DOI: 10.1161/CIRCGENETICS.113.000103 The Novel Desmin 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; ViVVirginiairginia KKimonis,imonis, MMDD3; HendrikHendndriikk MMilting,iillting, PhDPhhDD1 1Erich &H Hannaannna KlKlessmannessmann InInstnst ffo forr CaCardiovascularardr iovasccullarr RReseesearcheearch & 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 CCA;A; 4DivisionDiD visiion ofof 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 DanishDanish 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 intermediate filament protein desmin is encoded by the gene 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 withwiith 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 cytoplasmcytoplascytoplasmicm 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 anyany 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- proteins 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 DESDEES-mutations-mutations disdistributtribbuuted ooverver ththee wholewhole sequence areree known,known, whwhichiich leadleead iinn tththee mammajorityjo rityrrityy ofof cacasesc sessses too filamentfillammeent formationfformationrmation 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 genes coding for desmosomal plaque proteins cause ARVC 17–19 and rare forms of dilated cardiomyopathy (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 titientennt (III:24)(IIII::24) presentedp resentedressenntedd wwithitth atrialattrir 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