title: Functional analysis of a unique troponin c mutation, GLY159ASP, that causes familial dilated cardiomyopathy, studied in explanted heart muscle.
abstract: BACKGROUND
  Familial dilated cardiomyopathy can be caused by mutations in the proteins of the muscle thin filament. In vitro, these mutations decrease Ca(2+) sensitivity and cross-bridge turnover rate, but the mutations have not been investigated in human tissue. We studied the Ca(2+)-regulatory properties of myocytes and troponin extracted from the explanted heart of a patient with inherited dilated cardiomyopathy due to the cTnC G159D mutation.
  
  
  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 Ca(2+)-activated force was similar in cTnC G159D and donor myocytes, but the Ca(2+) sensitivity of cTnC G159D myocytes was higher (EC(50) 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 Ca(2+) sensitivity (EC(50) G159D/donor=0.55 + or - 0.13), whereas the maximally activated sliding speed was unaltered. In addition, the cTnC G159D mutation blunted the change in Ca(2+) sensitivity when TnI was dephosphorylated. With wild-type troponin, Ca(2+) sensitivity was increased (EC(50) P/unP=4.7 + or - 1.9) but not with cTnC G159D troponin (EC(50) P/unP=1.2 + or - 0.1).
  
  
  CONCLUSIONS
  We propose that uncoupling of the relationship between phosphorylation and Ca(2+) 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.
authors.affiliation: NHLI, King's College London, United Kingdom.
authors.forename: Emma C
authors.initials: EC
authors.lastname: Dyer
authors.forename: Adam M
authors.initials: AM
authors.lastname: Jacques
authors.forename: Anita C
authors.initials: AC
authors.lastname: Hoskins
authors.forename: Douglas G
authors.initials: DG
authors.lastname: Ward
authors.forename: Clare E
authors.initials: CE
authors.lastname: Gallon
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Messer
authors.forename: Juan Pablo
authors.initials: JP
authors.lastname: Kaski
authors.forename: Michael
authors.initials: M
authors.lastname: Burch
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Kentish
authors.forename: Steven B
authors.initials: SB
authors.lastname: Marston
mesh_terms: D000199:Actins / Q000378:metabolism; D001224:Aspartic Acid; D002118:Calcium / Q000378:metabolism*; D002311:Cardiomyopathy, Dilated / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D002675:Child, Preschool; D003599:Cytoskeleton / Q000378:metabolism*; D005838:Genotype; D005998:Glycine; D006801:Humans; D009154:Mutation; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010641:Phenotype; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D014335:Tropomyosin / Q000378:metabolism; D019209:Troponin C / Q000235:genetics / Q000378:metabolism*; D019210:Troponin I / Q000378:metabolism; D020107:Troponin T / Q000378:metabolism
chemical_list: D000199:Actins; D011994:Recombinant Proteins; D014335:Tropomyosin; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T; D001224:Aspartic Acid; D002118:Calcium; D005998:Glycine