Ex vivo heart perfusion induces time dependent inflammatory activation in human donor hearts

Ex vivo heart perfusion (EVHP) has enabled the use of hearts donated after circulatory death (DCD), expanding the donor pool but with persistently higher rates of rejection and graft dysfunction. The molecular consequences of EVHP, however, remain incompletely defined. We quantified 11,083 proteins in perfusate from 20 human DCD hearts undergoing EVHP using an aptamer-based proteomic platform. Among the most strongly upregulated proteins were inflammatory cytokines IL-6 (log₂FC = 3.5, FDR = 8.9 × 10 −6 ), IL-8 (CXCL8; log₂FC = 2.8, FDR = 4.7 × 10 −6 ), and CCL2/MCP-1 (log₂FC = 2.8, FDR = 2.0 × 10 −6 ). Levels of these cytokines correlated strongly with perfusion duration (Spearman ρ = 0.77–0.78, FDR < 5 × 10 −5 ). Pathway analysis demonstrated enrichment of innate immune signaling, including interleukin-1 signaling (normalized enrichment score (NES) = 2.14, adjusted p = 0.0016) and leukocyte chemotaxis pathways. These cytokine findings were validated in an independent human cohort using multiplex immunoassay and reproduced in a swine DCD perfusion model. Together, these data identify a conserved, time-dependent innate immune response during EVHP and suggest that inflammatory activation may represent one mechanism linking prolonged perfusion to graft dysfunction after DCD heart transplantation.