuscripttoEMBO MolecularMedicine.Wehave now received theenclosed reportfrom theReviewerwho wasasked to re-assessit.Asyou willsee the s/he is now globally supportive and I am pleased to inform youthat we will be able to accept yourmanuscriptpending thefollowing finalamendments: AsperourAuthorGuidelines,thedescriptionofallreporteddatathatincludesstatisticaltesting muststatethenameofthestatisticaltestusedtogenerateerror bars and P values,the number (n) of independent experiments underlying each data point (not replicate measures of one sample), and the actualP value foreach test(notmerely 'significant'or'P < 0.05'). Pleasesubmityourrevisedmanuscriptwithintwo weeks. Needless to say, the sooner we receive the final,revised version,the soonerIwillbe able to formally acceptitforpublication. I look forward to reading a new revised version of your manuscriptas soon as possible. ***** Reviewer'scomments***** Referee#1(GeneralRemarks): Theauthorsrespondedadequatetomycomments. Nofurthercomments orquestions 3rd Revision - authors'response 26 July 2013 Wearehappytoknowthatourmanuscripthasbeenprovisionallyacceptedforpublication in EMBO MolecularMedicine. Asrequested,wehaveincludedthepvalues,N numbers,andstatistalmethodandsoftwareusedin the calculations. 4th Editorial Decision 30 July 2013 ThankyouforthesubmissionofyourrevisedmanuscripttoEMBO MolecularMedicine. I am afraid thatthe manuscriptis notyetacceptable for publiscation.In fact,the description of the reported data thatincludes statisticaltesting is stille incomplete. As I mentioned in my previous decision letter,theactualP valueforeach test(notmerely 'significant'or'P < 0.05')mustbestated, in each and every case. This is missing in your revised manuscript for a numebr of figures and incompletely stated forothers.Indeed,this version has even less details than the previous (V3)e.g. forfigures 5,6,7,8.. Pleasesubmityourcorrectly revised manuscriptassoon aspossibleand in any casewithin two weeks. I look forward to reading a new revised version of your manuscriptassoon aspossible. EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02055 © EMBO 11 4th Revision - authors'response 06 August2013 Weapologizeforthelackofdescriptivestatistics.Sincethesepvalueswereverylowweusedthe lower limit, for example p<0.001 in our manuscript. We hadremoved the comparison I some cases aswe thoughtitwasnotnecessary.We apologize forthe misunderstanding. The“*”onFigure8isnotneededasitisasingledata.Wehaverestoredthenotation(*)inFigure 7. Asrequested,wehaveincludedtheacctualpvalues,N numbers,detailsintherevisedversionofthe manuscript. WehopethatthismanuscriptwillnowbesuitableforformalacceptanceandpublicationinEMBO MolecularMedicine.