Annals of Clinical and Laboratory ResearchISSN 2386-5180

2015Vol. 3 No. 4:43

1© Copyright iMedPub | www.aclr.com.es

iMedPub Journalshttp://www.imedpub.com

Research Article

Elika Shabrokh1,John Kavanaugh2,Ryan McMillan2,3, Yaru Wu2, Matt Hulver2,3 and Madlyn Frisard2,3

1 Department of Surgery, University of Michigan Medical School, USA

2 DepartmentofHumanNutrition,Foods,and Exercise, Virginia Tech, Blacksburg, USA

3 The Metabolic Phenotyping Core at Virginia Tech, Blacksburg, VA, USA

Corresponding author: MadlynFrisard

frisardm@vt.edu

DepartmentofHumanNutrition,FoodsandExercise.1981KraftDrive,1085ILSB,Blacksburg,Virginia24060,USA

Tel:540-231-9994Fax: 540-231-5522

Citation: Shabrokh E, Kavanaugh J, McMillan Retal.Effectof theAdditionofHand-MixedGeneric Vancomycin on Bone Cement Strength. AnnClinLabRes.2015,3:4.

IntroductionSporadic Inclusion Body Myositis (sIBM) is an inflammatorymuscle disease that strikes individuals at random and accounts forapproximately1/3ofallidiopathicinflammatorymyopathies[1]. The disease is characterized by progressive weakness and wastingof theproximal anddistalmuscles ultimately resultingin restricted movement and mobility [2]. Individuals afflictedwiththedisordermayberestrictedfromperformingactivitiesofdailylivingandareoftenconfinedtotheuseofawalkingaidorwheelchair[2].Currently,thereisnoknowncauseorcureforsIBM,norarethereanylong-termtreatmentoptions[3].Patientsdonotgenerally respond to anti-inflammatory,immunosuppressant,or

immune modulatory drugs and treatment of the disease usually includessymptommanagementandutilizingtherapytomaintainmobilization [14,5]. The identification of novel mechanism(s)contributing todiseaseprogressionand/ormuscledefectsmayprovide new opportunities for prevention and/or treatment ofthe disorder.

MCK-APP mice, overexpress amyloid precursor proteinspecifically in skeletalmuscle anddisplay characteristicsof thedisease starting at approximately 10months of age [6]. MCK-APPmiceareconsideredanacceptedmodelofsIBMandhavebeen used to identify potential mechanism(s) contributing todisease pathology [6]. Recently, mitochondrial impairmentssuch as mitochondrial membrane depolarization and calcium

Substrate Metabolism and Mitochondrial Function in Skeletal Muscle of Amyloid Precursor Protein-Overexpressing Mice

Received: August28,2015, Accepted: December15,2015, Published: December 22, 2015

AbstractBackground: SporadicInclusionbodymyositis(sIBM)isanidiopathicinflammatorymyopathy that involves inflammation and damage to skeletal muscle tissue.Studies in humans demonstrate altered mitochondrial morphology in skeletal musclefrompatientsdiagnosedwithsIBMsuggestingmitochondrialdefectsmaybeasignificantcontributortodiseaseprogression.

Methods: MCK-APPmice, overexpress amyloid precursor protein specifically inskeletalmuscle, display characteristics of sIBM, and are an acceptedmodel tostudy disease pathology.

Results: Thecurrentstudiesdemonstrateasignificantreductioninfatoxidationand oxidative efficiency in white gastrocnemius muscle in 9-month-old MCK-APP.However, therewerenodifferences inmitochondrial bioenergeticsor theproductionofreactiveoxygenspeciesinredorwhitegastrocnemiusmusclein3,6,or9-month-oldMCK-APPmicecomparedtowild-typelittermates.

Conclusion: Functional alterations in mitochondria are not yet pronounced in3,6,and9-month-oldMCK-APPmiceprior tosymptomdevelopment;howeveralterationsinsubstratemetabolisminwhiteskeletalmusclemaybepresent.

Keywords: Amyloid precursor protein, Amyloid beta, Bioenergetics, Fatty acidoxidation, Mitochondrial metabolism, Skeletal muscle metabolism, Reactiveoxygen species

2015Annals of Clinical and Laboratory Research

ISSN 2386-5180 Vol. 3 No. 4:43

2 This Article is Available in: www.aclr.com.es

dysregulation have been observed in 3-month-old MCK-APPmice [7]. Furthermore, glutathione administration to reduceintracellular reactive oxygen species (ROS) concentrations incells cultured from this model also reduced calcium leak and restoredmembranepotentialimplicatingtheroleofintracellularROS concentrations in mitochondrial dysfunction and diseaseprogression. However, whether mitochondrial dysfunction,specificallymitochondrialbioenergetics,reactiveoxygenspeciesproduction, and dysregulated metabolism occurs in thesemice is not yet known [8,9]. The purpose of this study was to examinemitochondrialbioenergetics,substratemetabolism,andproductionof reactiveoxygen species inmitochondria isolatedfromskeletalmusclefrom3,6,and9-montholdMCK-APPmice.

Material and MethodsAnimal model MCK-APPmicewithmuscle-specificover-expressionoftheAPPgene were obtained from the original lineage of mice from Dr. Alex Shtifman and used for the experiments proposed in thisstudy [8,10,11]. The transgenic mice (MCK-APP) selectivelyoverexpress human APP and accumulate Aβ42 in affectedmuscle fibers, both of which are important features observedin sIBM patients [12,13]. A recent study has highlighted thatoverexpression of APP in a new lineage does not lead to the accumulationofamyloidbetanorthedevelopmentofsIBMlikesymptoms [14]. The mice used in the current work were from the original lineage and therefore exhibit accumulation of amyloidbeta. This animal model also shows motor impairments, which isusuallythepartialortotallossoffunctionoflimbsasaresultofmuscleweakness,becomingexacerbatedinanage-dependentmanner[6,10].BasedonpreviousstudiesthisanimalmodelisanacceptedmodelofsIBM[12,13].Three,six,andninemontholdMCK-APPmiceandtheirwildtypelittermateswereusedforthecurrent studies.

Immediately following a 12-hour fast, the animals weresacrificed using carbon dioxide asphyxiation. Skeletal musclewas harvested (gastrocnemius and quadriceps femorismuscle)for measures described below. Red and white skeletal muscle was manually separated based on visual detection. Skeletalmusclehomogenatepreparationswereused forassessmentoffattyacidoxidation,pyruvatedehydrogenaseactivity,metabolicflexibility, and oxidative efficiency. Mitochondria were isolatedfromskeletalmuscleformeasuresofmitochondrialrespiration,fatty acid oxidation, and reactive oxygen species production.Tissue samples were also collected and immediately stored for lateranalysisofmRNAexpressionandenzymeactivity.Allmousestudies were performed under an approved protocol by the InstitutionalAnimalCareandUseCommitteeatVirginiaTech.

Skeletal muscle whole homogenate preparationApproximately50mgoffreshmusclesampleswereimmediatelyplacedinto0.2mlofamodifiedsucroseEDTAmedium(SET)onice containing 250mM sucrose, 1mM EDTA, 10mM tris-HCl,and 1 mM ATP, pH 7.4. Muscle samples were then minced with scissorsandSETbufferwasadded toa20-folddiluted (wt:vol)suspension.ThemincedsampleswerehomogenizedinaPotter-

Elvehjem glass homogenizer at 10 passes across 30 secondsat 1,200 rpmwith amotor-driven teflon pestle, andmeasuresof fatty acid oxidation, and maximal enzyme activities wereperformed.

Fatty acid oxidationFattyacidoxidationwasassessedinredandwhitegastrocnemiusandquadricepsfemorismusclebymeasuringandsumming14CO2 production and 14C-labeled acid-soluble metabolites from theoxidation of [1-14C]-palmitic acid (Perkin Elmer,Waltham,MA),respectively. Briefly, samples were incubated in 0.5 uCi/ml of[1-14C]-palmiticacid for3hours.Mediawas then removedandexposedtoto200µl,of70%perchloricacidfor1hourtoliberate14CO2,whichwas trapped in a tube containing1MNaOH.TheNaOHwasthenplacedintoascintillationvialwith5mlscintillationfluid.Thevialwasthenplacedonascintillationcounter(LS4500,Beckman Coulter) and counted for the presence of 14C. Acid solublemetabolitesweredeterminedbycollectingtheacidifiedmedia and measuring 14C content.

Pyruvate dehydrogenase activity (PDH), metabolic flexibility and oxidative efficiencyPyruvate oxidationwas used to assess the activity of pyruvatedehydrogenase(PDH),theenzymethatcatalyzestheoxidationofpyruvateresultingintheprovisionofglucose-derivedacetylCoAtotheTCAcycle[15,16].[1-14C]-pyruvateoxidationwasassessedinasimilarmannertofattyacidoxidationwiththeexceptionthatpyruvatewassubstitutedforBSA-boundpalmiticacid[17,18].

Metabolicflexibilitywasassessedbymeasuring[1-14C] pyruvate oxidation±non-labeledBSA(0.5%)bound-palmiticacid.Flexibilityisdenotedbythepercentagedecreaseinpyruvateoxidationinthepresenceoffreefattyacid(e.g.ahigherpercentageisindicativeof greater metabolic flexibility). It is expressed as the ratio ofCO2productionwithlabeledpyruvateoverCO2productionwithlabeled pyruvate in the presence of palmitate.

Oxidative efficiency was calculated by dividing CO2 productionbyacidsolublemetabolite(ASMs)productionandexpressedasaratio.

Enzyme activityMaximalenzymeactivitieswereassessedinmusclehomogenatesprepared in a sample buffer containing modified sucroseEDTAmedium (SET) on ice containing 250mM sucrose, 1mMEDTA,10mMtris-HCl,and1mMATP,pH7.4.Citrate synthaseactivity was determined by the rate of DNTB reduction uponexposure to acetyl CoA at 412 nm. β-3-hydroxyacyl coenzymeA dehydrogenase (BHAD) and Malate dehydrogenase (MDH)activity was determined by the rate of NADH oxidation in thepresence of acetoacetyl coA or oxaloacetate, respectively (340nm).

Mitochondrial isolationMitochondria were isolated from red and white gastrocnemius and quadriceps femoris muscle as previously described withmodifications [19]. Freshly dissectedmusclewasplaced in ice-cold buffer 1 for mitochondrial isolation (IBM1) containing 67

3© Under License of Creative Commons Attribution 3.0 License

Annals of Clinical and Laboratory Research ISSN 2386-5180 Vol. 3 No. 4:43

2015

mMofsucrose,50mMTris/HCl,50mMKCl,10mMEDTA/Tris,and0.2%BSA.The fatandconnectivetissuewasremovedandthe muscle was minced into very small pieces (<10 mg). Thetissuewas transferred to a cell strainer, rinsedwith PBS/EDTA,andplaced in IMB1/0.05%trypsinfordigestionfor30minutes.Thesamplewasthencentrifugedat200gfor3minutesat4°C,the supernatant removed and the pellet was resuspended in IBM1. The samplewas homogenized using a Potter Ehlvejhemglass/teflon homogenizer (Thomas Scientific, Swedesboro, NJ),centrifugedat700gfor10minutesat4°C.Thesupernatantwastransferredtoapolypropylenetubeandwascentrifugedat8000gfor10minutesat4°C.Thesupernatantwascarefullyremovedandthepelletsuspendedinbuffer2formitochondrialisolation(IBM2)containing250mMSucrose,3mMEGTA/Tris,10mMTrisHCl)andthencentrifugedagainat8000gfor10minutesat4°C.Finally, the supernatantwas carefully discarded and the pelletwas resuspended in200µLof IBM2.Protein concentrationwasdetermined using the bicinchoninic acid (BCA) assay (ThermoScientific,Rockford,IL).

Respiration in isolated mitochondriaRespirometry measures of isolated mitochondria were performed using an XF24 extracellular flux analyzer (SeahorseBioscience, North Billerica, MA). Following mitochondrialisolation, mitochondria were plated on Seahorse cell cultureplatesataconcentrationof5ug/wellinthepresenceof10mMpyruvate(P5280;Sigma-Aldrich,St.Louis,MO)and5mMmalate(P5280;Sigma-Aldrich,St.Louis,MO).Experimentsconsistedof25secondmixingand4-7minutemeasurementcycles.Oxygenconsumption was measured under basal conditions in thepresenceofpyruvateandmalate (state2),ADP(5mM,Sigma-Aldrich,St.Louis,MO)stimulatedrespiration(State3),oligomycin(2µM)insensitiverespirationState4O),anduncoupled,maximalrespirationinthepresenceofFCCP(0.3μM)toassessrespiratorycapacity(State3u).Respiratorycontrolratio(RCR)wascalculatedas the ratio of ADP stimulated state 3 and oligomycin inducedstate 4 respiration.Oligomycin induced state 4 respirationwasusedinthisratiotoaccountforanycontaminatingATPaseactivitythatmayprevent the restorationof low respiration rates.Dataare expressed as pmol/min. All experiments were performed at 37°C.

Reactive oxygen species productionROS production in isolated mitochondria was assessed usingAmplex Red. Amplex® Red reagent is a colorless substrate that reactswith hydrogen peroxide (H2O2)with a 1:1 stoichiometryto produce highly fluorescent resorufin (excitation/emissionmaxima=570/585 nm). H2O2 is produced from the conversion of superoxide to H2O2 by endogenous superoxide dismutase (SOD) in thematrix [20,21]. Tomeasure ROS production fromcomplex 1, complex 3, and reverse electron transfer (REV),isolatedmitochondria were plated on a 96-well black plate ata concentration of 5 ug/well under three different conditions,respectively.Thethreeconditionswerepyruvate(20mM)/malate(10mM)/oligomycin (2μM)/rotenone (200nM) for complex1,pyruvate(20mM)/malate(10mM)/oligomycin(2μM)/SOD(400U/ml)/antimycinA(2μM)forcomplex3,andsuccinate(20mM)/

oligomycin(2μM)forreverseelectronflowtocomplex1(REV).Experiments were conducted in sucrose/mannitol solution inorder to maintain mitochondrial integrity. Experiments consisted of 1-minute delay and 1 minute reading cycles, followed bya 5 second mixing cycle performed every third reading. Allexperimentswereperformedat37°C.Measures forROS levelswere conducted on a microplate reader (Biotek synergy 2,Winooski,VT).FluorescenceofAmplexRedwasmeasuredusinga530nmexcitationfilteranda560nmemissionfilter.

Total RNA extraction and qRT-PCRTotal cellular RNA was extracted using an RNeasy Mini Kit (Qiagen)andDNaseItreatment(Qiagen,Valencia,CA),accordingto the manufacturer’s instructions. Target gene expressionwasnormalized toβ-actin rRNA levels,whichwere assayedbymultiplexing with the manufactures 5#VIC-labeled, primer-limited β-actin endogenous control premix. Primers and 5#FAM-labeled Taqman probes were purchased as pre-validatedassaysandqRT-PCRwasperformedusinganABI7900HT(AppliedBiosystems,Carlsbad,CA).Relativequantificationoftargetgeneswascalculatedusingthe2Δ−CTmethod.Derivationofthe2−ΔCTequationhasbeendescribedinAppliedBiosystemsUserBulletinNo.2(P/N4303859).

Circulating inflammatory cytokines:Interleukin6(IL6)andC-reactiveprotein inflammatorymarkersweremeasuredviaenzyme-linkedimmunosorbentassay(ELISA)from Alpco (Salem, NH) and R&D Systems (Minneapolis, MN)respectively,accordingtomanufacturer’sinstructions.

Statistical analysis: StatisticsResultswereanalyzedwitha2-wayANOVAwithaTukeypost-hoctestformultiplecomparisons.AlldatawastestedfornormalityusingtheShapiro-Wilknormalitytest.If itwasdeterminedthatthedatawasnotnormal,aMann-WhitneytestwasconductedinplaceofANOVA.Resultsarepresentedasmean±SD.Thelevelofsignificancewasseta prioriatP<0.05.

ResultsFatty acid oxidationAlthough there were no significant interactions, there weresignificantageeffects inboth redandwhite skeletalmuscle inbothwildtypeandtransgenicanimals(Figure 1).AlthoughCO2 productionwassignificantlyhigherinredmusclefrom9-month-oldmice (Figure 1A),ASMswere significantly lower in6and9montholdmice(Figure 1B),andtotaloxidationwassignificantlylower in 6-month-old mice compared to 3-month-old mice(Figure 1C).

In white muscle, CO2 production was significantly lower in9-month-old mice compared to 3-month-old mice (Figure 1D), ASMswere significantly lower in 6 and 9-month-oldmicecomparedto3-month-oldmice (Figure 1E),andtotaloxidationwas significantly lower in 9-month-old mice compared to3-month-old mice (Figure 1F). Additionally, there were trendsfor an effect of genotype in white skeletal muscle. Post hocanalysis revealed that both CO2 and total fatty acid oxidation

2015Annals of Clinical and Laboratory Research

ISSN 2386-5180 Vol. 3 No. 4:43

4 This Article is Available in: www.aclr.com.es

was significantly lower in 9-month- oldMCK-APP compared to9-month-oldwildtypemice(Figure 1D and Figure 1F).

Pyruvate dehydrogenase activity (PDH), metabolic flexibility, and oxidative efficiencyTherewerenosignificantinteractionsinpyruvatedehydrogenaseactivity,metabolicflexibility,oroxidativeefficiencyinredorwhiteskeletalmuscle(Figure 2).However,therewereageeffects.

Pyruvate dehydrogenase activity and metabolic flexibilitywas significantly higher in red muscle from 9-month-old mice(Figure 2A and 2B)comparedto3-month-old-mice.Additionally,oxidativeefficiencywassignificantlyhigherin9-month-oldmicecomparedtoboth3and6-month-oldmice(Figure 2C).

Inwhitemuscle,pyruvatedehydrogenaseactivitywassignificantlyhigher in 6-month-old mice compared to 3-month-old mice(Figure 2D).However,oxidativeefficiencywassignificantlylowerin 9-month-old mice compared to 6-month-old mice (Figure 2F).Additionally,therewasatrendforaneffectofgenotypeinwhite skeletal muscle. There was a trend for reduced metabolic flexibility in 6 and 9-month-old -MCK-APP mice compared towild typemice (Figure 2E).Additionally, 9-month-old-MCK-APPexhibited reduced oxidative efficiency compared to wild typemice(Figure 2F).

Metabolic enzyme activityMetabolicenzymaticactivityisdisplayedinFigure 3.Whiletherewerenosignificantinteractionsorgenotypeeffectswithregardsto Citrate Synthase (CS), β-hydroxyacyl-CoA dehydrogenase(β-HAD) and Malate Dehydrogenase (MDH) activity in red or

whiteskeletalmuscle,therewereageeffects.

In red muscle, citrate synthase was significantly lower in6-month-oldanimalscomparedtoboth3and9montholdmice(Figure 3A),β-HADwassignificantlyhigherin9montholdmice(Figure 3B)comparedto3and6month-old-mice,andMDHwassignificantlylowerin6-month-oldmicecomparedtoboth3and9-month-oldmice(Figure 3C).Therewasnoeffectofgenotypewith regards to enzyme activity in red skeletal muscle. Therewerealsonosignificantageorgenotypedifferencesobservedinwhiteskeletalmuscle(Figure 3D-3F).

Respiration in isolated mitochondriaMitochondrialoxygenconsumptionwasassessedinredskeletalmuscle only and is displayed in Figure 4.Therewerenosignificantinteractionswithanyof themeasuresofmitochondrialoxygenconsumption, however there were significant age effects. RCRand state three respirationwas significantly lower in 9-month-oldmice compared to3and6-month-oldmice (Figure 4A and 4C).State2respirationandstate3urespirationwassignificantlylower in 9-month-old mice compared to 6-month-old mice(Figure 4B and 4E).Therewerenosignificantdifferencesinstate4Orespiration(Figure 4D).

Reactive oxygen species generationTherewerenosignificantdifferencesinROSgenerationbetweentransgenic and wild type animals at any of the time pointsmeasured(Figure 5).Therewas,however,significantdifferencesin ROS production from complex I (Figure 5A) and complex III(Figure 5B),between6and9-month-oldmice(Figure 5C).

Figure 1 PalmitateoxidationinskeletalmusclefromMCK-APPandwildtypemice.(A)CO2productioninredskeletalmuscle,(B)Acidsolublemetaboliteproductioninredskeletalmusclemuscle,(C)Totaloxidationinredskeletalmuscle,(D)CO2productioninwhite skeletalmuscle, (E)Acid solublemetaboliteproduction inwhite skeletalmuscle, (F) Totaloxidation inwhite skeletalmuscle.

5© Under License of Creative Commons Attribution 3.0 License

Annals of Clinical and Laboratory Research ISSN 2386-5180 Vol. 3 No. 4:43

2015

mRNA content in red and white skeletal muscle from MCKβ-APP mice and their littermate controlsmRNA content for markers of autophagy was assessed in red and whiteskeletalmuscle(Figure 6).LC3mRNAwassignificantlyhigherinredmuscleof3and9-month-oldMCKβ-APP mice compared to wildtypemice(Figure 6A).BeclinmRNAwassignificantlyhigherin red and white muscle from MCKβ-APP3-monthand9-month-old MCKβ-APP micecomparedtowildtypemice(Figure 6B-6D).

Markers of systemic inflammation in MCK-APP mice and wild-type littermates Toassesssystemicinflammation,fastingmeasuresofC-reactiveprotein (CRP) and interlukin-6 (IL- 6) were measured and aredisplayed in Figure 7.Although,at3-monthsCRPconcentrationswere slightly lower in MCK-APP mice compared to wild typeanimals(p=0.08),therewerenosignificantdifferencesbetweengenotype observed at 6 or 9-months (Figure 7A). Serum IL-6measurementsshowednosignificantdifferencesbetweenMCK-APPandwild-typemiceatanytimepoints(Figure 7B).

DiscussionsIBM is themost prevalentmuscle disease among the elderlyand risk increases with age [1]. There is no known cause or successful treatment for the disorder leaving patients withlimitedoptionsfollowingdiagnosis[3].ThecurrentstudytestedwhethermitochondrialdysfunctionispresentinskeletalmuscleinamousemodelofsIBM.Contrarytoourhypothesis,theresultsdemonstratethatmitochondrialfunctionisnotdisruptedinMCK-

APPmice. Additionally, there were no differences in substratemetabolismorreactiveoxygenspeciesgenerationinredmusclefrom MCK-APP mice compare to the wild types. Conversely,decreasedfatmetabolismanddecreasedoxidativeefficiency inwhitemusclesfromMCK-APPmicecomparedtowildtypemicewasobserved in thecurrentstudy. IncreasedmRNAcontentofLC3,amarkerofautophagy,wasalsoreportedinMCK-APPmicecompared to wild type controls.

These results are in contrast to data reported by Boncompagni et al.[8]whichdemonstratedstructuralandfunctionalalterationsin mitochondria of 2-3month oldMCK-APPmice. Their studyreported disruption of TCA cycle activity, i.e., reductions inradiolabeled glutamate, and succinate, in MCK-APP micecompared to wild type controls. Increased ROS production inMCK-APP mice was also reported in this study. Differences infindingsbetweenthecurrentstudyandBoncompagnimaybeduetothedifferencesinmethodologiesusedtoassessmitochondrialfunction. While the current study assessed mitochondrialfunctionbymeasuringmitochondrialoxygenconsumption,fattyacid oxidation, and oxidative enzyme activity, Boncompagni’sstudy assessed structural andmorphological differenceswithinthemusclealongwithTCAcycleactivity(rateofappearanceanddisappearance of radiolabeled glutamate and succinate) andmembranepotential.Furthermore,thetwostudiesuseddifferenttechniques to assess reactive oxygen species production. TheprecisemeasurementofROS in cells andtissues is challengingbecauseofextremelylowconcentrationsandshortlifespan.Thecurrent study employed Amplex Red to assess H2O2 as a marker of ROSproduction.Amplexredishighlyspecificandsensitive,with

Figure 2 Pyruvatedehydrogenaseactivity,metabolicflexibility,andoxidativeefficiencyinskeletalmusclefromMCK-APPandwildtypemice.(A)Pyruvateoxidationinredskeletalmuscle,(B)Metabolicflexibilityinredskeletalmuscle,(C)Oxidativeefficiencyinredskeletalmuscle,(D)Pyruvateoxidationinwhiteskeletalmuscle,(E)Metabolicflexibilityinwhiteskeletalmuscle,(F)Oxidativeefficiencyinwhiteskeletalmuscle.

2015Annals of Clinical and Laboratory Research

ISSN 2386-5180 Vol. 3 No. 4:43

6 This Article is Available in: www.aclr.com.es

Figure 3 EnzymeactivityinskeletalmusclefromMCK-APPandwildtypemice.(A)Citratesynthaseactivityinredskeletalmuscle,(B)β-hydroxyacyl-CoAdehydrogenaseactivityinredskeletalmuscle,(C)Malatedehydrogenaseactivityinredskeletalmuscle,(D)Citratesynthaseactivityinwhiteskeletalmuscle,(E)β-hydroxyacyl-CoAdehydrogenaseactivityinwhiteskeletalmuscle,(F)Malatedehydrogenaseactivityinwhiteskeletalmuscle.

Figure 4 Mitochondrial bioenergetics in isolated mitochondria from red skeletal muscle from MCK-APP and wild type mice. (A)Respiratorycontrol ratio, (B)State2respirationrate, (C)State3 (ADPstimulated) respirationrate, (D) state4o (oligomycininsensitive)respirationrate,and(E)state3u(FCCP-stimulated)respirationrate.

alimitofdetectionof≈5pmolofH2O2. Also, the stoichiometry of Amplex Red and H2O2is1:1;thus,theassayresultsarelinearovertherangeofvaluesencounteredintissuesandcells[22].Ontheotherhand,BoncompagniassessedROSproductionbymeasuringintracellularROSconcentrationsusing5- (and6) chloromethyl-2′,7′-dichlorodihydrofluoresceindiacetate(DCFH-DA).TheDCFH-DA technique is often criticized since photoreduction of DCF

results in artificial production of a semiquinone radical that inturncanreduceoxygentofreeradicals,andtheoxidationofDCFHtotheDCFcanbeself-catalyzedbyperoxidases[23].Therefore,conditions thataltercellularperoxidase levelscouldaffectDCFfluorescenceindependentofactualcellularROSlevels[23,24].

There were also differences between the two studies withregards to the fiber types assessed. While Boncompagni

7© Under License of Creative Commons Attribution 3.0 License

Annals of Clinical and Laboratory Research ISSN 2386-5180 Vol. 3 No. 4:43

2015

Figure 5 Reactiveoxygenspecies(ROS)productioninisolatedmitochondriafromredskeletalmusclefromMCK-APPandwildtypemice.(A)ROSproduction fromcomplexone, (B)ROSproduction fromcomplex three, (C)ROSproduction from reverseelectrontransport.

Figure 6 GeneexpressionofautophagymarkersinredandwhiteskeletalmusclefromMCK-APPandwildtypemice.(A)LC3expressionin red skeletalmuscle, (B) Beclin expression in red skeletalmuscle, (C) LC3expression inwhite skeletalmuscle, (D) Beclinexpression in red skeletal muscle.

et al. [8] examined mitochondrial parameters in extensor digitorum longus (EDL) andflexordigitorumbrevis (FDB), bothpredominately white muscle types, the current study assessed quadricepsandgastrocnemiusmuscles,bothconsideredmixedmusclegroups.Wechosethesemusclegroupsbecausetheyaremorephysiologicallyrelevanttothemusclesaffectedinpatients

diagnosedwithsIBM(vastuslateralis)[25].Itisimportanttonotethat red and white muscle was separated for the current studies andwhilewedidnotmeasuremitochondriabioenergeticsfromwhite skeletal muscle we did note a decrease in fat oxidation(total oxidation and CO2 production) and oxidative efficiencyin white gastrocnemius and quadriceps femorismuscle. These

2015Annals of Clinical and Laboratory Research

ISSN 2386-5180 Vol. 3 No. 4:43

8 This Article is Available in: www.aclr.com.es

results also coincide with the previous human proteomic studies thatfoundreductionsinproteinsexclusivelyinthewhitemuscles[26]. Finally, Boncompagni et al. [8] identified and separated“amyloidbetaaffected”musclefibersfortheirstudy.Thesefiberswere characterized by amyloid beta accumulation, amorphousmaterial, the presence of vacuoles, and mitochondrial structural alterations.NotallmusclefiberswithinamuscleareaffectedbysIBMandinfactaffectedfibersareinterspersedbetweenmanyhealthyfibers[8].Itwasnotpossibleinthecurrentstudytoonlyselectaffectedfibers.Asaresult,thelackofdifferencesbetweenMCK-APPandwildtypemiceobservedinthecurrentstudycouldbe due to dilution of affected fibers interspersedwithinmanyhealthy fibers. Nonetheless, these data along with the workfrom Boncompagni et al. [8] suggest that other factors such as amyloidbetaaccumulationmayberequiredfortheinitiationofmitochondrialabnormalitiesinskeletalmuscleandarethereforemorelikelythecause(orcauses)ofsIBM.

Whiledatafromthecurrentstudydonotindicatemitochondriaas a primary factor in the initial development of sIBM, it ispossiblethatmitochondriamaystillbeacontributortodisease

progression. The current study specifically chose to employ3,6 and 9-month old MCK-APP mice to explore the effects ofmitochondrial dysfunction prior to the occurrence of sIBMsymptoms including inflammation, amyloid beta accumulation,and motor defects [27,28]. It is possible that mitochondrialdysfunctionoccursfollowingdiseaseonsetandmaycontributetothedecreasedfunctionalityoccurringwithdiseaseprogression.This idea is supported by previous work conducted in humans, in which mitochondrial abnormalities are observed followingdiagnosis[29-31].

Data from the current study demonstrate increase in LC3mRNA in MCK-APP mice, which is an indication of increasedautophagosome formation. During autophagy, LC3 is lipidated,and the LC3-phospholipid conjugate (LC3-II) is localized to theautophagosome [32]. The autophagosome travels through the cytoplasm of the cell to a lysosome, which then fuses with the autophagosomeresultingintheformationoftheautolysosome.As such, the LC3-phospholipid conjugate system is importantfor the development and transport of the autophagosome [33]. Additionally,anydisruptioninanyofthestepsofautophagy,i.e.,disruptioninlysosomeformationorfusionoftheautophagosomewiththelysosome,couldresultintheaccumulationofdamagedcellular debris including degraded cellular protein. While thecurrentstudysuggestsanincreaseinautophagosomeformation,it is not evident whether this results in the formation of theautolysosome or if there are defects in the formation of thelysosome or autolysosome.

There are some limitations to the current work. For example,whiletheMCK-APPanimalmodel isanacceptedanimalmodelforthestudyofsIBM,theroleofamyloidbetaprecursorproteinand subsequent accumulation of amyloid beta protein in thedevelopment of the disease is still up for debate. In addition,in order to understand the effects of age on the role of themitochondriainthedevelopmentofsIBM,additionaltimepointswitholderanimals (12,15,18months) shouldbe investigated.sIBMisanage-relateddiseaseandtheeffectofageisanimportantfactortobeinvestigatedinMCK-APPanimals.

SummaryThepresentworkdemonstratesthatmitochondrialabnormalitiesand ROS production are not observed in red gastrocnemiusandquadricepsfemorismuscleandthereforedonotappeartobe a primary cause of sIBM like symptoms in MCK-APPmice.Nonetheless,thereisasignificantreductioninfatmetabolismaswellasanupregulationofautophagicpathwayssuggestingthatcertainalterationsinskeletalmusclefromMCK-APPmiceoccurprior to the onset of classic disease symptoms.

Figure 7 Fasting, circulating inflammatory markers fromMCK-APPandwildtypemice.(A)C-reactiveproteinconcentration,(B)Interleukin-6concentration.

9© Under License of Creative Commons Attribution 3.0 License

Annals of Clinical and Laboratory Research ISSN 2386-5180 Vol. 3 No. 4:43

2015

References1 MachadoP,MillerA,Holton J,HannaM (2009)Sporadic inclusion

bodymyositis:anunsolvedmystery.ActaReumatolPort34:161-182.

2 GarleppMJ,Mastaglia FL (1996) Inclusionbodymyositis. JNeurolNeurosurgPsychiatry60:251-255.

3 Askanas V, Engel WK (2008) Inclusion-body myositis: muscle-fibermolecular pathology and possible pathogenic significance ofits similarity to Alzheimer's and Parkinson's disease brains. Acta Neuropathol116:583-595.

4 Chou SM (1993) Inclusion bodymyositis. Baillieres Clin Neurol 2:557-577.

5 Mastaglia FL, Laing BA, Zilko P (1993) Treatment of inflammatorymyopathies.BaillieresClinNeurol2:717-740.

6 KitazawaM,GreenKN,CaccamoA, LaFerla FM (2006)GeneticallyaugmentingAbeta42levelsinskeletalmuscleexacerbatesinclusionbodymyositis-likepathologyandmotordeficitsintransgenicmice.TheAmericanjournalofpathology168:1986-1997.

7 ShtifmanA,WardCW,LaverDR,BannisterML,LopezJR,etal.(2010)Amyloid-βproteinimpairsCa2+releaseandcontractilityinskeletalmuscle.NeurobiolAging31:2080-2090.

8 BoncompagniS,MoussaCE,LevyE,PezoneMJ,LopezJR,etal.(2012)Mitochondrial dysfunction in skeletalmuscleof amyloidprecursorprotein-overexpressingmice.JBiolChem287:20534-20544.

9 Rhein V (2009) Amyloid-beta and tau synergistically impair theoxidative phosphorylation system in triple transgenic Alzheimer'sdiseasemice.Proceedingsof theNationalAcademyofSciencesoftheUnitedStatesofAmerica106:20057-20062.

10 SugarmanMC,YamasakiTR,OddoS,EchegoyenJC,MurphyMP,etal.(2002)Inclusionbodymyositis-likephenotypeinducedbytransgenicoverexpression of beta APP in skeletal muscle. Proc Natl Acad Sci U SA99:6334-6339.

11 Vattemi G, Engel WK, McFerrin J, Askanas V (2003) Cystatin Ccolocalizes with amyloid-beta and coimmunoprecipitates withamyloid-betaprecursorprotein insporadic inclusion-bodymyositismuscles.JNeurochem85:1539-1546.

12 Fukuchi K, Pham D, HartM, Li L, Lindsey JR (1998) Amyloid-betadepositioninskeletalmuscleoftransgenicmice:possiblemodelofinclusionbodymyopathy.AmJPathol153:1687-1693.

13 Jin LW, Hearn MG, Ogburn CE, Dang N, Nochlin D, et al. (1998)Transgenicmiceover-expressingtheC-99fragmentofbetaPPwithan alpha-secretase site mutation develop a myopathy similar tohumaninclusionbodymyositis.AmJPathol153:1679-1686.

14 LuoYB,JohnsenRD,GriffithsL,NeedhamM,FabianVA,etal.(2013)Primary over-expression of AβPP in muscle does not lead to thedevelopmentofinclusionbodymyositisinanewlineageoftheMCK-AβPPtransgenicmouse.IntJExpPathol94:418-425.

15 GaoS,McMillanRPJacasJZhuQ,LiX,etal. (2014)Regulationofsubstrateoxidationpreferencesinmusclebythepeptidehormoneadropin.Diabetes63:3242-3252.

16 ZhangS(2014)Chickensfromlinesselectedforhighandlowbodyweight show differences in fatty acid oxidation efficiency andmetabolic flexibility in skeletal muscle and white adipose tissue.Internationaljournalofobesity38:1374-1382.

17 AndersonAS,RobertsPC,FrisardMI,McMillanRP,BrownTJ,etal.(2013) Metabolic changes during ovarian cancer progression astargetsforsphingosinetreatment.ExpCellRes319:1431-1442.

18 Power RA, Hulver MW, Zhang JY, Dubois J, Marchand RM, et al.(2007)Carnitinerevisited:potentialuseasadjunctivetreatmentindiabetes.Diabetologia50:824-832.

19 FrezzaC,CipolatS,ScorranoL(2007)Organelleisolation:functionalmitochondriafrommouseliver,muscleandculturedfibroblasts.NatProtoc2:287-295.

20 Quinlan CL, Gerencser AA, Treberg JR, Brand MD (2011) Themechanism of superoxide production by the antimycin-inhibitedmitochondrialQ-cycle.JBiolChem286:31361-31372.

21 QuinlanCL,TrebergJR,PerevoshchikovaIV,OrrAL,BrandMD(2012)Nativeratesofsuperoxideproductionfrommultiplesitesinisolatedmitochondria measured using endogenous reporters. Free radicalbiology&medicine5:1807-1817.

22 Dikalov S, Griendling KK, Harrison DG (2007) Measurement ofreactiveoxygenspeciesincardiovascularstudies.Hypertension49:717-727.

23 Rota C, Chignell CF, Mason RP (1999) Evidence for free radicalformation during the oxidation of 2'-7'-dichlorofluorescin to thefluorescentdye2'-7'-dichlorofluoresceinbyhorseradishperoxidase:possibleimplicationsforoxidativestressmeasurements.Freeradicalbiology&medicine27:873-881.

24 RotaC,FannYC,MasonRP(1999)Phenoxyl freeradical formationduringtheoxidationofthefluorescentdye2',7'-dichlorofluoresceinby horseradish peroxidase. Possible consequences for oxidativestressmeasurements.TheJournalofbiologicalchemistry27:28161-28168.

25 KobayashiYM,RaderEP,CrawfordRW,CampbellKP(2012)Endpointmeasures in themdxmouse relevant formusculardystrophypre-clinicalstudies.NeuromusculDisord22:34-42.

26 ParkerKC,KongSW,WalshRJ;Bch,SalajeghehM,MoghadaszadehB,etal.(2009)Fast-twitchsarcomericandglycolyticenzymeproteinlossininclusionbodymyositis.MuscleNerve39:739-753.

27 Askanas V, Engel WK, Alvarez RB (1992) Light and electronmicroscopiclocalizationofbeta-amyloidproteininmusclebiopsiesofpatientswithinclusion-bodymyositis.AmJPathol141:31-36.

28 SchmidtJ(2008)InterrelationofinflammationandAPPinsIBM:IL-1betainducesaccumulationofbeta-amyloidinskeletalmuscle.Brain:ajournalofneurology11228-1240.

29 Askanas V, Alvarez RB, Engel WK (1993) beta-Amyloid precursorepitopesinmusclefibersofinclusionbodymyositis.AnnNeurol34:551-560.

30 AskanasV,McFerrin J,AlvarezRB,BaquéS,EngelWK (1997)BetaAPP gene transfer into cultured humanmuscle induces inclusion-bodymyositisaspects.Neuroreport8:2155-2158.

31 AskanasV (1996) Transferofbeta-amyloidprecursorprotein geneusing adenovirus vector causes mitochondrial abnormalities incultured normal human muscle. Proceedings of the NationalAcademyofSciencesoftheUnitedStatesofAmerica91314-1319.

32 Tanida I, Minematsu-Ikeguchi N, Ueno T, Kominami E (2005)Lysosomalturnover,butnotacellularlevel,ofendogenousLC3isamarkerforautophagy.Autophagy1:84-91.

33 Jessup W, Bodmer JL, Dean RT, Greenaway VA, Leoni P (1984)Intracellular turnover and secretion of lysosomal enzymes.BiochemicalSocietytransactions1:529-531.