title: Troponin I Mutations R146G and R21C Alter Cardiac Troponin Function, Contractile Properties, and Modulation by Protein Kinase A (PKA)-mediated Phosphorylation.
abstract: Two hypertrophic cardiomyopathy-associated cardiac troponin I (cTnI) mutations, R146G and R21C, are located in different regions of cTnI, the inhibitory peptide and the cardiac-specific N terminus. We recently reported that these regions may interact when Ser-23/Ser-24 are phosphorylated, weakening the interaction of cTnI with cardiac TnC. Little is known about how these mutations influence the affinity of cardiac TnC for cTnI (KC-I) or contractile kinetics during β-adrenergic stimulation. Here, we tested how cTnI(R146G) or cTnI(R21C) influences contractile activation and relaxation and their response to protein kinase A (PKA). Both mutations significantly increased Ca(2+) binding affinity to cTn (KCa) and KC-I. PKA phosphorylation resulted in a similar reduction of KCa for all complexes, but KC-I was reduced only with cTnI(WT). cTnI(WT), cTnI(R146G), and cTnI(R21C) were complexed into cardiac troponin and exchanged into rat ventricular myofibrils, and contraction/relaxation kinetics were measured ± PKA phosphorylation. Maximal tension (Tmax) was maintained for cTnI(R146G)- and cTnI(R21C)-exchanged myofibrils, and Ca(2+) sensitivity of tension (pCa50) was increased. PKA phosphorylation decreased pCa50 for cTnI(WT)-exchanged myofibrils but not for either mutation. PKA phosphorylation accelerated the early slow phase relaxation for cTnI(WT) myofibrils, especially at Ca(2+) levels that the heart operates in vivo. Importantly, this effect was blunted for cTnI(R146G)- and cTnI(R21C)-exchanged myofibrils. Molecular dynamics simulations suggest both mutations inhibit formation of intra-subunit contacts between the N terminus and the inhibitory peptide of cTnI that is normally seen with WT-cTn upon PKA phosphorylation. Together, our results suggest that cTnI(R146G) and cTnI(R21C) blunt PKA modulation of activation and relaxation kinetics by prohibiting cardiac-specific N-terminal interaction with the cTnI inhibitory peptide.
authors.affiliation: From the Department of Bioengineering, University of Washington, Seattle, Washington 98105, the National Biomedical Computational Resource and.
authors.forename: Yuanhua
authors.initials: Y
authors.lastname: Cheng
authors.affiliation: From the Department of Bioengineering, University of Washington, Seattle, Washington 98105.
authors.forename: Vijay
authors.initials: V
authors.lastname: Rao
authors.affiliation: From the Department of Bioengineering, University of Washington, Seattle, Washington 98105.
authors.forename: An-Yue
authors.initials: AY
authors.lastname: Tu
authors.affiliation: Pharmacology, University of California at San Diego, La Jolla, California 92093, and.
authors.forename: Steffen
authors.initials: S
authors.lastname: Lindert
authors.affiliation: From the Department of Bioengineering, University of Washington, Seattle, Washington 98105.
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.affiliation: From the Department of Bioengineering, University of Washington, Seattle, Washington 98105.
authors.forename: Lucas
authors.initials: L
authors.lastname: Oxenford
authors.affiliation: the National Biomedical Computational Resource and Departments of Bioengineering and.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: McCulloch
authors.affiliation: the National Biomedical Computational Resource and Pharmacology, University of California at San Diego, La Jolla, California 92093, and.
authors.forename: J Andrew
authors.initials: JA
authors.lastname: McCammon
authors.affiliation: From the Department of Bioengineering, University of Washington, Seattle, Washington 98105, the Center for Cardiovascular Biology, University of Washington, Seattle, Washington 98105 mregnier@uw.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Regnier
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics; D005998:Glycine / Q000235:genetics; D006801:Humans; D008297:Male; D056004:Molecular Dynamics Simulation; D009154:Mutation; D009200:Myocardial Contraction / Q000235:genetics / Q000502:physiology*; D009210:Myofibrils / Q000502:physiology*; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D051381:Rats; D017207:Rats, Sprague-Dawley; D019210:Troponin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D019210:Troponin I; D001120:Arginine; D017868:Cyclic AMP-Dependent Protein Kinases; D003545:Cysteine; D002118:Calcium; D005998:Glycine
keywords: C-I interaction; cardiomyopathy; contraction; kinetics; molecular modeling; mutant; myofibril; relaxation; troponin; β-adrenergic