International review of cell and molecular biology, volume 321

The C-terminal variable region of TnT resides in theTnI–TnT interface in troponin complex and is in the proximity of TnCTakedaetal.,2003;Vinogradovaetal.,2005,whereasitsfunctionalsignif-

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Evolution, Regulation,

International Review of Cell and Molecular Biology, Volume 321

1

to the physiologic and pathophysiologic significances in modifying the contractility of skeletal and cardiac muscles during development and in adaptation to stress and disease conditions, the hyperplasticity of the N-terminal region of TnT demonstrates

an informative example for the evolution of protein three-dimensional structure and provides insights into the molecular evolution and functional potential of proteins.

Thecontractilemachineryofstriatedmuscles(representedbyskeletalandcardiacmusclesofvertebrates) isthemyofibrilsthatconsist oftandemrepeatsofsarcomeres.Asarcomereiscomposedofoverlappingmyosinthickfilamentsandactinthinfilaments.Contractionispoweredbyactin-activatedmyosin ATPase-catalyzedATP hydrolysis during actomyosin cross-bridgecycling.Thisprocessisregulatedbythethinfilament-associatedregulatoryproteinstroponinunderthecontrolofcytosolicCa2+(Gordonetal.,2000).Residing at ∼37-nm intervals along the thin filament in the form ofF-actin-tropomyosin double helices (Galinska-Rakoczy et al., 2008;Lehmanetal.,2009; Ohtsukietal.,1967),thetroponincomplexconsists

of three protein subunits: theCa2+-binding subunit troponin C (TnC1),actomyosinATPase-inhibitingsubunittroponinI(TnI),andtropomyosin-bindingsubunittroponinT(TnT)(GreaserandGergely,1971).ToconvertthecellularsignalofcytosolicCa2+transientoriginatedfromsarcolemmalelectrical activity to myofilament movements during each excitation–contraction–relaxationcycle,troponinfunctionsthroughcooperativeinter-actionsamongthethreesubunitsandwithtropomyosin(Gordonetal.,2000;Tobacman,1996).WhereasTnCisarelativeofthecalmodulingenefamily(Collins,1991)andfunctionsastheCa2+receptorofthethinfilamentregu-latory system in striated muscle, TnI and TnTare striated-muscle-specificproteinsencodedbycloselylinkedgenesandhavecoevolvedintothreepairs

offiber-type-specificisoforms(ChongandJin,2009; Jinetal.,2008)

Inadditiontoanchoringthetroponincomplextothethinfilament,TnTdirectly interacts with multiple proteins in the thin filament regulatorysystem to play an organizer role in the troponin complex (Perry, 1998).Throughisoformgeneregulation,alternativeRNAsplicing,andposttrans-lationalmodifications,structuralandfunctionalvariationsofTnTprovideamechanismtomodulatestriatedmusclecontractionandrelaxation

To understand thestructure–function relationshipof TnT, thisreviewoutlines the evolution of muscle type-specific TnT isoform genes, the

multiplealternative spliceforms,thedevelopmental regulation ofisoformexpression and alternativesplicing, and theposttranslationalmodificationsduringphysiologicandpathophysiologicadaptations,withafocusontheN-terminal segment that is an evolutionarily diverged regulatory structure(ChongandJin,2009;Jinetal.,2008;WeiandJin,2011).Forbackgroundinformation, comprehensive summaries of striated muscle thin filamentregulation andthefunctionsofTnC,TnI,and tropomyosincanbe found

inseveralpreviouslypublishedreviews(Collins,1991;Gordonetal.,2000;Jin et al., 2008; Perry, 1998, 1999, 2001; Solaro and Rarick, 1998;Tobacman,1996;WeiandJin,2011;ShengandJin,2014)

TnT is a 30–35-kDa protein The sizes of vertebrate TnT withsequence information available range from 223 to 305 amino acids Thislargesize variation isalmost entirelydueto thevariable lengthoftheN-terminalregion,fromnearlyabsentincertainfishfastskeletalmuscleTnTtomorethan 70aminoacidslonginavian andmammalian cardiacTnT(Jin

etal.,2008;WeiandJin,2011).ThehypervariablenatureoftheN-terminaldomain ofTnT is furtherdemonstrated bythepresenceof 4–9 repeatingsequencemotifsinthebreastmusclefastTnTofavianordersGalliformesandCraciformes (Jin and Smillie, 1994) These five amino acid repeats form aclusterofhigh-affinity transitionmetal bindingsitesthatareonlyfound intheadultbreastmuscleofthesebirds(Jinand Samanez,2001; Ogutetal.,

1999).WhiletheN-terminalregionofTnTishypervariableinlengthandamino acid sequences, the amino acid sequences of the middle and C-terminal regions of TnTare highly conserved among the three muscle-type-specific isoforms and across vertebrate species (Jin et al., 2008; WeiandJin,2011)

Electron microscopic studies showed that the TnT molecule has anextended conformation (Cabral-Lilly et al., 1997; Wendt et al., 1997).ThefunctionaldomainsofTnThavebeen extensivelystudiedusing pro-teinfragmentsgeneratedfromlimitedchymotrypticandCNBrdigestions.Protein-bindingstudiesfoundthatthe∼100aminoacidsC-terminalchy-motryptic fragment T2 interacts with TnI and TnC and binds to themiddle regionoftropomyosin (Heeleyetal.,1987;Schaertl etal., 1995).ThechymotrypticfragmentT1thatcontainsboththeN-terminalvariableregion and the middle conserved region of TnT binds the head–tail

junctionof tropomyosinsin theactin thin filament(Heeley etal., 1987).The tropomyosin-binding activity of the T1 fragment resides in the 81aminoacidsCNBrfragmentCB2ofrabbitfastskeletalmuscleTnT,whichrepresentsthemiddle conservedregionofTnT.TheN-terminalsegment

ofTnT(e.g.,theCNBrfragmentCB3inrabbitfastskeletalmuscleTnT)isthe hypervariable region and does not bind any known thin filamentproteinsinthesarcomere(Perry,1998)

Consistentwiththeprotein-bindingdata,X-raycrystallographyminedthepartialstructureofcardiacandskeletalmuscletroponincomplexshowingthattheassociationsofTnTwithTnIandTnCarethroughtheC-terminalT2region(Takedaetal.,2003;Vinogradovaetal.,2005).However,thecrystallographydataonlydeterminedthestructureforaportionoftheTnT–T2regioninthetroponincomplex.TheentireT1regionandtheveryC-terminal13 amino acidsof TnTwere missing from theresolvedhigh-resolutionstructures(Takedaetal.,2003;Vinogradovaetal.,2005).The13aminoacidC-terminalendsegmentencodedbythelastexon oftheTnTgeneishighlyconservedamongisoformsandacrossspecies(Jinetal.,2008;WeiandJin,2011).DeletionoftheC-terminal57aminoacidsoffastTnT(Jhaetal.,1996)orslowTnT(JinandChong,2010)hadnosignificanteffect

deter-onthebindingaffinityofTnTfortropomyosin.However,pointmutations

inthissegment havebeen foundto causefamilialhypertrophicopathy(ShengandJin,2014);thus,itsroleinthestructureandfunctionofTnTremainstobeinvestigated

cardiomy-The high-resolution structural data showed that the main TnT–TnIinterface in thetroponin complex is a coiled-coil structure (i.e., the I-Tarm)formedbythesegmentsofL224–V274incardiacTnTandF90–R136incardiacTnI inhuman cardiactroponin complex (Takeda et al., 2003) or

E199–Q245infastTnTandG55–L102infastTnIinchickenfastskeletalmuscletroponin(Vinogradovaetal.,2005).TheaminoacidsequencesofTnTandTnIinthiscoiled-coilinterfacearebothhighlyconservedamongisoformsandacrossvertebratespecies(Jinetal.,2008;WeiandJin,2011)

Whereasgrossmappingofthetropomyosin-bindingsitesofTnTusingthechymotrypsinandCNBrfragmentshadservedinguidingthestudiesofTnTfunctionandthin filamentregulation ofmuscle contractionfor overthreedecades(Perry,1998),thepreciselocalizationsofthetwotropomyosin-bindingsitesof TnTwerenot determineduntil recentlyusinggeneticallyengineeredTnTfragments(JinandChong,2010).Analysis ofserialdele-tionsofTnTproteinandmappingusingsite-specificmonoclonalantibodyepitopeprobesshowedthattheT1regiontropomyosin-bindingsiteofTnT

involving a large content of α-helix interactions (Pearlstone et al., 1976,

1977)correspondsmainlytoa39aminoacidssegmentinthebeginningoftheconservedmiddleregion(JinandChong,2010).Ontheotherhand,theT2regiontropomyosin-bindingsitedependsonasegmentof25aminoacidsneartheverybeginningoftheT2fragment(JinandChong,2010).Aminoacidsequencesinthetwotropomyosin-bindingsitesare bothhighlycon-servedinthethreemuscle-typeTnTisoformsandacrossvertebratespecies(Jinetal.,2008;WeiandJin,2011)

Although the N-terminal variable region of TnT does not containbinding sites for TnI, TnC, or tropomyosin (Ohtsuki et al., 1984; Pan

et al., 1991; Pearlstone and Smillie, 1982), its structure is regulated byalternativesplicingduring lateembryonicandearlypostnataldevelopment

oftheheart(JinandLin,1988)andskeletalmuscles (WangandJin,1997),andinpathologicadaptation(Larssonetal.,2008).Thesedevelopmentalandadaptive regulations suggested functional significances of the N-terminalvariable region of TnT To investigate the molecular mechanism for theN-terminalvariableregiontoaffectTnTfunction,wedevelopedanepitopeconformationalanalysisusingmonoclonalantibodiesrecognizingthemiddleand C-terminal regions of TnTas three-dimensional structure-sensitiveprobes The studies demonstrated thatlocal structural changes in theN-terminalregionofTnT,suchasthatinducedbyZn2+-bindingtoatransitionmetalion(Cu(II),Ni(II),Co(II),andZn(II))bindingclusterinanα-helixintheN-terminalvariableregionofchickenbreastmuscle fastTnT,andthealternativesplicingofN-terminalcodingexonsincardiacTnT,alteredthestructuralconformationofremoteregionsandalteredthebindingaffinityforTnIand tropomyosin(Biesiadeckietal.,2007; Ogutand Jin,1996;WangandJin,1998).Fluorescencespectrometrystudiesfurtherdemonstratedthat

Cu2+-binding to the N-terminal metal-binding cluster in chicken breastmusclefastTnTalteredthefluorescenceintensityandanisotropyofTrp234,Trp236, Trp285, and fluorescein-labeled Cys263 in the C-terminal region(JinandRoot,2000).Theselong-rangeconformationaleffectsindicatethattheN-terminalvariableregionofTnTplaysaroleinmodulatingtheoverallmolecularconformationandfunctionofTnT

ThestructuralandfunctionaldomainsofTnTaresummarizedinFigure1

In addition to the hypervariable N-terminal region, there are two othervariable regions in the TnT polypeptide chain The C-terminalregion offastskeletalmuscleTnTcontainsasegmentof13aminoacidsencodedbyapairofmutuallyexclusive exons(exons16and 17).Thisregion alsoshowsdiversitybetweenmammalianandaviancardiacTnT,wheretheaviancardiac

TnTgenecontainsanadditionalexonencodingtwoaminoacids(CooperandOrdahl, 1985) The C-terminal variable region of TnT resides in theTnI–TnT interface in troponin complex and is in the proximity of TnC(Takedaetal.,2003;Vinogradovaetal.,2005),whereasitsfunctionalsignif-icanceandtheregulationofitsalternativesplicingrequiremoreinvestigation.There is another minor variable region between the middle and C-terminal regionsof TnT(i.e.,between theT1and T2 fragments),where

analternativelysplicedexon(exon13)isfoundinmammaliancardiacTnTencodingashortsegmentof2or3aminoacids(Jinetal.,1992,1996).Thealternative splicing of this exon involves exclusion, complete, and partialinclusions(Jinetal.,1996).Thefunctionalsignificanceofthisminorvariableregion and the regulation of its alternative splicing also remain to beinvestigated

Threehomologousgeneshaveevolvedinmammalianandavianspeciesencoding TnTisoforms in cardiac muscle (TNNT2), slowskeletal muscle

Restricted calpain I cleavage

TnT

Tnl TnC

T1

Ch ymotr ypsin clea vage

T2 N-terminal variable region

H2N

HOOC

Tropomyosin-binding site 1 Tropomyosin-binding site 2

Figure 1 Structural and functional domains of TnT The diagram summarizes the structural and functional regions of TnT The high-resolution structure of partial troponin complex including a C-terminal segment of TnT that interacts with TnI and TnC is redrawn from published crystallography data ( Takeda et al., 2003 ) The arrows indicate the chymotryptic cleavage site between the T1 and T2 fragments ( Perry, 1998 ) and the calpain I cleavage site for the selective removal of the N-terminal variable region

of cardiac TnT ( Zhang et al., 2006 ) The two tropomyosin-binding segments ( Jin and Chong, 2010 ) are also outlined.

(TNNT1), andfastskeletalmuscle(TNNT3) (Breitbart andNadal-Ginard,1986;CooperandOrdahl,1985;Farzaetal.,1998;Hiraoetal.,2004;Huang

etal.,1999b;Jinetal.,1992).ExpressionofthethreeTnTisoformgenesinadultcardiacandskeletalmusclesiscontrolledratherstrictlyinamusclefibertype-specificmanner.KnockoutofthecardiacTnTgeneresultedinembry-oniclethality(Nishiietal.,2008).AnonsensemutationinhumanslowTnTgenethat truncatestheproteinataminoacid180anddeletestheTnI-andTnC-bindingsitestogetherwithoneofthetropomyosin-bindingsitesintheC-terminalT2region(Jinetal.,2003;Johnstonetal.,2000)resultinginthelossofmyofilamentincorporationandrapiddegradationofslowTnT1–179inthemusclecells(Jinetal.,2003;Wangetal.,2005)andaclinicalphenotypeofrecessivenemalinemyopathywithinfantilelethality(Johnstonetal.,2000).Therefore,thethreeTnTisoformsplaynonredundantlycriticalrolesinthethreetypesofstriatedmuscle

Theprimarystructural diversityofthethreemuscle fibertype-specificTnTisoformsismainlyintheN-terminalregion(ChongandJin,2009;Jin

etal., 2008; WeiandJin, 2011;Sheng andJin, 2014) Thisobservationisconsistentwith theregulatoryfunctionof theN-terminal variableregionthatprovidesastructuralbasisforadaptationtovariousfunctionaldemands

indifferenttypesofmuscle,indifferentspecies,atdifferentstagesofopment,andunderpathologicconditions.Ontheotherhand,themiddleandC-terminalregionsofTnTarehighlyconservedamongthethreemuscletype-specificTnTisoformsandacrossvertebratespecies

devel-Investigatingtheevolutionarylineage ofthethree TnTisoform geneshelpstounderstandthestructure–functionrelationshipofTnT aswellasthephysiologicsignificance of theN-terminal hypervariable region Materialremains of ancestor nucleotides and proteins are largely unavailable forevolutionarystudies,thus,likeothermolecularevolutionarybiologystudies,nucleotideandaminoacidsequencecomparisonsamonghomologousTnTisoformgenesinpresent-dayorganismswereemployedtoprovidethecore

ofourcurrentknowledgefor themolecularevolutionofTnT Itisworthemphasizing thatthevariation in protein three-dimensional structure is abasisforfunctionaldiversity.Therefore,thestudyoftheevolutionofthree-dimensionalstructuresforTnTisoformsisa novelapproachtoenrichourknowledgeontroponinfunctionandthethinfilamentregulationofmusclecontraction

Usingmonoclonalantibodiesassite-specificepitopeprobes,wedetectedtherestorationofancestor-likeconformationinTnT afterremovingcertainevolutionarily added “suppressor” structure, for example, theN-terminal

ofrestoringancestralconformationsthathavebeenallostericallysuppressedbythe evolutionary addition of a modulatory structure The results revealedthree-dimensional structural evidence for the evolutionary relationshipbetween TnIand TnT, twosubunits ofthe troponin complex,andamongthethreemusclefibertype-specificTnTisoforms(ChongandJin,2009).Consistent with sequence analysis that suggested a distant homology

of the genes encoding TnI and TnT, the epitope analyses demonstratedrestorationof TnI-likethree-dimensionalstructuresinTnT,supportingthatthese two subunits of troponin arose from a TnI-like ancestor protein(Chong and Jin, 2009) This common ancestor wouldhave had functions

inbothanchoringtotheactin-tropomyosinfilamentandinhibitingmyosinATPase.TnI andTnT havediverged prior tothe emerging ofvertebrates(ChongandJin,2009).Itremainstobeinvestigatedwhetheranypresent-dayproteincouldrepresentthe commonancestorof TnIandTnT,possiblyininvertebratespecies

FurthersupportingthenotionthatTnIandTnTgenesareduplicatesofacommonancestralgene,TnIisalsopresentinthree musclefibertypeiso-formsandthesixTnIandTnTisoformgenesarecloselylinkedinthreepairs(fastTnI–fastTnT,slowTnI–cardiacTnT,andcardiacTnI–slowTnT)inthegenomeofvertebrates(Chong andJin,2009;Jinetal.,2008).Embryoniccardiacmuscle expressessolelyslowskeletalmuscle TnIthatisreplacedbycardiac TnI during late embryonic and early postnatal development (Jin,1996;Sagginetal.,1989).ThefunctionalpairingofslowTnIandcardiacTnTinembryonicheartindicatesthattheevolutionarilylinkedTnI–TnTgenepairs,including theseemingly scrambled slowTnI–cardiac TnTandcardiacTnI–slowTnTgenepairs,representoriginallyfunctionallinkages

Inaddition to thegenomic linkages,TnIandTnTalsohavestructuralalikenessthatsupportstheiroriginationbygeneduplication.Likethestruc-ture of TnT, the N-terminal region of TnI is also a variable structure ascardiac TnI has an evolutionarily additive N-terminal extension that is aheart-specificregulator (Parmacek and Solaro, 2004; Perry, 1999) to finetune the conformation and function of cardiac TnI in physiologic andpathophysiologicadaptations(Akhteretal.,2012;Jinetal.,2008)

Byrevealingsuppressedthree-dimensional structures,wefurtheronstrated an evolutionary lineage of fast to cardiac to slow TnT isoformgenes(ChongandJin,2009).DifferentfromTnIandTnTthathaveevolvedintothree isoforms for thethree fiber typesof vertebratestriatedmuscle,TnCispresentinonlytwoisoforms:fastTnC(GahlmannandKedes,1990)

dem-andslow–cardiacTnC(ParmacekandLeiden,1989).Theundifferentiatedutilization of the same TnCisoform in cardiac and slow skeletal musclessupportsthehypothesisthattheemergenceofthecardiacandslowTnI–TnTgenepairswasarelativelyrecenteventandthelinkedcardiacTnI–slowTnTgenes are the newest pair (Figure 2) (Chong and Jin, 2009) The latestemergence of the cardiac TnI–slow TnT gene pair is supported by thepresenceof theunique N-terminal extension in cardiacTnI (Chong andJin,2009;ParmacekandSolaro,2004).SummarizedinFigure2,thispattern

is consistent with the [fast skeletal (slow skeletal, cardiac)] phylogeneticrelationshipindicatedbysequenceanalysisofother muscleproteins(Oota

1998,2008).SuchconservationpatternindicatesthattheevolutionofTnTisoformgeneswasdrivenprimarilybyearlyadaptationstothedifferentiatedfunctionsofcardiac,fast,andslowskeletalmuscles.Thecriticalroleofmusclefibertype-specificTnTisoforms,thefunctionofskeletalmuscle,forexam-ple, slow TnT that is the newestisoform of TnT, is demonstrated by the

A duplication event later resulted in the emergence of a cardiac TnT-like gene that was further duplicated to give rise to the present-day cardiac TnT and slow TnT genes.

human slow TnT Glu180 nonsense mutation (Jin et al., 2003) that causessevere nemaline myopathy with infantiledeath (Johnston etal.,2000) andconfirmedbyknockingdownoftheexpressionofslowTnTgeneexpression

indiaphragm muscletoproduceatrophy,slow-to-fatfibertypeswitch,andreducedresistancetofatigueinmousemuscles(Fengetal.,2009b)

WereportedthattheheartofadulttoadsBufoexpressesexclusivelyslowskeletal muscle TnT instead of cardiac TnTwhile all other myofilamentproteinsremain to be the cardiac isoforms including normal cardiac TnIandcardiacmyosin(Fengetal.,2012).Thisuniquebiochemicalcontentoftoad cardiacmuscle is correlated to a striking physiologic feature of toadheart,thatis,itishighlytoleranttolargechangesinthevolumeofbodyfluidandbloodbetweenrainyanddryseasons(BoralandDeb,1970)andmuchmoreresistanttothelossofbloodvolumethanthatofthecloselyrelatedfrogheartunderexperimental conditions(Debet al.,1974) Theaortic bloodflowrateoftoaddidnotdropuntilabloodlossofmorethan5%ofthebodyweight, whereasblood loss of 2%of thebodyweight caused a decline ofaorticbloodflowrateinfrog(HillmanandWithers,1988).Wedemonstratedthattoadheartshadfastercontractileandrelaxationvelocitiesandasignif-icantly higher tolerance to afterload (Feng et al., 2012) These findingsindicatethattheuniqueutilizationofslowskeletal muscleTnTto replacecardiac TnTin toad cardiacmuscle was an evolutionary adaptionwith asignificant fitness value during natural selection, further supporting thedifferentiatedfunctionalitiesofTnTisoforms

Asdiscussed earlier,themaindifferencesamongthethreemuscle-typeTnTisoformsisintheN-terminalvariableregion(Jinetal.,2008;WeiandJin,2011;ShengandJin,2014)thatfinetunesthemolecularconformationandfunctionofTnT,thusrepresentsamajordrivingforceoftheevolution-arydiversityofTnTisoforms

AlternativeRNAsplicinggeneratesmultipleproteinspliceformsfromthetranscriptsofeachofthethreemuscletype-specificTnTgenes(Jinetal.,2008;Wei and Jin,2011).The mammalian cardiac TnT gene contains 14constitutivelyexpressedexonsand3alternativelysplicedexons,twoofwhichencodesegments in the N-terminalvariable region (Farza et al., 1998;Jin

et al., 1992, 1996) Exon5 of cardiac TnT gene, which encodes 9 or 10aminoacidsintheN-terminalvariableregion,isincludedinembryonicbut

notadult cardiacTnT(JinandLin, 1989) Exon 4ofcardiac TnT geneisalternatively splicedindependent of developmentalstages (Jinetal., 1996).TheaviancardiacTnTgenecontains16constitutivelysplicedexonsandonly

1 alternative exon (the embryonic exon 5) (Cooper and Ordahl, 1985).Correspondingly, four mammalianand twoavian cardiacTnTN-terminalalternativesplicingvariantshavebeenfoundinnormalcardiacmuscle.MammalianfastskeletalmuscleTnTgenecontains19exons,ofwhichexons4, 5,6, 7,8,anda fetalexonencodingsegments intheN-terminalvariableregionarealternativelyspliced(BreitbartandNadal-Ginard,1986;BriggsandSchachat,1993;WangandJin,1997).Thesealternativeexonsarenotincludedorexcluded randomlyand notallpossible splicingcombina-tionsareatasignificantleveldetectablebycDNAcloning.Accordingly,only

13mousefastTnTmRNA variantsand11chickenfastTnTmRNA variantsdiffering intheN-terminalvariableregionhave beenactuallyfound withsequenceinformationtorepresentthesplicingpathwaysforsignificantlevels

ofproteinproductsandphysiologicfunctions(OgutandJin,1998;Smillieetal.,1988;WangandJin,1997)

Inadditiontoexons4–8,several uniqueN-terminalalternativecodingexons are found in avian fast skeletal muscle TnT genes Seven P exonslocated between exons 5 and 6 encode a unique Tx segment (Jin andSamanez, 2001; Miyazaki et al., 1999; Smillie et al., 1988) consisting ofseventandemrepeatsofpentapeptides(AHH[A/E]E)arefoundinchickenfastTnTgene.Awexonandayexonarefoundbetweenexons4–5and7–8,respectively,furtherincreasingthediversityofavianfastTnT(Schachatetal.,

1995).Asdiscussedearlier,theTxsegmentencodedbythePexonsinthefastTnTgeneofbirds inavianordersof GalliformesandCraciformescontains acluster of high-affinity transitionmetal ion binding sites (Jin and Smillie,

1994) Nohomologouscounterpart wasfound inmammalian TnTgenesandthebiologicsignificanceoftheTxelementremainstobeinvestigated.OneofitsspecificphysiologicfunctionsistoserveasaCa2+reservoir(Zhang

etal.,2004),whichmayconfercertainfunctionsrequiredfortheavianflightmuscles

TheslowskeletalmuscleTnTgenehasasimplerstructurethanthatofthefastskeletalmuscleandcardiacTnTgenes.Thereareonly14exonsintheslowTnTgeneandoneof whichis alternativelyspliced.Withanexon–-intronorganizationsameasthatofthemammalianslowTnTgenes(∼9kb),chicken slow TnT gene is significantly smaller (3kb) by having shorterintron sequences (Hirao et al., 2004; Huang et al., 1999b) Alternativesplicing of exon 5 in the N-terminal region generates 2 variants of slow

TnT(Gahlmannetal.,1987;Huangetal.,1999b;Jinetal.,1998).Splicingattwoalternativeacceptorsites inintron5ofmouseslow TnTgenefurthergenerates a single amino acid variation in the exon 6-encoded segment(Huangetal.,1999b) Thesamepatternwas foundfor theintron4–exon

5splicingofchickenslowTnTgenetranscript(Hiraoetal.,2004).The molecular mechanism that regulates the alternative splicing ofTnTmRNAisnotfullyunderstood.Bothcisandtransregulatory factorshavebeenimplicatedtoaffectthealternativesplicingofcardiacTnT(Laddand Cooper, 2002) Alternative splicing of fast TnTwas found duringmyogenesis Muscle-specific trans regulatory factors were required forappropriate splicing and incorporation of constitutive and alternativeexons of fast TnT during myotube differentiation in culture (BreitbartandNadal-Ginard,1987)

The N-terminal alternatively spliced TnT variants have been shownwithfunctional impacts Skinned fibers of adult chicken pectoral musclecontaining alternatively spliced fast TnTwith more negatively chargedresidues intheN-terminal variableregion exhibited higher myofilamentcalcium sensitivity than control muscle fibers containing alternativelyspliced TnTwith less N-terminal negative charges (Ogut et al., 1999;Reiseretal.,1992,1996).Whenreconstitutedintoskinnedcardiacmusclestrips,embryoniccardiacTnT withmorenegativeN-terminalchargesalsoincreased Ca2+ sensitivity of myosin ATPase and force development incomparison to that of the less negatively charged adult cardiac TnT(Gomes et al., 2002) Similarly, studies using reconstituted myofilamentsshowedthattheembryoniccardiacTnTproducedhigherCa2+sensitivityascomparedwiththatofadultcardiacTnT(Gomesetal.,2004).Embryonicandneonatalcardiac musclecontainingembryoniccardiacTnTexhibitedhighertolerancetoacidosis(Solaroetal.,1988).Incontrast,overexpression

offast skeletal muscleTnT that hasa less negativelycharged N-terminalsegment than that of cardiac TnT decreased thetolerance to acidosis intransgenicmousecardiacmuscle(Noseketal.,2004)

No pathogenic point mutation has been identified in the N-terminalvariableregionofTnT,whereasmultiplesuchmutationshave beenfoundimmediatelyoutsidetheN-terminalvariableregion(for exampleI79Nofadult cardiac TnT that causes familial hypertrophic cardiomyopathy(Knollmannetal.,2001)).ThisobservationmayindicatethehighlyplasticnatureoftheN-terminalvariableregionofTnT

Nonetheless, larger structural variations such as aberrant splicing inthe N-terminal variable region of cardiac TnT have been reported in

cardiomyopathies.Inturkeyhearts,abnormalskippingofexon8incardiacTnTwas found ininherited dilated cardiomyopathy (Biesiadeckiand Jin,

2002).Counterpartofthisexon(exon7)inmammaliancardiacTnTwasalsospliced out indog heartswith dilated cardiomyopathy(Biesiadecki etal.,

2002).ThisexonencodesanormallyconstitutivesegmentincardiacTnT(Jinetal.,2008; Weiand Jin,2011) Itsaberrantsplice-out indilatedcar-diomyopathy turkey and dog hearts indicates a causal relationship to thepathogenesis.Supportingthisnotion,transgenicmousestudiesshowedthatoverexpression of exon 7-deleted cardiac TnT in adult cardiac muscledecreased systolic function of theheart (Wei etal., 2010) In addition tothesplice-outofexon7,thedilatedcardiomyopathydogheartsalsohadanabnormal inclusionof theembryonicexon 5 incardiac TnTintheadultcardiacmuscle(Biesiadeckietal.,2002).Thepathophysiologicsignificance

ofembryoniccardiacTnTinadultcardiacmusclewillbediscussedlater.Alternativesplicingof exon4thatencodes4–5 aminoacidsintheN-terminalvariableregionofcardiacTnTisalsorelatedtodiseaseconditions.SignificantexpressionoflowmolecularweightcardiacTnTexcludingexon

4 was found in failing human hearts (Anderson et al., 1995; Rouilleretal.,1997),diabeticrathearts(Akellaetal.,1995),andhypertro-phic rat hearts(McConnell et al., 1998) (it is worth mentioning that theabnormallyincreasedexclusionofexon4incardiacTnTwasmisinterpretedandquotedinsomecardiologytextbooks asre-expression offetalcardiacTnTinfailinghumanhearts)

Mesnard-Thealternativesplicing-generateddecreasesinsizeandnegativechargeoftheN-terminalvariableregionofcardiacTnTimplyafunctionaladaptation

tothesepathologicconditions.Supportingthishypothesis,thelowmolecularweightslowTnTwith the exon5-encodedsegmentspliced outwasupre-gulatedinthe musclesoftype1(demyelination)but nottype2(axonloss)Charcot–Marie–Toothdisease,suggestingafunctionalsignificanceinskeletalmuscleadaptationtoneuromusculardisorders (Larssonetal.,2008).TheaberrantsplicingoftheN-terminalvariableregionofcardiacTnTdoesnotabolishthecorefunctionofTnTandadultskeletalmusclesnormallycontain multipleN-terminal alternativelysplicedvariants of fastand slowTnT(Jinetal.,2008;WeiandJin,2011).Therefore,themechanismfortheaberrantlysplicedcardiacTnTtocontributetothepathogenesisofdilatedcardiomyopathy in turkeys and dogs raised a key question regarding thestructure–function relationship of TnT in cardiac muscle An importantfeature of vertebrate hearts is the synchronized and uniform ventricularcontraction activated as an electrical syncytium Accordingly, uniform

TnTfunctionis beneficialfor therhythmpumpingfunctionoftheheart.Thisisdifferentfromthefunctionofskeletalmuscle,inwhichmultipleTnTisoformsarepresenttofittheneedofbroadertwitchesforfusionintotetaniccontractions Based on this observation, we tested a hypothesis that theabnormalityofaberrantN-terminalsplicingofcardiacTnTisnotasimplelossoffunctionbutthechronicpresenceofmorethanoneclassofTnTinthethinfilamentsofadultcardiacmuscle(FengandJin,2010).

Inthishypothesis,desynchronizedactivationofventricularmuscleatthemyofilamentlevelduetothecoexistenceofTnTvariantsthatproducesplit

Ca2+sensitivitywoulddecreasetheefficiencyofcardiacpumping.Toonstratethismechanism,wefirstcreatedtransgenicmouseheartsthatcoex-pressawild-typefastskeletalmuscleTnTandtheendogenouscardiacTnT.ThecoexistenceoftwononmutantTnT’sinadultcardiacmusclealteredtheoverall cooperativity of Ca2+-activated force production (Huang et al.,1999a),decreasedcardiacfunction,andproducedmyocardialdegeneration(Huangetal.,2008).WethentestedintransgenicmouseheartstheeffectsofexpressingoneortwoofthecardiacTnTsplicingvariantsfoundinturkeyand canine-dilated cardiomyopathy together with endogenous wild-typeadultcardiacTnToncardiacefficiency.Theresultsshowedthatthecoex-istence of more than one forms of cardiac TnT in adult cardiac musclesignificantly decreased cardiac pumping efficiency proportional to thedegreeofTnTheterogeneity(FengandJin,2010)thatsplitsthinfilamentcalciumsensitivity(BiesiadeckiandJin,2002)

dem-Itisworth notingthatabnormalinclusionoftheembryonicexon5inadultcardiacTnTwasalsofoundincatandGuineapighearts(Biesiadecki

etal.,2002).Inaddition,theGuineapigheartsexpresscardiacTnT withanexclusionofalargersegmentintheN-terminalregionencodedbyexon6(Biesiadeckiet al.,2002) Catsand Guineapigs arebothreported to havehigh incidence of inherited cardiomyopathy and heart failure(Hasenfuss,1998;Tilleyetal.,1977).Therefore,impropersplicingofN-terminalexons

ofcardiacTnTmightbeacommonpathogenicmechanism

ThealternativelysplicedN-terminalcoding exonsoffast, cardiac,andslowTnTgenesaresummarizedinFigure3andTable1.Thelargenumber

ofalternativelysplicedTnTvariantsdifferingintheN-terminalregionmayprovideacapacityofmodifyingmusclecontractilitywhereasretainingthecorefunctionsofTnT

AveryinterestingobservationisapointmutationofturkeycardiacTnI(R111C) in the TnI–TnT interface (Biesiadecki et al., 2004), whichbluntedthefunctionaleffectofproteinkinaseAphosphorylationofcardiac

TnI(Weietal.,2010)had mutuallyrescuingeffectswhenitcoexistswiththeexon7-deletedcardiacTnT(Biesiadeckietal.,2004;Weietal.,2010)

in the hearts of double transgenic mice (Wei et al., 2010) This findingsuggests thatthe TnI–TnTinterface isa pivotal site intransmittingCa2+signals during striated muscle contraction and relaxation as well as inmediatingthefunctionaleffectsoriginatingfrom theN-terminal variableregion ofTnT(Jinetal.,2008;WeiandJin, 2011;Sheng andJin, 2014)

TheexpressionofTnTisoformgenesinembryonicstriatedmuscleswasnotasrestrictedtofibertypesasthatintheadultanimal.CardiacTnTisexpressed at significant levels in embryonic and neonatalskeletal muscles

Fast

Cardiac

Slow

N-terminal variable region Conserved regions

Tropomyosin-binding site 1 Tropomyosin-binding site 2

Exons 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16/17 18

Chymotrypsin

An additional exon in chicken

Alternative acceptor sites in intron 5

y X(P) 7

Figure 3 Alternatively spliced exons of mammalian and avian fast, cardiac, and slow TnT genes The linear maps of fast, cardiac, and slow TnT illustrate the segments encoded by each exon The alternatively spliced exons are indicated by the filled boxes, among which the developmentally regulated exons are in solid black The w, x (P), and y exons illustrated in the fast TnT structure are only found in avian species The alternative acceptor site involved in the splicing of exon 6 in slow TnT gene is indicated with an arrowhead The C-terminal and middle regions of TnT are well conserved among the three muscle-type-specific isoforms and across species whereas the N-terminal region is highly variable The calpain I cleavage site for the selective removal of the N-terminal variable region of cardiac TnT in stress conditions ( Zhang et al., 2006 ) and the chymotrypsin cleavage site dividing the T1 and T2 fragments of fast TnT ( Perry, 1998 ) are indicated with arrowheads.

Table 1 Modifications and regulations of the N-terminal variable region of TnT Exons of TnT genes, which are alternatively spliced under physiologic

conditions

Exons 4, 5, 6, 7, and 8 in avian and mammalian fast TnT

A fetal exon in mammalian fast TnT

P exons (up to 7) in avian fast TnT

Exons w and y in avian fast TnT

Exon 5 in avian and mammalian cardiac TnT

Exon 4 in mammalian cardiac TnT

Exon 5 in mammalian slow TnT

Alternative acceptor sites in intron 5 of mouse slow TnT and intron 4 of chicken slow TnT

Developmental regulations that result in isoform switches

Alternative splicing of exon 5 in cardiac TnT

Alternative splicing of exons 4, 6, 7, 8, and fetal in fast TnT

Postnatal inclusion of P exons in avian pectoral muscle fast TnT

Posttranslational modifications

Constitutive phosphorylation of Ser 2 at the N terminus

Selective removal of the N-terminal variable region by restrictive proteolysis in adaptation to stress conditions

Functional significance

Variable N-terminal negative charges determine the overall charge of TnT N-terminal structures modulate the conformation and function of the middle and C-terminal regions of TnT

N-terminal variation in TnT alters thin filament Ca2+-sensitivity and force production

N-terminal acidic residues of TnT provide a potential reservior of Ca2+Deletion of the N-terminal variable region of cardiac TnT moderately decreases systolic velocity of the heart and increases ejection time and stroke volume Deletion of the N-terminal variable region of TnT restores an ancestral conformation

Pathologic alternative splicing

Splice-out of exon 4 in adult cardiac TnT in human heart failure

Splice-out of exon 4 in adult cardiac TnT in diabetic and hypertrophic rat hearts

Splice-out of exon 8 or exon 7 in cardiac TnT in turkey and dog dilated cardiomyopathies

Splice-in of exon 5 in adult cardiac TnT in dog dilated cardiomyopathy Splice-out of exon 6 in cardiac TnT of Guinea pig heart

Splice-out of exon 5 in slow TnT in type 1 Charcot–Marie–Tooth disease The table summarizes the alternative splicing and posttranslational modifications of the N-terminal variable region of vertebrate TnT The relevant developmental regulation, physiologic and pathologic significances are listed.

1981).InsituhybridizationstudiesfoundthattheexpressionofcardiacTnT

inthedevelopingheartbeginsatday7.5postcoitumandinskeletalmuscles

atday11.75postcoitum(Wangetal.,2001).TheexpressionofcardiacTnTgeneisdownregulatedinskeletalmusclesduringpostnataldevelopmentandceasesintheadult(Jinet al.,2003; Sabryand Dhoot,1991; Sagginetal.,

1990) Thedevelopmental switchingfrom cardiacTnTto skeletal muscleTnT is seen in both avian and mammalian skeletal muscles (Cooper andOrdahl,1984;Jin,1996;SwiderskiandSolursh,1990;ToyotaandShimada,

1981),demonstratingafunctionalexchangeabilitybetweenthemusclespecific TnTisoforms.On theotherhand, thedevelopmentallyregulatedswitchofTnTisoformsindicatesdifferentiatedfunctionoftheTnTisoforms

type-indifferenttypesofadultstriatedmuscles

WhiletheexpressionofcardiacTnTgeneisdownregulated,thesion of slow TnT is upregulated in postnatal slow skeletal muscles Thisprocess is concurrent withtheonset of theAmishnemaline myopathy inwhich the affected infant’s lackof slow TnT in their skeletal muscle areapparentlynormalinskeletalmusclefunctionatbirthbutsoondevelopthediseasephenotypeswhilecardiacTnTceasesexpressioninskeletalmuscles(Jinetal.,2003).ThisobservationsuggeststhatcardiacTnTmayfunctioninplaceofslowTnTinembryonicandgrowingskeletalmuscles,ahypothesisthatisworthtestingforthedevelopmentoftargetedtherapeuticapproaches

expres-ofAmishnemalinemyopathy

Transientexpression ofslow TnT, but notfast TnT, wasfound in theembryonicheart.Atday13.5postcoitum,expressionsofallthreeTnTgenesweredetectedinthedevelopingtongueandthiscoexpressioncontinuedtoday16.5postcoitumwithfastTnTbeingpredominant.CardiacTnTtran-scriptwas alsodetectablebyinsituhybridizationintheembryonicurinarybladder,wherepresumablysmoothmusclewaspresent(Wangetal.,2001).Itremains to be investigated whether this low-level expression of TnT insmoothmusclehasaphysiologicsignificance

Inchicken skeletal muscle, cardiacTnC was coexpressedwith cardiacTnTinearlydevelopmentalstages(ToyotaandShimada,1981).Duringthedevelopmentof avian skeletalmuscle, thedownregulationofcardiac TnTandcardiacTnCandtheupregulationoftheadultformofskeletaltroponinsubunitsweredependent ondiffusible neurohumoralfactorsbut indepen-dentoffunctionalinnervation(ToyotaandShimada,1983)

As discussed earlier, the alternative splicing of cardiac TnT switchespattern during avian and mammalian heart development Embryonic and

neonatalheartsexpressembryoniccardiacTnTwiththeinclusionofa9or10amino acid segment encoded by exon 5 in the N-terminal region TheembryoniccardiacTnTislaterreplacedwithadultcardiacTnTbyexcludingthe exon 5-encoded segment (Cooper and Ordahl,1985; Jin etal., 1992,1996; Jin and Lin, 1989) The exon 5-encoded segment is highly acidic(negativelychargedatphysiologicpH)and,therefore,thisalternativesplicingregulationrepresentsalargetosmallandmoreacidictolessacidictransition

ofthephysicalpropertiesofcardiacTnT(JinandLin,1988).Thetimecourse

ofthisdevelopmentalswitchhasbeendescribedforchicken,mouse,andrathearts Cardiac TnT cDNAs with the same embryonic and adult splicingpatternsarealsofoundinhumanhearts(Townsendetal.,1995)andthesameproteinisoformswitchwasseenindevelopinghumanskeletalmuscleswherecardiacTnT istransientlyexpressed(Jinetal.,2003)

Complexalternativesplicingoffast skeletalmuscle TnToccursduringskeletal muscle development involving multiple coding exons for theN-terminal variable region Similar to that of cardiac TnT, a fetal exonlocatedbetweenthealternativeexon8andconstitutiveexon9isfoundinmammalianfast TnT genes(Briggs andSchachat, 1993) Inclusion ofthefetalexon-encodedsegmentintheembryonicfastTnThad aninhibitoryeffectonmyosinATPaseactivityinreconstitutedmyofilaments(Chaudhuri

etal.,2005).InvolvingthefetalexonandmultipleotherN-terminalnative exons (exons 4, 6, 7, and 8) encoding mainly acidic residues, theexpression offast TnTalsoexhibits a developmental switching from highmolecular weightacidic isoformsto low molecular weight basic isoforms(WangandJin,1997)

alter-Whereas,mostoftheN-terminalalternativelysplicedexonsoffastandcardiac TnT genes exhibit decreased inclusion during heart and skeletalmuscledevelopment(Jinetal.,1996; ShengandJin, 2014;Wangand Jin,1997;WeiandJin,2011),auniquecaseinthedevelopmentalregulationoffastTnTgeneistheposthatchinginclusionofsevenPexonsencodingtheTxsegmentintheN-terminalregionofavianpectoralbutnotlegmuscles(JinandSamanez,2001; Ogutand Jin,1998) While thelargenumberofGluresiduesencodedbythePexonsmightserveasacalciumreservoirinavianpectoralmusclethinfilaments(Zhangetal.,2004)andthenegativechargesaddedtotheN-terminalvariableregionofTx-positivefastTnTcorrelated

to an increased tolerance to acidosis (Ogut and Jin, 1998), the biologicsignificanceoftheTxsegmentanditsdevelopmentallyregulatedexpression

inadultavianpectoralmuscles,especiallyits capacityofbindingtransitionmetalions,remainstobeinvestigated

ThedevelopmentallyregulatedalternativeN-terminalcodingexonsofthe three muscle fiber type TnT genes are summarized in Table 1 TheN-terminalvariableregionconfersthemostsignificantdifferencebetweentheembryonicandadultisoformsandplaysaroleinmodulatingtheoverallconformationofTnTandtheinteractionswithTnI,TnC,andtropomyosin.Altogether,thedevelopmentalregulationofTnTgeneexpressionandalter-nativesplicingprovidesadaptivemodificationsforthecontractilityofcardiacandskeletalmuscles(Jinetal.,2008;WeiandJin,2011).

Thecellularmechanism(s)thatregulatesTnTalternativesplicingduringdevelopment remains to be established When cardiac TnT is naturallyexpressed in embryonic and neonatal skeletal muscles, itssplicing pattern

issynchronizedwiththedevelopmentalswitching intheheart(Jin,1996).Thisobservationindicatestheroleofasystemicbiologicalclockindepen-dentoftheverydifferentfunctionaladaptationsduringthepostnataldevel-opmentofcardiacandskeletalmuscles

MorerecentstudiesdemonstratedthatmicroRNAsplayaroleinlating striated muscle development and pathophysiologic remodeling(Tatsuguchi etal.,2007; vanRooijetal., 2009;Williams etal., 2009).Inadultmouseheart,thedeletionofmiR-208aincreasedtheexpressionsoffastTnIandfastTnT,whichcouldbecorrectedbyoverexpressionofmiR-499(vanRooijetal.,2009).Inskeletalmuscle,doubledeletionofmiR-208bandmiR-499leadtodecreasednumberofslowfibers(vanRooijetal.,2009)

Posttranslational modification of proteins provides rapid functionalregulations.TheposttranslationalregulationofTnThasbeenmainlyinves-tigatedfortherolesofphosphorylationandrestrictedproteolysis.Incontrast

tothechronicmechanismsofTnTisoformgeneregulationandalternativeRNAsplicing,themodificationofTnTstructurethroughphosphorylationand restrictedproteolysisareacute mechanismsfor themuscle to adapttofunctionaldemandsandstressconditions

VariousinvitroandexvivoexperimentalconditionsproducedtionofcardiacTnTatmultiplesites.Forexample,Thr197,Ser201,Thr206,andThr intheC-terminalregionofcardiacTnTwerereportedtobeprotein

phosphoryla-kinaseC(PKC)phosphorylationsites(Jideamaetal.,1996;NolandandKuo,1991; Sumandea et al., 2004) It was also reported that reactive oxygenspeciesexerted negative inotropic effect on rat cardiac myocytes throughphosphorylationofcardiacTnTatThr194andSer198byapoptosissignalingkinase1(Heetal.,2003).However,theseobservationsremaincontroversialandrecentmassspectrometrydatashowedthatadultcardiacTnTinratheartunderbasalin vivoconditionis100%monophosphorylatedatSer2,excludingalloftheother possiblesitesbeyondaminoacid 30(Marstonand Walker,2009;SanchoSolisetal.,2008).Consistently,constitutivephosphorylation

ofSer2attheNH2terminusofTnT wasreportedpreviously(Perry,1998)

Wefurther found that when embryonicmouse cardiacTnTwas pressedinadultheart,Ser25encodedbyexon5wasalsofullyphosphorylated(Zhangetal.,2011).ThehighlyefficientphosphorylationofSer2andSer25

overex-intheN-terminalvariableregionofcardiacTnTisaninterestingtionandfurtherstudiesarerequiredto identifythefunctionalsignificanceand the kinase(s) responsible, as well as the regulatory mechanisms thatsustaintheseN-terminalspecificphosphorylations

Severalover-andunderexpressionexperimentalmodelsdemonstratedthatmyofilamentincorporationdetermines thestoichiometryoftroponinsub-unitsincardiacmyocytesinvivo(Fengetal.,2009a).ItwasdeterminedinadultdogheartsthatTnTandTnIbothhaverapidturnoverratesincardiacmusclewithahalf-lifeofapproximately3.5daysthatwasshorterthanthe5.3dayshalf-lifeofTnC(Martin,1981).TheeffectiveremovalofsurplusTnTand TnIoverexpressed in transgenicmouse hearts under thestrongalphamyosinheavychainpromoterindicatesthatapotentproteolyticclearanceofnonmyofilament-incorporatedTnTandTnIiscriticaltomaintaintheinteg-rityandproteinstoichiometryofthethinfilamentregulatorysystem(Feng

etal.,2009a).Consistently,nosignificantcytoplasmicpoolofment-incorporatedcardiacTnT wasdetected(Martin,1981)

nonmyofila-It was reported that hypoxia in canine diaphragm muscle produced atruncated28-kDaTnTfragment(Simpsonetal.,2000).AcleavageofcardiacTnT by caspase 3 in apoptotic rat cardiomyocytes generated a 25-kDafragment with a deletion of the N-terminal variable region plus a partialdestructionofthemiddleconservedregion.Thisdestructivemodificationofcardiac TnTsignificantly decreased the maximummyosinATPase activityandmyofibrilforcegeneration(Communaletal.,2002)

ofcardiacmuscle.Differentfromthe destructivecleavagebycaspase3,thisrestrictive proteolysis selectively removes only the N-terminal variableregion and completely preserves the conserved regions of cardiac TnT(Figures 1and3) Therestrictive N-terminaltruncationof cardiacTnTisfoundinmouse(removingaminoacid1–71),rat,andpigheartsduringacuteischemia–reperfusion(Zhangetal.,2006)andpressureoverload(Fengetal.,

2008).Myofilament-associatedcalpainI(Golletal.,2003)hasbeenindicatedwitharoleintherestrictiveproteolysisofcardiacTnT(Zhangetal.,2006).TheselectiveremovaloftheN-terminalvariableregiondoesnotabolishthe function of TnT but alters the binding affinities for TnI, TnC, andtropomyosin(Biesiadeckietal.,2007).Studiesbyseverallaboratoriesshowedthat selective removal of the N-terminal variable region of TnT slightlydecreasedthemaximummyosinATPaseactivityandmyofibrilforcegener-ationwithout affectingthin filamentcalcium sensitivityand cooperativity(Chandraetal.,1999;Fujitaetal.,1992;Panetal.,1991)

Experiments using exvivoworking heartsfrom transgenic mice expressingN-terminaltruncatedcardiacTnTinthecardiacmuscleshowedmoderatelyreducedvelocityofventricularcontractionwithoutdecreasesinthe maximum left-ventricular pressure (Feng et al., 2008) However, thesmalldecreaseincontractilevelocitysignificantlyprolongedtherapidejec-tionphaseoftheventricularpumpingcycletoincreasestrokevolume.Thisnovel mechanism provides a plausible adaptation to compensate for thedecreaseinsystolicfunction againstworkloadsuch asthatoccursinmyo-cardialischemiaorventricularpressureoverload(Fengetal.,2008)

over-In vitroischemia–reperfusion-liketreatmentoftransgenicmousecardiacmuscle coexpressing fastskeletal muscle TnTand theendogenous cardiacTnT-inducedrestrictiveN-terminaltruncationsof bothcardiacTnT andfastTnTdespitetheirdifferentaminoacidsequencesatthecleavagesites(Zhang

et al., 2006) Therefore, the restrictive calpain I cleavage of cardiac TnTunderstressconditionsislikelyregulatedby thecalpainaccessibilityand/ormolecularconformationandcalpainsensitivityofTnTinthe myofilamentsotherthanbythe levelofcalpainactivationincardiomyocytes

The alternatively spliced exons and posttranslational modifications ofTnTareillustratedinFigure3.Table1summarizestheknownN-terminalvariationsandmodificationsofTnT,includingthetruncationbyrestrictiveproteolysis, along with their regulation and physiologic and pathologicrelevances

7 CONCLUSIONANDPERSPECTIVES

AcentralplayerinthethinfilamentCa2+regulatorysystemofstriatedmuscles,TnThasevolvedintothreefibertype-specificisoformgenesandmultiplealternativesplicingvariants withdiverged physiologicand patho-physiologicfunctions.Afterover fourdecades ofextensive studies carriedoutbyseveralgenerationsofdedicatedinvestigators,wearenowinposses-sionofagreatdealofknowledgefortheevolution,regulation,andfunction

of TnT However, many important questions concerning the molecularevolution,developmental andadaptiveregulations,andstructure–functionrelationshipsofTnTstillremainunanswered.Amongthekeyquestions,itwouldbeimportanttoknowthemechanismsthatregulatetheexpressionofTnT isoform genes and alternative splicing, the precise position of theN-terminal variable regionof TnT in muscle thin filament, how theN-terminalvariableregionproduceslong-rangeeffectsontheoverallconfor-mationandfunctionofTnT,whetherthephosphorylationintheN-termi-nalvariableregionregulatesthefunctionofcardiacTnT,theprimaryfunc-tionoftheTxelementintheavianpectoralmusclefastTnT,andthecellularmechanism thatregulates the restrictive N-terminal truncation of cardiacTnTinstressadaptations Moreresearchtoseekultimate answerstothesequestionswillenhanceourunderstandingofmusclecontraction

ACKNOWLEDGMENTS

I sincerely thank my current and past lab members and collaborators for their outstanding and continuing contributions to our troponin studies I want to also thank my mentors, especially Prof Jim Lin at the University of Iowa and Prof Larry Smillie at the University of Alberta, for their guidance and support during my scientific career This work was supported in part by grants from the National Institutes of Health (AR048816 and HL098945) to J.-P.J.

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ElizabethCalzada1,OumaOnguka1,StevenM.Claypool*

Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

*Corresponding author E-mail: sclaypo1@jhmi.edu.

1 Elizabeth Calzada and Ouma Onguka have contributed equally.

Contents

3.2 Mitochondrial Phosphatidylserine Decarboxylase (Psd) Pathway 38 3.3 PE as Precursor for Other Lipids and Substrate for Posttranslational Modifications 47

International Review of Cell and Molecular Biology, Volume 321

29

for important posttranslational modifications, influencing membrane topology, and promoting cell and organelle membrane fusion, oxidative phosphorylation, mitochon- drial biogenesis, and autophagy The importance of PE metabolism in mammalian health has recently emerged following its association with Alzheimer’s disease, Parkinson ’s disease, nonalcoholic liver disease, and the virulence of certain pathogenic organisms.

Phosphatidylethanolamine (PE) is a multifunctional phospholipidrequiredformammaliandevelopmentthatisessentialforavarietyofcellularprocesses.PEisanonbilayerformingphospholipidcontainingasmallpolarhead group diameter in proportionto its fatty-acid chains The intrinsicbiophysical properties of this cone-shaped lipid induces the formation ofhexagonalphaseswithinthemembraneand,insodoing,promotesmem-brane fusion and fission events, proteinintegration into membranes, andconformationalchangesinproteinstructure(DowhanandBogdanov,2009;vandenBrink-vanderLaanetal.,2004).PEisthesecondmostabundantphospholipidinthecell,comprising15–25%oftotalphospholipidsinmam-maliancells(Vance,2015).However,PEisnotsimplyapassivemembraneconstituentbutisfunctionallyassociatedwithproteinbiogenesisandactivity(Beckeretal.,2013;BogdanovandDowhan,1995,1998,1999),oxidativephosphorylation (Bottinger et al., 2012; Tasseva et al., 2013), autophagy(Ichimuraetal.,2000),membranefusion(Verkleijetal.,1984),mitochon-drial stability (Birner et al., 2001; Steenbergen et al., 2005; Storey et al.,

2001),andisanimportantprecursorofotherlipids(BremerandGreenberg,1961;MenonandStevens,1992)

FourbiosyntheticpathwaysproducePEinthecell,andnotably,oneofthesepathwaysresides withinthemitochondrion Theredundancyin PEbiosyntheticpathwaysisnotsufficienttoallowfornormalcellularfunction

intheabsenceof eitherofthetwomajorPE-producingpathways(Birner

etal.,2001; Fullerton etal.,2007; Steenbergen etal.,2005; Storeyet al.,

2001) This suggests that different pools of PE are required for specifiedpurposes in the cell The abundance of PE varies in the membranes ofdifferent tissues and cells in mammals and organelles of both yeast andmammals (Bleijerveld et al., 2007; Colbeau et al., 1971; Nelson, 1967;Van Deenen and De Gier, 1974; Vance, 2015; Zinser et al., 1991) Thisreviewwill focus on thenumerous biological functionsconferred bythe

intrinsic properties of PE Recently, disturbances in PE metabolism havebeenimplicated inbothchronicandinfectiousdisease(Chen etal.,2010;Deleault et al., 2012; Nesic et al., 2012; Wang et al., 2014) PhenotypiccharacterizationofthecellbiologyofthesediseasesusingavarietyofmodelorganismscollectivelyrevealsavitalroleforPEinmammalianhealth.

Biologicalmembranesformthebarriersthatdefinecellsandseparatespecifiedcellularfunctionsintodistinctbutinterconnectedcompartments.Beyondtheirabilitytodelineatedifferentcellandorganellemorphologies,cellularmembranesarealsomultifunctionalplatformsinvolvedinsignaling,regulation of solute, metabolite, and protein transport; and are necessarymediumsforproteinsthatrequireahydrophobicenvironmentforenzymaticfunctionandstability.Thewiderangeofbiologicalprocessesmediatedacrossmembranescanbeattributedtothemixtureofproteins,lipids,andcarbo-hydrates thatconcomitantly interactto give riseto specialized membraneenvironments.Greaterthan1000lipidspeciesarepresentinthecellandover30%of anorganism’stranslated genomeisdedicatedto theproductionofalphahelicalmembraneproteins(StevensandArkin,2000;Sudetal.,2007).Withrespecttocarbohydrates,thereareinnumerablestructures,conforma-tions, and combinations of sugars that can be formed in the cell, whichfurtheraddtothediversityofthemembraneenvironment

Themajorclassesoflipidsinthecellincludephospholipids,sterols,andsphingolipids.Therigidity,thickness,hydrophobicity,andfunctionofcel-lularmembranesaredependentuponthepresenceandrelativeabundanceofthesedifferentclassesoflipid.Glycerophospholipids,sterols,andsphingoli-pidscomprise∼75%,12–14%,and8–12%oflipidsinthecell,respectfully(Drin,2014).Phospholipidsareaccountablefortheformationofthemem-brane bilayer;thedifferent classes ofphospholipidin a membrane furthermodulatemembraneidentityandfluidity.Sterols,cholesterolinmammals,andergosterol inyeast,decreasecellpermeability byincreasingmembranethicknessandrigidity.Interestingly,thelevelofcholesterolishighestattheplasmamembrane(20–40%),moderateintheGolgi(8%)andendoplasmicreticulum (ER) (6%), and scarcely detected in mitochondria (4%) Thepresenceofsterolsinconjunction withsphingolipidsontheplasmamem-braneisimportantforcell-to-cellsignalingevents.AsPEisthefocusofthisreview,thebiologicalimportanceofsterolsandsphingolipidsisbeyondour

scopebuthasbeendiscussedinextensivedetailinseveralfantasticreviews(Cowartand Obeid,2007; Espenshade and Hughes,2007; Futerman andHannun, 2004; Hannun and Obeid, 2008; Mouritsen and Zuckermann,2004;Ohvo-Rekilaetal.,2002;VanceandVandenBosch,2000).

Phospholipids are the predominant lipid components of most cellularmembranesandaretypicallycharacterizedbyaglycerolbackbonecontain-ingtwoester linkedfattyacid chainsat thesn-1 and sn-2 positionsand aphosphatehead groupat thesn-3 position(Figure1;VanDeenen andDeGier,1974).Theheadgroupattachedatthesn-3positiondistinguishesthedifferentclassesofphospholipidwhilesubspeciesofeachphospholipidclassalsoarisefromdifferencesintheiracylchaincomposition.Themajorgly-cerophospholipidsinthecellincludephosphatidylcholine(PC),PE,phos-phatidylserine(PS),phosphatidylinositol(PI),phosphatidicacid(PA),phos-phatidylglycerol (PG), and cardiolipin (CL) The distribution of eachphospholipidcanvaryondifferentleafletsofthemembranebilayer,betweenorganellar membranes, and by cell type and organism (Bretscher, 1972;

Phosphatidylethanolamine

sn-3 sn-1

sn-2

O O O

O NO O O O OH OH

O OH

OH OH HO

OH

O O O

O

O OO

R R

O O

O O

R

P

Phosphatidylethanolamine Phosphatidic acid Phosphatidylcholine Phosphatidylserine Phosphatidylglycerol Phosphatidylinositol

Cardiolipin

Figure 1 The glycerophospholipids (A) Diagram of phosphatidylethanolamine structure The spheres represent different atoms present in the phospholipid structure tan: carbon, red: oxygen, orange: phosphate, and blue: nitrogen (hydrogen atoms are not represented) (B) General glycerophospholipid structure Fatty acids are linked to the glycerol backbone at the sn-1 and sn-2 positions while the phosphate headgroup is linked at the sn-3 position Different variations of headgroups are shown (for cardiolipin, R indicates additional acyl groups attached at these positions).

Colbeauetal.,1971;VanDeenenandDeGier,1974;Zinseretal.,1991).Atypical mammalian cell contains approximately45–55% PC, 15–25% PE,10–15%PI,5–10%PS,2–5%CL,and1–2%PA(Vance,2015).PCisfoundequallydistributedacrosscellularmembraneswhilePSandPEareprimarilyfound on theinner but not theouterleaflet of theplasma membrane.Inaddition,PE and CL areparticularlyabundant intheinner membraneofmitochondria(Vance,2015).Further,CLisabsentinothernonmitochon-drialmembranesofthecell.Enrichmentoflipidsindifferentcornersofthecellcan beattributed tonumerous factorsincludingtheirdifferent sitesofsynthesis, interconversion,acyl chainremodeling, traffickingmechanisms,anddegradation.

TheERistheprimarysiteofsynthesisforthemajorityoflipidsinthecell.ManyessentialcellprocessesaresequesteredintheER,andassuch,thisorganelle has compartmentalized some of these functions into distinctdomains (Vance, 2014) Initial studies on the subcellular localization ofphospholipidsynthesizingenzymeslocalizedthemtomicrosomalfractions,butsomemicrosomalvesiclescontaininghighPSsynthaseactivitywerenotenriched for the known ER-specific marker, NADPH-cytochrome-creductase (Dennis and Kennedy, 1972; van Golde et al., 1974; Zinser

et al., 1991) Subsequently, the mitochondrial-associated membrane(MAM) of the ER was identified as a distinct site of lipid synthesis thatharborsmultiplephospholipidbiosyntheticenzymes,includingPSsynthase,

PIsynthase,andPEmethyltransferase(Cuietal.,1993;Gaiggetal.,1995;Vance,1990).Additionally,theMAMisanimportantdepotforthetransport

ofsubstratesrequired forthebiosynthesisofPE,PA,CDP-DAG,PG,and

CL inmitochondria (transportmechanisms for PS and PEare covered inSections3.2.3and3.2.4)althoughCDP-DAGandPAcanbesynthesizedinboth theER and mitochondria (Chen etal., 2006; Colbeau etal., 1971;Kuchleretal.,1986;Tamuraetal.,2013;vanGoldeetal.,1974;WirtzandZilversmit,1968;Yetetal.,1993)

There are four independent pathwaysby which PEis generated ineukaryoticcells(Figure2).TheCDP-ethanolaminepathway(HjelmstadandBell,1991;Ishidateetal.,1985;Mancinietal.,1999;vanHellemondetal.,1994;WittenbergandKornberg,1953),acylationoflyso-PE(Riekhofetal.,

ERMES complex

EMC complex

PE

MICOS complex