Conclusion
Our study demonstrates that MLKL is directly involved in myocardial segmental necroptosis by interacting with macrophages through inflammatory adhesion, and possibly independently of RIPK3.
Methods
MLKL knockout mice (MLKLKO), RIPK3 knockout mice (RIPK3KO), and macrophage-specific MLKL conditional knockout mice (MLKLM-KO) 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.
Purpose
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.
Results
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 (MLKLM-KO) reduced the occurrence of MSN. This reduction was reversed upon reinfusion of wild-type macrophages. Additionally, myocardial injury was significantly ameliorated in MLKLM-KO mice following AMI. In a macrophage-cardiomyocyte co-culture system, MLKLM-KO 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.