A novel mutant cardiac troponin C disrupts molecular motions critical for calcium binding affinity and cardiomyocyte contractility.
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.
Lim
Chee Chew
CC
Department of Cardiovascular Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA. chee.lim@vanderbilt.edu
Yang
Haijun
H
Yang
Mingfeng
M
Wang
Chien-Kao
CK
Shi
Jianru
J
Berg
Eric A
EA
Pimentel
David R
DR
Gwathmey
Judith K
JK
Hajjar
Roger J
RJ
Helmes
Michiel
M
Costello
Catherine E
CE
Huo
Shuanghong
S
Liao
Ronglih
R
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*
D019209:Troponin C; D002118:Calcium