Integrated single-cell transcriptomic and experimental analyses identify macrophage-associated STAT3 signaling contributing to ferroptosis-related injury in septic cardiomyopathy

Septic cardiomyopathy (SCM) is a major complication of sepsis, but how inflammatory immune cells contribute to ferroptosis-related myocardial injury remains unclear. We investigated ferroptosis-associated transcriptional remodeling in SCM and the functional contribution of macrophage STAT3 signaling.

Single-cell RNA sequencing, cell-cell communication analysis, and independent mouse and human transcriptomic cohorts were integrated to characterize ferroptosis-associated changes. Driver and suppressor signatures were analyzed separately. STAT3 was silenced in LPS-stimulated RAW264.7 macrophages, followed by conditioned-medium transfer to H9C2 cardiomyocytes. Ferroptosis-related injury was evaluated using lipid peroxidation, Fe²⁺ accumulation, molecular markers, Ferrostatin-1 (Fer-1) intervention, and an SCM mouse model.

Ferroptosis-associated transcriptional features showed marked cell-type heterogeneity. Inflammatory macrophages exhibited prominent STAT3-related inflammatory signaling and extensive predicted communication with cardiomyocytes. Independent cohorts revealed context-dependent changes in ferroptosis driver and suppressor programs. Conditioned medium from LPS-stimulated macrophages induced cardiomyocyte injury, lipid peroxidation, Fe²⁺ accumulation, decreased GPX4, and increased ACSL4. Macrophage STAT3 knockdown partially attenuated these changes, reduced inflammatory responses, and altered Cp expression. Fer-1 further alleviated conditioned-medium-induced injury. Consistent ferroptosis-related molecular alterations were observed in SCM mice.

Macrophage STAT3 signaling contributes to a paracrine inflammatory environment associated with ferroptosis-related cardiomyocyte injury, linking inflammatory macrophages to myocardial injury in SCM.