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Site-specific acetyl-mimetic modification of cardiac troponin I modulates myofilament relaxation and calcium sensitivity.

ABSTRACT: OBJECTIVE:Cardiac troponin I (cTnI) is an essential physiological and pathological regulator of cardiac relaxation. Significant to this regulation, the post-translational modification of cTnI through phosphorylation functions as a key mechanism to accelerate myofibril relaxation. Similar to phosphorylation, post-translational modification by acetylation alters amino acid charge and protein function. Recent studies have demonstrated that the acetylation of cardiac myofibril proteins accelerates relaxation and that cTnI is acetylated in the heart. These findings highlight the potential significance of myofilament acetylation; however, it is not known if site-specific acetylation of cTnI can lead to changes in myofilament, myofibril, and/or cellular mechanics. The objective of this study was to determine the effects of mimicking acetylation at a single site of cTnI (lysine-132; K132) on myofilament, myofibril, and cellular mechanics and elucidate its influence on molecular function. METHODS:To determine if pseudo-acetylation of cTnI at 132 modulates thin filament regulation of the acto-myosin interaction, we reconstituted thin filaments containing WT or K132Q (to mimic acetylation) cTnI and assessed in vitro motility. To test if mimicking acetylation at K132 alters cellular relaxation, adult rat ventricular cardiomyocytes were infected with adenoviral constructs expressing either cTnI K132Q or K132 replaced with arginine (K132R; to prevent acetylation) and cell shortening and isolated myofibril mechanics were measured. Finally, to confirm that changes in cell shortening and myofibril mechanics were directly due to pseudo-acetylation of cTnI at K132, we exchanged troponin containing WT or K132Q cTnI into isolated myofibrils and measured myofibril mechanical properties. RESULTS:Reconstituted thin filaments containing K132Q cTnI exhibited decreased calcium sensitivity compared to thin filaments reconstituted with WT cTnI. Cardiomyocytes expressing K132Q cTnI had faster relengthening and myofibrils isolated from these cells had faster relaxation along with decreased calcium sensitivity compared to cardiomyocytes expressing WT or K132R cTnI. Myofibrils exchanged with K132Q cTnI ex vivo demonstrated faster relaxation and decreased calcium sensitivity. CONCLUSIONS:Our results indicate for the first time that mimicking acetylation of a specific cTnI lysine accelerates myofilament, myofibril, and myocyte relaxation. This work underscores the importance of understanding how acetylation of specific sarcomeric proteins affects cardiac homeostasis and disease and suggests that modulation of myofilament lysine acetylation may represent a novel therapeutic target to alter cardiac relaxation.

SUBMITTER: Lin YH

PROVIDER: S-EPMC7363438 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Site-specific acetyl-mimetic modification of cardiac troponin I modulates myofilament relaxation and calcium sensitivity.

Lin Ying H YH Schmidt William W Fritz Kristofer S KS Jeong Mark Y MY Cammarato Anthony A Foster D Brian DB Biesiadecki Brandon J BJ McKinsey Timothy A TA Woulfe Kathleen C KC

Journal of molecular and cellular cardiology 20200122

<h4>Objective</h4>Cardiac troponin I (cTnI) is an essential physiological and pathological regulator of cardiac relaxation. Significant to this regulation, the post-translational modification of cTnI through phosphorylation functions as a key mechanism to accelerate myofibril relaxation. Similar to phosphorylation, post-translational modification by acetylation alters amino acid charge and protein function. Recent studies have demonstrated that the acetylation of cardiac myofibril proteins accel ...[more]

PMID: 31981571

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Project description:Protein kinase A (PKA) phosphorylation of myofibril proteins constitutes an important pathway for β-adrenergic modulation of cardiac contractility and relaxation. PKA targets the N-terminus (Ser-23/24) of cardiac troponin I (cTnI), cardiac myosin-binding protein C (cMyBP-C) and titin. The effect of PKA-mediated phosphorylation on the magnitude of contraction has been studied in some detail, but little is known about how it modulates the kinetics of thin filament activation and myofibril relaxation as Ca(2+) levels vary. Troponin C (cTnC) interaction with cTnI (C-I interaction) is a critical step in contractile activation that can be modulated by cTnI phosphorylation. We tested the hypothesis that altering C-I interactions by PKA, or by cTnI phosphomimetic mutations (S23D/S24D-cTnI), directly affects thin filament activation and myofilament relaxation kinetics. Rat ventricular myofibrils were isolated and endogenous cTn was exchanged with either wild-type cTnI, or S23D/S24D-cTnI recombinant cTn. Contractile mechanics were monitored at maximum and submaximal Ca(2+) concentrations. PKA treatment of wild-type cTn or exchange of cTn containing S23D/S24D-cTnI resulted in an increase in the rate of early, slow phase of relaxation (kREL,slow) and a decrease in its duration (tREL,slow). These effects were greater for submaximal Ca(2+) activated contractions. PKA treatment also reduced the rate of contractile activation (kACT) at maximal, but not submaximal Ca(2+), and reduced the Ca(2+) sensitivity of contraction. Using a fluorescent probe coupled to cTnC (C35S-IANBD), the Ca(2+)-cTn binding affinity and C-I interaction were monitored. Ca(2+) binding to cTn (pCa50) was significantly decreased when cTnI was phosphorylated by PKA (ΔpCa50 = 0.31). PKA phosphorylation of cTnI also weakened C-I interaction in the presence of Ca(2+). These data suggest that weakened C-I interaction, via PKA phosphorylation of cTnI, may slow thin filament activation and result in increased myofilament relaxation kinetics, the latter of which could enhance early phase diastolic relaxation during β-adrenergic stimulation.

Dataset Information

Site-specific acetyl-mimetic modification of cardiac troponin I modulates myofilament relaxation and calcium sensitivity.

Publications

Site-specific acetyl-mimetic modification of cardiac troponin I modulates myofilament relaxation and calcium sensitivity.

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