New insights into the functional significance of the acidic region of the unique N-terminal extension of cardiac troponin I. Biochim Biophys Acta 2013 Apr;1833(4):823-32
Date
09/04/2012Pubmed ID
22940544Pubmed Central ID
PMC3548050DOI
10.1016/j.bbamcr.2012.08.012Scopus ID
2-s2.0-84875076162 (requires institutional sign-in at Scopus site) 11 CitationsAbstract
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.
Author List
Henze M, Patrick SE, Hinken A, Scruggs SB, Goldspink P, de Tombe PP, Kobayashi M, Ping P, Kobayashi T, Solaro RJMESH terms used to index this publication - Major topics in bold
Adenosine TriphosphatasesAnimals
Calcium
Cardiomyopathy, Dilated
Gene Expression
Humans
Isometric Contraction
Kinetics
Male
Mice
Mice, Transgenic
Muscle Tonus
Mutation
Myocardium
Myofibrils
Phosphorylation
Protein Isoforms
Protein Kinase C-epsilon
Protein Structure, Tertiary
Recombinant Proteins
Troponin I
