NLRP3 inflammasome activation by turbulent shear stress drives right ventricular outflow tract fibrosis in pulmonary regurgitation

Figures

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  Figure 1. Visualization of RV flow dynamics and TSS during the cardiac cycle. (A) The left inset displays a coronal view of the RV include RVIT, AT and RVOT. The right inset illustrates a sequence of transverse section slices from apical to basal region, with yellow lines outlining the endocardium. (B) Time-resolved velocity field within the RV at different time points during the cardiac cycle. (C) Turbulent shear stress distribution on the RV walls at corresponding time points.

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  Figure 2. Of activate NLRP3 inflammasome in cardiomyocytes (n = 3 per group). (A) Representative images of hiCMs after 4 h of cultivation under different shear stress conditions (scale bar: 500 μm). These images illustrate the morphological changes in response to varying shear stress levels. (B) Western blot analysis showing the expression levels of NLRP3 and Caspase-1 after 4 h of cultivation under the same shear stress conditions as in (A) (C,D) Quantification of NLRP3 and caspase-1 expression relative to GAPDH from the Western blot analysis in (B) (E) Average concentrations of IL-18 and IL-1β in the culture medium after 4 h of cultivation under different shear stress conditions. Data are represented as mean ± SEM. Statistical significances were determined by Kruskal–Wallis one-way ANOVA (C,D). *P < 0.05.

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  Figure 3. The effects of oF at 15 dyn/cm2 on hiCMs morphology and expression levels of NLRP3 and caspase-1 over time. All microscopy images were acquired at identical magnification (20×) to ensure spatial comparability across time points. (A) The time-course imaging revealed a spindle-shaped morphology alongside a progressively dispersed cellular distribution (scale bar: 200 μm). (B) Western blot bands for NLRP3 and Caspase-1 at 4, 8, and 12 h of OF treat as depicted in (A) (C,D) The quantification of NLRP3 and Caspase-1 expression levels, normalized to GAPDH, was derived from the Western blot bands illustrated in (B). (E) Average concentrations of IL-18 and IL-1β over 4, 8, and 12 h. Data are represented as mean ± SEM. Statistical significances were determined by single-factor repeated design ANOVA (C,D). *P < 0.05.

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  Figure 4. Comparison of RV functional and volumetric para meters between control and intervention groups. Data are represented as mean±SEM. Statistical significances were determined by single-factor repeated design A NOVA (A-F) and Spearman correlation (G). *P<0.05, **P< 0.01.

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  Figure 5. Analysis of RV relative NLRP3 expression at different follow-up time points (n = 6 per group). (A) Immunohistochemistry stain of NLRP3 in RVIT, AT and RVOT at each follow-up time point; Scale bar: 200 μm and 20 μm. (B) Quantification comparison of global RV relative NLRP3 expression in control and intervention groups. (C) Quantification comparison of regional RV relative NLRP3 expression in control and intervention groups. Data are represented as mean ± SEM. Statistical significances were determined by single-factor repeated design ANOVA (B) and Kruskal–Wallis one-way ANOVA (C) *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 6. Analysis of myocardial inflammation of RV at different follow-up time points via H-E stain (n = 6 per group). (A) 4 weeks after PR surgery. (B) 8 weeks after PR surgery. (C) 12 weeks after PR surgery. Scale bar: A, B, C, 100 μm. (D) Comparison of inflammation scores of global RV. (E) Comparison of inflammation scores of regional RV in intervention groups. Data are represented as mean ± SEM. Statistical significances were determined by single-factor repeated design ANOVA (D) and Kruskal–Wallis one-way ANOVA (E) *P < 0.05, **P < 0.01. Red arrow: peri-microvascular inflammatory cells; Yellow arrow: wall thickening of microvessels; Green arrow: inflammatory cells in extracellular matrix.

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  Figure 7. Fibrosis correlates with RV remodeling and systolic dysfunction (n = 6). (A) Comparison of fibrosis in the three regions of RV in control and intervention groups. (B) Quantitative analysis global RV fibrosis in control and intervention groups. (C) Quantitative analysis of regional RV fibrosis in control and intervention groups. (D) Correlation between the global RV fibrosis percentage and RV inflammation score with NLRP3 expression. (E) Correlation between the global RV fibrosis percentage and RVEDVi with RV longitudinal PSR. Statistical significances were determined by single-factor repeated design ANOVA (B) and Kruskal–Wallis one-way ANOVA (C) *P < 0.05, **P < 0.01.