Melatonin Ameliorates MI-Induced Cardiac Remodeling and Apoptosis through a JNK/p53-Dependent Mechanism in Diabetes Mellitus

Figures

• 

  Figure 1. Effect of melatonin treatment on cardiac contractile function in post-MI diabetic mice. (a) Representative M-mode echocardiographic images from four respective mouse groups. Cardiac function was assessed using echocardiography at the first and fourth weeks post-MI. (b) Ejection fraction. (c) Fractional shortening. (d) LVIDs: left ventricular internal dimension during systole (end-systolic diameter). (e) LVIDd: left ventricular internal dimension during diastole (end-diastolic diameter). (f) Body weight. (g) Heart weight. (h) Heart weight-to-body weight (HW/BW) ratio. CON: normal control; DCM: diabetic cardiomyopathy; DMI: diabetes mellitus with MI; DMI-MEL: diabetes mellitus with MI treated with melatonin. Mean ± SEM, n = 4-6. ∗p < 0.05 vs. CON group, #p < 0.05 vs. DMI group, and @p < 0.05 vs. DCM group.

• 

  Figure 2. Effect of melatonin on the cardiomyocyte cross-sectional area and cardiac fibrosis with high-fat diet and/or myocardial infarction. (a) H&E staining and Masson's trichrome staining. The upper panel exhibits representative images of H&E staining of cardiac tissues. The lower panel depicts representative images of cardiac interstitial fibrosis with Masson's trichrome staining. (b) Pooled data of the cardiomyocyte cross-sectional area (n = 20-25 cells per group). (c) Pooled data of the interstitial fibrotic area of myocardial tissues. CON: normal control; DCM: diabetic cardiomyopathy; DMI: diabetes mellitus with MI; DMI-MEL: diabetes mellitus with MI treated with melatonin. Mean ± SEM, n = 3-4 per group. ∗p < 0.05 vs. CON group, #p < 0.05 vs. DMI group, and @p < 0.05 vs. DCM group. Scale bar = 100 μm.

• 

  Figure 3. Effect of melatonin treatment on myocardial TUNEL apoptosis in post-MI diabetic mice. (a) Representative images of TUNEL staining 4 weeks post-MI in diabetic mice. Representative TUNEL staining images are displayed in the upper panel while the images with DAPI staining are displayed in the middle panel demonstrating nuclei of cells. The lower panel exhibits the overlay images to illustrate apoptotic cells. (b) Pooled data displaying the percentage of apoptotic cells. Mean ± SEM, n = 3-5 per group. ∗p < 0.05 vs. CON group, #p < 0.05 vs. DMI group, and @p < 0.05 vs. DCM group. Scale bar = 200 μm.

• 

  Figure 4. Effect of melatonin on JNK and apoptotic-related protein expressions with or without melatonin treatment in diabetic MI hearts. (a) Representative images of western blot 4 weeks post-MI in diabetic mice. (b) p-JNK to total JNK ratio. (c) p53 level. (d) Bcl-2 level. (e) Bax level. (f) Caspase-3 level. Mean ± SEM, n = 3-5 per group. ∗p < 0.05 vs. CON group, #p < 0.05 vs. DMI group, and @p < 0.05 vs. DCM group.

• 

  Figure 5. The effect of melatonin treatment on cellular apoptosis in H9c2 myocytes challenged by high glucose/high fat under hypoxia. (a) Representative images of TUNEL staining of H9c2 cells with or without melatonin and anisomycin treatment. The images with TUNEL staining are displayed in the upper panel. Images with DAPI staining are displayed in the middle panel depicting nuclei of cells. Lower panel indicates the overlay images to illustrate the apoptotic cells. (b) Pooled data depicting the percentage of apoptotic cells. Mean ± SEM, n = 3 per group. ∗p < 0.05 vs. CON group, #p < 0.05 vs. HF/HG-hypoxia group, †p < 0.05 vs. HF/HG-hypoxia-MEL group. Scale bar = 100 μm.

• 

  Figure 6. Effect of melatonin and the JNK activator anisomycin on protein levels of apoptotic-related proteins in H9c2 cells challenged by high glucose/high fat under hypoxia. (a) Representative images of western blot of H9c2 cells with or without melatonin and anisomycin treatment. (b) p-JNK to total JNK ratio. (c) p53 level. (d) Bcl-2 level. (e) Bax level. (f) Caspase-3 level. Mean ± SEM, n = 3. ∗p < 0.05 vs. CON group, #p < 0.05 vs. high-glucose/high-fat treatment under hypoxia, and †p < 0.05 vs. HF/HG-hypoxia-MEL group.

• 

  Figure 7. Effect of melatonin and the JNK activator anisomycin on contractile function of isolated cardiomyocytes. (a) Resting cell length. (b) Peak shortening (PS, normalized to resting cell length). (c) Maximal velocity of shortening (+dL/dt). (d) Maximal velocity of relengthening (−dL/dt). (e) Time-to-peak shortening (TPS). (f) Time-to-90% relengthening (TR90). Mean ± SEM, n = 39 cells per group. ∗p < 0.05 vs. CON group, #p < 0.05 vs. HF/HG-hypoxia group, and †p < 0.05 vs. HF/HG-hypoxia-MEL group.

• 

  Figure 8. Schematic diagram depicting the proposed signaling pathways involving JNK, p53, and apoptosis behind the melatonin-offered protective effect against post-MI injury in diabetes.