To investigate the myocardial expression of S100 calcium-binding proteins A8/A9 (S100A8/A9) in gene-edited pig donors before and after heterotopic cardiac xenotransplantation, and to delineate their relationship with macrophage-mediated inflammatory responses.

Three Bama gene-edited pigs were selected as donors and three adult male rhesus macaques served as recipients; heterotopic cardiac xenotransplantation was performed. Western blotting (WB) was employed to quantify the protein levels of S100A8/A9, inducible nitric-oxide synthase (iNOS), arginase-1 (Arg-1), TNF-α, IL-18, TGF-β, and IL-10. Immunohistochemical staining was performed to evaluate the tissue expression of CD3, CD19, and CD68. Between-group comparisons of normally distributed continuous data were performed using the independent-samples t-test, whereas the Wilcoxon signed-rank test was used for non-normally distributed continuous data. A P-value <0.05 was considered statistically significant.

Both donor and recipient hearts were successfully reperfused. WB analysis revealed that the relative expression levels of S100A8 in porcine cardiac tissue before and after transplantation were (1.000±0.000) and (3.234±0.790), respectively, while those of S100A9 were (1.000±0.000) and (2.796±0.617), respectively; the differences were statistically significant (t=-4.893 and -5.042, all P<0.05). HE staining showed extensive cardiomyocyte necrosis, hemorrhage, and inflammatory-cell infiltration in the transplanted gene-edited pig hearts. Immunohistochemical staining showed positive CD68 expression in the myocardial tissue of the gene-edited pig after transplantation. In all three recipient monkeys, the peripheral blood monocyte ratio did not exhibit a sustained postoperative increase. The relative expression levels of iNOS in porcine cardiac tissue before and after transplantation were (1.000±0.000) and (13.763±0.229), respectively, while those of Arg-1 were (1.000±0.000) and (0.143±0.071), respectively; the differences were statistically significant (t=-96.518 and 20.930, all P<0.05). Before transplantation, the relative expression levels of TNF-α, IL-18, TGF-β and IL-10 in porcine cardiac tissue were all (1.000±0.000); and the relative expression levels of them were (1.447±0.108), (3.656±0.596), (0.741±0.034), and (0.682±0.049) after transplantation, with statistically significant differences (t=-7.126, -7.725, 13.164 and 11.178, all P<0.05).

The expression of S100A8/A9, iNOS, and pro-inflammatory cytokines in gene-edited pig donor hearts was markedly up-regulated, whereas anti-inflammatory markers were down-regulated, indicating that S100A8/A9 may exacerbate the inflammatory response in cardiac xenografts by promoting macrophage M1 polarization.