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
Schlossarek et al., unpublished
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
Cardiac myosin-binding protein-C knock-in (KI) mice
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
Schlossarek et al., unpublished
Cardiac myosin-binding protein-C knock-in (KI) mice
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
Schlossarek et al., unpublished
Cardiac myosin-binding protein-C knock-in (KI) mice
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
Ubiquitinated proteins Chymotrypsin-like activity
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
Schlossarek et al., unpublished
Cardiac myosin-binding protein-C knock-in (KI) mice
UPS impairment in human heart failure and HCM
Predmore JM et al. Circulation 2010
Ubiquitinated proteins Chymotrypsin-like activity
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
↑ ubiquitinated proteins
↑ 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)
↑ ubiquitinated proteins
↑ 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