Heat Stress Modulates Both Anabolic and Catabolic Signaling Pathways Preventing

15 Pages·2016·2.37 MB·English

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ORIGINAL RESEARCH ARTICLE
JJoouurrnnaall ooff
CCeelllluullaarr
PPhhyyssiioollooggyy
Heat Stress Modulates Both
Anabolic and Catabolic Signaling
Pathways Preventing
Dexamethasone-Induced Muscle
Atrophy In Vitro
WAKAKO TSUCHIDA,1,2 MASAHIRO IWATA,1,2 TAKAYUKI AKIMOTO,3 SHINGO MATSUO,1,2
YUJI ASAI,1 AND SHIGEYUKI SUZUKI2*
1Department of Rehabilitation,Facultyof Health Sciences,Nihon FukushiUniversity,Handa, Aichi, Japan
2Program in PhysicalandOccupational Therapy,GraduateSchool of Medicine,NagoyaUniversity, Nagoya,Aichi, Japan
3Faculty ofSport Sciences, WasedaUniversity,Tokorozawa, Saitama,Japan
Itisgenerallyrecognizedthatsyntheticglucocorticoidsinduceskeletalmuscleweakness,andendogenousglucocorticoidlevelsincreasein
patientswithmuscleatrophy.Itisreportedthatheatstressattenuatesglucocorticoid-inducedmuscleatrophy;however,themechanisms
involvedareunknown.Therefore,weexaminedthemechanismsunderlyingtheeffectsofheatstressagainstglucocorticoid-induced
muscleatrophyusingC2C12myotubesinvitro,focusingonexpressionofkeymoleculesandsignalingpathwaysinvolvedinregulating
proteinsynthesisanddegradation.Thesyntheticglucocorticoiddexamethasonedecreasedmyotubediameterandproteincontent,and
heatstresspreventedthemorphologicalandbiochemicalglucocorticoideffects.HeatstressalsoattenuatedincreasesinmRNAsof
regulatedindevelopmentandDNAdamageresponses1(REDD1)andKruppel-likefactor15(KLF15).Heatstressrecoveredthe
dexamethasone-inducedinhibitionofPI3K/Aktsignaling.Thesedatasuggestthatchangesinanabolicandcatabolicsignalsareinvolvedin
heatstress-inducedprotectionagainstglucocorticoid-inducedmuscleatrophy.Theseresultshaveapotentiallybroadclinicalimpact
becauseelevatedglucocorticoidlevelsareimplicatedinawiderangeofdiseasesassociatedwithmusclewasting.
J.Cell.Physiol.232:650–664,2017.(cid:1)2016TheAuthors.JournalofCellularPhysiologypublishedbyWileyPeriodicals,Inc.
Skeletalmusclehasacrucialroleinmovement,breathing,and involvedintheanti-anaboliceffectofGCbecause
glucosehomeostasis,aswellasenergyhomeostasisand dephosphorylationofGSK3bsuppressesproteinsynthesis
thermogenesis.Variouschronicdiseasesresultinreduced (ProudandDenton,1997).GC-inducedmuscleatrophyalso
skeletalmusclemass(muscleatrophy),whichcompromises involvesincreasedmuscleproteolysissignaling,especially
patientqualityoflife.Therefore,preventingandattenuating muscleRINGfinger1(MuRF1)andAtrogin-1/muscleatrophy
skeletalmuscleatrophyisanimportantclinicalgoal. F-box(MAFbx),bothmuscle-specificE3ubiquitinligases
Glucocorticoids(GC)aresteroidhormoneswithwide- (Bodineetal.,2001;Sachecketal.,2004;Wadaetal.,2011)that
rangingregulatoryeffectsondevelopmentandmetabolism targetseveralmuscleregulatoryandmyofibrillarproteinsfor
(RevolloandCidlowski,2009)aswellaspotentanti- degradationviatheubiquitin-proteasomesystem(UPS).In
inflammatoryandimmunosuppressiveactivities,andnewGC
havebeensynthesizedfortherapeuticuse.However,GC
induceadverseeffectsincludinghyperglycemia,weightgain,
hypertension,osteoporosis,depression,decreased
immunologicalfunction,andskeletalmuscleweakness ThisisanopenaccessarticleunderthetermsoftheCreative
(RamamoorthyandCidlowski,2013).Manypathological CommonsAttribution-NonCommercial-NoDerivsLicense,which
permitsuseanddistributioninanymedium,providedtheoriginal
conditionscharacterizedbymuscleweakness,includingsepsis,
workisproperlycited,theuseisnon-commercialandno
cachexia,starvation,metabolicacidosis,andsevere
modificationsoradaptationsaremade.
insulinopenia,areassociatedwithincreasedcirculatingGC
levels(WingandGoldberg,1993;Tiaoetal.,1996;Huetal., Conflictsofinterest:Theauthorsdeclarethattheyhaveno
competinginterests.
2009;Braunetal.,2011,2013),suggestingtheymightbe
involvedinmuscleatrophy. Contractgrantsponsor:JapanSocietyforthePromotionofScience
GC-inducedmuscleatrophyresultsfromdecreasedtherate (JSPS);
ofproteinsynthesisandincreasedtherateofprotein Contractgrantnumbers:26870691,26350639.
breakdown(Tomasetal.,1979;Goldbergetal.,1980;Lofberg *Correspondenceto:ShigeyukiSuzuki,PrograminPhysicaland
etal.,2002).GCdecreasetherateofproteinsynthesisin OccupationalTherapy,GraduateSchoolofMedicine,Nagoya
skeletalmusclebyenhancingthetranscriptionof“regulatedin University,1-1-20Daikominami,Higashi-ku,Nagoya,Aichi461-
developmentandDNAdamageresponses1”(REDD1)and 8673,Japan.E-mail:[email protected]
“Kruppel-likefactor15”(KLF15)thatrepressesmammalian ManuscriptReceived:21February2016
targetofrapamycincomplex1(mTORC1)(Shimizuetal., ManuscriptAccepted:19September2016
2011).Inaddition,GCmediateanti-anabolicactionsby
AcceptedmanuscriptonlineinWileyOnlineLibrary
inhibitingphosphatidylinositol3-kinase(PI3K)/Akt-dependent (wileyonlinelibrary.com):20September2016.
mTORC1signaling(Kuoetal.,2012).Phosphorylationof DOI:10.1002/jcp.25609
glycogensynthasekinase3b(GSK3b)byAktmayalsobe
© 2016 THE AUTHORS. JOURNAL OF CELLULAR PHYSIOLOGY PUBLISHED BY WILEY PERIODICALS, INC.
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skeletalmuscle,MuRF1andAtrogin-1aretranscriptionally 0.1%(v/v).ControlcellsweretreatedwithDMSOalone.After
activatedbydephosphorylationofforkheadboxO1and3a treatments,cellswereharvestedinappropriatebuffersforvarious
(FoxO1andFoxO3a)(Kameietal.,2004;Sandrietal.,2004; analysesorwerefixedformorphologicalanalysis.
Senfetal.,2008;Waddelletal.,2008).ActivationofPI3K/Akt
signalingrescuedGC-mediatedatrophythroughthe
Heattreatment
phosphorylationandinactivationofFoxO1/FoxO3a(Sandri
etal.,2004;Stittetal.,2004;Latresetal.,2005).KLF15alsohas Heatstresswasperformedaspreviouslydescribedwith
catabolicactivitybytrans-activationsofMuRF1andAtrogin-1 modifications(Welcetal.,2012).C2C12myotubeswere
(Shimizuetal.,2011). maintainedat37°Corweretreatedinawater-jacketedhumidified
Heatstressiswidelyusedasanadjuvanttophysical incubator(MCO-18AIC,Sanyo)presettoanelevated
rehabilitationforcontrollingpainandrelievingmusclespasms temperature(41°Cor,onlywhereindicated,othertemperatures)
(Lehmannetal.,1974).Recently,thebenefitsofheatstressfor andthen,after60minexposuretothehighertemperature,were
muscleatrophyundervariousconditionsweredescribed returnedtotheoriginal37°Cincubator.Oncecellswereplacedin
(Naitoetal.,2000;Luoetal.,2001;SelsbyandDodd,2005; anincubatorat41°C,itrequiredabout42.5minforthe
Ichinoseki-Sekineetal.,2014;Morimotoetal.,2015;Tamura temperatureofmediuminthedishestoreachthedesired
etal.,2015;Yoshiharaetal.,2015).HeatstressattenuatedGC- experimentaltemperature(SupplementalFig.S1A).Controlcells
inducedmuscleatrophyinrats(Morimotoetal.,2015)and weremaintainedat37°Cfortheentireexperiment.Afterheat
preventedGC-induceddegradationofproteinsinculturedL6 treatment,mediuminthecellsreturnedto37°Cafter37.5min.
myotubes(Luoetal.,2001).However,themechanismsofheat Wepreviouslyreportedthat72-kDaheatshockprotein(Hsp72)
stress-inducedsuppressionofGC-inducedmuscleatrophy proteinexpressioninC2C12myotubeswassignificantlyincreased
remainunclear. after4–24hofheattreatment,peakingat6h(Tsuchidaetal.,
Inthisstudy,weexaminedthemechanismsinvolvedinthe 2012).Thus,inthecurrentstudy,a1-hheattreatmentperiodwas
preventiveeffectofheatstressforGC-inducedmuscleatrophy, used,beginningat7hpriortoDextreatment.
focusingonanabolicandcatabolicsignalingpathways.Weuseda
well-establishedinvitrosystemofculturedC2C12myotubesto
Myotubemorphologicalanalysis
modelskeletalmuscleatrophy.Heatstresspreventedthe
deleteriouseffectsofGCbymodifyingREDD1andKLF15 AftertreatingC2C12myotubesfor12or24hwithDex,the
expressionandrecoveringGC-affectedPI3K/Aktsignaling. myotubeswerefixedinmethanolandstainedinGiemsa(109204,
Therefore,heatstressmodulatesbothanabolicandcatabolic MerckMillipore)accordingtothemanufacturer’sinstructions.
signalingpathwaysinskeletalmuscle. Imageswerecapturedat100(cid:1)magnificationunderaphase
contrastmicroscope(AxioObserverA1,CarlZeiss,Oberkochen,
Germany)withamicroscopiccamerasystem(AxioCamHRm,
MaterialsandMethods
CarlZeiss).Averagediametersofatleast50myotubeswere
Cellculture
measuredforeachconditionatthreelocations50mmapartalong
ThemurineskeletalmusclecelllineC2C12wasobtainedfromthe thelengthofthemyotubesusingimageeditingsoftware(Adobe
EuropeanCollectionofCellCultures(Salisbury,UK),and PhotoshopCS5,AdobeSystems,CA)aspreviouslydescribed
maintainedasdescribedpreviously(Iwataetal.,2009).C2C12 (Stittetal.,2004).Measurementswereconductedinablinded
myoblastswereculturedat5(cid:1)103cells/cm2ontypeIcollagen fashiononcodedimages(control,Dex-,orheatstressþDex-
coated60-mmdishes(JapanBD,Tokyo,Japan)with4mlgrowth treatedmyotubes)ofmyotubesfromeachgroup.
mediumconsistingofhighglucose(4.5g/l)Dulbecco’sModified
Eagle’sMedium(DMEM,Sigma–Aldrich,St.Louis,MO)containing
Proteinisolation
15%(v/v)heat-inactivatedfetalbovineserum(Gibco,Carlsbad,
CA)andantibiotics(100U/mlpenicillinand100mg/ml Aftertheindicatedtreatments,C2C12myotubeswerewashed
streptomycin,Gibco)forapproximately48hinawater-jacketed twicewithice-coldphosphatebufferedsaline(PBS,Sigma–Aldrich)
humidifiedincubator(MCO-18AIC,Sanyo,Osaka,Japan) andwereharvestedwithcellscrapersinwhole-celllysatebuffer
equilibratedwith5%CO and95%air,at37°C,untilthemyoblasts composedof200mlice-coldRIPAbuffer(R0278,Sigma–Aldrich)
2
reachedabout90%confluence.Toinducespontaneous containing10%(v/v)proteaseinhibitorcocktail(P8340,
differentiationbygrowthfactorwithdrawal(YaffeandSaxel, Sigma–Aldrich)and1%(v/v)phosphataseinhibitorcocktail(524625,
1977),growthmediumwasreplacedwithdifferentiationmedium Calbiochem,Darmstadt,Germany).Celllysatesweretreatedwith
consistingoflowglucose(1g/l)DMEMcontaining2%(v/v)heat- anultrasonicdisintegrator(BioruptorUCD-200T,CosmoBio,
inactivatedhorseserum(Gibco).Inthismedium,themyoblasts Tokyo,Japan)andthencentrifugedat8,000gfor10minat4°C.The
fusedtoformelongated,multi-nucleatedmyotubes.After5days, supernatantswerecollectedascellextracts.Thetotalprotein
whenapproximately90%ofthecellshadfusedintomyotubes, contentineachcellextractwasmeasuredusingtheBCAprotein
culturesweretreatedwith10mMcytosineb-D-arabinofuranoside assaykit(23225,ThermoFisherScientific,Waltham,MA)according
hydrochloride(Ara-C;C6645,Sigma–Aldrich)for48htoremove tothemanufacturer’sinstructions.
dividingmyoblasts.Myotubesreachedawell-differentiatedstate
showingcross-striation,withafewmyotubesdisplaying
Westernblotanalysis
spontaneouscontractions.Mediumwasreplenishedevery48h
duringthe5-daydifferentiation,thenonceevery24hafterthe Eachextractwasadjustedtoaconcentrationof2mgproteinper
additionofAra-Candagain2hbeforeheattreatment.Toinduce ml with an appropriate volume of RIPA buffer for SDS–PAGE.
muscleatrophyinvitro,cellsweretreatedwiththesyntheticGC, ThesesampleswereeachaddedtoanequalvolumeofEzApply
dexamethasone(Dex;D2915,Sigma–Aldrich)indifferentiation (Atto, Tokyo, Japan) containing 100mM Tris–HCl buffer (pH
mediumfortheindicatedtimesandconcentrations.Insome 8.8), 2% SDS, 20% sucrose, 0.06% bromophenol blue and
experiments,cellsweretreatedwiththePI3Kinhibitor 100mM DTT, and heated at 95°C for 5min. For SDS–PAGE,
wortmannin(681675;MerckMillipore,Billerica,MA)2hbefore samples containing 10–30ml protein were loaded per lane and
heattreatment.Wortmanninwasmaintainedthroughoutthe separatedonprecastpolyacrylamidegradient(7.5–12%,Bio-Rad,
experiment.Wortmanninwaspreparedindimethylsulfoxide Hercules, CA) gels at 200V for 30min. Proteins were then
(DMSO)anddilutedindifferentiationmediumtotheindicated transferred from the gel to 0.2-mm polyvinylidene difluoride
concentrations.ThefinalconcentrationofDMSOneverexceeded membranes (Bio-Rad) by electro-blotting at a constant current
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of 1.3A for 7min using a rapid transfer system (Trans-Blot deviation(SD).ThedatawereanalyzedusingtheMann–Whitney
Turbo, Bio-Rad). After transfer, western blot analysis was U-testforcomparisonsbetweentwoconditions,theKruskal–
performed with a protein detection system (SNAP i.d. 2.0, WallistestfollowedbySteel’stestforcomparisonswithcontrols,
MerckMillipore).Theblotswereblockedwith0.5%(w/v)nonfat andtheSteel–Dwasstestformultiplecomparisons.Differences
dried milk or 1% (w/v) bovine serum albumin diluted in Tris- betweenconditionswereconsideredstatisticallysignificantat
bufferedsalinecontaining0.1%(v/v)Tween20(TBS-T),toblock P<0.05.
nonspecificreactions,andthenincubatedovernightat4°Cwith
the indicated primary antibodies. These antibodies were
Results
directed against Hsp72 (ADI-SPA-812, Enzo Life Sciences,
DextreatmentinducesC2C12myotubeatrophy
Farmingdale, NY), phosphorylated Akt (Thr308; #2965, Cell
Signaling Technology, Danvers, MA), Akt (#4691, Cell Signaling Weexaminedthedose-dependenteffectsofDexbyassessing
Technology),phosphorylatedp70ribosomalproteinS6kinase1 myotubediameter.Comparedwithuntreatedcontrols
(p70S6K1)(Thr389;#9206,CellSignalingTechnology),p70S6K1 (n¼55,myotubediameter18.7(cid:3)4.5mm),fullydifferentiated
(#2708, Cell Signaling Technology), phosphorylated GSK3b C2C12myotubestreatedwithDexfor24hshowedadistinct
(Ser9; #5558, Cell Signaling Technology), GSK3b (#9832, Cell atrophicphenotype(Fig.1A),withadose-dependentdecrease
Signaling Technology), phosphorylated mitogen-activated inmyotubediameter(Fig.1B).InmyotubestreatedwithDex
protein kinase kinase 1/2 (MEK1/2) (Ser217/221; #9154, Cell 0.01mM,therewerenosignificanteffects,althoughatrend
Signaling Technology), MEK1/2 (#9122, Cell Signaling towarda2%decreaseindiameter(n¼54,18.3(cid:3)4.9mm),with
Technology), phosphorylated extracellular signal-regulated asimilartrendofa10%decreasewith0.1mMDex(n¼53,
kinase 1/2 (ERK1/2) (Thr202/Tyr204; #4370, Cell Signaling 16.8(cid:3)4.2mm)wasobserved.Myotubediameterswere
Technology), ERK1/2 (#4696, Cell Signaling Technology), significantlydecreasedby18%with1mM(n¼51,
phosphorylated FoxO1 (Thr256; #9461, Cell Signaling 15.4(cid:3)3.1mm)andby23%with10mM(n¼54,14.4(cid:3)3.5mm)
Technology), FoxO1 (#2880, Cell Signaling Technology), or100mM(n¼54,14.5(cid:3)3.5mm)Dex(P<0.05)(Fig.1B).
phosphorylated FoxO3a (Ser253; ab47285, Abcam, Cambridge, Basedonthis,10mMDexwasusedinsubsequentexperiments
MA), FoxO3a (#2497, Cell Signaling Technology), total myosin toinduceatrophyinC2C12myotubes.
heavy chain (MyHC) (MF20, Developmental Studies Hybridoma Next,weexaminedthetimedependenceoftheeffectofDex
Bank, Iowa, IA), MyHC fast (MyHC-f) (M4276, Sigma–Aldrich), (10mM)treatmentonC2C12myotubediameter.Compared
and b-actin (#5125, Cell Signaling Technology). The blots were withcontrols,thediametersofmyotubestreatedwithDex
washed,incubatedwiththerespectivesecondaryantibodies,and wereunchangedat12hbutshowedasignificantdecreaseof24%
washed again. The immunostained bands were visualized using at24h(Fig.1C).Similartoitseffectondiameter,Dextreatment
the ECL Prime Western Blotting Detection System (RPN2232, significantlydecreasedthetotalproteincontentby16%
GE Healthcare Japan, Tokyo, Japan) according to the (Fig.1D),MyHC-fcontentby21%(Fig.1EandF),andtotal
manufacturer’s instructions. Illumination patterns were MyHCcontentby24%at24h,comparedwithcontrols
measured using a high sensitivity cooled CCD camera (Light (Fig.1EandG),buthadnoeffectat12h.Thus,theinvitro
CaptureII,Atto)toobtainanimageofthetargetproteinbands. responseofC2C12myotubestoDexrecapitulatedtheinvivo
Subsequent quantification of band images was performed using responseofskeletalmuscle(HicksonandDavis,1981;Maetal.,
image analysis software (CS analyzer 3.0, Atto). 2003;Clarkeetal.,2007;Yamamotoetal.,2010).Inaddition,
thesedatawereconsistentwithpreviousstudiesreportingDex
decreasedmyotubediameter(ChromiakandVandenburgh,
RNAisolationandassessmentofmRNAexpressionby 1992;Sandrietal.,2004;Stittetal.,2004;Latresetal.,2005;
real-timequantitativeRT-PCR Menconietal.,2008;Kukretietal.,2013),totalprotein
(ChromiakandVandenburgh,1992;Sachecketal.,2004;Stitt
C2C12myotubeswerewashedtwicewithPBSandtotalRNAwas
1 etal.,2004;Polgeetal.,2011;Wadaetal.,2011),and/or
isolatedusingthePureLink RNAMiniKit(LifeTechnologies, myofibrillarproteincontent(ChromiakandVandenburgh,1992;
GrandIsland,NY)accordingtothemanufacturer’sinstructions.
Clarkeetal.,2007;Verheesetal.,2011)invitro.
RNAconcentrationsweremeasuredusingaNanodropND-1000
UV-Visspectrophotometer(ThermoScientific,Wilmington,DE).
Next,totalRNAwasreversetranscribedintocDNAusingtheHigh- DextreatmentincreasesREDD1,KLF15,FoxO1,
CapacityRNA-to-cDNATMKit(LifeTechnologies)anda2,720 FoxO3a,MuRF1,andAtrogin-1mRNAexpressionin
thermocycler(LifeTechnologies),accordingtothemanufacturer’s C2C12myotubes
instructions.Quantitationofgeneexpressionwasdeterminedby
It was previously shown that REDD1, REDD2, and KLF15,
real-timequantitativereversetranscriptionPCR(qRT-PCR).qRT-
upstream negative effectors of mTORC1 signaling, induced
PCRreactionscontained2mlcDNA(50mg/ml),10mlTaqManFast
muscleatrophyviatheinhibitionofproteinsynthesis(Wang
AdvancedMasterMix(LifeTechnologies),and1mlprimers.PCR
et al.,2006;Shimizuet al.,2011;Kelleher et al.,2013;Britto
reactionanalysiswasperformedbyaStepOnePlusReal-timePCR
et al., 2014). Furthermore, the key muscle proteolysis
system(LifeTechnologies)usingtheDDCtmethod.Theprimers
factors, MuRF1 and Atrogin-1, were transcriptionally
wereasfollows:Hsp72(Mm01159846_s1),REDD1
activatedbyKLF15,FoxO1,andFoxO3ainresponsetoDex
(Mm00512504_g1),REDD2(Mm00513313_m1),KLF15
(Kamei et al., 2004; Sandri et al., 2004; Waddell et al., 2008;
(Mm00517792_m1),FoxO1(Mm00490672_m1),FoxO3a
Shimizu et al., 2011). Therefore, we assessed the mRNA
(Mm01185722_m1),Atrogin-1(Mm00499523_m1),MuRF1
expression of these regulatory factors in fully differentiated
(Mm01185221_m1),andglyceraldehyde-3-phosphate
C2C12 myotubes undergoing Dex-induced skeletal muscle
dehydrogenase(GAPDH;Mm99999915_g1)byTaqManGene
atrophy.REDD1mRNAexpressionrapidlyincreasedby3.1-
ExpressionAssays(LifeTechnologies).GAPDHwasusedasan
fold at 1h, reached a peak of 3.6-fold increase at 3h and
endogenouscontroltonormalizethesamples. gradually decreased at 6–24h of Dex (10mM) treatment
(Fig. 2A). In contrast, REDD2 mRNA expression was
unchanged throughout the entire Dex treatment period
Statisticalanalysis
(Fig.2B).KLF15mRNAexpressionincreasedby3.2-and5.2-
StatisticalanalyseswereperformedusingSPSSversion15.0(SPSS foldat1and3h,respectively,andreacheda6.3-foldpeakat
Inc.,Chicago,IL).Allvaluesarepresentedasmeans(cid:3)standard 6h of Dex treatment (Fig. 2C). FoxO1 mRNA expression
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Fig.1. DextreatmentinducesatrophicresponsesinC2C12myotubes.FullydifferentiatedC2C12myotubesweretreatedwiththesynthetic
GCDex.A:RepresentativeimagesofC2C12myotubeswereacquiredafter24htreatmentwith10mMDex.Thescalebarsrepresent100or
200mm.B:Thegraphshowsmyotubediametersafter24hDextreatmentattheindicatedconcentrations.Dataaremeans(cid:3)SD(n(cid:4)51
myotubespercondition).C:Thegraphshowsmyotubediametersinresponseto10mMDextreatmentfor12or24h.Dataaremeans(cid:3)SD
(n(cid:4)118myotubespercondition),relativetothemeanvalueofthecontrolcondition,whichwassetas1.D:Thegraphshowstotalprotein
contentinresponseto10mMDex.Dataaremeans(cid:3)SD(n¼3dishespercondition).E–G:Effectsoftreatmentwith10mMDexonMyHC-f
andtotalMyHCcontentweremeasuredbywesternblot.b-Actinwasusedasaproteinloadingcontrol.MyHC-fandtotalMyHCcontentare
expressedasmeans(cid:3)SD(n¼4dishespercondition).aandbindicatesignificantdifferencesamongthedesignatedgroups(P<0.05),where
a>b.
was increased by 1.2-fold, although this increase was increased at 1h, but rapidly increased to 1.7-fold at 3h of
observed only at 1h of Dex treatment (Fig. 2D). FoxO3a Dextreatment,remainingstableat1.7–1.8-foldatlatertime
mRNAexpressionincreasedby1.3-foldat1handreacheda points(Fig.2G).Thus,thepeakexpressionofREDD1wasat
2.0-foldpeakat3hofDextreatment(Fig.2E).MuRF1mRNA 3h of Dex treatment and this time point was used to
expression gradually increased by 1.2- and 1.6-fold at 1 and investigate changes in the anabolic markers, REDD1, KLF15,
3h, respectively and reached a 2.0-fold peak at 6h of Dex Akt,p70S6K1,andGSK3b,duringDex-inducedatrophy.We
treatment (Fig. 2F). Atrogin-1 mRNA expression was not investigated gene expression at 3–12h, and myotube
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Fig.2. TimecourseofDexeffectsonREDD1,REDD2,KLF15,FoxO1,FoxO3a,MuRF1,andAtrogin-1mRNAexpressioninC2C12myotubes.
A–G:FullydifferentiatedC2C12myotubesweretreatedwithDex(10mM)fortheindicatedtimes.REDD1,REDD2,KLF15,FoxO1,FoxO3a,
MuRF1,andAtrogin-1mRNAexpressionsweremeasuredbyreal-timequantitativeRT-PCR(opencircles,control;filledcircles,Dextreated).
Horizontalbarsindicatemeanvalues(n¼3dishespercondition),relativetothemeanvalueofthecontrolconditionthatwassetas1.
diameter/proteinupto24h,becausethereisatemporalpeak proteinbreakdown,wasat6hofDextreatment.Therefore,
ofgeneexpressionbeforethephysiologicalresponseoccurs. we analyzed C2C12 myotubes at 6h of Dex treatment to
The peak of changes in MuRF1 and Atrogin-1, both investigate changes in the catabolic markers, KLF15, Akt,
considered rate-limiting enzymes of UPS-mediated muscle FoxO, MuRF1, and Atrogin-1, during Dex-induced atrophy.
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WenextinvestigatedhowREDD1andKLF15expression etal.,2001).TheseeffectswereattributedtoHsp72inducedby
wasregulatedduringthisprocessinmoredetail.Dexsignalsby heatstress.ToinvestigatewhetherheatstressinducedHsp72
bindingtoGCreceptor(GR),whichthentranslocatesintothe mRNAandproteinexpressionunderourexperimental
nucleustoregulatetheexpressionoftargetgenes(Kadmieland conditions,weexposedfullydifferentiatedC2C12myotubes
Cidlowski,2013).WeinvestigatedwhetherGRwasinvolvedin toheatstress.TreatingC2C12myotubesat39,40,or
theDex-mediatedupregulationofREDD1andKLF15mRNA 41°CincreasedHsp72mRNAexpressionby1.5-,3.5-,and6.0-
expression.WhenfullydifferentiatedC2C12myotubeswere fold,respectively,whenmeasuredimmediatelyafterheat
treatedwithaGR-specificantagonistRU486,Dex-induced treatment.Hsp72mRNAexpressiongraduallydecreasedover
increasesinREDD1andKLF15mRNAexpressionwere timereachingcontrollevelsat5hafterheattreatment
inhibited(SupplementalFig.S2AandB),indicatingREDD1and (SupplementalFig.S1B).Hsp72proteinexpressionwas
KLF15expressionwasmediatedbytheGRinDex-induced significantlyincreasedby2.2-and4.7-foldat39and41°C,
atrophy. respectively,at6hofheattreatment,comparedwithcontrol
myotubesmaintainedat37°C(SupplementalFig.S1CandD).
Thus,heatstress-inducedincreasesinHsp72mRNAand
HeatstresssuppressesDex-inducedmuscleatrophy
proteinexpressionweretemperature-dependent.
Inpreviousstudies,heatstressattenuatedtheDex-induced Accordingly,heatstressat41°C,whichinducedsufficientlevels
decreaseinmusclefiberdiameterofextensordigitorumlongus ofHsp72expression,wasusedforsubsequentexperimentsto
muscleinvivoinrats(Morimotoetal.,2015)andsuppressed investigatehowheatstressinfluencesDex-inducedatrophyin
Dex-inducedmuscleproteolysisinL6myotubesinvitro(Luo C2C12myotubes.FullydifferentiatedC2C12myotubes
Fig.3. HeatstresssuppressesDex-induceddecreasesinmyotubediameterandMyHC-fcontent.FullydifferentiatedC2C12myotubeswere
exposedtoheating(at41°Cfor60min)6hpriortoadding10mMDexandtreatingfor24h.Theexperimentwasconductedusingthree
conditionsinwhichC2C12myotubeswereuntreated(control),treatedwith10mMDex(Dex),orexposedtoheatingandthentreatedwith
10mMDex(HeatþDex).A:Thegraphshowsaveragemyotubediametersunderthethreeconditions.Dataaremeans(cid:3)SD(n(cid:4)69myotubes
percondition),relativetothemeanvalueofthecontrolconditionwhichwassetto1.aandbindicatesignificantdifferencesamongthe
designatedgroups(P<0.01).B:Frequencyhistogramshowingdistributionofmyotubediameters.C–D:MyHC-fcontentmeasuredby
westernblottingwithb-actinasaproteinloadingcontrol.MyHC-fcontentisexpressedasmean(cid:3)SD(n¼7dishespercondition).aandb
indicatesignificantdifferencesamongthedesignatedgroups(P<0.01),wherea>b.
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Fig.4. HeatstressincreasesHsp72proteinexpressioninC2C12myotubesduringDextreatment.AandB:FullydifferentiatedC2C12
myotubeswereexposedtoheating(at41°Cfor60min)6hpriorto10mMDextreatmentfor3,6,12,and24h,asindicated.The
experimentwasconductedusingthreeconditions,control,Dex,andHeatþDexconditions,asdescribedforFigure3.Hsp72proteinlevels
weremeasuredbywesternblottingandareexpressedasmeans(cid:3)SD(n¼4–6dishespercondition),relativetothemeanvalueofthe
controlconditionthatwassetas1.aandbindicatesignificantdifferencesamongthedesignatedgroups(P<0.05),wherea>b.
exposedtoheatstress(at41°Cfor60min)ending6hpriorto Thiseffectwasinhibitedbyheatstress(Fig.5C–E).Wealso
treatmentwithDex(10mM)for24hhada25%decreasein examined mTORC1 activity by evaluating phosphorylation
diameterwhenmaintainedatnormaltemperature,butthiswas ofp70S6K1atthemTOR-specificsiteThr389(Brownetal.,
abolishedwhensubjectedtoheatstress(Fig.3A).Compared 1995; Brunn et al., 1997). Similar to the observations with
withuntreatedcontrols,C2C12myotubestreatedwithDex Akt, Dex treatment resulted in a 29% decrease in
hadalowerpercentageoflargemyotubes(>20mm)anda phosphorylatedp70S6K1atThr389,withnochangeintotal
higherpercentageofsmallmyotubes(>10mm)(Fig.3B).Both p70S6K1 protein, and heat stress prevented this effect
Dex-inducedeffectsweresuppressedinmyotubessubjectedto (Fig. 5C, F, and G). Because Akt reduces GSK3b kinase
heatstress.Furthermore,a28%lossofMyHC-fcontentin activity through the phosphorylation of Ser9 (Manning and
C2C12myotubesinducedbyDextreatmentwasalsoabolished Cantley,2007)andincreasesmRNAtranslationandprotein
byheatstress(Fig.3CandD).Therefore,heatstresssuppressed synthesis (Proud and Denton, 1997), we analyzed
Dex-induceddecreasesinbothmyotubediameterand phosphorylated GSK3b levels in Dex-treated C2C12
myofibrillarproteincontent.Inaddition,C2C12myotubes myotubes, with/without heat stress. We observed a 34%
exposedtoheatstress(at41°Cfor60min)6hpriortoDex decrease in phosphorylated GSK3b on Ser9 after Dex
treatment showed Hsp72 protein expression was increased treatment,withnochangein totalGSK3b protein(Fig. 5C,
3.0-,2.9-,3.4-,and2.1-foldat3,6,12,and24h,respectively,of H,andI).Thiseffectwasinhibitedbyheatstress(Fig.5C,H,
Dextreatment(Fig.4AandB)indicatingthatheatingat41°Cfor and I).
60mininducedasustainedincreaseinHsp72proteinexpression
throughouttheperiodofDextreatmentinC2C12myotubes.
DextreatmentandheatstressdonotalterMEK1/2and
ERK1/2signalsinC2C12myotubes
HeatstresssuppressesDex-inducedincreasesinREDD1
The MEK/ERK signaling pathway enhanced protein synthesis
andKLF15mRNAexpressionanddecreasesin
phosphorylatedAkt,p70S6K1,andGSK3bproteinlevels by regulating ribosomal RNA gene expression (Stefanovsky
et al., 2006) and might contribute to the maintenance of
inC2C12myotubes
skeletal muscle mass by modulating Akt signaling (Shi et al.,
To elucidate the effects of heat stress on Dex-mediated 2009).Therefore,weexaminedthephosphorylationstatesof
inhibition of anabolic pathways, we measured mRNA MEK1/2 at Ser217/221 sites and the downstream effector
expression of REDD1 and KLF15 (Shimizu et al., 2011; ERK1/2 at Thr202/Tyr204 sites in our atrophy model.
Britto et al., 2014), in C2C12 myotubes treated with Dex Treatment of C2C12 myotubes with Dex for 3h did not
(10mM) for 3h. Dex-treated C2C12 myotubes showed a affect the levels of phosphorylated and total MEK1/2 and
3.6-foldincreaseinREDD1mRNAanda4.8-foldincreasein ERK1/2(Fig.6A–E)consistentwithapreviousstudyshowing
KLF15 mRNA expression, both of which were blunted by Dex had no effect on the signaling pathway (Gwag et al.,
heat stress (Fig. 5A and B). We next examined Akt 2013). Similarly, heat stress did not alter the levels of
phosphorylated on Thr308, because REDD1-mediated phosphorylatedandtotalMEK1/2andERK1/2inDex-treated
inhibition of mTORC1 involves Akt dephosphorylation at C2C12 myotubes (Fig. 6A–E), suggesting the MEK/ERK
this site (Britto et al., 2014). Dex treatment resulted in a signaling pathway is not involved in the Dex-induced
30% decrease in phosphorylated Akt on Thr308 in inhibition of protein synthesis and heat stress-induced anti-
myotubes with no change in total Akt protein (Fig. 5C–E). atrophic effects under the present experimental conditions.
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Fig.5. HeatstressnormalizesDex-inducedincreasesinREDD1andKLF15mRNAexpressionanddecreasesinphosphorylatedAkt,p70S6K1,and
GSK3b.FullydifferentiatedC2C12myotubeswereexposedtoheating(at41°Cfor60min)6hpriorto10mMDextreatmentfor3h.Theexperiment
wasconductedusingthreeconditions,control,Dex,andHeatþDex,asdescribedforFigure3.AandB:REDD1andKLF15mRNAexpressionswere
measuredbyreal-timequantitativeRT-PCR.Horizontalbarsindicatemeanvalues(n¼6dishespercondition),relativetothemeanvalueofthe
controlconditionthatwassetas1.C–I:LevelsofphosphorylatedandtotalAkt,p70S6K1,andGSK3bweremeasuredbywesternblottingandare
expressedasmeans(cid:3)SD(n¼6dishespercondition).a–cindicatesignificantdifferencesamongthedesignatedgroups(P<0.05),wherea>b>c.
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Fig.6. DextreatmentandheatstressdonotalterthelevelsofphosphorylatedandtotalMEK1/2andERK1/2.FullydifferentiatedC2C12
myotubeswereexposedtoheating(at41°Cfor60min)6hpriorto10mMDextreatmentfor3h.Theexperimentwasconductedusingthree
conditions,control,Dex,andHeatþDex,asdescribedforFigure3.A–E:LevelsofphosphorylatedandtotalMEK1/2andERK1/2were
measuredbywesternblottingandareexpressedasmeans(cid:3)SD(n¼6dishespercondition).
HeatstressnormalizesdecreasesinphosphorylatedAkt, phosphorylated Akt, p70S6K1, and GSK3b with no change
p70S6K1,andGSK3bbyDexthroughPI3K-dependent in each total protein level (Fig. 7A and C–H). Therefore,
mechanisms heat stress may normalize the Dex-induced inhibition of
anabolic pathways through PI3K-dependent mechanisms.
Recent studies have shown that Dex exerted anti-anabolic Wealsoconfirmedthatwortmannindidnotinhibittheheat
actions by inhibiting PI3K/Akt-dependent mTORC1
stress-induced increase in Hsp72 protein expression in
signaling (Kuo et al., 2012). To determine whether heat
Dex-treated C2C12 (Fig. 7A and B).
stress suppressed the Dex-induced inhibition of PI3K/Akt
signaling, we determined basal phosphorylation status of
Akt (Thr308), p70S6K1 (Thr389), and GSK3b (Ser9) in
HeatstresspartiallyattenuatesDex-induceddecreasesin
non-Dex treated myotubes with/without a PI3K inhibitor,
myotubediameterthroughPI3K-dependentmechanisms
wortmannin. Lower concentrations of wortmannin
(10–100nM) had no effect, whereas higher concentrations Becausewortmanninblockedtheinhibitoryeffectofheat
(0.5–10mM) decreased phosphorylated Akt, p70S6K1, and stressonDex-induceddecreasesofphosphorylatedAkt,
GSK3b (Supplemental Fig. S3A–G). Therefore, 100nM p70S6K1,andGSK3b,weexaminedwhetherPI3Kwas
wortmannin was used for all further experiments to requiredfortheanti-atrophiceffectsofheatstress.Fully
investigate whether PI3K is required for the heat stress differentiatedC2C12myotubesweretreatedwith100nM
suppression of Dex-induced decreases in phosphorylated wortmannin2hbeforeheattreatment,andwereexposedto
Akt, p70S6K1, and GSK3b. Fully differentiated C2C12 heatstress(at41°Cfor60min)ending6hpriortotreatment
myotubesweretreatedwith100nMwortmannin2hbefore withDexfor24h.Wortmannindidnotaffectthediameterof
heat treatment, and then exposed to heat stress (at C2C12myotubeswith/withoutDextreatment(Fig.8AandB).
41°C for 60min) ending 6h prior to treatment with Dex However,wortmanninattenuated,butdidnotabolish,the
for 3h. Wortmannin completely abolished the inhibitory inhibitoryeffectofheatstressonDex-induceddecreasesin
effect of heat stress on Dex-induced decreases in myotubediameter(Fig.8AandB).Therefore,heatstressmay
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Fig.7. HeatstressnormalizesDex-induceddecreasesinphosphorylatedAkt,p70S6K1,andGSK3bthroughPI3K-dependentmechanisms.Fully
differentiatedC2C12myotubesweretreatedwithwortmannin(100nM)attheindicatedconcentrations2hbeforeheattreatment,andwere
exposedtoheating(at41°Cfor60min)6hpriorto10mMDextreatmentfor3h.Wortmanninwasmaintainedthroughouttheexperiment.The
experimentwasconductedusingfourconditions,control,Dex,HeatþDex,andwortmanninþHeatþDex(WþHeatþDex).A–H:Hsp72
proteinexpressionandlevelsofphosphorylatedandtotalAkt,p70S6K1,andGSK3bweremeasuredbywesternblottingandareexpressedas
means(cid:3)SD(n¼8dishespercondition),relativetothemeanvalueofthecontrolconditionthatwassetas1.aandbindicatesignificantdifferences
amongthedesignatedgroups(P<0.05),wherea>b.
partiallyattenuateDex-induceddecreasesinmyotube etal.,2011).Overexpressionofconstitutivelyactiveformsof
diameterthroughPI3K-dependentmechanisms. KLF15increasedFoxO1andFoxO3aexpressions(Shimizu
etal.,2011).OurresultsshowedthatKLF15wasincreased6.2-
foldinC2C12myotubesat6hDextreatment(Fig.9A),
HeatstresssuppressesDex-inducedincreasesinKLF15and correspondingwithincreasedtotalFoxO1(1.2-fold,
MuRF1mRNAexpression,decreasesinphosphorylated P¼0.0644)andFoxO3a(1.5-fold,P<0.05)proteinlevels
Akt,FoxO1,andFoxO3aproteinlevels,andincreasesin (Fig.9B,F,andH).Theseincreaseswerepreventedbyheat
totalFoxO1andFoxO3ainC2C12myotubes stress.
DephosphorylationandtheactivationofFoxO1and
DexinducesKLF15,whichinteractswiththepromoterregions FoxO3abydephosphorylatingandinactivatingAkt,resultedin
ofMuRF1andAtrogin-1toinducetheirexpression(Shimizu MuRF1andAtrogin-1mRNAexpression,inducingatrophy
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