Macrophage-Derived Exosomal Mir-155 Regulating Cardiomyocyte Pyroptosis and Hypertrophy in Uremic Cardiomyopathy

Figures

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  Figure 1. Cardiomyocytes Pyroptosis Is Involved in the Process of UCM (A) Overview of the experimental in vivo procedure. (B) Representative immunohistochemical staining of caspase 1. (C) Quantification of caspase-1 expression in heart tissues (n = 6 per group). (D) The level of caspase-1, IL-1β, IL-18, and Gsdmd p30 protein. (E) Graphic presentation shows the relative abundance levels of caspase-1, IL-1β, IL-18, and Gsdmd p30 after normalization with GAPDH (n = 6 per group). (F) TUNEL assay; the yellow arrows indicate TUNEL-positive cardiomyocytes. (G) Quantification of TUNEL-positive cardiomyocytes (n = 6 per group). (H) Gross morphology of heart. (I) Summary of heart weight (n = 6 per group) and (J) heart weight/body weight (n = 6 per group). (K) Representative micrographs of sagittal sections (HE). (L) Summary of myocyte size (n = 6 per group). (M) Representative micrographs of left ventricular sections (Trichrome). (N) Summary of semiquantification of the Trichrome-positive area (n = 6 per group). Scale bars: 2 mm, (F); 50 μm (B, K, M). #p < 0.05 versus control, *p < 0.05 versus uremic group. DAPI = 4’,6’-diamidino-2-phenylindole; HE = hematoxylin and eosin stain; IL = interleukin;…

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  Figure 2. Foxo3a, But Not Foxo1, Is Downregulated in Uremic Hearts (A) Representative Western blot for Foxo1 and FoxO3a. (B) Bar graph showing the fold change (n = 6 per group). (C) The expression of FoxO-specific target genes, including Atrogin-1, MuRF-1, Bnip-3, p21, and Pdk4 (n = 6 per group). (D) FoxO3a mRNA levels in uremic hearts and that of the control group (n = 6 per group). #p < 0.05 versus control. FoxO3a,O1 = forkhead transcription factors of the O class.

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  Figure 3. Overexpression of Foxo3a Attenuated Cardiomyocytes Pyroptosis and Improved UCM AAV-FoxO3a-GFP was generated to overexpress FoxO3a in the hearts of uremic mice through a single tail injection (1 day after the second surgery). (A) Overview of the experimental in vivo procedure. (B) Representative immunohistochemical staining of caspase-1. (C) Quantification of caspase-1 expression in heart tissues (n = 6 per group). (D) Levels of Gsdmd p30, caspase-1, IL-1β, IL-18, and FoxO3a proteins. (E) Graphic presentation shows the relative abundance levels of Gsdmd p30, caspase-1, IL-1β, IL-18, and FoxO3a after normalization with GAPDH (n = 6 per group). (F) TUNEL assay; the yellow arrows indicate TUNEL-positive cardiomyocytes. (G) Quantification of TUNEL-positive cardiomyocytes (n = 6 per group). (H) Gross morphology of heart. (I) Summary of heart weight (n = 6 per group) and (J) heart weights/body weights (n = 6 per group). (K) Representative micrographs of sagittal sections (HE). (L) Representative micrographs of left ventricular sections (Trichrome). (M) Summary of myocyte size (n = 6 per group). (N) Summary of semiquantification of the Trichrome-positive area (n = 6 per group). Scale…

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  Figure 4. Inhibiting Mir-155 Expression Attenuated Cardiomyocytes Pyroptosis and UCM by Targeting Foxo3a 3’-UTR miR-155 inhibitor was used to treat uremic mice through weekly tail injections for 8 weeks in wild-type uremic mice and miR-155 −/− mice. (A) Overview of the experimental in vivo procedure. (B) Representative immunohistochemical staining of caspase-1. (C) Quantification of caspase-1 expression in heart tissues (n = 6 per group). (D) Levels of Gsdmd p30, caspase-1, IL-1β, IL-18, and FoxO3a proteins. (E) Graphic presentation shows the relative abundance levels of Gsdmd p30, caspase-1, IL-1β, IL-18, and FoxO3a after normalization with GAPDH (n = 6 per group). (F) TUNEL assay; the yellow arrows indicate TUNEL-positive cardiomyocytes. (G) Quantification of TUNEL-positive cardiomyocytes (n = 6 per group). (H) Gross morphology of heart. (I) Summary of heart weight (n = 6 per group) and (J) heart weights/body weights (n = 6 per group). (K) Representative micrographs of sagittal sections (HE). (L) Summary of myocyte size (n = 6 per group). (M) Representative micrographs of left ventricular sections (Trichrome). (N) Summary of semiquantification of the Trichrome-positive area (n = 6 per…

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  Figure 5. Macrophage-Derived Exosome Transferred Mir-155 Into Cardiomyocytes and Worsened Cardiomyocytes Pyroptosis in Vivo Uremic miR-155−/− mice were injected with macrophage original exosomes through weekly tail injections for 8 weeks. (A) Overview of the experimental in vivo procedure. (B) Representative immunohistochemical staining of caspase-1. (C) Quantification of caspase-1 expression in heart tissues (n = 6 per group). (D) Levels of Gsdmd p30, caspase-1, IL-1β, IL-18, and FoxO3a proteins. (E) Graphic presentation shows the relative abundance levels of Gsdmd p30, caspase-1, IL-1β, IL-18, and FoxO3a after normalization with GAPDH (n = 6 per group). (F) TUNEL assay; the yellow arrows indicate TUNEL-positive cardiomyocytes. (G) Quantification of TUNEL-positive cardiomyocytes (n = 6 per group). (H) Gross morphology of heart. (I) Summary of heart weight (n = 6 per group) and (J) heart weights/body weights (n = 6 per group). (K) Representative micrographs of sagittal sections (HE). (L) Summary of myocyte size (n = 6 per group). (M) Representative micrographs of left ventricular sections (Trichrome). (N) Summary of semiquantification of the Trichrome-positive area (n = 6 per group). Scale…

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  Figure 6. Macrophage-Derived Exosome Transfer of Mir-155 Into Cardiomyocytes and Worsened Pyroptosis in Vitro (A) Exosomes harvested from the medium of cultured macrophages (RAW264.7) were labeled with PKH67 and added to primary cardiomyocytes for 24 h. (B) Macrophage original exosome was verified by transmission electron microscopy, ranging in size from 80 to 130 nm. (C) Western blot for Alix, CD9, and CD63 exosome markers. (D) PKH67 labeled exosomes introduced into primary cardiomyocytes. (E) qRT-PCR analysis of miR-155 relative folds to U6 expression is shown (n = 3 per group). (F) Levels of Gsdmd p30, Caspase 1, IL-1β, IL-18, and FoxO3a protein. (G) Graphic presentation shows the relative abundance levels of Gsdmd p30, caspase-1, IL-1β, IL-18, and FoxO3a after normalization with GAPDH (n = 3 per group). (H) Pyroptosis, as evidenced by caspase-1 and PI double-positive cells in flow cytometry experiments in cardiomyocytes treated with macrophage-derived exosomes in the presence or absence of miR-155 inhibitor (n = 3 per group). Scale bars: 100 nm (B). #p < 0.05 versus 5% US, *p < 0.05 versus 5% US plus macrophage exosome. PI = propidium iodide; qRT-PCR = quantitative real-time polymerase…

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  Figure 7. Blockade of Exosome Secretion With GW4869 Improves Cardiomyocytes Pyroptosis and UCM (A) Uremic mice were injected with GW4869 to block exosome secretion. Overview of the experimental in vivo procedure. (B) Representative immunohistochemical staining of caspase 1. (C) Quantification of caspase-1 expression in heart tissues (n = 6 per group). (D) Levels of Gsdmd p30, caspase-1, IL-1β, and IL-18 proteins. (E) Graphic presentation shows the relative abundance levels of caspase-1, IL-1β, IL-18, and Gsdmd p30 after normalization with GAPDH (n = 6 per group). (F) TUNEL assay; the yellow arrows indicate TUNEL-positive cardiomyocytes. (G) Quantification of TUNEL-positive cardiomyocytes (n = 6 per group). (H) Gross morphology of heart. (I) Summary of heart weight (n = 6 per group) and (J) heart weights/body weights (n = 6 per group). (K) Representative micrographs of sagittal sections (HE). (L) Summary of myocyte size (n = 6 per group). (M) Representative micrographs of left ventricular sections (Trichrome). (N) Summary of semiquantification of the Trichrome-positive area (n = 6 per group). Scale bars: 2 mm (F); 50 μm (B, K, M). #p < 0.05 versus control, *p < 0.05 versus uremic group.…

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  Figure 8. Working Model of Macrophage-Derived Mir-155–Containing Exosomes Promote Cardiomyocyte Pyroptosis by Directly Targeting Foxo3a in UCM miR-155 was synthesized and loaded into exosomes in increased infiltration of macrophages in uremic heart. The released exosomal fusion with the plasma membrane leads to the release of miR-155 into the cytosol and translational repression of FoxO3a in cardiomyocytes. Finally, macrophage-derived miR-155–containing exosomes promoted cardiomyocyte pyroptosis and UCM changes (cardiac hypertrophy and fibrosis) by directly targeting FoxO3a in uremic mice. Abbreviations as in Figures 1 and 2.