Wei Lijiang, Wan Naifu, Zhu Wentong, Liu Chenchen, Chen Zeyu, Rong Wuwei, Zhang Lujun, Xie Meifeng, Qin Yueqi, Sun Ting, Jing Qing, Lyu Ankang
Department of Vascular & Cardiology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
CAS Key Laboratory of Tissue Microenvironment and Tumor, Innovation Center for Intervention of Chronic Disease and Promotion of Health, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China.
Cell Commun Signal. 2025 Jan 17;23(1):32. doi: 10.1186/s12964-025-02031-3.
Cardiomyocyte death is a major cytopathologic response in acute myocardial infarction (AMI) and involves complex inflammatory interactions. Although existing reports indicating that mixed lineage kinase domain-like protein (MLKL) is involved in macrophage necroptosis and inflammasome activation, the downstream mechanism of MLKL in necroptosis remain poorly characterized in AMI.
MLKL knockout mice (MLKL), RIPK3 knockout mice (RIPK3), and macrophage-specific MLKL conditional knockout mice (MLKL) were established. AMI was induced by coronary artery ligation. The role of MLKL in regulating myocardial morphological necroptosis was evaluated using immunofluorescence staining, flow cytometry, qRT-PCR, Western blot, CCK-8 assay, and ELISA.
Our findings revealed that myocardial segmental necroptosis (MSN), a unique morphological characteristics of cell death observed post-AMI, was promoted by intercellular inflammatory adhesion mediated by MLKL. The key features of MSN included localized cytomembrane perforation, segmental attenuation of myofilaments, MLKL-mediated filling, and macrophage inflammatory adhesion. In a mouse model of AMI, we observed MSN, which was absent in immunosuppressed mice. Pharmacological depletion of macrophages or genetic knockout of macrophage-specific MLKL (MLKL) reduced the occurrence of MSN. This reduction was reversed upon reinfusion of wild-type macrophages. Additionally, myocardial injury was significantly ameliorated in MLKL mice following AMI. In a macrophage-cardiomyocyte co-culture system, MLKL attenuated hypoxia-induced MSN and inhibited macrophage-mediated inflammatory adhesion. Furthermore, MLKL was found to trigger the formation of membrane pores and the polymerization of integrin αvβ1, thereby enhancing inflammatory adhesion in the co-culture system. Notably, MLKL-enhanced inflammatory adhesion was not entirely dependent on RIPK3.
Our study demonstrates that MLKL is directly involved in myocardial segmental necroptosis by interacting with macrophages through inflammatory adhesion, and possibly independently of RIPK3.
心肌细胞死亡是急性心肌梗死（AMI）中的主要细胞病理学反应，涉及复杂的炎症相互作用。尽管现有报道表明混合谱系激酶结构域样蛋白（MLKL）参与巨噬细胞坏死性凋亡和炎性小体激活，但在AMI中，MLKL在坏死性凋亡中的下游机制仍未得到充分表征。
建立了MLKL基因敲除小鼠（MLKL-/-）、RIPK3基因敲除小鼠（RIPK3-/-）和巨噬细胞特异性MLKL条件性基因敲除小鼠（MLKLΔM）。通过冠状动脉结扎诱导AMI。使用免疫荧光染色、流式细胞术、qRT-PCR、蛋白质免疫印迹、CCK-8检测和酶联免疫吸附测定（ELISA）评估MLKL在调节心肌形态学坏死性凋亡中的作用。
我们的研究结果表明，心肌节段性坏死性凋亡（MSN）是AMI后观察到的一种独特的细胞死亡形态特征，由MLKL介导的细胞间炎症粘附所促进。MSN的关键特征包括局部细胞膜穿孔、肌丝节段性衰减、MLKL介导的填充以及巨噬细胞炎症粘附。在AMI小鼠模型中，我们观察到了MSN，而在免疫抑制小鼠中未观察到。巨噬细胞的药理学清除或巨噬细胞特异性MLKL（MLKLΔM）的基因敲除减少了MSN的发生。在重新输注野生型巨噬细胞后，这种减少得以逆转。此外，AMI后MLKL-/-小鼠的心肌损伤明显改善。在巨噬细胞-心肌细胞共培养系统中，MLKL减轻了缺氧诱导的MSN，并抑制了巨噬细胞介导的炎症粘附。此外，发现MLKL触发膜孔的形成和整合素αvβ1的聚合，从而增强共培养系统中的炎症粘附。值得注意的是，MLKL增强的炎症粘附并不完全依赖于RIPK3。
我们的研究表明，MLKL通过炎症粘附与巨噬细胞相互作用，可能独立于RIPK3，直接参与心肌节段性坏死性凋亡。
