Abstract
Arrhythmogenic cardiomyopathy (ACM) is an inherited progressive cardiac disease. Many patients with ACM harbor mutations in desmosomal genes, predominantly in plakophilin-2 (PKP2). Although the genetic basis of ACM is well characterized, the underlying disease-driving mechanisms remain unresolved. Explanted hearts from patients with ACM had less PKP2 compared with healthy hearts, which correlated with reduced expression of desmosomal and adherens junction (AJ) proteins. These proteins were also disorganized in areas of fibrotic remodeling. In vitro data from human-induced pluripotent stem cell-derived cardiomyocytes and microtissues carrying the heterozygous PKP2 c.2013delC pathogenic mutation also displayed impaired contractility. Knockin mice carrying the equivalent heterozygous Pkp2 c.1755delA mutation recapitulated changes in desmosomal and AJ proteins and displayed cardiac dysfunction and fibrosis with age. Global proteomics analysis of 4-month-old heterozygous Pkp2 c.1755delA hearts indicated involvement of the ubiquitin-proteasome system (UPS) in ACM pathogenesis. Inhibition of the UPS in mutant mice increased area composita proteins and improved calcium dynamics in isolated cardiomyocytes. Additional proteomics analyses identified lysine ubiquitination sites on the desmosomal proteins, which were more ubiquitinated in mutant mice. In summary, we show that a plakophilin-2 mutation can lead to decreased desmosomal and AJ protein expression through a UPS-dependent mechanism, which preceded cardiac remodeling. These findings suggest that targeting protein degradation and improving desmosomal protein stability may be a potential therapeutic strategy for the treatment of ACM.
| Original language | English |
|---|---|
| Article number | eadd4248 |
| Journal | Science Translational Medicine |
| Volume | 15 |
| Issue number | 688 |
| DOIs | |
| Publication status | Published - 22 Mar 2023 |
Bibliographical note
Funding: This work was supported by the Dutch CardioVascularAlliance with support of the Dutch Heart Foundation, DCVA2017-18 ARENA-PRIME (E.v.R.);
Dutch CardioVascular Alliance with support of the Dutch Heart Foundation, DCVA2015-12
eDETECT (T.A.B.v.V. and C.A.R); Dutch CardioVascular Alliance with support of the Dutch Heart
Foundation, DCVA2018-30 PREDICT2 (T.A.B.v.V. and C.A.R.); Fondation Leducq Transatlantic
Network of Excellence, 17CVD02 (C.A.R.); Vici grant from the Dutch Research Council (NWO),
project 09150181910020 (E.v.R.); Gravitation Program “Materials-Driven Regeneration” funded
by the Netherlands Organization for Scientific Research, 024.003.013 (C.L.M.); Netherlands
Organisation for Health Research and Development (ZonMW), MKMD project 114022504
(C.L.M.); European Research Council, ERC-CoG Mini-HEART 101001746 (M.B.); and EU’s H2020
research and innovation programme under Marie Skłodowska-Curie cofund RESCUE grant,
801540 (E.v.R.).
Funding Information:
We thank F. A. Moqadam and A. R. Leitoguinho for assisting in the design and selection of the gRNAs. This work was supported by the Dutch CardioVascular Alliance with support of the Dutch Heart Foundation, DCVA2017-18 ARENA-PRIME (E.v.R.); Dutch CardioVascular Alliance with support of the Dutch Heart Foundation, DCVA2015-12 eDETECT (T.A.B.v.V. and C.A.R); Dutch CardioVascular Alliance with support of the Dutch Heart Foundation, DCVA2018-30 PREDICT2 (T.A.B.v.V. and C.A.R.); Fondation Leducq Transatlantic Network of Excellence, 17CVD02 (C.A.R.); Vici grant from the Dutch Research Council (NWO), project 09150181910020 (E.v.R.); Gravitation Program “Materials-Driven Regeneration” funded by the Netherlands Organization for Scientific Research, 024.003.013 (C.L.M.); Netherlands Organisation for Health Research and Development (ZonMW), MKMD project 114022504 (C.L.M.); European Research Council, ERC-CoG Mini-HEART 101001746 (M.B.); and EU’s H2020 research and innovation programme under Marie Skłodowska-Curie cofund RESCUE grant, 801540 (E.v.R.).
Funding Information:
Acknowledgments:W ethankF .A.MoqadamandA.R.Leitoguinhoforassistinginthedesign andselectionofthegRNAs.Funding:ThisworkwassupportedbytheDutchCardioVascular AlliancewithsupportoftheDutchHeartFoundation,DCV A2017-18 ARENA-PRIME(E.v.R.); DutchCardioVascularAlliancewithsupportoftheDutchHeartFoundation,DCV A2015-12 eDETECT(T .A.B.v.V .andC.A.R);DutchCardioVascularAlliancewithsupportoftheDutchHeart Foundation, DCV A2018-30 PREDICT2 (T .A.B.v.V . and C.A.R.); Fondation Leducq Transatlantic NetworkofExcellence,17CVD02(C.A.R.);VicigrantfromtheDutchResearchCouncil(NWO), project09150181910020(E.v.R.);GravitationProgr am “Materials-DrivenRegeneration”funded bytheNetherlandsOrganizationforScientificResearch,024.003.013(C.L.M.);Netherlands OrganisationforHealthResearchandDevelopment(ZonMW),MKMDproject114022504 (C.L.M.); European Research Council, ERC-CoG Mini-HEART 101001746 (M.B.); and EU’s H2020 research and innovation progr amme under Marie Skłodowska-Curie cofund RESCUE grant, 801540(E.v.R.).Authorcontributions:H.T .performedallmolecularbiologyexperiments related to the animal studies and human studies and maintained the mouse colony. S.J.v.K. performedtheCRISPR-Cas9targetingexperimentsfortheanimalmodelandthehiPSCmodel. S.J.v.K.,S.J.H.,andJ.M.-K.maintainedthehiPSClinesandperformedtheexperimentsonhiPSC-CMs.V .M., M.B.,andC.L.M.wereinvolvedwithMTexperiments.W .B.v.H., S.C.,A.B.,G.A.M.,C.A.R., andT .A.B.v.V .wereinvolvedwithelectrophysiologyexperiments.A.V .andP .v.d.K. supplied patienttissuesamplesandperformedhistologicalstainingonhumansamples.M.M.andX.Y . performedtheglobalproteomicsanalysis.J.E.performedthebulkRNAsequencinganalyses. D.V .performedtheechocardiologymeasurementsonanimalsandmaintainedthemouse colony.L.W .v.L. andJ.G.provideduswithhumanclinicaldata.S.J.K.andG.J.P .L.K. performedthe karyotypingexperiments.H.d.R.performedthemolecularexperiments.J.A.A.D.andK.B. performedtheubiquitinproteomicsanalysis.H.T ., S.J.v.K.,andE.v.R.plannedallexperiments andwrotethemanuscript.Competinginterests:E.v.R.isaconsultantforT ena yaTherapeutics andNovoNordiskandisChiefScientificOfficerofPhloxTherapeutics.Allotherauthorsdeclare thattheyhavenocompetinginterests.Dataandmaterialsavailability:Alldataassociated withthisstudyarepresentinthepaperortheSupplementaryMaterials.ThebulkRNA sequencingdatasetshavebeendepositedwiththeGeneExpressionOmnibusrepository(GEO) underaccessionnumbersGSE199925(animalstudy)andGSE160289(hiPSC-CMstudy).The massspectrometryproteomicsdatasetshav ebeendepositedintheProteomeXchange ConsortiumviathePRIDEpartnerrepositorywiththedatasetidentifierPXD020605(global proteomics analysis) and PXD032964 (ubiquitination diglycine remnant containing peptides proteomicsanalysis).Allsubmitteddatasetsarelistedundertheprojectname“Desmosomal proteindegradationasunderlyingcauseofarrhythmogeniccardiomyopathy.”ThehiPSClines areavailablefromM.B.bymaterialtransferagreementwithLeidenUniversityMedicalCenter. Additionaldetailsandmaterialscanbemadeavailablefromthecorrespondingauthorson reasonable request.
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