【背景】

心肌梗死（myocardial infarction, MI）是全球范围内威胁人类健康的主要疾病，发病率高，死亡率高。探索缺血后心脏损伤和修复重塑的病理生理机制或将有助于MI患者预后风险的评估和治疗靶点的确定。Toll样受体介导的通路在MI炎症反应和心脏重塑发病机制中起重要作用，白细胞介素-1受体相关激酶（Interleukin-1 receptor-associated kinase，IRAK）-M作为Toll样受体的调节因子，在急性MI病理生理过程中发挥的作用尚不完全清楚有待系统研究。

【目的】

本研究旨在探讨急性MI患者中IRAK-M的表达特征和参与的病理生理过程，通过对IRAK-M基因敲除小鼠构建MI模型来明确IRAK-M缺失对MI生物学功能和代谢通路的影响，结合体内和体外功能实验探索IRAK-M对MI后纤维化、基质重塑和巨噬细胞极化的影响和可能机制。

【方法】

1、在GEO数据库检索急性MI患者的基因数据集，分析MI急性期IRAK-M表达量的变化，整合数据集中的差异基因进行功能富集分析并构建分子互作网络，比较IRAK-M上调MI患者与IRAK-M正常MI患者的生物学过程差异。

2、野生型（WT）小鼠和IRAK-M基因敲除（KO）小鼠构建MI模型，MI第3天和第7天对梗死心脏进行iTRAQ蛋白定量，蛋白组学数据进行GO和KEGG富集分析、GSEA分析、蛋白表达趋势分析、加权基因共表达网络分析和免疫细胞浸润等生物信息学分析。

3、运用马松染色和天狼猩红染色比较WT小鼠和KO小鼠MI后纤维化面积和胶原沉积比例，MI第7天提取WT和KO小鼠心脏的肌成纤维细胞，以及构建IRAK-M过表达的NIH 3T3细胞系，比较各组间的成纤维细胞收缩能力和α-SMA表达量，MMPs和TIMPs比例，Smad2和NF-kB磷酸化水平，以及NF-kB抑制剂对MMP9/TIMP1比例的影响。

4、运用免疫荧光比较WT小鼠和KO小鼠心梗部位的M1和M2巨噬细胞数量，免疫组化比较S100A8/A9表达量，MI第7天提取WT和KO小鼠骨髓来源巨噬细胞，以及构建IRAK-M过表达/敲低的RAW264.7细胞进行缺氧培养，比较各组间的M1和M2巨噬细胞标记物的变化，S100A8/A9和HIF-1α表达量变化，以及S100A9阻断剂对巨噬细胞极化的影响。

【结果】

1、IRAK-M在心梗患者的表达特点和参与生物学过程

GEO数据集分析发现IRAK-M在急性MI患者表达上调，并随MI时间演变而变化，IRAK-M表达量与急性冠脉综合征的类型有关；MI急性期白细胞活化和适应性免疫反应等生物学过程激活，IRAK-M作为关键分子之一，与TLR4、MMP9和Arg1等构成分子互作网络，共同参与调控MI的病理生理过程；IRAK-M上调患者较IRAK-M正常患者的炎症反应更剧烈，且IRAK-M表达量与S100A8/A9等炎症因子表达量呈正相关，提示IRAK-M的上调与MI急性期炎症反应的严重程度密切相关。

2、IRAK-M敲除小鼠心梗后主要蛋白和生物学过程的改变

小鼠心梗蛋白组学结果显示WT小鼠在MI第3天以免疫反应激活为主，第7天以细胞外结构和胶原纤维组织激活为主，并伴有细胞呼吸抑制；相比于WT小鼠，KO小鼠的急性炎症反应和线粒体呼吸功能障碍程度更重、时间更长，同时伴有巨噬细胞活化增加和胶原合成增加；另外，IRAK-M的缺失显著影响MI后的免疫细胞浸润，KO组在MI第3天出现M0巨噬细胞占比增加，并在第7天表现为M2巨噬细胞的增加；IRAK-M缺失后S100A8/A9、MMP9和Arg1等蛋白显著上调并形成蛋白互作网络，可能在上述病理生理过程的调节中发挥关键作用。

3、IRAK-M对心梗后纤维化和基质重塑的影响及机制探讨

IRAK-M敲除虽未影响MI第7天的纤维化面积，但却导致I型/III型胶原比例上调，肌成纤维细胞的收缩能力和α-SMA表达量显著下降，这可能与TGF-β1刺激后Smad2磷酸化水平下降有关；此外，IRAK-M缺失引起MMP9/TIMP1比例失衡，从而影响MI后基质蛋白重塑，这可能与iNOS和NF-kB表达量上调有关，iNOS和/或NF-kB抑制剂能够缓解IRAK-M缺失导致的MMP9/TIMP1比例上调；NIH 3T3成纤维细胞系中IRAK-M的过表达能够显著上调α-SMA表达量并降低MMP9/TIMP1比例，进一步验证IRAK-M对MI后纤维胶原和基质重塑的影响。

4、IRAK-M对心梗后巨噬细胞极化的影响及机制探讨

体内实验表明IRAK-M缺失确实导致心梗第7天的M2巨噬细胞数量增多，体外实验进一步验证缺氧培养条件下IRAK-M的过表达能促进M1极化并降低M2极化；IRAK-M缺失引起S100A8/A9表达量的增加，而S100A9阻断剂能够显著减弱IRAK-M缺失对巨噬细胞极化的影响，提示IRAK-M至少部分通过调控S100A8/A9来发挥介导巨噬细胞极化的作用；另外，缺氧条件下HIF-1α表达量在IRAK-M敲低组上调，而在IRAK-M过表达组下调，推测IRAK-M可能通过介导HIF-1α来调控S100A8/A9的表达。

【结论】

IRAK-M在急性MI患者表达上调，并随MI时间演变而变化，其表达量与MI急性炎症反应的严重程度密切相关，IRAK-M作为分子互作网络的关键分子之一，在MI病理生理过程中发挥重要作用：控制急性炎症反应的程度和时间，减轻线粒体呼吸功能障碍，同时调节心梗部位I型/III型胶原比例、MMP9/TIMP1比例和M1/M2巨噬细胞比例的平衡，从而影响MI后心脏修复重塑。

Background

Myocardial infarction (MI) is one of the major diseases threatening human health worldwide and contributes to substantial morbidity and mortality. Exploring the pathophysiological mechanisms of cardiac injury and repair remodeling after ischemia will contribute to the assessment of prognostic risk and the identification of therapeutic targets for MI patients. Toll-Like receptors (TLRs) mediated pathways are critically involved in the pathogenesis of inflammatory response and cardiac remodeling post-MI. Interleukin-1 receptor-associated kinase (IRAK)-M is a key regulator of TLRs-mediated responses, however, its potential role in the pathophysiology of acute MI is not yet fully understood and remains to be systemically studied.

Objective

1. To investigate the expression characteristics and biological processes of IRAK-M in patients with acute MI.

2. To identify the effects of IRAK-M deletion on the biological functions and metabolic pathways during MI via constructing MI model using wildtype and IRAK-M^-/- mice.

3. To explore the effects and underlying mechanisms of IRAK-M on post-MI fibrosis, matrix remodeling and macrophage polarization in vivo and in vitro experiments.

Methods

1. The gene datasets related to MI patients was searched in GEO database to compare the IRAK-M expression in MI patients with that in healthy controls. Differentially expressed genes from different datasets were integrated for functional enrichment analysis and molecular interaction network. MI patients were further divided into high-IRAK-M and normal-IRAK-M subgroups to compare the differences in biological processes during MI.

2. Wild-type (WT) and IRAK-M gene knockout (KO) mice were used to construct the acute MI model. The protein quantification of infarcted hearts at MI 3d and MI 7d were carried out via iTRAQ labeling and liquid chromatography-tandem mass spectrometry. Subsequently, GO and KEGG enrichment analysis, Gene Set Enrichment Analysis, Short Time-Series Expression Miner Analysis, Weighted Gene Co-expression Network Analysis and immune cell infiltration analysis were performed based on proteomics data.

3. Masson staining and Sirius Red staining were performed to compare the fibrotic area and collagen deposition following MI between WT and KO mice. Myofibroblasts were extracted from the infarcted heart at MI d7 and NIH 3T3 cell line was used to generate IRAK-M-overexpressing fibroblasts. Then contractile capacity and α-SMA expression of fibroblasts, MMPs and TIMPs ratio, Smad2 and NF-kB phosphorylation level were measured and compared within each group. Furthermore, we explored the effect of NF-kB inhibitor on MMP9/TIMP1 ratio of IRAK-M^-/- fibroblasts.

4. The number of M1 and M2 macrophages and the expression of S100A8/A9 in infarcted myocardium between WT and KO mice was compared via immunofluorescence and immunohistochemistry, respectively. Furthermore, the effect of S100A9 inhibitors on macrophage polarization was explored in vivo experiments. Bone marrow derived macrophages were extracted at MI 7d and RAW264.7 cell line was used to construct with IRAK-M-overexpressing or IRAK-M-knockdown cells. Then we measured the changes of M1 and M2 macrophage markers and the expression of S100A8/A9 and HIF-1α in a hypoxic environment.

Results

1. Expression characteristics and biological processes of IRAK-M in MI patients

IRAK-M was up-regulated in patients with acute MI and changed in a time-dependent manner during MI progression according to GEO datasets. The level of IRAK-M was also related to the type of acute coronary syndrome. During the acute stage of MI, biological processes such as leukocyte activation and adaptive immune response were markedly activated. IRAK-M was found to be one of the key molecules and participated in the regulation of the pathophysiological processes during MI through forming molecular interaction network with TLR4, MMP9 and Arg1. Of note, inflammatory responses were more severe in MI patients with high IRAK-M than those with normal IRAK-M, and the expression of IRAK-M is positively correlated with the level of inflammatory factors such as S100A8/A9, suggesting that the upregulation of IRAK-M is closely related to the severity of inflammation in acute MI.

2. Effect of IRAK-M deletion on key proteins and biological processes following MI

The proteomics data of infarcted hearts from WT mice showed that immune responses were activated at MI 3d, whereas extracellular structure and collagen fibers was upregulated at MI 7d, accompanied with inhibited cellular respiration. Compared with WT mice, KO mice had more severe inflammatory response and mitochondrial respiratory dysfunction, accompanied by increased macrophage activation and collagen synthesis. In addition, the loss of IRAK-M significantly affected the immune cell infiltration after MI. KO mice presented higher proportion of M0 macrophages at MI 3d and higher proportion of M2 macrophages at MI 7d, compared to WT group. IRAK-M deletion led to significantly increased S100A8/A9, MMP9 and Arg1, which formed protein interaction networks to regulate the above pathophysiological processes post-MI.

3. Effect of IRAK-M on fibrosis and matrix remodeling during MI and underlying mechanism

IRAK-M knockout did not change the fibrotic area at MI 7d, but resulted in the imbalance of type I /III collagen ratio, reduced contractility and α-SMA expression of myofibroblasts, which might be related to the decrease of Smad2 phosphorylation level after TGF-β1 stimulation. Additionally, imbalanced MMP9/TIMP1 ratio was an obvious performance of cardiac matrix remodeling in KO group, which might be related to the up-regulation of iNOS and NF-kB expression. The inhibition of iNOS or NF-kB could alleviate the imbalance of MMP9/TIMP1 ratio caused by IRAK-M deficiency. Overexpression of IRAK-M could significantly increase α-SMA expression and decrease MMP9/TIMP1 ratio in NIH 3T3 cell line, further confirming the effect of IRAK-M on fibrosis and matrix remodeling after MI.

4. Effect of IRAK-M on macrophage polarization after MI and underlying mechanism

Indeed, IRAK-M deletion led to increased number of M2 macrophages at MI 7d in vivo experiments. In contrast, IRAK-M overexpression in RAW264.7 cells could promote M1 polarization and reduce M2 polarization in hypoxia culture. Moreover, the loss of IRAK-M increased the expression of S100A8/A9 in infarcted heart, and S100A9 inhibitor significantly attenuated the effect of IRAK-M absence on macrophage polarization, suggesting that IRAK-M mediated macrophage polarization at least partly by regulating S100A8/A9 expression. In particular, HIF-1α expression was observed to be up-regulated in IRAK-M-knockdown group while down-regulated in IRAK-M-overexpressing group under hypoxia. It was thus speculated that IRAK-M may regulate the expression of S100A8/A9 by mediating HIF-1α.

Conclusions

In MI patients, the expression of IRAK-M was up-regulated and changed in a time-dependent manner during MI progression. As one of the key molecules in the protein interaction network, IRAK-M played an important role in regulating the pathophysiological processes following MI. IRAK-M could limit the severity and duration of acute inflammatory response, ameliorate mitochondrial respiratory dysfunction and regulate the balance of type I/III collagen ratio, MMP9/TIMP1 ratio and M1/M2 macrophage ratio, thereby affecting cardiac repair and remodeling post-MI.