Melatonin prevents doxorubicin-induced cardiotoxicity through suppression of AMPKα2-dependent mitochondrial damage

Figures

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  Fig. 1. Effects of doxorubicin and melatonin on AMPKα2 and apoptosis in H9c2 cardiomyoblasts. H9c2 cardiomyoblasts were treated with doxorubicin (DOX) and melatonin (MLT) for 24 h. Cell viability assays (a), western blotting (b), RT-PCR (c), and luciferase reporter assays for AMPKα2 promoter activity (d) or E2F activity (e) were performed. FACS analysis of Annexin V-PI double-positive H9c2 cells overexpressing AMPKα2WT or AMPKα2DN (f).

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  Fig. 2. Effects of doxorubicin and melatonin on AMPKα2 and mitochondrial function in H9c2 cardiomyoblasts. H9c2 cells overexpressing AMPKα2WT or AMPKα2DN were treated with doxorubicin (1 μM) and melatonin (1 mM) for 24 h. Under these conditions, mitochondrial membrane potential (a), cellular and mitochondrial ROS levels (b), mitochondrial OXPHOS complex protein levels (c), mitochondrial DNA contents (d), and cellular ATP levels (e) were measured. The level of AMPKα2 in whole-cell extracts (WCE), nonmitochondrial (non-Mito), and mitochondrial fractions (Mito) (f). Fluorescence images of AMPKα2, mitochondria, and nuclei, which were labeled with Texas Red dye, MitoTracker green, and DAPI, respectively (g).

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  Fig. 3. Effects of doxorubicin and melatonin on AMPKα2 and mitochondrial function in AMPK knockout MEFs. WT, Ampkα1−/−, Ampkα2−/−, and Ampkα1−/−α2−/− double-knockout mouse embryonic fibroblasts (MEFs) were treated with doxorubicin (1 µM) and melatonin (1 mM) for 24 h. The protein levels of AMPKα1 and AMPKα2 in whole-cell extracts (WCE) and mitochondrial fractions (Mito) (a), apoptosis rate (b), mitochondrial membrane potential (c), cellular and mitochondrial ROS levels (d), cellular ATP levels (e), and mitochondrial length (f) were measured. The pDsRed-Mito vector was used to label mitochondria (f). n.s., not significant.

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  Fig. 4. Effects of doxorubicin and melatonin on mitochondria-specific AMPKα2 and mitochondrial function in H9c2 cardiomyoblasts. H9c2 cells were treated with melatonin (1 mM) and/or doxorubicin (1 µM) for 24 h. a Endogenous AMPKα2 was immunoprecipitated and analyzed by western blotting. b The structure of mito-ABKAR. c After transfection with the mito-ABKAR plasmid, representative CFP images, YFP images, and FRET images were obtained by confocal microscopy under the indicated conditions. FRET efficiency was represented as pseudocolor images of FRET/CFP. d The correlation between mitochondrial length and FRET signal in the context of melatonin and doxorubicin treatment. e MFF phosphorylated at Ser146 (P-MFF) in H9c2 cells transfected with AMPKα2WT & DN. P-MFF (f), cellular ROS level (g), mitochondrial length (h), and cell viability (i) were measured in H9c2 cells transfected with mChF-Tom20-AIP.

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  Fig. 5. Role of ROS in the induction of AMPKα2 in the context of doxorubicin and melatonin treatment. H9c2 cells were treated with doxorubicin and melatonin in the presence of NAC (5 mM). Cellular and mitochondrial ROS levels (a), western blot analysis of immunoprecipitated Myc-AMPKα2 after transfection into H9c2 cells (b), western blot and RT-PCR (c), apoptosis rate (d) were measured or performed. e H9c2 cells were treated with doxorubicin and melatonin in the presence of the indicated concentration of 4-P-PDOT, and western blot analyses were performed.

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  Fig. 6. AMPKα2 in the heart tissue of mice treated with doxorubicin and/or melatonin. Mice were intraperitoneally injected with doxorubicin (2.5 mg/kg) and/or melatonin (1 mg/kg) every 2 days for 2 weeks. a After performing the experiments, the heart tissues of the mice were extracted, and H&E and Masson’s trichrome staining were performed. The arrows indicate the thickness of the myofibrillum. b The expression of AMPKα2 and the number of TUNEL-positive cells in heart tissue were identified by immunohistochemical staining. c Western blot analysis of the proteins from the heart tissue of each mouse group. Each image and western blot band were quantified using ImageJ software. LV, left ventricle; RV, right ventricle.