MicroRNA‐146b‐5p promotes atrial fibrosis in atrial fibrillation by repressing TIMP4

Figures

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  FIGURE 1. A, Representative surface ECG showing AF in an ATP dog and sinus rhythm in a sham dog. Surface ECG showing the lack of P waves and irregular RR intervals. B and C, AF inducibility and duration expanded substantially in ATP dogs. ** p < 0.001 versus sham. AF, Atrial fibrillation

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  FIGURE 2. A, Hot map and volcano plot showed that miR‐146b‐5p is the most pronounced change among the 20 differentially expressed miRNAs in atrial cardiomyocytes from dogs with AF. B, The volcano plot showed fibrotic genes were significantly dysregulated in atrial cardiomyocytes from dogs with AF. C, Pathway enrichment analysis predicted the TGF‐β1pathway was the most significant involved pathway based on the mRNA profiling results. D, PCR analysis showed a significant upregulation of miR‐146b‐5p, MMP9, TGFB1and COL1A1, with a significant downregulation of TIMP4 expression in dogs with AF. E, Western blot analysis showed protein expression of TIMP4, MMP9, TGF‐β1 and COL‐I in atrial cardiomyocytes from dogs with AF and sinus rhythm. F, Histopathological changes and the collagen volume fraction in left atrium after staining with Masson's trichrome stain from dogs with AF and sinus rhythm. ** p < 0.001 versus sham. AF, Atrial fibrillation

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  FIGURE 3. A, hiPSC‐aCMs‐fibroblast contact co‐culture. B and C, Decreased action potential period was found in hiPSC‐aCMs after RES. D‐F, ICa , L traces, present density‐voltage linkage, and Cav1.2 expression critically reduced after RES. G‐I, Regularized e ICa , L activation, inactivation and recovery curve. RES, rapid electrical field stimulation

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  FIGURE 4. A, Quantitative RT‐PCR analysis showed mRNA expression changes in hiPSC‐aCMs‐fibroblast contact co‐culture model after transfection of miR‐146b‐5p with or without RES. B, Western blot analysis showed protein expression in hiPSC‐aCMs‐fibroblast contact co‐culture model after transfection of miR‐146b‐5p with or without RES. C, The collagen fibres were accumulated in extracellular space after rapid depolarization of miR‐146b‐5p overexpressed hiPSC‐aCMs‐fibroblast co‐culture cellular model by RES (X4000). * p < 0.05 versus control; ** p < 0.001 versus control; # p < 0.001 versus RES (‐). NC, negative control; RES, rapid electrical field stimulation

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  FIGURE 5. A, Comparison Kaplan‐Meier survival curves after MI in mice during an 8‐week follow‐up period. B, Representative M‐mode echocardiograms of a mouse from a different group at baseline, 4 weeks and 8 weeks after MI. (C) The 4‐ and 8‐week echocardiographic data for ejection fraction (EF), (D) LVID at systole (LVIDs), (E) LVID at diastole (LVIDd) and (F) LAD. G, Representative surface ECG pacing‐induced AF in mice. (H) AF inducibility increased significantly in in both MI mice and antagomiR‐NC pretreated MI mice. * p < 0.05 versus WT; ** p < 0.001 versus WT; # p < 0.05 versus MI+antagomir‐146. LAD, left atrial diameter; LVID, left ventricular internal diameter; MI, myocardial infarction; NC, negative control; WT, wild type

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  FIGURE 6. A, Examples of whole hearts showing evidence of atrial dilatation in mice after MI. B, Whole heart long‐axis images of haematoxylin‐eosin staining. C, Atrial weight/tibia length (AW/TL) ratios of WT and other 3 groups mice 8 weeks after MI. D‐E, Representative Masson's trichrome images and quantification of atrial (D), and ventricle (E) fibrosis after 8 weeks of MI compared with WT. Quantification expressed as collagen volume fraction. F, Representative immunofluorescence images showing COL1A1 (green) and DAPI (blue) in WT and other 3 groups hearts 8 weeks after MI. G, PCR analysis showed mRNA expression of miR‐146b‐5p, MMP9, TGFB1and COL1A1. H, Western blot analysis showed protein expression of TIMP4, MMP9, TGF‐β1 and COL‐I in cardiomyocytes. * p < 0.05 versus WT; ** p < 0.001 versus WT; # p < 0.05 versus MI+antagomir‐146; ## p < 0.001 versus MI+antagomir‐146. MI, myocardial infarction; NC, negative control; WT, wild type

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  FIGURE 7. Proposed working model of the miR‐146b‐5p mediated atrial fibrosis in atrial fibrillation