Regulatory effect of transcription factor E4BP4 on pathological myocardial fibrosis through the AMPK-TGF-β1/SMAD3 signaling pathway

Huang Derong, Wen Qing, Su Yuchen

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Journal of Chongqing Medical University ›› 2025, Vol. 50 ›› Issue (05) : 640-648. DOI: 10.13406/j.cnki.cyxb.003762
Basic research

Regulatory effect of transcription factor E4BP4 on pathological myocardial fibrosis through the AMPK-TGF-β1/SMAD3 signaling pathway

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Abstract

Objective To explore the effects of transcription factor adenovirus E4 promoter-binding protein（E4BP4） in regulating pathological myocardial fibrosis through the adenosine monophosphate-activated protein kinase（AMPK）-transforming growth factor （TGF）-β1/Smad homolog 3（SMAD3） pathway. Methods A mouse model of myocardial fibrosis was established，and the expression of E4BP4 was determined in the model group and the sham-operation group. Primary cardiac fibroblasts were isolated，cultured，activated by angiotensin Ⅱ（Ang Ⅱ），and divided into the following groups：Ang Ⅱ+E4BP4 group （transfected with E4BP4 overexpression plasmids），Ang Ⅱ+siE4BP4 group （transfected with E4BP4 interfering plasmids），Ang Ⅱ group，and control group（without Ang Ⅱ treatment）. The fluorescence intensity of ɑ-smooth muscle actin（α-SMA） was determined by the immunofluorescence assay，the cell viability by the cell counting kit，the expression of E4BP4，α-SMA，collagen type Ⅰ（collagen Ⅰ），and collagen type Ⅲ（collagen Ⅲ） by polymerase chain reaction，and the protein expression of TGF-β1，AMPK，and SMAD3 by Western blot. Results Compared with the sham-operation group，the model group showed significantly increased myocardial fibrosis degree（38.46±1.21 vs. 3.39±0.39，t=-78.564，P=0.000） and E4BP4 protein expression（0.96±0.03 vs. 0.75±0.03，t=-11.480，P=0.000）. In vitro experiments found that the mean fluorescence intensity（0.05±0.01 vs. 0.42±0.03，F=677.591，P=0.000），cell viability（91.30±2.39 vs. 123.74±2.60，F=132.696，P=0.000），and the levels of α-SMA（1.26±0.09 vs. 3.59±0.86，F=52.274，P=0.000），collagen Ⅰ（1.16±0.11 vs. 3.79±0.89，F=55.336，P=0.000），collagen Ⅲ（1.23±0.13 vs. 2.92±0.36，F=119.929，P=0.000），TGF-β1（0.66±0.04 vs. 0.96±0.02，F=142.954，P=0.000），and p-SMAD3/SMAD3（0.81±0.03 vs. 1.37±0.02，F=739.609，P=0.000） in the Ang Ⅱ+siE4BP4 group were significantly lower than those in the Ang Ⅱ+E4BP4 group. The expression of p-AMPK/AMPK in the Ang Ⅱ+siE4BP4 group was significantly higher than that in the Ang Ⅱ+E4BP4 group（0.89±0.01 vs. 0.58±0.02，F=284.541，P=0.000）. Conclusion E4BP4 plays a crucial role in the regulation of fibrosis. Inhibition of E4BP4 expression exerts an anti-fibrotic effect by activating AMPK and inhibiting TGF-β1/SMAD3 pathway.

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myocardial fibrosis / adenovirus E4 promoter-binding protein / adenosine monophosphate-activated protein kinase / transforming growth factor-β / Smad homolog 3

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Huang Derong , Wen Qing , Su Yuchen. Regulatory effect of transcription factor E4BP4 on pathological myocardial fibrosis through the AMPK-TGF-β1/SMAD3 signaling pathway. Journal of Chongqing Medical University. 2025, 50(05): 640-648 https://doi.org/10.13406/j.cnki.cyxb.003762

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