*Author ([email protected]) for correspondence Arizona University,Biological Sciences, Northern Flagstaff, AZ86011-5640,USA. NAWI Graz,Karl-Franzens-University Graz,A-8010Austria. AB, Canada,T2N1N4. © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,3629-3636doi:10.1242/jeb.105361 Received 13March 2014;Accepted11 August 2014 1 exceeds thepredictionsofforce–lengthrelationship(Abbott and isometric steady-stateforceproducedatagivensarcomerelength 1966). However, followingeccentric,orlengthening, contractionsthe is generallyadheredtoduringisometriccontractions(Gordon et al., 1966). Thisconstitutestheforce–sarcomerelengthrelationship,which and thinfilaments(HuxleyNiedergerke, 1954;Gordonetal., form, whichcorrespondstotheamountofoverlapbetween thick muscle shouldbeproportionaltothenumberofcross-bridgesthat can of activation,theisometricsteady-stateforcegeneratedbyanactive sarcomere (HuxleyandSimmons,1971).Therefore,foragiven level bridges cyclicallyformbetweenthethickandthinfilamentsin the model. Thismodelpredictsthatactiveforceisproducedwhencross- force productioninmuscleistheslidingfilament-basedcross-bridge The current,andgenerallyaccepted,theorywhichexplainsactive bridge theory, Eccentriccontractions KEY WORDS:, Skeletalmuscle,Forceenhancement,Cross- titin–thin filamentinteractioninactivelystretchedsarcomeres. in themousemodel,futurestudieswillaimtoelucidateproposed titin bindingtothethinfilament.Withthisenhancedforceconfirmed that theremainingunexplained85%ofthisextraforceresultsfrom the PEVKsegmentandselectedIgdomainsegments.We suggest stiffening oftheproteinuponcalciumbindingtoE-richregion be attributedtodirectcalciumeffects ontheprotein,presumablya results alsodemonstratedthat15%oftheenhancedstatetitincan during activestretchisaninherentpropertyofskeletalmuscle.Our distinct animalmodels,suggestingthatmodulationoftitin-basedforce Enhanced titin-basedforcehasnowbeendemonstratedintwo times thatofpassiveforceduringactivestretchisolatedmyofibrils. calcium tothisforce.Titin-based forcewasincreasedbyuptofour force isobservedinamousemodelandquantifythecontributionof active stretch.Specifically, weconfirmthatthisenhancedstateof study wastofurtherinvestigatetheenhancedstateoftitinduring be explainedbytheslidingfilamenttheory. Theaimofthepresent to lengthswhichexceededfilamentoverlap.Thisobservationcannot enhanced titin-basedforceinmyofibrilsthatwereactivelystretched filament overlap.Previousstudiesfromourlaboratorydemonstrated produced duringmuscleactivationisproportionaltotheamountof Within theconstraintsofthistheory, isometric,steady-stateforce as themeansbywhichmusclesgenerateforceduringactivation. The slidingfilamenttheoryofmusclecontractioniswidelyaccepted Krysta Powers Titin forceisenhancedinactivelystretchedskeletalmuscle RESEARCH ARTICLE Walter Herzog INTRODUCTION ABSTRACT Human Performance Laboratory, Kinesiology, Calgary, Faculty of University of 1, 1 2 * , GudrunSchappacher-Tilp Department for Mathematics andScientificComputing, Department 3 2 Department of Department , AzimJinha 1 among themany processesbywhichthestiffness ofI-bandtitincan al., 2014)anddisulfidebonding (Alegre-Cebolladaetal.,2014)are Krüger etal.,2009),smallheat shockproteininfiltration(Kötteret (Granzier andLabeit,2004).Phosphorylation (Hidalgoetal.,2009; adjusted inresponsetomechanical demandsonthesarcomere Goulding, 1996).Thespringproperties ofI-bandtitincanberapidly particularly thePEVKsegment, passivelyresiststension(Gauteland band oftitintotheA-band.Duringstretch,elongationI-band, segment (Wang etal., 1991). ThedistalIgdomainconnectstheI- PEVK (Pro-Glu-Val-Lys) spring,whicharelinkedby theN2A 1).TheI-bandregion oftitiniscomposedaproximalIgand (Fig. separated intoastructuralA-bandandanextensibleI-bandregion signalling events(GranzierandLabeit,2004).Titin isfunctionally 1987), sarcomerestability(Herzogetal.,2012c),andvarious al., 1999),centeringofthethickfilament(HorowitsandPodolsky, production (Wang etal., 1991),myofibrillarassembly(Gregorioet to manydifferent aspectsof muscle function,includingpassiveforce level (Wang etal.,1991). Titin isadynamicproteinthatcontributes element inthepropertyofforceenhancement. Labeit etal.,2003;Joumaa2008),alludingtoapassive in musclesfollowingactivestretch(HerzogandLeonard,2002; al., 1982).Additionalstudieshaveobservedenhancedpassiveforce on thedescendinglimbofforce–lengthrelationship(Edmanet magnitude ofstretchandisgreaterasthemusclestretchedfurther sarcomere. Residualforceenhancementincreaseswithincreased properties thatalludetoapassiveforcecontributioninthe 2012; Nishikawaetal.,2012).Residualforceenhancementexhibits 2002; Herzogetal.,2012a;2012c;Monroy (Edman etal.,1982;Forcinito1998;HerzogandLeonard, result ofapassiveelementbecoming‘engaged’ duringactivestretch has beensuggestedthatresidualforceenhancementmaybethe is providedbytheinteractionsbetweenthickandthinfilaments,it attempt toexplainhowsarcomerescanproduceforcebeyondwhat produced increasesexponentially(GranzierandLabeit,2004).Inan length relationship,thecontributionoftitintototalforce increased stretchonthedescendinglimbofforce–sarcomere when sarcomeresarestretchedbeyondacertainrestinglength.With contraction. Titin isaspring-likeproteinthat carriespassiveforce thin filamentsareunderstoodtogenerateactiveforceduringmuscle of actinandregulatoryproteins.Interactionsbetweenthethick primarily oftheproteinmyosin,andthinfilamentiscomposed (), andthetitinfilaments.Thethickfilamentiscomposed contribute toforceproductioninmuscle:thethick(),thin 2012). Therearethreemainfilamentsatthesarcomerelevelthat 1982; HerzogandLeonard,2002;Leonardetal.,2010;Rassier, to thesarcomerelevel(AbbottandAubert,1952;Edmanetal., sliding filament-basedcross-bridgetheory. termed residualforceenhancement,providesadirectchallengetothe Herzog etal.,2006;LeonardandHerzog,2010).Thisproperty, Aubert, 1952;Edmanetal.,1978;1982;Morgan, 1994; , Tim Leonard Titin istheprimarycontributortopassiveforceatmyofibrillar Residual forceenhancementhasbeenobserved 1 , KiisaNishikawa 3 and in vivo and down 3629

The Journal of Experimental affects titin functionally. However, wholemuscle experiments and wild-typemice,itremains unclearhowthe 2005). Despitethevisibledifferences inphenotypebetween musculoskeletal systemofmice (Garveyetal.,2002;Huebsch source oftitin-basedforce. the PEVKdomainismain segments oftitin.TheproximalIg,N2A,PEVKanddistalIgdomainstitincanextendduringsarcomerestretch.Extension Z-discandcontainstheextensiblespring band titinrunsthelengthofthickfilamentandismainlystructural.I-bandattachedtothinat 3630 protein. Nevertheless,the deletion consideringthe~33,000 aminoacidsencompassingthetitin skeletal muscleinmice(Garvey etal.,2002).Thisisamodest N2A andproximalPEVKresultsinprogressivedegenerationof al., 2002).Thedeletionofapredicted83aminoacidsfrom distal a deletionofaminoacidsfromtheN2A regionoftitin(Garveyet model isageneticmutationinthemousegenomewhichresults in during activestretch.Themusculardystrophywithmyositis( insight intotheregionoftitinthatmodulatestitin-basedstiffness using musclesfrommicewithamutatedtitinproteinmayprovide by whichthismayoccurremainstobediscovered. Leonard andHerzog,2010;Nishikawaetal.,2012),amechanism previously suggested(Edmanetal.,1982;Herzog2006; and Herzog,2010).Whiletitinbindingtothethinfilamenthas been thin filamentduringstrongcross-bridgebindingtoactin(Leonard modulation oftitin-basedforcemayoccurwhentitinbindstothe cross-bridge-based activeforceswasobserved.Theyspeculatedthat was observed,asnoeffect ofcalciumactivationintheabsence mechanism otherthancalciumtoproducetheincreaseinforcethat the increaseintitin-basedstiffness mustemployanadditional beyond filamentoverlap.Thefindingsofthatstudyindicated investigated modulationoftitin-basedforceduringactivestretch et al.,2012). Herzog, 2010;Herzogetal.,2012a;2012c;Nishikawa stretch (Edmanetal.,1982;Herzog2006;Leonardand support thespeculationthattitinmaybeengagedduringactive observed (KellermayerandGranzier, 1996).Theseobservations interactions betweentitinandthethinfilamenthavealsobeen intrinsic changestothestiffness oftitin,calcium-dependent Ig domains(Labeitetal.,2003;DuVall etal.,2013).Inadditionto of titinincreaseswhencalciumionsbindtothePEVKregionand Specifically, ithasbeenshownthatthestiffness oftheI-bandregion changed inthepresenceofcalcium(Tatsumi etal.,2001). Herzog, 2010).TheconfigurationoftheextensibleI-bandtitinis et al.,2003;Bianco2007;MonroyLeonardand activation (Tatsumi etal.,2001;CampbellandMoss,2002;Labeit demonstrate tuningofthespringpropertiestitinduringcalcium of titinisnowbeingchallengedwithnumerousstudieswhichalso traditionally beenlimitedtopassivestretch,thisconventionalview While thecontributionoftitin-basedstiffness tomuscleforcehas be modulatedtoprotecttheproteinfromdamageduringstretch. Fig. RESEARCH ARTICLE Recent experiments(Monroyetal.,2012;Nishikawa2012) Recently, LeonardandHerzog(LeonardHerzog,2010)

.Schematicrepresentationofasarcomereincludingthick,thinandtitinfilaments. 1. Z-disc mdm deletion hasprofoundeffects onthe Distal Ig Proximal Ig N2A PEVK mdm mutation mdm) mdm thick filament Sarcomere A-band

M-line A-band titin within theregion offilamentoverlap(<4.0 Therefore calcium-activatedmyofibrils producedmoreforceboth measured sarcomerelengths (2.5–6.0 monoxime, BDM)andtroponin C(TnC)-depletedmyofibrilsatall compared withpassive,cross-bridge inhibited(2,3-butanedione 4.07 μm, respectively. stretched toanaveragesarcomerelengthof6.0 shortest sarcomeresmeasuredinactiveandpassivemyofibrils the endofstretch(Fig. present, allmeasuredsarcomereswerebeyondfilamentoverlap at sarcomeres. Althoughsarcomerelengthnon-uniformities were passive experimentstoensurethatfilamentoverlapwaslost in all measured attheendofstretchinasubsetthreeactiveand three of calciumontitinstiffness. increase instiffness wouldnotbeexplainedentirelybytheeffects increased inactivelystretchedmouseskeletalmuscleandthatthe of titin.We hypothesizedthattitin-basedstiffness wouldbe to quantifythecontributionofcalciumeffects totheenhancedforce et al.,2008;DuVall etal.,2013).Thusthisstudyspecificallyaimed shown toincreasethestiffness oftitin(Labeitetal.,2003;Joumaa high variationoftheirdata.Thisissurprisingascalciumhasbeen of calciumontheincreaseinstiffness oftitin,possiblyduetothe Herzog (LeonardandHerzog,2010)wereunabletoresolveaneffect (Granzier, 2010).Inaddition,theexperimentsbyLeonardand confirmed inanotheranimalmodel,aswaspreviouslysuggested rabbit psoasmyofibrils(LeonardandHerzog,2010)needstobe active stretchhasonlybeendirectlyobservedinexperimentsusing enhancement duringactivestretch.Enhancedtitin-basedforcewith titin stiffness tofurtherelucidatethemechanismoftitin-basedforce normal, mouseandtoquantifytheeffects ofcalciumactivationon of titin-basedforceduringactivestretchoccursinthewild-type,or be carefullydetermined. bridge-based activeforcesonthestiffness oftitinandforceneedto and thecontributionofcalciumactivationinabsencecross- actively stretchedmusclesneedtobeconfirmedinthemousemodel, experiments, thepropertiesoftitin-basedforceenhancementin to investigatethispossibility. However, priortoembarkingonsuch in demonstrate thatmodulationoftitin-basedstiffness doesnotoccur passive experiments(Fig. The steady-stateforcefollowingstretchwasgreaterinactive than force occurswithinthedeletedregionof thereby suggestingthepossibilitythatmodulationoftitin-based RESULTS Calcium-activated myofibrilsproduced moreforceduringstretch The purposeofthisstudywastodeterminewhethermodulation mdm soleus duringcalciumactivation(Nishikawaetal.,2012), The thickandthinfilamentsarethemaincontractileproteins.A- The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105361 Thin filament

2B) (averagesarcomerelength6.0 I-band titin

2A). Individualsarcomerelengthswere μm) (P

mdm Z-disc μm) andbeyond the titin. We wouldlike μm were4.15and <0.01) (Fig. μm). The 3).

The Journal of Experimental Biology RESEARCH ARTICLE and BDM-treated experimentalobservations. without N2A thinfilamentbindingcloselyresembledTnC-depleted Predicted force–elongationcurves ofactivelystretchedsarcomeres filament binding)andpassively stretchedmyofibrils(~12%). increase inpredictedforcebetween actively(withoutN2A thin were deficientintitin-basedforce enhancement.Therewasasmall for activelystretchedsarcomeres withoutN2A thinfilamentbinding filament bindingexceededpassiveforcepredictions.Forcespredicted Forces predictedinactivelystretchedsarcomereswithN2A thin region andthedistalIgdomainsasfreespringelements(Fig. region oftitinbindstothethinfilament,leavingonlyPEVK myofibrils didnotdiffer inforce atanysarcomerelength. normal (non-inhibited)myofibrils.BDM-treatedandTnC-depleted myofibrils averaged~15%ofthetotalincreaseobservedin the overlap, theforceincreaseinBDM-treatedandTnC-depleted ( stretched myofibrilsacrossallsarcomerelengthsexceeding3.5 ( less forcethancalcium-activatedmyofibrilsatallsarcomerelengths (TnC) orbyinhibitingstrongcross-bridgebinding(BDM). production wasprevented,eitherbydepletionofaregulatoryprotein myofibrils thatwereeithernotactivated,oractivatedbutforce region ofthick–thinfilamentoverlap(~4.0–6.0 during theimplementedstretch. measured sarcomeresarestretchedbeyondfilamentoverlap(greyarea) state. Individualsarcomerelengths(B)arenon-uniform;however, all (blue) forthedurationofstretchandfollowingforcerelaxationtoasteady myofibrils (red)remainshigherthantheforceofnon-activated stretch aredenotedbytheverticaldashedlines.Theforce(A)ofactivated and activated(red)myofibrils.Theinitiationofactivationduration and (B)individualsarcomerelengthsfromexemplarynon-activated(blue) Fig. P P Simulations usingathree-filamentmodelassumedthattheN2A BDM-treated andTnC-depletedmyofibrilsproducedsignificantly <0.05) (Fig. <0.01). However, theyproducedmoreforce thanthepassively

.Forceandsarcomerelengthtraces. 2. Sarcomere length (μm) Stress (nN μm–2) 200 400 1 3 5 7 0 2 040 20 0 –20 2 040 20 0 –20 B A 3). Forsarcomerelengthsbeyondmyofilament Activate Time (s) Time Stretch (A) ForcetracesinnN μm) inmousepsoas μm

− Beyond overlap 2 μm

4). passive myofibrilsataveragesarcomerelengthsof3.5–6.0 chemical depletionoftroponin-C(TnC-X,triangles),andinalowCa cross-bridge inhibitor(BDM,squares),inactivationsolutionfollowing activation solution(active,diamonds),inwithBDM,a Fig. active comparedwithpassivestretchinmousepsoasmyofibrils. present studydemonstratethattitin-basedforceisalsogreaterduring modulation (LeonardandHerzog,2010).Theresultsfromthe not beexplainedwithanyknownmechanismofsarcomereforce stretching. Theincreaseintitin-basedforcefromthesestudiescould titin-based forceismuchgreaterinactivecomparedwithpassive psoas myofibrilsshowedthatatlengthsbeyondfilamentoverlap, mouse skeletalmusclemyofibrils.Previousexperimentsinrabbit force isincreasedduringactivecomparedwithpassivestretchof The firstaimofthisstudywastodeterminewhethertitin-based ( sarcomere lengthsbothwithinandbeyondtheregionoffilamentoverlap significantly moreforcethanBDM,TnC-Xandpassivemyofibrilsatall relaxing solution(passive,circles).Activatedmyofibrilsgenerated Fig. be attributedtocalcium stiffening ofthePEVK andIgdomainsoftitin. increase inpredicted forcefrompassivelystretchedsarcomeres, whichcan in theabsenceofN2A thinfilamentbindingisnearlydiminished,withasmall sarcomeres (opencircles).Predicted forceofactivelystretchedsarcomeres filament binding(greydiamonds)and predictedforcesofpassivelystretched predicted titin-basedforcesofactively stretchedsarcomereswithoutN2A thin sarcomeres withN2A thinfilamentbinding(blackdiamonds)exceeds data. (nN μm conditions. DISCUSSION designated bythedashedline.MyofibrilswerestretchedinaCa average finalsarcomerelengthof6.0 were stretchedfromanaverageinitialsarcomerelengthof2.5 P <0.01). BDMandTnC-Xmyofibrilsgeneratedsignificantlymoreforcethan

.Predictedforce 4. .A comparisonofmyofibrilforce 3. The predictedtitin-basedforce(grey area)ofactivelystretchedskeletal −

μ –2 2 Predicted stress (nN m ) ) –2

μ

± Stress (nN m ) 185 370 185 370

s.e.m. asafunctionofaveragesarcomerelength( Force wasnormalizedtomyofibrilcross-sectionalarea 0 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105361 0 23456 23456 Passive binding N2A without Active binding Active with N2A Passive TnC-X BDM Active – sarcomere lengthrelationshipsfromsimulated Average sarcomere sarcomere Average ( length Average sarcomere sarcomere Average ( length μm. Thelossoffilamentoverlapis – length curvesfromexperimental μm) μm) μm (P μ μ m). Myofibrils m toan 2+ <0.01). -rich 2+ 3631

The Journal of Experimental Biology 3632 ~15% ofthetotal increaseobservedinactively (butnon-force BDM-treated andTnC-depleted myofibrilsincreasedtitinforceby beyond myofilamentoverlap(4.0–6.0 strong cross-bridgebinding(Tesi etal.,2002).Atsarcomere lengths from thenucleotidepocketof the cross-bridge,therebyinhibiting in theweaklyboundstate,but BDMpreventsphosphaterelease can becalciumactivated,andallow forcross-bridgebindingtoactin cross-bridges (Mossetal.,1985).Similarly, BDM-treatedmyofibrils the bindingsiteofcross-bridgeonactinremainsinaccessible to calcium bindingtotheregulatoryTnCproteinisnotpossible, thus myofibrils canbecalciumactivatedbutproducenoactiveforce as based stiffness inactively stretchedmyofibrils.TnC-depleted identify themechanismsunderlyingobservedincreasein titin- using BDMandTnC-depletionwereconductedtopotentially contribution ofcalciumtotheenhancedstatetitin.Experiments stretch inthemouse,subsequentexperimentssoughttoquantify the confirmation thattitin-basedforcewasenhancedduringactive force observedduringactivestretchofmyofibrils.Therefore with titin-based stiffness wasalikely contributortotheenhancedtitin et al.,2003;DuVall etal.,2013).Thiscalcium-initiatedincreasein sarcomere lengthnon-uniformitymustbeatwork. A completelydifferent mechanismthanmyofilamentoverlapor overlap,itcouldnotexplaintheresultsobservedhere. whether asingleorhalf-sarcomeremaintainssomeremnant is limitedbytheweakesthalf-sarcomere.Thusindependentof same forceasallother(half-)sarcomeres,andthistransmission that eachhalf-sarcomereandsarcomeremusttransmitexactlythe arranged sarcomeres.Fromamechanicalpointofview, thatmeans for thisassertionisthatamyofibril,bydefinition,consistsofserially was optimal(activestretch,2.5 with theactiveforcesobtainedwhenaveragesarcomerelength it definitelycouldnotexplainthefourtimeshigherforcescompared per sarcomere)comparedwiththepassivelystretchedmyofibrils,and observed inactivelystretchedmyofibrilsatthefinallength(6.0 this non-uniformitycouldnotexplainthefourtimeshigherforces half-sarcomeres stillhadoverlapbetweenactinandmyosinfilaments, force oncethefinalstretchlengthwasreached.Ifsomesarcomeresor half-sarcomeres couldnotproduceanyactive,cross-bridge-based stretch tests.Thereforewefeelconfidentthatmostsarcomeresand actin–myosin filamentoverlap(4.0 measured inselectedexperimentswerestretchedbeyond in length.Despitethisnon-uniformity, allindividualsarcomeres and Herzog,2011; PanchangamandHerzog,2012)arenon-uniform 2008), fibres(PageandHuxley, 1963)andmyofibrils(Panchangam contributor tomyofibrilforce(Herzogetal.,2012b). cross-bridges cannotformandtitinisvirtuallytheexclusive overlap (>4.0 μm)(LeonardandHerzog,2010).Attheselengths, calcium-activated myofibrilsthatarestretchedbeyondfilament cannot beusedtoexplaintheincreaseofforceproductionin contractile filamentsactinandmyosin.However, cross-bridgeforces active forcesproducedbycross-bridgeinteractionsbetweenthe produce moreforcethannon-activatedmyofibrilsbecauseofthe within filamentoverlap(<4.0 sarcomere lengths.Onewouldexpectthatatlengths force observedinpassivelystretchedmyofibrilsmatchedatall sarcomere bywhichactivestretchislimited. filament overlapandprovidesaprotectivemechanismwithinthe force-generating capabilityduringactivestretchtolengthsbeyond This modulationoftitinforceallowsthesarcomeretomaintainits RESEARCH ARTICLE In thepresenceofcalcium,titin-basedstiffness increases(Labeit It iswellknownthatsarcomeresinmuscles(Llewellynetal., In activelystretchedmousepsoasmyofibrils,forcesexceededthe μm persarcomere;Fig. μm), calcium-activatedmyofibrils μm) inouractiveandpassive μm), calciumactivationin

3). Thereason μm experiments in rabbit longissimusdorsimuscle, whichdemonstrate support fortitin–thinfilament interactionsisprovidedby based forcecouldbemodulated duringactivation.Experimental energetically efficient and reversiblemechanismbywhichtitin- This hypothesisisparticularly attractiveasitprovidesan available springlengthoftitin inanactivatedsarcomere(Fig. are exposed,allowingtitin–thin filamentbindingtodecreasethe of cross-bridgecycling,one(ormany)site(s)onthethinfilament we nowspeculatethatwiththeinfluxofcalciumandinitiation myofibrils (presumedmovementofregulatoryproteins).Therefore myofibrils (nomovementofregulatoryproteins)andBDM-treated because therewasnodifference inforcebetweenTnC-depleted is notsufficient toinitiatetitin-based forceenhancement.Thisis from thepresentstudysuggestthatmovementofregulatoryproteins Herzog etal.,2012a;2012c).However, theresults proteins withtheinfluxofcalcium(LeonardandHerzog,2010; filament maybeexposedduringthemovementofregulatory further investigation. precise locationandcharacteristicsofthismechanismwillrequire filament interactionscouldincreasethestiffness oftitinandthe filament. We acknowledgethevastpossibilitiesbywhichtitin–thin concerning theinteractionbetweenskeletalmuscletitinandthin (Kellermayer andGranzier, 1996);however, muchlessisknown filament–titin interactionsarewelldocumentedincardiacmuscle when cross-bridgesbindstronglytothethinfilament.Thin enhancement isbyassumingthattitinbindstothethinfilament mechanisms. However, thesimplestwaytoexplaintitin-basedforce Shortening ofthetitinspringcouldoccurbyanynumber length oftitinmustdecreaseinactivelystretchedsarcomeres. conceptually alignswithourassumptionthattheavailablespring (Nishikawa etal.,2012).Whilelogisticallydiscreet,thishypothesis thin filaments,windingtitinuponthefilamentduringactivation enhanced whencyclingcross-bridgesbothtranslateandrotatethe findings. Itwasrecentlyhypothesizedthattitin-basedforcemaybe must becomeshorterinactivelystretchedsarcomerestoexplainour up to400%.Therefore,weareoftheimpressionthattitinspring unaware ofanymechanismbywhichtitinstiffness isincreasedby stiffness canberapidlyandreversiblyadjusted,wearecurrently work. Ofthevariousknownmechanismsbywhichtitin-based which titin-basedforceisenhancedduringactivestretchmustbeat in titinstiffness duringactivestretch,anadditionalmechanismby observed inintact,non-inhibitedactivelystretchedmyofibrils. sufficient toallowforthefulltitin-basedforceenhancement configurational changesintheregulatoryproteinsdonotseem myofibrils. Thusweakcross-bridgebindingandsome produce titinforcebeyondthatobservedintheTnC-depleted wouldbeexpected,buteventheseeventsdonot configurational changesintheregulatoryproteinstroponinand weak cross-bridgebindingisthoughttooccur, andsmall enhancement issmall,asshownabove.InBDM-treatedmyofibrils, thought tobenocross-bridgeattachmentactin,andtitin beyond filamentoverlap.InTnC-depletedmyofibrils,thereis depleted myofibrilswerethesamewhensarcomeresstretched active forceproductionandstrongcross-bridgebindingtoactin. which wewillrefertoas‘titin-basedforceenhancement’ requires this effect isminimal.Thefullamountofincreasedtitin-basedforce, stiffness totitin,andthusincreasedforceuponsarcomerestretching, it appearsthatalthoughcalciumactivationprovidesincreased inhibited) comparedwithpassivelystretchedmyofibrils.Therefore We previouslyproposed thatabindingsitefortitinonthethin If calciumactivationaloneisnotsufficient toexplaintheincrease Interestingly, themyofibril forces inBDM-treatedandTnC- The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105361

5).

The Journal of Experimental Biology during Ca (bottom panel, RESEARCH ARTICLE titin–thin filamentbindingisbasedonstudiesinthe (Nishikawa etal.,2012).ThehypothesisthatN2A titinisthesiteof force PEVKregiontoberecruitedimmediatelyuponstretch domain fromextendinguponstretch(Fig. mechanically advantageousasitwouldpreventtheproximal Ig al., 2005).BindingattheN2A segmentoftitinwouldbe player innumerousskeletalmusclesignallingevents(Huebschet sarcomere lengths(GranzierandLabeit,2004),isalsoamain mechanical roleinpassiveskeletalmuscle,resistingstretchatlong basis forafocusedinvestigation.TheN2A segmentoftitinhasa of thinfilament–titinbinding(Nishikawaetal.,2012)providesthe thin filamentduringcalciumactivation. these resultsprovidesupportfortheideathattitinmaybindto Granzier, 1996).Whilethissegmentoftitinhasnotbeenidentified, actin filamentsinacalcium-dependentmanner(Kellermayerand studies anunidentifiedsegmentoftitindemonstratedbindingto calcium-dependent changesintitin–actininteractions.Inthese the availablespringlengthofpassivetitin(toppanel, hypothetically attheN2A segment.Thisbindingwouldsignificantlyreduce cross-bridge cyclingwhentitinreversiblybindstothethinfilament, the half-sarcomere. Fig. ( would besubstantiallylessthanthatforthebottompanelhalf-sarcomere length of mechanism iscorrect.Ifthesarcomerewasstretchedwithanactivespring N2A titin–thinfilament interactiontodetermine whetherthis (Yamasaki etal.,2001). expresses N2A titinandcardiacmusclethatexpressesN2B difference intitin–actininteractionsbetweenskeletalmuscle that filament bindingisprovidedby reportsthatsuggestafundamental altered inmdm achieves anenhancedstateofstiffness duringcalciumactivationis 2012). Theseresultssuggestthatthemechanismbywhich titin soleus musclesofmdm soleus muscleswhilenochangeinlengthorstiffness isobservedin soleus muscles,thetitinspringisshorterandstiffer thaninpassive titin (Garveyetal.,2002).Duringactiveunloadingofwholemouse model, whichischaracterizedbyadeletionintheN2A regionof L a Recent speculationthattheN2A segment oftitinmaybethesite To investigatethishypothesisfurther, wemodelledtheproposed ), evenforstretchesofidenticalmagnitude.

.Proposedmechanismbywhichtitin-based forceisenhancedin 5. L 2+ p rather than activation andcross-bridgecyclingtheactivetitinspringlength L a ) wouldbemuchshorterandstiffer, iftheproposed titin. FurthersupportforN2A asthesiteoftitin–thin The mechanismproposedoccursattheinitiationof L a , thepassiveforceintoppanelhalf-sarcomere mice duringactiveunloading(Monroyetal., L p Proximal Ig Half-sarcomere L a N2A PEVK

5) andallowthehigh- mdm L p ). Thus mouse stretch, itisimportant toconsiderthatwiththe exception ofcalcium filament inexplainingenhanced titin-basedforceduringactive involved inthemechanismoftitin-based forceenhancement. are astrongindicationthatN2A titinisabindingsiteorotherwise a morefocusedinvestigation.Nevertheless, thesepreliminarydata that bindstothethinfilamentduring calciumactivationwillrequire the interpretationoftheseresults.WhetherN2A isthesiteoftitin however, additionalexperimentsandconsiderations arerequiredin that N2A titinisinvolvedinthemodulationoftitin-basedforce; mdm suggesting thattitin-basedforceenhancementisaffected bythe extent thaninactivelystretchedwild-typemyofibrils(Fig. enhancement waspresentin sarcomere lengths.Duringactivestretch,titin-based force compared withwild-typemyofibrilsactivatedatcorresponding force–length relationship,contractionforcewasdecreasedin Despite ourexpectations,whenactivatedattheplateauof the material andforce-producingcapabilityasinwild-typemyofibrils. thus wereexpectedtocontainthesamequantityofcontractile Isolated the same experimentalprotocoldescribedbelowtodeterminewhether activated ( In apreliminaryinvestigation,weisolatedandstretchedcalcium- investigate theroleofN2A titinin titin-basedforceenhancement. muscle, futureexperimentscanutilizethe filament atordistaltotheN2A segment. observed inmousemyofibrilssupportstitinbindingtothethin mouse model.Nevertheless,theconceptualpremiseofresults experimental resultsonemightneedtofittheparametersfor filament duringactivation.To reproducetheabsolutevaluesof results andproposedmechanismthattitinisboundtothethin Overall, thesesimulationsconceptuallyreflecttheexperimental Ting etal.,2012;DuVall etal.,2013),butonlytoasmallextent. the PEVKandIgdomains(Labeitetal.,2003;Joumaa2008; exceeded passiveforces,presumablyduethebindingofcalciumto force ofactivelystretchedmyofibrilswithouttitinbindingstill deficient intitin-basedforceenhancement(Fig. bridge-inhibited (TnC-depletedandBDM)sarcomeres,whichwere model predictionsoftitin-basedforcecoincidedcloselywithcross- were performedintheabsenceofN2A thinfilamentbinding,the would belocatedatordistaltotheN2A segment.Whensimulations would besubstantiallyhigher, suggestingthatthebindingsiteontitin By shiftingthebindingsiteontitintowardsM-line,forcegain and Herzog,2010),whichexceedstheforcepredictedbymodel. approximately threetimesgreaterinrabbitpsoasmyofibrils(Leonard based forceinactivelystretchedmousepsoasmyofibrilsand Experimental resultsdemonstratethreetofourtimesgreatertitin- by themodelwasdoubledinactivelystretchedsarcomeres. binding. With N2A thinfilamentbinding,titin-basedforcepredicted purely passiveforcesandpredictedforactivationwithoutN2A a substantialforcegainatlongsarcomerelengthsvastlyexceeding actively stretchedsarcomereswithN2A thinfilamentbindingshow force predictionsconceptually(Fig. mouse, wecanstudytheimpactofN2A thinfilamentbindingon on therabbitpsoasmodeldiffer fromtheexperimentalresults spring elements.Whilethepredictedabsolutevaluesofstressbased leaving onlythePEVKregionanddistalIgdomainsasfree assumed thattheN2A regionoftitinbindstothethinfilament, mechanism couldexplaintitin-basedforceenhancement.We When consideringaninteraction betweentitinandthethin With theenhancedforceoftitinconfirmedinmouseskeletal mdm deletion. Thesepilotresultsareinaccordancewiththeidea mdm deletion inN2A titinaffects titin-basedforceenhancement. N The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105361 =1) andnon-activated( myofibrils didnotdiffer indiameterorstructureand mdm N (Fig. =1) 4). Forcespredictedduring mdm 6) buttoamuchlesser mdm myofibrils usingthe 3). Thepredicted model tofurther 3633 mdm

3),

The Journal of Experimental Biology collagen andadditional structuresalsocontribute totheresistiveforce actively stretchedintactsarcomeres. Whileintermediatefilaments, designed tospecificallyaddress titin-basedforceenhancementinthe level (Leonardetal.,2010;Rassier, 2012),theseexperimentswere residual forceenhancementhas been observedatthesinglesarcomere results referonlytotitin-based mechanicsinthesarcomere.As providing protectionagainststretch-induced sarcomereinjury, our by collagenandintermediatefilaments,whichareintegral in force (Wang etal.,1993). Intheabsenceofresistiveforcesprovided bearing structure,contributingaboutone-quarterofthetotalpassive sarcomere lengths,theintermediatefilamentnetworkactsasaforce- by thesolidverticalline.Theforceofactivated final sarcomerelengthof6.0 3634 structural damage(>4.5 filaments whichresistsarcomerestretchtolengthsthat cause sarcomeres arealsosurroundedbyanetworkofintermediate contributors topassiveforce(GranzierandIrving,1995).Individual Within workingmusclefibrelengths,titin andcollagenarethemain the sarcomeres(andtitin)frompotentiallyhazardouselongation. the variousadditionalstructuresthatprovideresistiveforcestoprotect within thecontextofanintactmusclefibreitisimportanttoconsider overlap duringactivestretchisanimportanttopicforfuturestudies. whether titincontributestotheforcegeneratedwithinregionof titin toactiveforcewithintheregionofoverlap,question While thepresentstudycouldnotdisseminateanycontributionof force producedduringactivestretchspansthefulllengthofstretch. activation occurs.Thisindicatesthatthecontributionoftitinto cross-bridge cycling,andthusatsarcomerelengthswhereinitial results wouldsuggestthattheinteractionoccurswithonsetof partially responsiblefortheenhancementoftitin-basedforce,these (Leonard andHerzog,2010).Iftitin–thinfilamentinteractionsare long sarcomerelengths,withlessthanoptimalfilamentoverlap enhancement wasdecreasedwhenthemyofibrilactivatedat from rabbitpsoasmyofibrilsshowedthattitin-basedforce presence ofcross-bridgecyclingandactiveforce.Previousresults the enhancedstateoftitinisatleastinpartdependenton strong cross-bridgecyclingwasinhibited.Thisresultsuggeststhat stiffening oftitin,titin-basedforceenhancementwasabolishedwhen Fig. RESEARCH ARTICLE but toalesserextentthaninactivelystretchedwild-typemyofibrils(Fig. the durationofstretch.Titin-based forcewasincreasedduringactivestretch, remains higherthantheforceofnon-activated stretched fromanaverageinitialsarcomerelengthof2.5 a singleactive(diamond)andpassive(circle) sectional area(nN passively stretched When consideringthepropertyoftitin-basedforceenhancement

.Pilotdatacomparingforce– 6.

Stress (nN μm–2) 150 75 0 23456 μm Passive Active mdm − 2 ) asafunctionofaveragesarcomerelength( mdm Average sarcomere sarcomere Average ( length mdm myofibrils. μm) (Wang etal.,1993). Attheselong μm. Thelossoffilamentoverlapisdesignated length curvesfromactiveand Force normalizedtomyofibrilcross- mdm mdm mdm myofibril. Myofibrilswere myofibrils (diamond) μm) myofibrils (circle)for μ m toanaverage μ m) for

2). include thecontributionoftitininactivelystretchedskeletalmuscle. our understandingofforceproductionshouldbegintoexpandand and presentstudiesdemonstratingarolefortitininactivemuscle, to thethinfilamentwithonsetofcross-bridgecycling.With past initiated whenasiteontitin(atordistaltotheN2A segment)binds based forceenhancementoccurs,ourresultssuggestthatitis experiments areneededtoelucidatethemechanismbywhichtitin- achieve enhancedforcefollowingactivestretch.Whileadditional could potentiallyprovideanexplanationforhowskeletalmuscles additional sourceofforceproductionduringactivestretch,which stiffness. Thismechanismwouldprovidethesarcomerewithan available springlengthresultinginadrasticincreaseits active force,titinbindstothethinfilamentanddecreasesits therefore weproposethatwiththeonsetofcross-bridgecyclingand of theincreaseintitin-basedforcebeyondfilamentoverlap; .Calciumactivationalonecouldonlyexplain15% effects ofcalciumisaninherentpropertyactivelystretched this property, weproposethat enhancedtitin-basedforcebeyondthe filament overlap.With studiesfromrabbitandmousedemonstrating actively stretchedmousepsoasmyofibrilsatlengthsbeyond resistance toactivelystretchingsarcomeres. property ofthesarcomeresthemselves,whichprovidesaninherent that titin-basedforceenhancementisafundamentalmechanical limiting sarcomerestretchwithinamusclefibre,ourresultsindicate measure theforce producedbythemyofibril. used tomanipulate thelengthofmyofibril,andacantilever usedto Fig. Force wascalculatedfromthemeasureddisplacementandknown lever second referencearmwasmeasuredusingacustomMATLAB program. Displacement ofonecantileverarmattachedtothemyofibrilrelative tothe at oneendandwrappedaroundastiff glassneedleattheotherend. 7).Myofibrilswereglued(DowCorning3145)to oneoftheleverarms (Fig. silicon nitridecantileverswithtwoarmsandastiffness of21or68 immersion. 1.3) witha×2.5Optovar(LeonardandHerzog,2010)under×100oil 200M) equippedwitha×100oilimmersionobjective(numericalaperture All testswereconductedusinganinvertedmicroscope(ZeissAxiovert previously (LeonardandHerzog,2010). contribution oftitintoforceproductioninsarcomeresasdescribed series. Therefore,myofibrilexperimentsareidealfortheinvestigationof measured mustbegeneratedbytheproteinscomprisingsarcomeresin single myofibril,extracellularconnectivetissuesareabsentandallforce natural architectureofthecontractileapparatus(Yang etal.,1998).Ina A myofibril isthesmalleststructuralunitofmusclethatmaintains Experiment set-up Sample preparations MATERIALS ANDMETHODS Myofibril forcesweredeterminedusingcustom-builtnanofabricated In summary, wefoundthat titin-basedforceisincreasedin

.Experimental set-up. 7. Needle The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105361 Myofibril A single myofibrilisattachedtoaglassneedle Cantilever 10 μm

pN nm − 1

The Journal of Experimental Biology ~4.0 μm) (Huxley, 1957;Gordonetal.,1966;LeonardandHerzog,2010). cease toexistatsarcomerelengthsbeyondmyofilamentoverlap (i.e. solution andthenstretched.Active,actin–myosin-basedcross-bridge forces excluded fromstatisticalanalyses. All experimentswhichdidnotexhibittheestablishedcriteria(~18%)were to visiblyshortenuponactivationfortheexperimentbeconsideredviable. experiments, inadditiontothetwocriteriastatedabove,allsarcomereshad stretch fortheentiredurationofstretch.Inactivelystretchedmyofibril successful if(1)allsarcomereslengthenedand(2)theforceincreasedwith determined andstrictlyadheredto.A myofibrilexperimentwasconsidered were heldisometricallyuntilasteady-stateforcewasreached. at aspeedof0.1 achieving lengthsthatfarexceededfilamentoverlap.Stretchingwasapplied RESEARCH ARTICLE electrophoresis and forcemeasurements,respectively (Joumaaetal.,2008). preventing anycross-bridge basedforces,hasbeen verifiedusinggel previously anditseffectiveness ineliminatingTnCfromactin, thereby solution. TheTnC-depletionprotocol usedinthisstudyhasbeendescribed of TnC(Joumaaetal.,2008)andthen stretchedincalcium-ATP activation solution (pH Myofibrils (N Myofibrils (N of thepassivetitinspring. solution containingATP. Thisgroupdemonstratesthemechanicalproperties Myofibrils (N from anaveragesarcomerelengthof2.5 of themyofibrilbynumbersarcomeres.Allmyofibrilswerestretched stretch toconfirmthelossoffilamentoverlap. sarcomeres. Individualsarcomerelengthsweremeasuredattheendof the restofmyofibril,havetomatchthoseproducedinremaining myofilament overlap,andthustheirforces,whicharestrictlyinserieswith assume thatsomesarcomeresandhalf-sarcomeresarepulledbeyond average sarcomerelengthswithinamyofibrilexceed4.0 sarcomere lengthsof~3.95 myofilament overlapinmousepsoaswouldbeexpectedtolostat psoas is~1.11 μm (BurkholderandLieber, 2001;Bangetal.,2009)and the plateauregionandthenstretched.Thethinfilamentlengthinmouse enhancement inthepresentstudy, myofibrilswereactivatedslightlyabove 2010). Therefore,tomaximizethemagnitudeoftitin-basedforce dependent componenttotitin-basedenhancedforce(LeonardandHerzog, sarcomere lengthof2.5 force (Figs between aneedleforcontrolledlengthchangesandcantilevertomeasure Single myofibrilswithfourtotwelvesarcomeresinseriesweremounted (Complete in arigor-glycerol solution( to woodenstripspreservethe Strips ofmousepsoasmusclewereextractedfromeuthanizedanimalsandtied stiffness andexpressedinunitsofstress(nN μm measured forcetothecross-sectionalareaofeachmyofibril. Ethics CommitteeoftheUniversityCalgary. Ethics approvalwasgrantedbytheLifeandEnvironmentalSciencesAnimal calcium ionsinanactivationsolution(pCa Herzog, 2010).Activationofmyofibrilswasachievedbydirectapplication were homogenizedinarigorsolutionat4°Candtested20°C(Leonard for 12–17 Group 3:TnCdepleted Group 2:active Group 1:passive Experimental groups Experimental protocol Materials The inclusioncriteriaforsuccessfulmyofibrilexperimentswere Average sarcomerelengthsweredeterminedbydividingthetotallength days (LeonardandHerzog,2010).Forexperiments,musclestrips ® , RocheDiagnostics,Montreal,QB,Canada)andstoredat 2, 3).Myofibrillengthswereadjustedtoaninitialaverage 7.8) for10 =10) werepassivelystretchedinanon-activating(pCa =7) wereactivatedinacalcium-ATP (pCa =8) wereincubatedinalowionic strengthrigor-EDTA μm persarcomeresecond.Afterstretch,myofibrils min toinhibitcross-bridgeformation bydepletion μm. Previousexperimentsdemonstratedaforce- μm (Herzogetal.,1992).Thereforewhenthe in situ − 20°C, pH length. Themusclestripswereplaced μm tolengthsof~6.0

3.5) (LeonardandHerzog,2010). 7.0) withproteaseinhibitors − 2 ) bynormalizingthe μm, wecansafely

3.5) activation μm, thereby − 20°C

8.0) (Joumaa etal.,2008;LeonardandHerzog,2010). et al.,2002).Allsolutionsusedinthisstudyhavebeenpreviouslypublished expressed aspredictedstress(nN μm model predictionswerescaledtoexperimentalmousepsoasvaluesand psoas titin(Linkeetal.,1998;Linke2002;Prado2005).The rather usedwell-knownmodelparametersfromtheliteraturebasedonrabbit filament bindingmodel,werefrainedfromfittingtheexperimentaldata,but cross-sectional area.Inordertoeliminateanybiastowardsthetitin–thin titin strands,andnormalizedtothepassiveforcesinasarcomereof1 force. ThelatteriscalculatedbyMonteCarlosimulationsperformedfor200 the sumofcross-bridge-basedactiveforceandvariabletitin-basedpassive influences passiveforce.Thetotalforceexertedbyasarcomereisgiven binding betweenactinandtitindoesnotgenerateforcebutdirectly rigid thinfilament,therebyreducingthefreespringlengthoftitin.The upon activation,weassumethattheN2A regionoftitinbindstothevirtually production basedonelongationoftitin.Inthecaseactin–titinbinding (Rayment etal.,1993;PiazzesiandLombardi,1995)passiveforce consider activeforceproductionbasedonafive-statecross-bridgemodel submitted asaseparatemanuscriptinthefuture.Briefly, thesimulations description ofthemathematicalmodelisnotprovidedhereandwillbe previous (LeonardandHerzog,2010)presentobservations.A detailed determine whetheritcouldexplaintitin-basedforceenhancementin The proposedN2A titin–thinfilamentinteractionwassimulatedto 20 Myofibrils (N Foundation (NSFIOS1025806)andtheAustrianScienceFund[(FWF):T478-N13]. and EngineeringResearchCouncilofCanada(NSERC),theNationalScience Foundation, CanadianInstitutesofHealthResearch(CIHR),theNaturalSciences This researchwassupportedbytheCanadaResearchChairProgram,TheKillam experimental design,conceptualdevelopment,interpretationandfinalediting. conceptual development,interpretationandediting;W.H. contributedto carried outtheconception,experimentaldesignandediting;K.N.contributed to performed themodelling;A.J.analysis,programmingandediting; T.L. K.P. performeddatacollectionandanalysis,preparedthepaper;G.S.-T. The authorsdeclarenocompetingfinancialinterests. (Ithaca, NY, USA),whichissupportedbytheNationalScienceFoundation. measurements werebuiltbyusattheNanoScaleFacilityofCornellUniversity version ofthismanuscript.Thenanofabricatedcantileversusedforforce Joumaa, UniversityofCalgary, fordiscussionswhichcontributedtothefinal collection. Inaddition,theauthorswouldliketothankScottSiboleandVenus Tahir fromNorthernArizonaUniversityfortheirassistancein The authorswouldliketoacknowledgeJennaMonroy, ReneFuquaandUzma with aMann–Whitney A Kruskal–Wallis non-parametricANOVA wasperformedandfollowed-up determined at0.05andusedforallanalyses. produced duringstretchdiffered betweenmyofibrilgroups.Significancewas bot .C n uet X.M. Aubert, and B.C. Abbott, Modelling Group 4:BDM References Funding Author contributions Competing interests Acknowledgements Statistical analysis ino . ay . ege,A,Samr,D,Mrofli . ue,T and T. Huber, Z., Mártonfalvi, D., Szatmári, A., J., Kengyel, Chen, A., R.L., Nagy, Lieber, P., R., Bianco, Littlefield, E., Brunello, M., J.A., Caremani, Rivas-Pardo, M.-L., Bang, E., Eckels, D., Giganti, P., Kosuri, J., Alegre-Cebollada, during andafterchangeoflength. domain measuredwith opticaltweezers. M.S.Z. Kellermayer, M. actin–myosin interactions. Linari, and V. Lombardi, J.M. Fernández, and W. A. Linke, protein folding.Cell R.J., Solaro, C.M., (2014). S-glutathionylationofcrypticcysteines enhancestitinelasticitybyblocking Warren, N., Hamdani, nmol l − 1 BDM, aninhibitorofstrongcross-bridgebindingtoactin(Tesi The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105361 =8) werestretchedinacalcium-ATP activationsolutionwith 156, 1235-1246. U (2007). Interactionforces betweenF-actinandtitinPEVK FASEB J. group-wise comparisontodeterminehowtheforce (2009). Nebulinplaysadirectroleinpromoting strong (1952). Theforceexertedbyactivestriatedmuscle J. Physiol. 23, 4117-4125. − Biophys. J. 2 ) byaveragesarcomerelength( 117 , 77-86. 93, 2102-2109. mdm tissue 3635 μ μm m). 2

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