The ubiquitin-proteasome system in hypertrophic cardiomyopathy

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Mearini G, Schlossarek S, Poggesi C, Marston et al., unpublished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