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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
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D
A
CE
BHCM DCM
ARVC
Cardiac Myocyte
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Lemola, K et al. Heart 2005;91:1167-1172
ARVC: Survival
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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
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Naxos Disease
Norgett EE. Et al. Human Mol. Genet. 2000;9:2761-2766 Yannis Protonotarios Foundation, Medical Center of Naxos, Naxos 84300, Greece
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Naxos Disease: Plakoglobin (JUP)
McKoy G et al. The Lancet 2002
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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
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Pathogenesis of ARVC
?
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The Enigma of Reverse Cardiac Alchemy
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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
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Competitive Inhibition of Canonical Wnt/β‐cateninSignaling by Nuclear Plakoglobin
Wnt
Frz
Nucleus
Tcf7l2/Lef1
β Catenin
β Catenin
PG
PG
Free PG
Desmosome
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Targeted Disruption of Desmosomes Assembly
Modified from Nature Reviews Molecular Cell Biology 1; 208‐216 2000
DSP
PG
PKP2
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Stable Suppression of Expression of DP in HL‐1 Cells Using Sequence‐Specific siRNAs
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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.
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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
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Transcription Switch To Adipogenesis in DP-Deficient HL-1 Cells
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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
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Untreated Dex + Ins + IBMX
Con
trol
DP
siR
NA
799
Higher Magnification
Suppression of Expression of DPAccumulation of Fat Droplets in HL-1 Cells
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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
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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
/
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A
B
Cre‐Mediated Deletion of Dsp Gene
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Dsp+/+
Survival Rate
Log Rank Chi Sq =73.2p<0.0001
Dsp+/+Dsp+/-
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Non-Transgenic
Nkx2.5-Cre:DspW/F Mef2C-Cre:DspW/F
α-MyHC-Cre:DspW/F
Cardiac Dysfunction in Cardiac‐Restricted DP‐Deficient Mice
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Ventricular Tachycardia in DP‐Deficient Mice
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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
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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
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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
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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
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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
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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
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Akio Kanazaw et al BBRC 2005 F.H.J. van Tienen et al. BBRC 2009
Wnt5b Suppressed canonical Wnt Signaling and enhances adipogenesis
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The Enigma of ARVC: Cellular Origin of Adipocytes in ARVC
H&E Masson trichrome
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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