Tongguan capsule‐derived herb reduces susceptibility to atrial fibrillation by inhibiting left atrial fibrosis via modulating cardiac fibroblasts

Figures

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  Figure 1. Tongguan capsule‐derived herb (TGD) inhibits atrial fibrillation (AF) inducibility and duration in post‐myocardial infarction (MI) rats. A, An example of an induced AF episode. Before the burst (a), the rat was in sinus rhythm. After termination of the burst (b), the rat displayed an irregular atrial rhythm, with an irregular ventricular response. After seconds (c), the AF episode terminated spontaneously, and the sinus rhythm resumed. B, An example of a non‐induced AF episode. After termination of the burst (b), the rat also displayed sinus rhythm. C, TGD inhibits AF inducibility in post‐MI rats (n = 15 rats/group). D, TGD decreases AF duration in post‐MI rats (n = 15 rats/group). HR, heart rate; RR, R‐wave to R‐wave interval; AR, atrial rate. *P < 0.05 vs sham group; # P < 0.05 vs MI group

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  Figure 2. Tongguan capsule‐derived herb (TGD) improves left atrial conduction function in post‐myocardial infarction (MI) rats. A, Representative isochronous maps from multielectrode array recordings. Areas of isochronal crowding were found in the MI group. The degree of crowding decreased in the TGD‐treated rats. The black pulse symbol indicates the site of the stimulus electrode. The white dashed lines indicate the areas of conduction block. B, Representative conduction heterogeneity maps. Conduction was more heterogeneous in the rats with MI compared with the sham and TGD groups. C, Conduction velocities in the four groups. TGD improves the conduction velocity (n = 5 rats/group). **P < 0.01 vs sham group; ## P < 0.01 vs MI group

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  Figure 3. Tongguan capsule‐derived herb (TGD) inhibits left atrial fibrosis in post‐myocardial infarction (MI) rats. A, Representative images of left atrial (LA) fibrosis (Masson staining, which stains fibrosis blue and viable muscle red, scale bar: 100 μm). B, Statistical results of LA fibrosis. TGD reduces fibrosis‐positive area (n = 5 rats/group). C, Western blot analysis of the protein expression of collagen I. TGD reduces the protein level of collagen I in the left atrium (n = 5 rats/group). D, Western blot analysis of the protein expression of collagen III. TGD reduces the protein level of collagen III in the left atrium (n = 5 rats/group). *P < 0.05, **P < 0.01 vs the sham group; # P < 0.05, ## P < 0.01 vs the MI group

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  Figure 4. Tongguan capsule‐derived herb (TGD) inhibits cardiac fibroblasts (CFs) collagen expression and proliferation in vitro. A, Western blot analysis of the protein expression of collagen I. TGD reduces the collagen I protein expression in vitro (n = 5 independent cell samples/group). B, Western blot analysis of the protein expression of collagen III. TGD reduces the collagen III protein expression in vitro (n = 5 independent cell samples/group). C, The bar graph of the proliferation rate. TGD inhibits the proliferation of CFs (n = 6 independent samples/group). D,E, Flow cytometry analysis of cell cycle revealed that TGD treatment induces cell cycle arrest in the G1 phase in CFs (n = 4 independent samples/group). F, CFs apoptosis in different experimental groups detected using flow cytometry. CFs were labelled with Annexin V–FITC and propidium iodide. Representative images are shown. Q1, Q2, Q3, and Q4 represent necrosis, late‐stage apoptosis, early‐stage apoptosis and normal cells respectively. G, Q2 and Q3 were chosen to analyse the change in the apoptosis rate in different experimental groups (n = 4 independent samples/group). *P < 0.05, **P < 0.01 vs the control group

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  Figure 5. Tongguan capsule‐derived herb (TGD) inhibits foetal bovine serum (FBS)‐induced cardiac fibroblasts (CFs) migration. A, Representative images of CFs migration detected using the Transwell migration assay (scale bar: 100 μm). B, Quantitative data for CFs migration (n = 4 independent samples/group). **P < 0.01 vs the control group. ## P < 0.01 vs the FBS group

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  Figure 6. Tongguan capsule‐derived herb (TGD) inhibits TGF‐β1–induced myofibroblast transformation. A, Representative images of the expression and distribution of myofibroblast‐specific marker α‐SMA (scale bar: 30 μm). Fluorescence immunohistochemistry (using a specific α‐SMA first antibody, followed by a secondary antibody conjugated with FITC) was performed to demonstrate TGF‐β1–induced myofibroblast transformation. Nuclei were stained with DAPI. B, Western blot analysis of the protein expression of α‐SMA. TGD reduces the expression of α‐SMA protein in cardiac fibroblasts (n = 5 independent samples/group). C, Western blot analysis of the protein expression of TGF‐β1. TGD reduces the expression of TGF‐β1 protein in cardiac fibroblasts (n = 5 independent samples/group). D, Western blot analysis of the protein expression of Smad3. TGD has no effect on the protein level of Smad3 (n = 5 independent samples/group). E, Western blot analysis of the protein expression of MMP‐9. TGD reduces the protein level of MMP‐9 (n = 5 independent samples/group). F, Western blot analysis of the protein expression of TIMP‐1. TGD increases the protein level of TIMP‐1 (n = 5 independent samples/group). *P <…

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  Figure 7. Tongguan capsule‐derived herb (TGD) inhibits cardiac fibroblasts secretion in vitro. A, Hydroxyproline assay in the cell culture supernatants. TGD inhibits collagen secretion (48 h, n = 5 independent samples/group). B‐E, ELISA of MCP‐1, IL‐6, TGF‐β1 and TNF‐α in the cell culture supernatants. TGD reduces the level of MCP‐1, IL‐6 and TGF‐β1, without TNF‐α (48 h, n = 5 independent samples/group). *P < 0.05, **P < 0.01 vs the control group