Macrophage Notch1 drives septic cardiac dysfunction by impairing mitophagy and promoting NLRP3 activation

Sepsis is a life-threatening condition with limited therapeutic options, characterized as excessive systemic inflammation and multiple organ failure. Macrophages play critical roles in sepsis pathogenesis. Although numerous studies support the critical role of Notch signaling in most inflammatory diseases, the function of Notch1 signaling in macrophages activation and its underlying molecular mechanism during sepsis has not been fully elucidated.

We evaluated Notch1 expression in a lipopolysaccharide (LPS)-induced model of septic cardiac dysfunction. Using macrophage-specific Notch1 knockout mice (NOTCH1^ΔMyelo) in conjunction with AAV-F4/80-mediated NICD1 overexpression, we investigated the impact of Notch1 on septic cardiac injury. LPS-stimulated bone marrow-derived macrophages (BMDMs) were analyzed by flow cytometry and ELISA to assess mitochondrial damage and inflammasome activation. Mitophagy flux and related protein levels were quantified, and a mitophagy inhibitor was applied to further delineate Notch1's in vivo role. Downstream targets of Notch1 were identified and validated via ChIP-qPCR and luciferase reporter assays.

Intraperitoneal injection of LPS markedly impaired cardiac function, increased macrophage infiltration, and elevated Notch1 expression compared with PBS-treated controls. Notch1 expression was inversely correlated with cardiac performance in LPS-treated mice. Notably, macrophage-specific deletion of Notch1 significantly improved cardiac function, whereas NICD1 overexpression worsened LPS-induced cardiac injury. NOTCH1^ΔMyelo macrophages showed reduced mitochondrial damage and diminished activation of NLRP3-dependent caspase-1. Moreover, LPS induced mitophagy, an effect that was further enhanced by Notch1 knockout. Mechanistically, ChIP-seq and qPCR analyses revealed that NICD1 upregulates Mst1 transcription. Furthermore, overexpression of Mst1 counteracted the increased mitophagy in Notch1-deficient macrophages, resulting in elevated mitochondrial reactive oxygen species production, inflammatory cytokine secretion, and caspase-1 activation during prolonged LPS stimulation.

Our study uncovers a novel role for Notch1 in exacerbating LPS-induced septic cardiac dysfunction by suppressing mitophagy in macrophages. These findings suggest that targeting Notch1 may offer a promising therapeutic strategy to mitigate sepsis-induced inflammation by restoring proper mitophagy.