the ubiquitin-proteasome system in hypertrophic cardiomyopathy
TRANSCRIPT
Frontiers in Cardiovascular Biology 201230.3 – 1.4.2012
London, UK
The ubiquitin-proteasome system in hypertrophic cardiomyopathy
Lucie Carrier
Department of Experimental Pharmacology and ToxicologyCardiovascular Research Center, UKE, Hamburg, Germany
Inserm U974-CNRS UMR7215, Paris, France
NO CONFLICT OF INTEREST
Hypertrophic cardiomyopathy (HCM)
Elliott P et al., Eur Heart J 2008;Maron BJ et al., Circulation 2006
Schlossarek S et al., J Mol Cell Cardiol 2011 (review)
HCM : 19 genes and > 500 mutations
Cardiac myosin-binding protein C (cMyBP-C)
Schlossarek S et al., J Mol Cell Cardiol 2011 (review)
Human MYBPC3 mutation
13% of unrelated HCM patients
18% of myofilament-positive patients
30% of MYBPC3-positive patients
From Olivotto I et al., Mayo Clinic Proc 2008
G>A transition on the last nucleotide of exon 6
Human MYBPC3 mutationG>A transition on the last nucleotide of exon 6
Mearini G, Schlossarek S, Poggesi C, Marston et al., unpublished
Absent
Vignier N*, Schlossarek S* et al., Circ Res 2009
Absent
Knock-in of the human MYBPC3 mutation into the mouse genome : Mybpc3-targeted KI mice
Molecular mechanisms of Mybpc3 mutation
Mutantallele
Nonsense mRNAs Mutant proteins
??????
Molecular mechanisms of Mybpc3 mutation
Mutantallele
Nonsense mRNAs Mutant proteins
??????
Translation inhibitors: Emetine, CHXProteasome inhibitors: MG132, MG262, EpoxomicinIn cardiac myocytes or in vivo
Molecular mechanisms of Mybpc3 mutation
Mutantallele
Nonsense mRNAs Mutant proteins
Nonsense-mediatedmRNA decay Ubiquitin-proteasome system
Sarikas A et al., Cardiovasc Res 2005; Mearini G et al., Cardiovasc Res 2010;Vignier N*, Schlossarek S* et al., Circ Res 2009
Molecular mechanisms of Mybpc3 mutation
Mutantallele
Wild-typeallele
Wild-type proteinNonsense mRNAs Mutant proteins
Nonsense-mediatedmRNA decay Ubiquitin-proteasome system
Haploinsufficiency
LVH, diastolic dysfunction ?
Vignier N*, Schlossarek S* et al., Circ Res 2009
LVM/BW
WT Het KI0
2
4
6
8***
9 109
(mg/
g)
FAS
WT Het KI0
10
20
30
40
50
**
9 109(%
)
Fraysse B*, Weinberger F*, Bardswell S* et al., J Mol Cell Cardiol 2012, in press
Heterozygous KI mice do not develop LVH and systolic dysfunction
**p<0.01 and ***p<0.001 vs WT
Increased myofilament Ca2+ sensitivity
Fraysse B*, Weinberger F*, Bardswell S* et al., J Mol Cell Cardiol 2012, in press
7.0 6.5 6.0 5.5 5.0
0.0
0.2
0.4
0.6
0.8
1.0
WT Het
pCa
Rel
ativ
e Fo
rce
Het-KI WT
7.0 6.5 6.0 5.5 5.0
0.0
0.2
0.4
0.6
0.8
1.0
WT KIR
elat
ive
Forc
epCa
Hom-KI WT
pCa50: p<0.05 vs WT pCa50: p<0.05 vs WT
PWD
WTE
A
Het KI
TDIE‘
A‘
WT Het KI
E/A
WT Het KI0.0
0.5
1.0
1.5
2.0
9
***
109
**
E´/A´
WT Het KI0.0
0.5
1.0
1.5
2.0
9 109
*** ***
E/E´
WT Het KI0
10
20
30
40
50
9 109
**
Fraysse B*, Weinberger F*, Bardswell S* et al., J Mol Cell Cardiol 2012, in press
Diastolic dysfunction
*p<0.05, **p<0.01 and ***p<0.001 vs WT
Molecular mechanisms of Mybpc3 mutation
Mutantallele
Wild-typeallele
Wild-type proteinNonsense mRNAs Mutant proteins
Nonsense-mediatedmRNA decay Ubiquitin-proteasome system
Haploinsufficiency
Increased Ca2+ sensitivity and diastolic dysfunction
Fraysse B*, Weinberger F*, Bardswell S* et al., J Mol Cell Cardiol 2012, in press
Why do heterozygous mice not develop LVH?
LVM/BW
WT Het KI0
2
4
6
8***
9 109
(mg/
g)
Schlossarek S et al., J Muscle Res Cell Motil 2012
Adrenergic stress induced proteasome impairment in heterozygous Mybpc3-targeted KI mice
Wild-type and heterozygous mice
Combination of isoprenaline and phenylephrine (ISO/PE) or NaCl for 1 week
Ventricular-to-body weight ratio
0
1
2
3
4
5
6
#
8 87 8
*** ***
(mg/
g)
Chymotrypsin-like activity
0.0
0.2
0.4
0.6
0.8
1.0
1.2
8 7 8 8
#
***(AU
)
Mybpc3-knock-in mice (KI):G>A transition in exon 6
10% of mutant cMyBP-Cs
Mybpc3-knock-out mice (KO):transcriptional knock-out
No cMyBP-C
Is the UPS altered in Mybpc3-targeted KI mice ?
UbG76V GFP
Schlossarek S et al., Basic Res Cardiol 2012
~1 yr-old Mybpc3 miceWT KO KI
012345678
*****
14 7 7
**p<0.01 and ***p<0.001 vs WT
HW
/BW
(mg/
g)
GAPDH
UbG76VGFP
WT KO WT KI
WT KO KI0
1
2
3
4
5
14 7 7
***###
***p<0.001 vs WT; ###p<0.001 vs KO
Protein
UbG
76V -G
FPpr
otei
n le
vels
(AU)
WT KO KI0
1
2
3
4
5
14 7 7
mRNA
UbG
76V -G
FP m
RN
A le
vel (
AU)
Schlossarek S et al., Basic Res Cardiol 2012
Impairment of the UPS in Mybpc3-targeted KI mice
WT + UbG76V-DsRed M7t + UbG76V-DsRed
MG132Saturation?
Impairment of the UPS in cardiac myocytes
Adenoviral gene transfer in rat cardiac myocytesSarikas A et al., Cardiovasc Res 2005
UPS impairment in human HCM
From Predmore JM et al., Circulation 2010Human myocardial tissue
Mutantallele
Wild-typeallele
Wild-type proteinNonsense mRNAs Mutant proteins
Nonsense-mediatedmRNA decay Ubiquitin-proteasome system
HaploinsufficiencyPoison peptides
Proposed mechanisms upon stress
Adrenergic stress or aging
Increased Ca2+ sensitivity and diastolic dysfunctionProteotoxicity and LVH
Improving proteasome function attenuated cardiac hypertrophy and delayed premature death
Transgenic mice overexpressing the proteasome activator PA28αX
CryABR120G miceFrom Li J et al., JCI 2010
LisaKrämer
DoreenKhajetoorians
AlbertoPorto
SophiaMaron
FriederikeSchürmann
GiuliaMearini
TiloThottakara
BirgitGeertz
SaskiaSchlossarek
AileenSchwalm
FrederikFlenner
VerenaBehrens-Gawlik
SilkeReischmann
FelixFriedrich
Christina Gedicke-Hornung
MarkusSauer
LisaKrämer
DoreenKhajetoorians
AlbertoPorto
SophiaMaron
FriederikeSchürmann
GiuliaMearini
TiloThottakara
BirgitGeertz
SaskiaSchlossarek
AileenSchwalm
FrederikFlenner
VerenaBehrens-Gawlik
SilkeReischmann
FelixFriedrich
Christina Gedicke-Hornung
MarkusSauer
Florian Weinberger
ThomasEschenhagen
Inserm U974Paris
Bodvael FrayssePascale RichardNicolas Vignier
EU-BIG-HeartSteve Marston
Corrado PoggesiCharles Redwood
Jolanda van der VeldenHugh Watkins
King‘s CollegeLondon
Metin AvkiranSonya C. Bardswell
Friederike CuelloJonathan C. Kentish
LeducqProteotoxicityMathias Gautel
Jeff RobbinsJoe Hill
Marco SandriCam Patterson
HamburgCardiology
Monica PattenJulia Münch