miR-181b targets semaphorin 3A to mediate TGF-β–induced endothelial-mesenchymal transition related to atrial fibrillation
Ying-Ju Lai, … , Wei-Jan Chen, Yung-Hsin Yeh
Ying-Ju Lai, … , Wei-Jan Chen, Yung-Hsin Yeh
Published July 1, 2022
Citation Information: J Clin Invest. 2022;132(13):e142548. https://doi.org/10.1172/JCI142548.
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Research Article Cardiology

miR-181b targets semaphorin 3A to mediate TGF-β–induced endothelial-mesenchymal transition related to atrial fibrillation

Abstract

Atrial fibrosis is an essential contributor to atrial fibrillation (AF). It remains unclear whether atrial endocardial endothelial cells (AEECs) that undergo endothelial-mesenchymal transition (EndMT) are among the sources of atrial fibroblasts. We studied human atria, TGF-β–treated human AEECs, cardiac-specific TGF-β–transgenic mice, and heart failure rabbits to identify the underlying mechanism of EndMT in atrial fibrosis. Using isolated AEECs, we found that miR-181b was induced in TGF-β–treated AEECs, which decreased semaphorin 3A (Sema3A) and increased EndMT markers, and these effects could be reversed by a miR-181b antagomir. Experiments in which Sema3A was increased by a peptide or decreased by a siRNA in AEECs revealed a mechanistic link between Sema3A and LIM-kinase 1/phosphorylated cofilin (LIMK/p-cofilin) signaling and suggested that Sema3A is upstream of LIMK in regulating actin remodeling through p-cofilin. Administration of the miR-181b antagomir or recombinant Sema3A to TGF-β–transgenic mice evoked increased Sema3A, reduced EndMT markers, and significantly decreased atrial fibrosis and AF vulnerability. Our study provides a mechanistic link between the induction of EndMT by TGF-β via miR-181b/Sema3A/LIMK/p-cofilin signaling to atrial fibrosis. Blocking miR-181b and increasing Sema3A are potential strategies for AF therapeutic intervention.

Authors

Ying-Ju Lai, Feng-Chun Tsai, Gwo-Jyh Chang, Shang-Hung Chang, Chung-Chi Huang, Wei-Jan Chen, Yung-Hsin Yeh

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Figure 3

TGF-β induces miR-181b via SMAD2/3 signaling in vitro.

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TGF-β induces miR-181b via SMAD2/3 signaling in vitro. (A) qRT-PCR analy...
(A) qRT-PCR analysis of mature (miR-181b), precursor (Pre-miR-181b), and primary (Pri-miR-181b) transcripts of miR-181b in AEECs treated with TGF-β for 0–48 hours (n = 3). (B) miR-181 RNA expression in AEECs treated with or without TGF-β. 18S rRNA served as the loading control. (C) Left: Linear map schematic of the putative SBEs at the promoter of the miR-181b gene, with the mutated luciferase (Luc) constructs. Right: Graph shows the luciferase assay results. AEECs were transiently transfected with mutant promoter constructs and treated with or without 5 ng/mL TGF-β for 8 hours. miR-181b promoter characterization by luciferase assays showed the fold change in luciferase activity of the WT SBEs and mutated SBE1 and SBE2 (Mut 1 and Mut 2; left) with and without 5 ng/mL TGF-β or 10 μM SD-208. (D) Representative qRT-PCR analysis of miR-181b in AEECs with siRNA-mediated SMAD3 knockdown and TGF-β treatment. (A, B, and D) *P < 0.05, **P < 0.01, and ***P < 0.001 versus the untreated group, by 1-way ANOVA with Dunnett’s post hoc test (n = 3). (C) *P < 0.05, by 2-tailed Student’s t test (n = 3).