The constant beat: cardiomyocytes adapt their forces by equal contraction upon environmental stiffening.

1.1 MB·English

by  Hersch, Nils

Research Article 351
The constant beat: cardiomyocytes adapt their forces
by equal contraction upon environmental stiffening
Nils Hersch, Benjamin Wolters, Georg Dreissen, Ronald Springer, Norbert Kirchgeßner*, Rudolf Merkel and
Bernd Hoffmann`
InstituteofComplexSystems,ICS-7:Biomechanics,ForschungszentrumJu¨lichGmbH,52425Ju¨lich,Germany
*Presentaddress:InstituteofAgriculturalSciences,ETHZu¨rich,Universita¨tsstraße2,8092Zu¨rich,Switzerland
`Authorforcorrespondence([email protected])
BiologyOpen2,351–361
doi:10.1242/bio.20133830
Received13thDecember2012
Accepted23rdDecember2012
Summary
Cardiomyocytesareresponsibleforthepermanentbloodflow contraction amplitude remains stable, which in turn leads to
by coordinated heart contractions. This vital function is increased force levels allowing cells to adapt almost
accomplished over a long period of time with almost the instantaneously to changing environmental stiffness.
sameperformance,althoughheartproperties,asitselasticity, Furthermore, our data strongly indicate specific adhesion to
change drastically upon aging or as a result of diseases like flat substrates via both costameric and focal adhesions. The
myocardialinfarction.Inthispaperwehaveanalyzedlaterat generalappearanceofthecontractileandadhesionapparatus
embryonic heart muscle cells’ morphology, sarcomere/ remainsalmostunaffectedbysubstratestiffness.
costamere formation and force generation patterns on
substratesofvariouselasticitiesrangingfrom,1to500kPa, (cid:2)2013.PublishedbyTheCompanyofBiologistsLtd.Thisis
n which covers physiological and pathological heart stiffnesses. an Open Access article distributed under the terms of the
e
p Furthermore,adhesionbehaviour,aswellassinglemyofibril/ CreativeCommonsAttributionNon-CommercialShareAlike
O sarcomere contraction patterns, was characterized with high License (http://creativecommons.org/licenses/by-nc-sa/3.0).
y spatial resolution in the range of physiological stiffnesses
g
o (15 kPato90 kPa).Here,sarcomereunitsgenerateanalmost Keywords: Cardiomyocyte, Tractionforce microscopy, Cell
ol stable contraction of ,4%. On stiffened substrates the adhesion, Mechanoresponse, Myofibril,Sarcomere
Bi
Introduction processes of those cells go along with extensive functional
Mechanical properties of tissues strongly affect embedded changes. In contrast, myocardial embedded cardiomyocytes,
mammalian cells. The consequences extend from simple, small responsible for the regular heartbeat, do not seem to adapt
adaptations of rather isolated cellular structures all the way to morphologicallyandfunctionallytosuchenvironmentalchanges.
initiation of very complex differentiation pathways. One of the Cardiomyocytes of healthy myocardial tissue interact
most potent mechanical signals is the elasticity of the mechanically with their environment via costameric adhesions
environment. Various cell types respond with simple to surrounding extracellular matrix molecules (ECM) and via
reinforcements of adhesion and cytoskeletal structures upon cell–cell contacts at intercalated disks to other myocytes. In
substratestiffening(Giannoneetal.,2003;Krishnanetal.,2009). disease affected tissues adhesion structures of cardiomyocytes
More extensive adaptations have been described for elasticity become remodeled. As a consequence, adhesion sites of
dependent stem cell differentiation. Here, cell fate could be cardiomyocytes increase at sites of scar formation to the ECM
directed by substrate stiffness in direction of neuronal, bone or aswellastonon-spontaneouslycontractingcellslikeendothelial
muscle progenitors(Discher et al., 2009; Engleret al., 2006). In cells or myofibroblasts (van den Borne et al., 2010). Resulting
addition, various other cell types undergo further differentiation changes in adhesion and heart tissue elasticity can affect the
processes after environmental stiffening without changing their volume of blood filling the heart and therefore also the stroke
tissue localization. Prominent examples can be found in volume according to the Frank–Starling mechanism and the
mammalian heart myocardial tissue, especially during aging force–velocityrelation(Fukudaetal.,2001;MartynandGordon,
andafterinfarctionorothercoronaryheartdiseases(Herrmannet 2001;RassierandPavlov,2010).Elevatedheartloadgoesalong
al., 2003; Hinz, 2009; Koltai et al., 1984; Mazhari et al., 2000). with enhanced forces during cardiomyocyte contraction due to
Whileelasticitiesofhealthymyocardialtissuesrangefrom10to cellstretchingandthereforeincreasedsarcomerelengthsasforce
30 kPa, stiffnesses in heart stroke affected areas increase to generating subunit of myofibrils from originally ,1.9 mm to
values of up to 150 kPa (Berry et al., 2006; Gupta et al., 1994; extended lengths of up to 2.4 mm (Rassier and Pavlov, 2010).
Jacot et al., 2010). Triggered by this stiffening myocardial Even isolated neonatal cardiomyocytes recognize substrate
fibroblasts differentiate together with bone marrow stem cells elasticity as very potent signal inducing maturation and
intomyofibroblasts,leadingtoscartissueformationwithdrastic functional development. This was shown, for example,
changes of myocardial physical properties. Differentiation examining cells grown on substrates mimicking the natural
Elasticity-driven muscle forces 352
elasticity of ,10 kPa and on stiffer ones (50 kPa). In the latter cardiomyocytes adhere during maturation or how and where
case sarcomere alignment was reduced in comparison to the remodeled adhesion sites transmit traction forces. Adaptations in
former. Additionally, cellular forces were reported to be highest morphology, structure and composition of adhesion structures to
on 10 kPa substrates and reduced on softer and stiffer materials substrate elasticity are also not fully elucidated. In this paper we
(Jacot et al., 2008; Jacot et al., 2010). For neonatal haveaddressedtheseimportantquestionsofadhesionbehaviorand
cardiomyocytes grown on elastomeric micropillars, twitch forcegeneration/transmissionofratlateembryoniccardiomyocytes.
forces increased on stiffened posts while twitch velocities Cellsweregrownonwell-characterizedsiliconerubbersubstratesof
leveled down. Elevated forces were accompanied by increased various elasticities in the natural range. Traction force
sarcomere lengths and z-band widths (Rodriguez et al., 2011). measurements were performed and analyzed in depth using two
Other studies indicate that beating frequency and fraction of independent algorithms. Special care was taken to achieve high
isolated cardiomyoctes corresponded best to natural spatialresolutiontodeterminetheforcestransmittedateverysingle
characteristics on substrates resembling native myocardial putative adhesion structure. Moreover, the local contractile strain
elasticities (Bajaj et al., 2010; Engler et al., 2008). Remarkably, alongsinglemyofibrilswasdetermined.
several aspects of how cardiomyocytes respond to substrate In summary our data prove that cultured late embryonic rat
elasticity are still under debate since other studies indicate a cardiomyocytes exhibit very stable contraction amplitudes of
completelossofmyofibrilsonsoft1 kPasubstrates(Bajajetal., sarcomeric units along separate myofibrils. This behavior
2010),anelevated number of beating myocytesafter maturation generates increased force values upon substrate stiffening. At all
on very soft substrates (below 1 kPa) (Shapira-Schweitzer and substrate elasticities analyzed adhesion and force transmission
Seliktar, 2007) or increasing force values upon substrate occuralongthewholelengthofmyofibrilsatsitesofcostameres
stiffening to up to 144 kPa (Bhana et al., 2010). Furthermore, and at remodeled myofibril ends. Interestingly, in the
atomic force microscopy measurements indicate a completely physiological elasticity range substrate stiffness did not affect
unaffected cell elasticity of cardiomyocytes on substrate thelengthsofrelaxedorcontractedsarcomericunits.Thisisnotin
elasticities ranging from 8 to 50 kPa (Shi et al., 2011). agreementwiththeFrank–Starlingmechanismandindicatesthat,
Afterisolationfromtissueandgrowthasisolatedcellsonflat despite it being well established in tissue, various regulatory
substratesmyocytesdisplayanalteredadhesionpattern(Hilenski mechanisms connected with this mechanism might be
et al., 1992; Simpson et al., 1993). This adaptation includes incompletelyactiveinisolatedcardiomyocytes.
n
e reorganization of costameric structures, resulting in adhesive
p sites that are structurally similar to focal adhesions (FA), which Results
O
are typically found in non-muscle cells (Decker et al., 1990). The sarcomericforce generationsystem isformed
y
g Simultaneously, intercalated disks at the end of myofibrils independentlyof environmental elasticity
o remodel to adherens junctions as well as to FA like structures. In order to analyze contractile and adhesive behavior of
ol
Bi Allthesestructuresprovidestrongadhesionsitesresponsiblefor cardiomyocytes in an elasticity range of ,1 to 500kPa silicone
mechanicalcommunicationbetweentheECMandthecontractile rubber substrates were fabricated. In a first step, late embryonic
machinery of the cultured cells (Goncharova et al., 1992; myocytes were grown for three days on substrate elasticities
Simpson et al., 1993). The disassembly of the contractile spanning the physiological range of 15 to 150kPa and,
apparatus upon isolation and a reassembly from pre-myofibrils additionally, on artificially very soft (,1kPa) or very stiff
afterattachmenttocellculturesubstratesseemtobedecisivefor substrates (500kPa) and analyzed for differences in cell shape.
this environmentally controlled adaptation (Du et al., 2003; When grown on substrate elasticities of 15kPa and stiffer a large
Sanger et al., 2005). percentageofcellswerecharacterizedbyashapeindexsibelow0.8,
Previous work has shown that both environmental stretch and correspondingtoamoreangularshape(Fig.1A,B).Just30%ofall
cell contractility increase size and frequency of adhesion cellsexhibitedaroundish,mostlyconvexshape(siabove0.8).On
structures (Miller et al., 2000; Sharp et al., 1997; Simpson et verystiffsubstratesalmostallcellswereangularinshape(fractionof
al.,1993).Furthermore,inearlyworkDanowskiandco-workers roundish cells below 10%). Only on very soft substrates (,1kPa)
cultivated adult cardiomyocytes on a thin and flexible silicone theratiobetweenroundandangularshapedmyocyteswasbasically
membrane and observed pleat-like wrinkles with a spacing of invertedwith,80%ofcellsexhibitingaconvexoutline(Fig.1A,B).
,1.9 mm (Danowski et al., 1992). Pleats coincided with the Sinceedgecurvaturedependsonthemechanicalstressformed
distribution and distance of z-disks. These observations clearly withinthenetworkofinneractinfibers(Bischofsetal.,2009),we
indicated the transmission of mechanical force at sites of analyzed whether sarcomeres, the force generating units of
costameric adhesions. Later traction force microscopy myocytes, were differently formed or organized depending on
measurements were evaluated with the assumption that forces substrate stiffness. To this end we used a-actinin as marker
were exclusively applied at FAs at the ends of myofibrils and protein (Fig. 2). Interestingly, myocytes always displayed
stress fibers. These analyses resulted in force values of ,15 nN regularly striated myofibrils irrespective of substrate elasticity
per FA and force dipole values in the 6 pNm range (Balaban et (,1 kPato500 kPa).Thisobservationwasconfirmedwithactin
al., 2001; Cesa et al., 2007). The force dipole, also called as marker (Fig. 2) indicating that the whole force generating
generalizedfirstmoment,isjustthesumofthescalarproductsof system is functional and largely unaffected by changes in
the vectors force and its position. For a cell of the size of a environmental elasticity. More elaborate analyses of myofibril
cultivated cardiomyocyte a force dipole of 6 pNm roughly formation within the most relevant physiological range of
corresponds to overall contractile forces in the 200 nN range. substrate elasticities between 15 and 90 kPa revealed that
Although cardiomyocytes have been intensively analyzed for neither the number of costameres per cell, nor the mean area
more than 20 years, several important questions are still not yet pera-actininclusterorthefractionofthecellarealabeledbya-
answered. For example, it is not entirely clear how neonatal actinin was significantly affected by substrate elasticity (Fig. 3).
Elasticity-driven muscle forces 353
Fig.1. Substrateelasticitydependentcellshapeadaptationofmyocytes. Fig.3. Substrateelasticityindependentformationofcostameres.GFP-a-
Freshlyisolatedratcardiomyocyteswereincubatedfor3daysonsilicone actinintransfectedcells(A)weregrownfor3dayson15kPa,30kPaand
rubbersubstratesrangingintheirelasticityfrom,1to500kPaasgiven. 90kPasubstrates.Livecellimagesweresegmentedusingimageprocessing
n Subsequently,cellswereanalyzedforeitherround(shapeindex,si.0.8)or (B).Scalebar:10mm.Meana-actininclustersize(C),relativecoverageof
e elongated,angular(si#0.8)cellshape(A)usingimageprocessing.Left cellswitha-actininclusters(D)andthetotalnumberofa-actininclustersper
Op micrographsi50.96,rightmicrographsi50.45.Scalebars:50mm.Distribution cell(E)weredetermined.Allresultsareindicatedwiththeirrespectives.d.;
betweenbothshapesisgiveninB.Roundcellshape5darkgreyandelongated n516cellsfor15kPa,20cellsfor30kPaand13cellsfor90kPa.
y shapedcells5lightgrey.n5numberofanalyzedcells.Meanvalues(black)
g
includings.d.(grey)ofshapeindicesforroundandelongatedcellsaregiven
o
ol nexttothebars. With respect to the cytoskeleton, the only effect observed on
Bi artificially soft ,1 kPa substrates was a slightly reduced order
andalignmentofindividualmyofibrilswithrespecttoeachother.
Since reduced order of myofibrils might indicate an altered
coupling of costameres to intracellular structures and also to the
outer environment, we additionally analyzed the localization of
plectin. This protein is vital for the mechanical coupling of
sarcomericborders(z-bands)totheintermediatefilamentprotein
desminand,viathislink,forthecouplingbetweenmyofibrilsand
the whole dystrophin/glycoprotein complex embedded in the
plasma membrane (Konieczny et al., 2008). These analyses
revealedalocalizationofplectinatsitesofsarcomereseparating
z-bands on all substrate elasticities tested. Interestingly, while
two distinct small plectin spots were found on both sides of z-
bands on ,1 kPa, these spots visibly enlarged in size in an
elasticity dependent manner (Fig. 4A). This structural
reinforcement resulted in plectin signals co-localizing with
actinalongitsfullsarcomericlengthonstiff500 kPasubstrates.
The structural studies performed above suggest a functional
force generation system as well as a proper connection of
myofibrils to the plasma membrane. However, whether plasma
membrane connected costameres also interacted with the
extracellular matrix was still unclear. We therefore cultivated
myocytesonsubstrateswithelasticitiesfrom,1 kPato500 kPa
and stained them for vinculin as marker for FA as well as for
integrin-dependentcostamerelinkedadhesionsites.Asshownin
Fig.2. Stableformationoftheforcegeneratingsystemofmyocytes. Fig. 4B,thesemicrographsdisplaydistinctsignalsatbothendsof
Cardiomyocytesweregrownon,1kPa,15kPaand500kPastiffelastomeric
every single myofibril. Furthermore, vinculin co-localized with
substratesfor3daysandanalyzedafterfixationformyofibrilformationusing
costameres. These signals were variable in intensity between
a-actinin(left)andactin(middle)asmarkerproteins.Overlayimagesare
given(right).Scalebars:50mm. individual cells and slightly blurred on very soft substrates.
Elasticity-driven muscle forces 354
Fig.4. Myofibrilalignmentandplasmamembraneattachment.
Cardiomyocytesweregrownfor3daysonsubstratesofindicatedelasticities.
Subsequently,cellswerefixedandstainedforplectinandactin(A)orvinculin
andactin(B).InsetsinAshowenlargementsoftheareaswithinthewhite
squares.Notetheelasticitydependentreinforcementofplectinandvinculin
aroundsitesofz-bands.Scalebars:20mm.
Nevertheless, these vinculin stainings might argue for an
interaction with integrin, and therefore with the environment, at
n sites of costameres on each substrate elasticity tested.
e
p
O Forceistransmittedatsitesofcostamericandfocaladhesions
y Tofurthertesttheadhesivebehaviorofcardiomyocytes,traction
og force microscopy was performed on spontaneously contracting Fig.5. Costamericandfocaladhesiondependenttractionforcesof
ol GFP-a-actinin transfected cells grown on 15 kPa, 30 kPa, and myocytes.GFP-a-actinintransfectedcardiomyocytes(A–E)andGFP-VASP
transfectedmyofibroblasts(F–J)weregrownonfluorescentbead
Bi 90 kPa stiff substrates (see also supplementary material Movie micropatterned15kPa(exemplarilyshownhere),30kPaand90kPa
1).Here,forcecalculationwasbasedontheassumptionofpoint elastomericsubstrates.Spontaneouslycontractingmyocyteswereanalyzedby
forces acting at interactively chosen adhesion sites. From the livecellmicroscopytovisualizethesubstratedeformationfield(A,green
arrows)uponcontraction.Usinginteractivelychosenforceinductionpointsand
forces determined in this way substrate deformation fields were
elasticitytheorycellforces(notshown)andtheresultingbackcalculated
back calculated (Fig. 5, top) and compared with the actual substratedeformations(A,yellowarrows)weredeterminedforeachtimepoint.
measured displacements. From those calculations residual Asresultwereceivedaresidualdisplacementvectorforeachmarkerbead
displacements for each tracked marker could be constructed. (B–D,right,bluearrows).Choosingasforceinductionpoints(yellowsquares)
eitherFAsonly(B),costameresonly(C)orbothtogether(D)backcalculations
WheneverexclusivelyFAswerechosenasforceinductionpoints
reproducedtheactualdeformationfieldwithverydifferentaccuracy.Frame
major residual displacements remained present below the center rate517Hz.Ascontrol,measuredsubstratedeformationsandbackcalculated
of cells (Fig. 5B,E). Vice versa, choosing only costameres as substratedeformationsofstablyadheredmyofibroblasts(F)weredeterminedas
force induction points resulted in back calculated displacements describedformyocytesusingFAs(G),alatticemimickingthelengthoftwo
sarcomeres(,4mm)alongstressfibers(H)andboth(I)asforceinduction
that fitted the measured ones well below the interior of the cell
points.Here,fordisplacementcalculationanimageofthesamesubstratearea
while major residuals remained at the periphery. (Fig. 5C,E).
wastakenaftercellremoval.Foreachbackcalculationthenormalizedsquared
Only combining FAs and costameres resulted in low residual deviation(x2)betweenfitresultandmeasureddatawasdeterminedas
displacements over the whole field (Fig. 5D,E). exemplarilygiveninE,J.Notethatformyocytesresidualdeformationsarelow
onlyifbothFAsandcostameresaretakenasforceinductionpoints,whilefor
As additional control we identically analyzed similar traction
myofibroblastsFAsalonearefullysufficient.Scalebarsfordisplacements
force experiments on myofibroblasts, a cell type well known to
(yellowandgreen)inA,F:0.5mm;forthecellsize(white):10mm;andfor
adhereexclusivelyviaFAswithstrongcontractilestressfibersin residualdeformation(lightblue):0.2mm.
between.WheneverweusedasforceinductionpointseitherFAs
alone (Fig. 5G,J), or together with them a fictitious pattern, adhesion and force transmission of cardiomyocytes at sites of
similartotheonesusedinmyocytesforcostameres,alongstress FAs and costameres.
fibers (Fig. 5I,J), we found very low residual displacements. In
contrast, high residual displacements resulted if exclusively Forcegeneration increaseswith substrate stiffening
pointsalongstressfiberswereused(Fig. 5H,J).Interestingly,for Based on the results given above we evaluated the sum of all
this cell type combining all types of force induction points was contractile forces of GFP-a-actinin transfected cells using either
not further reducing the residual displacements. Thus our both FAs and costameres as force induction points or,
implementation of traction force microscopy was well able to alternatively, a hexagonal lattice of fictitious points with a
discriminate between the different modes of force application. lattice constant of 5 mm, covering the whole area of the cell
Taken together, these data clearly prove the simultaneous (Fig. 6A,B). The latter choice was necessary to increase the
Elasticity-driven muscle forces 355
In cell force retrieval algorithms based on force fields the
completeinterfacebetweencellandsubstrateisusedasputative
forceinductionarea.Here,thesameelasticitydependentincrease
of generated cell forces was found albeit with slightly different
absolute values (Fig. 7D; supplementary material Fig. S1B).
Furthermore, substrate deformation and force fields of GFP-a-
actinintransfectedcells(Fig. 7A)werenotjustlocalizedmainly
at FA positions but also along most parts of myofibrils
independent on substrate elasticity (Fig. 7B,C). These data
confirm once more that adhesion of myocytes occurs simulta-
neously at FAs and costameres.
Contractionsof separatemyofibril sarcomeric unitsarestable
Elevating force values on stiffening substrates with a virtually
unaffectedcellforcegeneratingsystemopenedthepossibilityof
elasticity adapted contractile amplitudes of myofibrils. If this
increaseinforcewasbasedonaFrank–Starlinglikemechanism,
it should be accompanied by elasticity dependent sarcomere
Fig.6. Stiffnessdependentforcegenerationofmyocytes.Aftergrowthof lengths and contractions (de Tombe et al., 2010; Hanft et al.,
myocytesfor3daysonbeadmicropatternedsubstrates(A)withelasticities 2008). For this reason we analyzed the contraction patterns of
rangingfrom,1to500kPa(D),tractionforcemicroscopywasperformedon discretemyofibrilsoverseveralcontractions(exemplarilyshown
spontaneouslycontractingcells.Applyingelasticitytheorybasedonsubstrate
inFig. 8A).Thiswasdoneforcellsgrownonsubstratesspanning
deformations(B,yellowarrows)withahexagonalgridusedasforceinduction
the most relevant natural elasticity range of 15 to 90 kPa.
points(A),forcespergridpoint(B,redarrows)weredeterminedforevery
imageofthetimestack.Scalebars:10mm.Thetimecourseofthesumofall Foreverymyofibrilwecouldidentifyacentralspotthatbarely
contractileforcesfortheexemplarilygivencellisindicated(C).Maximum movedduringcontraction(Fig. 8B,C,blackarrowhead).Onboth
valuesofthesumofallcontractileforceswereaveragedforallcellsper
sides GFP-a-actinin labeled z-bands displaced upon contraction
n substrateelasticityandareindicatedwiththerespectives.d.inDusinga
e logarithmicsubstratestiffnessscale.Thedottedcurvedisplaysafitted towardsthemyofibrilcenter.Thisz-banddisplacementalongthe
Op proportionalforceincrease.Thedashedcurveindicatesafittedlinearforce filament(Fig. 8C)revealedastablecontractilestrain,i.e.relative
increase.n>15cellsperelasticity. shortening, for about the central half of each myofibril. As
y
g contractionssumupalongthefibrilthiscorrespondstoalinearly
o increasing displacement of the more outside located sarcomeres
ol number of analyzable cells since transfection rates of vital
Bi myocytesarealwaysverylow.Onallsubstrateelasticitiestested within the inner part of myofibrils (Fig. 8B,C). Interestingly,
displacementsofoutmostsarcomericunitsoftenleveleddownto
bothmethodsrevealedhighlycomparablecontractileforceswith
a plateau like displacement caused most likely by simple
surprisingly small differences (Table 1). We therefore analyzed
transmission of inner myofibril displacement without additional
cell forces of single myocytes on substrate elasticities in the
contractility (Fig. 8C).
range of ,1 to 500 kPa using point forces on such a hexagonal
In order to characterize the exact contractile behavior of
grid. Independent of substrate elasticity, data prove stable
discrete myofibrils, we automatically detected the linear region
spontaneous myocyte contractions over time (Fig. 6C). In good
of each displacement-position curve. Within this region a line
agreement with former analyses (Jacot et al., 2008), these data
was fitted to the displacements. The slope of this line is the
revealed low force generation of myocytes grown on soft contractile strain (relative shortening) of the myofibril (see
substrates (Fig. 6D; supplementary material Fig. S1A). With Materials and Methods). Averaging slope values of 2 to 4
increasing stiffness, contractile forces rose continuously from independent myofibrils per cell characterized itsmean myofibril
valuesofonly10 nN(s.d.36 nN,n522)on,1 kPasubstratesto contractile behavior (Fig. 8D). Mean values for various cells
more than 800 nN (s.d. 311 nN, n514) on 500 kPa stiff grown on 15 kPa, 30 kPa or 90 kPa revealed a large spread in
substrates. Within the physiological range from 15 to 150 kPa cellaveragedmyofibrilcontractilestrainbetween2%and8%for
thisincreasewasalmostlinear.Increasingcontractileforcesupon 15 kPa substrates (n57 cells with 15 myofibrils), 2% and 12%
substratestiffeningwent alongwithunchangedaverage contrac- for30 kPasubstrates(n513cellswith29myofibrils)and1%and
tion frequencies, with 38 (s.d. 18) spontaneous contractions per 6% for 90 kPa substrates (n58 cells with 20 myofibrils)
minute on soft (,1 to 15 kPa, n530 cells) and 36 (s.d. 18) on (Fig. 8D). However, with contraction values of ,4% (4.7% s.d.
stiff (150 to 500 kPa, n530 cells) substrates. 2.1% for 15 kPa, 4% s.d. 2.9% for 30 kPa, 3% s.d. 1.7% for
Table 1. Comparison of the averaged sum of all contractile cell forces using either FA and costameres or alternatively a
hexagonal lattice as force induction points for cells grown on 15, 30 and 90 kPa stiff substrates. s.d. is given in parentheses. n
indicates number of cells analyzed.
Elasticity[kPa] FA+costamer[nN] Hexagonallattice[nN] Difference[%]
15n516 74(24) 74(26) 3.9(8.4)
30n520 174(94) 175(93) 2.3(1.9)
90n513 262(82) 261(80) 2.3(3.3)
Elasticity-driven muscle forces 356
markedlydifferentappearanceofthecontractileapparatus.Such
elasticity independent behavior is in contrast to results reported
formanyothercelltypes:fibroblastsaswellasendothelialcells,
for example, respond to increasing substrate elasticities with
reinforcement processes of cytoskeletal stress fibers as well as
adhesion structures (Goffin et al., 2006). Furthermore, several
cell types as stem cells or cardiac fibroblasts differentiate in a
substrateelasticitydependentmannerwithprominentchangesin
almostalltypesofcytoskeletalandadhesionstructures(Engleret
al., 2004; Engler et al., 2006; Gilbert et al., 2010).
Atthismomentwecanonlyspeculateaboutthereasonforthis
constant behavior of myocytes. Since we have used late
embryonic rat myocytes (d19) most isolated cells are already
partially differentiated and able to contract spontaneously as
single cells. Beating itself is a mechanical signal that has been
shown to stimulate further muscle cell differentiation and
myofibril formation (Damon et al., 2009; Miller et al., 2000). It
might therefore be possible that myocytes beyond a certain
Fig.7. Forcefieldsalongthecontractileapparatus.GFP-a-actinin
differentiation state generate their own mechanical late
transfectedmyocytes(A)weregrownfor3daysonbeadmicropatterned
substrateswithelasticitiesasindicatedin(D).Substratedeformationfields differentiation signal causing the observed elasticity
weredetermined(B)andentireforcefieldswereretrieved(C).Scalebar: independent myofibril formation. Such a behavior would be
10mm.Thesumofallmaximumcontractileforceswereaveragedoverallcells physiologically advantageous. In the myocard cardiomyocytes
analyzedpersubstrateelasticityandareindicatedwiththerespectives.d.inD
arepermanentlyformed,althoughatlowrates,bydifferentiation
usingalogarithmicsubstratestiffnessscale.Thedottedcurvedisplaysafitted
proportionalforceincrease.Thedashedcurveindicatesafittedlinearforce of tissueembeddedprecursor cells. Thishappensthroughoutthe
increase.n>20cellsperelasticity. whole lifetime of the organism. Because on one hand
cardiomyocyte function critically depends on the structure of
n
e 90 kPa, Fig. 8D) overall averaged myofibril contractile strains theirforce producingapparatusand,ontheother hand,myocard
p stiffness varies enormously during aging or disease progression,
O showed no significantly different values for all substrate
myofibril formation should not depend on environmental
y elasticities analyzed. Notably, substrate elasticity did also not
g influence sarcomeric length for the inner myofibril region. No stiffness. However, it needs to be mentioned that published
o results on substrate elasticity dependent functional and
ol matter which elasticity was analyzed, contraction started from
morphological adaptations of cardiomyocytes are inconsistent
Bi basically identical sarcomeric lengths of 1.9 mm in relaxed state
to some extent (Bajaj et al., 2010; Bhana et al., 2010; Engler et
(s.d. 0.13 mm for 15 kPa, s.d. 0.17 mm for 30 kPa, s.d. 0.11 mm
al.,2008;Jacotetal.,2008;Shietal.,2011).Itmightwellbethat
for 90 kPa).
species of origin, exact time point of isolation and even the
These data clearly point to a morphological and functional
chemical nature of the cell culture substrate have unexpectedly
highly stable myofibril contractile mechanism that is basically
strong impact.
unaffectedandrobustagainstchangesinenvironmentalstiffness.
If plated on soft substrates, contractile forces of our
cardiomyocytes were very low. This result is in very good
Discussion
agreementwithvariousotherstudies(Engleretal.,2008;Jacotet
Cardiomyocytes are the mechanically most active cell type in
al., 2008). With increasing substrate elasticities contractile cell
mammals. Over decades these cells pump almost constant
forces rose. Force values did not peak at a distinct elasticity but
volumes of blood although environmental conditions within the insteadgraduallyincreased.Thisbehaviorresultedin,80times
myocard can change drastically. For example, the molecular
highercontractileforceson500 kPasubstratescomparedtovery
compositionof the extracellular matrix and the relative amounts soft ,1 kPa ones. Even for the more natural elasticity range
of cell types populating it change substantially during between 15 and 90 kPa this increase was still ,4-fold. This
embryogenesis, aging or diseases like heart strokes. Most of almostlinearbehaviorisinmarkedcontrastwithnon-monotonic
these changes go along with alterations in tissue elasticity behavior reported by Engler and co-workers and Jacot and co-
causingstiffeningfromelasticitiesinthe10 kParangefoundfor workers, who found a maximum of forces at close to nature
healthy young myocard to values of up to 100 kPa and substrate elasticity (Engler et al., 2008; Jacot et al., 2008). A
sometimes even higher (Berry et al., 2006; Gupta et al., 1994; probablereasonforthiscontrastisthattheircellswereisolatedat
Jacot et al., 2008). muchearlierstagesofembryonicdevelopmentcomparedtoours.
In light of this background it is even more surprising that Cardiomyocytes isolated at identical developmental state
cardiomyocytes are morphologically and functionally barely generated highly comparable contractile forces when tested on
affected by substrate elasticity. In the present study we could 12 kPa substrates (Balaban et al., 2001). Possibly due to the
clearly show that myocytes from late rat embryos form electricalstimulationperformedinexperimentsbyJacotandco-
contractile myofibrils by a very stable mechanism. These fibrils workers, forces generated by single myocytes reported by them
arenotvisiblyaffectedinnumber,lengthorregularityofstriation (Jacot et al., 2008) were higher on very soft substrates while
when cells are grown on elastomeric substrates with stiffnesses comparable to our results on stiffer ones.
covering the complete natural range. Even extreme elasticity Interestingly, with values in the range of 3 to 10 mN overall
valuesofverysoft,1 kPaorverystiff500 kPadidnotresultin forces reported for isolated skinned myocytes are much higher
Elasticity-driven muscle forces 357
(Diffee and Chung, 2003; Korte and McDonald, 2007) than the myocardial tissues. Such cardiomyocytes are characterized by
highest contractile force of 1 mN observed here on stiff well-aligned myofibrils spanning the whole cell in a defined
substrates. This discrepancy is most likely due to several direction and are therefore different from the prenatal
aspects. Most importantly, effective Ca2+ concentrations cardiomyocytes used here. Finally, traction force microscopy is
available to induce myosin activity are clearly different and not well suited to determine the highest possible contraction
optimalforskinnedcellscausingmaximumcontractionsinthose forces of cardiomyocytes. These forces are reached under
cells. Furthermore, cardiomyocytes used for skinned cell isometric conditions where the contraction amplitude vanishes.
preparations are usually isolated from adult, fully differentiated While this situation might be approached using very stiff
substrates, the resulting tiny substrate deformations cannot be
detected anymore.
Inadditiontoforces,wealsocalculatedtheworkperformedby
myocytes during one contraction according to Butler and co-
workers(Butleretal.,2002).However,averyhighscatterofthe
resultingvalues(about1to5 fJ)maskedallpossibledependence
on substrate stiffness.
Our results point at the elasticity independent presence and
functionofmyofibrilsasreasonforthecontinuousforceincrease
with substrate stiffening. These fibrils are already present in rat
cardiomyocytes isolated at day 19 of embryonic development.
Subsequent incubation on any substrate elasticity used here had
no significant effect on morphology or function of this force
generating structure. Although primary cardiomyocytes are by
nature a highly heterogeneous cell pool and, additionally, our
functional analyses were performed on the mechanically most
activesubfraction,wenotednodependenceoftheoverallnumber
ofmyocytesorthenumberofspontaneouslycontractingcellson
n
e substrateelasticity.Substrateelasticitydependentbeatfrequency
p analyses as done elsewhere (Bajaj et al., 2010; Engler et al.,
O
2008) were not performed here due to the naturally high
y
g differences of contraction rates between cardiomyocyte
o
subtypes (Joyner et al., 2007; Watanabe et al., 1983).
ol
Bi The only morphological difference that occurred only on the
very softest substrates was a more roundish shape of myocytes
with possibly slightly less aligned myofibrils. This phenotype is
mostlikelycauseddirectlybylowcellforcegenerationonthese
substrates. Bischofs and co-workers developed a theoretical
model that describes the relation between cell shape and cell
force distribution based on outer adhesion sites and connecting
inwardcurvedactinarcs(Bischofsetal.,2009).Softersubstrates
resultinsmallerforcesandtherefore,accordingtothismodel,in
less curvature. Consequently, reduction of outer adhesion sites
withconnectingactinbundlesasgivenforourcardiomyocyteson
very soft substrates (Fig. 4B) should result in rounded cell
shapes. The slightly reduced order of myofibrils on very soft
substrates fits very well with current knowledge. In independent
papers Englerandco-workersand Bajajandco-workersshowed
similar results for cardiomyocytes grown on very soft
polyacrylamide gels (Bajaj et al., 2010; Engler et al., 2008).
Fig.8. Constantsarcomericcontractilestrainuponsubstratestiffening.
ContractionofindividualmyofibrilsofGFP-a-actinintransfectedcellswas
analyzedovertimealongalinespanningtheirfulllength(A,left)andplotted
astime–spaceplot(A,right;blackarrowindicatesdirectionoftime).Scalebar:
10mm.Grayvaluesalongthemyofibrilatrelaxed(t1)andcontractedstate(t2)
showdisplacementstowardsthecenter(blackarrowheadinB,C)ofthe
myofibrilandenablecalculationofdisplacementsofz-bandsalongthe
myofibril(C).Notethealmostlineardependenceofdisplacementonpositionin
theinnerhalfofthemyofibril.Fittedlinescharacterizingthelinear
displacementsectionwereaveragedover2–4independentmyofibrilspercell.
Theresultingslopes(D)describetheaveragemyofibrilcontractilityofeach
cellon15kPasubstrates(n57),30kPa(n513)and90kPa(n58).Mean
contractilemyofibrilstrainaveragedoverallcellsperelasticityisgivenas
straightlineandseparatelyin(D),bottomright,withincludeds.d.
Elasticity-driven muscle forces 358
Although generated force values increase drastically with The model above includes cell adhesion all along the
substrate stiffening, not only myofibril structure, but also their contractile apparatus, which is well supported by our traction
contractility,seemtobebasicallyunaffectedbyelasticitywithin force experiments.Here,costameric andfocaladhesionsequally
the natural range of 15 to 90 kPa. Our quantitative analyses of participate in force transmission, especially on stiff substrates.
myofibrilcontraction(cf.Fig. 8)revealedrelativelystablestrain Only the very central sarcomere units show a clear contractility.
valuesofabout4%forcentrallylocalizedsarcomeres.Apossible Unfortunately, finding a clear correlation between contractile
tendency of reduced contractions on stiffer substrates was not length of myofibrils and substrate stiffness over all substrate
significant and even if it were, this behavior would indicate that elasticitiesanalyzedherewasnotpossibleduetolimitednumbers
cardiomyocytes generate elevating force values on stiffer of analyzable myofibrils of sufficient length. Such experiments
substrates with even less contraction. were out of the scope of this work due to the very low
Measurements of sarcomere lengths during single myofibril transfection rates achieved for cardiomyocytes but could finally
contractions (e.g. Fig. 8) very clearly showed different behavior prove or disprove our model in the future.
in the middle and at the ends of myofibrils. While centrally Most importantly our data argue for a force generating
located sarcomeres contracted, the outer ones were moved at apparatus in cardiomyocytes achieving stable contraction
constant length. From basic mechanics this is a very surprising amplitudes on varying substrate elasticities. Uncoupling
findingbecausealongthemyofibrilforcesmustaddupresulting cardiomyocytes from surrounding tissue tension leads to stable
inthehighesttensilestressatthecenterofthefibril.Atthesame sarcomerelengthsonphysiologicalsubstrateelasticitiesbetween
time traction forces transmitted by costameres from the elastic 15 and 90 kPa. Substrate stiffening sarcomeres have to generate
substrate to the myofibril were indeed low at the center and higher forces in order to keep their contraction value, which
increased towards fiber ends (e.g. Fig. 7). Exactly the same again argues for a force generating machinery never working at
behavior is found in technical mechanics for fibers of finite full load on softsubstrates.Such a regulatorymechanism would
length embedded in a strained elastic matrix (Hsueh, 1989). As therefore be an ideal system to respond instantaneously to
sarcomeric lengths were in the range of 1.9 mm where in tissue environmental changes or just stress factors causing temporal
sarcomere length raises with applied stress a passive material tissue stiffening connected with the necessity of enhanced force
should display contraction mostly at the ends and less in the values. Sarcomere length regulation as described by the Frank–
n middle.Takentogetherbasicmechanicsandourobservationscan Starlingmechanisminstiffermyocard,willcertainlyadditionally
e only be reconciled by assuming that in our experiments elevateforcesgeneratedbymyocytesbutisnotthecrucialfactor
p
O individual sarcomeres can sustain the same force at a whole for the force adaptation observed here.
y range of lengths. In turn, such a behavior would also imply that
g sarcomeresatstablelengthareabletogeneratearangeofforces
o
ol as we could clearly identify in our experiments (Fig. 8). Materials and Methods
Bi At first such a material behavior appears highly implausible. Isolationofratmyocytesandmyofibroblasts
Cardiomyocytesandcardiacfibroblastsweresimultaneouslyisolatedfrom19-day-
However, it should be seen in the context of the well-studied
oldWistarratembryos.PregnantratswereCO anesthetizedanddecapitated.The
2
spontaneous oscillations of isolated myofibrils (Fabiato and pupswereremovedandalsodecapitatedundersterileconditions.Theheartofeach
Fabiato, 1978; Linke et al., 1993). In this system spontaneous embryo was quickly isolated, washed in Hank’s balanced salt solution (HBSS,
Sigma, St. Louis, USA), cut into small pieces and digested in 8ml of a 0.5%
oscillations of sarcomeric lengths and even traveling density
Trypsin/0.2%EDTAsolutioninHBSStodisintegratethetissue.Thesupernatant
waves occur at intermediate levels of excitation, which lead to was discarded and resulting cell aggregates were incubated with 100ml DNase
the same conclusion as presented above (Anazawa et al., 1992). solution(10,000U/ml,Sigma,St.Louis,USA)for3minutesandthenfilledwith
As we observed spontaneous contractions in isolated cells 8ml of TE solution for further 5minutes. While cell aggregates precipitated
withoutcentrifugation,disintegratedsinglecellsremainedinthesupernatantand
excitation of the contractile system by cellular calcium
were transferred into TE-blocking solution (F10 Ham’s medium supplemented
oscillations was also merely at an intermediate level. with33%fetalbovineserum).ThedisintegrationstepandDNasetreatmentwas
In the abovementioned experiments on isolated myofibrils all repeatedonetotwomoretimesandsupernatantswerecombined.Cellswerethen
collected by centrifugation at 200g, maintained at 37˚C and 5% CO in a
sarcomeres within the fibril experience the same force and 2
humidifiedincubatorandculturedinF10Ham’smediumsupplementedwith10%
identical conditions. This is contrary to our case where traction fetal bovine serum, a 1:100 dilution of an antibiotic solution (10,000 units
forces transferred from the substrate to the fibril grow towards penicillinand10mg/mlstreptomycinin0.9%NaCl,Sigma)anda1:200dilution
the ends while tension in the fibril is maximal at center. This of solution containing insulin (1mg/ml), transferrin (0.55mg/ml), and sodium
selenite(0.5mg/ml)inEBSS(Sigma,St.Louis,USA).Myocyteswereseededon
could explain why we observed neither travelling waves nor fibronectin (2.5mg/cm2) (BD Biosciences, San Jose, CA) coated cross-linked
oscillations. In addition, since tensile stress within the myofibril siliconerubbersubstratesofdiverseandcontrolledelasticities.
and traction forces transmitted by costameres counteract, Forsomeexperimentscardiacfibroblastswereculturedforadditional3dayson
standardpolystyrenecellculturedishesinsupplementedF10Ham’smediumto
relatively small alterations in composition and structure might
inducetheirdifferentiationtomyofibroblasts.Differentiatedcellsweretrypsinized
be sufficient to modify the contractile behavior of myofibrils of andusedforcontrolexperimentsasdescribedbelow.
isolated cardiomyocytes. Evidence for such small differences
between centrally and distally located costameres was found in Celltransfection
vivo (Bennett et al., 2006; Bennett, 2012) as well as in vitro Cardiomyocytes and myofibroblasts were transfected using Nucleofector
technology (Lonza–Amaxa Biosystems, Cologne, Germany). 26106 cells were
(Goncharova et al., 1992; Sanger et al., 2005; Simpson et al.,
resuspendedin100mlNucleofectorsolution.2mgpurifiedplasmid-DNA(GFP-a-
1993). Taken together, suboptimal levels of excitation and actininformyocytesandGFP-VASPformyofibroblasts)wasaddedtothesample
gradual stress transfer from the substrate, perhaps together with andcellsweretransfectedaccordingtothemanufacturer’sinstructions(program
slightvariationsofmyofibrilcompositiontowardstheirends,are G09).Transfectionratesrangedbetween0.1and1%forcardiomyocytesandwere
,5% for myofibroblasts. After transfection, resuspended cells were seeded on
sufficient to reconcile our observations with present knowledge
fibronectincoatedcross-linkedsiliconerubbersubstrates atacelldensityof2–
on myofibril contractility. 46104perPetridish(ø3.5cm).
Elasticity-driven muscle forces 359
Fabricationofsiliconerubbersubstrates segmentation routine was developed. It started with bandpass filtering of the
For cell force analysis of cardiomyocytes, cells were seeded on cross-linked imagesbysubtractingtwobinomiallyfilteredimages(969binomialfilteredminus
elastomeric silicone rubber substrates (Sylgard 184, Dow Corning GmbH). 57657binomialfiltered).Pixelsizewas0.2mm.Subsequently,foreachpixelof
Substrate preparation and characterization of elastomer material properties thispreprocessedimagelocalaverageandlocalstandarddeviationwerecalculated
(Young’s modulusand Poisson’s ratio)were performed as described previously withina91691environment.Thesevalueswereusedtoreplaceeachpixelvalue
(Cesaetal.,2007).Inbrief,variousratios(seebelow)ofbase(vinylterminated bythez-score,i.e.thelocalaveragewassubtractedandtheresultnormalizedby
polydimethylsiloxane) and cross-linker (methylhydrosiloxane–dimethylsiloxane the local standard deviation. All connected regions exceeding a threshold size
copolymer)wereprepared.Theprepolymermixturesweredegassedandonepart (usually10pixels)andathresholdz-score(usually0.7)wereclassifiedasclusters.
of the mixture was mixed with red fluorescent beads (0.2mm diameter, In some micrographs highly elongated and obviously artificial clusters were
FluoSpheres Crimson carboxylate-modified beads, Invitrogen) and subsequently identifiedbythisroutine.Theseartifactswereremovedbythefollowingcriterion.
appliedonapreviouslysilanized(1H,1H9,2H,2H9-perfluorooctyl-trichlorosilane, Foreachclustertheellipsewithequalcentralsecondmomentswascalculated.The
Sigma)micro-structuredsilicondioxidemold.Themicrostructurewascomposed distributionofthesemi-majoraxesoftheseellipseswastransformedintothez-
of asquare lattice consistingof500nm highmicrodots withan edgelength of score(definitionseeabove).Allareasexceedinganinteractivelychosenthreshold
2.5mm and a lattice constant of 3.5mm. The layer thickness ofthe fluorescent value(typically15)wereconsideredartifactsandremoved.Fromtheremaining
bead/prepolymermixturewasreducedtolessthan500nmbywipingthesurface datawecalculatedaverageclustersize,thenumberofclusterswithinagivencell
with lint-free tissue. The remaining non-cross-linked prepolymer lacking andthefractionofthecellareacoveredbyclusters.Tothisendcellareaswere
fluorescent beads was added on top of the thin fluorescent bead/prepolymer interactivelymarkedbypolygonsasabove.
layer.Glassslides(80mmthick,MenzelGmbH,Germany)servedasspacersand
wereplacedonthesiliconemixturebesidethemicrostructure.Subsequently,they Cellforceretrieval
werecoveredwithanadditional80mmcoverslipontoptoobtainaconstantlayer
Traction forces applied by the cell to the elastic substrate caused substrate
thickness.Cross-linkingwasperformedat60˚Covernight.Elastomericsubstrates
deformations that were visualized by tracking fluorescent marker beads in the
weresubsequentlygluedtothebottomof3.5cmPetridishestocoverpredrilled
uppermost layer of the substrate (see above). From the displacements of these
1.5cmholes.Aftercuring,thesesiliconerubbersdisplayedaPoisson’sratioof0.5
particlescellforceswerecalculated.Thesecalculationsarebasedontheverywell
andaYoung’smodulusof500kPa(20:1ratio;basetocross-linkerw/w),150kPa
understoodbehavioroflayeredelasticmaterialsinresponsetoexternalforces(cf.
(30:1), 90kPa (35:1), 30kPa (45:1), 15kPa (50:1) and ,1kPa (70:1). The
Merkeletal.,2007;andreferencestherein).Thespecificsiliconeelastomersystem
elasticityfortheverysoft,1kPasubstratescouldnotbeaccuratelyanalyzeddue
usedherehasbeenshowntofulfilltheprerequisitesoftheabovementionedtheory,
totheirsoftness andstickiness. Therefore,theelasticity ofthosesubstrates was
i.e.theyarehomogeneousandisotropicmediaexhibitinglinearelasticityuptovery
estimatedbyextrapolatingexistingcalibrations.
largeextensions(Balabanetal.,2001;Cesaetal.,2007;GutierrezandGroisman,
2011). However, for rapidly contracting cells like cardiomyocytes viscoelastic
Microscopy material behavior is a concern, especially for the softest rubbers. We therefore
Livecellanalyseswereperformedat37˚Cand5%CO (cellincubatorXL2,Zeiss, measured the force relaxation after a rapid indentation step (0.2second) with a
2
Germany)usinganinvertedmicroscope(Axiovert200,Zeiss,Germany),equipped cylindricalpunch.Theratioofforceafterthedurationofanaveragecontraction
withaCCDcamera(Orcablack-and-white, Hamamatsu,Japan)utilizing a636 (,0.3second)andatverylongtimeswas2.1fortheverysoftestrubber(,1kPa)
n
e Antiflex EC Plan-Neofluar oil immersion objective (PH3, NA51.25, Zeiss, and1.3for15kParubber.Asstifferelastomersexhibitfasterforcerelaxationand
p Germany).ImagesweretakenusingtheimagingsoftwareOpenBox(version1.77, theoverallaccuracyofthetechniqueisabout30%,weconcludedthatviscoelastic
O Informationssysteme Schilling, Germany). Excitation of GFP-a-actinin and of materialbehaviorplaysasignificantroleforthesoftestelastomeronly.
fluorescentbeadswasdonewithamercuryarclamp(HBO100,Osram,Germany). Moreover,forisolatedcellsofthesizeofmyocytesalayerof80mmthickness
y
g FluorescentimagesofGFP-a-actininwereobtainedusinga505–530nmbandpass behavesalmostexactlylikeanelastichalfspace(Merkeletal.,2007).Thisgreatly
o filter,forthefluorescentbeadsa590nmlong-passfilterwasused. simplifies the underlying mathematics and enabled us to use the analytical
ol Immunofluorescentlylabeledcellswereanalyzedwithaninverseconfocallaser expressions derived first by Boussinesq (Boussinesq, 1885). These expressions
Bi scanningmicroscope(LSM710,Zeiss,Germany)usinga406Plan-Neofluaroil correctlydescribethedisplacementscausedbyapointforceactingonthesurface
immersionobjective(Ph3,NA1.3,Zeiss,Germany)anda636Plan-Apochromat of the elastomer. Our algorithms (see below) assume a test force distribution,
oil immersion objective (Ph3, NA 1.4, Zeiss, Germany). Confocal micrographs calculate the cumulative displacement caused by all these forces and iterally
weretakenusinganargonionlaser(488nm)witha490–530nmbandpassfilter improve the force distribution until it reproduces the measured displacements.
andahelium–neonlaser(543nm)witha550–812nmbandpassfilter. Unfortunately, in traction force microscopy this conceptually straightforward
approachinvolvessomelessthanintuitivemethodsofnumericalmathematics.For
detailsseee.g.Merkelandco-workersandSchwarzandco-workers(Merkeletal.,
Immunofluorescencetechniques
2007;Schwarzetal.,2002).Ourimplementationworksasdescribedbelow.
Myocytes (36104 cells per substrate) were seeded on silicone rubber substrates BeaddisplacementswerefirstdeterminedasdescribedbyMerkelandco-workers
with different stiffness ranging from ,1 to 500kPa and stained after 3 days. (Merkeletal.,2007).Inshort,thefirstimageofthesequencewasusedasreference
Myocyteswerefixedusing3.7%formaldehyde(Merck,Germany)incytoskeleton image.Asmallregionaroundarandomlychosenmicrobeadwasusedastemplate.
buffer(CB:150mMNaCl,5mMMgCl2,5mMEGTA,5mMglucose,1mg/ml Allotherbeadswerelocalizedbycross-correlatingthistemplatewiththeimageand
streptomycin,10mMMES,pH6.1)for20minutesat37˚C.Subsequently,cells finding local maxima of the normalized correlation coefficient with sub-pixel
werewashedoncewith30mMglycineinCBfor10minutes,permeabilizedwith accuracy.Onlyplaceswherethenormalizedcross-correlationcoefficientexceededa
5% Triton X-100 (Sigma, St. Louis, USA) in CB for 3minutes at room thresholdvalue(typically0.8)wereacceptedasbeadpositions.Insubsequentframes
temperature, rinsedtwice withCB and then treated with blocking solution (5% microbeadswerelocalizedusingbead-specifictemplatesandtheirdisplacements
skim milk powder (Sigma) in CB) for 45minutes at 37˚C. Primary antibodies withrespecttothereferenceimagewerecalculatedwithanaccuracyof25nm.
(anti-vinculin,clonehVin-1(Sigma,St.Louis,USA);anti-a-actinin,cloneEA53
Forceretrievalfromthesedisplacementsismorecomplicatedasmathematically
(Sigma, St. Louis, USA); anti-plectin, clone 10F6 (Santa Cruz Biotechnology,
thedisplacementvectorfieldoriginatesfromtheforcefieldbyaconvolutionwith
SantaCruz,CA)andsecondaryantibodiesconjugatedtoCy3(Dianova,Hamburg,
astiffnessdependentGreens’tensorthatexhibitsasingularityattheoriginand
Germany)werebothdiluted1:100in1%skimmilkpowderinCBandapplied
decaysasoneoverdistance.Invertingthisconvolutionisanumericallyill-posed
eachfor45minutesat37˚CwiththreeadditionalwashingstepswithCBbetween
problemrequiringregularizedleastsquaresfitorrelatedalgorithms(Demboand
theprimaryandsecondaryantibody.ForactinstainingAlexaFluor488phalloidin
Wang,1999;Merkeletal.,2007;Schwarzetal.,2002).
(Invitrogen,Carlsbad,USA)wasusedina1:200dilutioninCBtogetherwiththe
Inthisworkweusedtwodifferentalgorithms.Oneisbasedonasuperposition
secondaryantibodyfor45minutesat37˚C.
ofoptimallychosenpointforceslocalizedatmanuallypredefinedlocations(cell
adhesionsites),theothercalculatesentireforcefields.Thepointforcealgorithm
Cellshapedetermination wasdevelopedbySchwarzandco-workersandusedasdescribedbyCesaandco-
Forquantifyingcellroundnessweusedadimensionlessshapeindexsidefinedas workers(Schwarzetal.,2002;Cesaetal.,2007).Theforcefieldalgorithmusedis
si54pAP22whereAdenotestheareaofthecellandPitsperimeter.Foranyshape described in detail by Houben and co-workers (Houben et al., 2010). Both
siisinbetween1(circle)and0(line).Forcalculatingsitheshapeofacellwas algorithms yield force distributions. To assess the quality of these solutionswe
approximatedbyapolygonwithinteractivelychosencornerpoints(dependingon calculated the displacements resulting from the retrieved force distribution and
cell shape typically between 20 and 40). Cells exhibiting si above 0.8 were compareditwiththemeasuredbeaddisplacements.Besidesavisualcomparison
we calculated the normalized squared deviation, x2, between fit result and
classifiedasround,allothersasangular.
measured data according to x2~½2ðNB{NFÞs2(cid:2){1PNB ð~uuBi{~uuFiÞ2 where NB
DetectionofGFP-a-actininclustersinmicrographs signifies the total number of tracked beads with dispi~la1cements~uuBi (measured)
FluorescencemicrographsofcellstransfectedwithGFP-a-actininexhibitedbright and~uuFi (calculated).NFisthetotalnumberofforceinductionpointsandsthe
clusters indicating FAs and z-bands. For the localization of these clusters a standarddeviationofdisplacementmeasurement(here25nm).
Elasticity-driven muscle forces 360
For the characterization of cell contractions we calculated the sum of all Bajaj, P., Tang, X., Saif, T. A. and Bashir, R. (2010). Stiffness of the substrate
contractile forces. For the point force algorithm we proceeded as described by influencesthephenotypeofembryonicchickencardiacmyocytes.J.Biomed.Mater.
Merkelandco-workers(Merkeletal.,2007).Here,forceapplicationpointswere Res.A95A,1261-1269.
chosenonaregularhexagonalgrid(latticeconstant5mm)coveringthewholecell Balaban,N.Q.,Schwarz,U.S.,Riveline,D.,Goichberg,P.,Tzur,G.,Sabanay,I.,
area.Subsequently,thecellcenterwascalculatedasthecenterofgravityofthese Mahalu,D.,Safran,S.,Bershadsky,A.,Addadi,L.etal.(2001).Forceandfocal
points. The contractile parts of all forces, i.e. the force components pointing adhesion assembly: a close relationship studied using elastic micropatterned
substrates.Nat.CellBiol.3,466-472.
towardsthecellcenter,weresummeduptoyieldthetotalcontractileforce.This
Bennett,P.M.(2012).Frommyofibriltomembrane;thetransitionaljunctionatthe
procedureenablesthecalculationofoverallcellcontractionforcevaluesalthough
intercalateddisc.Front.Biosci.17,1035-1050.
accordingtoNewton’sprinciplesallforcevectorshereandforanyotherstably
Bennett,P.M.,Maggs,A.M.,Baines,A.J.andPinder,J.C.(2006).Thetransitional
adheredcellsumuptozero.
junction:anewfunctionalsubcellulardomainattheintercalateddisc.Mol.Biol.Cell
Intheothercase,theforcefieldalgorithm,thewholemicrographiscoveredbya 17,2091-2100.
mesh comprising almost the same number of vertices as beads were tracked Berry,M.F.,Engler,A.J.,Woo,Y.J.,Pirolli,T.J.,Bish,L.T.,Jayasankar,V.,
(typically2,000–4,000).Forcesarecalculatedateachvertex.Thereforewecould Morine, K. J., Gardner, T. J., Discher, D. E. and Sweeney, H. L. (2006).
calculate the contractile force in almost the same way as before. In effect the Mesenchymal stem cell injection after myocardial infarction improves myocardial
geometrical center of the cell was determined and within the cell area the compliance.Am.J.Physiol.HeartCirc.Physiol.290,H2196-H2203.
contractilepartsofallforceswereadded. Bhana,B.,Iyer,R.K.,Chen,W.L.K.,Zhao,R.,Sider,K.L.,Likhitpanichkul,M.,
For some control analyseswe performed traction force experiments on GFP- Simmons, C. A. and Radisic, M. (2010). Influence of substrate stiffness on the
VASP transfected myofibroblasts. Bead microstructured elastomer substrate phenotypeofheartcells.Biotechnol.Bioeng.105,1148-1160.
preparation, bead displacement determination and traction force analyses (point Bischofs,I.B.,Schmidt,S.S.andSchwarz,U.S.(2009).Effectofadhesiongeometry
forcealgorithm)wereperformedasdescribedaboveforcardiomyocytes.Inorder andrigidityoncellularforcedistributions.Phys.Rev.Lett.103,048101.
toobtainareferenceimageofthenon-deformedsubstratebeneaththeanalyzed Boussinesq, J. (1885). Application Des Potentiels A` L’e´tude De L’e´quilibre Et Du
cells, myofibroblasts were removed manually from the substrate using a glass MouvementDesSolideE´lastiques.Paris:Gauthier-Villars.
micropipettemountedonamicroinjector(InjectManNI2,Eppendorf,Germany). Butler,J.P.,Tolic´-Nørrelykke,I.M.,Fabry,B.andFredberg,J.J.(2002).Traction
fields, moments, and strain energy that cells exert on their surroundings. Am. J.
As force induction points we either used FAs labeled by GFP-VASP or an
Physiol.CellPhysiol.282,C595-C605.
interactively chosen lattice of ,4mm spacing beneath the central cell body to
Cesa, C. M., Kirchgessner, N., Mayer, D., Schwarz, U. S., Hoffmann, B. and
mimicadhesionsitesalongmyofibrilsofcardiomyocytes.
Merkel,R.(2007).Micropatternedsiliconeelastomersubstratesforhighresolution
analysisofcellularforcepatterns.Rev.Sci.Instrum.78,034301.
High-resolutionmyofibrilcontractionanalysis Damon,B.J.,Re´mond,M.C.,Bigelow,M.R.,Trusk,T.C.,Xie,W.,Perucchio,R.,
FluorescentmicrographsofcellstransfectedwithGFP-a-actininclearlyshoweda Sedmera,D.,Denslow,S.andThompson,R.P.(2009).Patternsofmuscularstrain
sarcomerestructure.Duetothecorrespondingbrightnessvariationalongthefibril intheembryonicheartwall.Dev.Dyn.238,1535-1546.
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Costameres are sites of force transmission to the substratum in adult rat
proceededasfollows.Theimagewasdenoisedwitha363binomialfilter(pixel
cardiomyocytes.J.CellBiol.118,1411-1420.
n size0.2mm).Subsequently,amyofibrilwastracedwithconnectingstraightlines.
deTombe,P.P.,Mateja,R.D.,Tachampa,K.,AitMou,Y.,Farman,G.P.and
e At distances closely corresponding to the original pixel size gray values were
p determinedbyinterpolationfromtheneighboringpixels.Inthefollowingwewill Irving,T.C.(2010).Myofilamentlengthdependentactivation.J.Mol.Cell.Cardiol.
O 48,851-858.
refertotheresultingvectoras‘‘profile’’.Grayvalueextractionwasrepeatedfor
Decker,M.L.,Simpson,D.G.,Behnke,M.,Cook,M.G.andDecker,R.S.(1990).
y eachimageofthesequencealongthesameline.Intheresultssectiontheseprofiles Morphologicalanalysisofcontractingandquiescentadultrabbitcardiacmyocytesin
og are shown in the space–time plane. Such plots are sometimes also called long-termculture.Anat.Rec.227,285-299.
ol kymographs. Dembo,M.andWang,Y.L.(1999).Stressesatthecell-to-substrateinterfaceduring
Bi Beforetheactualcalculationofsarcomeredisplacementseachprofilewasagain locomotionoffibroblasts.Biophys.J.76,2307-2316.
smoothedbyabinomialfilterofwidth3.Thefirstprofileofasequencewasused Diffee, G. M. and Chung, E. (2003). Altered single cell force-velocity and power
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Engler,A.J.,Carag-Krieger,C.,Johnson,C.P.,Raab,M.,Tang,H.Y.,Speicher,
numericallydeterminedinflectionpointsofthedisplacementprofile.Intheactual D. W., Sanger, J. W., Sanger, J. M. and Discher, D. E. (2008). Embryonic
experimentsthisregionwas5to10mmwide.Withinthisrangeastraightlinewas cardiomyocytes beat best on a matrix with heart-like elasticity: scar-like rigidity
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Fukuda, N., O-Uchi, J., Sasaki, D., Kajiwara, H., Ishiwata, S. and Kurihara, S.
Acknowledgements
(2001).Acidosisorinorganicphosphateenhancesthelengthdependenceoftensionin
We thank W. Rubner for helpful discussions and expert technical ratskinnedcardiacmuscle.J.Physiol.536,153-160.
support. We also thank E. Noetzel for critical reading of the Giannone,G.,Jiang,G.,Sutton,D.H.,Critchley,D.R.andSheetz,M.P.(2003).
manuscript and T. Jonas for cell isolation and helpful discussions. Talin1iscriticalforforce-dependentreinforcementofinitialintegrin–cytoskeleton
bondsbutnottyrosinekinaseactivation.J.CellBiol.163,409-419.
This work was supported by the German Bundesministerium fu¨r
Gilbert,P.M.,Havenstrite,K.L.,Magnusson,K.E.,Sacco,A.,Leonardi,N.A.,
BildungundForschung(BMBF,projecttitle:MechanoSys,BMBF- Kraft, P., Nguyen, N. K., Thrun, S., Lutolf, M. P. and Blau, H. M. (2010).
Nr.0315501A). Substrateelasticityregulatesskeletalmusclestemcellself-renewalinculture.Science
329,1078-1081.
Goffin,J.M.,Pittet,P.,Csucs,G.,Lussi,J.W.,Meister,J.J.andHinz,B.(2006).
Competing Interests Focaladhesionsizecontrolstension-dependentrecruitmentofa-smoothmuscleactin
Theauthors have nocompeting intereststodeclare. tostressfibers.J.CellBiol.172,259-268.
Goncharova, E. J., Kam, Z. and Geiger, B. (1992). The involvement of adherens
junction components in myofibrillogenesis in cultured cardiac myocytes.
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