Differential inflammatory responses of the native left and right ventricle associated with donor heart preservation

Figures

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  FIGURE 1. LV show worse function and more cell death during preservation. Murine donor hearts preserved in HTK with subsequent ex‐vivo reperfusion. Balloon transduced intracavitary pressures from the LV (red) and RV (blue) after 0 and 8 h of preservation demonstrating: (a) contractility (maximum dp/dt) and (b) relaxation (minimum dp/dt). n = 10–11 at each time point. (c) Representative images of TUNEL+cells (green) shown at 0, 4, and 8 h preservation in the LV and RV with DAPI (blue) staining. Magnification 200x. Scale bar 50 μm. (d) Quantification of percent TUNEL+ cells per average high‐ powered field (n = 4 per time point). (e) Western blot of cleaved caspase‐3 expression after 0, 4, and 8 h of preservation with reperfusion in the LV and RV. **p < 0.001, *p < 0.05, Kruskal–Wallis test or ANOVA with Tukey post hoc. LV, left ventricle; RV, right ventricle

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  FIGURE 2. Inflammatory mediator expression is greater in the LV. Western blot analysis of murine LV and RV samples following ex‐vivo perfusion with preservation at 0, 4, and 8 h to determine protein expression of (a) IL1β, (b) MyD88, (c) TNFα, (d) IL‐6, (e) MCP1, and (f) IL‐10. n = 4 per time point. **p < 0.001, *p < 0.05, Kruskal–Wallis test or ANOVA with Tukey post hoc, Relative fold change are normalized to 0 h. LV, left ventricle; RV, right ventricle

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  FIGURE 3. LV exhibit greater inflammasome activation. Western blot analysis of murine LV and RV samples following reperfusion with preservation at 0, 4, and 8 h to determine protein expression of inflammasome components (a) Nlrp3, (b) ASC, (c) cleaved Caspase‐1 p20, (d) p17‐IL‐1β, and (e) cleaved N‐GSDMD. n=3 per time point. **p < 0.001, *p < 0.05, Kruskal–Wallis test or ANOVA with Tukey post hoc, relative fold change are normalized to LV 0 h. (f) Immunohistochemistry for cleaved caspase‐1 (red) and troponin (green) in the LV and RV following preservation for 0, 4, and 8 h with reperfusion. White arrows indicate positive cleaved caspase‐1 staining (red). Magnification 400x. Scale bar 20 μm. LV, left ventricle; RV, right ventricle

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  FIGURE 4. Human donor hearts also show more pronounced inflammatory response in the LV with preservation. Human LV and RV were preserved with cold HTK and biopsied at 0, 4, and 8 h in the same donor heart. Western blot show the LV and RV expression of (a) Nlrp3, (b) IL‐1β, (c) TNFα, (d) IL‐6, (e) MCP1, and (f) IL‐10. **p < 0.001, *p < 0.05, # p < 0.1 Kruskal–Wallis test or ANOVA with Tukey posthoc, LV, left ventricle; RV, right ventricle

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  FIGURE 5. Inflammation response was transcriptionally activated in both murine LV and RV with cold preservation and reperfusion. (a) Venn diagram of upregulated and downregulated genes in LV show common regulated genes after 4 and 8 h storage followed by reperfusion. (b) Venn diagram of upregulated and downregulated genes in RV show common regulated genes after 4 and 8 h storage with reperfusion. GO analysis by panther classification system shows similar inflammation related GO gene sets were enriched in common upregulated genes of LV (c) and RV (d). Representative GSEA plot show that innate immune response gene set was enriched in LV (e) and RV (f) after 4 and 8 h preservation‐reperfusion compared to baseline 0 h. LV, left ventricle; RV, right ventricle; GO, Gene ontology