Melatonin Restores Autophagic Flux by Activating the Sirt3/TFEB Signaling Pathway to Attenuate Doxorubicin-Induced Cardiomyopathy

Figures

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  Figure 1. Echocardiography of melatonin with or without DOX treatment in S3 KO and wild-type mice. (A) Representative M-mode echocardiographic images of six respective mouse groups; (B) ejection fraction; (C) fractional shortening. WT, wild-type; DOX, doxorubicin; MT, melatonin; S3 KO, Sirt3 knockout. Mean ± SD, n = 4–6. * p < 0.05 vs. WT group, # p < 0.05 vs. WT-DOX group, & p < 0.05 vs. S3 KO group, @ p < 0.05 vs. WT-DOX-MT group.

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  Figure 2. Cardiac fibrosis was assessed by Masson’s trichrome staining in S3 KO and WT mice following DOX and melatonin treatment. (A) Representative Masson’s trichrome staining images of heart sections; (B) analysis of interstitial fibrotic area of myocardial tissues. WT, wild-type; DOX, doxorubicin; MT, melatonin; S3 KO, Sirt3 knockout. Mean ± SD, n = 4 per group. * p < 0.05 vs. WT group, # p < 0.05 vs. WT-DOX group, & p < 0.05 vs. S3 KO-DOX-MT group, @ p < 0.05 vs. WT-DOX-MT group. Scale bar = 50 μm.

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  Figure 3. Effects of melatonin treatment on autophagic protein expressions in DOX-injured mice hearts. (A) Representative blot images of protein expressions in DOX-injured mouse hearts with or without melatonin treatment; (B) Sirt3 level; (C) PGC-1α level; (D) TFEB level; (E) P62 level; (F) LC3II/I ratio; and (G) LAMP1 level. WT, wild-type; DOX, doxorubicin; MT, melatonin; S3 KO, Sirt3 knockout. Mean ± SD, n = 4 per group. * p < 0.05 vs. WT group, # p < 0.05 vs. WT-DOX group, & p < 0.05 vs. S3 KO-DOX-MT group, @ p < 0.05 vs. WT-DOX-MT group.

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  Figure 4. Contractile function of isolated cardiomyocytes with melatonin treatment and Sirt3 inhibitor 3-TYP. (A) Resting cell length; (B) maximal velocity of shortening (+dL/dt); (C) maximal velocity of relengthening (−dL/dt); (D) peak shortening (PS, normalized to resting cell length); (E) time-to-peak shortening (TPS); and (F) time-to-90% relengthening (TR90). Mean ± SD, n = 39 cells per group. * p < 0.05 vs. CON group, # p < 0.05 vs. DOX group, & p < 0.05 vs. DOX-MT group.

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  Figure 5. The effects of melatonin treatment on autophagic protein expressions in H9c2 myocytes treated with DOX. (A) Representative blot images of autophagic proteins with or without melatonin treatment; (B) Sirt3 level; (C) PGC-1α level; (D) TFEB level; (E) P62 level; (F) LC3II/I ratio; and (G) LAMP1 level. Mean ± SD, n = 3 per group. * p < 0.05 vs. CON group, # p < 0.05 vs. DOX group.

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  Figure 6. Evaluation of autophagic flux by ad-mRFP-GFP-LC3 infection with melatonin treatment in DOX-injured H9c2 cells. (A) Representative images of autophagosome and autolysosome in DOX-treated H9c2 cells with or without melatonin treatment; (B) autophagosome and autolysosome number in DOX-treated H9c2 cells with melatonin treatment; (C) autolysosome number in DOX-treated H9c2 cells with melatonin treatment. Scale bar = 10 μM, n = 10–15 cells per group. * p < 0.05 vs. CON group, ### p < 0.001 vs. DOX group. For total amount of autophagosomes and autolysosomes, &&& p < 0.001 vs. CON group, @ p < 0.05 vs. DOX group, *** p < 0.001 vs CON group.

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  Figure 7. Autophagic protein expressions in DOX-treated H9c2 cells with Sirt3 siRNA under melatonin treatment. (A) Representative blot images of autophagic proteins with or without Sirt3 knockdown; (B) Sirt3 level; (C) PGC-1α level; (D) TFEB level; (E) P62 level; (F) LC3II/I ratio; and (G) LAMP1 level. Mean ± SD, n = 3 per group. * p < 0.05 vs. CON group, # p < 0.05 vs. DOX + Scra siRNA group, & p < 0.05 vs. DOX + Scra siRNA + MT group.