As next generation sequencing for the genetic diagnosis of cardiovascular disorders becomes more widely used, establishing causality for putative disease causing variants becomes increasingly relevant. Diseases of the cardiac sarcomere provide a particular challenge in this regard because of the complexity of assaying the effect of genetic variants in human cardiac contractile proteins.
In this study we identified a novel variant R205Q in the cardiac troponin T gene (TNNT2). Carriers of the variant allele exhibited increased chamber volumes associated with decreased left ventricular ejection fraction. To clarify the causal role of this variant, we generated recombinant variant human protein and examined its calcium kinetics as well as the maximally activated ADP release of human β-cardiac myosin with regulated thin filaments containing the mutant troponin T. We found that the R205Q mutation significantly decreased the calcium sensitivity of the thin filament by altering the effective calcium dissociation kinetics.
The development of moderate throughput post-genomic assays is an essential step in the realization of the potential of next generation sequencing. Although technically challenging, biochemical and functional assays of human cardiac contractile proteins of the thin filament can be achieved and provide an orthogonal source of information to inform the question of causality for individual variants.
Hershberger RE, Morales A, Siegfried JD. Clinical and genetic issues in dilated cardiomyopathy: a review for genetics professionals. Genet Med. 2010; 12:655-67.
Lakdawala NK, Funke BH, Baxter S, Cirino AL, Roberts AE, Judge DP et al.. Genetic testing for dilated cardiomyopathy in clinical practice. J Card Fail. 2012; 18:296-303.
McNally EM, Golbus JR, Puckelwartz MJ. Genetic mutations and mechanisms in dilated cardiomyopathy. J Clin Invest. 2013; 123:19-26.
Hershberger RE, Pinto JR, Parks SB, Kushner JD, Li D, Ludwigsen S et al.. Clinical and functional characterization of TNNT2 mutations identified in patients with dilated cardiomyopathy. Circ Cardiovasc Genet. 2009; 2:306-13.
Pan S, Caleshu CA, Dunn KE, Foti MJ, Moran MK, Soyinka O et al.. Cardiac structural and sarcomere genes associated with cardiomyopathy exhibit marked intolerance of genetic variation. Circ Cardiovasc Genet. 2012; 5:602-10.
Mogensen J, Murphy RT, Shaw T, Bahl A, Redwood C, Watkins H et al.. Severe disease expression of cardiac troponin C and T mutations in patients with idiopathic dilated cardiomyopathy. J Am Coll Cardiol. 2004; 44:2033-40.
Exome Variant Server [http://evs.gs.washington.edu/EVS/]. Accessed January 20th, 2014.
Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc. 2009; 4:1073-81.
Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P et al.. A method and server for predicting damaging missense mutations. Nat Methods. 2010; 7:248-9.
Cooper GM. Distribution and intensity of constraint in mammalian genomic sequence. Genome Res. 2005; 15:901-13.
Sommese RF, Nag S, Sutton S, Miller SM, Spudich JA, Ruppel KM. Effects of troponin T cardiomyopathy mutations on the calcium sensitivity of the regulated thin filament and the actomyosin cross-bridge kinetics of human β-cardiac myosin. PLoS ONE. 2013; 8:e83403.
Willott RH, Gomes AV, Chang AN, Parvatiyar MS, Pinto JR, Potter JD. Mutations in Troponin that cause HCM, DCM AND RCM: what can we learn about thin filament function? J Mol Cell Cardiol. 2010; 48:882-92.
Davis JP, Norman C, Kobayashi T, Solaro RJ, Swartz DR, Tikunova SB. Effects of thin and thick filament proteins on calcium binding and exchange with cardiac troponin C. Biophys J. 2007; 92:3195-206.
Davis JP, Tikunova SB. Ca(2+) exchange with troponin C and cardiac muscle dynamics. Cardiovasc Res. 2008; 77:619-26.
McKillop DF, Geeves MA. Regulation of the interaction between actin and myosin subfragment 1: evidence for three states of the thin filament. Biophys J. 1993; 65:693-701.
Gordon AM, Homsher E, Regnier M. Regulation of contraction in striated muscle. Physiol Rev. 2000; 80:853-924.
Deacon JC, Bloemink MJ, Rezavandi H, Geeves MA, Leinwand LA. Erratum to: Identification of functional differences between recombinant human α and β cardiac myosin motors. Cell Mol Life Sci. 2012; 69:4239-55.
Mirza M, Marston S, Willott R, Ashley C, Mogensen J, McKenna W et al.. Dilated cardiomyopathy mutations in three thin filament regulatory proteins result in a common functional phenotype. J Biol Chem. 2005; 280:28498-506.
Norton N, Robertson PD, Rieder MJ, Züchner S, Rampersaud E, Martin E et al.. Evaluating pathogenicity of rare variants from dilated cardiomyopathy in the exome era. Circ Cardiovasc Genet. 2012; 5:167-74.
Jabbari J, Jabbari R, Nielsen MW, Holst AG, Nielsen JB, Haunsø S et al.. New exome data question the pathogenicity of genetic variants previously associated with catecholaminergic polymorphic ventricular tachycardia. Circ Cardiovasc Genet. 2013; 6:481-9.
Andreasen C, Nielsen JB, Refsgaard L, Holst AG, Christensen AH, Andreasen L et al.. New population-based exome data are questioning the pathogenicity of previously cardiomyopathy-associated genetic variants. Eur J Hum Genet. 2013; 21:918-28.
Manning EP, Tardiff JC, Schwartz SD. A model of calcium activation of the cardiac thin filament. Biochemistry. 2011; 50:7405-13.
Stehle R, Iorga B. Kinetics of cardiac sarcomeric processes and rate-limiting steps in contraction and relaxation. J Mol Cell Cardiol. 2010; 48:843-50.
McDonald KS, Herron TJ. It takes “heart” to win: what makes the heart powerful? News Physiol Sci. 2002; 17:185-90.
Lakdawala NK, Thune JJ, Colan SD, Cirino AL, Farrohi F, Rivero J et al.. Subtle abnormalities in contractile function are an early manifestation of sarcomere mutations in dilated cardiomyopathy. Circ Cardiovasc Genet. 2012; 5:503-10.
Sommese RF, Sung J, Nag S, Sutton S, Deacon JC, Choe E et al.. Molecular consequences of the R453C hypertrophic cardiomyopathy mutation on human β-cardiac myosin motor function. Proc Natl Acad Sci. 2013; 110:12607-12.
Szczesna D, Zhang R, Zhao J, Jones M, Guzman G, Potter JD. Altered regulation of cardiac muscle contraction by troponin T mutations that cause familial hypertrophic cardiomyopathy. J Biol Chem. 2000; 275:624-30.
Pan BS, Potter JD. Two genetically expressed troponin T fragments representing alpha and beta isoforms exhibit functional differences. J Biol Chem. 1992; 267:23052-6.
Sheng Z, Pan BS, Miller TE, Potter JD. Isolation, expression, and mutation of a rabbit skeletal muscle cDNA clone for troponin I. The role of the NH2 terminus of fast skeletal muscle troponin I in its biological activity. J Biol Chem. 1992; 267:25407-13.
Szczesna D, Guzman G, Miller T, Zhao J, Farokhi K, Ellemberger H et al.. The role of the four Ca2+ binding sites of troponin C in the regulation of skeletal muscle contraction. J Biol Chem. 1996; 271:8381-6.
Smillie LB. Preparation and identification of alpha- and beta-tropomyosins. Methods Enzymol. 1982; 85 Pt B:234-41.
La Cruz De EM, Ostap EM. Kinetic and equilibrium analysis of the myosin ATPase. Methods Enzymol. 2009; 455:157-92.
Dweck D, Reyes-Alfonso A, Potter JD. Expanding the range of free calcium regulation in biological solutions. Anal Biochem. 2005; 347:303-15.
Liu B, Tikunova SB, Kline KP, Siddiqui JK, Davis JP. Disease-related cardiac troponins alter thin filament Ca2+ association and dissociation rates. PLoS ONE. 2012; 7:e38259.
Kouyama T, Mihashi K. Fluorimetry study of N-(1-pyrenyl)iodoacetamide-labelled F-actin. Local structural change of actin protomer both on polymerization and on binding of heavy meromyosin. Eur J Biochem. 1981; 114:33-8.