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Department of Experimental and Clinical Pharmacology and Toxicology,

Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf,

Hamburg, Germany

The ubiquitin proteasome system in

cardiovascular disease

Basic mechanisms

Saskia Schlossarek

ECS Congress 2010

Stockholm, Sweden

28 Aug 2010 – 01 Sep 2010

No financial disclosure

Cardiac protein turnover

Cellular homeostasis

Protein

degradation

Protein

synthesis

Cardiac hypertrophy

Protein

synthesis

Protein

synthesis

Protein

degradationProtein

degradationProtein

degradation

The ubiquitin-proteasome system (UPS)

E2

E3K

UbUb

UbUb

UbK

UbUb

UbUb

Ub

E1

UbUb

KUb

Ub

Ub

UbUb

ATP

Ub

Ubiquitination Deubiquitination/Degradation

ATPATP

Base

Base

α-ring

α-ring

β-ring

β-ring

Chymotrypsin-like activity (b5)

Trypsin-like activity (b2)

Caspase-like actvity (b1)

Carrier et al. Cardiovasc Res 2009 (review)

Mearini et al. Biochemica Biophysica Acta 2008 (review)

Muscle-specific E3 ubiquitin ligases

E3 ubiquitin ligase Type Substrate

Atrogin-1 (MAFbx) RING finger, SCF

complex

CnA, MyoD, FoxO1, FoxO3

MuRF1 (Trim63) RING finger, single

subunit

cTnI, PKC, M-CK, β-MHC

MuRF3 (Trim54) RING finger, single

subunit

FHL2, γ-filamin, β-MHC

CHIP U-box misfolded proteins, Hsp70,

GATA4

Mdm2 RING finger, single

subunit

p53, β-arrestin2, Tcap

Ozz RING finger, VCB

complex

β-catenin

Nedd4-like HECT domain Nav1.5, KCNQ1

Trim32 RING finger Actin

Is the UPS altered in cardiac disease?

E2

E3K

UbUb

UbUb

UbK

UbUb

UbUb

Ub

E1

UbUb

KUb

Ub

Ub

UbUb

ATP

Ub

ATPATP

Is the UPS a good target for cardiac disease therapy?

Is the UPS altered in cardiac disease?

Deubiquitination?

Deubiquitinating enzymes?

E2

E3K

UbUb

UbUb

UbK

UbUb

UbUb

Ub

E1

UbUb

KUb

Ub

Ub

UbUb

ATP

Ub

ATPATP

Ubiquitination?

Ubiquitinated proteins?

Ubiquitinating enzymes?

Degradation?

Components?

Activities?

UPS alteration in human end-stage heart failure

Weekes J et al. Proteomics 2003

UPS activation in human dilated cardiomyopathy (DCM)

Ubiquitinated proteins

Chymotrypsin-like activity

Birks EJ et al. Cardiovasc Res 2008

0

1000

2000

3000

4000

5000

6000

7000

8 8

***

Chym

otr

ypsin

-lik

e

activity (

RF

U)

WT KI

UPS activation in a mouse model of hypertrophic

cardiomyopathy (HCM)

WT KI

Ubiq

uitin

ate

d p

rote

ins

Vignier N / Schlossarek S et al. Circ Res 2009

Cardiac myosin-binding protein-C knock-in (KI) mice

Ubiquitinated proteins Chymotrypsin-like activity

WT (n=8)

KI (n=8)

0.0

0.5

1.0

1.5

2.0

*** ******

*

***

mR

NA

(fo

ld c

hanges)

Nedd4 EBE3C Ozz Mdm2 ASB2 Trim32 Stub1 MuRF1 Atrogin-1

Schlossarek et al., unpublished

UPS activation in a mouse model of HCM

Cardiac myosin-binding protein-C-knock-in (KI) mice

Transcript levels of E3 ubiquitin ligases

MG132 suppressed hypertrophy in myocytes,

Velcade® reduced hypertrophy in hypertensive

Dahl-salt sensitive ratsMeiners S et al. Hypertension 2008

PS-159 reduced isoprenaline-induced hypertrophy in mice Stansfield WE et al. Am J Physiol 2008

Epoxomicin reversed TAC-induced hypertrophy in miceHedhli N et al. Am J Physiol 2008

Is the UPS a good target for cardiac disease therapy?

Epoxomicin partly rescued cardiac dysfunction in mice with HCMSchlossarek et al., unpublished data

Treatment of cMyBP-C KI mice with epoxomicin


Epoxomicin (0.5 mg/kg/d) or NaCl for 1 week

NaCl

WT

NaCl NaClEpox Epox Epox

Het KI

25

b5-subunit

0.0

0.2

0.4

0.6

0.8

1.0

1.2

4 4444 4

p<0.001

Ch

ym

otr

yp

sin

-lik

e

ac

tivit

y (

AU

)

p<0.001

p<0.01


BEFORE and AFTER NaCl

WT b

asal

NaC

l

WT a

fter NaC

l

HET b

asal

NaC

l

HET a

fter

NaC

l

KI b

asal

Nac

l

KI a

fter

NaC

l0

1

2

3

4

5

6

7P<0.01

5 5 5 54 4

LV

M/B

W (

mg

/g)

No regression of LV hypertrophy

by proteasome inhibition



BEFORE and AFTER Epoxomicin

0

1

2

3

4

5

6

7

5 5 5 54 4

P<0.01

LV

M/B

W (

mg

/g)

Improvement of cardiac function

by proteasome inhibition



BEFORE and AFTER NaCl

0

10

20

30

40

50

60

70

5 5 5 54 4

P<0.01

FA

S (

%)

BEFORE and AFTER Epoxomicin

0

10

20

30

40

50

60

70

*

*p<0.05 vs Basal

5 5 5 54 4

P<0.01

FA

S (

%)

K

UbUb

UbUb

UbK

UbUb

UbUb

Ub

E

1

UbUb

K

E

2

E

3 Ub

Ub

Ub

UbUb

AT

P

AT

P

AT

P

Ub

The UPS is altered in cardiac disease.

Proteasome inhibition may reverse cardiac disease.

However ….

Tsukamoto O et al. Biochem Biophys Res Commun 2006

UPS impairment in experimental heart failure


Transverse aortic constriction in mice

15 mg/Kg/d isoprenaline and phenylephrine (Iso/PE) or NaCl for 1 week

Altered UPS upon adrenergic stress

in a mouse model of HCM

***p<0.001 vs. NaCl

#p<0.05 vs. WT

0

1

2

3

4

5

6

8 7 8 8

******#

VW

/BW

(m

g/g

)

0.0

0.5

1.0

1.5

78 8 8Chym

otr

ypsin

-lik

e

activity (

AU

)

***

#

***p<0.001 vs. NaCl

#p<0.05 vs. WT



UPS impairment in human heart failure and HCM

Predmore JM et al. Circulation 2010


K

UbUb

UbUb

UbK

UbUb

UbUb

Ub

E

1

UbUb

K

E

2

E

3 Ub

Ub

Ub

UbUb

AT

P

AT

P

AT

P

Ub

The UPS is impaired in other studies of cardiac disease.

Proteasome inhibition may rather worsen than improve the phenotype.

Disease UPS alterations

Human heart failure ↑ ubiquitinated proteins

↑ E2 (Ubc2)

↑ deubiquitinases

Experimental cardiac hypertrophy and failure

induced by TAC

↑ ubiquitinated proteins

↑ E2 (UbcH2)

↑ E3 (atrogin-1, MuRF1)

↑ proteasome subunits

↓ or ↑ proteasome activities

Experimental myocardial ischemia ↑ ubiquitinated proteins

↓ proteasome activities

Modifications of 20S subunits

Experimental cardiac atrophy induced by

heterotopic heart transplantation


↑ E2 (UbcH2)

↓ E3 (atrogin-1, MuRF1)

Doxorubicin-induced cardiomyopathy ↑ E3 (atrogin-1)

↑ proteasome activities

Experimental hyperglycemia ↑ ubiquitinated proteins

↓ 26S proteasome activities

↑ 11S subunit of the proteasome

FHC cell model (truncated cMyBP-C) ↓ truncated cMyBP-C level in

cardiomyocytes; impaired UPS

DRM mouse models (CryABR120G or mutant

desmin)


↑ proteasome activities

↑ 20S subunits

↓ 19S subunits

Alterations of the UPS in cardiac disease

Mearini et al. Biochemica Biophysica Acta 2008 (review)

→ Proteasome inhibition as a therapeutic option?

T. Eschenhagen

(MD, PhD)

S. Schlossarek

(PhD)

C. Gedicke

(PhD student)

V. Behrens-Gawlik

(PhD)

M. Sauer

(MD student)

L. Carrier

(PhD)

A. Schwalm

(MD student)

S. Reischmann

(Technician)

D. Khajetoorians

(PhD student)

Department of Experimental and Clinical Pharmacology and Toxicology

B. Geertz

(Technician)F. Friedrich

(MD)

T. Thottakara

(MD student)

G. Mearini

(PhD)

E. Krämer

(Technician)

D. Juhr

(Technician)

F. Schürmann

(MD student)

ParisInserm U974

Nicolas Vignier

Ketty Schwartz†

FundingEU Marie Curie Excellence Team Grant

DFG Forschergruppe 604 (CA 618)

StockholmKarolinska Institute

Nico Dantuma

Chapel Hill, NCUniversity of North Carolina

Monte Willis

Vermillion, SDUniversity of South Dakota

Xuejun Wang

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