The ubiquitin proteasome system in cardiovascular disease Basic mechanisms

Transcription

1 ECS Congress 2010 Stockholm, Sweden 28 Aug Sep 2010 The ubiquitin proteasome system in cardiovascular disease Basic mechanisms Saskia Schlossarek Department of Experimental and Clinical Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany No financial disclosure

2 Cardiac protein turnover Cellular homeostasis Protein synthesis Protein degradation Cardiac hypertrophy Protein synthesis Protein degradation Protein degradation Protein degradation

3 The ubiquitin-proteasome system (UPS) iquitination Deubiquitination/Degradation E1 ATP E2 E3 ATP Base α-ring β-ring β-ring α-ring Base ATP Chymotrypsin-like activity (b5) Trypsin-like activity (b2) Caspase-like actvity (b1) Carrier et al. Cardiovasc Res 2009 (review)

4 Muscle-specific E3 ubiquitin ligases E3 ubiquitin ligase Type Substrate Atrogin-1 (MAFbx) MuRF1 (Trim63) MuRF3 (Trim54) RING finger, SCF complex RING finger, single subunit RING finger, single subunit CnA, MyoD, FoxO1, FoxO3 ctni, PC, M-C, β-mhc FHL2, γ-filamin, β-mhc CHIP U-box misfolded proteins, Hsp70, GATA4 Mdm2 Ozz RING finger, single subunit RING finger, VCB complex p53, β-arrestin2, Tcap β-catenin Nedd4-like HECT domain Na v 1.5, CNQ1 Trim32 RING finger Actin Mearini et al. Biochemica Biophysica Acta 2008 (review)

5 Is the UPS altered in cardiac disease? E1 E2 E3 ATP ATP ATP Is the UPS a good target for cardiac disease therapy?

6 Is the UPS altered in cardiac disease? Deubiquitination? Deubiquitinating enzymes? E1 E2 E3 ATP ATP ATP iquitination? iquitinated proteins? iquitinating enzymes? Degradation? Components? Activities?

7 UPS alteration in human end-stage heart failure Weekes J et al. Proteomics 2003

8 UPS activation in human dilated cardiomyopathy (DCM) iquitinated proteins Chymotrypsin-like activity Birks EJ et al. Cardiovasc Res 2008

9 iquitinated proteins Chymotrypsin-like activity (RFU) UPS activation in a mouse model of hypertrophic cardiomyopathy (HCM) iquitinated proteins Chymotrypsin-like activity WT I *** WT I Cardiac myosin-binding protein-c knock-in (I) mice Vignier N / Schlossarek S et al. Circ Res 2009

10 mrna (fold changes) UPS activation in a mouse model of HCM Transcript levels of E3 ubiquitin ligases *** *** * *** *** WT (n=8) I (n=8) Nedd4 EBE3C Ozz Mdm2 ASB2 Trim32 Stub1 MuRF1 Atrogin-1 Cardiac myosin-binding protein-c-knock-in (I) mice Schlossarek et al., unpublished

11 Is the UPS a good target for cardiac disease therapy? MG132 suppressed hypertrophy in myocytes, Velcade reduced hypertrophy in hypertensive Dahl-salt sensitive rats Meiners 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 mice Hedhli N et al. Am J Physiol 2008 Epoxomicin partly rescued cardiac dysfunction in mice with HCM Schlossarek et al., unpublished data

12 Treatment of cmybp-c I mice with epoxomicin Chymotrypsin-like activity (AU) Epoxomicin (0.5 mg/kg/d) or NaCl for 1 week p<0.001 p<0.001 p< b5-subunit 25 WT Het I NaCl Epox NaCl Epox NaCl Epox Cardiac myosin-binding protein-c knock-in (I) mice Schlossarek et al., unpublished

13 LVM/BW (mg/g) No regression of LV hypertrophy by proteasome inhibition LVM/BW (mg/g) BEFORE and AFTER NaCl BEFORE and AFTER Epoxomicin 7 6 P< P< WT basal NaCl WT after NaCl HET basal NaCl HET after NaCl I basal Nacl I after NaCl Cardiac myosin-binding protein-c knock-in (I) mice Schlossarek et al., unpublished

14 FAS (%) Improvement of cardiac function by proteasome inhibition FAS (%) BEFORE and AFTER NaCl P< BEFORE and AFTER Epoxomicin P<0.01 * *p<0.05 vs Basal Cardiac myosin-binding protein-c knock-in (I) mice Schlossarek et al., unpublished

15 The UPS is altered in cardiac disease. E 1 AT P E 2 E 3 AT P AT P Proteasome inhibition may reverse cardiac disease.

16 However.

17 UPS impairment in experimental heart failure iquitinated proteins Chymotrypsin-like activity Transverse aortic constriction in mice Tsukamoto O et al. Biochem Biophys Res Commun 2006

18 VW/BW (mg/g) Altered UPS upon adrenergic stress in a mouse model of HCM Chymotrypsin-like activity (AU) 15 mg/g/d isoprenaline and phenylephrine (Iso/PE) or NaCl for 1 week *** # *** # *** ***p<0.001 vs. NaCl #p<0.05 vs. WT ***p<0.001 vs. NaCl #p<0.05 vs. WT Cardiac myosin-binding protein-c knock-in (I) mice Schlossarek et al., unpublished

19 UPS impairment in human heart failure and HCM iquitinated proteins Chymotrypsin-like activity Predmore JM et al. Circulation 2010

20 The UPS is impaired in other studies of cardiac disease. E 1 AT P E 2 E 3 AT P AT P Proteasome inhibition may rather worsen than improve the phenotype.

21 Alterations of the UPS in cardiac disease Disease Human heart failure Experimental cardiac hypertrophy and failure induced by TAC Experimental myocardial ischemia Experimental cardiac atrophy induced by heterotopic heart transplantation Doxorubicin-induced cardiomyopathy UPS alterations ubiquitinated proteins E2 (c2) deubiquitinases ubiquitinated proteins E2 (ch2) E3 (atrogin-1, MuRF1) proteasome subunits or proteasome activities ubiquitinated proteins proteasome activities Modifications of 20S subunits Proteasome inhibition as a therapeutic option? Experimental hyperglycemia FHC cell model (truncated cmybp-c) DRM mouse models (CryAB R120G or mutant desmin) ubiquitinated proteins E2 (ch2) E3 (atrogin-1, MuRF1) E3 (atrogin-1) proteasome activities ubiquitinated proteins 26S proteasome activities 11S subunit of the proteasome truncated cmybp-c level in cardiomyocytes; impaired UPS ubiquitinated proteins proteasome activities 20S subunits 19S subunits Mearini et al. Biochemica Biophysica Acta 2008 (review)

22 S. Schlossarek (PhD) T. Thottakara (MD student) F. Friedrich (MD) T. Eschenhagen (MD, PhD) M. Sauer (MD student) B. Geertz (Technician) A. Schwalm (MD student) G. Mearini (PhD) L. Carrier (PhD) C. Gedicke (PhD student) V. Behrens-Gawlik (PhD) S. Reischmann (Technician) D. hajetoorians (PhD student) E. rämer (Technician) D. Juhr (Technician) F. Schürmann (MD student) Department of Experimental and Clinical Pharmacology and Toxicology

23 Paris Inserm U974 Nicolas Vignier etty Schwartz Stockholm arolinska Institute Nico Dantuma Chapel Hill, NC University of North Carolina Monte Willis Vermillion, SD University of South Dakota Xuejun Wang Funding EU Marie Curie Excellence Team Grant DFG Forschergruppe 604 (CA 618)