Untargeted metabolomics identified kynurenine as a predictive prognostic biomarker in acute myocardial infarction

Figures

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  Figure 1. High levels of Kyn as a prognostic indicator in STEMI patients. (A) Kaplan-Meier plots indicated the association between quartile levels of Kyn and major adverse cardiovascular events, the numbers of STEMI patients in the different quartile groups were included in each graph. (B) Receiver operating characteristic curves for the patients with STEMI vs. control subjects for Kyn. (C) Restricted cubic spline modeling indicated the relationship between the plasma Kyn levels and the risk of MACCE. The solid line indicated the hazard ratios and the shaded areas, the 95% confidence intervals. (D) Forest plots showed that Kyn levels were associated with the prognosis in STEMI patients of subgroups according the STEMI prognostic analysis cohort data, after adjusting for per-standard deviation odds ratio (95% confidence interval) for the plasma.

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  Figure 2. High IDO1 and Kyn expression in thrombi of coronary arteries. (A) Distribution of IDO1 plasma level in control subjects(n=10), SAP patients(n=10), NTSTEMI patients(n=34) and STEMI patients(n=35), NS, not significant, *P<0.05 vs con subjects, **P<0.01 vs con subjects. (B) Coronary artery thrombi were obtained from the STEMI patients presenting to the cardiac catheterization laboratory. (a) Angiogram demonstrating thrombotic occlusion (arrow) of the right coronary artery in the STEMI patient, dashed line indicated the occluded vessels. (b) Final angiogram showed a widely patent of the right coronary artery with no significant luminal narrowing. (c, d) Thrombectomy catheter aspirate after passage through filter showing coronary artery thrombi (arrow) (scale bar, 1mm). (C) Representative immunofluorescence staining for CD68 and IDO1 in the coronary artery thrombi of STEMI patients. Immunofluorescence image showing CD68+(red) macrophages and IDO1 (green) expression; merged image was included. Nuclei was counterstained with DAPI. Co-localization of IDO1 and CD68+ macrophages was depicted in the overlay. The square indicated the macrophage cells expressing IDO1(scale bar, 40 μm).

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  Figure 3. IDO1 expression in the cardiac macrophage cells and the plasma of mice remodeling after MI. (A) Induction of MI by the permanent ligations of the left anterior descending artery (MI) at 1d, 3d, 7d, 14d, and 28d points or sham operations without ligation were performed on the experimental animals as described previously. Immunohistochemical analyses showed that IDO1 and CD68 were co-localized in the infarct and non-infarct area at different time points after MI, indicating that IDO1 was highly expressed in macrophage cells especially during 1−3 days after MI (scale bar, 1000 μm, scale bar, 40 μm). (B) IDO1 expression in heart was determined at different time points after MI using western blot analysis, NS, not significant, *P<0.05 vs sham group, **P<0.01 vs sham group, n=3. (C) IDO1 expression in the infarct and non-infarct area after MI was showed in the western blot analysis, *P<0.05 vs sham group, **P<0.01 vs sham group, n=3. (D) The colocalization of IDO1 with CD68 demonstrated with immunofluorescence in macrophage cells. (E) Distribution of Trp metabolites between the sham and MI group at different time points. Trp, tryptophan; Kyn, kynurenine; 3-HK, 3-hydroxykynurenine; KA,…

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  Figure 4. Kyn induced expression of inflammatory factors and increased the oxidative stress levels in macrophage cells and induced Sirt3. (A) Kyn expression were observed in the supernatant of macrophages, endothelial cells, VSMCs and myocardial cells stimulated with 20 ng/ml IFNγ for 24 h. (B, C) Mitochondria-derived ROS stimulated by Kyn after 24 h was detected using MitoSox-probe (Red) and the nuclei were stained with Hoechst stain (Blue), (scale bar, 10 μm),*P<0.05 vs DMSO, **P<0.01 vs DMSO, n=3. (D) SOD activity was determined using a detection kit. Kyn increased the mitochondrial oxidative stress activity in macrophage cells, *P<0.05 vs DMSO, n=3. (E) Interaction of Kyn with Sirt3, the AutoDock predicted binding modes of Kyn with Sirt3, in which the carboxyl group at the Kyn terminus formed salt-bonding interactions with Arg-158 and hydrogen-bonding interactions with Phe-157 and Arg-158. The benzoyl group and the protonated amino group formed a cross-linked hydrogen bond network with Gly-319, Thr-320 and Ser-321. The binding energy predicted by autodock was displayed. (a) Two dimensional structure diagram, (b) Three dimensional structure diagram, (c) Overall structure diagram. (F, G)…

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  Figure 5. Kyn enhanced MI lesion size, and reversely Sirt3 activation alleviated the inflammatory response in the lesion area of MI. (A) Immunohistochemical analyses showed that Sirt3 and CD68 co-localized in the infarct area after MI, indicating that Sirt3 decreased in macrophages especially during the 1−3 days after MI, scale bar, 40 μm. (B) Sirt3 expression levels were analyzed in both infarct and non-infarct area after MI, n=3. NS, not significant, **P<0.01 vs sham. (C) Representative images showed H&E and Masson staining of myocardial tissue sections from control (sham) and MI mice subjected to Kyn (15μmol) treatment. Kyn supplementation deteriorated the cardiac function in MI mice in vivo, and the infiltration of CD68+ macrophages in the region of MI was aggravated. Treating mice with the Sirt3 activator can significantly reduce the area of MI and improve the cardiac function after MI; in addition, the infiltration of macrophages at the site of MI was reduced, indicating that the Sirt3 activator (Viniferin) can decrease the inflammatory response in the area of MI, scale bar, 10 μm, scale bars 40 μm.

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  Figure 6. Downregulation and overexpression of Sirt3 altered the levels of inflammatory factors and oxidative stress levels in macrophages induced by Kyn. (A, B) Immunofluorescence staining showed that Sirt3 downregulation reduced the levels of mitochondria-derived ROS induced by Kyn. Mitochondria-derived ROS was detected using the MitoSox-probe and imaged using confocal microscopy. SOD activity was detected using the superoxide dismutase test kit. Macrophages transfected with sicon or siSirt3 were treated with Kyn. sicon was the negative control siRNA, while siSirt3 referred was the siRNA against Sirt3, scale bar, 10 μm, *:P < 0.05 vs.si-con; #P < 0.05 vs. Si-Sirt3, n=3. (C) Representative blots and quantification of acSOD2, Sirt3, IL-1β, and DRP-1 expression following Kyn stimulation, transfected with negative control, *:P < 0.05 vs.si-con; #P < 0.05 vs. to Si-Sirt3, n=3. (D, E) Sirt3 upregulation decreased the levels of mitochondria-derived ROS induced by Kyn, as observed through immunofluorescence. Mitochondria-derived ROS and SOD activity was detected using MitoSox-probe and imaged using confocal microscopy. SOD activity was detected using the superoxide dismutase test kit. The…

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  Figure 7. Representative western blots of SOD2 acetylation, SOD2, and Sirt3 expression in PBMCs of STEMI subjects and control subjects. Representative western blots of SOD2 acetylation, SOD2, and Sirt3 expression in STEMI patients compared with that in control subjects. Human peripheral blood mononuclear cells (PBMCs) were obtained from STEMI patients and control subjects. SOD2 acetylation was measured using anti-acetyl-SOD2, *:P < 0.05 vs. control subjects.