w w w . e l s e v i e r . c o m / l o c a t e / b j i d
The
Brazilian
Journal
of
INFECTIOUS
DISEASES
Original
article
Role
of
FAK
signaling
in
chagasic
cardiac
hypertrophy
Amanda
R.
Tucci
a,
Francisco
O.
R.
de
Oliveira
Jr
a,
Guilherme
C.
Lechuga
a,
Gabriel
M.
Oliveira
b,
Ana
Carolina
Eleuterio
a,
Liliane
B.
de
Mesquita
a,
Priscila
S.G.
Farani
c,
Constanc¸a
Britto
c,
Otacílio
C.
Moreira
c,
Mirian
Claudia
S.
Pereira
a,∗aInstitutoOswaldoCruz,LaboratóriodeUltraestruturaCelular,Fiocruz,RiodeJaneiro,RJ,Brazil bInstitutoOswaldoCruz,LaboratóriodeBiologiaCelular,Fiocruz,RiodeJaneiro,RJ,Brazil
cInstitutoOswaldoCruz,LaboratóriodeBiologiaMoleculareDoenc¸asEndêmicas,Fiocruz,RiodeJaneiro,RJ,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received29April2020
Accepted16August2020
Availableonline12September2020
Keywords: Chagasdisease Trypanosomacruzi Cardiachypertrophy Endothelin-1 FAKsignaling
a
b
s
t
r
a
c
t
CardiachypertrophyanddysfunctionareasignificantcomplicationofchronicChagas
dis-ease,withheartfailure,stroke,andsuddendeathrelatedtodiseaseprogression.Thus,
understandingthesignalingpathwaysinvolvedinthechagasiccardiachypertrophymay
providepotentialtargetsforpharmacologicaltherapy.Herein,weinvestigatedthe
implica-tionoffocaladhesionkinase(FAK)signalingpathwayintriggeringhypertrophicphenotype
duringacuteandchronicT.cruziinfection.C57BL/6miceinfectedwithT.cruzi(Brazilstrain)
wereevaluatedforelectrocardiographic(ECG)changes,plasmalevelsofendothelin-1(ET-1)
andactivationofsignalingpathwaysinvolvedincardiachypertrophy,includingFAKand
ERK1/2,aswellasexpressionofhypertrophymarkerandcomponentsoftheextracellular
matrixinthedifferentstagesofT.cruziinfection(60–210dpi).Heartdysfunction,evidenced
byprolongedPRintervalanddecreaseinheartratesinECGtracing,wasassociatedwithhigh
plasmaET-1level,extracellularmatrixremodelingandFAKsignalingactivation.
Upregula-tionofbothFAKtyrosine397(FAK-Y397)andserine910(FAK-S910)residuesphosphorylation
aswellasERK1/2activation,leadtoanenhancementofatrialnatriureticpeptide gene
expressioninchronicinfection.OurfindingshighlightFAK-ERK1/2signalingasaregulator
ofcardiachypertrophyinTrypanosomacruziinfection.Bothmechanicalstress,inducedby
cardiacextracellularmatrix(ECM)augmentandcardiacoverload,andET-1stimuli
orches-tratedFAKsignalingactivationwithsubsequentactivationofthefetalcardiacgeneprogram
inthechronicphaseofinfection,highlightingFAKasanattractivetargetforChagasdisease
therapy.
©2020SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.Thisis
anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
∗ Correspondingauthor.
E-mailaddress:mirian@ioc.fiocruz.br(M.C.Pereira).
https://doi.org/10.1016/j.bjid.2020.08.007
1413-8670/©2020SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.ThisisanopenaccessarticleundertheCC
Introduction
Chagasdisease(CD),aneglectedtropicaldiseasecausedby
Trypanosomacruzi,isconsideredanimportantpublichealth
problem responsible for high rates of morbidity and
mor-tality in Latin America.1 Epidemiological surveillance has
estimated that 8 million people are chronically infected
worldwide,with morethan 10,000 deathsper year.2 Acute
infectionismostlyasymptomatic,whereaschronicinfection
mayremainwithoutsigns(indeterminateform),in60–70%of
infectedpeople,orprogresstodistinctclinicalsymptomsas
neurological,digestive(megacolonandmegaesophagus)and
heartmanifestations.3–5 Cardiomyopathyisarelevant
man-ifestationof CD,affecting 30% ofinfected individuals,and
endemiccountriessuchasBrazil (1.2 millioninfected
peo-ple)andArgentina(1.5millioninfectedpeople)concentrate
42% ofall Chagasic cardiopathy cases.6 Cardiac damage is
progressiveandclassified intodifferent stages(A,B,Cand
D)accordingtotheseverityofcardiacdysfunction.7
Chroni-callyinfectedindividualswithanunalteredelectrocardiogram
(ECG)areconsideredintheindeterminatephaseofCD(stage
A)whilediseaseprogressionleadstoECGabnormalities(stage
B),including arrhythmiasor conduction disorders (B1)and
decreasedleftventricular(LV)ejectionfraction(B2),andalso
signsofheartfailure(stageCandD).7Suddencardiacdeath
due to CD cardiomyopathy reaches annual mortalityrates
between0.2and19.2%,8highlightingtheurgencyofmanaging
thissilentdiseaseinpublicpolicies.
Myocardialhypertrophy, an important pathological
out-come of chronic chagasic cardiomyopathy (CCC), is an
adaptativeresponsetocardiachemodynamicoverload
trig-gered by multifactorial processes, including cardiac injury
due to severe myocarditis and fibrosis.9 Multiple signaling
pathways,includingNF-kB,mediatedbyToll-Likereceptor2
(TLR2)thatmodulatespro-inflammatorycytokines(IL-1),10,11
phosphatidylinositide3-kinases(PI3K)/AKT/Nitricoxide(NO)
elicited by increased tumor necrose factor-alpha (TNF-␣)
and/or NF-kB12 and also mitogen-activated protein kinase
(MAPK)throughextracellularsignal-regulatedproteinkinases
1and2(ERK1/2)13–15orCa2+/Calcineurin/nuclearfactorof
acti-vated T cells (NFAT)-dependent mechanism16 mediated by
endothelin-1(ET-1),havebeenimplicatedintheupstream
sig-nalingofcardiac hypertrophiceffectorsinduced byT. cruzi,
resultingintheactivationofhypertrophic-responsivegenes
thatregulatecardiacgrowth.17
ET-1,avasoactivepeptidethatactsthroughG-protein
cou-pledreceptors(GPCRs), endothelinreceptorA(ETAR)and B
(ETBR),is overexpressed in bothacute and chronicphases
ofCD showingelevatedplasmalevelsand highcardiac
tis-sue concentration.18,19 ET-1, that acts in an autocrine and
paracrinemanner,potentiatesthepro-inflammatoryprocess
byup-regulatingcellularadhesionmolecules(ICAM-1,
VCAM-1,and e-selectin), favoring immunesystem cell trafficand
therebyexacerbatingmyocardialcelldamage.17Persistenceof
theparasiteinthechronicphaseofthediseasemodulatesthe
inflammatoryresponsethatsustainshighlevelsofET-1,
capa-bleofinducingcardiachypertrophyandfibrosis.Aremarkable
featureisthatET-1alsomodulateshypertrophicphenotype
indilatedidiopathiccardiomyopathy(DCM)triggeredbyfocal
adhesionkinase(FAK).20However,theparticipationoftheFAK
signalingpathwayinchagasiccardiachypertrophyhasyetnot
beenevaluated.
FAK,anon-receptorproteintyrosinekinaseessentialfor
cardiac physiology, hasemerged asa protagonistmolecule
ofhypertrophyandheartfailure.21,22FAKsignalingregulates
mechanotransduction and sustains cardiac hemodynamics
through integrinsignaling pathway23 but may alsobe
acti-vatedthroughtransmembranereceptorsasheparansulfate
proteoglycans(HSPG)24andGPCRs,25thelatterhasbeen
sug-gestedasanactivatorofthehypertrophicresponsemediated
byET-1.26FAKisamultidomainscaffoldproteincomposedof
aband4.1,ezrin, radixin,moesin(FERM)domain,acentral
kinasedomainandafocaladhesiontargeting(FAT)domain.
TheautophosphorylationofFAKtyrosineresidue397(Y397)
triggers the recruitment and activation ofSrc-kinase
fam-ily amplifying kinase activity through the phosphorylation
ofother tyrosineresidues(Y576/577,Y861andY925),
regu-latingbiologicaleventsasadhesion,migration,proliferation,
andsurvival.27,28Additionally,FAKserineresidues
phospho-rylation(S722,S843,S846,andS910)modulateshypertrophic
response29 and,interestingly,ET-1 isakeyinducerof
FAK-S910 phosphorylation promoting sarcomere reorganization
andcardiachypertrophy,20thusraisingquestionsaboutthe
role ofFAK signalingcascadeintriggeringchagasic cardiac
hypertrophy.
Inthisstudy,weevaluatedwhetherFAKsignalingmayact
asaregulatorofcardiachypertrophyinChagas
cardiomyopa-thy.FAKactivationwasassessedbyFAK-Y397andFAK-S910
phosphorylationand itsdownstreameffectorinbothacute
andchronicphasesofthemurinemodelofT.cruziinfection.
Materials
and
methods
T.cruziexperimentalinfection
C57BL/6lineagemalemice(15–20g),obtainedfromthe
Insti-tuteofScienceandTechnologyinBiomodels(ICTB,Fiocruz),
were housedata maximumoffiveanimals percage, kept
in a specific-pathogen-free (SPF) room at20–22◦C under a
12/12hlight/darkcycle,50–60%humidityandprovided
ster-ilized water and chow ad libitum. All experimental animal
procedureswereperformedfollowingthelicense(L–015/2017)
approved by the Ethics Committee for Animal Use of the
OswaldoCruzInstitute(CEUA/IOC)andalsofollowing
Brazil-ianLaw11.794/2008andregulationsoftheNationalCouncil
of Animal Experimentation Control (CONCEA). In order to
preserveanimalwelfare,adailyobservationwassupervised
byaPhDveterinarydoctoraimingtoavoidanimalsuffering
andpain.Animalswerehumanelyeuthanizedwheneverearly
endpointsofanimalsufferingbecameapparentsuchasmotor
disturbance,exploratoryactivityand/ormoribundcondition.
Atotalof42micewasdividedintotwogroups:noninfected
(12) andT. cruzi-infected (30)animals. Micewere
intraperi-toneally(i.p.)infectedwithtrypomastigotesformsofT.cruzi
Brazilstrain(105parasites/animal),whilenoninfectedanimals
wereinoculatedwithsterilephosphate-bufferedsaline(PBS).
ParasitemiawasevaluatedbyPizzi-Brenermethod.30Briefly,
Table1–PrimersandstandardcurveparametersforhypertrophicmarkeramplificationinC57Bl/6miceinfectedwith Trypanosomacruzi.
Mouse gene target
Primersequences Reference Amplicon length
Slope Intercept Coefficientof linearity(r2)
Amplification efficiency(%)
GAPDH Fw5’-GGTTGCCAAACCTTATCA GAAATG-3’ Ribeiro-Romãoetal. [50] 169bp −3.34 11.71 0.99 99.3 Rv5’-TTCACCTGTTCCACAGCC TTG-3’ HPRT Fw5’ TCCCAGCGTCGTGATTAGCGATG-3’ Barreto-Albuquerque [51] 178bp −3.44 21.15 0.98 95.3 Rv 5’-GGCCACAATGTGATGGCCTCCC-3’ ANP Fw 5’-GGATTTCAAGAACCTGCTAGACC-3’ Thisstudy 167bp −3.27 19.69 0.99 102.1 Rv 5-CGTCTCTCAGAGGTGGGTTG-3
thetailveinandexaminedbylightmicroscopy.The
circulat-ingparasiteloadwasmonitoreddailyfrom5thdayofinfection
until50dayspost-infection(dpi).Acuteinfection(30–60dpi)
wasdeterminedbypositiveparasitemiaandtissueparasitism.
Thesurvivinganimalsweremonitoredduringthecourseof
thechronicinfection(90–210dpi).31Theheartwasharvested
frombothnoninfectedandT.cruzi-infectedmicefrom60to
210days.Afterlongitudinallysectioned,halfofthecardiac
tissuewascryopreservedwithTissue-Tekfor
immunohisto-chemicalanalysisandtheremainingtissuewasfragmented
andprocessedforproteinandRNAextraction.Two
indepen-dentexperimentswereperformed.
Electrocardiographicanalysis
Electrocardiographic parameters were evaluated in
non-sedatedmicebyusingtransducerscarefullyplacedunderthe
skin,inaccordancewithachosenpreferentialderivation(DII).
Traceswererecordedwithadigitalsystem(PowerLab2/20)
connectedtoabio-amplifierat2mVfor1s (PanLab
Instru-ments).Thefilterswerestandardizedbetween0.1and100Hz,
and the traces were analyzedby using Scopesoftware for
WindowsV3.6.10(PanLabInstruments).Wemeasuredcardiac
frequency(beatsper minute,bpm)and duration ofthe PR,
QRS,andQTintervalsinmilliseconds(ms)through(i)Pwave:
atriumchambers,(ii)PRinterval:atrioventricularelectric
con-duction,(iii)QRSinterval:ventriculardepolarization,(iv)QT
interval:heartrepolarization,and(v)heartrate,corresponding
tothecardiacfrequency(bpm).Moreover,ECGtracesallowed
foraqualitativeanalysisofheartratechanges(arrhythmias).
WeindividuallyassessedtherelationsbetweentheQT
inter-val and the RR interval to obtain physiologically relevant
valuesforheartrate-correctedQTinterval(QTc)bygivinga
normalizedRRinterval(RR100=RR0/100ms).Next,thevalue
oftheexponent(y) inthe formulaQT0=QTc×RRy100was
assessed,inwhichQT0istheobservedQTandbothQTand
QTcaregiveninmilliseconds(ms).Bytakingthenatural
loga-rithmofeachsideoftheformula(QT0)=In(QTc)+yln(RR100),
theslopeofthelinearrelationbetweenthelog-transformed
QTand RR100 thusdefined the exponenttowhich the RR
intervalratioshouldberaisedtocorrecttheQTfortheheart
rate.32
Indirectimmunofluorescence
Samplesofcardiactissue,frombothnoninfectedandT.
cruzi-infected mice, were frozen inTissue-Tek O.C.T Compound
(Sakura,Zoeterwoude,TheNetherlands)andstoredinliquid
nitrogen.Heartcryosectionswerefixedincoldacetone,
air-driedandstoredat-20◦C.SectionswerewashedinPBSand
thenblockedwithPBScontaining4%serumalbuminbovine
(BSA;PBS+BSA)beforeimmunostaining.Thesampleswere
incubatedovernightat4◦Cwithanti-fibronectin(1:600,Sigma
ChemicalCo.,StLouis-USA)ortype1Collagen(1:400;Life
TechnologiesCo.,Carlsbad,CA-USA)antibodiesfollowedby
washingwithPBS+BSA.Theantigen-antibodycomplexwas
detectedbyincubationfor1hat37◦CwithTRITC-conjugated
secondaryantibody(1:400,Sigma).Afterwashing,thesections
wereincubatedwith4,6-diamidino-2-phenylindole(DAPI),a
DNA staining, and mounted withthe anti-fading DABCO®
(Sigma). Images were acquired under a Zeiss Axio Imager
M2fluorescencemicroscopeusingAxioVision(Zeiss)digital
imageprocessingsoftware.
ForthetypeIcollagenbioimageanalysis,atleastfive
dif-ferentfieldswerecapturedfromeachgroup(uninfectedand
infected),andfluorescenceimagesweresegmentedandthe
meanintensitymeasuredusingKnimeworkflow.33
Proteinextractionandimmunoblottingassays
Noninfected and T. cruzi-infected cardiac tissue
frag-ments were extracted with lysis buffer (Tris 50mM, NaCl
150mM, Triton X-100 1%, pH 8,0) containing protease
([4-(2-Aminoethyl)-benzenesulfonyl-fluoride hydrochloride
(AEBSF),aprotinin,bestatin,Antipain-hydrochloride,
E-64–[N-(trans-Epoxysuccinyl)-l-leucine 4-guanidinobutylamide,
Leupeptin hemisulfate salt, Pepstatin A; Sigma Chemical
Co.) and phosphatase (Cantharidin, (–)-p-Bromolevamisole
oxalateandCalyculinA;Sigma)inhibitors.Asmallaliquotof
Fig.1–EvaluationofcardiacelectricalconductionsysteminuninfectedandBrazilstrainT.cruzi-infectedC57BL/6mice.(A) Schemeofcardiacelectricalimpulsesshowingtheatrialconduction(Pwave),thetransitionbetweenatriumandventricle (PRinterval),ventriculardepolarization(QRSinterval)andcardiacrepolarization(QTinterval).ECGanalysisofuninfected (circle)andT.cruzi-infectedmice(squares)demonstratednosignificantdifferenceinPwave(B),QRS(D)andQTc(E)intervals duringthecourseofinfection.However,asignificantincreaseinthePRinterval(C)andaslightdecreaseinheartrate(F) wereevidencedininfectedanimals.Statisticalsignificancerelativetop≤0.01betweeninfectedanduninfectedmice.
the Follin–Lowry method, and the loading buffer (62.5mM
Tris–HCl,pH6.8;2%SDS;10%glycerol; 0.01%bromophenol
blue;and5%-mercaptoethanol)addedtotherestofthe
pro-teinextractfollowedbyheatingfor5minat100◦C.Atotalof
20–40gofproteinwasresolvedbySDS–PAGE(10%)according
to the antigen analyzed. The proteins were transferred to
nitrocellulosemembranes(BioRad,Hercules,CA)intransfer
buffer(25mMTris–HCl192mMglycineand10%methanol,pH
8.3).Membraneswereblockedfor40minwith5%skimmilk
inTris-buffered saline (TBS;50mM Tris-Cl, pH 7.5,150mM
NaCl)containing0.05%Tween20(TBST)priortoanovernight
incubation(4◦C)withspecificantibodies(anti-FAKand
anti-ERK 1/2 antibodies (1:1000) and anti-fibronectin antibody,
1:2500),exceptforphosphorylatedproteinswhoseantibodies
(anti-FAK-Y397,anti-FAK-S910oranti-pERK1/2;1:1000)were
incubatedinTBSTcontaining1%BSA.Then,themembranes
were incubated for 1h with the appropriated horseradish
peroxidase-conjugated second antibody (1:20,000). The
antigen-antibody complex was detected by
chemilumines-cencekit reagents (PIERCE;ThermoScientific, Rockford, IL)
and the membraneswere exposed toHyperfilmTM ECL(GE
Healthcare). All assays were normalized by glyceraldehyde
3-phosphatedehydrogenase(GAPDH)expression,usedasthe
internalcontrol.Densitometricanalysiswasperformedusing
ImageJquantificationsoftwaretomeasuretherelativeband
Fig.2–ElectrocardiographicanalysisofC57BL/6miceinfectedwithT.cruziBrazilstrain.Heartarrhythmiastypificationby ECGtraces:(A)uninfectedanimals(NI);(B)T.cruzi-infectedmicewithatrioventricularblock(AVB;blackcircle),(C)sinus bradycardia(BRA;blackline),(D)ventricularextrasystole(VE;arrow)and(E)sinusarrhythmia(SA:tracedline).Quantitative analysisofarrhythmiaincidenceinuninfectedandT.cruzi-infectedmice(F).Amarkedincreaseincardiacarrhythmiaswas noticedduringthecourseofT.cruzichronicinfection.
RNAextractionandreversetranscription
Thehypertrophicmarkeratrialnatriureticpeptide(ANP)gene
expressionwasevaluatedonheartfragmentsusinga
combi-nationofTRIzol® (Invitrogen,California,USA)andRNeasy®
mini kit (Qiagen, Austin, Texas, USA) for RNA extraction.
Cardiacfragments(10mg)wereincubatedwithTRIzol®
(Invit-rogen)andstoredatliquidnitrogenuntiluse.Afterward,the
cardiactissuewashomogenizedwiththeUltra-Turraxtissue
Dispenser(IKA,Wilmington,USA)for30s.After200l
chloro-formadditionandvigoroushomogenizing,RNAwasextracted
fromaqueousphaseusingaRNeasykit,accordingtothe
man-ufacturer’srecommendations.TheamountoftotalRNAwas
determinedbymeasuringtheabsorbanceat260nmandpurity
at260/280 and 260/230nm ratios, using a
Spectophotome-terPico200(PicodropLtd.,SaffonWalden,UnitedKingdom).
Then,RNA(2g)wasreversetranscribedusingtheSuperScript
IIIcDNASynthesisKit(Invitrogen)accordingtothe
manufac-turer’sprotocol.ThecDNAconcentrationwasmeasuredusing
Qubit® ssDNAAssayKit(LifeTechnologies,Eugene,Oregon,
USA)andadjustedto10ng/Lfinalconcentration.
Real-timequantitativePCR
ANP gene expression was compared between noninfected
and T. cruzi-infected cardiac tissues using Power SYBR®
GreenMasterMix(LifeTechnologies,CA,USA)RT-qPCR
sys-tem. Forward and reverse primer sequences are shown in
Table 1. PCRcyclingconditions were:thefirst stepat95◦C
for10min,followedby40 cyclesat95◦Cfor15s and60◦C
for 1min. The relative quantification of amplified
prod-ucts was calculated bythe comparative Ct method(Ct)
using glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
andhypoxanthine-guaninephosphoribosyltransferase(HPRT)
as endogenous controls.34 Theamplifications were carried
out inan ABI Prism7500 Fastdevice (Applied Biosystems,
USA).Geneexpressionwasexpressedasfoldchange(2-Ct),
comparedtosamplesfromuninfectedmiceusedas
calibra-tors.Fortheselectionofreferencegenecandidatesandgene
expressionanalysis,theExpressionSuiteSoftwarev1.1(Life
Statisticalanalysis
StatisticalanalysisandgraphicswereperformedusingR
(ver-sion3.6.0)andRStudio.Statisticaldifferencewasconsideredif
p-value≤0.05usingt-test.AllRT-qPCRassayswereperformed
withbiologicaltriplicatesintwoindependentexperiments.
Resultswereexpressedasthemeans±standarddeviations.
StatisticaltestsfromtheCtvalues(studentt-testor
Mann-Whitneyrank-sumtest)wereperformedwithGraphPadPrism
softwareversion5.00forWindows(GraphPadSoftware,San
Diego,CA,USA).
Results
InanattempttounraveltheroleoftheFAKsignalingpathway
inT.cruzicardiomyopathy,C57BL/6micewereinfectedwith
T.cruzi Brazilstrainand FAKsignalingand its downstream
effector(ERK1/2),actingoncardiachypertrophyprogram,were
analyzed on an acute and chronic infection. The peak of
parasitemia(7×104 parasites/mL) wasobserved at26days
post-infection (dpi) and then declined to an undetectable
level after 45 dpi (data not shown). All mice survived the
acuteinfection(30–60dpi)andprogressedtochronic
infec-tion,showingalterationsinthecardiac electricconduction
systemobservedbyECGparameters.Themain
electrocardio-graphicchangewastheslowingofatrioventricular(AV)nodal
conduction,resultinginaprolongedPRinterval(Fig.1).A
sig-nificantincreaseinthedurationofthePRinterval(p≤0.05)
wasobservedinT. cruzi-infectedmice(43.7±10.2ms)
com-paredtouninfectedgroups(37.1±5.0ms),reflectingadelayin
electricalconductionthroughAVnodeinchronicallyinfected
C57BL/6mice.Anotherstrikingchangewastheincidenceof
atrioventricularblocks(AVB)in67%oftheinfectedmiceat150
dpi(Fig.2).Atrendtowardsadecreaseinheartrate,orsinus
bradycardia(BRA),wasalsoobservedbetweenthegroupsof
uninfectedanimals(627±42 bpm) and thoseinfected with
T.cruzi(598±140bpm),with50%ofinfectedanimalsat180
dpi(Fig.2).Theotherparametersevaluated(Pwave,QRSand
QTintervals)showednosignificantdifferenceinchronically
infectedmice,remainingsimilartouninfectedanimals(Fig.1).
Whenassessingthekineticsofinfection,wefoundalow
incidenceofcardiacarrhythmias(20%)attheacuteinfection
(60dpi)withaprevalenceofBRAandsinusarrhythmia(SA)
amonginfectedanimalgroups.Progressively,thepresenceof
cardiacarrhythmiasbecamemoreprevalentinthe
electrocar-diographictraces,rangingfrom50to67%from120dpiwith
BRA,AVBandsupraventricularextrasystole(VE)asrelevant
eventsinchronicinfection(Fig.2).
Inviewofthefactthatourelectrocardiographicfindings
wereindicativeofheartfailure,revealedbytheimpairment
ofthecardiacelectricalconductionsystemduringthechronic
phaseoftheinfection causingseverearrhythmias, suchas
adecrease inheart rate, sinus bradycardiaand promoting
cardiovascularshock,weconductedouranalysisforthe
detec-tionofendothelin-1(ET-1),ahypertrophicagonistcapableof
activatingtheFAKsignalingpathway.Then,weassessedthe
plasmaticlevelofET-1duringthecourseofT.cruziinfection
byELISA.ElevatedplasmalevelsofET-1weredetectedinboth
acuteandchronicT.cruziinfections (Fig.3).Infectiontimes
Fig.3–ET-1plasmalevelinBrazilstrainT.cruzi-infected
C57BL/6mice.ElevatedET-1levelwasobservedintheacute (60dpi)andchronic(90–210dpi)phasesofT.cruziinfection. A3.4-foldincreaseofET-1level(4.5pmol/mL)wasdetected at120dpicomparedtotheuninfectedanimals(1.3
pmol/mL).ET-1levelswereexpressedasmean(±SD)pMin theplasmaofinfectedanduninfectedC57BL/6mice. Student’st-test:*p<0.05,**p<0.01.
from60to180dpishowedasignificantincreaseinET-1plasma
levelcomparedtothebaselineleveloftheuninfectedgroup
(1.3 pmol/mL).Plasma levelsofET-1 increased3.4-fold and
peakedat120dpi,reachingamaximalvalueof4.5pmol/mL
(Fig.3).
Wefurtherassessediftheabnormalitiesincardiac
hemo-dynamics and the increase of plasma ET-1 level induced
activation ofthe FAK signaling pathway,sincethis protein
tyrosine kinase (PTK) has been highlighted in response to
changesinmechanotransductionmediatedbycellstretching
andcardiacoverload.Ourfindingsdemonstratedthe
activa-tionofFAKsignalingincardiactissueofT.cruzi-infectedmice,
evaluatedbyphosphorylationofbothtyrosine397(Y397)and
serine 910(S910)residues (Fig.4). AlthoughtotalFAK level
wasunalteredinT.cruzi-infectedheart,FAK
autophosphoryla-tionattyrosineresidue397(Y397)wassignificantlyincreased
after90dpi(0.9±0.2),showinganactivationpeakat120dpi
(1.04±0.3) with3.4-fold increasedphosphorylated levelsof
FAK-Y397(Fig.4).AsimilarFAKactivationcurvewasobserved
forphosphorylatedFAK-S910expression.However,highlevels
ofFAK-S910werenoticedat120(1.0±0.1)and180(1.12±0.2)
dpi,increasing3.3to3.6-fold,respectively,comparedto
stan-dardactivationlevelsofuninfectedanimals(0.3±0.08)(Fig.4).
Interestingly,FAKactivationcorrelateswiththeET-1plasma
levelelevationandtheenhancementofbradycardia(50%)in
infected mice. Thus, sinceERK1/2, a member of
mitogen-activated protein kinases (MAP kinase) signaling pathway,
hasbeenimplicatedinthehypertrophicresponsemodulated
byFAK, theexpressionoftotaland phosphorylatedERK1/2
(pERK1/2)werealsoanalyzed.TotalERK1expressionwas
prac-ticallyinvariable,exceptforthedeclineat210dpi,whileits
levelofphosphorylationincreasedthroughoutthecourseof
Fig.4–ActivationofFAKsignalinginT.cruzichronicinfection.(A)RepresentativeimmunoblotimagesofFAK,FAK-Y397 andFAK-S910.(B)AnincreaseinFAKautophosphorylation(FAK-Y397)inthecardiactissueoccurredattheearlychronic infection(90dpi)andpeakedat120dpi.(C)HighlevelsofphosphorylatedFAK-S910wasnoticedfrom120to180dpi withoutalterationintotalFAKexpression(D).(E)RepresentativeimmunoblotimagesofERK1andphospho-ERK1/2
(pERK1/2).(F)pERK1/2expressionincreasedduringthecourseofT.cruziinfection(60–210dpi),peakingat180dpi.However, nochangeinERK1expressionwasobservedinthecardiactissueofinfectedanimals,exceptthedeclineinERK1levelat 210dpi(G).GAPDHwasusedasaninternalloadingcontrol.Student’st-test:ns-notsignificant,*p≤0.05;**p≤0.01; ***p≤0.001;****p≤0.0001.
Fig.5–Trypanosomacruzichronicinfectioninduces hypertrophicgeneexpression.IncreasedANPmRNA expression,evaluatedbyRT-qPCR,wasobservedinthe myocardiumofC57BL/6micechronicallyinfectedwithT. cruzi(150–210dpi).HPRTandGAPDHgeneswereusedas endogenouscontrols.ThemeanCtvaluesbetweencontrol andinfectedgroupswereevaluatedusingStudent’st-test
ornon-parametricMann-Whitneyranksumtest.*p≤0.05, **p≤0.01.
theactivation ofERK1/2occurred priortotheactivation of
FAK,butcoincidedwiththeET-1plasmalevelincreases.
Phos-phorylatedERK1/2(pERK1/2)levelpeakedat180dpi(4.7±0.7),
coincidingwiththeFAK-S910peak(Fig.4).
Toaddresswhetherthecardiachypertrophyprogramwas
triggeredinresponsetotheactivationoftheFAK(Y397and
S910residues)—ERK1/2signalingcascade,weinvestigatedthe
atrialnatriureticpeptides(ANP)mRNAexpression,a
hyper-trophic marker, by RT-qPCR. Marked upregulation of ANP
mRNAexpressionwasrevealedinthemyocardiumofinfected
mice. Our results demonstrated that the ANP mRNA level
tendedtoincreaseat120dpi,butasignificant2-foldincrease
intheexpressionofthishypertrophicmarkerwasnoticedin
theBrazilstraininfectedmicefrom150dpionwards(Fig.5).
Additionally, theremodeling ofthe cardiac extracellular
matrix(ECM)hasbeenshowntobeassociatedwith
myocar-dial hypertrophy and heart failure.35 Thus, the myocardial
fibrosis profile was also analyzed in the myocardium of
T.cruzi-infected C57BL/6mice.Thespatial distribution and
ECMcomponentsexpression,astype1collagen(COL1)and
fibronectin(FN),were assessedinthe cardiactissue.Athin
depositionofCOL1(Fig.6)and FN (Fig. 7)wasobserved in
themyocardial interstitiumofuninfectedanimals. Aslight
increase in COL1 deposition, as revealed by fluorescence
images,wasobservedintheinfectedtissueat60and90dpi,
becomingadensefibrillarCOL1fluorescencesignalatalater
timeofinfection(120–210dpi)(Fig.6).Thequantitative
analy-sis,usingKnimebioimagetools,alsoconfirmedtheaugment
intheCOL1expressioninthemyocardiumoftheinfectedmice
(Fig.6).Likewise,FNspatialdistributionanalysisalsorevealed
anintensefluorescentsignal,mainlyafter60dpi,withadense
FNdepositionintheinfectedcardiactissue(Fig.7).Infected
animalsalsoshowedenhancementinFNlevel,demonstrated
byimmunoblottingassay,showingasignificantincrease in
FNexpressionfrom 60dpicomparedtocardiactissuefrom
uninfectedmice(Fig.7).
Discussion
Aplethoraofsignalingpathwayshavebeenimplicatedinthe
pathophysiologyofcardiachypertrophy.Thus,delineatingthe
cellularandmolecularmechanismsunderliningthechagasic
cardiachypertrophyshedslightonChagasdisease
pathogen-esis.Inthisstudy,weaddressedtheroleofFAKsignalinginT.
cruziinducedcardiachypertrophyandheartfailure.
FAKsignalingemergedasakeycardiachypertrophic
reg-ulator in response to biomechanical stress and humoral
stimuli.36Ourdatasuggestthatbothmechanicaland
hyper-trophic agonist stimuli are involved in T. cruzi cardiac
hypertrophic response. Integrin, a transmembrane
recep-torinvolvedinforce transmission,connectsthecontractile
muscleapparatustoextracellularmatrixcomponents(ECM),
regulating the mechanical force. FAK, a key molecule in
mechanosignalling,interactswithintegrinthroughstructural
proteins, astalin and vinculin,in focal adhesionsites and
costameres.37PressureoverloadinducesFAKtyrosine
phos-phorylation and promotes c-Src activation in addition to
downstreameffectors ofFAK, asERK1/2andAKT viaGRB2
andphosphatidylinositol3-kinase(PI3K).21,26,38,39Ourfindings
demonstrated that changes inintegrin-mediated
mechani-cal force, induced by fibrosis leading to pressure overload,
activatesFAKsignalinganditsdownstreameffectorinvolved
in hypertrophic response. The abnormal ECM deposition,
revealed by enhancement of COL-1 and FN expression in
cardiac tissue,causes itsstiffnessand disturbsmechanical
homeostasis, altering integrin-mediated
mechanotransduc-tion.Additionally,disruptionofvinculincostameresinT.cruzi
infected myocardium may alsocontribute toalterations in
the transmissionofcardiaccontraction force.40 Inthis
sce-nario,elevatedFAK-Y397phosphorylationlevelcoincideswith
theECGabnormalities,showingreducedheartrate,prolonged
PRintervalandincreasedrateofarrhythmias(from90dpi)
suggestiveofcardiachypertrophyandheartfailure.
Interest-ingly,aninvitromodelofacuteinfectionbyT.cruziwithhigh
parasiticloadrevealedlossofspatialorganizationand
down-regulationofmechanosensitiveproteinsincardiomyocytes,
suchastalinandpaxilin,inducingadownregulationofthe
FAKsignaling pathwayactivationand therefore,suggesting
adisruptionofintegrin-dependentsignalingtransduction.41
FAKsignalingactivationhasalsobeenshowntobea
benefi-cialeventinischemia/reperfusion,promotingcardiomyocytes
survivalunderstressconditions.ActivationoftheNF-kB
sur-vival signaling pathway, as downstream ofFAK activation,
modulatesFAK-dependentcardioprotectionbyreducing
pro-apoptoticgeneslevel.42
Inadditiontointegrin-regulatedFAKactivation,this
tyro-sinekinaseisalsoactivatedbyET-1,thehypertrophicagonist.
ET-1, a potent vasoconstrictor involved in Chagas disease
pathogenesis,hasbeenreportedtotriggerFAKactivationand
promotes cardiac hypertrophic remodeling.43 As expected,
Brazil strain C57BL/6 mice infection induced ET-1 plasma
level increase and cardiac dysfunction in both acute and
Fig.6–Enhancementoftype1collagen(COL1)expressioninthemyocardiumofT.cruzi-infectedmice.(A)Distributionof COL1(red)inthecardiactissueofuninfectedmice.AgradualincreaseinCOL1depositionwasnoticedinthemyocardiumat lateracute(60dpi;B)andearlychronicinfection(90dpi;C).DenseCOL1fibrilswereclearlyseenat120(D),150(E),180(F) and210dpi(G).TheincreaseinCOL1expressionwasconfirmedbymeasuringtheCOL1fluorescencesignalwithKnime workflow(H).DAPI(blue)stainednucleus.Bar=10m.Student’st-test:**p≤0.01;***p≤0.001;****p≤0.0001.
in patients with Chagas cardiomyopathy44 and also in a
mouse model of T. cruzi chronic infection (Brazil strain),
which evolvedto right ventricular enlargement.15,19 In our
findings, elevated ET-1 levels were associated with ERK1/2
phosphorylation up-regulation, still in the acute phase of
infection (60 dpi). Also, increased ERK1/2 activation
pre-cededFAK-S910 phosphorylation (120 dpi), suggestingthat
FAKundergoesFAK-S910phosphorylationviaERK1/2
signal-ingaspreviouslyreportedfordilatedcardiomyopathy(DCM).20
ET-1stimulatedneonatalratventricularmyocyte(NRVM)
cul-tures, inan ETAR-dependentprotein kinase Cdelta (PKC␦)
activation,inducedERK1/2activationthroughRaf-1—MEK1/2
effectors,resultinginFAK-S910phosphorylationand
sarcom-ere assembly during cardiac myocyte hypertrophy.20 Also,
bi-directional crosstalk between Src and PKC␦ has been
proposedwithET-1stimulatingPKC␦-MEK1-ERK1/2and
Src-MEK1-ERK1/2 pathways20,45 or Src-MEK5-ERK5.20 Sustained
MEK1-ERK1/2activationhasbeenshowntopromotecardiac
hypertrophyandanti-apoptoticeffect.46Additionally,ET-1has
alsobeenhighlighted inT.cruzi infectionduetoits role in
triggeringtheCa2+/calcineurin(Cn)/nuclearfactorofactivated
Tcells (NAFT) signaling pathway,causingthe activation of
fetalgeneprogram.16C57BL/6andBalbcmiceinfectedwith
T.cruziYstrainshowedhighlevelsofET-1andeicosanoids
(cyclooxygenase-2; COX2)associatedwithhigh tissue
para-sitismandmyocarditis.ThecooperativeactionbetweenET-1
andT.cruziinfectionregulatestheincreaseinCOX2expression
byactivatingtheCn/NAFTc4signalingpathwayinHL-1atrial
myocytes,leadingtoANPproduction.16Curiously,ourresults
demonstrated asignificant increase in ANP mRNA
expres-siononlyfrom150dpi, butwithatendencytoincreaseby
120dpi.Althoughhypertrophyhasbeenwellestablishedin
BrazilstraininfectedC57BL/6mice,thisexperimentalmodel
ofT.cruziinfectiongraduallydevelopsdilated
cardiomyopa-thy withan increasein rightventricular internaldiameter
after100dpi.15Incontrast,astudyanalyzingthe
electrocar-diographicprofileofdifferentmicelineageandT.cruzistrains,
asamodelforexperimentalchagasiccardiomyopathy,didnot
showsignificantchangesintheECGofB6miceinfectedwith
Brazilstrain,probablyduetotheshortperiodofinfection
ana-lyzed(upto13weeks).47
Activationofhypertrophicresponseprograminresponse
toT. cruziinfection hasbeen showntodependon Toll-like
Fig.7–Fibronectin(FN)expressioninchronicallyinfectedC57BL/6mice.(A)FNspatialdistributioninthemyocardiumof uninfectedmice.(B–G)ThickdepositionofFNwasobservedinthecardiactissueofT.cruzi-infectedmice.Intense fluorescencesignalinthemyocardiuminterstitiumwasobservedat60(B),90(C),120(D),150(E),180(F)and210dpi(G). RepresentativeimmunoblottingimageanddensitometricanalysisshowingasignificantincreaseofFNexpressioninacute (60dpi)andchronicinfection(90–210dpi)(H).DAPI(blue)stainedthenucleus.Bar=10m.GAPDHwasusedasaninternal controlforproteinnormalization.Student’st-test:*p≤0.05;**p≤0.01;***p≤0.001.
IL-1 leveland cardiomyocyte hypertrophy (Petersenet al.,
2005).11 On theother hand,PI3K-AKT/nitricoxide(NO)
sig-nalinghasbeendemonstratedtobeimplicatedinthecardiac
myocyteremodelingofchagasiccardiomyopathy.12 C57BL/6
miceinfectedwithColombianstrain inducedPI3K-AKT/NO
signaling pathwayactivation withcardiac hypertrophy
evi-dencedintheacute(30dpi)andtheearlychronicphase(90
dpi)ofT. cruzi infection,showingincreased mRNAlevel of
hypertrophicmarkers.12AremarkablefeatureisthatERK1/2
andAKThavebeenhighlightedasadownstreameffectorof
FAKactivation37,48 andFAKsignalingmayalsoregulate
NF-kBactivation,49suggestingthatchagasiccardiachypertrophy
mayberegulatedbycomplexregulatoryinteractionsinvolving
interlacednetworksofmultiplesignalingpathways.
Conclusion
Our data contribute with new insights on the regulation
of cardiac hypertrophy induced by T. cruzi infection. We
demonstratedthattheFAKsignalingpathwayregulatesthe
hypertrophic process in chronic chagasic cardiomyopathy.
Cardiac overload induced by ET-1-mediated
extracellu-lar matrix remodeling and tissue damage triggers the
hypertrophicresponseviaET-1-ERK1/2-FAKS910and
integrin-FAKY397-ERK1/2, leading to activation of the fetal gene
program,heartfailureandhypertrophy.Furtherstudieswill
becarriedouttoevaluatetheeffectofFAKsignalinginhibitors
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
Thiswork wassupportedbygrantsfrom theOswaldoCruz
InstituteoftheOswaldoCruzFoundation(Fiocruz),Programa
EstratégicodeApoioàPesquisaemSaúde(PapesVI)/Conselho
NacionaldeDesenvolvimentoCientíficoeTecnológico(CNPq),
Brazil(grant421856/2017-3and424015/2018-8),Fundac¸ãode
AmparoàPesquisadoEstadodoRiodeJaneiro(FAPERJ)(grant
E-26/010.101050/2018)andCoordenac¸ãodeAperfeic¸oamento
dePessoaldeNívelSuperior-Brasil(CAPES)-FinanceCode
001. Theauthors thankAlanderson da RochaNogueira for
technicalsupportandthePlatformofRealTimePCRRPT09A
fromFiocruz,bytheuseoftheequipmentandsupport.
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