Mixed lineage kinase 3 contributes to myocardial ischemia/reperfusion injury by regulating neutrophil activation

Mixed lineage kinase 3 (MLK3), a member of the MAP3K family, is known to participate in cellular stress and inflammatory responses, but its role in neutrophil-mediated myocardial ischemia-reperfusion (I/R) injury remains unclear. In this study, we investigated the function and downstream signaling of MLK3 in neutrophils using genetically modified mouse models with neutrophil-specific MLK3 knockout or overexpression. MLK3 deficiency in neutrophils reduced infarct size, improved cardiac function, and decreased neutrophil infiltration, NET formation, and pro-inflammatory cytokine release following I/R. Transcriptomic profiling revealed that MLK3 promotes the expression of the antimicrobial peptide CRAMP by stabilizing and activating the transcription factor C/EBPβ. Administration of exogenous CRAMP abolished the protective effects of MLK3 deletion, confirming its functional relevance. Furthermore, treatment with CEP-1347, a small-molecule MLK3 inhibitor, attenuated myocardial injury, reduced apoptosis, and limited adverse remodeling in vivo. In acute myocardial infarction (AMI) patients, elevated levels of phosphorylated MLK3 (pMLK3) in circulating neutrophils were associated with increased levels of MPO-DNA, cTnT, and CK-MB, as well as a trend toward higher rates of cardiovascular rehospitalization. These findings identify a neutrophil-intrinsic MLK3-C/EBPβ-CRAMP axis that amplifies myocardial inflammation and injury, and suggest MLK3 as a promising therapeutic target and potential biomarker for ischemic heart disease.