SeminarsinCell&DevelopmentalBiology73(2018)4–12
ContentslistsavailableatScienceDirect
Seminars
in
Cell
&
Developmental
Biology
jo u r n al h om ep age : w w w . e l s e v i e r . c o m / l o c a t e / s e m c d b
Review
Novel
insights
into
cardiomyocytes
provided
by
atomic
force
microscopy
Daniele
Borin
a,1,
Ilaria
Pecorari
a,1,
Brisa
Pena
b,c,
Orfeo
Sbaizero
a,∗ aDepartmentofEngineeringandArchitecture,UniversityofTrieste,ViaValerio10,34127,Trieste,ItalybCardiovascularInstitute,UniversityofColoradoDenver,E17thPl,Aurora,CO80045,UnitedStates cBioengineeringDepartment,UniversityofColoradoDenver,E17thPl,Aurora,CO80045,UnitedStates
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received30May2017
Receivedinrevisedform29June2017 Accepted3July2017
Availableonline4July2017 Keywords:
Atomicforcemicroscopy Cardiomyocyte Biomechanicalproperties Mechanotransduction Beatingactivity
a
b
s
t
r
a
c
t
Cardiovasculardiseases(CVDs)arethenumberonecauseofdeathglobally,thereforeinterestinstudying aetiology,hallmarks,progressandtherapiesforthesedisordersisconstantlygrowing.Overthelast decades,theintroductionanddevelopmentofatomicforcemicroscopy(AFM)techniqueallowedthe studyofbiologicalsamplesatthemicro-andnanoscopiclevel,hencerevealingnoteworthydetailsand pavingthewayforinvestigationsonphysiologicalandpathologicalconditionsatcellularscale.
Thepresentworkisaimedtocollectandreviewtheliteratureoncardiomyocytes(CMs)studiedbyAFM, inordertoemphasisethenumerouspotentialitiesofthisapproachandprovideaplatformforresearchers inthefieldofcardiovasculardiseases.OriginaldataarealsopresentedtohighlighttheapplicationofAFM tocharacterisetheviscoelasticpropertiesofCMs.
©2017ElsevierLtd.Allrightsreserved.
Contents
1. Introduction...4
2. AFMasimagingmachineappliedtocardiomyocytes...5
3. AFMasmechanicalmachineappliedtocardiomyocytes ... 5
3.1. Assessmentofelasticbehaviourofcardiomyocytes...5
3.2. Assessmentofadhesivebehaviourofcardiomyocytes...7
3.3. Assessmentofplasticityindexoncardiomyocytes...7
3.4. Viscoelasticitycharacterizationofcardiomyocytes...8
3.5. AFMquantificationofbeatingactivity...8
4. OtherapplicationsofAFMoncardiomyocytes...9
5. Conclusion...10
Conflictofintereststatement ... 10
Fundings...10
Acknowledgements ... 10
References...10
1. Introduction
Inthelate1980s,theatomicforcemicroscope(AFM)was
devel-opedby Binniget al.[1] toinvestigate surfacesof insulatorsat
theatomicscale,whileavoidingdamagestothespecimens.AFM
∗ Correspondingauthorat:DepartmentofEngineeringandArchitecture Univer-sityofTrieste,PiazzaleEuropa1,34127Trieste,Italy.
E-mailaddress:sbaizero@units.it(O.Sbaizero). 1 Shareco-authorship.
isascanningprobetechnique,whichexploitsthedeflectionsofa
cantileveredspringtoimageandprobedifferentkindofsamples.
FundamentalcomponentsandworkingprinciplesoftheAFMare
showninFig.1.
Theatomicforcemicroscopehasproventobeaversatiletool
ofinvestigation,sinceitcaneitheracquireasampletopographyor
measurevariousmechanicalproperties,suchasstiffness,adhesive
andviscoelasticbehaviour;intheformercase,theAFMworksas
animagingmachine,whereasinthelatteritcanbeconsidereda
mechanicalinstrument[2].Inaddition,astheAFMcanworkin
aqueousenvironments,itwasforthwiththoughttobeusedfor
Fig.1.AFMdiagramandworkingprinciple.Thetipmountedonthecantileverscansandprobethesample.Movementsofcantilever-tipsystemin(X,Y,Z)directionsare controlledbyapiezo-scanner.Whileinteractingwiththespecimen,thecantileverbends;itsdeflectionsarerecordedbyanopticalsystem(lasersource,focallens,mirror andphotodiodedetector).Thecontrollerelectronicsprovidesafeedbackloopthatpermitstoadjustthetip-sampleseparation.
studyingbiologicalspecimenseitherclosetoorunderphysiological
conditions.
Cardiovascular diseases are still the leading cause of death
worldwide.Particularly,accordingtoGillespieetal.[3],heart
dis-easeandstrokearethefirstandfourthcausesofdeath,respectively,
intheUnitedStates.Theproperfunctioningoftheheartispivotal
forlivingorganisms,andcardiologyresearchismainlyfocusedon
thethoroughcomprehensionofbio-chemo-mechanicalprinciples
underlyingthephysiologicalbehaviouroftheheart.Yet,theeffects
ofinheritedorincidentalcardiacdysfunctionsareunder
investiga-tion,likewiseanypossibletherapy,suchasdrugadministration
and tissue regeneration/transplantation. Thestudy of aetiology,
hallmarksandprogressofacardiacpathologycanbeconsidered
a multidisciplinary task, since it involvesphysicians, biologists,
andmorerecentlybiophysicistsandbioengineers.Altogether,they
investigate cardiac behaviour both at the macroscopic, e.g. via
echocardiographyorventriculography,andmicroscopiclevel.
Inrecentyears,moreandmoreresearchersfocusonthebasic
constituent of cardiac tissue, mainlyon cardiomyocytes(CMs),
whicharethecoreunitoftheheart,generatingandtransmittingthe
contractileforce.Inthisscenario,AFMcanplayakeyrole,duetoits
highresolution,bothintopographicandforceterms.Thepresent
workaimstoreviewthestudiesthatinvolvedAFMtechnologyto
investigatethestructureandfunctionofCMs.Inaddition,original
dataarepresentedasfurthercharacterizationofcardiomyocytesin
termsofplasticityindexandstressrelaxationbehaviour.
2. AFMasimagingmachineappliedtocardiomyocytes
Overtheyears,variousbiologicalsampleshavebeen
character-izedfortheirtopographybyAFM,asthoroughlyreviewedin[4,5].
Regardingtheaimofthepresentwork,in1999,Perez-Terzicetal.
[6]imagedsarcolemma-strippedCMsfromneonatalrats,
focus-ingonthenuclearporecomplexes(NPCs).Datashowedthatboth
Ca2+andATP/GTPdepletioninducedasignificantdecreaseinthe
percentageofopenNPCscomparedtountreatedCMs,thesecond
treatmentresultingalsoinmorerelaxedpores(biggerouter
diam-eter).
In2001,Davisetal.[7]resolvedbothexternalandsub-surface
featuresoffixed,intactcardiomyocytesfromtheventriclesofadult
guineapigs,thuspavingthewayforfurtherAFMinvestigationsof
wholecells.Morerecently,thenanoscopicresolutionofthis
tech-niqueallowedtodetectchangesintheplasmamembranesurfaceof
neonatalmurineCMsinducedbyadministrationofaldosterone[8].
Similarly,neonatalratCMsweretreatedwithGCIP-27,asynthetic
polypeptidethoughttopreventcardiachypertrophy,and
exhib-itedanincreasedsurfaceroughnesscomparedtothenot-treated,
controlgroup[9].Anotherstudyanalysedthecellsurfaceand
con-firmedtheabsenceoft-tubulesinmouseandhumanembryonic
stem cell-derived cardiomyocytes(ESC-CMs),which were
com-paredwithadultguineapigventricularCMs[10].Moreover,AFM
revealedthecellsurfacepropertiesofcardiomyocytesfromhealthy
andfailingmicehearts:theformerwerecharacterizedbyaperiodic
successionofcrestsandhollows,whilethelattershowedageneral
lossofthesestructures[11].
Ina ground-breakingstudy,AFMwasappliedtoobservethe
cytoskeleton of murine CMs stimulated by lipopolysaccharide
(LPS),whichisknowntoberelatedtosepsisand,therefore,to
car-diacdysfunction.Theacquiredimageshighlightedthecytoskeleton
reorganizationandthemorphologicalchangesinducedbyLPS,thus
provingtheuseofAFMtostudythefunctionalpropertiesthrough
theobservationofstructuralinformation[12].
The atomic forcemicroscopy was then exploitedto
investi-gate infarcted porcine myocardium treated with mesenchymal
stemcells(MSCs),eitherwithorwithoutglucagon-like
peptide-1(GLP-1)expression[13].Interestingly,Wrightetal.[13]found
thatcollagenfibrildiametersignificantlyincreasedaftertreatment
withMSCs,furtherdemonstratingthattheMSCstherapyoption
canhaveadramaticeffectonthescardepositedasaconsequence
ofmyocardialinfarction.
3. AFMasmechanicalmachineappliedtocardiomyocytes
3.1. Assessmentofelasticbehaviourofcardiomyocytes
Earlyafteritsinvention,theatomicforcemicroscopewasused
toassessthemechanicalproperties,namelyhardness,elasticand
plasticbehaviour,bynano-indentationoftheinvestigatedsample
[14].ThemechanicalanalysisbyAFMattheatomiclevelhasraised
6 D.Borinetal./SeminarsinCell&DevelopmentalBiology73(2018)4–12
Fig.2. Whenprobingthemechanicalpropertiesofasample,theAFMworksintheso-calledforcespectroscopymode.Dwelltimeisthetimepassingbetweentheendof theindentation(loading)(blackcurve,leftpanel)ofthespecimenandthebeginningoftheunloadingprocess(redcurve,leftpanel).Ifdwelltimeisnull,stiffness,adhesion andplasticityindexcanbeassessed.Stiffnessisevaluatedfittingtheportionofloadingcurveafterthecontactpoint“C”withanappropriatemechanicalmodel.Adhesion workisgenerallyreferredtothegreyareaaboveunloadingcurve(leftpanel).Plasticityindexiscalculatedfromtheareasunderloadingandunloadingcurves,namelyas (1-BÔD/BÔC)(leftpanel).Not-nulldwelltimeallowsinferringtheviscoelasticpropertiesofasample,bothwithstressrelaxationandcreepcomplianceexperiments.Inthe former,forceisdetectedwhileZ-positionofthetipiskeptconstant;thelatterimpliesmaintainingtheforceappliedtothetipandrecordingthevariationsintheZ-position oftheprobe(rightpanel).
studyofcellularandmolecularprocessesthatconvertmechanical
cuesintobiochemicalsignals[15].
Alargenumberofstudiesontheelasticpropertiesoflivingcells,
commonlydescribedasYoung’smodulus,hasbeenissued.Young’s
modulusisameasureofthematerial’sstiffness,thatistheaptitude
ofasampletobedeformed.Basically,theAFMcanactasan
inden-ter,thuscanperformforcemeasurementsonadesiredportionofa
cellwhiledetectinghowthespecimendeformsinresponsetothe
appliedforce(seeFig.2).Nevertheless,theestimationofcell
stiff-nessisalwaysaffectedbyseveralfactors,forinstancethechoiceof
themostsuitablemechanicalmodeltofittheexperimentaldata,
theassumptionsonwhichthatmodelrelies,andtheprecisionin
determiningthecontactpoint(i.e.thepointintheforcecurveat
whichtheAFMprobereachesthespecimen).Athoroughdiscussion
aboutthistopicisprovidedin[16].
Adultratatrialmyocyteswerenano-mechanically
character-izedbyAFMbothintheirquiescentandcontractilestatus,along
theiroverallsurface(i.e.nuclearandperipheralregions),andunder
differentconditions (i.e. either withdifferent extracellular
cal-ciumconcentrationsorafterfixation)[17].Alsoadultmalemouse
CMsweretested intheirrelaxedand contractile state,
exploit-ingeitherhigh-K+ solutionorlow-Ca2+ solutionwithEGTAand
2,3-butanedionemonoxime[18].Spontaneouslybeating,
neona-talrat cardiomyocytes were studiedwith AFM in theirsystole
anddiastolebyAzelogluandCosta[19],revealingthespatial
het-erogeneityofmechanical propertieswithinasinglecellandthe
increaseofCMsstiffnessduringcontractionthanrelaxation.The
same study demonstrated that administration of blebbistain, a
specificinhibitor ofactin-myosin interactionin cardiacmuscle,
reducedtheelasticmodulusofCMstodiastolicvalues;theauthors
alsooutlinedthatincreasingtheculturetimeofcellsresultedinthe
stiffeningofbothsystolicanddiastolicmoduli,althoughinthelast
case,thevariationwasnotstatisticallysignificant.Saenz-Cogollo
andcolleagues[20]grewaconfluentlayerofratembryosCMson
microelectrodearrays(MEA)andcoupledthesystemwithAFM.
Suchsetupallowedtherecordingofextracellularactionpotential
signalandthesynchronizationofthecontractilecyclewith
mor-phologicalandmechanicalmeasurements.Similarlytowhatwas
reportedin[19],asubstantialstiffeningofcellsduringcontraction
wasobserved,followedbysofteningintherelaxationphase.
Dague etal. [11] studiedthe effects inducedby formamide,
whichisknowntocausedetubulation,bothontopographyand
elasticpropertiesofmurinecardiomyocytes.Theyfoundthatthe
responseofcellstovariousconcentrationsofformamidemimics
thephenotypeof CMsfromfailed heartsat differentstages. In
anotherstudy,CMsderivedfromchickenembryoswereanalysed
afterdosingcytochalasinB,inordertodeterminetheinfluenceof
theactinnetworkontheYoung’smodulusofthesecells[21].Here,a
consistentdecreaseoftheelasticmoduluswasobservedafteractin
depolymerization.
Anticancerdrugsarewell-knowntocausecardiotoxicity,ergo
primary adult murine CMs were exposed to an anthracycline
anticancerdrug(doxorubicin)andacardioprotectiveagent
(dexra-zoxane),andcellstiffnesswasassessedtoelucidatetheeffectsof
thesetwodrugsatthecellularlevel[22].DoxorubicintreatedCMs
displayedthesamestiffnessofcontrolCMswithinthefirst50min,
followedbyasignificantsoftening,whereaspre-treatmentwith
dexrazoxaneavoidedthesevariations.
Besides the outcome of various chemical substances in the
mechanical properties of CMs, theeffect of a gradient of
sub-straterigidityintheelasticityofneonatalratventricularmyocytes
(NRVMs)wasinvestigated[23],resultinginanon-significant
influ-ence.
The AFM was also exploited to study the role of different
cytoskeletalproteinsinmaintainingthestructureandfunctionof
cells,asinthecaseofvinculin.Cardiomyocytesfromvinculin
het-erozygousnull miceweresofterthancellsfromtheirwildtype
littermate;yet,rescueofvinculinrestoredtheelasticmodulusto
controllevels,asdemonstratedbyTangneyetal.[24].AdultCMs
fromephrin-B1knock-outmicewerefoundtobestifferthantheir
counterpartderivedfromwildtypeanimals,asresultedfromthe
destabilizationofthelateralmembraneinducedbytheabsenceof
Tangneyetal.[24]investigatedvinculinbecauseitsmutations
arerelated tohypertrophicand dilatedcardiomyopathy(DCM),
thussuggestingtheAFMasavaluabletooltoinvestigate
patho-logicalconditionsandtheeffectsofdrugtherapies.Severalother
studiessupportthisapplication:forinstance,ageingwasrelatedto
leftventriculardiastolicdysfunctionandwasdemonstratedtobe
crucialforthestiffnessofratCMs;valuesfor30-months-oldmale
animalswerehigherthanthosefrom4-months-oldrats[26].The
increaseofcellstiffnesswithagewasconfirmedalsoinintact,
enzy-maticallyisolatedcardiomyocytesderivedfromagedand young
murineheartsunderCa2+-freeconditions[27].Similarly,diabetes
inducedastiffeninginadultmiceCMs,asobservedin[28].In
addi-tion,theYoung’smodulusofNRVMswasassessedtoelucidatethe
effectsofgeneticmutationsinA-typelamin,whichareresponsible
foraparticularclassofpathologiescalledlaminopathies.Lanzicher
etal.[29]showedthatNRVMsinfectedwithhumanD192GLMNA
werestifferthanwildtype(expressingwildtypehumanlamin)
andcontrol(not-treated)counterparts.Moreover,cellstiffnesswas
investigatedinhypertrophiedhearts:adultmaleratsweretreated
witheitheravehicle(control)orisoproterenol(ISO),resultinginan
increaseoftheelasticmodulusoftheISOgroupcomparedtothe
controlone[30].Anothercardiopathologicalcondition,theheart
failure,wasstudiedfromamechanicalpointofview,exploiting
theuniquepropertiesoftheAFM.Indeed,Dagueetal.[11]
com-paredtheelasticity ofcardiomyocytesfrom healthyand failing
micehearts,findingthatearlyafterheartfailure,CMsdecreased
theirYoung’smodulus,whereasatlaterstages,cellsbecamestiffer
thancontrols.Likewise,Liuetal.[31]exploitedtheAFMtocompare
thepropertiesofinducedpluripotentstemcellsderived
cardiomy-ocytes(iPSC-CM)fromhealthysubjectsandpatientsaffectedby
dilatedcardiomyopathy.TheauthorsdemonstratedthatDCMled
toadecreaseiniPSC-CMselasticity.Itisnoteworthytostressthat
inthelaststudy,cardiomyocyteswerederivedfromhumanskin
biopsies,whereasmostofthereportedworksusedanimalmodels.
Growingeffortshavebeenrecentlymadetodevelopprotocolsfor
obtainingCMs,e.g.iPSC-CM,thatresemblesthosefoundinnative
humanhearttissue.Indeed,thisapproachcouldovercomethe
lim-itationsimposedbythedifferencesbetweenhumanandanimal
modelsandtheobviouschallengesfacedtoobtainCMsfromheart
biopsies.
Itisalsonoteworthytomentionthatthedeformabilityof
car-diomyocyteswasassessedevenundernonconventionalconditions,
likeafterantiorthostaticsuspension[32,33]or2-gcentrifugation
[34].
Some of the reviewed works applied the Hertz model or
its variants to quantify the Young’s modulus of cardiac cells
[11,23,24,29,30,35].Nevertheless,thesuitabilityofthesemodels
forbiological specimenshasbeenquestioned.A better
descrip-tionofcellularstiffnesswasproposedbySoufivandandcolleagues
[36],usinghyperelasticmodelstofitdatafromembryonicmurine
CMs.Furthermore,nonlinearelasticbehaviourofrabbitCMswas
alreadyproveninthepastbyMathuretal.usingAFM[37].Itisthe
opinionoftheauthorsthatassumptionofelasticbehaviour(within
thelimitsofapplicability)anduseofHertzmodelcouldbe
com-monlyacceptedforcomparativeinvestigations,asinthecaseofthe
presentedstudies.
3.2. Assessmentofadhesivebehaviourofcardiomyocytes
FromthefirststudiesdemonstratingAFMtohavethesensitivity
forinvestigatingintermolecularforces[38],tothedevelopmentof
singlecellprobeforcespectroscopy[39],biologicalsampleshave
beenwidelycharacterizedfortheiradhesiveproperties(seeFig.2).
Inparticular,thecelladhesionofCMshasbeenreportedinsomeof
theaforementionedstudies[18,24,29].Notably,inthestudyofWu
etal.[18],substratecoatingdidnotaffectcelladhesiveproperties
Fig.3.PlasticityindexforNRVMsexpressingthehumanD192Glaminmutation comparedtowildtype(WT)andcontrol(NT)cells.Datawereacquiredonn>40 cellsforeachcondition.StatisticalsignificancewasprobedwithKruskal-Wallis non-parametrictestandDunn’scorrectionformultiplecomparisons.Ap-value<0.05 wasassumedasstatisticallysignificant.n.s.=notsignificant.
inadultmurineCMs.Ontheotherhand,itwasdemonstratedthat
thegeometryofthetip(sharpversusmicrobead)andthestatusof
thecell(contractedversusrelaxed)influencetheadhesionofCMs,
duetothedifferentcontactareabetweentipandsampleandto
thevariationofactiveintegrinreceptorsandcostamerecomplexes
duringcontractilecycle,respectively[18].
Vinculinwasshownnottoaffecttheinitialpeakadhesionforce,
whiletheprobabilityofadhesionbetweenAFM-fibronectincoated
probeandacelldecreasedsignificantlyinvinculinnullmurineCMs
comparedtowildtypespecimens[24].Inthesamestudy,therescue
ofvinculinexpressionrestoredtheadhesivepropertiestothelevel
ofwildtypecontrols.Anotherprotein,thehumanmutantLMNA,
zeroedtheadhesionworkinNRVMs,whereaswildtypeandcontrol
cellsexhibitedsimilarvalues,asdemonstratedbyLanzicheretal.
[29].
3.3. Assessmentofplasticityindexoncardiomyocytes
Themechanicalmodelsusedtoestimatetheelasticpropertiesof
acellrelyonstrictassumptions,suchasisotropyandhomogeneity
of the investigated sample, thus severelyaffecting the
reliabil-ityofYoung’smodulus estimation.Toovercomethis limitation,
Klymenko et al. [40] introduced theso-called index of
plastic-ity,exploitingthehysteresisbetweenloadingandunloadingforce
curves(Fig.2).Suchdifferenceisattributedtoanenergydissipation
duetocytoskeletalorganization.Tothebestofourknowledge,this
parameterwasmeasuredbyothergroupsinlivingcells[41,42],but
notincardiomyocytes.Inparticular,intheworkofMendezetal.
[42],theplasticityindexprovidedadditionalinformationto
elu-cidatetheroleofvimentinintheviscoelasticresponseofmouse
embryonic fibroblasts,thus demonstratingtherelevance ofthis
mechanicalproperty.
We exploited the plasticity index to highlight the different
behaviourofalivingcellinducedbyageneticmutation.Thesame
protocolreportedin[29,43]wasusedtoextract,infectand
cul-tureNRVMsexpressingthehumanmutantD192GLMNAgeneor
thewildtypeform.Notinfected(NT)NRVMswereusedasfurther
control.Measurements wereperformeddirectlyonPetridishes
keptatphysiological temperature.Cells werethenindented on
thenuclearregionusingagoldsphericalprobe(diameteraround
5m)attachedtoatriangularcantilever(nominalspringconstant
8 D.Borinetal./SeminarsinCell&DevelopmentalBiology73(2018)4–12
data)andshowthatNRVMsexpressingthemutantLMNAwere
significantlyalteredcomparedtocontrols.
3.4. Viscoelasticitycharacterizationofcardiomyocytes
Besidesthe applications reported so far,the AFM hasbeen
usedtocharacterisecellsfortheirviscoelasticbehaviour,
indent-ingthesampleandkeepingeithertheheight(stressrelaxation)
ortheforce(creep)constantforafixedperiodoftime[44](see
Fig.2).Alternatively,afterindentation,thecantilevercanoscillate
atsmallamplitudeandvariablefrequencytodescribethe
viscoelas-ticbehaviourintermsofstorageandlossmoduli[45,46].Despite
thispotentiality,tothebestofourknowledge,onlyfewexamplesof
AFMrheologicalcharacterizationofcardiomyocyteswerereported
intheliterature.
In theworkof Lanzicher, Martinelliet al. [43], stress
relax-ationexperimentswereperformedonNRVMsinfectedwithhuman
LMNAD192Gmutantgene.Relaxationdatawerefittedwiththe
standardlinearsolid(SLS)model,alsoknownasZener’smodel.As
comparedtocontrolsandcellsexpressingwildtypehumanLMNA,
mutantsamplesshowedanincreasedpurelyelasticcontribution,
inaccordancewithYoung’smodulusvalues[29].Moreover,cells
withmutantA-typelaminexhibitedareducedviscosity,whichwas
attributedtoanimpairmentoflengthandthicknessoftheactin
cytoskeletonelementsinducedbythelaminmutation.
Theeffectofdifferentsubstrates(i.e.fibronectin-or
collagen-coated,alignedornot)andofageing(from1to15daysofculture)
ontheviscoelasticbehaviourofneonatalratCMswereassessedby
Deitchetal.[47]throughstressrelaxationexperiments.Here,the
(normalized)relaxationdatawerefittedbothwiththe
Quasilin-earViscoelastic(QLV)andtheSLSmodelstoevaluatethepercent
relaxationafter60sofload,thefirstresultinginabetterfitofthe
experimentaldata.Adecreaseofthepercentrelaxationfromday1
to3inallsampleswasobserved,thenstabilizingtoplateauvalues.
CMsonalignedsubstratesdisplayedgenerallyalowerrelaxation
thanonrandomcoating,sincetheactinfibresweremoreoriented,
thusbettercounteractingtheappliedstress.
Ina recent workby Robisonet al.[48],AFM contributedto
demonstratetheeffectofdetyrosinationonbucklingorslidingof
microtubulesduringcontractioninadultratventricularmyocytes,
wheremicrotubulesactasforcebearing structuresanchoredto
sarcomericZ-disksbydesmin. Inparticular,theSLS modelwas
usedtodescribetheelasticandviscouscomponentsasfunction
oftheindentationrate.Loweringthedetyrosinationlevel, both
elasticityand viscosityofCMs weresignificantlyreduced
com-paredtocontrols.Depletionofdesminexpressionstronglyreduced
cellstiffnessbecauseofthemissingconnectionbetween
detyrosi-natedmicrotubulesandsarcomeric Z-disks.On theotherhand,
ondesmin-depletedCMs,viscositydidnotvaryafterreductionof
detyrosinationlevels.
Stressrelaxationdatacanbeusedtopredicttherheological
behaviour of cells as function of frequency, assuming that the
imposeddeformationiswithinthelinearregime.Inthelinear
vis-coelastictheory,theBoltzmannsuperposition isapplicable and
therelaxationmoduluscanbeconsideredapredictiveconstituent
parameterofthemodelforanyarbitrarydeformationhistory[49].
RelaxationdatacanbedescribedusingtheGeneralizedMaxwell
model,namelyapurelyelasticelement(aspring)inparallelwith
Maxwellelements(aspringinserieswithadashpot).Considering
theintegralformofthelineargeneralizedviscoelasticmodeland
anoscillatingdeformationofangularfrequency,thestorage(E’)
andthelossmoduli(E”)arecalculatedas:
E(ω) =Ee+ni=1Ei ω 22 i 1+ω22 i (1)
Fig.4.Representativecurvesofstorage(E’,continuouslines)andloss(E”,dashed lines)modulusprofilesvs.theangularfrequencyforNRVMsbearingamutation inthelamingene(N195K,redcurves)andinthenativeform(NT),usedascontrol (blackcurves).
E(ω) =n
i=1Ei1+ωωi22 i
(2)
whereEeistherelaxationmodulusatequilibrium(t→∞),nisthe
totalnumberofMaxwellelementsusedtofittherelaxation
pro-file,Eiandiaretheelasticmodulusandtherelaxationtimeofthe
Maxwellelementi.Thus,theelasticandviscouscontributionsof
themechanicalresponseareeasilyseparatedandcompared.Asan
example,weappliedthismodeltopredicttheviscoelasticresponse
ofNRVMsafterinfectionwithhumanmutantN195Klamin,using
thesameprotocolreportedin§3.3.Resultswerecomparedtothose
obtainedonnot-treatedsamples(NT).Cellsatphysiological
tem-peraturewereindentedonthenuclearregionwiththespherical
probepreviouslydescribed,at20%oftheirheight.Relaxationforce
datawereconvertedtomoduliandfittedwithamodelconsisting
ofonepureelasticelementinparallelwithtwoMaxwellelements,
whichdemonstratedabetterdescriptionoftheinitialpartofthe
curvethanasingleMaxwellmodel.Resultingstorageandloss
mod-uliarereportedinFig.4(original,unpublisheddata),whereahigher
elasticcontributionatbothlowandhighfrequencyisdisplayedfor
mutantcellscomparedtocontrols.Eventheviscouscontribution
forfrequenciesaround1rad/sismoremarkedformutantcells.
3.5. AFMquantificationofbeatingactivity
Monitoringthedeflectionofthecantileverpositionedona
beat-ingcardiomyocyte(oronaclusterofthem)givesinformationabout
contractileforce,frequencyandverticaldisplacementofthecell.
Tothebestofourknowledge,thefirstcharacterisationofbeating
profilewasperformedbyDomkeandco-authors[50],who
mea-suredtheheightchangeandtheverticaldeflectionduetoeach
pulse.ConsideringthatcontractionofadherentCMsisprimarily
a lateraldisplacement inducinga heightchange, Liuet al. [51]
suggestedthelateral(torsional)deflectionofthecantileverasa
morereliablesignaltomeasurecontractileactivity.Nonetheless,
mostof thereportedstudiesconsidered theverticaldeflection,
sincetheassociatedforcecanbeeasilycalculatedbymultiplying
thedeflectionsignalbythesensitivityandthespringconstantof
thecantilever.Changetal.[52]introducedtheshort-termFourier
transform to identify even small variations in thebeating
fre-quency,andsuggesteditsapplicationtodetectCMsdynamicsafter
drugadministration.
TheconfluencelevelofCMcultureisakeyparameteraffecting
thebeatingprofile:indeed,thefrequencywasshowntobemuch
moreregularonconfluentcultures thanonsub-confluentones,
whereastheeffectonthebeatingamplitudeseemscontroversial
Adeepercharacterisationofthecontractilebehaviourof
car-diomyocytes has been obtained coupling the AFM with other
techniques.Veryoften,theatomicforcemicroscopyismounted
oninvertedmicroscopes,inordertoperformsimultaneousoptical
andmechanicalinvestigationofthecells.Theintracellular
oscil-lationofCa2+ionscanbemonitoredloadingCMswitha proper
calciumsensitivemedium, as Ca2+plays a fundamental role in
contractionofthesarcomericunitsthatformthecontractile
appa-ratusofcardiomyocytes[53].TheaforementionedworkofLiuand
co-workers[51]demonstratedalinearrelationshipbetweenthe
torsionalforce and thecalcium flux intensity,whereas vertical
deflectiondidnotshowsuchdependence.Burridgeetal.[54]
syn-chronizedthemechanicalandfluorescentsignals,thusdescribing
thetime-dependencebetweencalciumfluxandforcegeneration.
AFMbeatingmeasurementwascombinedwithdigitalholographic
microscopyanalysisbyYueetal.[22],overcomingthelimitsin
resolutionthataffecttheclassicalopticalmethodsusedtomonitor
contraction.In2016,Tianandcolleagues[55]coupledtheAFMwith
amicroelectrodearray(MEA),thusproposinganovelandpowerful
methodtosimultaneouslycharacterisetheelectricaland
mechan-icalactivityofcontractileCMs.Although,somelimitationswere
identified,particularlythepresenceoffalsepeaksintheforceand
extracellularfieldpotentialprofilesproducedbycellclusters.These
falsepeakswereabsentwhenasingleCMwasdepositedonthe
MEA.
AFMhasbeenwidelyusedtoassesstheroleplayed by
spe-cific genesin the beatingactivity,as theirmutations areoften
associatedwithcardiomyopathies.Nguyenetal.[56]showedthat
theknock-downofthestromalinteractionmolecule1(STIM1),a
transmembraneproteinonthesarcoplasmicreticulumactingasa
Ca2+sensor,decreasedthebeatingregularityofHL-1cardiaccells.
Theinhibition of theprotein kinaseC isoform(PKC),which
isassociatedwithadecreasedphosphorylationoftroponinTin
ischaemicheartfailure,wasdemonstratedtoreducethe
contrac-tionamplitudeinneonatalratCMs[57].Mutationsinthecardiac
troponinTcanleadtodilatedcardiomyopathies,thusSunetal.
[58]studiedtheeffectsofapointmutationoftheTNNT2gene,
car-riedbyacohortofpatients.Authorsobtainedcardiomyocytesfrom
patient-specificiPSCsandstudiedthemusingAFM.DCM-derived
CMsexhibitedsimilarbeatingfrequencyanddurationcompared
tohealthydonors,butthecontractionforcewasweaker.Thiswas
restoredbyinducingtheoverexpressionofsarcoplasmicreticulum
Ca2+adenosinetriphosphatase(Serca2a).Liuetal.[31]compared
thebeatingprofilesofsingleiPSCandESCderivedcardiomyocytes:
despitethehighvariability,whichresultedlowerinCMsclusters,
thetwocelllinesexhibitedcomparablebeatingforce,while
iPSC-CMsdemonstrated a slightly higher beat duration and a lower
beatratethanESC-CMs.Aprotocoltoimprovethehomogeneity
ofembryoidbodies(EBs)toobtainbeatingcardiomyocytesboth
fromhumanESCsandiPSCswasproposedbyPesletal.[59].Evenin
thiscase,AFMmeasurementsofthebeatingprofileofEBs
demon-stratedcomparablebeatingforceinthetwomodels,whilethebeat
ratewashigherforiPSCsderivedCMs.Recently,thesamegroup
[60]proposedtheuseofEBscoupledtoanAFMcantileverasa
sen-sortoprobetheeffectsofdifferentenvironmentalconditions(i.e.
appliedforce,temperature,calciumlevelanddrugtreatments)on
beatingforceandrate.Authorssuggestedthisnovelbiosensoralso
forbiomechanicalcharacterisationofheartdiseasesanddrug
ther-apies,sinceEBsareclosertophysiologicalconditionsthansingle
CMson2Dcultures.
AFMstudieswereconductedtoevaluatealsoviabilityand
beat-ingactivityofCMsonsupportspurposelydesignedtodeliverthese
cells to theinjured cardiac tissue and repair it. Burridgeet al.
[54]usedAFMtoverifythatinteractionofiPSC-CMswithother
celltypesina3Dhydrogeldidnotinterferewiththecontractile
activity.Linandco-workers[61]investigatedtheeffectofsizeand
orientationofelectro-spunfibrouspatchesofgelatine-coated
poly-acrylonitrileonthebeatingfrequencyand forceofneonatalrat
myocytes.AuthorsfoundthatCMsonalignednanofibresbeatfaster
andstrongerthanthoseonrandomlyorientedones.Theco-culture
withendothelialcellsalsoincreasedthefrequencyandforceofthe
beating,whereastheuseofmicron-sizedfibresstronglyreduced
theadhesionandthebeatingforce,asnanofibreshavedimensions
closertonativecardiacextracellularmatrix.
Detectionofcardiomyocytecontractileactivitycanbealso
fruit-fullyusedtoevaluatetheeffectsand/orcytotoxicityofdrugsat
the single celllevel. Severalstudies demonstrated the
reliabil-ityofAFMinrecordingthechronotropic(variationoffrequency)
and/or theinotropic (variationof beatingforce) actions of
cer-tainsubstances,suggestingitsuseforpreclinicaltestsofcandidate
treatments.StudiesreportedintheliteratureuseddifferentCMs
models,concentrationsandadministrationapproaches,sothata
real quantification and comparison of the effects of each drug
onthecontractionforceandthebeatrateiscumbersome.Asa
generalobservation,drugsactivating␣1and1adrenergic
recep-torssuchasepinephrine(ornorepinephrine,ademethylatedform
ofepinephrine)ledtoanincreaseofboththebeatrateandthe
contractionforce[31,52,55].Thesameeffectwasproducedby
iso-proterenol[55,57,59,60],areceptoragonist,whichwasshownto
augmentboththebeatrateandcontractionforceafterinhibition
ofSer208phosphorylation[57],ortreatmentswith1receptor
blockers,suchasmetoprolol[59,60]or propranolol[55].
More-over,caffeineenhancedtheCa2+releasefromsarcomericreticulum
byactivationoftheRyR2pathway,resultinginafastincreaseof
thecontractionforce,followedbyaprogressivedecreaseand
lead-ingtohighlyirregularbeatfrequency(arrhythmia)[59,60].Other
testedblockingagentswereesmololhydrochlorideanddoxazosin
mesylate,actingontheand␣adrenergicreceptors,respectively,
andleading toa decreaseofboththebeatrateand contraction
amplitude[52].Ibutilide,adrugusedtotreatarrhythmia,caused
anincreaseofbeatingfrequencyandareductionofthecontraction
force[51].
Aspreviouslystated,atomicforcemicroscopycanbeusedto
evaluatethetoxicityof drugsonsingleCMsand toidentifythe
bestadministrationstrategytominimizetheadverseeffects.For
instance,treatmentwithdoxorubicinwasdemonstratedto
signif-icantlyaffectthebeatingpropertiesofcardiomyocytes.Yueetal.
[22]provedanincreaseofthebeatdurationandaquick
intensifica-tionofthebeatingforceinmurinemyocytes,followedbyastrong
decrease.Meanwhile,Changetal.[52]foundhighirregularityand
reducedcontractileforceinchickenembryonicmyocytes.Inthe
firstcase,theactionofdexrazoxaneascardioprotectivedrugwas
proved,showingthatitsadministration30minbeforedoxorubicin
treatmentreducedthecardiotoxiceffects.IntheworkofChangand
colleagues[52],theantioxidantactionofvitaminCwasshownto
restorethecontractionforceofcardiomyocytes.
4. OtherapplicationsofAFMoncardiomyocytes
Beyondthealreadydiscussedassessmentofcontractileactivity,
AFMwasemployedtoinducecontractioninlivingcells,activating
theirmechano-sensitiveionchannels.ThiswasexploitedbyHe
etal.[62]todemonstratetheformationofmembranenanotubes
asatransportmechanismbetweenNRVMsandratfibroblaststhat
arenotstrictly connectedbygapjunctions. Usingsimultaneous
fluorescenceimaging,authorsshowedthattheCa2+signalfrom
induced contraction propagated through the membrane
nano-tubes,thusleadingtheconnectedcellstocontract.In2015,Galie
andco-workers[63]studiedtheeffectsofindentationdepthand
frequencyforAFMinducedcontractionon3Dsuspendedconstructs
10 D.Borinetal./SeminarsinCell&DevelopmentalBiology73(2018)4–12
(from1to3m)didnotchangeeitherthecontractionforceorthe
contractilevelocity,whichwasshowntoincreasewiththe
inden-tationrate(from0.5to2Hz).
Ossolaandcolleagues[64] exploitedhollowcantilevers
con-nectedwitha pressurecontrol system(FluidFM technology)to
developanAFM-basedpatch-clampdevice,thatcancharacterise
bothelectrophysiologicalandmechanical activityof contracting
CMs.ThissetuptookadvantagefromtheforcecontrolloopofAFM
foramorestablecouplingbetweenthehollowtipandthebeating
cellcomparedtothecommonpatchclamptechnique.The
repeata-bilityandthepotentialuseofthistechnologyasmicroinjectortool
wasdemonstrated,whilethelimitationscomingfromthelow
elec-tricalresistanceinthecell-tipcouplingwereaddressedforfuture
improvements.
Recently,Smolyakovetal.[65]optimizedtheapplicationofa
novelmultiparametricAFMmodeformorphologicaland
mechan-icalinvestigationoflivingandfixedadultmurinecardiomyocytes.
IntheclassicaltopologicalmappingwithAFM,theforce-volume
method is exploited, where each pixel is probed by a
force-deformationcurve.Thealternativetechniqueuseshighfrequency
oscillatingcantileversformultipleprobingonthesamepoint.Such
improvementallowshigherresolution,reducedacquisitiontime
andbettercontrastforcellularsubstructuresintermsofheight,
stiffness,adhesion(relatedtothesurfacechemistry)and
dissipa-tion(relatedbothtoviscousandchemicaleffects)comparedtothe
classicalforce-volumemethod.In thecited study,authorswere
abletoclearlycharacteriseevensubsarcolemmalstructures,such
asmitochondriaandsarcomericapparatus,identifyingbothZ-lines
andM-bands.
5. Conclusion
The interest in cardiovascular research is constantly
fos-teredbythestrikingamountofdeathscausedbycardiovascular
diseases, therefore new methods and approaches are being
pursued to better understand the pathophysiological
condi-tions of the heart, both at the macro- and microscopic
level.
Thepresentworkhasbeenfocusedmainlyonstudiesperformed
onsinglecellsandcellularclusters,andreviewedmostofthe
lit-eratureaboutcardiomyocytesinvestigationviaAFM,rangingfrom
thefirstworksonCMstopographytothemostrecentones
exploit-ingadvancednanomechanicalmapping.Reporteddataprovideda
thoroughinsightintocardiacphysiologicalandpathological
condi-tions,aswellastheeffectsoftherapeuticapproachesatthecellular
scale.
Nowadays, thetopics herebydiscussed areamong themost
pressing issues, and many open questions still require to be
answered.Researchersin thefieldcouldtakeadvantageof this
reviewandthepotentialofAFMtechniqueinordertotesttheir
hypotheses,achieveadeepercomprehensionofcardiacbehaviour
and,hopefully,findnewandeffectivetherapiesforcardiac
patholo-gies.
Conflictofintereststatement
Theauthorshavenoconflictofinteresttodeclare.
Fundings
Thisworkwassupportedbythe“FondationLeducq”,
Transat-lanticNetworkofExcellence(grantnumber14-CVD03).
Acknowledgements
AuthorsgratefullythankThomasLanzicherforhiscontribution
withtheplasticityindexdataonNRVMsandDr.Valentina
Mar-tinelliforprovidingtheNRVMsusedinthisstudy.
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