Lipopolysaccharide-induced DNA damage response activates DNA-PKcs to drive actin cytoskeleton disruption and cardiac microvascular dysfunction in endotoxemia.

Rationale: Sepsis-induced cardiomyopathy is characterized by microvascular injury, which is linked to lipopolysaccharide (LPS)-induced DNA damage response (DDR). This study investigates the role of DNA-PKcs, a key enzyme in the DDR pathway, in driving actin disruption and microvascular dysfunction following LPS exposure. Methods: We analyzed diverse transcriptomic datasets from septic human and murine models using bioinformatics tools to assess DDR pathway activation, correlations, and prognosis. In vivo, LPS-challenged mice were treated with inhibitors of DNA-PKcs or mitochondrial fission, and we evaluated cardiac function, microvascular integrity, mitochondrial status, and actin polymerization. Results: Bioinformatic analyses consistently revealed significant activation of the DDR pathway and upregulation of key genes across diverse septic models. Notably, elevated DDR pathway activity was significantly correlated with poor 28-day survival in human sepsis patients. Single-cell analysis localized this DDR gene upregulation predominantly to cardiac endothelial cells (ECs), fibroblasts, and macrophages during sepsis. Within septic capillary ECs, DDR pathway activity scores strongly correlated spatially and functionally with heightened mitochondrial fission and cytoskeletal remodeling pathway activities. In vivo experiments confirmed that LPS induced severe systolic and diastolic dysfunction, microvascular damage, and mitochondrial fragmentation, as well as significant actin depolymerization. Inhibition of DNA-PKcs with NU7441 markedly attenuated all these LPS-induced pathologies, improving cardiac function, preserving microvascular structure, preventing mitochondrial fragmentation, and normalizing related gene expression and actin cytoskeleton stability. Additionally, inhibiting mitochondrial fission with Mdivi-1 significantly ameliorated LPS-induced cardiac dysfunction and microvascular injury. Conclusions: Our findings suggest that LPS triggers a DNA-PKcs-dependent DDR that promotes mitochondrial fragmentation and actin disruption, particularly in cardiac ECs, contributing to sepsis-induced cardiomyopathy. Targeting DNA-PKcs or mitochondrial fission may hold therapeutic potential for the treatment of sepsis-induced cardiomyopathy.