The Downregulation of ADAM17 Exerts Protective Effects against Cardiac Fibrosis by Regulating Endoplasmic Reticulum Stress and Mitophagy

Figures

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  Figure 1. ADAM17 is upregulated in fibrosis heart tissues and mCFs treated with TGF-β1. Mice were divided into two groups: sham and MI. (a, b) The mRNA and protein levels of ADAM17, collagen I, and α-SMA in fibrosis heart tissues (n = 4). (c, d) mCFs were divided into two groups: control and TGF-β1 treatment (5 ng/mL, 48 h). The mRNA and protein levels of ADAM17, collagen I, and α-SMA in the process of mCF activation (n = 5). Data in (a–d) are expressed as mean ± SEM. ∗ indicates P < 0.05 and ∗∗ indicates P < 0.01 vs. sham or control groups.

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  Figure 2. ADAM17 knockdown attenuated TGF-β1-induced mCF activation and the upregulation of ECM proteins. (a) The mRNA expression of ADAM17 after three siRNA chain transfections (n = 3); siRNA3 was the most efficient chain. (b, c) NC or siRNA3 transfection in the presence or absence of TGF-β1 (5 ng/mL, 48 h). The mRNA and protein levels of ADAM17, collagen I, and α-SMA in activated mCFs after transfection with NC or siRNA3 (n = 5). (d) The migration of mCFs (determined by the areas of cells protruding from the wound border) after transduction with NC or siRNA3 (n = 3). (e) The proliferation of mCFs was assayed using CCK-8 (n = 5). Data in (a–e) are expressed as mean ± SEM. ∗ indicates P < 0.05, and ∗∗ indicates P < 0.01 vs. the NC group; # indicates P < 0.05, and ## indicates P < 0.01 vs. the NC+TGF-β1 group.

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  Figure 3. ADAM17 regulates ER stress and mitophagy to promote the activation of mCFs. (a) Protein expressions of Smad2/3, p-Smad2/3, p-PERK, ATF4, p-IRE1, XBP1s, c-ATF6, and GRP78 in mCFs with NC or siRNA3 transfection in the presence or absence of TGF-β1 (n = 5). (b) The protein levels of the ER stress branch, the ATF6 and GRP78, after transfection of NC or siRNA3 in the presence or absence of TGF-β1 or TG (n = 5). (c, d) Wound healing and CCK-8 assays for migration and proliferation in mCFs transfected with NC or siRNA3 in the presence or absence of TGF-β1 or TG (n = 5). (e) Protein expression of PINK1 and Parkin with NC or siRNA3 transfection in the presence or absence of TGF-β1 in mCFs (n = 5). Data in (a–e) are expressed as mean ± SEM. ns: not statistically significant; ∗ indicates P < 0.05, and ∗∗ indicates P < 0.01 vs. the NC or NC+TGF-β1 groups.

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  Figure 4. Inhibition of ADAM17 with TMI-005 reduced the degree of post-MI fibrosis and enhanced cardiac function in vivo. C57BL/6 mice were randomly divided into 4 groups, including control, TMI-005, MI, and MI treated with TMI-005. MI groups had left anterior descending ligation for 28 days to induced cardiac fibrosis. (a) Schema of the TMI-005 treatment protocol. (b) ADAM 17 activity in heart tissues was determined by measuring cleavage of the internally quenched fluorogenic substrate MCA-KPLGLDpa-AR-NH2 (n = 6). (c, d) In the control and TMI-005 groups, Masson's trichrome staining and M-mode images were used to assess the collagen deposition and cardiac function. LVESD, LVEDD, LVEF, and LVFS were quantified via echocardiography. (e, f) In the MI and MI treated with TMI-005 groups, Masson's trichrome staining and M-mode images were used to assess the degree of fibrosis and cardiac function. LVESD, LVEDD, LVEF, and LVFS were quantified via echocardiography. (g) Collagen I and α-SMA expression levels were quantified using western blotting. n = 6 in each group. Data in (a–g) are expressed as mean ± SEM. ∗ indicates P < 0.05, and ∗∗ indicates P < 0.01 vs. the control or MI groups.