High-Fat-Diet-Induced Obesity Produces Spontaneous Ventricular Arrhythmias and Increases the

15 Pages·2017·1.2 MB·English

International Journal o f
Molecular Sciences
Article
High-Fat-Diet-Induced Obesity Produces Spontaneous
Ventricular Arrhythmias and Increases the Activity of
Ryanodine Receptors in Mice
GinaSánchez1,†,FelipeAraneda2,†,JuanPedroPeña3,JoséPabloFinkelstein2,
JaimeA.Riquelme4,LuisMontecinos2,GenaroBarrientos2,PaolaLlanos5,
ZullyPedrozo2,4,MatildeSaid6,RicardoBull2 ID andPaulinaDonoso2,*
1 ProgramadeFisiopatología,InstitutodeCienciasBiomédicas,FacultaddeMedicina,UniversidaddeChile,
8380453Santiago,Chile;[email protected]
2 ProgramadeFisiologíayBiofísica,InstitutodeCienciasBiomédicas,FacultaddeMedicina,
UniversidaddeChile,8380453Santiago,Chile;[email protected](F.A.);
jfi[email protected](J.P.F.);[email protected](L.M.);[email protected](G.B.);
[email protected](Z.P.);[email protected](R.B.)
3 EscueladeCienciasVeterinarias,UniversidaddeViñadelMar,2572007ViñadelMar,Valparaíso,Chile;
[email protected]
4 AdvancedCenterforChronicDiseases,FacultaddeCienciasQuímicasyFarmacéuticas,
UniversidaddeChile,8380494Santiago,Chile;[email protected]
5 InstitutodeInvestigaciónenCienciasOdontológicas,FacultaddeOdontología,UniversidaddeChile,
8380492Santiago,Chile;[email protected]
6 CentrodeInvestigacionesCardiovasculares,CCT-CONICETLaPlata,FacultaddeMedicina,
UniversidadNacionaldeLaPlata,1900LaPlata,Argentina;[email protected]
* Correspondence:[email protected];Tel.:[phone]
† Theseauthorscontributedequallytothiswork.
Received:9January2018;Accepted:7February2018;Published:10February2018
Abstract: Ventriculararrhythmiasareacommoncauseofsuddencardiacdeath,andtheiroccurrence
is higher in obese subjects. Abnormal gating of ryanodine receptors (RyR2), the calcium release
channelsofthesarcoplasmicreticulum,canproduceventriculararrhythmias. Sinceobesitypromotes
oxidativestressandRyR2areredox-sensitivechannels,weinvestigatedwhethertheRyR2activitywas
alteredinobesemice. Micefedahighfatdiet(HFD)becameobeseaftereightweeksandexhibiteda
significantincreaseintheoccurrenceofventriculararrhythmias. SingleRyR2channelsisolatedfrom
theheartsofobesemiceweremoreactiveinplanarbilayersthanthoseisolatedfromtheheartsofthe
controlmice. Atthemolecularlevel,RyR2channelsfromHFD-fedmicehadsubstantiallyfewerfree
thiolresidues,suggestingthatredoxmodificationswereresponsibleforthehigheractivity. Apocynin,
providedinthedrinkingwater,completelypreventedtheappearanceofventriculararrhythmias
inHFD-fedmice,andnormalizedtheactivityandcontentofthefreethiolresiduesoftheprotein.
HFDincreasedtheexpressionofNOX4,anisoformofNADPHoxidase,intheheart. Ourresults
suggestthatHFDincreasestheactivityofRyR2channelsviaaredox-dependentmechanism,favoring
theappearanceofventriculararrhythmias.
Keywords: calciumreleasechannels;reactiveoxygenspecies(ROS);redoxmodifications;ventricular
tachycardia;NADPHoxidase
1. Introduction
Suddencardiacdeath(SCD)representsahighproportionofallnaturaldeaths[1]. Ventricular
arrhythmias,suchasventriculartachycardiaandventricularfibrillation,thatinterferewiththenormal
Int.J.Mol.Sci. 2018,19,533;doi:10.3390/ijms19020533 www.mdpi.com/journal/ijms
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bloodpumpingactivityoftheheart,areprevalentcausesofSCD,andobesesubjectsareatahigher
riskofSCDthannon-obesesubjects[2–4]. Obesityisfrequentlyassociatedwithdyslipidemia,insulin
resistance,andtype2diabetes,andalltheseconditionsfurtherincreasetheriskofarrhythmias[5].
Cardiac contraction is initiated by an action potential that spreads through the membrane of the
cardiomyocytes.Thedurationandshapeoftheventricularactionpotentialdependsonthecoordinated
interplayofmultipleoutwardandinwardioniccurrents. Changesinelectrochemicalgradients,or
abnormal gating of any of the ion channels responsible for these currents, can be involved in the
generationofarrhythmias.
Disruptionofcalciumhomeostasisisinvolvedinthepathogenesisofventriculararrhythmias.
Ryanodinereceptors(RyR2),thesarcoplasmicreticulum(SR)calciumreleasechannels,providemost
ofthecalciumneededforheartcontractionduringsystole,andtheiropeningistightlycontrolledby
theL-typecalciumcurrentthatentersthecellduringtheactionpotential. Duringdiastole,RyR2must
closetoallowforthereuptakeofcalciumintotheSR[6]. Aberrantreleaseofcalciumduringdiastole,
duetothefailureofRyR2tocloseproperly,activatesitsextrusionviathesodium-calciumexchanger,
generating a net inward current that can depolarize the cardiomyocyte cell membrane (delayed
after depolarization) and cause arrhythmias. Genetic studies have shown that catecholaminergic
polymorphic ventricular tachycardia (CPVT), a severe form of arrhythmia induced by emotional
stressorexerciseinpatientswithoutpreviouscardiacdisease,iscausedbymissensemutationsof
the RyR2 gene that disrupt the interactions between the protein domains responsible for channel
closing[7,8]. Post-translationalmodificationsofRyR2canalsopreventthecorrectchannelclosing.
RyR2containshyperreactivecysteinesthatareoxidativelymodifiedbyreactiveoxygenspecies(ROS),
especiallyinthepresenceofanimbalanceofROSinthecell[9]. InvitrooxidationofRyR2increases
thechannelresponsetocytoplasmiccalciumconcentrationandfavorsthecalciumreleaseinisolated
cardiomyocytes,generatingcalciumwavesandarrhythmias[10,11].
Obesity is characterized by an increased generation of ROS [12,13], which may impact RyR2
functionandaffecttheheartrhythm. TheRyR2activityintheheartsofobeseanimalshasnotyet
beeninvestigated.
FeedingC57BL/6micewithahighfatdiet(HFD)foreightweeksisawell-establishedmethodto
induceobesity[14]. Therefore,usingthismodel,westudiedtheresponsetocytoplasmiccalciumof
singleRyR2channelsincorporatedinplanarbilayersandquantifiedtheoccurrenceofspontaneous
arrhythmiasthroughelectrocardiogram(ECG)recordings. Wealsoinvestigatedtheeffectofapocynin
ontheactivityofthesechannels. Apocynin(acetovanillone)isacathecol-derivedmoleculeproduced
intherootsofPicrorhizakurroa,aplantusedintraditionalIndianmedicineasananti-inflammatoryand
antioxidant. ApocyninhasROSscavengingpropertiesandisapotentinhibitorofNADPHoxidase,
isoform2(NOX2),whoseactivityincreasesinseveraltissuesinobesityandhyperglycemia[15–17].
WepreviouslyshowedthatapocyninpreventstheincreaseinRyR2activitycausedbyreversibleredox
modificationsoftheproteinafterashortepisodeofischemia-reperfusioninrathearts[18].
2. Results
2.1. MiceBodyWeight,Glucose,andPlasmaLipids
Mice fed with an HFD became obese after eight weeks and had an average 35% increase in
bodyweightwithconcomitantincreasesinfastingglucoseandtotalcholesterol. Plasmatriglycerides,
however,weresimilarinbothcontrolandHFD-fedmice. Theadditionofapocynintodrinkingwater
(1.5mmol/L)duringthelastfourweeksoftreatmentdidnotproducesignificantchangesinthese
metabolicparameters(TableS1).
2.2. ECGRecording
We recorded surface ECG (Lead I) under light anesthesia with isoflurane. A representative
ECG record obtained in a mouse fed with the control diet is shown in Figure 1A. Control mice
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had normal sinus rhythms exhibiting only occasional or no ventricular extrasystoles during the
recording period. Exceptionally, one mouse had a single episode of spontaneous ventricular
six-minuterecordingperiod. Exceptionally,onemousehadasingleepisodeofspontaneousventricular
tachycardia (VT), regaining normal rhythm after 1 s. In contrast, 6 out of 12 mice fed the HFD showed
tachycardia(VT),regainingnormalrhythmafter1s. Incontrast,6outof12micefedtheHFDshowed
different arrhythmic events during the six-minute recording period. These events ranged from
differentarrhythmiceventsduringthesix-minuterecordingperiod.Theseeventsrangedfromfrequent
frequent ventricular extrasystoles (Figure 1B) to one or more episodes of monomorphic ventricular
ventricularextrasystoles(Figure1B)tooneormoreepisodesofmonomorphicventriculartachycardia
tachycardia (Figure 1C). Two mice had sustained ventricular tachycardia during most of the
(Figure1C).Twomicehadsustainedventriculartachycardiaduringmostoftherecordingperiod: one
recording period: one with monomorphic ventricular tachycardia and another with bidirectional
withmonomorphicventriculartachycardiaandanotherwithbidirectionalventriculartachycardia
ventricular tachycardia (Figure 1D). Apocynin greatly reduced the number of premature ventricular
(Figure1D).Apocyningreatlyreducedthenumberofprematureventricularbeats,bothincontrol
beats, both in control and HFD-fed mice (Figure 1E) and completely eliminated the tachycardia
andHFD-fedmice(Figure1E)andcompletelyeliminatedthetachycardiaeventsinHFD-fedmice
events in HFD-fed mice (Figure 1F).
(Figure1F).

Figure 1. A high fat diet (HFD) increases the occurrence of spontaneous ventricular arrhythmias.
Figure 1. A high fat diet (HFD) increases the occurrence of spontaneous ventricular arrhythmias.
Representative electrocardiographic records obtained in (A) control and (B–D) HFD-fed mice:
Representative electrocardiographic records obtained in (A) control and (B–D) HFD-fed mice: (A)
(A) normal sinus rhythm, (B) premature ventricular beats (PVB, arrows), (C) non-sustained
normal sinus rhythm, (B) premature ventricular beats (PVB, arrows), (C) non-sustained
monomorphicventriculartachycardia(VT),and(D)sustainedbidirectionalVT.(E)Averagenumberof
monomorphic ventricular tachycardia (VT), and (D) sustained bidirectional VT. (E) Average number
PVBduring5minofrecordingincontrol(n=11)andHFD-fedmice(n=12),withorwithoutapocynin.
of PVB during 5 min of recording in control (n = 11) and HFD-fed mice (n = 12), with or without
(F)Numberofventriculartachycardia(VT)episodespermouseincontrolandHFD-fedmice,with
apocynin. (F) Number of ventricular tachycardia (VT) episodes per mouse in control and HFD-fed
orwithoutapocynin. *p<0.05comparedtoallotherconditions,Kruskal–Wallistestfollowedby
mice, with or without apocynin.* p < 0.05 compared to all other conditions, Kruskal–Wallis test
Dunnstest.
followed by Dunns test.
2.3. EchocardiographicMeasurements
2.3. Echocardiographic Measurements
TToo aasssseessss tthhee eeffffeecctt ooff eeiigghhtt wweeeekkss ooff HHFFDD oonn ccaarrddiiaacc ffuunnccttiioonn,, wwee ddeetteerrmmiinneedd tthhee ffrraaccttiioonnaall
sshhoorrtteenniinngg ooff tthhee lleefftt vveennttrriiccllee iinn eecchhooccaarrddiiooggrraammss oobbttaaiinneedd iinn ccoonnsscciioouuss mmiiccee.. CCoommppaarreedd ttoo ccoonnttrroollss,,
HHFFDD--ffeedd mmiiccee hhaadd aa ssiiggnniiffiiccaannttllyy hhiigghheerr hheeaarrtt rraattee,, wwhhiicchh wwaass nnoott cchhaannggeedd bbyy aappooccyynniinn ((TTaabbllee 11)),,
bbuutt tthhee vveennttrriiccuullaarr iinntteerrnnaall ddiimmeennssiioonnss iinn ssyyssttoollee oorr ddiiaassttoollee wweerree nnoott ddiiffffeerreenntt ffrroomm ccoonnttrroollss,, ssoo nnoo
cchhaannggee wwaass ffoouunndd iinn tthhee sshhoorrtteennininggf rfaracctitoionn,,i ninddiciactaitningga an onromrmalasly ssytsotloiclifcu fnucnticotnioanf taefrteeri gehigthwt ewekesekosf
oHfF HDF(DTa (bTlaeb1le). 1).
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Table1.Heartrateandfractionalshorteninginconsciousmice.
Control HFD ControlwithApocynin HFDwithApocynin
n
10 10 8 8
Heartrate(BPM) 668±113 778±142* 637±83 745±70*
LVIDdiastole,mm 1.58±0.27 1.66±0.58 1.74±0.17 1.74±0.34
LVIDsystole,mm 0.78±0.17 0.77±0.32 0.90±0.18 0.96±0.30
FS% 49±12 50±16 47±14 51±11
LVID:Leftventricularinternaldiameter;FS:fractionalshortening;HFD:highfatdiet.Valuesaregivenasmean±SD.
*p<0.05comparedtocontrol.
2.4. SingleRyR2ChannelActivityfromMouseVentricularMuscle
We incorporated single RyR2 channels from cardiac muscle of mice fed with HFD into
planar bilayers to study their response to different cytoplasmic free Ca2+ concentrations ([Ca2+]).
We previously showed that single RyR2 channels isolated from the hearts and brains of rats and
rabbitsrespondtoCa2+withlow,moderate,orhighactivation,dependingontheredoxstateofthe
protein[18–20]. RyR2channelsisolatedfrommiceheartsexhibitedthesameresponsestocytoplasmic
[Ca2+] (Figure 2A,B). Low and moderate activity channels displayed a bell-shaped response to
cytoplasmic[Ca2+]intheconcentrationrangeof5to500µM,reachingmaximalP valuesof0.05and
o
0.5,respectively,at30µM[Ca2+]andsignificantinhibitionathigher[Ca2+](Figure2BandTable2).
Incontrast,highactivitychannelswereactivatednear1µM[Ca2+],reachedaP near1.0intherangeof
o
5to500µM[Ca2+],andshowednoCa2+-dependentinhibition(Figure2BandTable2). RyR2channels
obtainedfromcontrolmiceexhibitedmoderateactivitywiththehighestfrequency(Figure2C).Outof
the26channelsfromthecontrolmice,4,19,andonly3displayedlow,moderate,andhighactivity.
Ofthe28channelsanalyzedinmicefedanHFD,onlyonechannelhadlowactivity,14channelshad
moderateactivity,and13channelsshowedhighactivity. Therefore,theHFDcausedasignificantshift
inthedistributionofRyR2channelresponsestocytoplasmic[Ca2+],favoringresponseswithhigher
channelactivity(Figure2C).Thesupplementationofdrinkingwaterwithapocyninpreventedthe
increasedactivityofRyR2inHFD-fedmice,asonly3outof33channelsanalyzedshowedhighactivity,
24hadmoderateactivity,and6hadlowactivity(Figure2C).Apocynindidnotchangethedistribution
of channel responses to cytoplasmic [Ca2+] in mice fed the control diet (Figure 2C), suggesting a
balancedredoxstate.
Whenhighactivitychannelswereincubatedwithdithiothreitol(DTT),athiolreducingagent,
whenstillincorporatedinthebilayer,theiractivityat10µM[Ca2+]significantlydecreasedfroma
P valueof0.96±0.02toaP valueof0.46±0.02,whichcorrespondstothemoderateresponseof
o o
RyR2 channels (p < 0.05, n = 3). An example of such a channel is shown in Figure 2D. This result
suggeststhatredoxmodificationsofthiolresiduesareresponsiblefortheincreasedfrequencyofthe
highactivityresponsetocytoplasmic[Ca2+].
Table2.Fittingparametersofthethreeresponsestocytoplasmic[Ca2+]ofsingleRyR2channels.
ChannelActivity Ka(µM) nHill Ki(µM)
Low 47±16 1.5# 7.0±2.4
Moderate 9.3±1.7* 1# 177±28$
High 1.1±0.1*,$ 3# 5000#
1ValueswereobtainedfromthebestnonlinearfittoEquation(1)ofthevaluesobtainedwithsinglechannelsfrom
controlheartsthatdisplayedlow,moderate,orhighactivityresponses.*p<0.05vs.moderateorlow;$p<0.001vs.
low.#Parameterwasfixedtotheindicatedvaluefordatafitting.
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amamplpifliiefide dvevretirctaicl aslcsaclea.l eS.oSliodl ildinleisn erseprreepsreenste nthtet hbeesbt ensotnn-olinn-elianre faitrs fittos EtoquEaqtiuoant i1o n(s1ee( sSeeectSioecnt i4o.n6).4 .6).
FiFttiitntign gpapraarmameteertes rasraer deedpeicptiecdte idn iTnaTbaleb l2e; 2(C;()C T)hTeh ferefqreuqeunceyn coyf othfet hinecionrcpoorrpaotiroanti oonf cohfacnhnaenlsn ewlsithw ith
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HHFDFD-fe-fde dmmiciec derdinrikniknign wgawteart ewriwthiothuot uotr owriwthi tahpaopcyoncyinn dinudriunrgi nWgeWekese k5–s85.– *8 p. *< p0.<050,. 0ch5,i cshquisaqreu aterset;t est;
(D(D) )RReperperseesnetnattaivtiev ceucrurerrnetn rtecreocrodrindginsg osf oafna RnyRRy2R c2hachnanneln oebltoabintaeidn efrdofmro tmhet hheeahret aorft moficme ifceedf weditwh ith
ana nHHFDFD wwithitohuotu atpaopcoycnyinni tnhtaht astpsopnotanntaenoeuosluys ldyisdpilsapyleady ehdighhi gahctaivcittiyv ibtyefboerefo (ruep(pueprp reercorerdco) radn)da anfdtearf ter
didthitihoitohtrherietoitlo (lD(DTTT)T ()lo(lwowere rrerceocrodr)d. ).
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 6 of 15
Table 2. Fitting parameters of the three responses to cytoplasmic [Ca2+] of single RyR2 channels.
Channel Activity K (µM) n K (µM)
a Hill i
Low 47 ± 16 1.5 # 7.0 ± 2.4
Moderate 9.3 ± 1.7 * 1 # 177 ± 28 $
High 1.1 ± 0.1 *,$ 3 # 5000 #
1 Values were obtained from the best nonlinear fit to Equation (1) of the values obtained with single
channels from control hearts that displayed low, moderate, or high activity responses. *: p < 0.05 vs.
moderate or low; $: p < 0.001 vs. low. # Parameter was fixed to the indicated value for data fitting.
2.5. RyR2 Content and Phosphorylation
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Increases in RyR2 content or phosphorylation status could account for the ventricular
arrhythmias observed in HFD-fed mice and the increased activity observed in isolated channels.
2.5. RyR2ContentandPhosphorylation
However, we did not find changes in RyR2 mRNA expression or protein abundance in mice hearts
IncreasesinRyR2contentorphosphorylationstatuscouldaccountfortheventriculararrhythmias
after eight weeks of HFD (Figure 3A,B, respectively). Additionally, no changes occurred in the
observedinHFD-fedmiceandtheincreasedactivityobservedinisolatedchannels. However,wedid
phosphorylation of RyR2 at Ser-2808 or Ser-2814 (Figure 3C,D), the PKA- and CaMKII-dependent
notfindchangesinRyR2mRNAexpressionorproteinabundanceinmiceheartsaftereightweeksof
sites, resHpeFcDti(vFeigluyr e[231A–,B2,3r]e. sTpehcetisvee lrye)s.uAldtds istiuongaglelys,tn tohcahta nngeeisthoeccru irnrecdreinasthese pinh oRspyhRo2ry claotniotnenoft RnyoRr2 changes
in its phaotsSpehr-o28r0y8laotrioSner -s2t8a1t4u(sF igpularey3 aC ,Dro),leth einP KthAe- aonbdsCeravMeKdI Ia-dcetipveantdieonnt soitfe sR,yreRsp2e. ctTivoe lcyo[n21fi–r2m3]. that no
TheseresultssuggestthatneitherincreasesinRyR2contentnorchangesinitsphosphorylationstatus
activation of PKA or CaMKII occurred in our mice, we determined the phosphorylation of
play a role in the observed activation of RyR2. To confirm that no activation of PKA or CaMKII
phospholamban at Ser-16, the PKA site (Figure 3E) and Thr-17, the CaMKII site (Figure 3F). The
occurredinourmice,wedeterminedthephosphorylationofphospholambanatSer-16,thePKAsite
unchanged phosphorylation state at these two sites suggests that no activation of these kinases
(Figure3E)andThr-17,theCaMKIIsite(Figure3F).Theunchangedphosphorylationstateatthesetwo
occurreds iateftsesru gegigeshtts twhaetenkos aocfti vHatFioDn. ofthesekinasesoccurredaftereightweeksofHFD.

Figure 3.F iHguFrDe3 d. HoeFsD ndoote smnootdmifoyd RifyyRRy2R c2ocnotnetnent toorr pphhoosspphhoroyrlyatliaotnio. (nA. )(RAy)R R2ymRR2N mARdeNteArm dineetderbmyined by
quantitatqiuvaen triteaatilv-teirmeael- tpimoelypmolyemraesreas cehchaainin rreeaacctitoinon(q R(qT-RPTC-RP)C(nR=)6 ();n( B=– D6));R (eBp–reDse)n tRateivperWeseesntetrantbivloet sWestern
oftotalandphosphorylatedformsofRyR2atser-2808orser-2814;(E,F)RepresentativeWesternblotsof
blots of total and phosphorylated forms of RyR2 at ser-2808 or ser-2814; (E,F) Representative Western
totalandphosphorylatedphospholamban(PLB)atthr-17oratser-16.Barsrepresentthemean±SEM
blots of total and phosphorylated phospholamban (PLB) at thr-17 or at ser-16. Bars represent the mean
ofsixdeterminationslikethoseshownontop,obtainedindifferenthearts.
± SEM of six determinations like those shown on top, obtained in different hearts.
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Int.J.Mol.Sci. 2018,19,533 7of15
2.6. Effect of HFD on the Free Thiol Content of RyR2
2.6. EffectofHFDontheFreeThiolContentofRyR2
Because single channel experiments suggest a more oxidized RyR2 state, we determined the
content oBf efrceaeu sSeHs irnegslieducheasn onfe tlheex ppreoritmeienn btsys iungcguebsattaiomn owreitohx bidioiztiend lRabyeRl2edst Nat-ee,twhyeld-metaerlemiminieddet (hNeEM-
bioticno)n. tAenst sohfofwrene SinH Friegsuidruee 4s, oRfythRe2 pcrhoateninneblsy firnocmub aHtiFonD-wfeidth mbiioctein inlacboerlpeodrNat-eedth syilg-mniaflieciamnitdlye less
(NEM-biotin). AsshowninFigure4,RyR2channelsfromHFD-fedmiceincorporatedsignificantly
NEM-biotin than RyR2 channels from controls, indicating a significantly reduced number of free SH
lessNEM-biotinthanRyR2channelsfromcontrols,indicatingasignificantlyreducednumberoffree
residues. Supplementation of drinking water with apocynin prevented the decrease in free thiol
SHresidues. Supplementationofdrinkingwaterwithapocyninpreventedthedecreaseinfreethiol
residues of RyR2 in HFD-fed mice (Figure 4).
residuesofRyR2inHFD-fedmice(Figure4).

FiguFreig 4u.r eH4F.DH FdDecdreecarseeass ethseth ceocnotnetnetn toof ffrfreeee tthhiiooll rreessiidduueessi ninR RyRy2R.2R. eRperpesreensetanttivaetivWee sWteersntebrlont sbolofts of
NEM-biotinincorporationinRyR2incontrolandHFD-fedmice.Barsrepresentthemean±SEMofsix
NEM-biotin incorporation in RyR2 in control and HFD-fed mice. Bars represent the mean ± SEM of
determinationslikethoseshownontop,obtainedinsixdifferentsarcoplasmicreticulum(SR)fractions.
six determinations like those shown on top, obtained in six different sarcoplasmic reticulum (SR)
EachSRfractionwaspreparedfromapoolofthreetofivehearts. *p<0.05comparedtocontrols,
fractions. Each SR fraction was prepared from a pool of three to five hearts. * p < 0.05 compared to
one-wayanalysisofvariance(ANOVA),followedbyTukeytest.
controls, one-way analysis of variance (ANOVA), followed by Tukey test.
2.7. NOXIsoformsintheHeartsofHFD-FedMice
2.7. NOX Isoforms in the Hearts of HFD-Fed Mice
To investigate whether the expression of NOX isoforms was altered in mice fed an HFD, we
To investigate whether the expression of NOX isoforms was altered in mice fed an HFD, we
measured the mRNA expression of NOX2 by qPCR and the protein abundance in Western blots.
measWuerefodu tnhde nmoRdNiffAer eenxcperbeestswioene nofH NFDO-Xfe2d bmyi cqePaCnRd aconndt rtohles ipnrtohteeimnR aNbuAnedxapnrecses iionn W(Feigstuerren5 bAl)ootsr. We
founcdo nntoe ndtiofffetrheisnpcero bteeitnw(Feeignu rHeF5DB)-.fLeidk emwiicsee, athneda mcoonutnrotlosf ipn4 7tphheo mx,RaNreAgu elaxtporryescsyitoons o(lFicigsuubreu n5iAt ) or
conteonftN oOf Xth2i,sa pssrooctieaitne d(Fwigituhreth 5eBm).e Lmikbreawniesefr,a tchtieo namdeocurneat soefd ,ps4u7gpgheosxti,n ag rtehgatutlhatisorsyu bcuyntoitswolaics snuobtunit
of NrOecXr2u,i taedssotocitahteedm ewmitbhr atnhee amndemthberreafnoer efrNaOctXio2nw daescnreoatsaecdti,v sautegdgiensttihnegh tehaartts tohfisH sFuDb-ufenditm wicaes not
(Figure5C).Incontrast,wefoundasignificantincreaseinbothmRNA(Figure5D)andtheprotein
recruited to the membrane and therefore NOX2 was not activated in the hearts of HFD-fed mice
contentofNOX4(Figure5E)inHFD-fedmice,althoughneithermRNAnorproteincontentofNOX4
(Figure 5C). In contrast, we found a significant increase in both mRNA (Figure 5D) and the protein
weresubstantiallymodifiedbyapocynin.
content of NOX4 (Figure 5E) in HFD-fed mice, although neither mRNA nor protein content of NOX4
were substantially modified by apocynin.
Int.J.Mol.Sci. 2018,19,533 8of15
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 8 of 15

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[[CCaa22++]] ffoorr hhaallff--mmaaxxiimmaall aaccttiivvaattiioonn aanndd aann iinnccrreeaasseedd [[CCaa22++]] ffoorr hhaallff--mmaaxxiimmaall iinnhhiibbiittiioonn,, aass hhaass bbeeeenn
previously reported [18–20]. The in vitro effect of DTT on channel activity and the decreased free
thiol content of RyR2 support this idea.
Int.J.Mol.Sci. 2018,19,533 9of15
previouslyreported[18–20]. TheinvitroeffectofDTTonchannelactivityandthedecreasedfreethiol
contentofRyR2supportthisidea.
Alargeamountofevidence,obtainedinhumansandanimals,showsthatoxidizedRyR2produces
arrhythmias[24]. ObesityischaracterizedbyincreasedoxidativestressduetothegenerationofROS
invariousmetabolicpathways[25,26]. NADPHoxidasesareimportantsourcesofROSintheheart.
TheheartexpressestwoisoformsofNADPHoxidase,NOX2andNOX4. WhereasNOX2requires
the recruitment of several cytosolic subunits for activation [27], NOX4 is constitutively active and
regulatedatthetranscriptionalleveloftheprotein[27]. Tothebestofourknowledge,theeffectsof
anHFDintheactivationofNOXinthehearthavenotbeenreported,butseveralstudiesshowthat
NOX2isactivatedinresponsetoanHFDinendothelialcells[28],skeletalmuscles[29],thebrain[30],
thekidney[31]andaorticsmoothmuscles[32]. Therefore,thisisoformisconsideredtoberesponsible
for the oxidative stress and damage induced by an HFD in these different tissues. In contrast to
thesereports,wefoundthatNOX2activitydidnotincreaseinthecardiactissueofHFD-fedmice.
NodifferenceswerefoundinthemRNAcontentorproteinabundanceoftheNOX2catalyticsubunit,
gp91phox,orintheassociationoftheregulatorysubunitp47phoxtothemembranefraction,which
wouldhavebeenanindicatorofactivation. Instead,wefoundanincreaseinthemRNAandprotein
contentofNOX4,whichistheNADPHisoformthatresidesmostlyintheinternalmembranesofthe
cell. Theheartparticularlyrespondstometabolicstresswithadifferentisoformthanmosttissues,
whichcouldbeinherenttoheartfunction. Theheartundergoescyclicchangesinvolumeandpressure,
so its membranes are under continuously varying mechanical stress. We have previously shown
that NOX2 is activated by rapid pacing or exercise in dog hearts [33,34], two conditions that infer
asignificantmechanicalstressforcardiomyocytes. Additionally,NOX2israpidlyactivatedwithin
1minofreperfusionofratheartssubjectedexvivoto5minofischemia,butactivationdisappears
after5–15minofreperfusion[18],suggestingthatfastactivationisfollowedbyfastdeactivationof
theenzyme. Infact,theactivation–deactivationofNOX2mayalsooccuronabeat-to-beatbasisin
isolatedcardiomyocytes[35,36]. Inthepresenceofachronicstimulus,asinthiswork,inwhichthe
animalsreceivedanHFDforseveralweeks,differentpathwaysmighthavebeenactivated,resulting
intheexpressionofadifferentisoform. Apocynin(4-hydroxy-3-methoxyacetophenone)isanaturally
occurring anti-inflammatory compound that has been widely used as an NOX2 inhibitor invivo
becauseitinhibitstheassociationoftheregulatorycytosolicsubunitp47phoxtothemembrane[15].
However,apocyninisalsoanantioxidant[16]andaperoxidescavenger[17];therefore,viaitsROS
scavengeractivity,apocynincanoverridetheeffectsofbothisoforms,NOX2andNOX4.
Increased RyR2 activity, evidenced by increased sparks frequency, has been reported in the
fructose-induced obesity model [37]. In this model, the authors demonstrated that the increased
activityofRyR2isduetoCaMKII-dependentphosphorylationofthechannelprotein. Inthiswork,we
didnotobservedifferencesinthephosphorylationofRyR2inserine2814inmicefedwithanHFD
comparedtocontrols.Furthermore,weobservednoincreaseinthephosphorylationofphospholamban
atthreonine17,aclassicaltargetofCaMKII,suggestingthatthiskinasewasnotactivatedandtherefore
wasnotresponsiblefortheincreasedRyR2activity. Additionally,proteinkinaseAwasnotactivated,
evidenced by the lack of phosphorylation at serine 2808 of RyR2 and of phospholamban at serine
16. Rather,theactivationofRyR2isduetotheoxidativemodificationoftheproteinintheheartsof
HFD-fedmice,assuggestedbytheinvitroinhibitoryeffectofDTTonchannelactivityandbythe
significantdecreaseinthefreethiolresiduesofRyR2.
HyperreactivecysteinesinRyR2canundergodiverseredoxmodificationsbyreactiveoxygen
andnitrogenspecies[38]. Inthisstudy,wedidnotinvestigatethenatureoftheredoxmodification
responsiblefortheincreasedRyR2activity. WepreviouslydemonstratedthatS-gluthationylationcan
reversiblyactivateRyR2-mediatedCa2+fluxesinSRvesicles[33,34],andcalciumchannelsinplanar
bilayers[18]. Likewise, S-nitrosylationalsoincreasesRyR2activity[39]. BothRyR2modifications,
S-glutathionylationandS-nitrosylation,increaseduringreperfusionofischemichearts,andselective
inhibitionofanyofthemfurtherincreasesthefrequencyofreperfusionarrhythmias[40],suggesting
Int.J.Mol.Sci. 2018,19,533 10of15
a protective effect against arrhythmias in the acute setting of ischemia-reperfusion. Therefore, it
isunlikelythatS-glutathionylationorS-nitrosylationofRyR2areresponsibleforthegenerationof
arrhythmiasinthisstudy. Nevertheless, thesearejusttwooftheseveralpossiblereversibleredox
modificationsoffreethiolsthatcanoccursimultaneously. ThecharacterizationofRyR2modifications
isbeyondthescopeofthepresentwork.
HFD-inducedcardiachypertrophyorheartfailureoccursaftertimeperiodsmuchlongerthan
theeightweeksusedinthisstudy[41–44]. TheincreaseinRyR2activityshownhereisanearlyevent
induced by the HFD, that may have a role, not only in the generation of ventricular arrhythmias
suggestedhere,butalsointhelong-termgenerationofcardiachypertrophyinducedbyHFD.
4. MaterialsandMethods
4.1. EthicalApproval
AllproceduresinthisworkwereperformedinaccordancetotheGuidefortheCareandUseof
LaboratoryAnimals,publishedbytheU.S.NationalInstitutesofHealth(NIHPublication,8thEdition,
2011),andapprovedbytheInstitutionalEthicsCommitteeoftheSchoolofMedicine,Universidadde
Chile(ProtocolCBA0819FMUCH,approvedon2June2016).
4.2. AnimalsandDietaryModel
MaleC57BL/6mice(21daysold)werefedwithaHFD(60%caloriesfromfat,CatN◦ D-12492,
ResearchDiets,NewBrunswick,NJ,USA)orwitharegulardiet(10%caloriesfromfat,Champion®,
Santiago,Chile)for8weeks. Duringthisperiod,micewerekeptatatemperatureof23±2◦Canda
12:12hlight–darkcyclewithfreeaccesstofoodandwater. Somemicereceivedapocynin(1.5mM)in
thedrinkingwater,fromthestartofWeek5totheendofWeek8. WechoseWeek5toinitiateapocynin
treatmentbecausethisiswhendifferencesinweightgainbetweencontrolandHFD-fedmicearefirst
noticeable. Apocynindidnotcausedifferencesintheamountofwaterdrunkbytheanimals.
4.3. SurfaceElectrocardiogram(ECG)
At8weeks,micewereanesthetizedwithisoflurane(inductionwith1.0–1.5%andmaintenance
with0.5%inair). Electrodeswereattachedtotheforelimbs(LeadI)andconnectedthroughanML136
AnimalBioAmptoaPowerLab2/26modelML-826dataacquisitionsystem(www.adinstruments.
com). Rectal temperature was monitored and maintained at 36 ± 1 ◦C with the help of a heating
pad. After stabilization, ECG was recorded for 5 min to quantify the occurrence of arrhythmia or
tachycardiaepisodes.
4.4. EchocardiographicDeterminations
Transthoracic M-mode images of the left ventricle were obtained in conscious mice with an
echocardiographequippedwithan8MHztransducer(ATL5000ultrasoundmachine). Theinternal
cavitysizesoftheleftventriclesattheendofdiastole(LVIDd)andsystole(LVIDs)weremeasured,
andthefractionalshorteningoftheleftventriclewascalculatedas((LVIDd−LVIDs)/LVIDd)×100.
Mice were trained on three consecutive days previous to the echocardiographic examination as
described[45].
4.5. PreparationofCardiacSubcellularFractions
Aftereightweeks,micewereeuthanizedbycervicaldislocation;theheartswereharvestedand
cleanedofbloodwithKrebssolution. Theventricleswereimmediatelyfrozeninliquidnitrogento
preparewholehomogenatesorsarcoplasmicreticulum(SR)-enrichedfractionsasdescribedindetail
previously[18,46]. Hearthomogenateswerepreparedfromsinglehearts. SR-enrichedfractionswere
preparedfromapoolof5hearts.