generation ofanintracellularCa hyperpolarization impedesbothexpression andactivity ofmyogenin (Fischer-Lougheed etal.,2001;Liu2003).Preventing this of theirmembranerestingpotentialtoapproximately–70 mV muscle-specific promoters. (MEF2A-D) thatbindtoaconsensussequencepresentinseveral family (BlackandOlson,1998).MEF2family hasfourmembers also requirestheirinteractionwithtranscriptionfactors ofthe MEF2 activation ofmuscle-specificgenesby myogenicbHLHproteins differentiation ofmyogeniccells.Duringmyoblastdifferentiation, 1989). Thesefactors areimplicatedinthespecificationand inthe 1989; Davis etal.,1987;RhodesandKonieczny, 1989;Wright etal., factors, whichincludesMYOD, MYF5andMRF4(Braunetal., the family ofmyogenicbasichelix-loop-helix(bHLH)transcription the expression ofanearlymarker, myogenin.Myogeninbelongsto level, theinductionofdifferentiation processisassociatedwith myotubes canbeinducedbyserumwithdrawal. Atthemolecular months inculture,andterminaldifferentiation andfusioninto single satellitecells.Humanmyoblastscanproliferateforseveral major transcriptionfactors ofthedifferentiation process. hyperpolarization andtheexpression ofmyogeninand MEF2,two uncover themolecularlink betweentheKir2.1-induced to al., 2001;Liuet2003).Thegoalofthepresentwork was linked hyperpolarizationwasblocked.TheCaMKpathway, althoughCa of thisstudy. Ofthecalcineurin,p38-MAPK,PI3KandCaMKpathways,onlycalcineurinpathwaywasinhibitedwhenKir2.1- *Author forcorrespondence (e-mail:[email protected]) Hospital, Geneva,Switzerland. hyperpolarization-induced Ca the differentiation process,myogeninandMEF2.Blockinghyperpolarizationinhibitsmyoblastdifferentiation. Thelinkbetween resulting fromtoKir2.1K of humanmyoblastdifferentiation isamembranehyperpolarization and repair. We have shown previously thatoneoftheearliestevents Myoblasts differentiation isakey stepforskeletal musclegrowth INTRODUCTION KEY WORDS:Myogenesis,Calcineurin,Hyperpolarization,Humanmyoblasts In humanmyoblaststriggeredtodifferentiate, ahyperpolarization,resultingfromK Stéphane Konig induced Ca The calcineurinpathwaylinkshyperpolarization(Kir2.1)- Development 133,3107-3114(2006)doi:10.1242/dev.02479 Accepted 6June2006 1 expression/activity ofmyogeninandMEF2. hyperpolarization triggershumanmyoblastdifferentiation viatheactivationofcalcineurinpathway, which,inturn,induce myoblasts andtheyareunaffected byhyperpolarizationorKir2.1channelblock.We concludethattheKir2.1-induced potential orblockofKir2.1channels.Concerningthep38-MAPKandPI3Kpathways,theiractivityispresentalreadyinprolifer 1211 Geneva4,Switzerland. fusion Department ofBasicNeurosciences, UniversityMedicalCenter, RueMichelServet1, Differentiation ofhumanmyoblastsrequiresahyperpolarization We areusingprimaryhumanmyoblastculturesderived from 1, *, AnneBéguet 2 Department ofClinicalNeurosciences, University 2+ + channel activity (Fischer-Lougheed et signals tohumanmyoblastdifferentiation and 2+ signals andthefourmainregulatorypathwaysinvolvedinmyoblastdifferentiation wastheobject 2+ signal. Thissignalinducesanincreaseinexpression/activityoftwokeytranscriptionfactors 1 , CharlesR.Bader 2 and Laurent Bernheim 2005) activity isstronglycontrolledbycytoplasmic Ca differentiation CaMK(Chin,2005)andcalcineurin(Stiberetal., 2000). Althoughitiswellknown thatduringmyoblast inducing thetranscriptionalactivity ofMEF2(Tamir andBengal, p38-MAPK, CaMKandcalcineurinpathways appearcapableof 1999; Fridayetal.,2003;Xu2002;Zetser1999).The dependent kinase(CaMK)andcalcineurin(CuendaCohen, cytoplasmic freeCa hyperpolarization initiatesthedifferentiation processbyincreasing (Konig etal.,2004).We proposedthatKir2.1-linked and MEF2,indicatingthatitisaprerequisitefordifferentiation expression ofmyogeninandMEF2.We find,inaddition,that p38- p38-MAPK, PI3KandCaMKpathways arealsorequiredforafull through theselective activation ofthecalcineurin pathway, although hyperpolarization controlsthe onset ofthedifferentiation process known. are modulatedbethemembrane hyperpolarization,however, isnot are involved inhumanmyoblastdifferentiation, andwhetherthey 2005; Stokoe etal.,1997).Whetherthesefoursignaling pathways both dependonPI3Kactivity (Alessietal.,1996;Sarbassov etal., requires aphosphorylationattwo sitesbytwo separate kinasesthat AKT (proteinkinaseB).Fullactivation ofAKTbyinsulinorIGF1 Kandel andHay, 1999).Theprincipaldownstream target ofPI3Kis tyrosine kinasereceptor(Jiangetal.,1998;Kaliman1996; activation ofPI3Kiscoupledtoinsulin growth factor (IGF1) MKK3 andMKK6(Derijardetal.,1995;Han1996), and Activation ofp38-MAPKislinked todirectphosphorylationsby MAPK), phosphatidyl-inositol3-kinase(PI3K),Ca different pathways: p38mitogen-activated proteinkinase(p38- differentiation, hasbeensuggestedtoberegulated byatleastfour human myoblastdifferentiation? pathways downstream ofthiscytoplasmic Ca Liu etal.,2003).Thequestioniswhatarethesignaltransduction Ca In thepresentstudy, weshow thattheKir2.1-induced In mousemyoblasts,myogeninexpression, anearlymarker for 2+ 2+ in theactivation ofp38-MAPKand PI3Kislessclear. dependent, isunaffected bychangesinmembrane + channel (Kir2.1)activation,allowsthe 2+ (Arnaudeau etal.,2006;Bijlenga2000; 1 RESEARCH ARTICLE 2+ signal thatinitiate 2+ -calmodulin- 2+ , theroleof ating 3107 s

DEVELOPMENT the maximumCaMKII activity ofthemyoblast sample)was assessedfrom EGTA and 0.5 conditions ofculture)was assessedinabuffer solutioncontaining5mM activity (whichrepresentsthelevel ofCaMKIIactivity ofmyoblastsineach in abuffer solutioncontainingthespecificpeptideforCaMKII.Endogenous supernatant werecollectedforeach assayandincubated2minutesat30°C (Novagen) andspunfor5minutes(13,000 Myoblasts werelyzedusing100 the Metamorph5.0software (Universal Imaging,West Chester, PA). Scientific, Trenton, NJ).Imageacquisitionandanalysiswas performedwith 12 bitsTE/CCDinterlinedCoolsnapHQcamera(Photometrics,Roper and 602±50nm(XF3063Omega Optical,Brattleboro,VT)usingacooled, fluorescence emissionwererespectively acquiredat460±30nm,520±40nm through aXF2050dichroicmirror(Omega Optical,Brattleboro,VT)andthe from anOptoscanMonochromator(CairnResearch,Faversham, UK) respectively excited bythe360±10nm,488±10nmand546±10line AG, Feldbach, Switzerland). DAPI, Alexa 488andAlexa 546were microscope usinga40 plated on25mmcoverslips was imagedwithaZeissAxiovert S100TV SantaCruz Biotechnology).Immunostainingfluorescencefrommyoblasts Biosciences) andMEF2usingarabbitpolyclonalantibody(1/300,Sc-313 Myogenin was revealed usingamousemonoclonalantibody(1/1000,BD Immunostaining was performedaspreviously described(Konig etal.,2004). Immunostaining medium was complementedwith10 medium (differentiation medium,DM).Whenindicated,differentiation (growth medium)andinducedtodifferentiate intomyotubes inaserum-free (clonal culture).Myoblastswereamplifiedinserum-containingmedium cell/well) ofa96wellscontainer(BectonDickinson)usingmicropipette under themicroscopeonasizecriteria,transferredintosinglewells(one cells obtainedaftermuscleenzymaticdissociationweremanuallycollected according totheguidelinesoflocalethicalcommittee.Singlesatellite samples wereobtainedfromchildrenduringcorrective orthopedicsurgery were preparedandgrown aspreviously described(Liu et al.,1998).Muscle Primary culturesofhumanmyoblasts(progenitorsfromsinglesatellitecell) Cell cultures andimmunocytochemistry myoblast proliferationthroughaCa proliferation, andthatCaMKactivation canbeinducedduring MAPK andPI3Karealreadyactivated duringmyoblast 3108 Ca CaMK activity was quantifiedusingtheradioactive assaySignaTECT CaMKII assay (1/1000, CellSignalingTechnology #9215);phopho-AKT rabbit monoclonalantibodyagainstphospho-p38-MAPK Biosciences) andp38-MAPK(1/1000,CellSignalingTechnology #9212); used: mousemonoclonalantibodyagainstmyogenin(1/2000, BD orthovanadate). AntibodiesweredilutedinTTBSwith5%BSA. out inthepresenceofphosphataseinhibitors(50mMNaFand1sodium al., 2004),except thatsaturationwith5%non-fat milkinTTBSwas carried 3 minutes.Western blotswereperformedaspreviously described (Konig et After addinganequivalent volume ofLaemmli2 Myoblasts werelyzedusingPhosphoSafeExtractionBuffer (Novagen). Western blots (Calbiochem) or5 differentiation bygeneratingaCa hyperpolarization thatactsasamolecularswitch,forcing process inhumanmyoblastsisinitiatedbyamembrane related tothehyperpolarization.We proposethatthedifferentiation MATERIALS ANDMETHODS specific activation ofthecalcineurinsignalingpathway. CellSignaling Technology #4058);phopho-AKT CellSignaling Technology, #4377). CellSignaling Technology, #4056);andp42/44MAPK LY194002 (CellSignaling),30 2+ /Calmodulin-Dependent ProteinKinaseAssaySystem(Promega). RESEARCH ARTICLE ␮ Ci of ␮ M FK-506(A.G.Scientific). ϫ ␥ 32 Fluar 1.3NA oil-immersionobjective (CarlZeiss P-ATP. Total CaMKIIactivity (whichrepresents ␮ M KN-93(Calbiochem),7 ␮ ␮ M SB202190(Calbiochem),50 l PhosphoSafeExtractionBuffer 2+ 2+ -dependent mechanismnot signal responsibleforthe g ) at4°C.Five microlitresof ϫ , extracts wereboiledfor Thr202/Tyr204 Thr308 Ser473 Thr180/Tyr182 ␮ (1/1000, (1/1000, (1/1000, M CsA ␮ M specific antibody. recATF-2 was detectedbyimmunoblottingusingaphospho-ATF2 (Thr71)- and usingrecombinantATF-2 (recATF-2) asasubstrate.Phosphorylated the immunoprecipitatedphospho-p38-MAPKinpresenceof200 monoclonal antibody. Aninvitrokinaseassaywas performeddirectlyon (with LY294002) hadastronger effect onmyoblast differentiation, (Promega) asrecommendedbythemanufacturer. times, cellswereprocessedwiththeDual-Luciferasereporterassaykit luciferase [phRL-TK-luc,Promega (Konig etal.,2004)].Attheindicated 9NFAT-luc) togetherwith1pmolcontrolplasmidencodingtheRenilla transfected with2pmolfireflyluciferaseencodingplasmid(3MEF2-lucor external K illustrates thattheinhibitionisnotduetoatoxiceffect ofhigh mM) toprevent membranehyperpolarization.Thisfigurealso Using electroporation(Espinosetal.,2001),2 Luciferase assay 200 active formofp38-MAPKwas immunoprecipitated(overnight at4°C)from times, cellslysiswas carriedoutwith500 MAPKinase AssayKit(CellSignalingTechnology, #9820).Attheindicated p38-MAPK activity was assessedusingthenon-radioactive p38- p38-MAPK assay 1 differentiation isinducedinpresenceofhighextracellular K expressing myogeninandMEF2isstronglyinhibited when differentiation markers. Fig.1Ashows thatthepercentageofnuclei methods), andmyogeninMEF2expression were usedas growth mediumbydifferentiation medium(seeMaterialsand myoblast todifferentiate. Differentiation was inducedbyreplacing Membrane hyperpolarizationisacrucialearlystepthatallows myoblast todifferentiate? Which regulatory pathwaysinducehuman RESULTS respectively. Inhibitionofthehyperpolarization with10mMCs and 71%ofthenucleiwerepositive for myogeninandMEF2, (6%) orMEF2(7%).After3-4daysindifferentiation medium,69% proliferating myoblasts,very few nucleiwere positive formyogenin expressing myogeninorMEF2over thetotalnumberofnuclei.In differentiation was assessedusingthepercentageofnuclei involved inhumanmyoblastdifferentiation (Fig.1B).Myoblast investigated whetherany of theseregulatory pathways were during murinemyoblastdifferentiation (Xuetal.,2002).We first have beensuggestedtoregulate myogenin andMEF2expression pathways thatcontrolmyogeninandMEF2expression. hyperpolarization prevents theactivation ofoneorseveral regulatory observation, wehypothesizethataninhibitionofthe this by treatingcellswith10mM Cs myogenin andMEF2expression tothesamelevel asthatobtained MAPK (withSB202190)and CaMK(withKN-93)reduced fold (Konig et al.,2004).Fig.1Balsoshows thatinhibitingp38- Kir2.1 blocker) reducedmyogenin andMEF2expression by1.9- 15 obtained withmyoblastsmaintainedindifferentiation mediumcontaining CaMKII remainsnearlyconstant(notshown). For eachexperiment, theratio modification oftheCaMKIIactivity inculturedmyoblastsasthetotal the specificactivation ofCaMKIIthroughouttheexperiments reflectsa endogenous activity inculturedmyoblastsandthetotalactivity. Variation of ATP). Specificactivation ofCaMKIIwas calculatedastheratiobetween activation invitro(5mMCaCl replaced inadifferentiation mediumcontaining5mMK limiting amountsofCa ␮ CaMK, PI3K,calcineurinandp38-MAPKregulatory pathways ␮ l ofsupernatant(toavoid saturation)inabuffer solutioncontainingnon- M BAPTA-AM was setto1. ␮ l cellextracts with20 + as myoblastsarestillabletofullydifferentiate when 2+ and calmodulintoallow maximalCaMKII ␮ 2 l phospho-p38-MAPK(Thr180/Tyr182) , 5 ␮ M calmodulinand0.5 + . However, inhibitionofPI3K ␮ l ofprovided lysisbuffer. The ϫ 10 Development 133(16) 6 human myoblastswere ␮ Ci of ␮ + . From M ATP + ␥ (116 32 + P- (a

DEVELOPMENT whether itsactivity iscontrolledbyaCa phosphatase involved inhuman myoblastdifferentiation, wetested or indifferentiation mediumcontaininghighK 2000; Liuetal.,2003).AscalcineurinisaCa differentiation toproceed(Arnaudeauetal.,2006; Bijlengaetal., intracellular Ca hyperpolarization generatedbyKir2.1channelsincreases Our previous work onhumanmyoblastssuggestedthatmembrane hyperpolarization myoblast differentiation requires amembrane Early activationofcalcineurinduringhuman hyperpolarization generatedbyKir2.1channels. moreofthesepathways arecontrolledbymembrane or one human myoblastdifferentiation. Whatislessclear iswhether PI3K andcalcineurinpathways areallinvolved intheearlystepsof 2.6-fold, respectively. strategy, myogeninandMEF2expression was reducedby3.7and required toinhibitcalcineurinactivity fully(seebelow). Usingthis reduce thetoxicityofeachdrug.Concentrationsusedwerethose pathway, weusedcyclosporin AandFK506mixed togetherto myogenin andMEF2,respectively. To inhibitthecalcineurin reducing thepercentageofpositive nucleibya3.8and4.1-foldfor Membrane hyperpolarizationcontrols calcineurin obtained inpresence ofthecalcineurin inhibitors clearly background level. ThestronginhibitionoftheNF-AT activity calcineurin inhibitors,maintained NF-AT activity toits combination ofcyclosporin AandFK-506,two known NF-AT activity onlydependson calcineurin activity asa NF-AT activity (by4.3-and2.8-fold, respectively), andalsothat Fig. 2Bshows that bothdepolarizingconditionsstronglyinhibited out after4daysofdifferentiation tomaximizeluciferasesignals. prevent membranehyperpolarization.Experiments werecarried supplemented with10mMCs differentiation medium,orindifferentiation medium plasmid wereinducedtodifferentiate eitherincontrol the calcineurinactivation, myoblasts expressing 9NF-AT-luc before MEF2. of myoblastdifferentiation, atthesametimeasmyogenicbHLHand we concludethatcalcineurinisactivated duringthevery earlysteps process. Activation ofMEF2is,however, slower. Fromtheseresults, pathway isactivated atthevery beginning ofthedifferentiation myogenic bHLH,suggestingthat,like myogenicbHLH,calcineurin Interestingly, kineticsofactivation ofNF-AT aresimilartothatof by eithermyogenicbHLHorMEF2transcriptionfactors). (myoblasts wereelectroporatedwithaluciferaseplasmidcontrolled was thencomparedwiththatofmyogenicbHLHandMEF2 10.0-fold after4days).Kineticsofactivation ofNF-AT (calcineurin) then increasesduringthefollowing days(4.7-foldafter3and myoblasts, isinducedduringthefirst24hoursofdifferentiation, and 2A. ItcanbeseenthatNF-AT activity isabsentinproliferating myoblasts expressing the9NF-AT-luc plasmidareillustratedinFig. Kinetics ofactivation ofNF-AT transcriptionfactor inelectroporated induced todifferentiate indifferentiation mediumfor1to4days. either maintainedinproliferationcondition(growth medium)or factors (9NF-AT-luc plasmid).Theelectroporatedmyoblastswere the controlofaspecificpromoterinduciblebyNF-AT transcription electroporated withaplasmidencodingtheluciferaseproteinunder transcription factor NF-AT. For thispurpose,humanmyoblastwere used itsabilitytodephosphorylateandtherebyactivate the induced bythehyperpolarization.To assesscalcineurinactivity we Taken together, theseresultssuggestedthatp38-MAPK,CaMK, To evaluate theimportanceplayedbyhyperpolarizationin 2+ and thatthisstepisessentialtoallow myoblast + to blockKir2.1channelactivity, 2+ signal thatcouldbe + (116 mM)to 2+ -dependent (DM) orinDMwhich116mMNa induced todifferentiate for3dayseitherindifferentiation medium myoblast maintainedingrowing medium(GM).Myoblastswere hyperpolarization. NeithermyogeninnorMEF2were detectedin concentration ofK in highK MEF2 activities throughtheinhibition ofthecalcineurinpathway. induced membranehyperpolarization inactivates myogeninand activity by3.0-and 17.5-fold,respectively. together reducedmyogenicbHLH andMEF2transcriptional that theinhibitionofcalcineurin bycyclosporin AandFK-506 affects thesetwo activities inhumanmyoblasts.Fig.2Cshows al., 2003),weexamined whetheraninhibitionofcalcineurin been relatedtocalcineurinactivity inmousemyoblasts(Fridayet shown). Finally, as myogenicbHLHandMEF2activities have inducing astrongtoxicitytothehumanmyoblast(data not used asasingledrugcouldnotfullyinhibitcalcineurinwithout to onlythecalcineurinactivation. Acombination ofinhibitorswas confirmed thattheactivity ofthetranscriptionfactor NF-AT isdue ( pathways are involvedinhumanmyoblastdifferentiation. Fig. 1.Calcineurin,CaMK,p38-MAPKandPI3Kregulatory block p38-MAPK,by30 Cs medium (DM).Myoblastdifferentiation ispartiallyinhibitedby10mM Myoblast were inducedtodifferentiate for3-4 daysindifferentiation expressed asapercentage ofmyogenin-andMEF2-positivenuclei. Alexa546, andnucleiwithDAPI.Scalebar:20 differentiated nicely. Myogeninwasrevealed withAlexa488,MEF2 days incontrol differentiation medium(washKCl) inwhichthey LY294002 (LY) toblockPI3K,andbyacombinationof7 A (CsA)and5 expressed asmean±s.e.m. A Induction ofmyogenicdifferentiation requires amembrane ) + Together, theseresultsshow thatsuppressionoftheKir2.1- (Cs) toblockthehyperpolarization,by10 + differentiation medium,myoblastswere replaced for3more ␮ M FK506(FK)toblockcalcineurin.Resultsare + to prevent thehyperpolarization(KCl).After3days ␮ M KN93(KN)toblockCaMK,by50 + was replaced byequivalent RESEARCH ARTICLE ␮ ␮ M SB202190(SB)to m. ( B ) Differentiation is ␮ M cyclosporin ␮ M 3109

DEVELOPMENT membrane hyperpolarization(10mMCs differentiation incontrolconditionsandthatprevent Endogenous CaMKIIactivity was evaluated duringmyoblast hyperpolarization, inthesameway ascalcineurinwas. activated byCa differentiation. We, thus,examined whetherthispathway was dependent pathway involved inthecontrolofhumanmyoblast 3110 As seeninFig.1B,CaMKregulatory pathway isasecondCa membrane hyperpolarization differentiation isnotlinkedtotheKir2.1-induced The role ofCaMKIIinhumanmyoblast differentiation (Konig etal.,2004). linked hyperpolarization, whichoccursafter6hoursof exposure todifferentiation medium,i.e.beforethedifferentiation- CaMKII activation couldalreadybedetectedaftera1-hour hyperpolarization. Inagreementwiththisresult,amaximum calcineurin, CaMKIIisnotregulated bytheKir2.1-linked agents (10mMCs However, thisincreasedactivity isinsensitive todepolarizing activity isincreasedby2.3-foldafter24hoursofdifferentiation. background) arepresentinproliferatingconditions,andthat this Fig. 3Ashows thatlow levels ofCaMKIIactivity (40%above activity obtainedinpresenceof15 to compareexperiments, wenormalizedtheresultstoCaMKII extracellular Ca for CaMKIIactivation, weexamined differentiation atalow C A B To investigate theoriginof thevery earlyCa Luciferase activity Luciferase activity Luciferase activity 0 1 2 3 4 5 6 7 8 9 16 10 12 14 RESEARCH ARTICLE 0 2 4 6 8 10 12 14 0 2 4 6 8 M2h4h7h96h 72h 48h 24h GM MDM GM MD Cs DM GM Myogenic bHLH bHLH Myogenic 2+ NFAT concentration (15 2+ + DM or 116mMK CsA+FK signals inducedbytheKir2.1-linked 0 1 2 3 4 5 6 18 10 12 14 16 NFAT 0 2 4 6 8 ␮ + ␮ MD CsA+FK DM GM M BAPTA-AM (background). M2h4h7h96h 72h 48h 24h GM ). Thisindicatesthat,unlike Myogenic bHLH bHLH Myogenic M insteadof1.8 mM),which + MEF2 or 116mMK KCl 2+ DM signal responsible CsA +FK + ). Inorder 60 10 20 30 40 50 0 M2h4h7h96h 72h 48h 24h GM 2+ - MEF2 1.8 mMCa However, activation oftheCaMKIIingrowth mediumcontaining with thatobtainedtheCaMK inhibitorKN-93(Fig.1A). and 1.8-foldrespectively (Fig.3B).Thisreductioniscomparable positive nuclei forbothmyogeninandMEF2was reducedby2.4 containing 0.7mMCa (i.e. toexpress myogeninandMEF2)indifferentiation medium it isunaffected byhyperpolarization. activity isdirectlylinked toextracellular Ca together, theseresultssuggestthat,inhuman myoblasts,CaMK to thatobtainedincontrolgrowth medium( hour ( different tothatobtainedincontroldifferentiation mediumafter1 medium containing1.8mMCa differentiation medium containing0.7mMCa expected, whenCaMK ismaintainedatalow activity in in growth mediumcontaining1.8mMCa ( similar tothatofproliferatingmyoblastskept ingrowth medium In thislow Ca differentiation mediumcontainingthesameCa Furthermore, whenmyoblastswereinducedtodifferentiate ina was reducedtothebackgroundlevel after1hour(Fig.3A). reduces massively allCa of myoblasts[fura-2Ca in thegrowth medium(0.7mMCa P =0.78). However, theCaMKIIactivity inmyoblastsproliferating DM We thentestedthecapacity ofhumanmyoblasttodifferentiate P =0.14) orafter24hours( 2+ 2+ did notallow any induction ofmyogeninorMEF2 -containing differentiation medium,CaMKIIactivity (DM) orinpresence of10mMCs differentiate for4dayseitherincontrol conditions plasmid. Humanmyoblastswere inducedto Calcineurin activitywasassessedusingNFAT-luc ( to 4daysindifferentiation medium(DM). myoblasts (GM)andfrom myoblastsmaintainedfor1 Luciferase extractswere prepared from proliferating myogenic bHLHandMEF2transcriptionfactors. expressed asmean±s.e.m. respectively. SameconditionsasinB.Resultsare was assessedusing4RE-lucand3MEF2-lucplasmid, factors activation.MyogenicbHLHandMEF2activity decreases myogenicbHLHandMEF2transcription cyclosporin A(CsA)and5 calcineurin activity. ( of NFAT transcriptionfactoractivityreflects Luciferase activityisinarbitraryunits.Increased level 9NFAT-luc or4RE-luc3MEF2-lucplasmids. myoblasts were transientlytransfectedwitheither myoblast differentiation activation ofcalcineurinpathwayduringhuman Fig. 2.Hyperpolarizationcontrols theearly high extracellularK ( B C Depolarizationinhibitscalcineurinactivation. ) Inhibitionofcalcineurinpathwaystrongly ) 2+ 2+ 2+ measurements (Arnaudeauetal.,2006)]. (CaMKII activity islow) oringrowth influxes throughtheplasmamembrane 2+ P (CaMKII activity isincreased).As =0.55), but significantlydifferent + A (KCl), oracombinationof7 2+ ) KineticsofactivationNFAT, ), theCaMKIIactivity was ␮ . Humanprimary 2+ M FK506(FK). 2+ Development 133(16) concentration andthat , was notstatistically 2+ 2+ + , thepercentageof P (Cs), 116mM concentration as =0.002). Taken ␮ M

DEVELOPMENT or proliferating myoblastwith1.8mMCa phosphatase inhibitorswere prepared from proliferating myoblast(GM) substrate inpresence of ability ofmyoblastextractstophosphorylateaspecificpeptide and 1.8mMCa increased) werestillabletoproliferateactively. Myoblasts(10 (activity incultured) wasassessedinabsenceofaddedCa extracellular Ca . mMCa 1.8 were seededingrowth mediumcontainingeither 0.7(control)or medium containing1.8mMCa expression. We alsoverified thatmyoblastsmaintainedingrowth Membrane hyperpolarizationcontrols calcineurin hyperpolarization. Fig. 3.CaMKIIactivationisunrelated tothemembrane containing 15 the ratioobtainedwithmyoblasts maintainedindifferentiation medium between theendogenousand totalactivity. Foreachexperiment, calmodulin. Specificactivationof CaMKII wascalculatedastheratio (4.7±0.3 proliferation ratewas notaffected byCaMKactivation modulated bychangesinCa correlate withKir2.1-linked membranehyperpolarizationbut canbe (DM), orinDMcontaining10mMCs induced todifferentiate for1or24hoursindifferentiation medium expressed asmean±s.e.m. a percentage ofmyogenin- andMEF2-positivenuclei.Resultsare does notinducemyoblastdifferentiation. Differentiation isexpressed as sample) wasassessedafteradditionof5mMCa calmodulin; totalCaMKIIactivity(maximumofthemyoblast mM Ca mean±s.e.m. ( B A

CaMK II activity ratio In conclusion,theseresultsshow thatCaMKIIactivity doesnot % of positive nuclei (endogenous/total) 2+ 10 20 30 40 50 60 70 1.0 1.5 2.0 2.5 3.0 0 ϫ (DM 10 2+ 5 B low MD sKlD DM DM KCl DMCs GM ␮ , andcountedafter48hours.We foundthatthe ) CaMKIIactivationinhighCa versus 4.6±0.3 M BAPTA-AM wassetto1.Resultsare expressed as 2+ ) or0.7mMCa 2+ MDM GM , respectively, ( concentration. Ourresultsalsoshow thatan A ) CaMKIIactivitywasassessedbymeasuringthe ␥ 32P-ATP. Total cellextractscontaining 4 1h 24h 2+ ϫ 2+ fluxes associatedwithchangesinthe 10 P 2+ (DM =0.74). 5 (myoblasts withCaMKactivity + myoblasts after48hoursin0.7 (Cs), 116mMKCl(KCl),15 0.7 ). EndogenousCaMKIIactivity 2+ (GM 2+ proliferating medium 2+ 1.8 DM and 5 ), andfrom myoblast 0.7 low ␮ 2+ M DM or 0.7 MEF2 Myogenin GM GM ␮ M 1.8 1.8 5 ) p38-MAPK activity, wefirstusedantibodiesspecificforits controlled bytheKir2.1-linked hyperpolarization.To evaluate differentiation (seeFig.1B),weexamined whetherthispathway is Given theimportancesuggestedforp38-MAPKinmyoblast myoblast differentiation hyperpolarization, norsufficient toinduce neither controlled bymembrane The observedactivitiesofp38-MAPKandPI3Kare differentiation. proliferation andthatitis,onitsown, notsufficient toinduce increase ofCaMKIIactivity doesnotstoporreduce myoblast depolarization inducedby10mMCs myoblasts (GM),ismaintainedindifferentiating myoblasts (DM)and (Thr180/Tyr182). Phospho-p38-MAPKispresent inproliferating using anantibodydirected againstphospho-p38-MAPK ( not controlled bythedifferentiation-linked hyperpolarization. Fig. 4.p38-MAPKandPI3Kare activatedinproliferation andare myogenin expression. ␮ in presence orabsenceof 10 mMCs myoblasts (GM),andfrom myoblastsdifferentiated for4and24hours Phospho-p38-MAPK wasimmunoprecipitated from proliferating detected withanantibodyspecificforphospho-ATF2 (Thr71). used toassessp38-MAPKactivity. ATF2 phosphorylationhasbeen recombinant ATF2 byimmunoprecipitated phospho-p38-MAPKwas were prepared attheindicatedtime.( with differentiation isreduced inpresence ofCs level ofphosphorylation.Bycontrast,myogeninexpression associated Phospho-AKT expression is notaffected by10mMCs myoblasts (GM)anddifferentiated for24hours(DM). insulin. Phospho-AKT(Ser473/Thr308) ispresent inproliferating blot.Myoblastswereby western grown inmediawithoutexogenous A M BAPTA-AM (BA).Myogenicdifferentiation isconfirmed by ) Activation of p38-MAPK pathway was detected by western blot ) Activationofp38-MAPKpathwaywasdetectedbywestern + + . ( B RESEARCH ARTICLE (Cs10 mM)doesnotaffect the ) Phosphorylationof C ) Detectionofphospho-AKT + . Protein extracts + (Cs10) or15 3111

DEVELOPMENT kinase activity was observed inpresenceof10mMCs myoblasts inducedtodifferentiate for4and24hours.Thesame immunoprecipitated fromproliferatingmyoblasts,aswellby recombinant ATF2 isphosphorylatedbyphospho-p38-MAPK phosphorylaterecombinantATF2. Fig.4Bshows that to from proliferatinganddifferentiating human myoblasts evaluating theabilityofimmunoprecipitatedphospho-p38-MAPK hyperpolarization duringhumanmyoblastdifferentiation. regulatory pathway arenotcorrelatedwiththe Kir2.1-induced activation andthemaintainedactivity ofthep38-MAPK the after calcineurinactivation. the increase ofintracellular Ca induced todifferentiate inthepresenceof10mM Cs MAPK isnotsensitive toCs This was confirmedbyshowing thatphosphorylationofp38- would becontrolledbythedifferentiation-linked hyperpolarization. MAPK duringproliferationmadeitunlikely thatthispathway differentiation process.Thepresenceofphosphorylated p38- that itremainedphosphorylatedduringthefirst24hoursof already phosphorylatedinproliferatingmyoblasts(Fig.4A)and phosphorylated (activated) form.We foundthatp38-MAPKwas 3112 influx through T-type Ca hyperpolarization. Calcineurinactivation isstrictlyassociatedwithKir2.1activityandtheonsetof differentiation proc illustration ofthemechanismsmyoblast differentiation, thatbeginswithcalcineurin activationasaconsequenceofKir2.1- Fig. 5.Schematicrepresentation oftherole ofmembranehyperpolarization duringhumanmyoblastdifferentiation. in presenceof10mMCs illustrated bythemarked downregulation ofmyogeninexpression affected. Theefficiency oftheCs hyperpolarization, theexpression ofphospho-p38-MAPKwas not be activatedbyanincrease inextracellularCa However, p38-MAPK,PI3KandCaMK are (orcanbe)activatedduringmyoblastproliferation withoutinducingmyoblastdifferentiat phosphorylated attwo sites,Ser473andThr308.Fig.4Cshows that phosphorylation. WhenthePI3Kpathway isactivated, AKTis kinase B).Thus,asreadoutforPI3Kactivity, weusedAKT media intheexperiments involving thispathway. PI3K, insulinwas omittedfrombothgrowth anddifferentiation Kandel andHay, 1999).Inordertoavoid unwanted stimulationof insulin/IGF1 signaling(Jiangetal.,1998;Kaliman1996; human myoblastdifferentiation. maintained activity ofthep38-MAPKregulatory pathway during Kir2.1-linked hyperpolarizationisnotinvolved intheactivation or by adirectassessmentofphospho-p38-MAPKactivity thatthe We alsoverified directlyphospho-p38-MAPKactivity by A key downstream moleculeactivated byPI3KisAKT(protein Activation ofthePI3Kregulatory pathway dependsupon RESEARCH ARTICLE 2+ channels (CaT)orstore-operated channels(SOCs),orrelease ofCa 2+ + + concentration responsible for myoblastdifferentiation etal.,2006). andcalcineurinactivation (Arnaudeau . Theseresultsdemonstratethat (Fig. 4A).Whenmyoblastswere + treatment inthisexperiment is 2+ concentration notlinkedtothedifferentiation process. MyogeninandMEF2expression isobserved + + , confirming to blockthe sustained elevations ofintracellularCa Calcineurin isaserine/threoninephosphatasethatactivated during in transcriptionalregulation ofslow fibergenes(Chinetal.,1998). In skeletal muscle,thecalcineurinpathway hasbeenfirstimplicated Calcineurin signalingpathway forces myoblasttodifferentiate andfusetoformmyotubes(Fig.5). resulting calcineurinactivation constitutethemolecularswitchthat hyperpolarization. proliferation, andisnotaffected bythedifferentiation-induced differentiation totake place,doesnotinterfere withmyoblast MAPK andPI3Kpathways, althoughrequiredforafull process. Thiswork alsoshows thatactivity oftheCaMK,p38- pathway istightlylinked totheinductionofdifferentiation activates theCa demonstrates thattheKir2.1-linked hyperpolarizationspecifically This work performedonprimaryculturesofhumanmyoblasts DISCUSSION required forafullmyoblastdifferentiation totake place. controlled bytheKir2.1-linked hyperpolarization,but they are pathways areactivated duringmyoblastproliferation.They arenot myogenin expression was markedly inhibited. BAPTA-AM, AKTphosphorylationwas notaffected although Cs experiment, adownregulation ofmyogeninexpression indicatedthat notmodulatingthePI3Kregulatory pathway. Inthesame is phosphorylation (Fig.4C),indicatingthatthehyperpolarization to inhibitKir2.1-inducedhyperpolarizationhadnoeffect onAKT proliferating humanmyoblast.Treating myoblastwith10mMCs shows that,like p38-MAPK,PI3Kisalreadyfullyactive in that ofmyoblastsinducedtodifferentiate for24hours. Thisresult myoblasts, andthatthephosphorylationlevel iscomparablewith AKT isalreadyphosphorylatedatthetwo sitesinproliferating the AKTpathway isnotregulated byCa Our modelisthattheKir2.1-linked hyperpolarizationandthe Taken together, theseresultsshow thatPI3Kandp38-MAPK + was efficient atinhibitingdifferentiation. Fig.4Calsoshows that 2+ 2+ for endoplasmicreticulum (stores), couldprovide -dependent calcineurinpathway, andthatthis 2+ 2+ (Klee etal.,1998),asoccur . Inthepresenceof15 ess. ActivationofCa Development 133(16) induced membrane Chronological ion. CaMKcan 2+ ␮ M +

DEVELOPMENT blockade ofKir2.1channelswith 10mMCs hyperpolarization tookplace)are statisticallyidentical.Furthermore, Kir2.1channels)orat24hours(longafter membrane of measured at1hourindifferentiation medium(beforeactivation to Kir2.1-inducedhyperpolarization. Indeed,CaMKIIactivities myoblast differentiation. CaMKIIactivation is,however, notlinked channels orareleaseofCa of Kir2.1channelswith10mMCs proliferating myoblast.Inaprevious work, weshowed thatblockade activity isreducedtoalevel close tothebasallevel measuredin can use,inadditiontoorassubstitutesforT-type Ca found that,asthey differentiate, different clonesofhumanmyoblasts on theearlystepsofdifferentiation process,whereasT-type Ca CaMK isanotherCa CaMK regulatory pathway expression/activity ofmyogeninandMEF2 transcriptionfactors. and/or ether-à-gogo-related geneK the signalsthey induce. activation isstrictlycontrolledbytheactivity ofKir2.1channelsand earlystagesofhumanmyoblastdifferentiation, calcineurin the fully inactivated by10mMCs Liu etal.,2003).Here,weshow thatcalcineurinactivity isalready receptors. However, alltheseCa through L-typeCa calcineurin isadownstream mediatorofIGF-1-inducedsignaling differentiating C2C12myoblasts,itwas clearly shown that myocytes (Graefetal.,1999;Perrier2004).Inaddition, in initiate calcineurinactivation inneuronsandratventricular hyperpolarization. InpresenceofeitherCs calcineurin activation islinked totheKir2.1linked membrane remained tobeexplained. beginning ofthedifferentiation process,i.e.priortoinnervation, However, thetriggerofachangeincalcineurinactivity atthe has alsobeensuggested(Fridayetal.,2000;Xu2002). for calcineurinatearlystepsofdifferentiation in mouse myoblasts factors NFAT andMEF2(Chinetal.,1998;Wu etal.,2000a).Arole implicated inthefast-to-slow fiberswitchincludethetranscription Serrano, 2002).Invivo, thedownstream substratesforcalcineurin in musclefibersbyslow typemotoneuronfiring (Schiaffino and Membrane hyperpolarizationcontrols calcineurin show thatactivation ofthe T-type Ca plays animportantrole(Bijlengaetal.,2000).Specifically, wecould channels activity inducedbytheKir2.1linked hyperpolarization hyperpolarization with116mMK membrane hyperpolarizationanditsassociatedCa of calcineurinthatlinkstheearlyactivation oftheKir2.1-induced prior totheinnervation ofmyotubes,thereisalreadyafirstactivation hyperpolarization isblocked. high external K to differentiate toapproximately–35mV(Konig etal.,2004),while myoblasts, however, inhibitionofL-typeCa type Ca showed thatallclonesofhumanmyoblastsdonotexclusively useT- that activate calcineurin(Kleeetal.,1998).However, werecently others sourcesofCa Ca potentials allows asustainedlow amplitudeincreaseofintracellular presence of10mMCs in thevicinityof0mV. Theremainingpotentialofabout–35mVin There areevidences thatL-typeCa In humanmyoblastsinducedtodifferentiate, we showed that Thus, weproposethat,indifferentiating humanmyoblastsand 2+ (Bijlenga etal.,2000)thatiscompatiblewiththeCa 2+ channels todifferentiate (Arnaudeauetal.,2006).We + (116 mM)clampstherestingmembranepotential 2+ channels (Spangenburg etal.,2004).Inhuman 2+ 2+ + such asaninfluxthroughstoreoperated is probablyduetoactivity ofether-à-gogo -dependent pathway involved inhuman 2+ from theendoplasmicreticulumviaIP3 2+ + signals aresuppressedwhenthe + + + , whichsuggeststhat,during channels (Bijlengaetal.,1998; have no effect ontheCaMKII depolarizes myoblastinduced 2+ 2+ channels athyperpolarized channels areessentialto + 2+ or highK channels hasnoeffect + or inhibitionofthe 2+ signals tothe + , calcineurin 2+ channels, 2+ signals 2+ nextracellular Ca on place. ItispossiblethatagradedCaMKactivation, depending differentiation but isrequiredforanoptimaldifferentiation totake that CaMKactivation onitsown doesnotinducemyoblast according toextracellular Ca it ispossiblethatCaMKIIactivated inproliferatingmyoblasts activation doesnotaffect therateofmyoblastproliferation.Invivo, suggesting thatthesetwo enzymesareactivated bydifferent Ca CaMK andcalcineurin.Thisisinagreementwithprevious work in itselftoinduce humanmyoblastdifferentiation. activation ofbothPI3Kandp38-MAPKpathways isnotsufficient (Gonzalez etal.,2004;Li 2000;Wu etal.,2000b),concerted mentioned that,incontrastto whatoccursinmousecelllines mechanism (RauchandLoughna, 2005).Itshouldalsobe secrete IGF1,orthatPI3Kisactivated viaanIGF1-independent methods), wehypothesizethat either humanmyoblastsareableto as ourculturesarepurelycomposedofmyoblasts(seeMaterials and media. Asdifferentiation mediumdoesnotcontainany serumand PI3K isactivated intheabsenceof added insulinintheculture however, that,bothinproliferatinganddifferentiating myoblasts, Ca during amassive myofiberdegeneration orafteramuscleinjury, differentiation mediumcontainingthesameCa CaMK activity issimilarinmyoblastskept ineithergrowth or is Ca linked hyperpolarization.We found,indeed,thatp38-MAPKactivity pathway was notaffected (downregulated) bythedifferentiation- myoblast differentiation toproceed,itwas important toshow thatthis et al.,2005).However, asp38-MAPKactivity isrequiredforafull proliferating myoblastsratherthanintomyotubes(Jones major roleintriggeringdifferentiation ofquiescentsatellitecellsinto agreement witharecentwork, suggestingthatp38-MAPKplaysa during theearlystepsofdifferentiation process.Ourresultsarein in proliferatingmyoblasts,andthatthispathway isnotregulated different approaches,wefoundthatp38-MAPKisalreadyactivated implication ofthispathway inhumancells.However, usingtwo myoblasts decreasesmyoblastdifferentiation, whichconfirmsan Zetser etal.,1999).Inhibitionofp38-MAPKinhumanprimary myoblast differentiation (CuendaandCohen,1999;Wu etal.,2000b; induction ofmyoblastdifferentiation andthatthisactivation promotes Several authorshave proposed thatp38-MAPKisactivated afterthe p38-MAPK andPI3Kregulatory pathways differentiation andmuscleregeneration. seems todependonlyonextracellular Ca induced eitherinproliferationordifferentiation conditions.It signals, i.e.transienthighamplitudeCa activity. TheseresultssuggestthattheCa least two different Ca the hyperpolarizationdonotcontrolCaMKIIactivity, andthatat extracellular Ca rather inducecalcineurinactivity (McKinsey etal.,2002). Ca is active inproliferatingmyoblastsandthat itsactivity isneither differentiation issimilartothatofp38-MAPK.We foundthatPI3K generated byKir2.1channelactivation. was testedfor0.7and1.8mMCa activate CaMKII,whereassustainedlow-amplitude Ca medium containingonly15 activity measuredinmyoblastskept for1hourindifferentiation Our resultsalsoshow thatCaMKIIactivation dependson Kinetics ofactivation ofPI3K during humanmyoblast 2+ 2+ -dependent norhyperpolarizationsensitive. Itshouldbenoted, concentration couldbelocallyincreased.Itisworth recalling 2+ -independent andinsensitive tothechange inpotential 2+ concentration (influx).We foundthattheCaMK 2+ 2+ concentration, couldoptimizemyoblast signals mustexist toactivate sequentially ␮ 2+ M Ca concentration. Indeed,weexpect that 2+ ). CaMKIIactivation canthusbe 2+ RESEARCH ARTICLE is massively reduced,andthat 2+ 2+ 2+ signals thatarelinked to concentration, andits spikes preferentially 2+ concentration (this 2+ increases 3113 2+

DEVELOPMENT hyperpolarization, itsassociatedCa fusion. Inourmodel,thetriadconstitutedbyKir2.1-linked linked totheinductionofdifferentiation leadingtomyoblast to take place,andthatonlythesignalactivating calcineurinisdirectly Alessi, D.R.,Andjelkovic,M.,Caudwell,B.,Cron, P., Morrice,N.,Cohen,P. References Française contre lesMyopathies. Maladies Musculaires, theFondationMarcel Levaillant, andtheAssociation number 3100A0-105331),theFondationSuissepourlaRecherche surles supported bytheFondsNationalSuissepourlaRecherche Scientifique(grant Molkentin forproviding the9NFAT-luciferase plasmid.Thisworkwas assistance, DrA.Kaelinforproviding humanmusclebiopsies,andDrJ. We thankP. Brawand,C.PomponioandP. Teta fortheirexcellenttechnical two different Ca As CaMKisactivated beforecalcineurin,itthusappearsthatatleast activated duringproliferationwithoutaffecting theproliferationrate. process. 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