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) Ca2+-binding proteins and is an essential component of the regulatory thin. lament complex. In a patient diagnosed with idiopathic dilated cardiomyopathy, we identified two novel missense mutations localized in the regulatory Ca2+-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 Ca2+ sensitivity and Ca2+ binding affinity, respectively. Furthermore, computational analysis of TnC showed the Ca2+-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 Ca2+-binding pocket of TnC that hinders the relay of the thin. lament 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.