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 Ca2+. In contrast, pseudo-phosphorylation at position 5 (S5D) had no effects, although S5A induced an increase in Ca2+-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.
Highlights
We investigated the functional significance of modifications in an acidic region comprised of residues 1–10 in the unique N-terminal peptide of cardiac troponin I. cTnI, in conjunction with the Ca-receptor, cardiac troponin C, the scaffolding protein, cardiac troponin T, and tropomyosin (Tm), functions in a molecular mechanism to switch on and modulate sarcomeric force and shortening [1, 2] In relaxed sarcomeres, two basic domains of cTnI are tethered to actin and together with the N-terminal extension of cTnT, hold Tm in a position to block force generating cross-bridges from reacting with thin filament actins
Results of recent studies have significantly altered thinking regarding the role of the unique N-terminus in control of cardiac function [7, 14, 15] The N-terminus did not resolve in the elucidation of the core crystal structure of cTnI [16], but Howarth et al [14] determined the NMR solution structure of cTnI and employed bioinformatic analysis together with data from small angle x-ray scattering experiments to model the N-terminal extension into the core crystal structure of cTn
To identify novel sites of cTnI phosphorylation in mouse myocardium that potentially play a role in the maladaptive functional response, sarcomeric sub-proteomes were analyzed via LC-MS/MS
Summary
We investigated the functional significance of modifications in an acidic region comprised of residues 1–10 in the unique N-terminal peptide of cardiac troponin I (cTnI). cTnI, in conjunction with the Ca-receptor, cardiac troponin C (cTnC), the scaffolding protein, cardiac troponin T (cTnT), and tropomyosin (Tm), functions in a molecular mechanism to switch on and modulate sarcomeric force and shortening [1, 2] In relaxed sarcomeres, two basic domains (an inhibitory peptide, Ip, and a second actin binding region [3, 4]) of cTnI are tethered to actin and together with the N-terminal extension of cTnT, hold Tm in a position to block force generating cross-bridges from reacting with thin filament actins. We investigated the functional significance of modifications in an acidic region comprised of residues 1–10 in the unique N-terminal peptide of cardiac troponin I (cTnI). Results of recent studies have significantly altered thinking regarding the role of the unique N-terminus in control of cardiac function [7, 14, 15] The N-terminus did not resolve in the elucidation of the core crystal structure of cTnI [16], but Howarth et al [14] determined the NMR solution structure of cTnI (aa 1–32) and employed bioinformatic analysis together with data from small angle x-ray scattering experiments to model the N-terminal extension into the core crystal structure of cTn. In the non-phosphorylated state, the model demonstrated a close interaction between the N-terminal extension with cTnC near L29 as previously shown in peptide-array experiments [13]. Our results indicate that phosphorylation and structural modifications depress myofilament tension and that the A2V mutation alters myofilament cooperative activation as well as responsiveness to phosphorylation of cTnI at S23/S24
Sign-in/Register to view highlights & summary 
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callMore From: Biochimica et Biophysica Acta (BBA) - Molecular Cell Research
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
