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

Abstract

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 actomyosin 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.

Lysine acetylation of F-actin decreases tropomyosin-based inhibition of actomyosin activity

“…In contrast, our data suggest that actin acetylation does not appreciably affect actomyosin activity except when Tpm is present. Recently, we found that introduction of cardiac TnI Ac-mimetic K132Q decreased Ca 2+ sensitivity of both RTFs and isolated myofibrils (62). In comparison, K328Q actin increased RTF Ca 2+ sensitivity.…”

Zebrafish Model for Studying Dexamethasone-Induced Muscle Atrophy and Preventive Effect of Maca (Lepidium meyenii)

“…The Tn complex in myofibers is composed of a calcium-binding subunit, troponin C (TnC); an actin-binding inhibitory subunit, troponin I (TnI); a tropomyosin (Tm)-binding subunit; and troponin T (TnT). TnC contains a Ca 2+ -binding site, which leads to increased calcium sensitivity and changes in the duration of contraction by enhancing calcium–TnC affinity, thereby reducing the calcium dissociation rate, which in turn stimulates the affinity of TnI for TnC, allowing actin to interact with myosin, resulting in muscle contraction [ 55 ]. In contrast, TnI binds to actin in the relaxed state, preventing muscle contraction by inhibiting the ATPase activity of actomyosin [ 56 ].…”

HDAC6 modulates myofibril stiffness and diastolic function of the heart

“…We previously demonstrated that ITF2357 and the related pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) improve diastolic function in murine and feline models of hypertension, aging, and chronic pressure overload in association with enhanced myofibril relaxation ( 21 , 38 ). The inability of pan-HDAC inhibition to alter titin compliance likely explains why ITF2357 and SAHA failed to exacerbate DD, and instead elicited protective actions in the heart through distinct mechanisms, such as enhancing the acetylation state of troponin I, which augments myofibril relaxation ( 39 ). The relative contributions of impaired myofibril relaxation and titin stiffening to the pathogenesis of DD remain to be determined.…”