Contractile Proteins
Professor Steve Marston, Head of Group
We study cardiac muscle contractility, its regulation by Ca2+ and its modulation by PKA-dependent phosphorylation
We use a quantitative in vitro motility assay to measure the unloaded contractility of synthetic thin filaments from purified actin, tropomyosin and troponin and we manipulate the phosphorylation levels of tropoinin I by PKA or phosphatase treatment. We have also developed the phosphate affinity SDS-PAGE method for quantitaive determination of phosphorylation levels of troponin I, MyBP-C and MLC-2 in situ in heart tissue.
Our main interest is the mechanism by which mutations in the contractile proteins lead to cardiomyopathy.
Hypertrophic cardiomyopathy is the most common inherited heart disease and is overwhelmingly due to mutations in sarcomeric proteins. At the single filament level we have found HCM mutations cause a higher Ca2+-sensitivity and incomplete relaxation.
Familial dilated cardiomyopathy may account for 30% of dilated cardiomyopathy cases and is a sigmificant cause of heart failure. When we studied recombinant proteins we found that Ca2+-sensitivity was decreased, but when we studied mutations in tissue samples, either from patients with DCM or in a transgenic mouse model of DCM (ACTC E361G mutation) we found that in fact the only consistent molecular phenotype in DCM is an uncoupling of the change in Ca2+-sensitivity normally associated with troponin I phosphorylation, which blunts the inotropic response.
The recombinant protein studies have indicated a clear molecular phenotype common to all familial DCM mutations (Red Line): lower Ca2+ sensitivity, crossbridge turnover rate and cooperativity compared with wild-type (black line).
The DCM molecular phenotype is the opposite of the molecular phenotype of HCM (green line) and also differs from acquired heart failure (purple line) where reduced crossbridge turnover rate is associated with higher Ca2+- sensitivity.
Current projects
We are working on the molecular mechanisms by which cardiomyopathy mutations in contractile proteins cause hypertrophic cardiomyopathy or dilated cardiomyopathy
Our working materials are a collection of human heart samples with identifed disease-causing mutations and transgenic mouse models of DCM (ACTC E361G) and HCM (ACTC E99K). We investigate regulation of contractility in the samples by in vitro motility assay, single myofibrils and isolated papilary muscle (for mouse). we also study post-translational modifications that impact on heart function, especially phosphorylation of Troponin I and myosin binding protein C by PKA and O-GlcNac modifications of alpha-actinin
Major funding comes from BHF grants including a programme Grant and from EU Framework 7 programme, BIG-Heart. To do this research we have built up collaborations with laboratories at Imperial, on the UK, Europe and elsewhere.
BHF Programme
In the BHF-funded programme we investigate the hypothesis that contractile apparatus dysfunction plays an important role in heart failure and is the initiating factor in cases of HCM and DCM caused by contractile protein mutations.
FP7 BIG-Heart
The overarching aim of the Big-Heart project is to test specific hypotheses that have emerged from reductionist studies of disease mechanisms in HCM as we believe that these will provide tractable targets for disease-modifying therapy.
We are also studying the molecular mechanism of congenital skeletal muscle myopathies that are caused by mutations in contractile proteins, notably actin (ACTA1) and tropomyosin (TPM2 and TPM3). We do these experiements in collaboration with clinicians and geneticists of the European Neuromuscular Centre
- Caroline Sewry, Francesco Muntoni (Great Ormond Street)
- Nigel Laing, Kristen Nowak (Perth, Australia)
- Alan Beggs (Harvard)
- Nigel Clarke, Cathy North (Sydney, Australia)
References
Copeland O; Sadayappan S; Messer AE; Stienen GJ; van der Velden J; Marston SB. (17 Sep 2010). Analysis of cardiac myosin binding protein-C phosphorylation in human heart muscle. J Mol Cell Cardiol. 49: 380-389.
Song W; Dyer E; Stuckey D; Leung MC; Memo M; Mansfield C; Ferenczi M; Liu K; et al. (Sep 2010). Investigation of a transgenic mouse model of familial dilated cardiomyopathy. J Mol Cell Cardiol. 49:380-389.
Dyer E, Jacques A, Hoskins A, Ward D, Gallon C, Messer A, Kaski J, Burch M, Kentish J, Marston S. Functional Analysis of a Unique Troponin C Mutation, Gly159Asp that Causes Familial Dilated Cardiomyopathy, Studied in Explanted Heart Muscle. Circulation: Heart Failure. 2009;2:456-64.
Marston S, Copeland O, Jacques A, Livesey K, Tsang V, McKenna WJ, Jalilzadeh S, Carballo S, Redwood C, Watkins H. Evidence from human myectomy samples that MYBPC3 mutations cause hypertrophic cardiomyopathy through haploinsufficiency. Circulation Research. 2009;105(3):219-22.
Messer A, Gallon C, McKenna W, Elliott P, Dos Remedios C, Marston S. The use of phosphate-affinity SDS-PAGE to measure the troponin I phosphorylation site distribution in human heart muscle. Prot Clin Appl. 2009;3(1371-1382):DOI: 10.1002/prca.200900071.
Jacques A, Hoskins A, Kentish J, Marston SB. From genotype to phenotype: a longitudinal study of a patient with hypertrophic cardiomyopathy due to a mutation in the MYBPC3 gene. Journal of muscle research and cell motility. 2009 02/14;29(6-8):231-8.
Marston S, deTombe P. Point/Counterpoint. Troponin phosphorylation and myofilament Ca2+-sensitivity in heart failure: increased or decreased? . J Mol Cell Cardiol. 2008;45:603-7.
Feng JJ, Marston S. Genotype-Phenotype Correlations in ACTA1 Mutations That Cause Congenital Myopathies. Neuromusc Disord. 2009;19(1):6-16.
Clarke NF, Ilkovski B, Cooper S, Valova VA, Robinson PJ, Nonaka I, Feng JJ, Marston S, North K. The pathogenesis of ACTA1-related congenital fiber type disproportion. Ann Neurol. 2007 Mar 23;61(6):552-61.
Messer AE, Jacques AM, Marston SB. Troponin phosphorylation and regulatory function in human heart muscle: Dephosphorylation of Ser23/24 on troponin I could account for the contractile defect in end-stage heart failure. J Mol Cell Cardiol. 2007;42(1):247-59.
D'Amico A, Graziano C, Pacileo G, Petrini S, Nowak KJ, Boldrini R, Jacques A, Feng JJ, Porfirio B, Sewry CA, Santorelli FM, Limongelli G, Bertini E, Laing N, Marston SB. Fatal hypertrophic cardiomyopathy and nemaline myopathy associated with ACTA1 K336E mutation. Neuromuscul Disord. 2006 Aug 29;16:548-52.
Mirza M, Marston S, Willott R, Ashley C, Mogensen J, McKenna W, Robinson P, Redwood C, Watkins H. Dilated cardiomyopathy mutations in three thin filament regulatory proteins result in a common functional phenotype. J Biol Chem. 2005 May 27;280:28498-506.
