Activating Transcription Factor 4 Drives the Progression of Diabetic Cardiac Fibrosis

Figure 1. ATF4 silencing improves cardiac function and relieves interstitial fibrosis in diabetic mice. (A, B) The mRNA and protein expression of ATF4 in heart tissues was determined by RT‐qPCR and western blotting. LVEF (C) and LVFS (D) were detected by M‐mode echocardiography. (E) Representative images of myocardial sections subjected to H&E staining and Masson's staining. (F) Apoptosis in cardiac tissues was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling assay. (G) Western blotting analysis of collagen I and collagen III protein abundance in heart tissues. Data are presented as the mean ± SD (n = 5). **P < 0.01, ***P < 0.001.

Figure 2. ATF4 depletion suppresses oxidative stress in mice with diabetic cardiomyopathy. (A) SOD activity, (B) GSH‐Px activity, (C) MDA level in heart tissues were assessed. (D) ROS production in hearts was evaluated by DHE staining. (E) Protein abundance of HIPK2, nuclear Nrf2 and HO‐1 in myocardial tissues was assessed by western blotting. Data are presented as the mean ± SD (n = 5). **P < 0.01, ***P < 0.001.

Figure 3. Inhibition of ATF4 attenuates apoptosis, oxidative stress, and fibrosis in HG‐exposed cardiomyocytes. (A) ATF4 level in HL‐1 cells after exposure to HG was determined by RT‐qPCR. (B) The mRNA level of ATF4 in HL‐1 cells transfected with sh‐ATF4 was detected by RT‐qPCR. (C) Cell viability was detected by MTT assay. (D) Flow cytometry analysis of cell apoptosis. (E) SOD activity, (F) GSH‐Px activity, (G) MDA level in HL‐1 cells were determined. (H) DCFH‐DA staining for measurement of ROS release from HL‐1 cells. Data are presented as the mean ± SD (n = 3). **P < 0.01, ***P < 0.001.

Figure 4. HIPK2 activates Nrf2/HO‐1 pathway to alleviate oxidative stress and fibrosis in HG‐exposed cardiomyocytes. (A, B) RT‐qPCR analysis of HIPK2 mRNA expression in HL‐1 cells with different treatments. (C) MTT assay for detection of cell viability. (D) Apoptotic rate of HL‐1 cells was evaluated by flow cytometry. (E) SOD activity, (F) GSH‐Px activity, (G) MDA level in HL‐1 cells from various groups were detected. (H) Production of ROS from HL‐1 cells was determined by DCFH‐DA staining. Data are presented as the mean ± SD (n = 3). **P < 0.01, ***P < 0.001.

Figure 5. ATF4 knockdown restrains Smurf2‐mediated ubiquitination and degradation of HIPK2. (A) The mRNA level of Smurf2 after ATF4 silencing was assessed by RT‐qPCR. (B) ChIP and (C) dual‐luciferase reporter assays were carried out to determine the interaction between Smurf2 and ATF4. (D) Smurf2 mRNA expression after transfection with sh‐Smurf2 was assessed by RT‐qPCR. (E, F) The interplay between exogenous and endogenous Smurf2 and HIPK2 was validated by co‐immunoprecipitation assay. (G) Protein abundance of HIPK2 in Smurf2‐depleted HL‐1 cells was detected by western blotting. (H) HIPK2 protein level in MG132‐treated cells was determined by western blotting. Data are presented as the mean ± SD (n = 3). ***P < 0.001.

Figure 6. Smurf2 is involved in ATF4‐induced cardiac fibrosis and oxidative stress in HG‐stimulated cardiomyocytes. (A, B) The mRNA and protein levels of Smurf2 in HL‐1 cells were determined by RT‐qPCR and western blotting. (C) MTT assay was adopted to detect cell viability. (D) Apoptotic rate of HL‐1 cells was measured by flow cytometry. (E) SOD activity, (F) GSH‐Px activity, (G) MDA level in HL‐1 cells were assessed. Data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.