New insights into the functional significance of the acidic region of the unique N-terminal extension of cardiac troponin I Journal Article

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Authors: Henze, M.; Patrick, S. E.; Hinken, A.; Scruggs, S. B.; Goldspink, P.; de Tombe, P. P.; Kobayashi, M.; Ping, P.; Kobayashi, T.; Solaro, R. J.
Article 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-epsilon (PKCepsilon) 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.
Journal Title: Biochimica et biophysica acta
Volume: 1833
Issue: 4
ISSN: 0006-3002; 0006-3002
Publisher: Elsevier Inc  
Journal Place: Netherlands
Date Published: 2013
Start Page: 823
End Page: 832
Language: eng
DOI/URL:

10.1016/j.bbamcr.2012.08.012
Notes: LR: 20140402; CI: Copyright (c) 2012; GR: F32 HL099029/HL/NHLBI NIH HHS/United States; GR: HHSN268201000035C/PHS HHS/United States; GR: HL082923/HL/NHLBI NIH HHS/United States; GR: P01 HL062426/HL/NHLBI NIH HHS/United States; GR: P01 HL062426/HL/NHLBI NIH HHS/United States; GR: R01 HL022231/HL/NHLBI NIH HHS/United States; GR: R01 HL022231/HL/NHLBI NIH HHS/United States; GR: R01 HL082923/HL/NHLBI NIH HHS/United States; GR: T32 HL007692/HL/NHLBI NIH HHS/United States; GR: T32 HL007692/HL/NHLBI NIH HHS/United States; JID: 0217513; 0 (Protein Isoforms); 0 (Recombinant Proteins); 0 (Troponin I); EC 2.7.11.13 (Protein Kinase C-epsilon); EC 3.6.1.- (Adenosine Triphosphatases); SY7Q814VUP (Calcium); NIHMS407371; OID: NLM: NIHMS407371; OID: NLM: PMC3548050; 2012/07/02 [received]; 2012/08/10 [revised]; 2012/08/18 [accepted]; 2012/08/25 [aheadofprint]; ppublish