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.