Inhibition of ADAM10 ameliorates doxorubicin-induced cardiac remodeling by suppressing N-cadherin cleavage

Figures

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  Figure 1. Construction of ADAM10-shRNA lentiviral vectors. (a) PCR products of LentiLox-ADAM10-shRNA-transfected cells. (b) Fluorescence images of H9C2 cells transduced with ADAM10-shRNA lentiviral vectors for 3 days; GFP was observed under light (left) or fluorescence (right) microscopy. Analysis of (c) ADAM10 mRNA levels and (d) protein expression in H9C2 cells transduced with or without ADAM10-shRNA lentiviral vectors for 3 days. P-values in (c and d) were determined using the Kruskal–Wallis test. All data are presented as the mean values ± SEM. Magnification ×200. ADAM10, disintegrin metalloproteinase 10; shRNA, short hairpin RNA; GFP, green fluorescent protein.

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  Figure 2. ADAM10-dependent N-cadherin cleavage in H9C2 cells. (a) Protein expression of N-cadherin and CTF1 in the H9C2 cells transfected with or without ADAM10-shRNA lentiviral vectors for 3 days. (b) Protein expression of N-cadherin and CTF1 in the H9C2 cells treated with various concentrations of ADAM10 inhibitor (GI254023X) or vehicle control (DMSO) for 4 h and analyzed by western blotting. P-values in (a and b) were determined using the Kruskal–Wallis test. All data are presented as the mean values ± SEM. ADAM10, disintegrin metalloproteinase 10; CTF1, C-terminal fragment 1; shRNA, short hairpin RNA.

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  Figure 3. Cadherin-mediated intercellular adhesion is upregulated in ADAM10-knockout H9C2 cells. (a) Detection of N-cadherin on the surface of H9C2 cells transfected with ADAM10-shRNA or ADAM10 inhibitor by flow cytometry. (b) The adhesion ability of H9C2 cells transfected with ADAM10-shRNA (left) or ADAM10 inhibitor (right). P-values in a and b were determined using the Kruskal–Wallis test. All data are presented as the mean values ± SEM. ADAM10, disintegrin metalloproteinase 10; CTF1, C-terminal fragment 1; shRNA, short hairpin RNA.

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  Figure 4. ADAM10-shRNA alleviates the DOX-induced mouse death and cardiac dysfunction. (a) Fluorescence images of primary cardiomyocytes isolated from rats in the ADAM10-shRNA group; GFP was observed under light (left) or fluorescence (right) microscopy. (b) ADAM10 expression in control and DCM groups. (c) Representative images of LV M-mode echocardiograms (left), cardiac function parameters measured by echocardiography and hemodynamic analysis (right). (d) Hematoxylin and eosin-stained myocardial samples. (e) Immunohistochemistry for N-cadherin expression in sections of cardiac tissues. (f) Western blotting analysis of N-cadherin and CTF1 levels in rats’ cardiac tissues in the control, DCM, ADAM10-shRNA, and vector groups. P-values in a, c, e, and f were determined using the Kruskal–Wallis test. All data are presented as the mean values ± SEM. Magnification ×200. ADAM10, disintegrin metalloproteinase 10; shRNA, short hairpin RNA; DOX, doxorubicin; GFP, green fluorescent protein; CTF1, C-terminal fragment1; DCM, dilated cardiomyopathy.

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  Figure A1. N-cadherin shedding was restored in H9C2 cells following transfection with wild-type ADAM10. Protein expression of N-cadherin and CTF1 in the H9C2 cells transfected with ADAM10 overexpression vectors for 3 days. P-values were determined using the Kruskal–Wallis test. All data are presented as the mean value ± SEM. ADAM10, disintegrin metalloproteinase 10; CTF1, C-terminal fragment1.