Molecular Pathogenesis of Arrhythmogenic Right Ventricular Cardiomyopathy

Ed Garcia‐Graz, Ph.D.Raffaella Lombardi, M.D., Ph.D.

AJ Marian, M.D.

Center for Cardiovascular GeneticsBrown Foundation Institute of Molecular Medicine

The University of Texas Health Science Centerand Texas Heart Institute, Houston, TX

Brown Foundation Institute of Molecular Medicine

for the Prevention of Human Diseases

Center for Cardiovascular Genetics

D

A

CE

BHCM DCM

ARVC

Cardiac Myocyte

Lemola, K et al. Heart 2005;91:1167-1172

ARVC: Survival

Dionysos, the God of vines and wine, who was born from a thigh of his father Jupiter, 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 Island

Naxos Disease

Norgett EE. Et al. Human Mol. Genet. 2000;9:2761-2766 Yannis Protonotarios Foundation, Medical Center of Naxos, Naxos 84300, Greece

Naxos Disease: Plakoglobin (JUP)

McKoy G et al. The Lancet 2002

DSP

(6‐16%)

DSG2(10‐12%)

PG

(Rare)

PKP2

(11‐43%)

DSC2 (1‐5%)

ARVC is a Disease of Desmosomal Proteins 

Modified from Dusek et al. J Derm Sci 2007

Pathogenesis of ARVC

?

The Enigma of Reverse Cardiac Alchemy

NH2 COOH

Armadillo Repeat ArmsAmino acid sequence identity = 88%

NH2 COOH

Plakoglobin Tcf/Lef‐1 interaction domain

β‐catenin

Plakoglobin is also Known as γ‐Catenin

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6 7 8 9 10 11 12 131 745

1 781

Competitive Inhibition of Canonical Wnt/β‐cateninSignaling by Nuclear Plakoglobin

Wnt

Frz

Nucleus

Tcf7l2/Lef1

β Catenin

β Catenin

PG

PG

Free PG

Desmosome

Targeted Disruption of Desmosomes Assembly

Modified from Nature Reviews Molecular Cell Biology 1; 208‐216 2000

DSP

PG

PKP2

Stable Suppression of Expression of DP in HL‐1 Cells Using Sequence‐Specific siRNAs

Nuclear localization of PG in DP-deficient HL-1 Cells

Control HL-1 cells DP-deficient HL-1 cells

PG:DAPI PG:DAPI

ControlsiRNADP‐1102

siRNADP‐1102Control

Cytosolic Nuclear

PG

β‐Cat.

α‐Tub.

0

5

10

15

20

25

F=48.7, p<0.001* p<0.001

* **

*

*

*

FOP FOP FOP FOPTOP TOP TOP TOPControl GFP siRNA DP siRNA-799 DP siRNA-1102

Fold

Cha

nge

Suppression of The Canonical Wnt/β-Catenin SignalingThrough Tcf/Lef1 Transcription Factor: TOP-Flash Assay

Transcription Switch To Adipogenesis in DP-Deficient HL-1 Cells

Con

trol

DP

siR

NA

110

2Enhanced Expression and Nuclear Localization of

PPAR-γ in DP-Deficient HL-1 Cells

PPAR-γ::DAPIPPAR-γ DAPI

PPAR-γ::DAPIPPAR-γ DAPI

Untreated Dex + Ins + IBMX

Con

trol

DP

siR

NA

799

Higher Magnification

Suppression of Expression of DPAccumulation of Fat Droplets in HL-1 Cells

PKP-2

α-Tubulin

Scramble sh

RNA

Control C

ells

PKP2-49-11

stable

line

PKP2-51 (le

nti-siRNA)

100

75

50

Suppression of Expression of PKP2:Accumulation of Fat Droplets in HL-1 Cells

PKP2-49-11 stable line PKP-51 (lenti-siRNA)

Control Cells Scramble shRNA

Floxed DspB BExon 2

LoxP LoxP

5’ 3’

Cre‐deleter mice Nkx2.5 Mef2Cα‐MyHC

X

/

Targeted Deletion of Dsp in the Heart 

Recombined DspB B

Cre Recombinase

ATG

A

B

C

Courtesy of MD Schneider, R. Schwartz, , B. Black

/

A

B

Cre‐Mediated Deletion of Dsp Gene

Dsp+/+

Survival Rate

Log Rank Chi Sq =73.2p<0.0001

Dsp+/+Dsp+/-

Non-Transgenic

Nkx2.5-Cre:DspW/F Mef2C-Cre:DspW/F

α-MyHC-Cre:DspW/F

Cardiac Dysfunction in Cardiac‐Restricted DP‐Deficient Mice

Ventricular Tachycardia in DP‐Deficient Mice

Wild

type

N

kx-C

re:

Dsp

W/F

Mef

2C-C

re:

Dsp

W/F

MTOROαM

yHC

-Cre

:D

spW

/F

H&E GWA‐TR

Increased Nuclear Localization of Plakoglobin and Enhanced Adipogenesis in the Heart in DP-Deficient Mice

Dsp+/+ Dsp+/- Dsp+/+ Dsp+/-

Cytosolic Nuclear

Plakoglobin

α-Tubulin

Dsp+/+ Dsp+/- Dsp-/-

C/EBP-α

Adiponectin

α-Tubulin

ORO H&E

MT IF:PG (pan-specific)

Phenotype in PG Transgenic Mice

PG cDNAα-MyHC 3’ UT BGH

ATG

Flag

A

100

75

B NTG PG Lines

100

75

Flag

Pan PG

Tubulin50

1 2 1 2 kDa

C

B Nucleus Cytosol Membrane

PGNTG PGNTG PGNTG

Anti-Pan PG ab

α-Tubulin

100

75

50

Anti-Flag ab

100

75

kDa

PG (Flag) PG (Flag):DNADNAA

Nuclear Localization of PG In Transgenic Mice Hearts

Wnt

Frz

Nucleus

Tcf7l2/Lef1

β Catenin

β Catenin

PG

PG

Free PG

Desmosome

NTG PG

IP:Tcf7l2IB:Flag-PG

IP:Tcf7l2IB:Pan-PG

100

75100

75

kDa

IP:Tcf7l2IB:β-catenin 100

Competitive Binding of Nuclear PG and β‐Catenin to Tcf7l2

P<0.001

cMycNTG PGRe

lativ

e Expression

 Levels

Relativ

e Expression

 Levels P<0.001

Wnt5bNTG PG

P<0.001

Relativ

e Expression

 Levels

CTGFNTG PG

Transcriptional Switch to Adipogenesis

P<0.001

BMP‐7

Relativ

e Expression

 Levels

NTG PG

Akio Kanazaw et al BBRC 2005 F.H.J. van Tienen et al. BBRC 2009

Wnt5b Suppressed canonical Wnt Signaling and enhances adipogenesis

The Enigma of ARVC: Cellular Origin of Adipocytes in ARVC

H&E Masson trichrome

Potential Cell Source of Adipocytes in Desmosomal ARVC

Cardiac Cells expressingMutant desmosomal proteins Extra‐Cardiac cells

Bone marrow derivedcirculating cells

FibroblastsMyofibroblasts

Smooth muscle cellsEndothelial cells

Other stromal cells

MatureMyocyte

Express desmosomal proteinsDo not express desmosomal proteins

Cardiac Cells

Paracrine Mechanisms

Primary Hypothesis Alternative Hypothesis

CardiacProgenitors

AutocrineMechanisms

Lineage Specific Promoters

X

Stop3’5’

EYFPB BExon 2

LoxP LoxP

Floxed DspB BExon 2

LoxP LoxP

5’ 3’Cre‐deleter mice

Floxed Dsp & EYFP

LoxP LoxP

ROSA26

Stop3’5’

ROSA26

LoxP LoxP

EYFP

Floxed EYFP

Nkx2.5 Mef2Cα‐MyHC

X

Lineage Tracing to Map The Cell Origin of Adipocytes in ARVC

Or Or

Recombined Dspand EYFP

B BROSA26

3’5’EYFP

Cre Recombinase

A B

C

D

ATG

ATG

E

Courtesy of F. Costantini

Courtesy of B. Black, Schwartz R and MD Schneider

Nkx‐Cre:Dsp

+/‐ :EYFP+

/+Wild

 type

 Expression of EYFP in Adipocytes in Nkx‐Cre:Dsp+/‐:EYFP+/+ Mice

Bright Field DAPI EYFP CEBP/α

EYFP:CEBP/α:DAPIEYFP:CEBP/α

DAPI

EYFP:DAPIEYFP

Bright Field CEBP/α CEBP/α:DAPI

EYFP:DNAEYFP EYFP:C/EBP‐α:DNA

C/EBP‐α C/EBP‐α:DNA

EYFP:CEBP/α

DNAOil Red O

Expression of EYFP in Adipocytes in Mef2C‐Cre:Dsp+/‐:EYFP+/+ Mice

Mef2C

DNA

Mef2C:DNA

C/EBP‐α C/EBP‐α:DNA

Mef2C:C/EBP‐α:DNAMef2C:C/EBP‐α

Bright Field

Co‐Expression of Second Heart Field and AdipogenicMarkers in Nkx‐Cre:Dsp+/‐:EYFP+/+ Mice

PPARγ

Isl1

PPARγ:Isl1:DNA

Isl1:DNA

PPARγ:Isl1

DNA

PPARγ:DNA

Oil Red O

Expression of Second Heart Field Markers in Adipocytesin Nkx‐Cre:Dsp+/‐:EYFP+/+ Mice

PPAR‐γ

Isl1:DNA

Isl1:PPAR‐γ:DNAPPAR‐γ:DNA

Isl1DNA

PPARγ:Isl1

Oil Red O

Co‐Expression of Second Heart Field and AdipogenicMarkers in Mef2C‐Cre:Dsp+/‐:EYFP+/+ Mice

C/EBP‐α:Mef2C:DNA

C/EBP‐α C/EBP‐α:DNA

Mef2C:DNAMef2C C/EBP‐α:Mef2C

DNABright Field

Co‐Expression of Second Heart Field and AdipogenicMarkers in Mef2C‐Cre:Dsp+/‐:EYFP+/+ Mice

Co‐Expression of Cardiac Progenitor Cells and AdipogenicMarkers in Human Hearts with ARVC

Bright Field C/EBP‐α Isl1 Isl1: C/EBP‐α:DAPI

Hum

an Heart with

 ARV

C

A

Bright Field Mef2C C/EBP‐α Mef2C:C/EBP‐α:DAPI

Normal Heart

Bright FieldC C/EBP‐α:DAPI Isl1: DAPIMef2C:DAPI

B Bright Field

Molecular Pathogenesis of ARVC

Adipocyte

Cardiac Progenitor Cell

ARVCmutation

Desmosome

PGPG

PGPG

Tcf/LefTcf/Lef

β‐Cateninβ‐Catenin

Wnt

GSK3β

β‐Cateninβ‐Catenin

Frizzled

FatFat

FatFat

FatFat

FatFat FatFat

FatFat

FatFat

FatFat

FatFat

FatFat

FatFat

FatFat

FatFat

FatFat

FatFat

PPAR‐γC/EBP‐αPPAR‐γC/EBP‐α

Expression of canonical Wntsignaling target genes

CTGFBMP7  BMP7  

Wnt5b   Wnt5b