Blockade of Wnt Secretion Attenuates Myocardial Ischemia–Reperfusion Injury by Modulating the Inflammatory Response

Figures

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  Figure 1. Timeline and animal use of in vivo experiments. Numbers in brackets represent the animals used in the experiments. In total, 52 mice underwent ischemia/reperfusion (I/R) surgery, of which five animals died during or shortly after the procedure. Blood was drawn from the surviving animals (47) for troponin T measurements. 18 (9 per group) animals were sacrificed for flow cytometric analysis, 6 (3 per group) were sacrificed for RNA seq experiments. In total, 19 animals (8 in the DMSO group and 11 in the LGK-974 group) were used for echocardiographic and histological evaluation; four animals had no elevated troponin T levels and were excluded from the analysis.

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  Figure 2. Heatmap of differentially expressed genes. Columns represent individual samples from the two groups. RNA-seq analysis revealed differential expression of 529 genes in heart tissue (from the infarct area) from animals treated with LGK-974 compared to DMSO (vehicle control). N = 3 per group.

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  Figure 3. LGK-974 treatment alters the inflammatory response following I/R injury. (a) Gating scheme excluding dead cells, cell debris, and doublets. (b,c) representative histogram of heart tissue leukocytes resp. myeloid cells 2 days after I/R injury. (d) Quantification of total leukocytes in heart tissue following I/R injury. (e) Quantification of myeloid cells in the heart following I/R injury. (f) and (g) representative images of flow cytometric analysis of heart tissue cell suspension following I/R injury in DMSO resp. LGK-974 treated animals on monocytes, macrophages, and neutrophils. (h–j) quantification of inflammatory monocytes, macrophages, and neutrophils two days after I/R injury. (k) cardiac troponin T plasma levels 24 h after I/R injury. N = 9 per group.

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  Figure 4. LGK-974 treatment during the inflammatory response following I/R injury improves heart function. (a) Representative M-Mode echocardiographic images of DMSO (top) and LGK-974 treated (bottom) animals. (b,c) Echocardiographic results from DMSO and LGK-974 treated animals. (d) Representative Masson trichrome staining of DMSO (left) and LGK974 (right) treated animals 4 weeks after I/R injury. (e) Quantification of the fibrotic area of the LV wall in DMSO resp. LGK974 treated animals. N = 8 for DMSO-treated animals and N = 11 for the LGK-974-treated animals.

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  Figure 5. LGK-974 inhibits non-canonical Wnt signaling in monocytes/macrophages and leads to a reduced inflammatory character. (a) representative Western blots of pATF2 expression in monocytes/macrophages stimulated with supernatant of heart muscle cells that were cultured under hypoxic conditions in the presents or absence of LGK-974 and (b) quantification. (c) Expression of the inflammatory markers TNFα and IL1β and (d) the matrix metalloproteinases MMP2 (top) and MMP9 (bottom) measured by qPCR in monocytes/macrophages stimulated with supernatant of heart muscle cells that were cultured under hypoxic conditions in the presents or absence of LGK-974. Results originate from three independent experiments. n.s.: not significant; *: p ≤ 0.05.