Background: S-nitrosylation (SNO), a prototypic redox-based posttranslational modification, is involved in the pathogenesis of cardiovascular disease. The aim of this study was to determine the role of S-nitrosylation of muscle LIM protein (MLP) in myocardial hypertrophy, as well as the mechanism by which SNO-MLP modulates hypertrophic growth in response to pressure overload. Methods: Myocardial samples from patients and animal models exhibiting myocardial hypertrophy were examined for SNO-MLP level using biotin-switch methods. S-nitrosylation sites were further identified through liquid chromatography-tandem mass spectrometry (LCMS/MS). Denitrosylation of MLP by the mutation of nitrosylation sites or overexpression of S-nitrosoglutathione reductase (GSNOR) was used to analyze the contribution of SNO-MLP in myocardial hypertrophy. Downstream effectors of SNO-MLP were screened through mass spectrometry (MS) and confirmed by co-immunoprecipitation. Recruitment of toll-like receptor 3 (TLR3) by SNO-MLP in myocardial hypertrophy was examined in TLR3 small interfering RNA (siRNA)-transfected neonatal rat cardiomyocytes (NRCMs) and in TLR3 knockout mouse model. Results: SNO-MLP level was significantly higher in hypertrophic myocardium from patients and in spontaneously hypertensive rats and mice subjected to transverse aortic constriction (TAC). The level of SNO-MLP also increased in angiotensin II (Ang II) or phenylephrine (PE)-treated NRCMs. S-nitrosylated site of MLP at cysteine (Cys) 79 was identified by LCMS/MS and further confirmed in NRCMs. Mutation of Cys79 significantly reduced hypertrophic growth in Ang II or PE-treated NRCMs and TAC mice. Reducing MLP Snitrosylation level by overexpression of S-nitrosoglutathione reductase greatly attenuated myocardial hypertrophy. Mechanistically, MLP S-nitrosylation stimulated TLR3 binding to MLP in response to hypertrophic stimuli, and disrupting this interaction by downregulating TLR3 attenuated myocardial hypertrophy. SNO-MLP also increased the complex formation between TLR3 and receptor-interacting protein kinase 3 (RIP3). This interaction in turn induced NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome activation, thereby promoting the development of myocardial hypertrophy. Conclusions: Our findings revealed a key role of SNO-MLP in myocardial hypertrophy and demonstrated TLR3-mediated RIP3 and NLRP3 inflammasome activation as the downstream signaling pathway, which may represent a novel therapeutic target for myocardial hypertrophy and heart failure.

中文翻译：

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肌肉 LIM 蛋白的 S-亚硝基化通过 Toll 样受体 3 介导的受体相互作用蛋白激酶 3 和 NLRP3 炎性体激活促进心肌肥大。

背景：S-亚硝基化（SNO）是一种原型的基于氧化还原的翻译后修饰，参与心血管疾病的发病机制。本研究的目的是确定肌肉 LIM 蛋白 (MLP) 的 S-亚硝基化在心肌肥大中的作用，以及 SNO-MLP 调节肥大生长以响应压力过载的机制。方法：使用生物素转换方法检查来自表现出心肌肥大的患者和动物模型的心肌样本的 SNO-MLP 水平。S-亚硝基化位点通过液相色谱-串联质谱 (LCMS/MS) 进一步鉴定。通过亚硝基化位点突变或S-亚硝基谷胱甘肽还原酶（GSNOR）过表达对MLP进行脱亚硝基化，分析SNO-MLP在心肌肥大中的作用。通过质谱 (MS) 筛选 SNO-MLP 的下游效应物，并通过免疫共沉淀确认。在 TLR3 小干扰 RNA (siRNA) 转染的新生大鼠心肌细胞 (NRCM) 和 TLR3 敲除小鼠模型中检测了 SNO-MLP 在心肌肥大中募集 toll 样受体 3 (TLR3)。结果：SNO-MLP 水平在患者肥厚心肌和自发性高血压大鼠和经受横向主动脉缩窄 (TAC) 的小鼠中显着升高。SNO-MLP 水平在血管紧张素 II (Ang II) 或去氧肾上腺素 (PE) 处理的 NRCM 中也增加。通过 LCMS/MS 鉴定 MLP 在半胱氨酸 (Cys) 79 处的 S-亚硝基化位点，并在 NRCM 中进一步证实。Cys79 的突变显着降低了 Ang II 或 PE 处理的 NRCM 和 TAC 小鼠的肥大生长。通过 S-亚硝基谷胱甘肽还原酶的过表达降低 MLP 亚硝基化水平可大大减弱心肌肥大。从机制上讲，MLP S-亚硝基化刺激 TLR3 与 MLP 结合以响应肥大刺激，并通过下调 TLR3 减弱心肌肥大来破坏这种相互作用。SNO-MLP 还增加了 TLR3 和受体相互作用蛋白激酶 3 (RIP3) 之间的复合物形成。这种相互作用进而诱导NOD样受体pyrin结构域含3（NLRP3）炎症小体活化，从而促进心肌肥大的发展。结论：我们的研究结果揭示了 SNO-MLP 在心肌肥大中的关键作用，并证明 TLR3 介导的 RIP3 和 NLRP3 炎性体激活是下游信号通路，