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A Novel Mutant Cardiac Troponin C Disrupts Molecular Motions Critical for Calcium Binding Affinity and Cardiomyocyte Contractility

Chee Chew Lim ^* , Haijun Yang , Mingfeng Yang , Chien-Kao Wang ^¶, Jianru Shi , Eric A. Berg , David R. Pimentel , Judith K. Gwathmey ^||, Roger J. Hajjar ^**, Michiel Helmes , Catherine E. Costello , Shuanghong Huo  and Ronglih Liao 

^* Department of Cardiovascular Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee; Department of Medicine and Biochemistry, Boston University School of Medicine, Boston, Massachusetts; Gustaf H. Carlson School of Chemistry and Biochemistry, Clark University, Worcester, Massachusetts; ^¶ Departments of Physiology and Biophysics, University of Washington, Seattle, Washington; ^|| Gwathmey Inc., Cambridge, Massachusetts; ^** Massachusetts General Hospital and Harvard Medical School, Cardiovascular Research Center, Charlestown, Massachusetts; IonOptix Europe, Wageningen, the Netherlands; and Cardiovascular Division, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts

Correspondence: Address reprint requests to Chee Chew Lim, PhD, Department of Cardiovascular Medicine, Vanderbilt University School of Medicine, 2220 Pierce Ave, PRB 359B, Nashville, TN 37232. Tel.: 615-936-2510; Fax: 615-936-1872, E-mail: chee.lim{at}vanderbilt.edu; or to Shuanghong Huo, PhD, Gustaf H. Carlson School of Chemistry and Biochemistry, Sackler Science Building, Rm. N200B, Clark University, 950 Main Street, Worcester, MA 01610. Tel: 508-793-7533, Fax: 508-793-8861, E-Mail: shuo{at}clarku.edu.

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 filament 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 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.