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.

中文翻译：

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心脏肌钙蛋白I的位点特异性乙酰基模拟修饰可调节肌丝松弛和钙敏感性。

目的心脏肌钙蛋白I（cTnI）是心脏松弛的重要生理和病理调节剂。对该调节至关重要的是，cTnI通过磷酸化的翻译后修饰是加速肌原纤维舒张的关键机制。与磷酸化相似，通过乙酰化进行的翻译后修饰会改变氨基酸电荷和蛋白质功能。最近的研究表明，心脏肌原纤维蛋白的乙酰化促进松弛，而心脏中的cTnI被乙酰化。这些发现突出了肌丝乙酰化的潜在意义。然而，尚不知道cTnI的位点特异性乙酰化是否会导致肌丝，肌原纤维和/或细胞力学改变。这项研究的目的是确定模仿cTnI（赖氨酸132； K132）的单个位点上的乙酰化对肌丝，肌原纤维和细胞力学的影响，并阐明其对分子功能的影响。方法为了确定cTnI在132处的假乙酰化是否能调节细丝对肌动蛋白相互作用的调节，我们重构了含有WT或K132Q的细丝（模拟乙酰化）cTnI，并评估了体外运动性。为了测试在K132上模拟乙酰化是否会改变细胞松弛，用表达精氨酸（K132R；防止乙酰化）替代cTnI K132Q或K132的腺病毒构建体感染成年大鼠心室心肌细胞，并测量细胞缩短和分离的肌原纤维力学。最后，为了确认细胞缩短和肌原纤维的变化直接是由于在K132处cTnI的假乙酰化所致，我们将含有WT或K132Q cTnI的肌钙蛋白交换为分离的肌原纤维，并测量了肌原纤维的机械性能。结果与用WT cTnI重构的细丝相比，含有K132Q cTnI的重构细丝表现出降低的钙敏感性。与表达WT或K132R cTnI的心肌细胞相比，表达K132Q cTnI的心肌细胞具有更快的延长时间，从这些细胞中分离出的肌原纤维具有更快的松弛以及降低的钙敏感性。离体与K132Q cTnI交换的肌原纤维显示出更快的松弛和降低的钙敏感性。结论我们的结果首次表明，模拟特定cTnI赖氨酸的乙酰化可加速肌丝，肌原纤维和肌细胞松弛。这项工作强调了理解特定肌节蛋白的乙酰化如何影响心脏稳态和疾病的重要性，并表明调节肌丝赖氨酸乙酰化可能代表改变心脏松弛的新治疗靶标。