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Description of the team, research area and previous work

Lucie Carrier is a French scientist (CNRS Director of Research) who has received a Marie Curie Excellence Grant to lead an European team at the Institute of Experimental and Clinical Pharmacology of the UKE in Hamburg between 2005 and 2009. Her team is also part of the French mixed research structure (Inserm U974-CNRS UMR7215, directed by Thomas Voit in Paris). She is involved in the genetics and pathophysiology of familial hypertrophic cardiomyopathy (HCM) for 20 years.
         HCM is a myocardial disease with the major feature of asymmetric septal hypertrophy. It is one of the most common monogenic diseases with a disease prevalence of 1:500 in young adults. It is the major cause of sudden death in the young and is associated with a significant risk of heart failure. It is an autosomal-dominant familial disease in most of the cases and involves mutations in at least 13 different genes encoding proteins of the sarcomere. The first disease gene for HCM, the beta-myosin heavy chain gene, was identified in the early 90s by the team of C. Seidman (Boston). This opened the way of genetic cardiology.
         In France, K. Schwartz and M. Komajda have initiated the genetic analyses of HCM in 1991, and L. Carrier has been involved from the beginning. A French INSERM network has been created in 1992, which has largely contributed to the discovery of the molecular causes of FHC. A panel of 297 French families with FHC has been recruited, and about 2000 blood samples have been collected. Two European networks have been created later on this project and L. Carrier has been always actively involved (the BIOMED project from 1996 to 2001 and the Eurogene Heart Failure Project-Leducq Foundation from 2000 to now). The latter one allowed recruiting 350 unrelated European families with HCM. The major discoveries were (1) identification of the CMH4 locus on chromosome 11 (Carrier et al., 1993), (2) identification of the FHC disease gene MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C; Bonne / Carrier et al., 1995), (3) determination of the complete structure and organization of the MYBPC3 gene and demonstration that most of the MYBPC3 mutations produce C-terminal truncated proteins (Carrier et al., 1997), (4) identification of 97 different mutations in 9 different HCM genes in 124 unrelated families, and evidence that 5% of the families present individuals with a complex genetic status who exhibit a more severe phenotype (Richard et al., 2003), (5) identification of a polymorphism in calmodulin III promoter that modifies the expression of FHC (Friedrich et al., 2009).
         Since the mechanisms by which MYBPC3 mutations lead to HCM was not known, a large part of our projects aims at elucidating the functional consequences of HCM mutations. The major findings were: (1) cMyBP-C is expressed exclusively in the heart during human and mouse development (Fougerousse et al, 1998), (2) human truncated cMyBP-C mutants are unstable and mis-incorporated in fetal rat cardiomyocytes (Flavigny et al., 1999), (3) recombinant human truncated cMyBP-C mutants do not interact with human beta-MHC in biosensor chips (Flavigny et al., 2003), (4) heterozygous cMyBP-C targeted mice develop asymmetric septal hypertrophy, therefore constituting the first model with the major feature of HCM (Carrier et al, 2004), (5) human homozygous R403W mutant cardiac myosin presents disproportionate enhancement of mechanical and enzymatic properties (Keller et al., 2004), (6) cMyBP-C is required for complete relaxation in diastole (Pohlmann et al., 2007), (7) truncated cMyBP-C mutants impair the ubiquitin-proteasome system, which could contribute to the pathogenesis of HCM (Sarikas/Carrier et al., 2005), (8) Atrogin-1 degrades truncated cMyBP-C but MuRF1 down-regulates the transcription of myosin-heavy chains (Mearini et al., 2010), (9) The nonsense-mediated mRNA decay and the ubiquitin-proteasome system regulate the levels of cMyBP-C in targeted Mybpc3-knock-in mice (Vignier/Schlossarek et al., 2009; Carrier et al., 2010).

Objectives

The major objective of the Carrier team is the understanding of the molecular mechanisms by which mutations in MYBPC3 lead to the development of HCM. However, a large part of the projects also evaluate new molecular tools for HCM therapy. Our projects are the following:

1. Screening for mutations in new genes encoding components of the cardiac sarcomere in patients with HCM, and investigation of their consequences in mouse cardiac myocytes and engineered heart tissue using adeno-associated virus (AAV). Collaboration with UKE-HEXT-AAV and O. Müller, Cardiology, Heidelberg.
2. Evaluation of the role of several protein quality controls (ubiquitin-proteasome system, autophagy and unfolded-protein response) in cardiac homeostasis, HCM and other cardiac diseases. Collaboration with G. Bonne, Inserm U974, Paris.
3. Evaluation of RNA-based therapies in a mouse model of HCM (Mybpc3-knock-in mice). The goal is to suppress the endogenous mutation by different strategies, such as exon skipping, exon inclusion and spliceosome-mediated RNA transplicing (in mouse cardiac myocytes, engineered mouse heart tissue and in vivo using AAV). Collaborations with Luis Garcia and Thomas Voit, Inserm U974, Paris.
4. Development of a clinical and experimental reference center for hypertrophic cardiomyopathy in Northen Germany: recruitment of patients and relatives with HCM (plus blood sample, septal myectomy, skin biopsy), deciphering of genetic causes, analysis of the pathophysiology and development of personalized therapy concepts (via reprogramming skin fibroblasts into iPS cells and differentiation in cardiac myocytes and engineered heart tissue). Collaborations with Dr Monica Patten, Cardiology UKE, HEXT-iPS, and EU-FP7-Health-BIG-Heart network, in which biopsies are shared for complementary physiological analyses.

Material and methods

• Transgenic mice: (1) targeted Mybpc3-knockout, (2) targeted Mybpc3-knock in (point mutation leading to 3 different mutant mRNAs and/or proteins), (3) Mybpc3-M7t transgenic (truncated protein), (4) Ub^G76V-GFP and GFPdgn transgenic mice as UPS-reporters in vivo, (5) MuRF1-KO mice (ubiquitin E3 ligase)
• Adenovirus encoding wild-type cMyBP-C, 2 truncated cMyBP-C mutants, atrogin-1, MuRF1, Ub^G76V-dsRed, ASB2beta.
• Adeno-associated virus encoding different components of the sarcomere.
• DNA bank of >350 unrelated patients with hypertrophic cardiomyopathy (EUROGENE Heart Failure Project; LeDucq Foundation)
• Mouse cardiac physiology (Echocardiography, measurement of cell and sarcomere length shortening and Ca²⁺ transient in adult mouse myocytes with the ionOptix system, measurements of cardiac muscle contractility)
• Cell biology and biochemistry (isolation and culture of neonatal and adult mouse cardiac myocytes, fluorescence and confocal microscopy, proteasome activities, immuno-precipitation.)
• Molecular biology (DNA and RNA extractions, production of recombinant proteins Protein extraction and fractionation (Rotofor), PCR, RT-PCR, RT-qCR on Taqman, Western-blot analysis. several antibodies including phospho-specific antibodies)
• Ubiquitin-proteasome system: Ubiquitination assays in vitro (E1, E2, and muscle-specific E3 ubiquitin ligases), measurement of the proteasome activities with fluoregenic substrates, antibodies directed against ubiquitin (FK2 and others) and against beta5-subunit of the proteasome (which shows 2 bands when the proteasome is blocked by epoxomicin in vivo), muscle-specific ubiquitin E3 ligase deficient mice (MuRF1-KO), adenovirus and mouse reporters for the proteasome function (Ub^G76V-GFP and GFPdgn transgenic mice), adenovirus encoding muscle-specific E3 ligase (atrogin-1, MuRF1, ASB2beta).

Current Collaborations

T. Arimura (University of Tokyo, Tokyo, Japan)
G. Bonne, B. Fraysse L. Garcia, N. Vignier and T. Voit (INSERM U974-CNRS UMR7215, Paris, France)
N. Dantuma (Karolinska Institute, Stockholm)
M. Gautel (King's College, London, UK)
J. Hill (UT Southwest Medical Center, Dallas, TX, USA)
S. Marston (Imperial's College, London, UK)
C. Patterson (UNC-CH School of Medecine, Chapel Hill, NC, USA)
C. Poggesi (University of Florence, Italy)
C. Redwood (University of Oxford, UK)
J. Robbins (Children's Hospital, Cincinnati, OH, USA)
M. Sandri (University of Padova, Italy)
J. van der Velden (VUMC, Amsterdam, The Netherlands)
X. Wang (University of South Dakota, SD, USA)
H. Watkins (University of Oxford, UK)
M. Willis (Carolina Cardiovascular Biology Center, Chapel Hill, NC, USA)

Finanzierung / Present funding

1. DFG Forschergruppe 604, 2005-2011: Signalling pathways in the healthy and diseased heart (Coordinator T. Eschenhagen, Principal investigator L. Carrier)
2. EU-FP7-Health-BIG-Heart, 2010-2012: Bench-to-bedside InteGrated approach to familial hypertrophic cardiomyopathy: to the HEART of the disease (coordinator C. Redwood, Oxford; principal investigator L. Carrier)
3. Fritz Thyssen Stiftung (Az. 10.09.1.139), 2010-2011: Familial hypertrophic cardiomyopathy: Evaluation of new molecular mechanisms and tools for therapy (principal investigator L. Carrier)

Ausgewählte Publikationen / Selected original publications (out of 70)

• Carrier L, Hengstenberg C, Beckmann JS, Guicheney P, Dufour C, Bercovici J, Dausse E, Berebbi-Bertrand I, Wisnewsky C, Pulvenis D, Fetler L, Vignal A, Weissenbach J, Hillaire D, Feingold J, Bouhour JB, Hagege A, Desnos M, Isnard R, Dubourg O, Komajda M, Schwartz K. Mapping of a novel gene for familial hypertrophic cardiomyopathy to chromosome 11. Nature Genet. 1993; 4:311-313.
• Bonne G / Carrier L, Bercovici J, Cruaud C, Richard P, Hainque B, Gautel M, Labeit S, James M, Beckmann JS, Weissenbach J, Vosberg HP, Fiszman M, Komajda M, Schwartz K. Cardiac myosin binding protein-C gene splice acceptor site mutation is associated with familial hypertrophic cardiomyopathy. Nature Genet. 1995; 11:438-440.
• Carrier L, Bonne G, Bährend E, Yu B, Richard P, Niel F, Hainque B, Cruaud C, Gary F, Labeit S, Bouhour JB, Dubourg O, Desnos M, Hagège AA, Trent RJ, Komajda M, Schwartz K. Organization and sequence of human cardiac myosin binding protein C gene (MYBPC3) and identification of mutations predicted to produce truncated proteins in familial hypertrophic cardiomyopathy. Circ Res 1997; 80:427-434.
• Fougerousse F, Delezoide AL, Fiszman MY, Schwartz K, Beckmann JS, Carrier L. Cardiac myosin binding protein C gene is specifically expressed in heart during murine and human development. Circ. Res. 1998; 82:130-133.
• Flavigny J, Souchet M, Sébillon P, Berrebi-Bertrand I, Hainque B, Mallet A, Bril A, Schwartz K, Carrier L. COOH-terminal truncated cardiac myosin-binding protein C mutants resulting from familial hypertrophic cardiomyopathy mutations exhibit altered expression and/or incorporation in fetal rat cardiomyocytes. J Mol Biol 1999; 294:443-456.
• Flavigny J, Robert P, Camelin J, Schwartz K, Carrier L, Berebbi-Bertrand I. Biomolecular Interactions Between Human recombinant beta-MyHC and cMyBP-Cs implicated in Familial Hypertrophic Cardiomyopathy. Cardiovasc. Res. 2003; 60:388-396.
• Richard P, Charron P, Carrier L, Ledeuil C, Cheav T, Pichereau C, Benaiche A, Isnard R, Dubourg O, Burban M, Gueffet JP, Millaire A, Desnos M, Schwartz K, Hainque B, Komajda M. Hypertrophic Cardiomyopathy: Distribution of disease genes, spectrum of mutations and implications for molecular diagnosis strategy. Circulation 2003; 107:2227-2232.
• Keller DI, Coirault C, Rau T, Cheav T, Weyand M, Amann K, Lecarpentier Y, Richard P, Eschenhagen T, Carrier L. Human homozygous R403W mutant cardiac myosin presents disproportionate enhancement of mechanical and enzymatic properties. J Mol Cell Cardiol 2004; 36:355-362 (Associated Editorial)
• Carrier L, Knöll R, Vignier N, Keller DI, Ambroisine ML, Bausero P, Prudhon B, Isnard R, Fiszman M, Ross J, Schwartz K, Chien KR. Asymmetric septal hypertrophy in heterozygous cardiac myosin-binding protein C null mice. Cardiovasc Res 2004; 63:293-304.
• Vu Manh TP, Mokrane M, Georgenthum G, Flavigny J, Carrier L, Sémériva M, Piovant M, Röder L. Expression of cardiac myosin binding protein C (cMyBP-C) in Drosophila as a model for the study of human cardiomyopathies. Hum Mol Genet 2005; 14:7-17.
• Sarikas A / Carrier L, Schenke C, Flavigny J, Lindenberg K, Eschenhagen T, Zolk O. Impairment of the ubiquitin-proteasome system by truncated cardiac myosin-binding protein C mutants. Cardiovasc Res 2005; 66:33-44 (Associated Editorial).
• Cazorla O, Szilagyi S, Vignier N, Salazar G, Krämer E, Vassort G, Carrier L, Lacampagne A. (2006) Length and PKA modulations of myocytes in cardiac myosin-binding protein C deficient mice. Cardiovasc Res., 69:370-380 (Associated Editorial).
• El-Armouche, A, Boknik, P, Eschenhagen, T, Carrier, L, Knaut, M, Ravens, U, Dobrev, D. (2006) Molecular determinants of altered Ca²⁺-handling in human chronic atrial fibrillation. Circulation 114:670-680.
• Pohlmann L, Kröger I, Vignier N, Schlossarek S, Krämer L, Coirault, C, Sultan KR, El-Armouche A, Winegrad, S, Eschenhagen T, Carrier L (2007) Cardiac myosin-binding protein C is required for complete relaxation in intact myocytes. Circ Res 101:928-938.
• Eijssen LM, van den Bosch BJ, Vignier N, Lindsey PJ, van den Burg CM, Carrier L, Doevendans PA, van der Vusse GJ, Smeets HJ (2008) Altered myocardial gene expression reveals possible maladaptative processes in heterozygous and homozygous cardiac myosin binding protein C knockout mice. Genomics 91:52-60.
• Lecarpentier Y, Vignier N, Oliviero P, Guellich A, Cortes-Morichetti M, Carrier L, Coirault C (2008) Cardiac myosin-binding protein C modulates the tuning of the molecular motors in the heart. Biophys J 95:1-9.
• Bahrudin U, Morisaki H, Morisaki T, Ninomiya H, Nanba E, Igawa O, Mizuta E, Miake J, Yamamoto Y, Shirayoshi Y, Kitakaze M Carrier L, Hisatome I (2008) Role of the ubiquitin-proteasome system in the development of heart failure in hypertrophic cardiomyopathy caused by a mutation of cardiac myosin binding protein C. J Mol Biol 284:896-907.
• Mearini G, Schlossarek S, Willis M, Carrier L (2008) The ubiquitin-proteasome system in cardiac dysfunction. Biochem Biophys Acta 1782:749-763.
• Van Dijk SJ, Dooijes D, dos Remedios C, Michels M, Lamers JMJ, Schlossarek S, Carrier L, ten Cate FJ, Stienen GJM, van der Velden J (2009). Cardiac myosin-binding protein C mutations and hypertrophic cardiomyopathy: haploinsufficiency, deranged phosphorylation and cardiomyocyte dysfunction. Circulation 119:1473-1483.
• Duncker DJ, Boontje NM, Merkus D, Versteilen A, Krysiak J, Mearini G, El-Armouche A, de Beer VJ, Lamers JJM, Carrier L, Walker LA, Linke WA, Stienen GJM, van der Velden J (2009) Prevention of myofilament dysfunction by beta-blocker therapy in post-infarct remodeling. Circ Heart Fail 2:233-242.
• Friedrich F, Bausero P, Sun Y, Treszl A, Kraemer E, Juhr D, Richard P, K. Wegscheider, Schwartz K, Brito D, Arbustini E, Waldenström A, Isnard R, Komajda M, Eschenhagen T, Carrier L (2009) A new polymorphism in human calmodulin III gene promoter is a potential modifier gene for familial hypertrophic cardiomyopathy. Eur Heart J 30:1648-55.
• Vignier N / Schlossarek S, Fraysse B, Mearini G, Krämer E, Pointu H, Mougenot N, Guiard J, Reimer R, Hohenberg H, Schwartz K, Vernet M, Eschenhagen T, Carrier L (2009) Nonsense-mediated mRNA decay and ubiquitin-proteasome system regulate cMyBP-C mutant levels in cardiomyopathic mice. Circ Res 105:239-248.
• Carrier L, Schlossarek S, Willis M, Eschenhagen T (2010) Ubiquitin-proteasome system and nonsense-mediated mRNA decay in hypertrophic cardiomyopathy. Cardiovasc Res 85:230-238
• Mearini G, Gedicke C, Schlossarek S, Witt, CC, Krämer E, Cao P, Gomes MD, Lecker SH, Labeit S, Willis MS, Eschenhagen T, Carrier L (2009) Atrogin-1 and MuRF1 regulate cMyBP-C level of via different mechanisms. Cardiovasc Res 85:357-366.
• Carrier L (2010) Too much of a good thing is bad: proteasome inhibition induces stressed hearts to fail. Cardiovasc Res 88:389-390 (Editorial).
• Schlossarek S, Mearini G, Carrier L (2011) Cardiac myosin-binding protein C in hypertrophic cardiomyopathy: mechanisms and therapeutic opportunities. J Mol Cell Cardiol 50:613-620.
• Schlossarek S, Carrier L (2011) The ubiquitin-proteasome system in cardiomyopathies. Curr Opin Cardiol 26: 190-195.