Genetics and pathophysiology of ARVC AJ Marian, M.D. Center for - PowerPoint PPT Presentation

8 th International SADS Foundation Conference: Preventing Unexpected Sudden Death in the Young Jointly Provided by: NYU Post Graduate Medical School (NYU ‐ PGMS) And Sudden Arrhythmia Death Syndromes Foundation (SADS) Genetics and pathophysiology of ARVC AJ Marian, M.D. Center for Cardiovascular Genetics B rown ¡ F oundation ¡ I nstitute ¡of ¡ M olecular ¡ M edicine ¡ for ¡ the ¡ P revention ¡ of ¡H uman ¡ D iseases ¡ Center ¡for ¡Cardiovascular ¡Genetics ¡

Rare Variant-Rare Disease

Arrhythmogenic ¡Cardiomyopathy ¡(AC) ¡ Normal AC

Causes ¡of ¡SCD ¡in ¡young ¡athletes ¡ U.S.A. Italy HCM HCM (46%) ARVC (22%) HCM ARVC DCM ARVC Mitral valve prolapse CCAA CAD Others Corrado D. N Engl J Med 1998 Maron BJ. JAMA 1996

The ¡Enigma ¡of ¡ARVC ¡ H&E ¡ Masson ¡trichrome ¡

The Island that Solved the Riddle of AC Naxos Island Dionysos, the God of vines and wine, who was born from a thigh of his father Jupiter (parthenogenesis ) , sits resting on a rock, his leg on the thigh of Ariadne, the daughter of Minos, a pose symbolic of ‘ sacred marriage ’ .(Bronze krater from Derveni, 320–300 BC).

Naxos Disease D Coonar AS Circulation. 1998

A Truncated Mutation in Junction Plakoglobin ( JUP ) Causes Naxos Disease McKoy G et al. The Lancet 2002

Known Causal Genes for ARVC Locus Gene Symbol Gene Prevalence 12p11 PKP2 Plakophilin 2 ~25% 18q12 DSG2 Desmoglein 2 ~10-15% 18q12 DSC2 Desmocollin 2 ~10-15% 6p24 DSP1 Desmoplakin <5% 17q21 JUP Plakoglobin Uncommon 3p23 TMEM43 Transmembrane protein 43 Rare 1q21.2 Lamin A/C Rare LMNA ¡ 14q24 TGFB3 ? Transforming growth factor β 3 Rare 6p22.1 Phospholamban Rare PLN? ¡ 11p15.5 Iks channels Rare KCNQ1? ¡

Desmosome Adherens junction (AJ) Gap junction (GJ) G C S Canonical Wnt S D CHD2 D Wnt GJA1 LRP5/6 PKP2 PKP2 PKP2 PKP2 β-cat Dvl JUP JUP β-cat DSP

Pathogenesis ¡of ¡ARVC ¡ ?

Human Heart Human Heart Molecular Remodeling Normal AC Normal AC 1 2 1 2 3 4 kD Of IDs in Human AC MT 100 JUP 75 JUP PKP2 100 75 DSC2 PKP2 JUP PKP2 DSC2 150 1.0 1.0 1.0 Fold change 0.8 0.8 0.8 0.6 0.6 0.6 DSG2 150 0.4 0.4 0.4 DSC2 *** *** 0.2 0.2 0.2 *** * 0.0 0.0 0.0 DSP 250 DSG2 DSP GJA1 1.0 1.0 1.0 DSG2 Fold change 0.8 0.8 0.8 * 0.6 0.6 ** 0.6 GAPDH 50 0.4 0.4 0.4 37 0.2 0.2 0.2 *** DSP 0.0 0.0 0.0 GJA1 50 AC Normal 37 GJA1 CDH2 150 100 50 GAPDH CDH2 37

Transcriptome ¡in ¡Human ¡AC ¡ Differentially expressed genes (FDR0.05) Human Heart Normal AC 1 2 3 5 1 2 3 Canonical Wnt signaling Human Heart Normal AC 1 2 3 5 1 2 3 Wnt9a Prickle1 Sfrp1 Glul Gadd45g Dixdc1 Jun Atm Ddx17 Akr1c3 Myh6 Ndnf Lrrc32 Ndrg2 Serpina3 Lmna Eif5a Ngfr Orc4

Plakophilin 2 (PKP2) knock down (KD) HL-1 Myocyte and Mouse Models Conditional Knockdown of PKP2 in mouse heart

YAP-TEAD Transcription in HL-1 Pkp2shRNA Cells RNA-Seq 15 HL-1 Pkp2-shRNA HL-1 WT Transcript copy number 1 2 1 -Log10 (q value) Ccnd1 Ctgf 10 20 4 Transcripts/Million Plac8 Inhba 15 3 Runx1 Serpine1 10 2 Dusp6 5 Runx1t1 * Adamts1 5 1 * q<0.05 Jak1 * ** * * * * ** Plk2 0 0 Adamts1 0 Runx1 Inhba Cyr61 Jun Ccnd1 Bmp4 Runxt1 Serpine1 Ctgf Plac8 -4 -2 0 2 4 Myocd Itgb2 Log2 (Fold change) Cenpa Slc2a4 Irs1 Smad1 TEAD-Luciferase 1.0 Eomes qPCR Bmp4 (Normalized Relative Ratio) Cav2 activity Sema3c ** Ankrd1 Ctgf Cyr61 Ccdn1 Inhba Ankrd1 Amotl2 1.2 1.2 1.2 1.2 0.5 1.2 Cav1 Nav3 0.8 0.8 0.8 0.8 0.8 Prss23 * *** Lcp1 *** *** 0.4 0.4 0.4 0.4 Cyr61 0.4 0.0 Cyp1b1 *** HL-1WT Adamts5 0.0 0.0 0.0 0.0 0.0 Myof HL-1Pkp2:shRNA −1 1 Row Z − Score

Row max Row min Differentially Expressed miRNAs, miRNA:mRNA Pathway Analysis PKP2 A C D HL-1 PKP2:shRNA P=0.03(N=5) : e P=0.04(N=3) 1.5 l b U6 cPPT hPGK Puro m Puro : 1 : 2 2 2 A a A A p p Fold Change r N s k N k N c B l R P P l S R R e h h h 1.0 C s s s P<0.001(N=5) Normalized Relative 100 1.5 P<0.001(N=3) PKP2 Pkp2 mRNA 0.5 75 1.0 0.0 TUBA1B 50 e Cells : : 0.5 l 2 2 b 1 2 p p m A A A k k a N N N P P r R R c R S h h h 0.0 s s s e : : Cells l 2 2 b 1 2 p p m A A k k A a P N P N N r R R c R S h h h s s F Fold s HL-1Pkp2:shRNA HL-1 Change E miR-184 -15.92 miR-881 -8.06 3 miR-574-3p -5.49 miR-465b-5p -5.35 miR-184 miR-100 -4.34 miR-470 -4.08 miR-671-3p -4.01 miR-10a -3.75 miR-741 -2.90 -log (p value) miR-34b-3p -2.76 2 miR-23b -2.74 miR-99a -2.71 miR-196b -2.69 miR-1939 -2.15 miR-196a -2.12 miR-338-5P -2.09 miR-1198 2.51 miR-133a 2.67 1 miR-200a 2.68 miR-351 2.71 miR-20b 2.71 miR-132 2.73 miR-342-3p 2.74 miR-362-5p 3.80 miR-1 4.06 miR-676 5.48 0 miR-429 5.53 -4 -2 0 2 4 miR-188-5p 6.69 miR-487b 7.39 log2 (fold change) miR-200b 7.79 G P<0.001 (N=14) 1.5 H I P<0.001 (N=4) Normalized Relative P<0.0001 (N=3) P<0.0001 (N=3) Normalized Relative Normalized Relative 1.0 miR-184 miR-200b miR-429 0.5 0.0 : e : : Cells 2 2 l 1 2 b p p A A m k k A N N P P a N R R r R c h h HL-1 Pkp2:shRNA HL-1 HL-1 Pkp2:shRNA HL-1 S h s s s

The Pathway(s) From PKP2 of Desmosomes to YAP/TEAD of the Hippo Pathway

Inactivation of YAP by Phosphorylation in AC: HL-1 Pkp2:shRNA Myocytes

Inactivation of YAP by Phosphorylation in AC: Mouse Models

Inactivation of YAP by Phosphorylation in AC: Human Hearts

Activation of The Hippo Pathway in AC: HL-1 Pkp2:shRNA Myocytes

Activation of The Hippo Pathway in AC: Mouse Models

Activation of The Hippo Pathway in AC: Human Heart

PKC α as a link between PKP2 and the Hippo Pathway Intercalated Intercalated Discs Discs Active Active Active Active Inactive TEAD Hippo

Localization and Level of pPKC α in AC: HL-1 Pkp2:shRNA Myocytes HL-1WT HL-1Pkp2:shRNA HL-1 Pkp2:shRNA WT kD PKP2 PKP2 PKP2 75 pPKC α 75 pPKC- α pPKC- α pPKC- α TUBA1B 50 Overlay Overlay

Localization and pPKC α Level in AC: Mouse Models : 5 . p 2 u x F J k : / 6 N Mouse models of AC W G G h T p T y N s M N D NTG Myh6:Jup Nkx2.5:DspW/F kD pPKC α 75 TUBA1B PKP2 50 PKP2 PKP2 PKP2 Mouse pPKC- α 1.2 1.2 p=0.003 p=0.002 1.0 1.0 (N=2) pPKC- α pPKC- α pPKC- α Fold change Fold change (N=2) 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 Overlay Overlay Overlay 0.0 0.0 NTG Myh6:Jup NTG Nkx2.5:DspW/F

Localization and Level of pPKC α in AC: Human Hearts Human Heart Normal AC Normal AC 1 2 1 2 3 4 kD pPKC α 75 50 GAPDH 37 PKP2 PKP2 Human pPKC- α 1.2 p=0.007 1.0 (N=2-4) Fold change pPKC- α pPKC- α 0.8 * 0.6 0.4 0.2 0.0 Normal AC Overlay Overlay

The Hippo Pathway Intercalated Intercalated Discs Discs Active Active Active Active Inactive TEAD Hippo

Suppression of the Canonical Wnt Signaling in the HL-1 Pkp2:shRNA Myocytes: Transcriptional Activity HL-1WT HL-1Pkp2:shRNA HL-1WT HL-1Pkp2:shRNA 1 2 1 2 Myc Bves Ctgf c-Jun Snai1 Ccnd1 Ccnd1 Sox2 Normalized Relative 1.2 1.2 1.2 1. 2 1. 2 1.2 Jun Ccnd1 mRNA Dvl3 0.8 0. 8 0.8 0.8 0. 8 0.8 *** *** *** Ppard *** LRP5 0.4 0. 4 0.4 0.4 0.4 0. 4 *** Tgfb3 *** Akt3 0. 0 0.0 0.0 0. 0 0.0 0.0 Rarb Gsk3b Fzd2 TCF-Luciferase Reporter Assay Wif1 1.2 Sfrp5 Relative luciferase Nlk Tle4 * 0.8 activity Ppp2r3a Tle1 0.4 Csnk2a1 Sox12 Wnt11 0.0 Wnt5b HL-1Pkp2:shRNA HL-1 WT Tab1 Csnk1e Row Z − Score Color Key 1 0 -1

Suppression of the Canonical Wnt Signaling in AC: p β -Catenin Localization and Levels in the HL-1 Pkp2:shRNA Myocytes HL-1 Pkp2:shRNA WT kD HL-1Pkp2:shRNA HL-1Pkp2:shRNA HL-1WT HL-1WT 100 pCTNNB1 CTNNB1 CTNNB1 pCTNNB1 pCTNNB1 100 CTNNB1 GSK3B 50 CTNNB1:DAPI CTNNB1:DAPI pCTNNB1:DAPI pCTNNB1:DAPI AXIN1 100 TUBA1B 50 100 pCTNNB1 Phos-Tag

Suppression of the Canonical Wnt Signaling in AC: p β -Catenin Localization and Levels in the Mouse Models NTG Myh6:Jup (PG) Nkx2.5:DspW/F Nkx2.5: Myh6:Jup pCTNNB1:DAPI DspW/F NTG NTG kD 100 pCTNNB1 100 CTNNB1:DAPI CTNNB1 50 TUBA1 pCTNNB1:DAPI pCTNNB1 CTNNB1 8.0 5.5 2.5 1.2 *** * 1.0 2.0 Fold Change Fold Change 6.0 0.8 CTNNB1:DAPI 3.5 1.5 4.0 0.6 * 1.0 0.4 1.5 2.0 0.5 0.2 0.0 0.0 0.0 0.0 NTG Myh6:Jup Nkx2.5:DspW/F

Suppression of the Canonical Wnt Signaling in AC: p β -Catenin Localization and Levels in the Human Hearts Normal AC 1 2 3 4 1 2 3 4 CTNNB1 pCTNNB1 kD 2.5 2.0 pCTNNB1 100 * 2.0 1.5 Fold Change Fold Change 1.5 1.0 100 CTNNB1 1.0 0.5 0.5 0.0 0.0 GAPDH Normal AC Normal AC 37 Normal AC pCTNNB1:DAPI CTNNB1:DAPI

Hippo/Wnt Interactions

Shared Origin of JUP (Plakoglobin, γ -Catenin) and β -Catenin Through Gene Duplication β-­‑catenin ¡ Plakoglobin ¡ (JUP) ¡ TCF7L2 Amino ¡acid ¡sequence ¡iden;ty ¡= ¡88% ¡