Kynurenic acid mediates epicardial fat-induced lymphatic metabolic dysfunction in atrial fibrillation

Figures

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  Fig. 1. Impaired atrial lymphangiogenesis in human atrial fibrillation. A Schematic illustration of the experiments in human left atrial appendage (LAA). LAA excised during open cardiac surgery were analysed. n = 16 for No AFib patients, n = 13 each for PAF and PerAF patients. Created in BioRender. Takahashi, M. (https://BioRender.com/5uy66i9). B Relative mRNA levels of lymphangiogenic genes in LAA from the patients with No AFib, PAF and PerAF. n = 16 for No AFib, n = 13 each for PAF and PerAF, biologically independent samples. Data are median with IQR. Error bars indicate the IQR.; p value was determined by the Kruskal-Wallis test followed by the Dunn’s post hoc test. C Relative mRNA levels of angiogenic genes in LAA from the patients with No AFib, PAF and PerAF. n = 16 for No AFib, n = 13 each for PAF and PerAF, biologically independent samples. Data are median with IQR. Error bars indicate the IQR.; p value was determined by the Kruskal-Wallis test followed by the Dunn’s post hoc test. D Relative mRNA levels of inflammatory genes in LAA from the patients with No AFib, PAF and PerAF. n = 16 for No AFib, n = 13 each for PAF and PerAF, biologically independent samples. Data are median…

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  Fig. 2. Kynurenic acid mediates EAT-induced lymphatic endothelial dysfunction. A Schematic illustration of experiments in LEC treated with EAT conditioned media from the patients with No AFib and AFib. Created in BioRender. Takahashi, M. (https://BioRender.com/v1w6u45). B Quantification of permeabilization for LECs treated with EAT conditioned media from the patients with No AFib and AFib. n = 8 for both groups, biologically independent samples. Data are mean ± SD.; p value was determined by two-tailed unpaired Student’s t-test. C Representative image of tube formation of LECs treated with EAT conditioned media from the patients with No AFib and AFib. Scale bar, 200 μm. D Relative number of branch formed by LEC treated with EAT conditioned media from the patients with No AFib and AFib. n = 3 for both groups, biologically independent samples. Data are mean ± SD.; p value was determined by two-tailed unpaired Student’s t-test. E Relative mRNA levels of lymphatic endothelial markers, mesenchymal markers, and mitochondria-encoded genes in LECs treated with EAT conditioned media from the patients with No AFib and AFib. n = 8 for both groups, biologically independent samples. Data are mean ±…

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  Fig. 3. Angiotensin II impairs atrial lymphangiogenesis and increases atrial fibrillation susceptibility. A Representative transmission electron microscopy image for the mice LECs. Similar ultrastructural features were observed in five biologically independent mice. Scale bars: 10 μm. Inset: magnified image. Scale bar, 1 μm. Yellow arrowheads indicate the button-like junction. L, lymphatic vessel. M, atrial cardiomyocyte. B UMAP from published single-cell RNA sequencing datasets in mice left atrial tissues from No AFib (n = 3) and AFib (n = 3, combining of angiotensin II, high-fat diet, and mitral valve regurgitation) models29. LECs population: No AFib 1.2%, AFib 0.5%. Partially created in BioRender. Takahashi, M. (https://BioRender.com/81qzk8d). C Schematic illustration of the experiment procedure in mice. Male C57BL/6J mice at 8 weeks old received vehicle, Angiotensin II and/or VEGFC by osmotic pump for 14 days. Created in BioRender. Takahashi, M. (https://BioRender.com/kmfm2vr). D Changes in heart weight (left) and Heart weight/Tibial length (right) treated with Angiotensin II and/or VEGFC for 14 days. n = 6 per group, biologically independent mice. Data are mean ± SEM.; p value was…

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  Fig. 4. VEGFC restores lymphangiogenesis and suppresses atrial fibrillation. A Schematic illustration of the experiment procedure in mice. Male C57BL/6J mice at 8 weeks old received vehicle, Angiotensin II and/or VEGFC by osmotic pump for 28 days. Created in BioRender. Takahashi, M. (https://BioRender.com/2s31w47). B Relative mRNA levels of lymphangiogenic genes in left atria from vehicle, Angiotensin II and/or VEGFC treated mice. n = 6 per group, biologically independent mice. Data are mean ± SEM.; p value was determined by one-way ANOVA followed by the Tukey-Kramer’s post hoc test. C Representative immunofluorescent staining for LYVE1 (Green)/WGA (Red) in left atria from vehicle, Angiotensin II and/or VEGFC treated mice. Scale bar, 100 μm. D Quantification of relative LEC density (Left) and LYVE1-positive area (Right) in left atria from vehicle, Angiotensin II and/or VEGFC treated mice. n = 6 per group, biologically independent mice. Data are mean ± SEM.; p value was determined by one-way ANOVA followed by the Tukey-Kramer’s post hoc test. E Representative Masson’s Trichrome staining for atrium from vehicle, Angiotensin II and/or VEGFC treated mice. Scale bar, 50 μm. F Quantification…

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  Fig. 5. Weight loss improves lymphangiogenesis and reduces atrial fibrillation susceptibility. A Schematic illustration of the experiment for HFD-induced obese mice followed by administration of LY3437943. Male C57BL/6J mice at 8 weeks old were given access to normal chow or High-fat diet (60 % fat) for 13 weeks. A subset of mice was then injected LY3437943 intraperitoneally every third day for 3 weeks. Created in BioRender. Takahashi, M. (https://BioRender.com/kc11e0c). B Changes in body weight and food intake in each group. Body weight sample sizes were n = 20 (NFD-vehicle), n = 18 (HFD- vehicle), n = 19 (NFD-LY3437943), and n = 20 (HFD- LY3437943), biologically independent mice. Food intake sample sizes were n = 20 (NFD-vehicle), n = 19 (HFD- vehicle), n = 20 (NFD-LY3437943), and n = 20 (HFD- LY3437943), biologically independent mice.Data are mean ± SEM.; p value was determined by two-way repeated-measures ANOVA followed by two-tailed unpaired Student’s t-test. C Representative image of mice fed High-Fat diet with or without LY3437943 treatment. Scale bar, 5 cm D Left: Glucose tolerance test in HFD-induced obese mice with or without LY3437943 treatment. After 6 h of fasting, mice…