August 2009

Description of the team, research area and previous work

Lucie Carrier is a French scientist (Director of Research CNRS) who has been team leader at the INSERM unit 582 in Paris for several years. She received a  Marie Curie Excellence Grant to lead an European team at the Institute of Experimental and Clinical Pharmacology of the UKE in Hamburg. Her team is also part of the new 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 (FHC) for more than 15 years.

FHC 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 twelve different genes encoding proteins of the sarcomere. The first disease gene for FHC, the b-myosin heavy chain gene, was identified in the early 90^th 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 FHC 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 to recruit 350 unrelated European families with FHC. The major discoveries were (1) identification of the CMH4 locus on chromosome 11 in 1993 (Carrier et al., 1993), (2) identification of the FHC disease gene MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C) in 1995 (Bonne / Carrier et al., 1995), (3) determination of the complete structure and organization of the cMyBP-C gene in 1997 and demonstration that most of the cMyBP-C mutations are expected to produce C-terminal truncated proteins (Carrier et al., 1997), (4) identification of 97 different mutations in 9 different FHC genes in 124 unrelated families, evidence that 5% of the families present individuals with a complex genetic status who exhibit a more severe phenotype (Richard et al., 2003), identification of a new polymorphism in human calmodulin III gene promoter as a potential gene modifier for FHC (Friedrich et al., 2009).

Since the mechanisms by which MYBPC3 mutations lead to FHC was not  known, a large part of our projects aims at elucidating the functional consequences of FHC 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 b-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 FHC (Carrier et al, 2004), (5) human homozygous R403W mutant cardiac myosin presents disproportionate enhancement of mechanical and enzymatic properties (Keller et al., 2004), (5) truncated cMyBP-C mutants impair the ubiquitin-proteasome system, which could contribute to the pathogenesis of FHC.

Objectives

The major objective of the EU Marie Curie team is the understanding of the molecular mechanisms by which mutations in cMyBP-C lead to the development of FHC. Particularly, we investigate two new concepts, the first one emerging from our recent data: (1) Impairment by truncated cMyBP-C mutants of the ubiquitin-proteasome system (UPS) as a pathogenic factor of FHC in vivo (2) Specific mobilization and differentiation of recently identified Isl-1 positive cardioblasts as a cause of asymmetric septal hypertrophy. The other objectives concern the role of cMyBP-C in cardiac myofibrillogenesis and its PKA-dependent phosphorylation in the regulation of cardiac contraction.

Material and methods

·          Transgenic mice: (1) targeted cMyBP-C knockout, (2) targeted cMyBP-C knock in (point mutation leading to both E256K and truncated mutants), (3) cMyBP-C 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.

·          DNA bank of >350 unrelated patients with familial 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, Langendorf-perfused heart)

·          Cell biology and biochemistry (isolation and culture of neonatal and adult mouse cardiac myocytes, fluorescence and confocal microscopy, proteasome activities, immuno-precipitation, pulse-chase analysis.)

·          Molecular biology (DNA and RNA extractions, production of recombinant protein, Protein extraction and fractionation (Rotofor), PCR, RT-PCR, quantitative RT-PCR on Taqman, Western-blot analysis. several antibodies including phospho)

·          Ubiquitin-proteasome system: Ubiquitination assays in vitro (E1, E2, and muscle-scecific E3 ubiquitin ligases), measurement of the proteasome activities with fluoregenic substrates, antibodies directed against ubiquitin (FK2 and others) and against b5-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, ASB2b).

Collaborations

G. Bonne, C. Coirault, B. Fraysse L. Garcia, N. Vignier and T. Voit (INSERM U974-CNRS UMR7215, Paris, France)

N. Dantuma (Karolinska Institute, Stockolm)

A.H. Guse (Institut für Biochemie und Molecularbiologie, UKE, Hamburg)

S. Labeit and C. Witt (Institut für Anästhesiologie und Operative Intensivmedizin, Mannheim University-Hospital, Mannheim)

A. Lacampagne and O. Cazorla (INSERM, U667, Montpellier, France)

S. Lecker (Beth Israel Diacones Medical Center, Boston, MA, USA)

X. Wang (University of South Dakota, SD, USA)

S. Winegrad (Department of Physiology, University of Pennsylvania, Philadelphia, PA, USA)

M. Willis (Carolina Cardiovascular Biology Center, Chapel Hill, NC, USA)

Finanzierung / Present funding

(1)   Leducq Foundation, 2005-2009: CaPTAA: Isl-1 positive cardioblasts for cardiac regeneration (Coordinators K. Schwartz, Paris & K.R. Chien, Boston, Core Member L. Carrier)

(2)   DFG Forschergruppe 604, 2005-2011: Signalling pathways in healthy and diseased heart (Coordinator T. Eschenhagen, Principal investigator L. Carrier)

Ausgewählte Publikationen / Selected papers (out of 62)

·         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 b-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, in press