DOI: 10.1161/CIRCGENETICS.113.000103

The Novel Mutant p.A120D Impairs Filament Formation, Prevents Intercalated Disk Localization and Causes Sudden Cardiac Death

Running title: Brodehl et al.; Characterization of a novel desmin mutation

Andreas Brodehl, PhD1,9*; Mareike Dieding, MSc2*; Bärbel Klauke, PhD1; Eric Dec, MD3; Shrestha Madaan, MD3; Taosheng Huang, MD, PhD4; John Gargus, MD, PhD3; Azra Fatima, PhD5; Tomo Šaric, MD, PhD5; Hamdin Cakar, PhD6; Volker Walhorn, PhD2; Katja Tönsing, PhD2; Tim Skrzipczyk, MSc1; Ramona Cebulla, TN1; Désirée Gerdes, TN1; Uwe Schulz, MD1; Jan Gummert, MD1; Jesper Hastrup Svendsen,dsen,n MMD,D,D DDMScMSc7,87,8; Morten Salling Olesen, PhD7,8; Dario Anselmetti, PhD2; Alex Hørby ChrChristensen,isstensen, MDMD,, PPhDh 7,8; VirginiaViVirginia Kimonis,Kimonis, MMDD3; HendrikHendndriikk MMilting,iillting, PhDPhhDD1

1Erich &H Hannaannna KlKlessmannessmann InInstnst ffo forr CaCardiovascularardr iovasccullarr RReseeseeaearchrch & DevelopmentDeD velopmmennt (EHKI),(EHKI), HeartHeH art & DiabetesDiabettes CenterC NRW, Ruhr UnivUnivv Bochum,Bochuh m,m BadBadd Oeynhausen;Oeynhn auses n; 2ExperimentalExE peerir mentala BiophysicsBioophysics & ApAppliedplied NaNanoscience,nooscience, FacultyFaculltyt ooff Physics & Bielefeld Instnst for BiophysicsBiophysicss & NanoscienceNanosccieence ((BBINASINAS),), BielefeldBieelefeld UUniv,niiv,v BBielefeld,ielefeld,, GGermany;ere many; 3DivisionDivision ofof GeneticsG & Metabolism,m, Dept ofof Pediatrics,Pediatrrics, UnivUniv ofo California,Califi orrnia, Irvine,Irvine,e CA;CA; 4DivisionDiD visiion ooff HumanHuman Genetics,Genen tics, DeptDept ofof Pediatrics,Ped Cincinatti Children’s HoHospital,sppital, CCincinnati,incinnati, OOH;H;; 5InstInst for Neurophysiology,Neuropphyysiologgy, Medical CenteCenter,r,, Univ of ColCologne,o Cologne;e; 6Physikalisch-TechnischePhysikallissch-Teechc niscs hee BBundesanstaltunndesas nstaltt ((PTB),PTTB), BrBraunschweig,auunsschc weig, GeGermany;rmmany; 7DeptDept of Cardiology,Cardiologg Rigshospitalet,pitaletpitalet CopenhagenCopenhagen UnivUniv HospitalHoHospital,spital 8TheThe Da Danishnish NNational ational Re Researchsearch F Foundationoundation Ce Centrentre ffor or C CardiCardiacarddi Arrhythmia, Copenhagen, Denmark; 9Present address: Libin Cardiovascular Inst of Alberta, Dept of Cardiac Sciences, Univ of Calgary, Calgary, AB, Canada *contributed equally

Correspondence: Andreas Brodehl, PhD Hendrik Milting, PhD Department of Cardiac Sciences E. & H. Klessmann Inst for Cardiovascular Libin Cardiovascular Institute of Alberta Research & Development (EHKI) University of Calgary Heart and Diabetes Center NRW 3280 Hospital Drive NW Ruhr-University T2N4Z6 Calgary, AB Bochum, D-32545 Bad Oeynhausen Canada Germany Tel: +1-403-210-7322 Tel: +49-5731-973510 Fax: none Fax: +49-5731-972476 E-mail: [email protected] E-mail: [email protected]

Journal Subject Codes: [16] Myocardial cardiomyopathy disease, [89] Genetics of cardiovascular disease, [137] Cell biology/structural biology, [178] Aggregation

1 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 DOI: 10.1161/CIRCGENETICS.113.000103

Abstract:

Background - The desmin is encoded by the DES and contributes to the mechanical stabilization of the striated muscle sarcomere and cell contacts within the cardiac intercalated disk. DES mutations cause severe skeletal and cardiac muscle diseases with heterogeneous phenotypes. Recently, DES mutations were also found in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC). Currently, the cellular and molecular pathomechanisms of the DES mutations leading to this disease are not exactly known. Methods and Results - We identified the two novel variants DES-p.A120D (c.359C>A) and -p.H326R (c.977A>G), which were characterized by cell culture experimentsp and atomic force microscopy. Family analysis indicated a broad spectrum of cardiomyopathiesioomyopathies wwithiith a striking frequency of arrhythmias and sudden cardiac deaths. The in vitroitro experimentsexxperiments of desmin-p.A120DA120DA120D evidencedeviv denccede a severe intrinsic ffilafilameilamentmentn formation defectdeffect causing cytoplascytoplasmcytoplasmicm aggregates inin ccecellll lines andand ofof thethe isolatedisolatted recombinantrrecoecommbinantmbbinaannt protein,pro rotein,tet in, respectively.rer sppece ectively.ttiveely. ModelMoM deel vavariantvariantsriana t of codon 120 iinindicatedndicac ted ththathata iionicoonicc iintenterractionsactions cocontributeonttrribbutte ttoo ththisis filamentfilament formationfoormmaation defect.ddefeect. ExE vivo analysis of ventricular tissuetisssue slicesslices revealedrer vealed a lossloso s ofo desmindesmiin stainingsts aia ningg withinwithin thethe intercalateintercalated disk and severe ccytoplasmicyty opplasmic aggregateaggregag te fformationormation wwhhehereasreas zz-band-bband llolocalizationcalizatiion waswas notnot affected.affected. The functional experimentsexperiments ooff desmin-p.H326Rdeddesmin-p.Hsmin-pp.H326R326RR diddidd notnott demonstratedemonstratdemonsttratete aanyny ddifferencesiffef rences ffromrom wiwildld type. Conclusions - Due to the functional in vivo and in vitro characterization DES-p.A120D has to be regarded as a pathogenic mutation, whereas DES-p.H326R is a rare variant with unknown significance, respectively. Presumably, the loss of the desmin-p.A120D filament localization at the intercalated disk explains its clinical arrhythmogenic potential.

Key words: cardiomyopathy, desmosome, death, sudden, desmosomes arrhythmia, desmin, intermediate filaments, intercalated disk

2 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 DOI: 10.1161/CIRCGENETICS.113.000103

The intermediate filament (IF) protein desmin is encoded by the gene DES and contributes to the mechanical stabilization of the sarcomeres and cell contacts within the cardiac intercalated disk

(ID). Desmin is the predominant IF-protein of striated muscles. It belongs to the type III IF- characterized by a uniform assembly mechanism. In the first step of the in vitro assembly two coiled-coil dimers form an antiparallel tetramer 1. These tetramers are the essential

building blocks of the IF. Eight tetramers anneal in lateral orientation into unit length filaments

(ULFs). In the longitudinal elongation step these ULFs are assembled and radially compacted

into IF 2. Since the first reports on DES-mutations 3–5 it became obviousus ththatat DESDES-mutations-mutationo cause skeletal myopathies and different forms of cardiomyopathies 6,7.

In thehe memeantimeantit mem mmoreoore ththanan 6600 ddifferentifferent DEDESES-m-mutationsutations disdistributtribbuuted ooverver ththee wholewhole

sequence areree kknown,nown, whwhichiich leleadead iinn tththee mammajorityjo rityrrityy ofof caccasessessses too filamentfillammeent formationfformrmationation defectsdefeeccts wiwithith

deposition ofof cytoplasmiccyytooplaasmiic desminddesmini aggregatesaggregates 8,98,9. However,HHoweverr, tthehe ppathomechanismsathomechc annisms ooff deddesminsminin

aggregationn leadingleading to skeletalskelel tal or cardiaccardiac myopatmmyyoyopathiespap thhieshiies are memechanisticallychhanisi tiically nonott understood iinn

detail. Moreover,eo er aggregateat formationffo atiio off mmutant tantt t desminsdde iin does ddo not ot explain e plallaini per se thethh arrhythmogenic phenotype of some cardiomyopathies.

Recently, different DES-mutations were also identified in patients with ARVC 10–15.

ARVC is an inherited cardiomyopathy clinically characterized by arrhythmias and predominately

right ventricular dilatation leading to cardiac syncope, heart failure, or even sudden cardiac death

16. It is well established that mutations in the coding for desmosomal plaque proteins cause

ARVC 17–19 and rare forms of (DCM) 20. In the cardiac muscle desmin is

found in costamers, the z-disk and -connected via plaque proteins- to the cardiac desmosome

within the ID. The molecular processes contributing to the destabilization of the ID through

desmin filaments are fragmentarily understood. Especially, it is not known, how and which of

3 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 DOI: 10.1161/CIRCGENETICS.113.000103 the desmin mutations impair the connection of the IF-system to the cardiac desmosome.

In this study, we report a novel pathogenic DES-mutation (c.359C>A, p.A120D), which appears to interfere particularly with the connection of desmin IF to the ID. Furthermore, we investigate if the DES-variants p.A120D and p.H326R (c.977A>G) affect the IF formation using ectopic expression cell culture systems and atomic force microscopy (AFM). These data reveal that desmin-p.A120D but not desmin-p.H326R inhibits the longitudinal assembly step confirming its pathogenic potential.

Material and Methods

Clinical descriptionscriptionscripption ofof ththehe patients

In family A tththee 34 yyearsea earsrss ooldlld femalefemmale inindexded x patientpap ti tientennt (III:24)(IIII::24) presentedp resentedressenntedd wwithitth atatrialtrir al fflutter,lul tter, variavavariableria atrioventricularcullarr cconductiononduuction (F(Fig.igg. S11) anandd didilatedilal teed atria.atria.. TheThee averageaveerar gee vventricularenntriccularr ffrequencyrequencyc wwas 64 beats per minuteminute (bpm) andand thethe atrialatrriai l frequencyfrequenccy wawasas 1201220 bpm.bppmm. InIn thethhe electrocardiogramellectrrocardiogram sosomsomem polymorphicicic prematurepremature ventricularventriicular cocontractionsnttracttioi ns (PVCs)(PVCsC ) wiwithith a frequencyfrequency ooff 455 – 111111 bpmbpm were detected (Fig. S1). The cardiological evaluation including 2D, M-mode, spectral and color

Doppler was performed. These investigations reveal normal left ventricular systolic function (left ventricular ejection fraction of 67%), a borderline concentric left ventricular hypertrophy.

Nevertheless, the left atrium was severely dilated and the right atrium was also dilated. She is a member of a large family with dilated cardiomyopathy (DCM) and several sudden cardiac deaths

(SCD) (Fig. 1A). The patient had no signs of a myopathy but received an implantable cardioverter defibrillator (ICD). She had one sister (III:21) and two brothers (III:22, III:23) who died from SCD as teenagers (aged 13, 17 and 13 years, respectively). Her father (II:13) and grandfather (I:3) died due to cardiomyopathy aged 33 and 45 years. Patient III:24 lost three aunts

(II:2 aged 34 years, II:4 aged 42 years and II:10 aged 50 years) by SCD. Another four members

4 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 DOI: 10.1161/CIRCGENETICS.113.000103 of family A died suddenly at age 13 (III:3, III:4, III:15) and at age 30 years (III:16). One paternal aunt (II:6) of the index patient (III:24) suffering from Ebstein anomaly (EA) was transplanted.

All children of II:6 were without cardiac disease. The index patient (III:24) and her aunt (II:6) were heterozygous for the DES mutation p.A120D (c.359C>A). Her son (IV:3) was wild-type and presented no signs of a muscle disease. The mutation was not found in the children of patient

II:6. One cousin of the index patient (III:7) was positive for the DES mutation p.A120D. The

ECG of patient (III:7) showed normal sinus rhythm with prolonged PR interval, T wave abnormalities and AV block. Furthermore, patient III:7 was examined by ecechocardiographyhocardiograaphy and magnetic resonance imaging (MRI). The patient revealed normal left vvententrriculariicullar ssizeizi e anandnd nnormal left ventricularularularr ssystolicystot liic functionfufunctiunnctionon (left ventricular ejectionejeection fractionfraction 64%64%% by Biplane). There werew no evidencesess forfor left veventricularnttriiculaar hyhypertrophy.yppe pertrortror phyy. InIn summary,ssummarry,, thethe spectralspep ctraal DopplerDoopppleer showedshs owedd nnonormalo pattern of LLVV diddiastolicasstolic fifilling.lliing.g NormalNoro mal rirightght veventricularntn riicullar sisizeze aandndd ssystolicyystot lic fufunctionnctiion werewere ddedetected.tete

Of note, thee rrightigght atrium was severeseverelylyl ddilated,ilatedd, sisimilarimiillar to his cousincousiin (p(patient( atatientiei nt III:24,III:24,, familfamilyy AA).

Nevertheless,ss bbe becabecause se off thethhe young o ng age off thisthhiis patientatiient andndd thethhe remarkablekkabl sisimilar imiilla phenotype phenotphh ot p compared to the index patient (III:24) it is expected that the clinical symptoms will potentially increase during the next decades. For that reason the patient received a pacemaker/ICD.

In family B (Fig. 1B) the index patient (IV:1) had experienced palpitations from an age of

25 years. Clinical workup showed a borderline ARVC phenotype based on non-sustained ventricular tachycardia of left bundle branch block (LBBB) morphology, a positive signal averaged-ECG, and a suspicious family history. Echocardiography and MRI were normal.

Review of her father’s (III:2) medical history revealed that he had been evaluated in the early eighties due to syncope. Workup had shown frequent premature ventricular contractions (PVC) of LBBB morphology. He died suddenly playing golf aged 38 years. The probands uncle (III:3)

5 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 DOI: 10.1161/CIRCGENETICS.113.000103 had experienced syncope during running at the age of 38 years and was diagnosed with a non- ischemic cardiomyopathy (ejection fraction 25%) and non-sustained ventricular tachycardia. His ejection fraction improved to 50% on therapy (Angiotensin-converting-enzyme inhibitor, beta blocker, digoxin, spironolactone, ICD) but he developed chronic atrial fibrillation. All three individuals (IV:1, III:2, III:3) were tested positive or obligate carriers of the DES-p.H326R

(c.977A>G) variant. The probands brother (IV:2) was phenotype and genotype negative (current age 37 years). No clinical or genetic data were available on individuals II:1 and II:2 as they had died more than 30 years ago. Individual III:5 was diagnosed with a DCMCM andand PVCsPVCs beforebeforer experiencing sudden cardiac arrest aged 36 years. Individual IV:4 was ininitiallyittiai lly examexaminediined aatt the age of nine yeyearsars duduee tot PPVCsVCV s and non-non-sustainedsustained ventnntricularricular tachtachycardia.ycy arrdid a. LaLater,ter, her left ventricleven was dilatedd ((l(lefteeft ventricuventricularlar enendnd diddiastolicasa toliic diiaamametereteer 6699 mmmm)m) andana d eejejectioneectionn ffractionractioon wawass at tthehhe lowest 25%% bbututt improvedimprroveed tto 55%55%% wwithith treatmentttreatmt ent (Angiotensin-conve(A(Angiotensin-cAngioi tennsin-converting-enzymeonnvertrting-enzymetiing-enzzymee iinhibitor,inhibitonhhibi ito beta blocker, amiodarone,miodarone,miodarone,, PPVCVCC ablation,aablabllattion,ion,, ICD).ICDC ).) BloodBlloodd was availableavailabble froffromromm individualindid viiddual IV:4 and shesshh did not carry thehe DESDES-p.H326Rp H326RH326R variant. ariantiiant NNoNone of f ththe h patienttii tsshadsignsofperipheralmsclas hadhadd signsiig off peripheralriiphh all muscularllar involvement.

Genetic analysis and mutation detection

Genomic DNA was isolated and purified from the affected individuals using the illustra blood genomicPrep Mini Spin Kit (GE Healthcare, Chalfont St. Giles, UK). The BigDye® Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA) and an ABI310 Genetic Analyser

(Applied Biosystems, CA, USA) were used for sequencing according to the manufacturer´s instructions. The sequences were analyzed with the Variant Reporter Software v1.0 (Applied

Biosystems, CA, USA). The allele frequencies of novel variants were determined in 394 healthy control individuals using the TaqMan SNP Genotyping Assay (Applied Biosystems, CA, USA).

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The consent of all participants was obtained and the study was approved by the ethics committee.

Cloning and site-directed mutagenesis

The sequence variants were inserted into the plasmids pET100D-Desmin 12 and pEYFP-N1-

Desmin 21 using appropriate primers and the QuickChange Lightning Kit (Agilent Technologies,

Santa Clara, USA) according to the manufacturer’s instructions (see Table S1). The DES coding regions were verified by sequencing using the BigDye® Terminator v1.1 Cycle Sequencing Kit

(Applied Biosystems, CA, USA). The reference cDNA of human desmin (NM_001927.3) was used for comparison.

Cell culture

H9c2-, HeLa-,La-,, CC2C12-2C122- anandnd SW-13-cells ((LGCLGC SStandards,taandn aards, Middlesex,Middlesex, USA)USA) were cultured in

DMEM (Invitrogen,nvitrogen,vvitrrogen, Carlsbad,Carllsbbadd, UUSUSA)A)A ssupsupplementeupplem ementedmennted withwiw thh 10%10% fetalfeetaal ccacalfaallff seserumerrum (F(FCS),CS S)), 44.5.55 gg/g/L/

Glucose andd PePenicillin/Streptomycin.nicilll iin/SSttreptot mycin.n TThehhe HHL-1LL-1 cecellslls (k(kindlyindly pprprovidedovidedd bbyy W.WW.C.C.C CClaycomb)lal ycomb)b were cultured in ClClaycombayycomb mmediumeddiium (Sigma-Aldrich,(S( igi ma-AAlddriichh, SStSt.. Louis,LoL uiis,, USA)USAA) supplementedsupplel mented with 10% FFCS,

2 mM L-Glutamine,lta taminemii 10010 0 nM M NorepineN Norepinephrineiin phrinehhriin and d PPePenicillin/Streptomycin Penicillin/Streptomniiciilliin/Str to ci in 22. CardiomyocytesCardiomCCarddiio oct

derived from human induced pluripotent stem cells (hiPS-CM) were generated and cultured as

previously described 23,24. Lipofectamin 2000 (Invitrogen, Carlsbad, USA) was used to transfect

the cells according to the manufacturer’s protocol.

Immunohistochemistry and fluorescence microscopy

Transfected cells were fixed with methanol (15 min, -20°C) and were then permeabilized with

0.1% Triton X 100 (20 min, RT). After blocking with 1% BSA/PBS the cells were incubated

with 7.5 μg/mL anti-desmin antibodies (R&D Systems, Minneapolis, USA) or 25 μg/mL anti-

antibodies (Sigma-Aldrich, Saint Louis, USA) over night at 4°C and were gently

washed with 1% BSA/PBS. Then the cells were incubated with Cy3-conjugated anti-goat-IgG or

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anti-mouse-IgG secondary antibodies (1:400, Jackson Immuno Research, West Grove, PA, USA)

for 1 h at RT. The nuclei were stained with 1 μg/mL 4',6-diamidino-2-phenylindole (DAPI, 5 min, RT). The cells were washed with PBS and fluorescence images were recorded with an

Eclipse TE2000-U microscope (Nikon, Tokyo, Japan) equipped with a Digital sight DS-2MV

CCD-camera (Nikon, Tokyo, Japan), YFP, Cy3 and DAPI filter sets (AHF, Tübingen, Germany)

and an oil immersion objective (Plan Apochromat 60x/1.40 Oil; Nikon, Tokyo, Japan).

The paraffin embedded sections (5 μm) were deparaffinized and rehydrated with a

standard technique using Xylene and Ethanol. The heart tissue was stainedained wwithith primaryprimaryy

antibodies over night at 4°C (see Table S2) and afterwards with secondarydary anantibodiestibbodies forfor oonen

hour at roomm temperature.temperatut ree.

Desmin expressionprresssion and pupurificationuriifficcationo

Bacteria (BL21-Star-DE3)BL211-StS ara -DDE3) transformedttransfformed withwithh desdessmiminin expressionexprressioon constructsconsttruccts (see(see TableTabblle S1)S1) werew

cultured in LB-LB-mediummedium ssupplementedupppllemented wiwithithh aampicillinmppiicili lil n (1((10000 μg/g/mL)./mLL).) TThehhe ddesminesmin expressionexprp ession was

induced withthh IsopropIsopropyl-I l ȕ-D1-thiogalactopyranosideD1 thiogalactopthi all to ranosidesiidde ( (IPTG(IPTG,IPTG 1 mM),mM)mMM) whenhenhhe theth h A 600 nm reachedreachh a

value of 0.6-0.8. After 4 h incubation at 37°C, bacteria were harvested by centrifugation and

were frozen at -80°C. The inclusion bodies were isolated as earlier described 12. Finally, the

proteins were dissolved (8 M Urea, 20 mM Tris-HCl, 100 mM NaH2PO4, pH 8.0) and supplied

to a HiTrap DEAE Sepharose Fast Flow column (GE Healthcare, Chalfont St Giles, UK) using

the Aktapurifier system (GE Healthcare, Chalfont St Giles, UK). Recombinant desmin was

eluted by a linear salt gradient (0-0.35 M NaCl), fractions were pooled and supplied to 5 mL

Ni2+-NTA (Qiagen, Hilden, Germany) over night at 4°C. The column was washed with buffer (8

M Urea, 20 mM Tris-HCl, 10 mM imidazole, pH 8.0) until A280 nm decreased to a constant value

below 0.01. Recombinant desmin molecules were eluted with imidazole containing buffer (8 M

8 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 DOI: 10.1161/CIRCGENETICS.113.000103

Urea, 20 mM Tris-HCl, 300 mM imidazole, pH 6.9). The fractions containing more than 95% recombinant desmin were pooled and stored at -80°C. The desmin concentration was determined

E\DEVRUSWLRQPHDVXUHPHQWDWQP İ 0-1 cm-1, www.expasy.org).

Proteolysis and mass spectrometry

The identity of purified desmin variants was proven by mass spectrometry. The desmin variants

(20 μg, 100 mM Tris-HCl, pH 8.5) were incubated with 0.08 mg DTT (30 min, 60°C) and were digested with trypsin or Lys-C (Sigma-Aldrich, St. Louis, USA) according to the manufacturer’s instructions. The lyophilized peptides were dissolved in 0.1% formic aacidcid anandd ididentifiedentified byb peptide mass fingerprinting using ESI-LC/MS and were confirmed by ESESI-LC/MS-MSI-LC/MS-MMS uususingin a micrOTOF-Q-Q hhybridybbrid massmam ss spectrospectrometermeter (Bruker, Bremen,Brremmen, GermGermanyGermany).any).).. A Jupiter 5u C18 (2.(2.0x150(2.00 mm, 300 Å)Å reversereeverse phasephase ccolumnolluumn waswwas usedusu ed forforr cchromatography.hromattoggrapphyy. TheThee sspectrometerpeecctromeeter wwasass rrununn in multiple reactionsactionss mmonitoringonittoriing modemode (20-25(20-25 eV)eVV) ffoforr MSMS-MS-MSM analysis.annalysis.s

Atomic forcece microscopymicroscoppy

After a stepwisep isei dialddialysisiial sissii intoiinto buffer b fferfffe withouti ithothh t urea rea ((5 5 mM M Tris-HCl,T Trisrii HClHCl 1 mM M DTT, DTTDTT pH H 8.4)8 4 4)) theth h filament formation of recombinant desmin was initiated by addition of an equal volume of sodium chloride buffer (200 mM NaCl, 45 mM Tris-HCl, pH 7.0) and subsequent heating to

37°C for 1 h as previously described 257KHDVVHPEOHGGHVPLQVSHFLHV ȝJP/ ZHUH applied to freshly cleaved mica substrates (Plano, Wetzlar, Germany), rinsed with deionized water to remove unbound desmin and dried under a gentle flow of nitrogen. Topographic AFM imaging was done with a Multimode AFM and Nanoscope IIIa controller (Bruker, Santa

Barbara, USA) as previously described 21.

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Results

Genetic analysis

We identified two unreported heterozygous sequence variants p.A120D (c.359C>A) and

p.H326R (c.977A>G) in the DES gene (Fig. 1C-D). Both variants were not present in 394

healthy control individuals. Furthermore, both variants were not present in publicly available

databases and in more than 12,500 alleles from the NHLBI Exome Sequencing Project (ref

http://evs.gs.washington.edu/EVS/).

The index patient (III:24) of family A (Fig. 1A) was tested for mutationsmutations inin ARVC-ARVC-

related genes DSG2, DSC2, JUP, PKP2, DSP, LMNA, TMEM43, PLNN aandndd iinn adadditionddition byby a gene

panel includingdinng LDLLDB3/ZASP,B3/Z/ ASSP, TNNT2TNNT2,, SGCD,SGCD, ACTC1,ACTC1,1 MYHMYH77,, TPMTPM1,1,, TTNNI3,NNI3, TAZTAZ,, TTR,TTR, MYBC3M

and LAMP22, whichwwhich representrepreseent ththehe mommostst frequentfreqqueent DDCDCMM gegeneenees. WWee identifiediidenntiifiedd in thethe indexinnddex patientppat of

family A (III:24)II:244) thethe PKP2PKP22-variant-variiant ((c.1577C>T,cc.1577C>TT, pp.T526M),.TT5226M)M , whwhichicch waswas earliereearllier ddedefinedfined as a non-

pathogenic sinsingleglg e nucleotidenucleotided ppolymorphismollymy orphisi m 26,2726,227. InI thetheh NHLBINHLLBII EExomexome SSequencingeqquencing PrProjectojject

(ESP) the PKP2PKP P2 variantariantrii t ((c(c.1577C>T, 1 1577C>T577C>TT pp.T526M)T5 T526M)26M) was as fofoundf ndd 4848x48 ini 12,958 12 958958 alleles llell (ref ( f

http://evs.gs.washington.edu/EVS/), which is by far above the expected prevalence of the

mutation if this was relevant for the disease. The Danish index patient (IV:1) of family B (Fig.

1B) has also been screened for mutations in above listed ARVC-genes without positive findings.

The p.A120D is part of the initial helix motive in coil 1 of the desmin protein and is absolutely conserved in different species (Fig. 2A-B). This mutation is localized in a b-position of the heptade sequence and therefore in close proximity to N116. The other sequence variant

p.H326R is localized at the heptade's f-position in coil 2 of the rod domain (Fig. 2B-C).

However, this amino acid is not completely conserved among IF-proteins (Fig. 2C) complicating its pathogenic interpretation.

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Filament or aggregate formation of the desmin variants in transfected cells

We investigated the filament formation of the different DES variants in transfected SW-13, HL-

1, H9c2, C2C12, HeLa and hiPS-CM. In these transfection experiments desmin p.H326R formed in all cell lines filaments comparable to the wild-type (Fig. 3). In contrast desmin-p.A120D

accumulated into cytoplasmic aggregates independently of the transfected cell line (Fig. 3).

We further investigated by immunohistochemistry if the endogenous IFs were impaired

by coexpression of the desmin mutants. These analysis revealed that the expression of desmin-

p.A120D influences the assembly of endogenous desmin in the musclee cellcell lineslines HL-1,HL-1, H9c2H9H c and

C2C12, indicating a dominant inhibiting defect (Fig. 4). Desmin and vimentinvimenttiin formform hetero-hheterro-

filaments, whenwheen cocoexpressedexprp esssed within the same cecellll 28. Therefore,ThT erefore, we ususedsede endogenously vivimentinm

expressing HeLaHeLLa cells to investigateinnvestigatet if thethe desmindesded smminminn mutantsmuutannts induceinducce a cocoaggregationoaaggregattioon withwwithh vvimentin.vimim

In contrast tto ddesmin-p.H326Resmmini -p.HH3226R andand -WT-WT thethe pp.A.AA12120D20D0 mutantmuttant inducediinduuced a papartiallyrtiallly coaggregationcoaggrega

with vimentinntin in transfectedtransfectedd HeLa-cells.HeLLa-celllls. Nevertheless,NNeverthhelless, thethhe vimentinviimentini networknetworkk was not stronglystrong

affected by thethh eexpression pressionsii off dde desmindesmin-p.A120D.iin p A120DA120D

In vitro desmin assembly using atomic force microscopy

To get better insights into the putative filament formation defects caused by these new desmin

variants, we purified the recombinant desmins by ion exchange and immobilized metal affinity

chromatography and analyzed the filament formation in vitro by AFM. In accordance with our

cell culture experiments we found that the variant p.H326R formed filaments similar to wild-type

desmin (Fig. 5). In contrast, desmin-p.A120D formed small accumulated fibrils (Fig. 5),

suggesting severe impairment of the filament elongation step by this mutation.

Investigation of different model mutants at position A120

Since it is not known, if the loss of the methylene group or the gain of the aspartate residue is

11 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 DOI: 10.1161/CIRCGENETICS.113.000103 causative for aggregation of desmin-p.A120D, we constructed different model variants

(p.A120E, p.A120K, p.A120R, p.A120V and p.A120L) to get more insights into the nature of the amino acid residue, which causes the filament formation defect of desmin-p.A120D. In transfected cells, the hydrophobic model mutants (p.A120V and p.A120L) formed filaments similar to wild-type desmin (Fig. 6). In contrast, the exchange of A120 against positive or negative amino acids induced desmin aggregation, with the exception of p.A120K (Fig. 6). In summary, these experiments reveal the essential role of a hydrophobic amino acid at position

120 for the filament formation.

Immunohistochemistry in cardiac tissue

Ventricularr mymyocardiumoco ardiumu ooff individual II:6 (family A)A was available ffromrom heart transplantatransplantationtransplantatt

(Mount Sinainaai Hospital,Hospital, LLososs Anngngeles,gellese , USUSA).SA). CoConfirmingnfn irming thethe inn vvitroittro rresultsessults ofof tthehe ccellelll cultureccultl u and

AFM experimentsriments wiwwithth ttheheh mmutauttanantt desmin-p.A120Ddesmin-pp.AA121 0DD we fofoundound a hihighighh ddensityeensiity ofof deddesminsmini aggregates within the ventricularventriicullar mymyocardium,yocardium, whwhichhiich was unundetectableddetectabble iinn ththehe ventricles of rejected donornor heartsh rt (F (Fig(Fig.iig 7) 7).

In addition, when the slices of the DES-p.A120D heart were costained for colocalization of desmin and desmoplakin could not be observed. Thus, desmin was detectable in the DES-p.A120D-heart of the mutation carrier in the z-bands and in prominent protein aggregates but not within the ID (Fig. 7).

Recently, remodeling for (JUP) 29 as well as connexin-43 (Cx43) 30 was described in AC patients. Therefore, we investigated if the localization of both proteins were affected in the patient with the DES-p.A120D mutation. These results demonstrate that plakoglobin as well as connexin-43 are localized in the ID similar like in healthy control persons

(Fig. S2-S3).

12 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 DOI: 10.1161/CIRCGENETICS.113.000103

Discussion

At the end of the 1990’s the first mutations in the human DES gene were published 3,4. It became obvious that mutations within the DES gene may lead to skeletal and/or cardiac myopathies with a broad spectrum of pathological muscle phenotypes even within the same family 31. Of note,

about 74% of the desminopathy patients develop a cardiac phenotype like conduction disease,

arrhythmias or cardiomyopathies 6. Nevertheless, the molecular pathomechanisms leading to

desmin related arrhythmogenic cardiomyopathies are not well understood.

In recent years, mutations of DES associated with an ARVC-relatedelated phenotypephenotype werewere found 10–15. In this study we identified and characterized the two novell heheterozygousterozygous DEDESS-

variants p.A120DA1220D aandndd pp.H326R..HH323 6R.

The variantvarriant p.A120Dp.A12200D iiss lolocalizedcallized withinwwitthinn thethhe hhighlyiig hlyhly conservedcoc nnsere vved IFIF-consensusF-ccoonsensn uss mmotifottiff atat the

N-terminal segmentsegmeent ofof cocoilill 1.1. TheThe amaminomino acacididi rresidueresiese idddueue A120A1220 is absolutelyabsooluttelly conservedcoonsn erveed ini ddiffedifferentiffe

human IF-proteinsproteins and amonamongg spspeciespeciei s 323 . EvenEvE en llaminamini ooff HydraHyddra attenuata ccontainsontains thithiss aminoamin

acid 33,34. Interestingly,nterestinglnte stiingll mutations m tationstatiio in i the thh homologous homologohho llo s positions iitiio of f thethh genes coding di for f kerakeratinsk a

K5 35,36, K10 37, K12 38, K86 39 and A/C 40 cause severe clinical diseases of eye, skin and muscle, respectively. The desmin variant p.A120D was not detectable in 788 control and revealed a severe filament formation defect in cell culture and AFM. These data were confirmed by immunostaining of the failing ventricular myocardium of the affected patient. Interestingly, the desmin-staining within the ID was undetectable in the mutation carrier.

It is known, that desmin is linked to the cardiac desmosome via the plaque protein desmoplakin

41. Based on yeast two hybrid analyses this protein interaction was claimed to be affected by the

desmin mutant p.I451M 41. However, the transgenic murine model of Mavroides et al. revealed

in contrast that this mutation affects the positioning of desmin to the z-bands but not in the

13 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 DOI: 10.1161/CIRCGENETICS.113.000103

intercalated disk 42. Thus, it remains an open question if a specific (sub-)domain of desmin is

linked to the ID via desmoplakin. The lack of desmin within the ID might explain the high prevalence of malignant arrhythmias in the affected family A. Of note, the pedigree of this family reveals a number of SCDs among teenaged family members, which might reflect the arrhythmogenic potential of desmin-p.A120D. Recently, remodeling of plakoglobin and connexin-43 were described in AC patients 29,30. Nevertheless, plakoglobin staining as well as

connexin-43 remodeling are controversially discussed in the literature since the absence could

not be detected in every patient 43–46. In this study, we demonstrate comparablemparable amounamountstss ofof

plakoglobin and connexin-43 within the ID in heart tissue of a healthyy controlcontrt oll personperson andannd oof a

patient withh tthehe DESDES-p.A120D-pp.AA121 0D mutation. Based onon thesetht eese exexperimentperimentss wew conclude that the

remodelingg ooff plplakoglobinakoglobiin andannd coconnexin-43nnn exin-43 dodoesese nnotoot playplaay a majormmajor rolerolle forfoor the ppathomechanismsaathomechhan

caused by thishis specificspeecific DESDED S mmutation.uttata ion.n

The mutation p.A120Dp.p A120D leadslleadds to an exchangeexchhangeg ofof a hyhydrophobicydrd opphoh bic amaminoini o acid side chaichainn

against an acidicciidi oneone. W We h hypothesized pothesithh i ed d tthat hhat a hhydrophobic ddr drophobichhobi amino iin acidid att positionsiitii 120120 isi esessential

for filament formation. For testing this hypothesis we constructed further model mutants with (i)

hydrophobic residues (p.A120L and p.A120V), (ii) negative (p.A120E) and (iii) positive charge

(p.A120R and p.A120K). Remarkably, ionic side chains at this position disturbed the filament

formation with the exception of the lysine residue. Whereas the experiments with hydrophobic

amino acid side chains reveal that even a larger mutant amino acid at this position does not

disturb filament formation. We conclude from these data that a steric hindrance of the side chain

can be excluded as a reason for the p.A120D filament formation defect. We assume that this

position within the desmin primary structure, which is in the neighborhood of the recently

published mutations p.N116S 12 and p.E114del 13, might be a hotspot for desminopathies

14 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 DOI: 10.1161/CIRCGENETICS.113.000103

associated with arrhythmias. It could be speculated that the intra-molecular interaction of this

part of the desmin coil 1 with its head domain, might be affected, which was recently shown for

vimentin 47,48.

In summary, although a cosegregation analysis was not possible in family A due to the

lack of genomic DNA from suddenly deceased members, we conclude from our experiments that

DES-p.A120D is indeed a disease causing mutation with a high potential for SCD.

The amino acid p.H326 is conserved among vertebrate desmins. However, arginine at this

position is also found in the human homologue vimentin. In addition thishis aallelellele was notnot fofoundu in

788 control chromosomes or in the Washington Exome Data. To assessss tthehe pathogenicpatthhogeniic popotentialte

of this variantant we iinvestigatednvestigated in vvitroitro the influeninfluencecee ooff p.H326R on ffilamentilaament formation anandd

performed a cocosegregationosegregatioon ananalysisnallysiss wwithinithih n tththehe ffafamily.mmilyy. WhWhenen testedtestet d inn ccellelll culturecultuuree experimentsexxperrimme

this recombinantbinantbinant desmindese min variantvariiant dididd notnoot revealreveeall aanyannyy filamentfilamennt formationformmattiion ddedefect,fect, whwhichici h waswas alaalso

supported byy AFM of the purifiedpup riifiedd recomrecombinantbiinant ddedesessmin.min.miin. Nevertheless,Neverthhelless,, we cannot exclude tthatthh

other relevantantnt f functions nctionsctiio of f desdde desminmin,iin likeli kke biomechanicalb biomechaniio chh icaliicall properties rtiie or protein-protein proteinteiin proteinteiin interaciinteractions,nt

i.e. with desmoplakin, might be disturbed by this variant. Of note, the variant did not completely

cosegregate within family B. In summary, we regard p.H326R as a rare variant of unknown

pathogenic significance.

Acknowledgements: We thank all participating patients. The authors are grateful to Birte Bohms for excellent technical assistance. Furthermore we thank Dr. Dawn Lombardo (University if Irvine) for her help in clinical investigations. In addition, we thank Dr. Bianca Werner (Heart and Diabetes Centre NRW, Germany) for providing the HeLa cells, Dr. Christine Mummery (Leiden University Medical Centre, Netherlands) for providing the END2 cells and Dr. William Claycomb (New Orleans, USA) for providing the HL-1 cells.

Funding Sources: H.M. was kindly funded by the Erich and Hanna Klessmann Foundation, Gütersloh, Germany and by FoRUM-grant (F649-2009) of the Ruhr-University Bochum. This work was also supported by the grant from the BMBF to T.Š. (grant no: 01 GN 0824). J.H.S. was

15 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 DOI: 10.1161/CIRCGENETICS.113.000103

funded by The Danish National Research Foundation, The John and Birthe Meyer Foundation, The Research Foundation of the Heart Centre, Copenhagen University Hospital and The Arvid Nilsson Foundation.

Conflict of Interest Disclosures: None.

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Figure Legends

Figure 1. Identification and verification of the DES-variants. Pedigrees of family A (A) and family B (B). Squares represent males and circles females. Deceased individuals are indicated by slashes. The index patients are marked by a red arrow. Genotypes are shown by present (+) or by absent (-) of the heterozygous DES mutations (p.A120D, family A; p.H326R family B).

Electropherograms showing the heterozygous alleles c.359C>A, p.A120D (C) and c.977A>G, p.H326R (D). Converted codons lead to the protein changes p.A120D (C) and p.H326R (D).

20 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 DOI: 10.1161/CIRCGENETICS.113.000103

Figure 2. Localization of the identified DES-mutations within the desmin sequence. (A)

Schematic domain structure of the desmin protein and the distribution of known disease causing mutations. Some DES mutations affecting the splice sites are not shown (e.g. 49,50). *This

sequence variants were also identified in healthy control persons 9. #p.K201RfsX20 and p.R429X

were both identified in the same patient 51. **p.A360P and p.N393I were both identified in the

same patients 3. ##In the same ARVC patient a pathogenic PKP2 mutation (p.T816RfsX10) was

identified 15. ***The mutation p.K240del was originally described as an insertion mutation 52.

However, the authors corrected the sequence analysis in 2007. ###Two ssiblingsiblings compoundcompounnd heterozygous for the mutations p.T76fsX21 and p.E108X were describedbed 53. (B, C)AAl Alignmentignnm of

the desmin/vimentin/vimmentin sequencessequueences of HomoHomo sapiens (hs),(hhs)s , MusMus mumusculussculus (mm),(mmm), RattusRattus norvegnorvegicusi

(rn), Xenopuspuus llalaevisevis (xl)(xl) aandnnd DaDarioario reriorreriio (d(dr)dr)). TheThT e hheheptadpptad ssequenceeqe uuencn e iss hhighlightedigghlh ightede iinn yeyellowellloww and

the positionsns off tthehe mmutationsutattiions iidentifiedddentifieed in tthishih s mammanuscriptnuscriiptt aarere mmarkedarrkked iin rred.ed.

Figure 3. Impairmentmpairmentmpairment ofof fifilamentlal ment formationformation bbyy dedesminsmin mutantsmuttantst inin transfectedtrt ansffectted cecells.lls.

Representative fluorescence images of transfected SW-13, H9c2, HL-1, C2C12 and HeLa cells

and hiPS-CM expressing desmin-eYFP constructs (yellow). hiPS-CM were identified by staining

for sarcomeric D- using AlexaFluor555-conjugated secondary antibodies (red). Nuclei

were stained with DAPI (blue). Scale bars represent 10 μm.

Figure 4. Endogenously expressed IF-proteins in cells transfected with desmin mutants.

Representative fluorescence images of transfected HL-1, H9c2, C2C12 and HeLa cells

expressing desmin-eYFP constructs (green), immunostained for desmin (HL-1, H9c2 and

C2C12) or vimentin (HeLa) using Cy3-conjugated secondary antibodies (red). The nuclei were

stained with DAPI (blue). Scale bars represent 10 μm.

21 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 DOI: 10.1161/CIRCGENETICS.113.000103

Figure 5. Filament formation of recombinant mutant desmin. Desmin was expressed in E. Coli,

purified, prepared on mica, and measured under ambient conditions in AFM tapping mode of operation . Representative AFM topography images of desmin-wt, -p.A120D and p.H326R are

VKRZQIURPOHIWWRULJKWLQ ȝP[ȝP $)0VFDQV WRSURZ DQG ȝP[ȝP $)0VFDQV

(bottom row) . Distinct filament structures could be discerned for desmin-wt and –p.H326R with

typical (averaged) dimensions of 500 nm (length), 30 nm (width) and 3-6 nm (height). The

apparent width of 30 nm is consistent with a real filament diameter of 8-10 nm that is broadened

by artifacts due to a finite AFM tip radius of ~ 20 nm. The reduced heightigght iiss aattributedttributed ttoo ssurfaceu

capillary force effects. In contrast, desmin-p.A120D exhibited completeete lolossss ooff fifilamentlal ment

structure, presentingresentingresenting moremom re globular structures with typicaltypip cal size of 85 nm.nmm. Representative AFMAAFF

topographyy imagesimmages are shshown.hoownn.

Figure 6. AAnalysisnalysis of modelmodel mummutantstaantn s forfor dedesmin-p.A120Dsmmin-pp.A1220D0 iinn differentdifff errene t cecelllll llines.ini es. Represen Representativet

fluorescencecee iimagesmages off ttransfectedransfecttedd SSWSW-13,W-13,-113, HH9c2,9c9 2, CC2C122C121 andand HeLaHeLa cellscells expressingexpressing indicatedindicaindica

mutant desmin-eYFP constructs (yellow). Nuclei were stained with DAPI (blue). Scale bars

represent 10 μm.

Figure 7. Immunohistological analysis of cardiac tissue heterozygous for the DES-p.A120D

mutation. (A-C) Representative fluorescence images of a control sample from a human non-

failing control heart, showing normal localization of desmin (red) and desmoplakin (green) at the

intercalated discs and at the z-bands. (D-F) Representative paraffin sections of cardiac tissue of

patient II:6 demonstrate strong accumulation of desmin (white arrowheads). Of note, the desmin

localization is completely lost at the ID. Scale bars represent 10 μm.

22 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 Downloaded from http://circgenetics.ahajournals.org/ by guest on November 9, 2013 SUPPLEMENTAL MATERIAL

Table S1: Designation of constructs and methods of cloning Primers (5’-3’): (1) agactcgaggccgtcaccatgagccaggcctactcgtccagcc; (2) acaggatccccgagcacttcatgctgctgctgtgtgg; (3) gctcaatgaccgcttcgacaactacatcgagaagg; (4) ccttctcgatgtagttgtcgaagcggtcattgagc; (5) gctcaatgaccgcttcgtcaactacatcgagaagg; (6) ccttctcgatgtagttgacgaagcggtcattgagc; (7) ggagctcaatgaccgcttcctaaactacatcgagaaggtgc; (8) gcaccttctcgatgtagtttaggaagcggtcattgagctcc; (9) agctcaatgaccgcttcgagaactacatcgagaaggtg; (10) caccttctcgatgtagttctcgaagcggtcattgagct; (11) ggagctcaatgaccgcttcaagaactacatcgagaaggtgc; (12) gcaccttctcgatgtagttcttgaagcggtcattgagctcc; (13) agctcaatgaccgcttccgcaactacatcgagaagg; (14) ccttctcgatgtagttgcggaagcggtcattgagct; (15) gatgatggaataccgacgccagatccagtcctcca; (16) tgtaggactggatctggcgtcggtattccatcatc; (17) caccatgagccaggcctactc; (18) ttagagcacttcatgctgctgct;

Construct No. Construct designation Source of cDNA/ method of cloning Restriction sides Primers

1 pEYFP-N1-Desmin PCR of pCMV6-AC-Desmin# / TOPO-TA XhoI, BamHI 1, 2

2 pEYFP-N1-Desmin-p.A120D SDM of pEYFP-N1-Desmin XhoI, BamHI 3, 4

3 pEYFP-N1-Desmin-p.A120V SDM of pEYFP-N1-Desmin XhoI, BamHI 5, 6

4 pEYFP-N1-Desmin-p.A120L SDM of pEYFP-N1-Desmin XhoI, BamHI 7, 8

5 pEYFP-N1-Desmin-p.A120E SDM of pEYFP-N1-Desmin XhoI, BamHI 9, 10

6 pEYFP-N1-Desmin-p.A120K SDM of pEYFP-N1-Desmin XhoI, BamHI 11, 12

7 pEYFP-N1-Desmin-p.A120R SDM of pEYFP-N1-Desmin XhoI, BamHI 13, 14

8 pEYFP-N1-Desmin-p.H326R SDM of pEYFP-N1-Desmin XhoI, BamHI 15, 16

10 pET100D-Desmin PCR of pCMV6-AC-Desmin# / TOPO-TA --- 17, 18

11 pET100D-Desmin-p.A120D SDM of pET100D-Desmin --- 3, 4

12 pET100D-Desmin-p.H326R SDM of pET100D-Desmin --- 15, 16

# Origene Technologies, Rockville, USA Table S2: Overview about the antibodies used for IHC

Antibody Manufacturer Specification Used concentration (over night, 4°C)

anti Desmin R&D Systems Inc. (Minneapolis, MN, USA) AF3844, polyclonal goat IgG 1:40 (25 ng/µL)

anti Desmoplakin Acris Antibodies GmbH (Herford, Germany) AM09122SU-N, monoclonal mouse IgG2b undiluted

anti Plakoglobin (JUP) Acris Antibodies GmbH (Herford, Germany) BM5100, monoclonal mouse IgG2b 1:5 (10 ng/µL)

anti Connexin-43 Abcam (Cambridge, UK) AB11370, polyclonal rabbit IgG 1:500 (1.22 ng/µL)

Supplemental Figure Legends:

Figure S1. Electrocardiogram (ECG) of patient III:24 (family A) with DES-p.A120D.

The atrial-ventricular conduction is variable (2:1 and 3:1 conduction). Atrial heart rate is approximately 120 bpm. The ventricular heart rate is approximately 64 bpm. Left axis deviation. Left anterior hemiblock is present with slow progression of R wave and pronounced S in the cheast leads. No premature ventricular beats (but polymorphic forms of

PVB in 24 h ECG). QRS duration 91 msec. QT/QTc in the normal range.

Figure S2. Immunohistological plakoglobin staining of cardiac tissue heterozygous for the DES-p.A120D mutation.

(A) Representative IHC analysis of paraffin sections of cardiac tissue of patient II:6 demonstrate no reduced expression of plakoglobin at the intercalated disc. (B) Representative fluorescence images of a control sample from a human non-failing control heart. Plakoglobin was stained with FITC-conjugated secondary antibodies (green). The nuclei were stained with

DAPI (blue). Scale bars represent 100 µm.

Figure S3. Immunohistological connexin-43 staining of cardiac tissue heterozygous for the DES-p.A120D mutation. (A) Representative IHC analysis of paraffin sections of cardiac tissue of patient II:6 exclude a severe remodeling of connexin-43 at the intercalated disc. (B)

Representative fluorescence images of a control sample from a human non-failing control heart. Connexin-43 was stained with Cy3-conjugated secondary antibodies (red). The nuclei were stained with DAPI (blue). Scale bars represent 100 µm.

Figure S1

Figure S2

Figure S3