title: New insights into the functional significance of the acidic region of the unique N-terminal extension of cardiac troponin I.
abstract: Previous structural studies indicated a special functional role for an acidic region composed of residues 1-10 in the unique N-terminal peptide of cardiac troponin I (cTnI). Employing LC-MS/MS, we determined the presence of phosphorylation sites at S5/S6 in cTnI from wild type mouse hearts as well as in hearts of mice chronically expressing active protein kinase C-ε (PKCε) and exhibiting severe dilated cardiomyopathy (DCM). To determine the functional significance of these phosphorylations, we cloned and expressed wild-type cTnI, (Wt), and cTnI variants expressing pseudo-phosphorylation cTnI-(S5D), cTnI(S6D), as well as cTnI(S5A) and cTnI(S6A). We exchanged native Tn of detergent-extracted (skinned) fiber bundles with Tn reconstituted with the variant cTnIs and measured tension and cross-bridge dynamics. Compared to controls, myofilaments controlled by cTnI with pseudo-phosphorylation (S6D) or Ala substitution (S6A) demonstrated a significant depression in maximum tension, ATPase rate, and ktr, but no change in half-maximally activating Ca(2+). In contrast, pseudo-phosphorylation at position 5 (S5D) had no effects, although S5A induced an increase in Ca(2+)-sensitivity with no change in maximum tension or ktr. We further tested the impact of acidic domain modifications on myofilament function in studies examining the effects of cTnI(A2V), a mutation linked to DCM. This mutation significantly altered the inhibitory activity of cTnI as well as cooperativity of activation of myofilament tension, but not when S23/S24 were pseudo-phosphorylated. Our data indicate a new functional and pathological role of amino acid modifications in the N-terminal acidic domain of cTnI that is modified by phosphorylations at cTnI(S23/S24). This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.
authors.affiliation: Department of Physiology and Biophysics, University of Illinois, Chicago, IL 60612, USA.
authors.forename: Marcus
authors.initials: M
authors.lastname: Henze
authors.forename: Stacey E
authors.initials: SE
authors.lastname: Patrick
authors.forename: Aaron
authors.initials: A
authors.lastname: Hinken
authors.forename: Sarah B
authors.initials: SB
authors.lastname: Scruggs
authors.forename: Paul
authors.initials: P
authors.lastname: Goldspink
authors.forename: Pieter P
authors.initials: PP
authors.lastname: de Tombe
authors.forename: Minae
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Peipei
authors.initials: P
authors.lastname: Ping
authors.forename: Tomoyoshi
authors.initials: T
authors.lastname: Kobayashi
authors.forename: R John
authors.initials: RJ
authors.lastname: Solaro
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002311:Cardiomyopathy, Dilated / Q000378:metabolism* / Q000473:pathology; D015870:Gene Expression; D006801:Humans; D007537:Isometric Contraction; D007700:Kinetics; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009129:Muscle Tonus; D009154:Mutation; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D009210:Myofibrils / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051744:Protein Kinase C-epsilon / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D020033:Protein Isoforms; D011994:Recombinant Proteins; D019210:Troponin I; D051744:Protein Kinase C-epsilon; D000251:Adenosine Triphosphatases; D002118:Calcium