title: pH-responsive titratable inotropic performance of histidine-modified cardiac troponin I.
abstract: Cardiac troponin I (cTnI) functions as the molecular switch of the thin filament. Studies have shown that a histidine button engineered into cTnI (cTnI A164H) specifically enhances inotropic function in the context of numerous pathophysiological challenges. To gain mechanistic insight into the basis of this finding, we analyzed histidine ionization states in vitro by studying the myofilament biophysics of amino acid substitutions that act as constitutive chemical mimetics of altered histidine ionization. We also assessed the role of histidine-modified cTnI in silico by means of molecular dynamics simulations. A functional in vitro analysis of myocytes at baseline (pH 7.4) indicated similar cellular contractile function and myofilament calcium sensitivity between myocytes expressing wild-type (WT) cTnI and cTnI A164H, whereas the A164R variant showed increased myofilament calcium sensitivity. Under acidic conditions, compared with WT myocytes, the myocytes expressing cTnI A164H maintained a contractile performance similar to that observed for the constitutively protonated cTnI A164R variant. Molecular dynamics simulations showed similar intermolecular atomic contacts between the WT and the deprotonated cTnI A164H variant. In contrast, simulations of protonated cTnI A164H showed various potential structural configurations, one of which included a salt bridge between His-164 of cTnI and Glu-19 of cTnC. This salt bridge was recapitulated in simulations of the cTnI A164R variant. These data suggest that differential histidine ionization may be necessary for cTnI A164H to act as a molecular sensor capable of modulating sarcomere performance in response to changes in the cytosolic milieu.
authors.affiliation: Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, Minnesota, USA.
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Palpant
authors.forename: Evelyne M
authors.initials: EM
authors.lastname: Houang
authors.forename: Yuk Y
authors.initials: YY
authors.lastname: Sham
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Metzger
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D005260:Female; D018014:Gene Transfer Techniques; D006639:Histidine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D016296:Mutagenesis; D032383:Myocytes, Cardiac / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering; D011522:Protons; D051381:Rats; D012518:Sarcomeres / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D011522:Protons; D019210:Troponin I; D006639:Histidine; D002118:Calcium