GCN2 deficiency ameliorates doxorubicin-induced cardiotoxicity by decreasing cardiomyocyte apoptosis and myocardial oxidative stress

Fig. 1. General control nonderepressible 2 (GCN2) deficiency attenuated doxorubicin (Dox)-induced left venticular (LV) dysfunction. Male Gcn2–/– and wild-type (WT) mice received intraperitoneal injections (5 mg/kg/week) for 4 weeks. The body weight (A), the heart weight to body weight ratio (B), and the mRNA levels of atrial natrurietic peptide (ANP) (C) were measured. Echocardiography was used to measure LV ejection fraction (D, E). n = 7–8, * indicates p < .05, * * indicates p < .01.

Fig. 2. GCN2 deficiency attenuated Dox-induced myocardial fibrosis and apoptosis. After exposure to Dox or control conditions for 4 weeks, representative heart sections from control and Dox-treated WT and Gcn2–/– mice were stained with Masson trichrome (upper panel, scale bar=50 µm), and DiO (green), DAPI (blue) and TUNEL assay kit (red) (lower panel, scale bar=10 µm, arrows point to TUNEL positive cells) (A). The fibrosis area (B) and TUNEL positive cells (C) were quantified. Heart tissue was collected from control and Dox-treated mice and lysates were subjected to casapse-3 activity assay (D) and Western blot for the expression of cleaved caspase-3, Bcl-2 and Bax. β-tubulin was used as a loading control (E). N = 3–5, * indicates p < .05, ** indicates p < .01, *** indicates p < .001.

Fig. 3. GCN2 deficiency attenuated Dox-induced myocardial oxidative stress and endoplasmic reticulum (ER) stress. Heart tissue was collected from WT and Gcn2−/− mice 4 weeks after Dox treatment or control conditions. Then, myocardial 3΄-nitrotyrosine (3΄-NT) (A), malondialdehyde (MDA) (B) levels, and the ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) ratio (C) were determined. The mRNA levels of anti-oxidative stress response genes were examined using real-time polymerase chain reaction (PCR) (D). Heart lysates were examined by Western blotting for the expression of total and phosphorylated eIF2α, activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP) and uncoupling protein 2 (UCP2). β-tubulin was used as a loading control (E). N = 3–4, * indicates p < .05, ** indicates p < .01.

Fig. 4. GCN2 affects Dox induced cell death, oxidative stress and ER stress in H9C2 cardiomyoblast cells. Scramble shRNA (shScr) and GCN2-specific shRNA (shGCN2) stably transfected cells were treated with 0–2 μM Dox for 24 h and the cell viability (A) was determined. After treatment with 1 μM Dox for 24 h, the intracellular reactive oxygen species (ROS) (B) and superoxide (C) levels in control and GCN2 knockdown cells were measured, and cell lysates were examined by Western blot (D). After treatment with 0–2 μM Dox for 24 h, the cell viability of control and GCN2 overexpressed cells was determined (E). Control and GCN2 overexpressed cells were treated with 1 μM Dox for 24 h, then the intracellular ROS (F) and superoxide (G) levels were measured, and cell lysates were examined by Western blot (H). N = 3–8, * indicates p < .05, ** indicates p < .01.

Fig. 5. CHOP affects Dox-induced cell death, oxidative stress and ER stress in H9C2 cells. Cells were infected with adenovirus with empty vector (Ad-Empty) or FLAG-tagged CHOP (Ad-CHOP), then treated with different concentrations (0–1 μM) of Dox for 24 h, and the cell viability (A) was determined. After treatment with 1 μM Dox for 24 h, the intracellular reactive oxygen species (ROS) (B) and superoxide (C) levels in Ad-Empty and Ad-CHOP transfected cells were measured, and cell lysates were examined by Western blot (D). After treatment with 0–2 μM Dox for 24 h, the cell viability of WT and Chop-/- cells was determined (E). WT and Chop-/- cells were treated with 1 μM Dox for 24 h; then, the intracellular ROS (F) and superoxide (G) levels were measured, and cell lysates were examined by Western blot (H). N = 3–8, * indicates p < .05, ** indicates p < .01.

Fig. 6. Knockdown of UCP2 exacerbates Dox-induced cell death and oxidative stress in H9C2 cells. ShScr and UCP2-specific shRNA (shUCP2) stably transfected cells were treated with 0–1 μM Dox for 24 h, and then the cell viability was determined (A). After treatment with 1 μM Dox for 24 h, the intracellular reactive oxygen species (ROS) (B) and superoxide (C) levels in control and UCP2 knockdown cells were measured, and cell lysates were examined by Western blot (D). Control or UCP2 knockdown cells were infected with Ad-Empty, Ad-GCN2, Ad-CHOP or Ad-GCN2 plus Ad-CHOP for 24 h, and the cell viability was determined (E). N = 3–8, * indicates p < .05, ** indicates p < .01, *** indicates p < .001, NS, not significant.