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ReviewMechanismsofwater-holdingcapacityofmeat:TheroleofpostmortembiochemicalandstructuralchangesElisabethHu-Lonergan*,StevenM.
LonerganDepartmentofAnimalScience,IowaStateUniversity,Ames,IA50011,USAAbstractUnacceptablewater-holdingcapacitycoststhemeatindustrymillionsofdollarsannually.
However,limitedprogresshasbeenmadetowardunderstandingthemechanismsthatunderliethedevelopmentofdriporpurge.
ItisclearthatearlypostmortemeventsincludingrateandextentofpHdecline,proteolysisandevenproteinoxidationarekeyininuencingtheabilityofmeattoretainmoisture.
Muchofthewaterinthemuscleisentrappedinstructuresofthecell,includingtheintra-andextramyobrillarspaces;therefore,keychangesintheintracellulararchitectureofthecellinuencetheabilityofmusclecellstoretainwater.
Asrigorpro-gresses,thespaceforwatertobeheldinthemyobrilsisreducedanduidcanbeforcedintotheextramyobrillarspaceswhereitismoreeasilylostasdrip.
Lateralshrinkageofthemyobrilsoccurringduringrigorcanbetransmittedtotheentirecellifproteinsthatlinkmyobrilstogetherandmyobrilstothecellmembrane(suchasdesmin)arenotdegraded.
Limiteddegradationofcytoskeletalproteinsmayresultinincreasedshrinkingoftheoverallmusclecell,whichisultimatelytranslatedintodriploss.
Recentevidencesuggeststhatdegradationofkeycytoskeletalproteinsbycalpainproteinaseshasaroletoplayindeterminingwater-holdingcapacity.
Thisreviewwillfocusonkeyeventsinmusclethatinuencestructuralchangesthatareassociatedwithwater-holdingcapacity.
2005ElsevierLtd.
Allrightsreserved.
Keywords:Water-holdingcapacity;Driploss;Calpain;Proteolysis;pHContents1.
Introduction1952.
Structureofskeletalmuscle1953.
Locationofwaterinmuscle1954.
Physical/biochemicalfactorsinmusclethataffectwater-holdingcapacity1964.
1.
Netchargeeffect1964.
2.
Geneticfactors1964.
3.
Stericeffects1975.
Postmortemproteolysis1985.
1.
Calpainsystem1985.
2.
Calpastatin2005.
3.
Roleofproteinoxidation.
2006.
Summary202Acknowledgments202References2020309-1740/$-seefrontmatter2005ElsevierLtd.
Allrightsreserved.
doi:10.
1016/j.
meatsci.
2005.
04.
022*Correspondingauthor.
Tel.
:+15152949125;fax:+15152949143.
E-mailaddress:elonerga@iastate.
edu(E.
Hu-Lonergan).
www.
elsevier.
com/locate/meatsciMEATSCIENCEMeatScience71(2005)194–2041.
IntroductionTheabilityoffreshmeattoretainmoistureisargu-ablyoneofthemostimportantqualitycharacteristicsofrawproducts.
Ithasbeenestimatedthatasmuchas50%ormoreoftheporkproducedhasunacceptablyhighpurgeordriploss(Kauman,Cassens,Scherer,&Meeker,1992;Stetzer&McKeith,2003).
Productweightlossesduetopurgecanaverageasmuchas1–3%infreshretailcuts(Oer&Knight,1988a)andcanbeashighat10%inPSEproducts(Melodyetal.
,2004).
Inadditiontothelossofsalableweight,purgelossalsoentailsthelossofasignicantamountofpro-tein(Oer&Knight,1988a;Oer&Knight,1988b).
Onaverage,purgecancontainapproximately112mgofproteinpermilliliterofuid;mostlywater-soluble,sar-coplasmicproteins(Savage,Warriss,&Jolley,1990).
Themajorityofwaterinmuscleisheldeitherwithinthemyobrils,betweenthemyobrilsandbetweenthemyobrilsandthecellmembrane(sarcolemma),be-tweenmusclecellsandbetweenmusclebundles(groupsofmusclecells).
Oncemuscleisharvestedtheamountofwaterandlocationofthatwaterinmeatcanchangedependingonnumerousfactorsrelatedtothetissueit-selfandhowtheproductishandled(Honikel,2004;Honikel&Kim,1986).
Overtheyearstherehavebeennumerousreviewsdevotedtofactorsthatinuencewater-holdingcapacity(Hamm,1986;Honikel,2004;Honikel&Kim,1986;Oer&Knight,1988a;Oer&Knight,1988b).
Thisreviewwillgiveanoverviewsomeofthemajorfactorsinuencingwater-holdingcapacityandwillspecicallyfocusonrecentdevelopmentsinunderstandinghowchangesinthestructureofpostmor-temmusclecaninuencedriploss.
2.
StructureofskeletalmuscleSkeletalmusclehasaverycomplexorganization,inparttoallowmuscletoecientlytransmitforceorig-inatinginthemyobrilstotheentiremuscleandulti-matelytothelimborstructurethatismoved.
Arelativelythicksheathofconnectivetissue,theepimy-sium,enclosestheentiremuscle.
Inmostmuscles,theepimysiumiscontinuouswithtendonsthatlinkmus-clestobones.
Themuscleissubdividedintobundlesorgroupingsofmusclecells.
Thesebundles(alsoknownasfasciculi)aresurroundedbyanothersheathofconnectivetissue,theperimysium.
Athinlayerofconnectivetissue,theendomysium,surroundsthemusclecellsthemselves.
Theendomysiumliesabovethemusclecellmembrane(sarcolemma)andconsistsofabasementmembranethatisassociatedwithanouterlayer(reticularlayer)thatissurroundedbyalayerofnecollagenbrilsimbeddedinamatrix(Bailey&Light,1989).
Whenmusclecellsareviewedunderamicroscope,veryregulartransversestriationsareseen.
Thesestria-tionsarecausedbyspecializedcontractileorganelles,themyobrils,foundinmuscle.
Thestriationsarisefromalternating,proteindenseA-bandsandlessdenseI-bandswithinthemyobril.
BisectingtheI-bandsaredarklinesknownasZ-lines.
TheareabetweentwoZ-linesisasarcomere.
ThelessdenseI-bandismadeupprimarilyofthinlamentswhiletheA-bandismadeupofthicklamentsandsomeoverlappingthinla-ments(Goll,Robson,&Stromer,1984).
Thebackboneofthethinlamentsismadeupprimarilyoftheproteinactinwhilethelargestcomponentofthethicklamentistheproteinmyosin.
Myosinconsistsofatailorrodre-gionthatformsthebackboneofthethicklamentandaglobularheadregionthatextendsfromthethickla-mentandinteractswithactininthethinlament.
Therigorcomplexformedbytheinteractionofmyosinandactinisoftenreferredtoasactomyosin.
Inelectronmicrographimagesofcontractedmuscleorofpost-rigormuscletheactomyosinlooksverymuchlikecross-bridgesbetweenthethickandthinlaments,indeed,itisoftenreferredtoassuch.
Inpostmortemmusclethesebondsbecomeirreversibleandarealsoknownasrigorbonds.
Theglobularheadofmyosinalsohasenzymaticactivity;itcanhydrolyzeATPandliberateenergy.
Inlivingmuscleduringcontraction,theATPaseactivityofmyosinprovidesenergyformyosinboundtoactintoswivelandultimatelypullthethinlamentstowardthecenterofthesarcomere.
Thisshortensthemyobril,themusclecellandeventuallythemuscletoproducecontraction.
ThemyosinandactincandisassociatewhenanewmoleculeofATPisboundtothemyosinhead(Golletal.
,1984).
Inpost-rigormuscle,thesupplyofATPisdepleted,resultingintheactomyosinbondsbecomingmoreorlesspermanent.
Withinthestructureofthemuscle,thereareseveral''compartments''fromwhichdripcouldoriginate.
Thesecouldincludethespacewithinthemyobril,theintra-cellularspaceoutsidethemyobrilandtheextracellularspace,includingthespacebetweenthemusclebundles.
Lossofwaterfromeachofthesecompartmentsmayin-volveslightlydierentmechanisms.
Inaddition,lossofwaterfromeachofthesecompartmentsmayoccuratdierenttimesduringstorage.
Forexample,itwouldbeeasytoenvisionthatwaterfoundintheextracellularspacescouldbelostmoreeasily,withdeepercompart-mentstakingmoretimeorforcetobereleased.
3.
LocationofwaterinmuscleLeanmusclecontainsapproximately75%water.
Theothermaincomponentsincludeprotein(approximately20%),lipidsorfat(approximately5%),carbohydrates(approximately1%)andvitaminsandminerals(oftenE.
Hu-Lonergan,S.
M.
Lonergan/MeatScience71(2005)194–204195analyzedasash,approximately1%).
Themajorityofwaterinmuscleisheldwithinthestructureofthemuscleandmusclecells.
Specically,withinthemusclecell,waterisfoundwithinthemyobrils,betweenthemyo-brilsthemselvesandbetweenthemyobrilsandthecellmembrane(sarcolemma),betweenmusclecellsandbe-tweenmusclebundles(groupsofmusclecells)(Oer&Cousins,1992).
Waterisadipolarmoleculeandassuchisattractedtochargedspecieslikeproteins.
Infact,someofthewaterinmusclecellsisverycloselyboundtoprotein.
Bydenition,boundwateriswaterthatexistsinthevicinityofnon-aqueousconstituents(likeproteins)andhasreducedmobility,i.
e.
doesnoteasilymovetoothercompartments.
Thiswaterisveryresistanttofreezingandtobeingdrivenobyconventionalheating(Fen-nema,1985).
Trueboundwaterisaverysmallfractionofthetotalwaterinmusclecells;dependingonthemea-surementsystemused,approximately0.
5gofwaterpergramofproteinisestimatedtobetightlyboundtopro-teins.
Sincethetotalconcentrationofproteininmuscleisapproximately200mg/g,thisboundwateronlymakesuplessthanatenthofthetotalwaterinmuscle.
Theamountofboundwaterchangesverylittleifatallinpost-rigormuscle(Oer&Knight,1988b).
Anotherfractionofwaterthatcanbefoundinmus-clesandinmeatistermedentrapped(alsoreferredtoasimmobilized)water(Fennema,1985).
Thewatermole-culesinthisfractionmaybeheldeitherbysteric(space)eectsand/orbyattractiontotheboundwater.
Thiswaterisheldwithinthestructureofthemusclebutisnotboundpersetoprotein.
Inearlypostmortemtissue,thiswaterdoesnotowfreelyfromthetissue,yetitcanberemovedbydrying,andcanbeeasilyconvertedtoiceduringfreezing.
Entrappedorimmobilizedwaterismostaectedbytherigorprocessandtheconversionofmus-cletomeat.
UponalterationofmusclecellstructureandloweringofthepHthiswatercanalsoeventuallyescapeaspurge(Oer&Knight,1988b).
Freewateriswaterwhoseowfromthetissueisunimpeded.
Weaksurfaceforcesmainlyholdthisfrac-tionofwaterinmeat.
Freewaterisnotreadilyseeninpre-rigormeat,butcandevelopasconditionschangethatallowtheentrappedwatertomovefromthestruc-tureswhereitisfound(Fennema,1985).
Themajorityofthewaterthatisaectedbytheprocessofconvertingmuscletomeatistheentrapped(immobilized)water.
Maintainingasmuchofthiswateraspossibleinmeatisthegoalofmanyproces-sors.
Someofthefactorsthatcaninuencethereten-tionofentrappedwaterincludemanipulationofthenetchargeofmyobrillarproteinsandthestructureofthemusclecellanditscomponents(myobrils,cytoskeletallinkagesandmembranepermeability)aswellastheamountofextracellularspacewithinthemuscleitself.
4.
Physical/biochemicalfactorsinmusclethataectwater-holdingcapacity4.
1.
NetchargeeectDuringtheconversionofmuscletomeat,lacticacidbuildsupinthetissueleadingtoareductioninpHofthemeat.
OncethepHhasreachedtheisoelectricpoint(pI)ofthemajorproteins,especiallymyosin(pI=5.
4),thenetchargeoftheproteiniszero,meaningthenum-bersofpositiveandnegativechargesontheproteinsareessentiallyequal.
Thesepositiveandnegativegroupswithintheproteinareattractedtoeachotherandresultinareductionintheamountofwaterthatcanbeat-tractedandheldbythatprotein.
Additionally,sincelikechargesrepel,asthenetchargeoftheproteinsthatmakeupthemyobrilapproacheszero(diminishednetnega-tiveorpositivecharge)repulsionofstructureswithinthemyobrilisreducedallowingthosestructurestopackmorecloselytogether.
Theendresultofthisisareduc-tionofspacewithinthemyobril.
PartialdenaturationofthemyosinheadatlowpH(especiallyifthetemper-atureisstillhigh)isalsothoughttoberesponsibleforalargepartoftheshrinkageinmyobrillarlatticespacing(Oer,1991).
4.
2.
GeneticfactorsAcceleratedpHdeclineandlowultimatepHarere-latedtothedevelopmentoflowwater-holdingcapacityandunacceptablyhighpurgeloss.
RapidpHdeclineresultinginultimateornearultimatepHwhilethemus-cleisstillwarmcausesthedenaturation(lossoffunc-tionalityandwaterbindingability)ofmanyproteins,includingthoseinvolvedinbindingwater.
Themostse-verepurgeordriplossisoftenfoundinPSE(Pale,Soft,andExudative)productfrompigsthathaveinheritedamutationintheryanodinereceptor/calciumreleasechannel(halothanegene)inthesarcoplasmicreticulum(Fujiietal.
,1991).
Thismutationresultsinimpairmentoftheabilityofthischanneltocontrolcalciumreleaseintothesarcoplasmofthemusclecell,particularlyunderperiodsofphysicalstress.
AcceleratedreleaseofcalciumcausesrapidcontractionandanincreaseintherateofmusclemetabolismandintherateofpHdecline(Ben-dall&Wismer-Pedersen,1962;Lundstrom,Essen-Gustavsson,Rundgren,Edforslilja,&Malmfors,1989).
Thisparticularmutationinthehalothanegenecanbeidentiedinparentstock.
Becauseacommercialtestforthismutationexists,theUnitedStatesindustryhasvirtuallyeliminatedthisgeneinmostcommercialherds.
TheHalothanegeneisbutoneexampleofacondi-tionthatcanresultinPSE.
OtherfactorscancausePSEmeattooccur.
Beforeharvest,short-termstressinnormalanimalscanacceleratetheirmetabolismenough196E.
Hu-Lonergan,S.
M.
Lonergan/MeatScience71(2005)194–204thatthepostmortemmetabolisminthemuscleisaccel-erated,causingamorerapidpHdeclinethanisseeninnon-stressedanimals.
WhiletheconditionmaynotbeassevereasthatcausedbytheHalothanegene,pro-teindenaturationdoesoccur,anddriplossescanbegreaterthaninmusclethathasanormal,slowerrateofpHdecline.
ItshouldbenotedthatwhilethepHofthesemusclesfallsfasterthannormal,theultimatepHmaynotbebelownormalranges(Rosenvold&Ander-sen,2003).
Othermetabolicstateandexistingconditionsofmus-cleoftendirecttheextentofpHdeclineinpostmortemmuscle.
Manystudieshavereportedtheeectoftreat-menttodecreaseglycogencontentinmuscletominimizelactateaccumulationinpostmortemmuscle(reviewedbyRosenvold&Andersen,2003).
Geneticfactorsinu-encingbasalmetabolismclearlyhavethepotentialtosimilarlyaectlactateaccumulationandextentofpHdecline.
Thediscovery(Milanetal.
,2000)ofanon-con-servedsubstitutioninproteinkinaseadenosinemono-phosphate-activatedc3-subunitgene(PRKAG3)hasexplainedthedominantmutation(denotedRN)thataccountedforlargedierencesinmeatqualityandpro-cessingyieldintheHampshirepigbreed(Monin&Sel-lier,1985).
Thesubstitution(R200Q)inthePRKAG3genecausesa70%increaseinmuscleglycogeninRNhomozygousandheterozygouspigs.
Thisincreaseinglycogendirectlyresultsingreaterproductionoflactateinpostmortemmuscle,alowerultimatepHandpoorerwaterholdingcapacityinfreshpork.
ThePRKAG3geneencodesoneisoformofoneoftheregulatorysubunits(c)inmammalianadenosinemono-phosphate(AMP)-activatedproteinkinase(AMPK).
Whensubjectedtonutritionalorenvironmentalstress,theAMP/ATPratioofeukaryoticcellswillrise,trigger-ingthe''AMPKcascade'',stimulatingthecellstocon-serveenergy(Thornton,Snowden,&Carling,1998)andtobeginATPsynthesis(Hardie,Carling,&Carl-son,1998).
Theprecisefunctionsofthebandcregula-torysubunitsofAMPKarestillunknown;however,bothareknowntobeimportantforkinaseactivity(Hardie&Carling,1997).
ThecregulatorysubunitalsomaybeinvolvedwiththeAMP-bindingsiteoftheAMPKheterotrimericcomplex(Cheung,Salt,Davies,Hardie,&Carling,2000).
WhilethedominantRNmutationthatisfoundintheHampshirebreedofpigsisanonconservativesubsti-tution(R200Q)inthePRKAG3genethatcauseshighglycogencontentinskeletalmuscleresultingindetri-mentaleectsonprocessingyield(Milanetal.
,2000),otheralleleswithinthesamegeneareassociatedwithlowermuscleglycogencontent,andimprovedmeatqualitytraits(Ciobanuetal.
,2001).
Fromthreemis-sensemutationsidentied(T30N,G52R,I199V)inpor-cinePRKAG3,least-squaresestimatesofgenotypemeansacrossvecommercialpigbreedsdemonstratesignicanteectsbetweentheanalyzedsubstitutionsandproductpHandMinoltaLvalues.
Inthesamestudy,onlyfourhaplotypeswerefoundinthevecom-mercialpopulations.
OneofthosehaplotypeswasfoundtobesignicantlyassociatedwithhigherpHanddarkermeatcolor(T30-G52-I199),whileasecondhadinterme-diatepHandcolorandthetwoothershadthelowestpHandthepoorestcolor(Ciobanuetal.
,2001).
TheseobservationsestablishageneticbasisforvariationinultimatepHacrossmanybreedsandcommerciallines,notjustHampshirepigs.
4.
3.
StericeectsMyobrilsmakeupalargeproportionofthemusclecell.
Theseorganellesconstituteasmuchas82–87%ofthevolumeofthemusclecell.
Asmentionedpreviously,muchofthewaterinsidelivingmusclecellsislocatedwithinthemyobril.
Infact,itisestimatedthatasmuchas85%ofthewaterinamusclecellisheldinthemyo-brils.
Muchofthatwaterisheldbycapillaryforcesarisingfromthearrangementofthethickandthinla-mentswithinthemyobril.
Inlivingmuscle,ithasbeenshownthatsarcomeresremainisovolumetricduringcontractionandrelaxation(Millman,Racey,&Matsu-bara,1981;Millman,Wakabayashi,&Racey,1983).
Thiswouldindicatethatinlivingmuscletheamountofwaterwithinthelamentousstructureofthecellwouldnotnecessarilychange.
However,thelocationofthiswatercanbeaectedbychangesinvolumeasmuscleundergoesrigor.
Asmusclegoesintorigor,cross-bridgesformbetweenthethickandthinlaments,thusreducingavailablespaceforwatertoreside(Oer&Trinick,1983).
IthasbeenshownthatasthepHofporcinemuscleisreducedfromphysiologicalvaluesto5.
2–5.
5(neartheisoelectricpointofmyosin),thedis-tancebetweenthethicklamentsdeclinesanaverageof2.
5nm(Diesbourg,Swatland,&Millman,1988).
Thisdeclineinlamentspacingmayforcesarcoplasmicuidfrombetweenthemyolamentstotheextramyobrillarspace.
Indeed,ithasbeenhypothesizedthatenoughuidmaybelostfromtheintramyobrillarspacetoin-creasetheextramyobrillarvolumebyasmuchas1.
6timesmorethanitspre-rigorvolume(Bendall&Swat-land,1988).
Duringthedevelopmentofrigor,thediameterofmus-clecellshasbeenshowntodecrease(Hegarty,1970;Swatland&Belfry,1985)andislikelytheresultoftrans-mittalofthelateralshrinkageofthemyobrilstotheen-tirecell(Diesbourgetal.
,1988).
Additionally,duringrigordevelopmentsarcomerescanshorten;thisalsore-ducesthespaceavailableforwaterwithinthemyobril.
Infact,ithasbeenshownthatdriplosscanincreaselin-earlywithadecreaseinthelengthofthesarcomeresinmusclecells(Honikel,Kim,Hamm,&Roncales,1986).
Morerecently,highlysensitiveloweldnuclearmagneticE.
Hu-Lonergan,S.
M.
Lonergan/MeatScience71(2005)194–204197resonance(NMR)studieshavebeenusedtogainamorecompleteunderstandingoftherelationshipbetweenmusclecellstructureandwaterdistribution(Bertram,Purslow,&Andersen,2002).
Thesestudieshavesug-gestedthatwithinthemyobril,ahigherproportionofwaterisheldintheI-bandthaninthemoreproteindenseA-band.
Thisobservationmayhelpexplainwhyshortersarcomeres(especiallyincold-shortenedmuscle)areoftenassociatedwithincreaseddriplosses.
Asthemyo-brilshortensandrigorsetsin,theshorteningofthesarcomerewouldleadtoshorteningandsubsequentlow-eringofthevolumeoftheI-bandregioninmyobrils.
Lossofvolumeinthismyobrillarregion(wheremuchwatermayreside),combinedwiththepH-inducedlateralshrinkageofthemyobrilcouldleadtoexpulsionofwaterfromthemyobrillarstructureintotheextramyo-brillarspaceswithinthemusclecell(Bendall&Swatland,1988).
Infact,recentNMRstudiessupportthishypothesis(Bertrametal.
,2002).
Itisthuslikelythatthegradualmobilizationofwaterfromtheintramyobr-illarspacestotheextramyobrillarspacesmaybekeyinprovidingasourceofdrip.
Allofthepreviouslymentionedprocessesinuencetheamountofwaterinthemyobril.
Itisimportanttonotethatshrinkageofthemyobrillarlatticealonecouldnotberesponsibleforthemovementofuidtotheextracellularspaceandultimatelyoutofthemuscle.
Themyobrilsarelinkedtoeachotherandtothecellmembraneviaproteinaceousconnections(Wang&Ra-mirez-Mitchell,1983).
Theseconnections,iftheyaremaintainedintactinpostmortemmuscle,wouldtransferthereductionindiameterofthemyobrilstothemusclecell(Diesbourgetal.
,1988;Kristensen&Purslow,2001;Melodyetal.
,2004;Morrison,Mielche,&Purslow,1998).
Myobrilshrinkagecanbetranslatedintocon-strictionoftheentiremusclecell,thuscreatingchannelsbetweencellsandbetweenbundlesofcellsthatcanfun-neldripoutoftheproduct(Oer&Knight,1988).
Extracellularspacearoundmuscleberscontinuallyin-creasesupto24hpostmortem,butgapsbetweenmuscleberbundlesdecreaseslightlybetween9and24hpost-mortem,perhapsduetouidoutowfromthesemajorchannels(Schafer,Rosenvold,Purslow,Andersen,&Henckel,2002).
Theselinkagesbetweenadjacentmyo-brilsandmyobrilsandthecellmembranearemadeupofseveralproteinsthatareassociatedwithintermediatelamentstructuresandstructuresknownascostameres.
Costameresprovidethestructuralframeworkresponsi-bleforattachingthemyobrilstothesarcolemma.
Pro-teinsthatmakeup,orareassociatedwiththeintermediatelamentsandcostameresinclude(amongothers)desmin,lamin,andsynemin.
dystrophin,talinandvinculin(Greaser,1991).
Ifcostamericlinkagesre-mainintactduringtheconversionofmuscletomeat,shrinkageofthemyobrilsasthemusclegoesintorigorwouldbetransmittedtotheentirecellviatheseprotein-aceouslinkagesandwouldultimatelyreducevolumeofthemusclecellitself(Kristensen&Purslow,2001;Mel-odyetal.
,2004;Oer&Knight,1988b).
Thus,therigorprocesscouldresultinmobilizationofwateroutnotonlyoutofthemyobril,butalsooutoftheextramyo-brilspacesastheoverallvolumeofthecelliscon-stricted(Fig.
1).
Infact,reductioninthediameterofmusclecellshasbeenobservedinpostmortemmuscle(Oer&Cousins,1992).
Thiswaterthatisexpelledfromthemyobrilandultimatelythemusclecelleventuallycollectsintheextracellularspace.
Severalstudieshaveshownthatgapsdevelopbetweenmusclecellsandbe-tweenmusclebundlesduringthepost-rigorperiod(Oer&Cousins,1992;Oeretal.
,1989).
Thesegapsbetweenmusclebundlesaretheprimarychannelsbywhichpurgeowsfromthemeat;someinvestigatorshaveactuallytermedthem''dripchannels''.
5.
PostmortemproteolysisSinceithasbeenhypothesizedthatproteinaceouslinkageswithinthecellmayinuencedripproduction,itisimportanttoinvestigatethefactorsregulatingpost-mortemproteolysisofkeyproteins.
5.
1.
CalpainsystemTheendogenouscalpainsystemplaysamajorroleinregulatingproteolysisofmuscleproteinsunderpost-mortemconditions(Koohmaraie,Schollmeyer,&Dut-son,1986;Lonergan,Hu-Lonergan,Wiegand,&Kriese-Anderson,2001;Maddock,Hu-Lonergan,Rowe,&Lonergan,2005).
Proteinsthataresubstratesofcalpainsincludeproteinslikedesmin,synemin,talinandvinculinthatformthecytoskeletalframeworkofthemusclecell(Bilaketal.
,1998;Evans,Robson,&ProteolysisNoProteolysisFig.
1.
Schematicoverviewingthepotentialchangesinmusclecelldiameterduringpostmortemagingasinuencedbyproteolysis.
198E.
Hu-Lonergan,S.
M.
Lonergan/MeatScience71(2005)194–204Stromer,1984;OShea,Robson,Huiatt,Hartzer,&Stromer,1979;Schmidt,Zhang,Lee,Stromer,&Rob-son,1999).
Thecalpainsystemiscomposedofseveralisoformsofcalcium-dependentcysteineproteases(calpains),andtheirspeciccompetitiveinhibitor,calpastatin(Goll,Thompson,Li,Wei,&Cong,2003).
Thetwobest-characterizedisoformsofcalpainsarel-calpainandm-calpain,whichbothdegradethesamespecicsetofmyobrillarandcytoskeletalproteinsthatarede-gradedasmuscleisconvertedintomeat(Geesink&Koohmaraie,1999;Hu-Lonerganetal.
,1996).
Asthepostmortemperiodprogresses,dramaticchangesoccurwithinthemicroenvironmentofthemusclecell(e.
g.
,de-clineinpH,increaseinionicstrength)(Winger&Pope,1981)thatcanaectcalpainactivity(Hu-Lonerganetal.
,1996;Maddocketal.
,2005).
Asmuscleisconvertedtomeat,manychangesoccur,including:(1)agradualdepletionofavailableenergy,(2)ashiftfromaerobictoanaerobicmetabolismfavoringtheproductionoflac-ticacid,resultinginthepHofthetissuedecliningfromnearneutralityto5.
4–5.
8,(3)ariseinionicstrength,inpart,becauseoftheinabilityofATP-dependentcalcium,sodium,andpotassiumpumpstofunction,and(4)anincreasinginabilityofthecelltomaintainreducingcon-ditions.
Bothl-calpainandm-calpainhaveslowerratesofactivityagainstmyobrillarproteinsubstratesatpHvaluesandionicstrengthssimilartothosefoundinpost-mortemmuscle(Geesink&Koohmaraie,1999;Hu-Lonergan&Lonergan,1999;Kendall,Koohmaraie,Arbona,Williams,&Young,1993).
AlterationsinpHand/orionicstrengthsmaycauseconformationalchangesthatallowanincreaseinthehydrophobicityandaggregationoftheenzyme.
Likewise,pH/ionicstrengthchangesmayaltertheconformationofsubstrateproteinsandrenderthemlesssusceptibletocleavagebyl-calpain(Hu-Lonergan&Lonergan,1999).
Experimentalevidenceexiststhatsupportstheideathatthatproteolysisofkeycytoskeletalproteinssuchastheintermediatelamentprotein,desminmaybere-latedtodripproduction.
Theseproteinshavebeenshowntobedegradedasearlyas45minto6hpostmor-teminsomemuscles(Melodyetal.
,2004).
Degradationoftheseproteinsatsuchanearlytimepostmortemwouldcertainlyallowwaterthatisexpelledfromtheintramyobrillarspacestoremaininthecellforalongerperiodoftime.
Thecorollarytothisisthatreduceddeg-radationofproteinsthattiethemyobriltothecellmembrane(suchasdesmin)resultsinincreasedshrink-ingofthemusclecell,whichisultimatelytranslatedintodriploss.
Certainly,desminisnottheonlycandidateproteintobeinvolved.
Otherproteinsassociatedwithintermediatelamentsandwiththecostamereshavebeenimplicated.
Theseincludetheproteinstalinandvinculin(Bee,Lonergan,&Hu-Lonergan,2004;Kris-tensen&Purslow,2001;Morrisonetal.
,1998).
Thesedierencesindriplossassociatedwithdecreasedprote-olysiscanbeseenasearlyas24–48hpostmortem(Mel-odyetal.
,2004)(Fig.
2).
Similarobservationshavebeenmadeinenhancedporkloins(Davis,Sebranek,Hu-Lonergan,&Lonergan,2004)wherereduceddegrada-tionofdesminwasassociatedwithincreasedpurgeloss.
Signicantcorrelationsbetweendesmindegradationat1and7dpostmortemwithporksirloinpurgelossover7daysinavacuumpackagehavebeenreported(Gardner,Hu-Lonergan,&Lonergan,2005).
Whendesmindeg-radationwasaddedtothestepwiseregressionmodelstopredictpurgeloss,desmindegradationat1daypost-mortemexplained24.
1%ofthevariationinpurgelossinthesirloin.
Infact,desmindegradationatday1post-mortemwastherstindependentvariabletoenterthemodelfromalistofvariablesincludingpHdecline,ulti-matepH,temperaturedecline,colorandrmness.
Thisobservationsupportsthehypothesisthatproteolysisofintermediatelamentproteinsearlypostmortemcanminimizetheowofwaterfromwithinthecelltothedripchannels.
Becausedesminisaknownl-calpainsubstrate(Hu-Lonerganetal.
,1996),itisreasonabletohypothesizethatcalpainautolysisandactivationmayexplainapor-tionofthevariationofdesmindegradationandcouldsubsequentlyinuencedriploss.
Inaneorttodeter-minewhatfactorsinuencedesmindegradation,l-cal-painautolysisinsamplesagedonedaywasdetermined.
Therelativeintensityofintactdesminat1and5dpostmortemwassignicantlycorrelated(r=.
295,and.
270,respectively)withtheproportionofl-calpainlargesubunitpresentastheunautolyzed80kDaproteinat1dpostmortem.
Further,relativeintensityofintactdesminat1,5and7dpostmortemwassignicantlycorrelated(r=.
349,.
385,and.
378,respectively)withtheproportionofl-calpainlargesubunitpresentastheautolyzed76kDaproteinFig.
2.
Relationshipbetweendesmindegradationandpercentagedriplossinporcinelongissimusdorsi.
Driplossovertherst5dayspostmorteminloinsfromfourdierentanimals.
Desminshownat1and5dayspostmortemforeachoftheanimals.
Lackofabandindicatesdesminhasbeendegraded.
E.
Hu-Lonergan,S.
M.
Lonergan/MeatScience71(2005)194–204199at1dpostmortem.
Ithasbeenreportedthat52.
5%ofthevariationdesmindegradationat1dpostmortemcanbeexplainedinamodelthatincludeslointempera-tureat4hpostmortem,lointemperatureat24hpost-mortemandpercentageofl-calpainlargesubunitpresentastheautolyzed76kDaproteinat1dpostmor-tem(Gardneretal.
,2005).
Anegativecorrelationbe-tweenthe76kDaautolysisproductandintactdesminindicatesthatwhenverylittleautolysisoccurredwithintherstdaypostmortem,alargeproportionofdesminremainedintact.
Autolysisofcalpainisthehallmarkforactivationinpostmortemmuscle.
Thus,itiscon-cludedthatconditionsfavorableforcalpainactivationaresimilartothosenecessaryfordesmindegradation.
5.
2.
CalpastatinSincerapidproteolysisofintermediatelamentpro-teins(likedesmin)inmeathasbeenassociatedwithim-provedwater-holdingcapacity(Melodyetal.
,2004;Morrisonetal.
,1998)andtenderness(Melodyetal.
,2004)inporkandbecausethereisstrongevidencethatthecalpainenzymesareresponsibleforpostmortemproteolysisobservedinpre-andpost-rigormuscle(Hu-Lonergan&Lonergan,1999;Hu-Lonerganetal.
,1996;Koohmaraie,1992)itisimportanttocon-sidertheendogenousinhibitorofl-andm-calpain,cal-pastatin.
Calpastatinisaneectiveregulatorofcalpainactivityinpostmortemmuscle(reviewedbyKoohma-raie,1992).
Post-rigorcalpastatinactivityhasexplainedahighproportionofthevariationofmeattenderness(Whippleetal.
,1990).
Thereisadocumentedlinkbe-tweenhighcalpastatinactivity,limitedpostmortemproteolysisoftroponin-T(indicatedbyalimitedappear-anceofa30kDatroponin-Tdegradationproduct)andhighshearforce(Koohmaraie,1992;Lonerganetal.
,2001).
Melodyetal.
(2004)documenteddierencesincalpastatinactivitybetweenmusclesmeasured6and24hpostmorteminsemimembranosus(highest),longiss-imusdorsi(intermediate),andpsoasmajor(lowest).
Thesedierencescorrespondedtosignicantdierencesindesmindegradationbetweenthepsoasmajorandsemimembranosus,providingevidencethatvariationincalpastatinactivitymayprovideapartialexplanationforvariationinobservedproteolysis.
Inmanycases,thephysiologicalexplanationofvariationincalpastatinactivityhasnotbeenexplained.
Ithasbeenhypothesizedthattheremaybeageneticcomponenttovariationincalpastatinactivityagainstcalpain.
Twomissensemuta-tionshavebeenidentiedinthecalpastatingene(R249KandS638R)toclassifyfourhaplotypes,threeofwhichaccountedformostofthegeneticvariationintheUScommercialindustrypopulationsexamined(Ciobanuetal.
,2004).
Haplotype1(249K-638R)wasfoundtobethefavorablehaplotypeasitwasassociatedwithmorejuicyandtenderpork.
Thesedataprovidecompel-lingevidencethatcalpastatingenevariantscontributetodierencesinfreshporkquality,presumablybyregulat-ingcalpainactivityinmeat.
5.
3.
RoleofproteinoxidationPostmortemchangesinmusclearealsoaccompaniedbyamarkedincreaseinindicesofoxidation(Harris,Hu-Lonergan,Lonergan,Jones,&Rankins,2001).
Anotherchangethatoccursinpostmortemmuscledur-ingagingofwholemuscleproductsisincreasedoxida-tionofmyobrillarproteins(Martinaudetal.
,1997).
Thisresultsintheconversionofsomeaminoacidresi-dues,includinghistidine,tocarbonylderivatives(Le-vine,Williams,Stadtman,&Shacter,1994;Martinaudetal.
,1997)andcancausetheformationofintraand/orinterproteindisuldecross-links(Martinaudetal.
,1997;Stadtman,1990).
Ingeneral,bothofthesechangesreducethefunctionalityofproteins(Xiong&Decker,1995).
Becausel-calpainandm-calpainenzymescon-tainbothhistidineandSH-containingcysteineresiduesattheiractivesites,theymaybeparticularlysusceptibletoinactivationbyoxidation.
Therefore,oxidizingcondi-tionsinpostmortemmusclemayleadtoinactivationormodicationofcalpainactivity.
Infact,evidencesuggestsoxidizingconditionsinhibitsproteolysisbyl-calpain,butmightnotcompletelyinhibitautolysis(Guttmann,Elce,Bell,Isbell,&Johnson,1997;Gutt-mann&Johnson,1998).
Inpostmortemmuscle,therearedierencesbetweenmusclesintheratethatpostmor-temoxidationprocessesoccur(Martinaudetal.
,1997).
Ithasbeennotedthatdierencesintherateofoxidationinmuscletissueareseenwhencomparingthesamemus-clesbetweenanimalsand/orcarcassesthathavebeenhandleddierently(Juncheretal.
,2001).
Thesedier-encesmayarisebecauseofdierencesindiet,breed,antemortemstress,postmortemhandlingofcarcasses,etc.
Infact,therehavebeenreportsofdierencesbe-tweenanimalsandbetweenmusclesintheactivityofsomeenzymesinvolvedintheoxidativedefensesystemofmuscle(Daun,Johansson,Onning,&Akesson,2001).
Therefore,theremaybegeneticdierencesinsus-ceptibilitytooxidationthatcouldbecapitalizedontoimprovemeatquality.
Itisthereforereasonabletohypothesizethatdierencesintheantioxidantdefensesystembetweenanimalsand/ormuscleswouldinuencecalpainactivity,proteolysis,andthusqualitycharacter-isticsinuencedbyproteolysissuchastendernessandwaterholdingcapacity.
Experimentalevidenceexiststhatindicateshighlevelsofantioxidantsinmeatcaninuenceproteolysisandearlypostmortemshearforce(Harrisetal.
,2001;Rowe,Maddock,Lonergan,&Hu-Lonergan,2004b).
Roweetal.
(2004b)showedtherewasasignicantincreaseinproteolysisoftropo-nin-Tat2daysofpostmortemaginginsteaksfromsteersfedhighlevelsof1000IUa-tocopherol(Fig.
3).
200E.
Hu-Lonergan,S.
M.
Lonergan/MeatScience71(2005)194–204Takentogether,thesedataindicatethatverylowlevelsofoxidationcaninuenceproteolysis.
Theoxidativestateofthetissue,whichgreatlyinu-encestheactivityofl-calpain(Guttmannetal.
,1997;Guttmann&Johnson,1998),andthepresenceofendog-enousnitricoxidereactionproducts(e.
g.
,peroxynitrite,S-nitrosothiols)arefactorslikelyinuencingcalpainthathavereceivedlittleattention(Koh&Tidball,2000;Maddocketal.
,2005;Rowe,Maddock,Trenkle,Lonergan,&Hu-Lonergan,2003).
Becausel-andm-calpainhaveanoxidizablecysteineresidueattheirac-tivesite,theyrequirereducingconditionstobeactive(Guttmannetal.
,1997).
ThisactivesitecysteinemayalsobereversiblyS-nitrosylatedleadingtoinactivationofthecalpain(Koh&Tidball,2000).
Therefore,oxidiz-ingandnitrosylatingspeciesinthetissuecouldinhibitcalpainsandserveasaregulatoryfactor.
Ithasbeenshownthatthepresenceofoxidizingspe-ciesdoessignicantlyimpedetheabilityofcalpainstodegradetheirsubstrates.
OxidationwithH2O2signi-cantlylimitsproteolyticactivityofl-andm-calpainagainsttheuorescentpeptideSuc-Leu-Leu-Val-Tyr-AMC,regardlessofthepHorionicstrength(Roweetal.
,2003)(Fig.
4).
Similarresultswereseenwhenusingpuriedmyobrilsasthesubstrate(Maddock,Hu-Lonergan,Rowe,&Lonergan,2004).
Inhibitionofcalpainwithhydrogenperoxide(demonstratedwithboththeuorescentsubstrateandthemyobrils)isreversibleasadditionofreducingagent(DTT)totheoxidizedsamplesrestoresactivity.
Inmuscletissuethathasbeenexposedtoirradiationtoinduceoxidation(Rowe,Maddock,Lonergan,&Hu-Lonergan,2004a)duringtheearlypostmortemperiod,degradationofsev-eralproteinsisarrested,includingdesmin(Fig.
5)(Roweetal.
,2004b;Roweetal.
,2003).
Invitrostudiesusingpuriedcalpaintodegrademyobrilsinthepresenceofhydrogenperoxide(oxidizingagent)havealsoshownthatoxidizingconditionsmaylimittheabilityofcalpaintodegradeitssubstratesincludingdesmin(Maddocketal.
,2004).
Inmuscletissue,oxidizingconditionsdoappeartoreversiblyinhibitcalpain.
Roweetal.
(2004b)usedirradiationasatooltostudycalpainactiv-ityinoxidizedearlypostmortemmeatusingcaseinzymography.
Thisactivityassayinvolvedrunningthesupernatantfractionextractedfromfresh(neverfrozen)steaksrightafterirradiationandateachofthedaysofagingonnon-reducing,non-denaturingpolyacrylamidegels.
Thesegelscontainedcaseinintheseparatinggelsolution.
Aftergelswererun,theywereincubatedatroomtemperatureina5mMcalciumchloridesolutioncontaining0.
1%2-mercaptoethanoltoreducereversiblyoxidizedcalpainandallowittodegradecasein.
Calpainactivitywasidentiedbytheappearanceofclearingzones(lightbands,Fig.
6)afterthegelwasstainedinCoomassieBrilliantBlue(Raser,Posner,&Wang,1995).
Becausecalpainlosesactivityafterextensiveautolysis,lossofcalpainactivityduringpostmortemagingofmeatindicatesprioractivation.
Calpainthatispre-ventedfrombeingactiveinthetissuewillnotfullyauto-lyzeandwillthusbeabletobeactivatedoncetheconditionsforactivityaresatised(forexample,amplecalciumandreducingconditionsasinthecaseingelas-01020304050607.
5,1657.
5,2955.
6,1655.
6,295Non-OxidizedOxidizedFlousercUecnenitspHandIonicStrength(mMNaCl)ConditionsabceddeeFig.
4.
Eectofoxidizingconditions(100lMH2O2)ontheactivityofl-calpainunderdierentpHandionicstrengthconditionsafter15minofincubationat25°C.
TheuorescentpeptideSuc-Leu-Leu-Val-Tyr-AMCwasusedasthesubstrate.
a–esignicantdierencesatP<0.
01.
Fig.
3.
(a)WesternblotofTroponin-Tinpuriedmyobrilsfromnon-irradiatedsteaksfromcontroldietfedanda-tocopherol(vitaminE)fedsteers.
(b)DensitometryofthedegradationproductofTroponin-T(30kDaband).
Anincreasedvalueindicatesmoredegradation.
Fig.
5.
Westernblotofdesmininpuriedmyobrilsfromirradiated(Irr)andnon-irradiated(NI)beeflongissimusdorsiaged7dayspostmortem.
E.
Hu-Lonergan,S.
M.
Lonergan/MeatScience71(2005)194–204201say).
Thisstudyshowedconsistentlythatl-calpaininnon-irradiatedsampleslostactivityfasterthaninirradi-atedsamplesfromthesameanimals.
Thel-calpainfromtheirradiatedsamplesshowedlittlechangeinactivityasagingtimeprogressed.
Infact,theirradiatedsampleshadmorel-calpainactivityat7daysafterirradiationthandidthenon-irradiatedsamplesfromthesamecar-casses(Fig.
6)potentiallyindicatingl-calpainwasnotactiveintheirradiatedmuscle.
However,oncecalpainwasremovedfromthetissueandsubjectedtoreducingconditionsinthecaseinzymogramassay,itwasactive;furtherevidencethatinthetissue,l-calpainmayhavebeeninactivated(butreversibly)byoxidation.
6.
SummaryThemechanismofwater-holdingcapacityiscenteredintheproteinsandstructuresthatbindandentrapwater,specicallythemyobrillarprotein.
ThereisagreatbodyofevidencethatdemonstratesadirecteectofpH,ionicstrength,andoxidationontheabilityofmyobrillarproteinandmyobrilsandmusclecellstoentrapwater.
Independentoftheseeects,itisclearthatthesamefactors(pHdecline,ionicstrength,oxidation)alsoaectproteolysisofkeycytoskeletalproteinsinpostmortemmuscle.
VariationinwaterholdingcapacityatgivenpH,andtemperatureofstorageisproposedtobeatleastpartiallyduetovariationinproteolysisandtheresultingmusclecellshrinkageandmobilizationofwatertotheextracellularspace.
Becauseofthis,investi-gationsofmetabolic,chemicalandgeneticsourcesofvariationinpostmortemproteolysisshouldprovidenewinsightintomechanismsgoverningwater-holdingcapacity.
AcknowledgmentsTheauthorsaregratefultotheUnitedStatesDepart-mentofAgricultureNationalResearchInitiativeCom-petitiveGrantsProgram(USDANRICGP)forfundingsomeoftheworkreviewedinthisdocument.
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204E.
Hu-Lonergan,S.
M.
Lonergan/MeatScience71(2005)194–204
LonerganDepartmentofAnimalScience,IowaStateUniversity,Ames,IA50011,USAAbstractUnacceptablewater-holdingcapacitycoststhemeatindustrymillionsofdollarsannually.
However,limitedprogresshasbeenmadetowardunderstandingthemechanismsthatunderliethedevelopmentofdriporpurge.
ItisclearthatearlypostmortemeventsincludingrateandextentofpHdecline,proteolysisandevenproteinoxidationarekeyininuencingtheabilityofmeattoretainmoisture.
Muchofthewaterinthemuscleisentrappedinstructuresofthecell,includingtheintra-andextramyobrillarspaces;therefore,keychangesintheintracellulararchitectureofthecellinuencetheabilityofmusclecellstoretainwater.
Asrigorpro-gresses,thespaceforwatertobeheldinthemyobrilsisreducedanduidcanbeforcedintotheextramyobrillarspaceswhereitismoreeasilylostasdrip.
Lateralshrinkageofthemyobrilsoccurringduringrigorcanbetransmittedtotheentirecellifproteinsthatlinkmyobrilstogetherandmyobrilstothecellmembrane(suchasdesmin)arenotdegraded.
Limiteddegradationofcytoskeletalproteinsmayresultinincreasedshrinkingoftheoverallmusclecell,whichisultimatelytranslatedintodriploss.
Recentevidencesuggeststhatdegradationofkeycytoskeletalproteinsbycalpainproteinaseshasaroletoplayindeterminingwater-holdingcapacity.
Thisreviewwillfocusonkeyeventsinmusclethatinuencestructuralchangesthatareassociatedwithwater-holdingcapacity.
2005ElsevierLtd.
Allrightsreserved.
Keywords:Water-holdingcapacity;Driploss;Calpain;Proteolysis;pHContents1.
Introduction1952.
Structureofskeletalmuscle1953.
Locationofwaterinmuscle1954.
Physical/biochemicalfactorsinmusclethataffectwater-holdingcapacity1964.
1.
Netchargeeffect1964.
2.
Geneticfactors1964.
3.
Stericeffects1975.
Postmortemproteolysis1985.
1.
Calpainsystem1985.
2.
Calpastatin2005.
3.
Roleofproteinoxidation.
2006.
Summary202Acknowledgments202References2020309-1740/$-seefrontmatter2005ElsevierLtd.
Allrightsreserved.
doi:10.
1016/j.
meatsci.
2005.
04.
022*Correspondingauthor.
Tel.
:+15152949125;fax:+15152949143.
E-mailaddress:elonerga@iastate.
edu(E.
Hu-Lonergan).
www.
elsevier.
com/locate/meatsciMEATSCIENCEMeatScience71(2005)194–2041.
IntroductionTheabilityoffreshmeattoretainmoistureisargu-ablyoneofthemostimportantqualitycharacteristicsofrawproducts.
Ithasbeenestimatedthatasmuchas50%ormoreoftheporkproducedhasunacceptablyhighpurgeordriploss(Kauman,Cassens,Scherer,&Meeker,1992;Stetzer&McKeith,2003).
Productweightlossesduetopurgecanaverageasmuchas1–3%infreshretailcuts(Oer&Knight,1988a)andcanbeashighat10%inPSEproducts(Melodyetal.
,2004).
Inadditiontothelossofsalableweight,purgelossalsoentailsthelossofasignicantamountofpro-tein(Oer&Knight,1988a;Oer&Knight,1988b).
Onaverage,purgecancontainapproximately112mgofproteinpermilliliterofuid;mostlywater-soluble,sar-coplasmicproteins(Savage,Warriss,&Jolley,1990).
Themajorityofwaterinmuscleisheldeitherwithinthemyobrils,betweenthemyobrilsandbetweenthemyobrilsandthecellmembrane(sarcolemma),be-tweenmusclecellsandbetweenmusclebundles(groupsofmusclecells).
Oncemuscleisharvestedtheamountofwaterandlocationofthatwaterinmeatcanchangedependingonnumerousfactorsrelatedtothetissueit-selfandhowtheproductishandled(Honikel,2004;Honikel&Kim,1986).
Overtheyearstherehavebeennumerousreviewsdevotedtofactorsthatinuencewater-holdingcapacity(Hamm,1986;Honikel,2004;Honikel&Kim,1986;Oer&Knight,1988a;Oer&Knight,1988b).
Thisreviewwillgiveanoverviewsomeofthemajorfactorsinuencingwater-holdingcapacityandwillspecicallyfocusonrecentdevelopmentsinunderstandinghowchangesinthestructureofpostmor-temmusclecaninuencedriploss.
2.
StructureofskeletalmuscleSkeletalmusclehasaverycomplexorganization,inparttoallowmuscletoecientlytransmitforceorig-inatinginthemyobrilstotheentiremuscleandulti-matelytothelimborstructurethatismoved.
Arelativelythicksheathofconnectivetissue,theepimy-sium,enclosestheentiremuscle.
Inmostmuscles,theepimysiumiscontinuouswithtendonsthatlinkmus-clestobones.
Themuscleissubdividedintobundlesorgroupingsofmusclecells.
Thesebundles(alsoknownasfasciculi)aresurroundedbyanothersheathofconnectivetissue,theperimysium.
Athinlayerofconnectivetissue,theendomysium,surroundsthemusclecellsthemselves.
Theendomysiumliesabovethemusclecellmembrane(sarcolemma)andconsistsofabasementmembranethatisassociatedwithanouterlayer(reticularlayer)thatissurroundedbyalayerofnecollagenbrilsimbeddedinamatrix(Bailey&Light,1989).
Whenmusclecellsareviewedunderamicroscope,veryregulartransversestriationsareseen.
Thesestria-tionsarecausedbyspecializedcontractileorganelles,themyobrils,foundinmuscle.
Thestriationsarisefromalternating,proteindenseA-bandsandlessdenseI-bandswithinthemyobril.
BisectingtheI-bandsaredarklinesknownasZ-lines.
TheareabetweentwoZ-linesisasarcomere.
ThelessdenseI-bandismadeupprimarilyofthinlamentswhiletheA-bandismadeupofthicklamentsandsomeoverlappingthinla-ments(Goll,Robson,&Stromer,1984).
Thebackboneofthethinlamentsismadeupprimarilyoftheproteinactinwhilethelargestcomponentofthethicklamentistheproteinmyosin.
Myosinconsistsofatailorrodre-gionthatformsthebackboneofthethicklamentandaglobularheadregionthatextendsfromthethickla-mentandinteractswithactininthethinlament.
Therigorcomplexformedbytheinteractionofmyosinandactinisoftenreferredtoasactomyosin.
Inelectronmicrographimagesofcontractedmuscleorofpost-rigormuscletheactomyosinlooksverymuchlikecross-bridgesbetweenthethickandthinlaments,indeed,itisoftenreferredtoassuch.
Inpostmortemmusclethesebondsbecomeirreversibleandarealsoknownasrigorbonds.
Theglobularheadofmyosinalsohasenzymaticactivity;itcanhydrolyzeATPandliberateenergy.
Inlivingmuscleduringcontraction,theATPaseactivityofmyosinprovidesenergyformyosinboundtoactintoswivelandultimatelypullthethinlamentstowardthecenterofthesarcomere.
Thisshortensthemyobril,themusclecellandeventuallythemuscletoproducecontraction.
ThemyosinandactincandisassociatewhenanewmoleculeofATPisboundtothemyosinhead(Golletal.
,1984).
Inpost-rigormuscle,thesupplyofATPisdepleted,resultingintheactomyosinbondsbecomingmoreorlesspermanent.
Withinthestructureofthemuscle,thereareseveral''compartments''fromwhichdripcouldoriginate.
Thesecouldincludethespacewithinthemyobril,theintra-cellularspaceoutsidethemyobrilandtheextracellularspace,includingthespacebetweenthemusclebundles.
Lossofwaterfromeachofthesecompartmentsmayin-volveslightlydierentmechanisms.
Inaddition,lossofwaterfromeachofthesecompartmentsmayoccuratdierenttimesduringstorage.
Forexample,itwouldbeeasytoenvisionthatwaterfoundintheextracellularspacescouldbelostmoreeasily,withdeepercompart-mentstakingmoretimeorforcetobereleased.
3.
LocationofwaterinmuscleLeanmusclecontainsapproximately75%water.
Theothermaincomponentsincludeprotein(approximately20%),lipidsorfat(approximately5%),carbohydrates(approximately1%)andvitaminsandminerals(oftenE.
Hu-Lonergan,S.
M.
Lonergan/MeatScience71(2005)194–204195analyzedasash,approximately1%).
Themajorityofwaterinmuscleisheldwithinthestructureofthemuscleandmusclecells.
Specically,withinthemusclecell,waterisfoundwithinthemyobrils,betweenthemyo-brilsthemselvesandbetweenthemyobrilsandthecellmembrane(sarcolemma),betweenmusclecellsandbe-tweenmusclebundles(groupsofmusclecells)(Oer&Cousins,1992).
Waterisadipolarmoleculeandassuchisattractedtochargedspecieslikeproteins.
Infact,someofthewaterinmusclecellsisverycloselyboundtoprotein.
Bydenition,boundwateriswaterthatexistsinthevicinityofnon-aqueousconstituents(likeproteins)andhasreducedmobility,i.
e.
doesnoteasilymovetoothercompartments.
Thiswaterisveryresistanttofreezingandtobeingdrivenobyconventionalheating(Fen-nema,1985).
Trueboundwaterisaverysmallfractionofthetotalwaterinmusclecells;dependingonthemea-surementsystemused,approximately0.
5gofwaterpergramofproteinisestimatedtobetightlyboundtopro-teins.
Sincethetotalconcentrationofproteininmuscleisapproximately200mg/g,thisboundwateronlymakesuplessthanatenthofthetotalwaterinmuscle.
Theamountofboundwaterchangesverylittleifatallinpost-rigormuscle(Oer&Knight,1988b).
Anotherfractionofwaterthatcanbefoundinmus-clesandinmeatistermedentrapped(alsoreferredtoasimmobilized)water(Fennema,1985).
Thewatermole-culesinthisfractionmaybeheldeitherbysteric(space)eectsand/orbyattractiontotheboundwater.
Thiswaterisheldwithinthestructureofthemusclebutisnotboundpersetoprotein.
Inearlypostmortemtissue,thiswaterdoesnotowfreelyfromthetissue,yetitcanberemovedbydrying,andcanbeeasilyconvertedtoiceduringfreezing.
Entrappedorimmobilizedwaterismostaectedbytherigorprocessandtheconversionofmus-cletomeat.
UponalterationofmusclecellstructureandloweringofthepHthiswatercanalsoeventuallyescapeaspurge(Oer&Knight,1988b).
Freewateriswaterwhoseowfromthetissueisunimpeded.
Weaksurfaceforcesmainlyholdthisfrac-tionofwaterinmeat.
Freewaterisnotreadilyseeninpre-rigormeat,butcandevelopasconditionschangethatallowtheentrappedwatertomovefromthestruc-tureswhereitisfound(Fennema,1985).
Themajorityofthewaterthatisaectedbytheprocessofconvertingmuscletomeatistheentrapped(immobilized)water.
Maintainingasmuchofthiswateraspossibleinmeatisthegoalofmanyproces-sors.
Someofthefactorsthatcaninuencethereten-tionofentrappedwaterincludemanipulationofthenetchargeofmyobrillarproteinsandthestructureofthemusclecellanditscomponents(myobrils,cytoskeletallinkagesandmembranepermeability)aswellastheamountofextracellularspacewithinthemuscleitself.
4.
Physical/biochemicalfactorsinmusclethataectwater-holdingcapacity4.
1.
NetchargeeectDuringtheconversionofmuscletomeat,lacticacidbuildsupinthetissueleadingtoareductioninpHofthemeat.
OncethepHhasreachedtheisoelectricpoint(pI)ofthemajorproteins,especiallymyosin(pI=5.
4),thenetchargeoftheproteiniszero,meaningthenum-bersofpositiveandnegativechargesontheproteinsareessentiallyequal.
Thesepositiveandnegativegroupswithintheproteinareattractedtoeachotherandresultinareductionintheamountofwaterthatcanbeat-tractedandheldbythatprotein.
Additionally,sincelikechargesrepel,asthenetchargeoftheproteinsthatmakeupthemyobrilapproacheszero(diminishednetnega-tiveorpositivecharge)repulsionofstructureswithinthemyobrilisreducedallowingthosestructurestopackmorecloselytogether.
Theendresultofthisisareduc-tionofspacewithinthemyobril.
PartialdenaturationofthemyosinheadatlowpH(especiallyifthetemper-atureisstillhigh)isalsothoughttoberesponsibleforalargepartoftheshrinkageinmyobrillarlatticespacing(Oer,1991).
4.
2.
GeneticfactorsAcceleratedpHdeclineandlowultimatepHarere-latedtothedevelopmentoflowwater-holdingcapacityandunacceptablyhighpurgeloss.
RapidpHdeclineresultinginultimateornearultimatepHwhilethemus-cleisstillwarmcausesthedenaturation(lossoffunc-tionalityandwaterbindingability)ofmanyproteins,includingthoseinvolvedinbindingwater.
Themostse-verepurgeordriplossisoftenfoundinPSE(Pale,Soft,andExudative)productfrompigsthathaveinheritedamutationintheryanodinereceptor/calciumreleasechannel(halothanegene)inthesarcoplasmicreticulum(Fujiietal.
,1991).
Thismutationresultsinimpairmentoftheabilityofthischanneltocontrolcalciumreleaseintothesarcoplasmofthemusclecell,particularlyunderperiodsofphysicalstress.
AcceleratedreleaseofcalciumcausesrapidcontractionandanincreaseintherateofmusclemetabolismandintherateofpHdecline(Ben-dall&Wismer-Pedersen,1962;Lundstrom,Essen-Gustavsson,Rundgren,Edforslilja,&Malmfors,1989).
Thisparticularmutationinthehalothanegenecanbeidentiedinparentstock.
Becauseacommercialtestforthismutationexists,theUnitedStatesindustryhasvirtuallyeliminatedthisgeneinmostcommercialherds.
TheHalothanegeneisbutoneexampleofacondi-tionthatcanresultinPSE.
OtherfactorscancausePSEmeattooccur.
Beforeharvest,short-termstressinnormalanimalscanacceleratetheirmetabolismenough196E.
Hu-Lonergan,S.
M.
Lonergan/MeatScience71(2005)194–204thatthepostmortemmetabolisminthemuscleisaccel-erated,causingamorerapidpHdeclinethanisseeninnon-stressedanimals.
WhiletheconditionmaynotbeassevereasthatcausedbytheHalothanegene,pro-teindenaturationdoesoccur,anddriplossescanbegreaterthaninmusclethathasanormal,slowerrateofpHdecline.
ItshouldbenotedthatwhilethepHofthesemusclesfallsfasterthannormal,theultimatepHmaynotbebelownormalranges(Rosenvold&Ander-sen,2003).
Othermetabolicstateandexistingconditionsofmus-cleoftendirecttheextentofpHdeclineinpostmortemmuscle.
Manystudieshavereportedtheeectoftreat-menttodecreaseglycogencontentinmuscletominimizelactateaccumulationinpostmortemmuscle(reviewedbyRosenvold&Andersen,2003).
Geneticfactorsinu-encingbasalmetabolismclearlyhavethepotentialtosimilarlyaectlactateaccumulationandextentofpHdecline.
Thediscovery(Milanetal.
,2000)ofanon-con-servedsubstitutioninproteinkinaseadenosinemono-phosphate-activatedc3-subunitgene(PRKAG3)hasexplainedthedominantmutation(denotedRN)thataccountedforlargedierencesinmeatqualityandpro-cessingyieldintheHampshirepigbreed(Monin&Sel-lier,1985).
Thesubstitution(R200Q)inthePRKAG3genecausesa70%increaseinmuscleglycogeninRNhomozygousandheterozygouspigs.
Thisincreaseinglycogendirectlyresultsingreaterproductionoflactateinpostmortemmuscle,alowerultimatepHandpoorerwaterholdingcapacityinfreshpork.
ThePRKAG3geneencodesoneisoformofoneoftheregulatorysubunits(c)inmammalianadenosinemono-phosphate(AMP)-activatedproteinkinase(AMPK).
Whensubjectedtonutritionalorenvironmentalstress,theAMP/ATPratioofeukaryoticcellswillrise,trigger-ingthe''AMPKcascade'',stimulatingthecellstocon-serveenergy(Thornton,Snowden,&Carling,1998)andtobeginATPsynthesis(Hardie,Carling,&Carl-son,1998).
Theprecisefunctionsofthebandcregula-torysubunitsofAMPKarestillunknown;however,bothareknowntobeimportantforkinaseactivity(Hardie&Carling,1997).
ThecregulatorysubunitalsomaybeinvolvedwiththeAMP-bindingsiteoftheAMPKheterotrimericcomplex(Cheung,Salt,Davies,Hardie,&Carling,2000).
WhilethedominantRNmutationthatisfoundintheHampshirebreedofpigsisanonconservativesubsti-tution(R200Q)inthePRKAG3genethatcauseshighglycogencontentinskeletalmuscleresultingindetri-mentaleectsonprocessingyield(Milanetal.
,2000),otheralleleswithinthesamegeneareassociatedwithlowermuscleglycogencontent,andimprovedmeatqualitytraits(Ciobanuetal.
,2001).
Fromthreemis-sensemutationsidentied(T30N,G52R,I199V)inpor-cinePRKAG3,least-squaresestimatesofgenotypemeansacrossvecommercialpigbreedsdemonstratesignicanteectsbetweentheanalyzedsubstitutionsandproductpHandMinoltaLvalues.
Inthesamestudy,onlyfourhaplotypeswerefoundinthevecom-mercialpopulations.
OneofthosehaplotypeswasfoundtobesignicantlyassociatedwithhigherpHanddarkermeatcolor(T30-G52-I199),whileasecondhadinterme-diatepHandcolorandthetwoothershadthelowestpHandthepoorestcolor(Ciobanuetal.
,2001).
TheseobservationsestablishageneticbasisforvariationinultimatepHacrossmanybreedsandcommerciallines,notjustHampshirepigs.
4.
3.
StericeectsMyobrilsmakeupalargeproportionofthemusclecell.
Theseorganellesconstituteasmuchas82–87%ofthevolumeofthemusclecell.
Asmentionedpreviously,muchofthewaterinsidelivingmusclecellsislocatedwithinthemyobril.
Infact,itisestimatedthatasmuchas85%ofthewaterinamusclecellisheldinthemyo-brils.
Muchofthatwaterisheldbycapillaryforcesarisingfromthearrangementofthethickandthinla-mentswithinthemyobril.
Inlivingmuscle,ithasbeenshownthatsarcomeresremainisovolumetricduringcontractionandrelaxation(Millman,Racey,&Matsu-bara,1981;Millman,Wakabayashi,&Racey,1983).
Thiswouldindicatethatinlivingmuscletheamountofwaterwithinthelamentousstructureofthecellwouldnotnecessarilychange.
However,thelocationofthiswatercanbeaectedbychangesinvolumeasmuscleundergoesrigor.
Asmusclegoesintorigor,cross-bridgesformbetweenthethickandthinlaments,thusreducingavailablespaceforwatertoreside(Oer&Trinick,1983).
IthasbeenshownthatasthepHofporcinemuscleisreducedfromphysiologicalvaluesto5.
2–5.
5(neartheisoelectricpointofmyosin),thedis-tancebetweenthethicklamentsdeclinesanaverageof2.
5nm(Diesbourg,Swatland,&Millman,1988).
Thisdeclineinlamentspacingmayforcesarcoplasmicuidfrombetweenthemyolamentstotheextramyobrillarspace.
Indeed,ithasbeenhypothesizedthatenoughuidmaybelostfromtheintramyobrillarspacetoin-creasetheextramyobrillarvolumebyasmuchas1.
6timesmorethanitspre-rigorvolume(Bendall&Swat-land,1988).
Duringthedevelopmentofrigor,thediameterofmus-clecellshasbeenshowntodecrease(Hegarty,1970;Swatland&Belfry,1985)andislikelytheresultoftrans-mittalofthelateralshrinkageofthemyobrilstotheen-tirecell(Diesbourgetal.
,1988).
Additionally,duringrigordevelopmentsarcomerescanshorten;thisalsore-ducesthespaceavailableforwaterwithinthemyobril.
Infact,ithasbeenshownthatdriplosscanincreaselin-earlywithadecreaseinthelengthofthesarcomeresinmusclecells(Honikel,Kim,Hamm,&Roncales,1986).
Morerecently,highlysensitiveloweldnuclearmagneticE.
Hu-Lonergan,S.
M.
Lonergan/MeatScience71(2005)194–204197resonance(NMR)studieshavebeenusedtogainamorecompleteunderstandingoftherelationshipbetweenmusclecellstructureandwaterdistribution(Bertram,Purslow,&Andersen,2002).
Thesestudieshavesug-gestedthatwithinthemyobril,ahigherproportionofwaterisheldintheI-bandthaninthemoreproteindenseA-band.
Thisobservationmayhelpexplainwhyshortersarcomeres(especiallyincold-shortenedmuscle)areoftenassociatedwithincreaseddriplosses.
Asthemyo-brilshortensandrigorsetsin,theshorteningofthesarcomerewouldleadtoshorteningandsubsequentlow-eringofthevolumeoftheI-bandregioninmyobrils.
Lossofvolumeinthismyobrillarregion(wheremuchwatermayreside),combinedwiththepH-inducedlateralshrinkageofthemyobrilcouldleadtoexpulsionofwaterfromthemyobrillarstructureintotheextramyo-brillarspaceswithinthemusclecell(Bendall&Swatland,1988).
Infact,recentNMRstudiessupportthishypothesis(Bertrametal.
,2002).
Itisthuslikelythatthegradualmobilizationofwaterfromtheintramyobr-illarspacestotheextramyobrillarspacesmaybekeyinprovidingasourceofdrip.
Allofthepreviouslymentionedprocessesinuencetheamountofwaterinthemyobril.
Itisimportanttonotethatshrinkageofthemyobrillarlatticealonecouldnotberesponsibleforthemovementofuidtotheextracellularspaceandultimatelyoutofthemuscle.
Themyobrilsarelinkedtoeachotherandtothecellmembraneviaproteinaceousconnections(Wang&Ra-mirez-Mitchell,1983).
Theseconnections,iftheyaremaintainedintactinpostmortemmuscle,wouldtransferthereductionindiameterofthemyobrilstothemusclecell(Diesbourgetal.
,1988;Kristensen&Purslow,2001;Melodyetal.
,2004;Morrison,Mielche,&Purslow,1998).
Myobrilshrinkagecanbetranslatedintocon-strictionoftheentiremusclecell,thuscreatingchannelsbetweencellsandbetweenbundlesofcellsthatcanfun-neldripoutoftheproduct(Oer&Knight,1988).
Extracellularspacearoundmuscleberscontinuallyin-creasesupto24hpostmortem,butgapsbetweenmuscleberbundlesdecreaseslightlybetween9and24hpost-mortem,perhapsduetouidoutowfromthesemajorchannels(Schafer,Rosenvold,Purslow,Andersen,&Henckel,2002).
Theselinkagesbetweenadjacentmyo-brilsandmyobrilsandthecellmembranearemadeupofseveralproteinsthatareassociatedwithintermediatelamentstructuresandstructuresknownascostameres.
Costameresprovidethestructuralframeworkresponsi-bleforattachingthemyobrilstothesarcolemma.
Pro-teinsthatmakeup,orareassociatedwiththeintermediatelamentsandcostameresinclude(amongothers)desmin,lamin,andsynemin.
dystrophin,talinandvinculin(Greaser,1991).
Ifcostamericlinkagesre-mainintactduringtheconversionofmuscletomeat,shrinkageofthemyobrilsasthemusclegoesintorigorwouldbetransmittedtotheentirecellviatheseprotein-aceouslinkagesandwouldultimatelyreducevolumeofthemusclecellitself(Kristensen&Purslow,2001;Mel-odyetal.
,2004;Oer&Knight,1988b).
Thus,therigorprocesscouldresultinmobilizationofwateroutnotonlyoutofthemyobril,butalsooutoftheextramyo-brilspacesastheoverallvolumeofthecelliscon-stricted(Fig.
1).
Infact,reductioninthediameterofmusclecellshasbeenobservedinpostmortemmuscle(Oer&Cousins,1992).
Thiswaterthatisexpelledfromthemyobrilandultimatelythemusclecelleventuallycollectsintheextracellularspace.
Severalstudieshaveshownthatgapsdevelopbetweenmusclecellsandbe-tweenmusclebundlesduringthepost-rigorperiod(Oer&Cousins,1992;Oeretal.
,1989).
Thesegapsbetweenmusclebundlesaretheprimarychannelsbywhichpurgeowsfromthemeat;someinvestigatorshaveactuallytermedthem''dripchannels''.
5.
PostmortemproteolysisSinceithasbeenhypothesizedthatproteinaceouslinkageswithinthecellmayinuencedripproduction,itisimportanttoinvestigatethefactorsregulatingpost-mortemproteolysisofkeyproteins.
5.
1.
CalpainsystemTheendogenouscalpainsystemplaysamajorroleinregulatingproteolysisofmuscleproteinsunderpost-mortemconditions(Koohmaraie,Schollmeyer,&Dut-son,1986;Lonergan,Hu-Lonergan,Wiegand,&Kriese-Anderson,2001;Maddock,Hu-Lonergan,Rowe,&Lonergan,2005).
Proteinsthataresubstratesofcalpainsincludeproteinslikedesmin,synemin,talinandvinculinthatformthecytoskeletalframeworkofthemusclecell(Bilaketal.
,1998;Evans,Robson,&ProteolysisNoProteolysisFig.
1.
Schematicoverviewingthepotentialchangesinmusclecelldiameterduringpostmortemagingasinuencedbyproteolysis.
198E.
Hu-Lonergan,S.
M.
Lonergan/MeatScience71(2005)194–204Stromer,1984;OShea,Robson,Huiatt,Hartzer,&Stromer,1979;Schmidt,Zhang,Lee,Stromer,&Rob-son,1999).
Thecalpainsystemiscomposedofseveralisoformsofcalcium-dependentcysteineproteases(calpains),andtheirspeciccompetitiveinhibitor,calpastatin(Goll,Thompson,Li,Wei,&Cong,2003).
Thetwobest-characterizedisoformsofcalpainsarel-calpainandm-calpain,whichbothdegradethesamespecicsetofmyobrillarandcytoskeletalproteinsthatarede-gradedasmuscleisconvertedintomeat(Geesink&Koohmaraie,1999;Hu-Lonerganetal.
,1996).
Asthepostmortemperiodprogresses,dramaticchangesoccurwithinthemicroenvironmentofthemusclecell(e.
g.
,de-clineinpH,increaseinionicstrength)(Winger&Pope,1981)thatcanaectcalpainactivity(Hu-Lonerganetal.
,1996;Maddocketal.
,2005).
Asmuscleisconvertedtomeat,manychangesoccur,including:(1)agradualdepletionofavailableenergy,(2)ashiftfromaerobictoanaerobicmetabolismfavoringtheproductionoflac-ticacid,resultinginthepHofthetissuedecliningfromnearneutralityto5.
4–5.
8,(3)ariseinionicstrength,inpart,becauseoftheinabilityofATP-dependentcalcium,sodium,andpotassiumpumpstofunction,and(4)anincreasinginabilityofthecelltomaintainreducingcon-ditions.
Bothl-calpainandm-calpainhaveslowerratesofactivityagainstmyobrillarproteinsubstratesatpHvaluesandionicstrengthssimilartothosefoundinpost-mortemmuscle(Geesink&Koohmaraie,1999;Hu-Lonergan&Lonergan,1999;Kendall,Koohmaraie,Arbona,Williams,&Young,1993).
AlterationsinpHand/orionicstrengthsmaycauseconformationalchangesthatallowanincreaseinthehydrophobicityandaggregationoftheenzyme.
Likewise,pH/ionicstrengthchangesmayaltertheconformationofsubstrateproteinsandrenderthemlesssusceptibletocleavagebyl-calpain(Hu-Lonergan&Lonergan,1999).
Experimentalevidenceexiststhatsupportstheideathatthatproteolysisofkeycytoskeletalproteinssuchastheintermediatelamentprotein,desminmaybere-latedtodripproduction.
Theseproteinshavebeenshowntobedegradedasearlyas45minto6hpostmor-teminsomemuscles(Melodyetal.
,2004).
Degradationoftheseproteinsatsuchanearlytimepostmortemwouldcertainlyallowwaterthatisexpelledfromtheintramyobrillarspacestoremaininthecellforalongerperiodoftime.
Thecorollarytothisisthatreduceddeg-radationofproteinsthattiethemyobriltothecellmembrane(suchasdesmin)resultsinincreasedshrink-ingofthemusclecell,whichisultimatelytranslatedintodriploss.
Certainly,desminisnottheonlycandidateproteintobeinvolved.
Otherproteinsassociatedwithintermediatelamentsandwiththecostamereshavebeenimplicated.
Theseincludetheproteinstalinandvinculin(Bee,Lonergan,&Hu-Lonergan,2004;Kris-tensen&Purslow,2001;Morrisonetal.
,1998).
Thesedierencesindriplossassociatedwithdecreasedprote-olysiscanbeseenasearlyas24–48hpostmortem(Mel-odyetal.
,2004)(Fig.
2).
Similarobservationshavebeenmadeinenhancedporkloins(Davis,Sebranek,Hu-Lonergan,&Lonergan,2004)wherereduceddegrada-tionofdesminwasassociatedwithincreasedpurgeloss.
Signicantcorrelationsbetweendesmindegradationat1and7dpostmortemwithporksirloinpurgelossover7daysinavacuumpackagehavebeenreported(Gardner,Hu-Lonergan,&Lonergan,2005).
Whendesmindeg-radationwasaddedtothestepwiseregressionmodelstopredictpurgeloss,desmindegradationat1daypost-mortemexplained24.
1%ofthevariationinpurgelossinthesirloin.
Infact,desmindegradationatday1post-mortemwastherstindependentvariabletoenterthemodelfromalistofvariablesincludingpHdecline,ulti-matepH,temperaturedecline,colorandrmness.
Thisobservationsupportsthehypothesisthatproteolysisofintermediatelamentproteinsearlypostmortemcanminimizetheowofwaterfromwithinthecelltothedripchannels.
Becausedesminisaknownl-calpainsubstrate(Hu-Lonerganetal.
,1996),itisreasonabletohypothesizethatcalpainautolysisandactivationmayexplainapor-tionofthevariationofdesmindegradationandcouldsubsequentlyinuencedriploss.
Inaneorttodeter-minewhatfactorsinuencedesmindegradation,l-cal-painautolysisinsamplesagedonedaywasdetermined.
Therelativeintensityofintactdesminat1and5dpostmortemwassignicantlycorrelated(r=.
295,and.
270,respectively)withtheproportionofl-calpainlargesubunitpresentastheunautolyzed80kDaproteinat1dpostmortem.
Further,relativeintensityofintactdesminat1,5and7dpostmortemwassignicantlycorrelated(r=.
349,.
385,and.
378,respectively)withtheproportionofl-calpainlargesubunitpresentastheautolyzed76kDaproteinFig.
2.
Relationshipbetweendesmindegradationandpercentagedriplossinporcinelongissimusdorsi.
Driplossovertherst5dayspostmorteminloinsfromfourdierentanimals.
Desminshownat1and5dayspostmortemforeachoftheanimals.
Lackofabandindicatesdesminhasbeendegraded.
E.
Hu-Lonergan,S.
M.
Lonergan/MeatScience71(2005)194–204199at1dpostmortem.
Ithasbeenreportedthat52.
5%ofthevariationdesmindegradationat1dpostmortemcanbeexplainedinamodelthatincludeslointempera-tureat4hpostmortem,lointemperatureat24hpost-mortemandpercentageofl-calpainlargesubunitpresentastheautolyzed76kDaproteinat1dpostmor-tem(Gardneretal.
,2005).
Anegativecorrelationbe-tweenthe76kDaautolysisproductandintactdesminindicatesthatwhenverylittleautolysisoccurredwithintherstdaypostmortem,alargeproportionofdesminremainedintact.
Autolysisofcalpainisthehallmarkforactivationinpostmortemmuscle.
Thus,itiscon-cludedthatconditionsfavorableforcalpainactivationaresimilartothosenecessaryfordesmindegradation.
5.
2.
CalpastatinSincerapidproteolysisofintermediatelamentpro-teins(likedesmin)inmeathasbeenassociatedwithim-provedwater-holdingcapacity(Melodyetal.
,2004;Morrisonetal.
,1998)andtenderness(Melodyetal.
,2004)inporkandbecausethereisstrongevidencethatthecalpainenzymesareresponsibleforpostmortemproteolysisobservedinpre-andpost-rigormuscle(Hu-Lonergan&Lonergan,1999;Hu-Lonerganetal.
,1996;Koohmaraie,1992)itisimportanttocon-sidertheendogenousinhibitorofl-andm-calpain,cal-pastatin.
Calpastatinisaneectiveregulatorofcalpainactivityinpostmortemmuscle(reviewedbyKoohma-raie,1992).
Post-rigorcalpastatinactivityhasexplainedahighproportionofthevariationofmeattenderness(Whippleetal.
,1990).
Thereisadocumentedlinkbe-tweenhighcalpastatinactivity,limitedpostmortemproteolysisoftroponin-T(indicatedbyalimitedappear-anceofa30kDatroponin-Tdegradationproduct)andhighshearforce(Koohmaraie,1992;Lonerganetal.
,2001).
Melodyetal.
(2004)documenteddierencesincalpastatinactivitybetweenmusclesmeasured6and24hpostmorteminsemimembranosus(highest),longiss-imusdorsi(intermediate),andpsoasmajor(lowest).
Thesedierencescorrespondedtosignicantdierencesindesmindegradationbetweenthepsoasmajorandsemimembranosus,providingevidencethatvariationincalpastatinactivitymayprovideapartialexplanationforvariationinobservedproteolysis.
Inmanycases,thephysiologicalexplanationofvariationincalpastatinactivityhasnotbeenexplained.
Ithasbeenhypothesizedthattheremaybeageneticcomponenttovariationincalpastatinactivityagainstcalpain.
Twomissensemuta-tionshavebeenidentiedinthecalpastatingene(R249KandS638R)toclassifyfourhaplotypes,threeofwhichaccountedformostofthegeneticvariationintheUScommercialindustrypopulationsexamined(Ciobanuetal.
,2004).
Haplotype1(249K-638R)wasfoundtobethefavorablehaplotypeasitwasassociatedwithmorejuicyandtenderpork.
Thesedataprovidecompel-lingevidencethatcalpastatingenevariantscontributetodierencesinfreshporkquality,presumablybyregulat-ingcalpainactivityinmeat.
5.
3.
RoleofproteinoxidationPostmortemchangesinmusclearealsoaccompaniedbyamarkedincreaseinindicesofoxidation(Harris,Hu-Lonergan,Lonergan,Jones,&Rankins,2001).
Anotherchangethatoccursinpostmortemmuscledur-ingagingofwholemuscleproductsisincreasedoxida-tionofmyobrillarproteins(Martinaudetal.
,1997).
Thisresultsintheconversionofsomeaminoacidresi-dues,includinghistidine,tocarbonylderivatives(Le-vine,Williams,Stadtman,&Shacter,1994;Martinaudetal.
,1997)andcancausetheformationofintraand/orinterproteindisuldecross-links(Martinaudetal.
,1997;Stadtman,1990).
Ingeneral,bothofthesechangesreducethefunctionalityofproteins(Xiong&Decker,1995).
Becausel-calpainandm-calpainenzymescon-tainbothhistidineandSH-containingcysteineresiduesattheiractivesites,theymaybeparticularlysusceptibletoinactivationbyoxidation.
Therefore,oxidizingcondi-tionsinpostmortemmusclemayleadtoinactivationormodicationofcalpainactivity.
Infact,evidencesuggestsoxidizingconditionsinhibitsproteolysisbyl-calpain,butmightnotcompletelyinhibitautolysis(Guttmann,Elce,Bell,Isbell,&Johnson,1997;Gutt-mann&Johnson,1998).
Inpostmortemmuscle,therearedierencesbetweenmusclesintheratethatpostmor-temoxidationprocessesoccur(Martinaudetal.
,1997).
Ithasbeennotedthatdierencesintherateofoxidationinmuscletissueareseenwhencomparingthesamemus-clesbetweenanimalsand/orcarcassesthathavebeenhandleddierently(Juncheretal.
,2001).
Thesedier-encesmayarisebecauseofdierencesindiet,breed,antemortemstress,postmortemhandlingofcarcasses,etc.
Infact,therehavebeenreportsofdierencesbe-tweenanimalsandbetweenmusclesintheactivityofsomeenzymesinvolvedintheoxidativedefensesystemofmuscle(Daun,Johansson,Onning,&Akesson,2001).
Therefore,theremaybegeneticdierencesinsus-ceptibilitytooxidationthatcouldbecapitalizedontoimprovemeatquality.
Itisthereforereasonabletohypothesizethatdierencesintheantioxidantdefensesystembetweenanimalsand/ormuscleswouldinuencecalpainactivity,proteolysis,andthusqualitycharacter-isticsinuencedbyproteolysissuchastendernessandwaterholdingcapacity.
Experimentalevidenceexiststhatindicateshighlevelsofantioxidantsinmeatcaninuenceproteolysisandearlypostmortemshearforce(Harrisetal.
,2001;Rowe,Maddock,Lonergan,&Hu-Lonergan,2004b).
Roweetal.
(2004b)showedtherewasasignicantincreaseinproteolysisoftropo-nin-Tat2daysofpostmortemaginginsteaksfromsteersfedhighlevelsof1000IUa-tocopherol(Fig.
3).
200E.
Hu-Lonergan,S.
M.
Lonergan/MeatScience71(2005)194–204Takentogether,thesedataindicatethatverylowlevelsofoxidationcaninuenceproteolysis.
Theoxidativestateofthetissue,whichgreatlyinu-encestheactivityofl-calpain(Guttmannetal.
,1997;Guttmann&Johnson,1998),andthepresenceofendog-enousnitricoxidereactionproducts(e.
g.
,peroxynitrite,S-nitrosothiols)arefactorslikelyinuencingcalpainthathavereceivedlittleattention(Koh&Tidball,2000;Maddocketal.
,2005;Rowe,Maddock,Trenkle,Lonergan,&Hu-Lonergan,2003).
Becausel-andm-calpainhaveanoxidizablecysteineresidueattheirac-tivesite,theyrequirereducingconditionstobeactive(Guttmannetal.
,1997).
ThisactivesitecysteinemayalsobereversiblyS-nitrosylatedleadingtoinactivationofthecalpain(Koh&Tidball,2000).
Therefore,oxidiz-ingandnitrosylatingspeciesinthetissuecouldinhibitcalpainsandserveasaregulatoryfactor.
Ithasbeenshownthatthepresenceofoxidizingspe-ciesdoessignicantlyimpedetheabilityofcalpainstodegradetheirsubstrates.
OxidationwithH2O2signi-cantlylimitsproteolyticactivityofl-andm-calpainagainsttheuorescentpeptideSuc-Leu-Leu-Val-Tyr-AMC,regardlessofthepHorionicstrength(Roweetal.
,2003)(Fig.
4).
Similarresultswereseenwhenusingpuriedmyobrilsasthesubstrate(Maddock,Hu-Lonergan,Rowe,&Lonergan,2004).
Inhibitionofcalpainwithhydrogenperoxide(demonstratedwithboththeuorescentsubstrateandthemyobrils)isreversibleasadditionofreducingagent(DTT)totheoxidizedsamplesrestoresactivity.
Inmuscletissuethathasbeenexposedtoirradiationtoinduceoxidation(Rowe,Maddock,Lonergan,&Hu-Lonergan,2004a)duringtheearlypostmortemperiod,degradationofsev-eralproteinsisarrested,includingdesmin(Fig.
5)(Roweetal.
,2004b;Roweetal.
,2003).
Invitrostudiesusingpuriedcalpaintodegrademyobrilsinthepresenceofhydrogenperoxide(oxidizingagent)havealsoshownthatoxidizingconditionsmaylimittheabilityofcalpaintodegradeitssubstratesincludingdesmin(Maddocketal.
,2004).
Inmuscletissue,oxidizingconditionsdoappeartoreversiblyinhibitcalpain.
Roweetal.
(2004b)usedirradiationasatooltostudycalpainactiv-ityinoxidizedearlypostmortemmeatusingcaseinzymography.
Thisactivityassayinvolvedrunningthesupernatantfractionextractedfromfresh(neverfrozen)steaksrightafterirradiationandateachofthedaysofagingonnon-reducing,non-denaturingpolyacrylamidegels.
Thesegelscontainedcaseinintheseparatinggelsolution.
Aftergelswererun,theywereincubatedatroomtemperatureina5mMcalciumchloridesolutioncontaining0.
1%2-mercaptoethanoltoreducereversiblyoxidizedcalpainandallowittodegradecasein.
Calpainactivitywasidentiedbytheappearanceofclearingzones(lightbands,Fig.
6)afterthegelwasstainedinCoomassieBrilliantBlue(Raser,Posner,&Wang,1995).
Becausecalpainlosesactivityafterextensiveautolysis,lossofcalpainactivityduringpostmortemagingofmeatindicatesprioractivation.
Calpainthatispre-ventedfrombeingactiveinthetissuewillnotfullyauto-lyzeandwillthusbeabletobeactivatedoncetheconditionsforactivityaresatised(forexample,amplecalciumandreducingconditionsasinthecaseingelas-01020304050607.
5,1657.
5,2955.
6,1655.
6,295Non-OxidizedOxidizedFlousercUecnenitspHandIonicStrength(mMNaCl)ConditionsabceddeeFig.
4.
Eectofoxidizingconditions(100lMH2O2)ontheactivityofl-calpainunderdierentpHandionicstrengthconditionsafter15minofincubationat25°C.
TheuorescentpeptideSuc-Leu-Leu-Val-Tyr-AMCwasusedasthesubstrate.
a–esignicantdierencesatP<0.
01.
Fig.
3.
(a)WesternblotofTroponin-Tinpuriedmyobrilsfromnon-irradiatedsteaksfromcontroldietfedanda-tocopherol(vitaminE)fedsteers.
(b)DensitometryofthedegradationproductofTroponin-T(30kDaband).
Anincreasedvalueindicatesmoredegradation.
Fig.
5.
Westernblotofdesmininpuriedmyobrilsfromirradiated(Irr)andnon-irradiated(NI)beeflongissimusdorsiaged7dayspostmortem.
E.
Hu-Lonergan,S.
M.
Lonergan/MeatScience71(2005)194–204201say).
Thisstudyshowedconsistentlythatl-calpaininnon-irradiatedsampleslostactivityfasterthaninirradi-atedsamplesfromthesameanimals.
Thel-calpainfromtheirradiatedsamplesshowedlittlechangeinactivityasagingtimeprogressed.
Infact,theirradiatedsampleshadmorel-calpainactivityat7daysafterirradiationthandidthenon-irradiatedsamplesfromthesamecar-casses(Fig.
6)potentiallyindicatingl-calpainwasnotactiveintheirradiatedmuscle.
However,oncecalpainwasremovedfromthetissueandsubjectedtoreducingconditionsinthecaseinzymogramassay,itwasactive;furtherevidencethatinthetissue,l-calpainmayhavebeeninactivated(butreversibly)byoxidation.
6.
SummaryThemechanismofwater-holdingcapacityiscenteredintheproteinsandstructuresthatbindandentrapwater,specicallythemyobrillarprotein.
ThereisagreatbodyofevidencethatdemonstratesadirecteectofpH,ionicstrength,andoxidationontheabilityofmyobrillarproteinandmyobrilsandmusclecellstoentrapwater.
Independentoftheseeects,itisclearthatthesamefactors(pHdecline,ionicstrength,oxidation)alsoaectproteolysisofkeycytoskeletalproteinsinpostmortemmuscle.
VariationinwaterholdingcapacityatgivenpH,andtemperatureofstorageisproposedtobeatleastpartiallyduetovariationinproteolysisandtheresultingmusclecellshrinkageandmobilizationofwatertotheextracellularspace.
Becauseofthis,investi-gationsofmetabolic,chemicalandgeneticsourcesofvariationinpostmortemproteolysisshouldprovidenewinsightintomechanismsgoverningwater-holdingcapacity.
AcknowledgmentsTheauthorsaregratefultotheUnitedStatesDepart-mentofAgricultureNationalResearchInitiativeCom-petitiveGrantsProgram(USDANRICGP)forfundingsomeoftheworkreviewedinthisdocument.
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