title: A novel mutant cardiac troponin C disrupts molecular motions critical for calcium binding affinity and cardiomyocyte contractility.
abstract: Troponin C (TnC) belongs to the superfamily of EF-hand (helix-loop-helix) Ca(2+)-binding proteins and is an essential component of the regulatory thin filament complex. In a patient diagnosed with idiopathic dilated cardiomyopathy, we identified two novel missense mutations localized in the regulatory Ca(2+)-binding Site II of TnC, TnC((E59D,D75Y)). Expression of recombinant TnC((E59D,D75Y)) in isolated rat cardiomyocytes induced a marked decrease in contractility despite normal intracellular calcium homeostasis in intact cardiomyocytes and resulted in impaired myofilament calcium responsiveness in Triton-permeabilized cardiomyocytes. Expression of the individual mutants in cardiomyocytes showed that TnC(D75Y) was able to recapitulate the TnC((E59D,D75Y)) phenotype, whereas TnC(E59D) was functionally benign. Force-pCa relationships in TnC((E59D,D75Y)) reconstituted rabbit psoas fibers and fluorescence spectroscopy of TnC((E59D,D75Y)) labeled with 2-[(4'-iodoacetamide)-aniline]naphthalene-6-sulfonic acid showed a decrease in myofilament Ca(2+) sensitivity and Ca(2+) binding affinity, respectively. Furthermore, computational analysis of TnC showed the Ca(2+)-binding pocket as an active region of concerted motions, which are decreased markedly by mutation D75Y. We conclude that D75Y interferes with proper concerted motions within the regulatory Ca(2+)-binding pocket of TnC that hinders the relay of the thin filament calcium signal, thereby providing a primary stimulus for impaired cardiomyocyte contractility. This in turn may trigger pathways leading to aberrant ventricular remodeling and ultimately a dilated cardiomyopathy phenotype.
authors.affiliation: Department of Cardiovascular Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA. chee.lim@vanderbilt.edu
authors.forename: Chee Chew
authors.initials: CC
authors.lastname: Lim
authors.forename: Haijun
authors.initials: H
authors.lastname: Yang
authors.forename: Mingfeng
authors.initials: M
authors.lastname: Yang
authors.forename: Chien-Kao
authors.initials: CK
authors.lastname: Wang
authors.forename: Jianru
authors.initials: J
authors.lastname: Shi
authors.forename: Eric A
authors.initials: EA
authors.lastname: Berg
authors.forename: David R
authors.initials: DR
authors.lastname: Pimentel
authors.forename: Judith K
authors.initials: JK
authors.lastname: Gwathmey
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Hajjar
authors.forename: Michiel
authors.initials: M
authors.lastname: Helmes
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Costello
authors.forename: Shuanghong
authors.initials: S
authors.lastname: Huo
authors.forename: Ronglih
authors.initials: R
authors.lastname: Liao
mesh_terms: D008841:Actin Cytoskeleton / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability; D005786:Gene Expression Regulation; D006801:Humans; D009068:Movement / Q000502:physiology*; D020125:Mutation, Missense; D009200:Myocardial Contraction / Q000235:genetics* / Q000502:physiology; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding; D016658:Psoas Muscles / Q000166:cytology / Q000378:metabolism; D011817:Rabbits; D012518:Sarcomeres / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D019209:Troponin C / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D019209:Troponin C; D002118:Calcium