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Original Articles

Functional Analysis of a Unique Troponin C Mutation, GLY159ASP, that Causes Familial Dilated Cardiomyopathy, Studied in Explanted Heart Muscle

Emma C. Dyer, PhD; Adam M. Jacques, MD; Anita C. Hoskins, PhD; Douglas G. Ward, PhD; Clare E. Gallon, PhD; Andrew E. Messer, PhD; Juan Pablo Kaski, MD; Michael Burch, MD; Jonathan C. Kentish, PhD and Steven B. Marston, DPhil, DSc

From the NHLI (E.C.D., A.M.J., C.E.G., A.E.M., S.B.M.), Imperial College London; Cardiovascular Division (A.C.H., J.C.K.), King’s College London; Great Ormond St Hospital (M.B.); University College London (J.P.K.); and School of Cancer Sciences (D.G.W.), Birmingham University, Birmingham, London, United Kingdom.

Correspondence to Steven Marston, DPhil, DSc, Cardiovascular Science, National Heart and Lung Institute, Imperial College London, Dovehouse Street, London SW3 6LY, United Kingdom. E-mail s.marston{at}imperial.ac.uk

Received August 29, 2008; accepted June 9, 2009.

Background— Familial dilated cardiomyopathy can be caused by mutations in the proteins of the muscle thin filament. In vitro, these mutations decrease Ca²⁺ sensitivity and cross-bridge turnover rate, but the mutations have not been investigated in human tissue. We studied the Ca²⁺-regulatory properties of myocytes and troponin extracted from the explanted heart of a patient with inherited dilated cardiomyopathy due to the cTnC G159D mutation.

Methods and Results— Mass spectroscopy showed that the mutant cTnC was expressed approximately equimolar with wild-type cTnC. Contraction was compared in skinned ventricular myocytes from the cTnC G159D patient and nonfailing donor heart. Maximal Ca²⁺-activated force was similar in cTnC G159D and donor myocytes, but the Ca²⁺ sensitivity of cTnC G159D myocytes was higher (EC₅₀ G159D/donor=0.60). Thin filaments reconstituted with skeletal muscle actin and human cardiac tropomyosin and troponin were studied by in vitro motility assay. Thin filaments containing the mutation had a higher Ca²⁺ sensitivity (EC₅₀ G159D/donor=0.55±0.13), whereas the maximally activated sliding speed was unaltered. In addition, the cTnC G159D mutation blunted the change in Ca²⁺ sensitivity when TnI was dephosphorylated. With wild-type troponin, Ca²⁺ sensitivity was increased (EC₅₀ P/unP=4.7±1.9) but not with cTnC G159D troponin (EC₅₀ P/unP=1.2±0.1).

Conclusions— We propose that uncoupling of the relationship between phosphorylation and Ca²⁺ sensitivity could be the cause of the dilated cardiomyopathy phenotype. The differences between these data and previous in vitro results show that native phosphorylation of troponin I and troponin T and other posttranslational modifications of sarcomeric proteins strongly influence the functional effects of a mutation.

Key Words: cardiomyopathy • contractility • heart failure

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