SNO-MLP（肌肉 LIM 蛋白的 S-亚硝基化）通过 TLR3（Toll 样受体 3）介导的 RIP3（受体相互作用蛋白激酶 3）和 NLRP3（含 NOD 样受体pyrin 域蛋白 3）炎性小体激活促进心肌肥大。
SNO-MLP (S-Nitrosylation of Muscle LIM Protein) Facilitates Myocardial Hypertrophy Through TLR3 (Toll-Like Receptor 3)-Mediated RIP3 (Receptor-Interacting Protein Kinase 3) and NLRP3 (NOD-Like Receptor Pyrin Domain Containing 3) Inflammasome Activation.
机构信息
Key Laboratory of Cardiovascular and Cerebrovascular Medicine, Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Medical University, Nanjing, China (X.T., L.P., S.Z., F.D., M.C., H.J., X.L., Z.L., H.C., Y.G., Q.L., L.X., Y.J.).
Department of Cardiology, the First Affiliated Hospital of Xiamen University, China (Z.H.).
出版信息
Circulation. 2020 Mar 24;141(12):984-1000. doi: 10.1161/CIRCULATIONAHA.119.042336. Epub 2020 Jan 6.
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 SNO of MLP (muscle LIM protein) 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. SNO sites were further identified through liquid chromatography-tandem mass spectrometry. Denitrosylation of MLP by the mutation of nitrosylation sites or overexpression of S-nitrosoglutathione reductase was used to analyze the contribution of SNO-MLP in myocardial hypertrophy. Downstream effectors of SNO-MLP were screened through mass spectrometry and confirmed by coimmunoprecipitation. Recruitment of TLR3 (Toll-like receptor 3) by SNO-MLP in myocardial hypertrophy was examined in TLR3 small interfering RNA-transfected neonatal rat cardiomyocytes and in a 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. The level of SNO-MLP also increased in angiotensin II- or phenylephrine-treated neonatal rat cardiomyocytes. S-nitrosylated site of MLP at cysteine 79 was identified by liquid chromatography-tandem mass spectrometry and confirmed in neonatal rat cardiomyocytes. Mutation of cysteine 79 significantly reduced hypertrophic growth in angiotensin II- or phenylephrine-treated neonatal rat cardiomyocytes and transverse aortic constriction mice. Reducing SNO-MLP level by overexpression of S-nitrosoglutathione reductase greatly attenuated myocardial hypertrophy. Mechanistically, SNO-MLP stimulated TLR3 binding to MLP in response to hypertrophic stimuli, and disrupted this interaction by downregulating TLR3-attenuated myocardial hypertrophy. SNO-MLP also increased the complex formation between TLR3 and RIP3 (receptor-interacting protein kinase 3). This interaction in turn induced NLRP3 (nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3) 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 therapeutic target for myocardial hypertrophy and heart failure.
背景
S-亚硝基化（SNO）是一种典型的基于氧化还原的翻译后修饰，参与了心血管疾病的发病机制。本研究的目的是确定 MLP（肌肉 LIM 蛋白）的 SNO 在心肌肥大中的作用，以及 SNO-MLP 调节对压力超负荷反应的肥大生长的机制。
方法
使用生物素转换方法检测患有心肌肥大的患者和动物模型的心肌样本中的 SNO-MLP 水平。通过液相色谱-串联质谱进一步鉴定 SNO 位点。通过突变硝化位点或过表达 S-亚硝基谷胱甘肽还原酶来使 MLP 去硝化，分析 SNO-MLP 在心肌肥大中的作用。通过质谱筛选 SNO-MLP 的下游效应物，并通过共免疫沉淀证实。在 TLR3 小干扰 RNA 转染的新生大鼠心肌细胞和 TLR3 敲除小鼠模型中检测 SNO-MLP 在心肌肥大中的 TLR3 募集情况。
结果
患者肥厚心肌和自发性高血压大鼠和接受横主动脉缩窄的小鼠中 SNO-MLP 水平明显升高。血管紧张素 II 或苯肾上腺素处理的新生大鼠心肌细胞中 SNO-MLP 水平也升高。通过液相色谱-串联质谱鉴定了 MLP 半胱氨酸 79 位的 S-亚硝基化位点，并在新生大鼠心肌细胞中得到证实。半胱氨酸 79 的突变显著减少了血管紧张素 II 或苯肾上腺素处理的新生大鼠心肌细胞和横主动脉缩窄小鼠的肥大生长。通过过表达 S-亚硝基谷胱甘肽还原酶降低 SNO-MLP 水平可显著减轻心肌肥大。在机制上，SNO-MLP 刺激 TLR3 与肥大刺激物结合到 MLP 上，并通过下调 TLR3 减轻心肌肥大来破坏这种相互作用。SNO-MLP 还增加了 TLR3 与 RIP3（受体相互作用蛋白激酶 3）之间的复合物形成。这种相互作用反过来又诱导 NLRP3（核苷酸结合寡聚结构域样受体含吡咯域 3）炎性小体的激活，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，并证实了 TLR3 介导的 RIP3 和 NLRP3 炎性小体激活作为下游信号通路，这可能成为心肌肥大和心力衰竭的治疗靶点。
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