Published Online
on July 6, 2009

A more recent version of this article appeared on September 1, 2009

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Original Article

Functional Analysis of a Unique Troponin C Mutation, Gly159Asp that Causes Familial Dilated Cardiomyopathy, Studied in Explanted Heart Muscle.

Emma C. Dyer¹; Adam M. Jacques¹; Anita C. Hoskins²; Douglas G. Ward³; Clare E. Gallon¹; Andrew E. Messer¹; Juan Pablo Kaski⁴; Michael Burch⁵; Jonathan C. Kentish² and Steven B. Marston^1,6

¹ Imperial College London, London, UK;
² King's College London, London, UK;
³ University of Birmingham, Birmingham, UK;
⁴ University College London, London, UK;
⁵ Great Ormond Street Hospital, London, UK

⁶ E-mail: s.marston{at}imperial.ac.uk

Background—Familial dilated cardiomyopathy (DCM) can be caused by mutations in the proteins of the muscle thin filament. In vitro these mutations decrease Ca²⁺-sensitivity and crossbridge turnover rate but the mutations have not been investigated in human tissue. We have studied the Ca²⁺-regulatory properties of myocytes and troponin extracted from the explanted heart of a patient with inherited DCM 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 non-failing 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) whilst 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).

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

Key Words: cardiomyopathy • contractility • heart failure