Matrine Inhibits the Wnt3a/β‐Catenin Signalling to Attenuate Pressure Overload‐Induced Atrial Remodelling and Vulnerability to Atrial Fibrillation

Figures

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  FIGURE 1. Activation of the Wnt3a/β‐catenin signalling pathway in the atrial tissue of patients diagnosed with AF. (A) Heatmap showing 297 up‐regulated and 256 down‐regulated DEGs in the TAC group versus the Sham group. (B) GO enrichment results including BP (n = 5). (C, D) Representative western blot images and relative atrial expression of Wnt3a, p‐GSK3β, active β‐catenin, β‐catenin (n = 6). Data were expressed as mean ± SEM. **p < 0.01, ***p < 0.001.

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  FIGURE 2. Potential of MAT to ameliorate atrial remodelling and mitigate tissue fibrosis induced by TAC. (A) Grouping of experimental animals and schematic diagram of the experiment. (B) Representative echocardiographic images of the atria and ventricle. (C–H) Echocardiographic parameters including left ventricular ejection fraction (LVEF), left ventricular shortening fraction (LVFS), left ventricular end‐diastolic diameter (LVIDd), left ventricular posterior wall diameter (LVPWd), left atrial diameter (LAD) and left atrial diastolic area (n = 5). (I) Overall cardiac morphology and representative Masson's trichromatic staining images (upper bar = 2 mm, lower bar = 100 μm). (J) Percentage of the total area with fibrosis (n = 5). (K–N) Representative western blot images and relative expression of COL1A1, COL3A1 and α‐SMA in each group of atria (n = 4). (O, P) Relative immunofluorescence intensity of α‐SMA (n = 4). Data were expressed as mean ± SEM. ***p < 0.001, versus Sham group; # p < 0.05, ## p < 0.01, ### p < 0.001, versus TAC group.

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  FIGURE 3. Potential of MAT to ameliorate atrial remodelling induced by TAC. (A) Representative ECG traces following burst pacing in the right atrium. The dashed line represents the atrial fibrillation rhythm. (B, C) Summary of AF induction and AF duration in each group (n = 8). (D) Langendorff electrophysiological mapping images. The bars represent the duration from the initial to the final measurement within one heartbeat (n = 5). (E–G) Summary of left atrial activation patterns. EMapScope 5.0 software was used to calculate the mean conduction velocity, absolute inhomogeneity and inhomogeneity index of the left atrium (n = 5). (H–J) Representative western blot images and relative atrial expression of Cx40 and Cx43 (n = 4). (K–M) WGA (green), Cx40 and Cx43 (red) and DAPI (blue) immunofluorescence stain representation and Cx40 and Cx43 relative immunofluorescence intensity (n = 4). Data were expressed as mean ± SEM. ***p < 0.001, versus Sham group; ns p > 0.05, # p < 0.05, ## p < 0.01, ### p < 0.001, versus TAC group.

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  FIGURE 4. Effects of MAT administration on Wnt3a/β‐catenin signalling and atrial inflammation and fibrosis in mice subjected to TAC. (A) Venn diagram of the Matrine targets and AF targets. (B, C) MAT component‐target‐pathway network diagram and the key targets screened out. (D) Heatmap showing 593 up‐regulated and 706 down‐regulated DEGs in the TAC + MAT group versus the TAC group. (E) GO enrichment results including BP, CC, MF (n = 5). (F–J) Representative western blot images and relative atrial expression of Wnt3a, p‐GSK3β, active β‐catenin and β‐catenin (n = 4). (K–M) α‐actinin/Vimentin (green), Wnt3a/active β‐catenin (red) and DAPI (blue) immunofluorescence stain representation and Wnt3a/active β‐catenin relative immunofluorescence intensity (n = 4). (N–P) Representative western blot images and quantification of active β‐catenin expression in the atrial nucleus or cytoplasm (n = 4). (Q–W) Representative western blot images and relative atrial expression of TNF‐α, IL‐1β, IL‐6, TGF‐β, MMP2 and MM9 (n = 4). Data were expressed as mean ± SEM. ***p < 0.001, versus Sham group; # p < 0.05, ## p < 0.01, ### p < 0.001, versus TAC group.

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  FIGURE 5. Reversal of the beneficial effect of MAT on atrial remodelling in TAC mice by activation of β‐catenin. (A) Grouping of experimental animals and schematic diagram of the experiment. (B) Representative echocardiographic images of the atria and ventricle. (C–H) Echocardiographic parameters including left ventricular ejection fraction (LVEF), left ventricular shortening fraction (LVFS), left ventricular end‐diastolic diameter (LVIDd), left ventricular posterior wall diameter (LVPWd), left atrial diameter (LAD) and left atrial diastolic area (n = 5). (I) Overall cardiac morphology and representative Masson's trichromatic staining images (upper bar = 2 mm, lower bar = 100 μm). (J) Percentage of the total area with fibrosis (n = 5). (K–N) Representative western blot images and relative expression of COL1A1, COL3A1 and α‐SMA in each group of atria (n = 4). Data were expressed as mean ± SEM. **p < 0.01, ***p < 0.001, versus TAC + MAT group; ns p > 0.05, ### p < 0.001, versus TAC group. ns, not significant.

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  FIGURE 6. Reversal of the beneficial impact of MAT on atrial electrical remodelling in TAC mice by activation of β‐catenin. (A) Representative ECG traces following burst pacing in the right atrium. The dashed line represents the atrial fibrillation rhythm. (B, C) Summary of AF induction and AF duration in each group (n = 8). (D) Langendorff electrophysiological mapping images. The bars represent the duration from the initial to the final measurement within one heartbeat (n = 5). (E–G) Summary of left atrial activation patterns. EMapScope 5.0 software was used to calculate the mean conduction velocity, absolute inhomogeneity and inhomogeneity index of the left atrium (n = 5). (H–J) Representative western blot images and relative atrial expression of Cx40 and Cx43 (n = 4). Data were expressed as mean ± SEM. **p < 0.01, ***p < 0.001, versus TAC + MAT group; ns p > 0.05, ### p < 0.001, versus TAC group. ns, not significant.

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  FIGURE 7. The impact of SKL2001 on the Wnt3a/β‐catenin signalling pathway, atrial fibrosis and inflammation and the mechanisms by which MAT prevents and treats AF. (A–E) Representative western blot images and relative atrial expression of Wnt3a, p‐GSK3β, active β‐catenin and β‐catenin in each group of atria. (F–H) α‐actinin/Vimentin (green), Wnt3a/active β‐catenin (red) and DAPI (blue) immunofluorescence stain representation and Wnt3a/active β‐catenin relative immunofluorescence intensity (n = 4). (I–K) Representative western blot images and quantification of active β‐catenin expression in the atrial nucleus or cytoplasm (n = 4). (L–R) Representative western blot images and relative atrial expression of TNF‐α, IL‐1β, IL‐6, TGF‐β, MMP2 and MM9. (S) Schematic diagram illustrating the mechanism by which MAT inhibits AF via the Wnt3a/β‐catenin signalling pathway. Data were expressed as mean ± SEM. n = 4 per group. ns p > 0.05, ***p < 0.001, versus TAC + MAT group; ns p > 0.05, ### p < 0.001, versus TAC group. ns, not significant.