Melatonin activates Parkin translocation and rescues the impaired mitophagy activity of diabetic cardiomyopathy through Mst1 inhibition

Abstract

Mitophagy eliminates dysfunctional mitochondria and thus plays a cardinal role in diabetic cardiomyopathy (DCM). We observed the favourable effects of melatonin on cardiomyocyte mitophagy in mice with DCM and elucidated their underlying mechanisms. Electron microscopy and flow cytometric analysis revealed that melatonin reduced the number of impaired mitochondria in the diabetic heart. Other than decreasing mitochondrial biogenesis, melatonin increased the clearance of dysfunctional mitochondria in mice with DCM. Melatonin increased LC3 II expression as well as the colocalization of mitochondria and lysosomes in HG-treated cardiomyocytes and the number of typical autophagosomes engulfing mitochondria in the DCM heart. These results indicated that melatonin promoted mitophagy. When probing the mechanism, increased Parkin translocation to the mitochondria may be responsible for the up-regulated mitophagy exerted by melatonin. Parkin knockout counteracted the beneficial effects of melatonin on the cardiac mitochondrial morphology and bioenergetic disorders, thus abolishing the substantial effects of melatonin on cardiac remodelling with DCM. Furthermore, melatonin inhibited Mammalian sterile 20-like kinase 1 (Mst1) phosphorylation, thus enhancing Parkin-mediated mitophagy, which contributed to mitochondrial quality control. In summary, this study confirms that melatonin rescues the impaired mitophagy activity of DCM. The underlying mechanism may be attributed to activation of Parkin translocation via inhibition of Mst1.

Keywords: diabetic cardiomyopathy; mammalian Ste20-like kinase 1, Mst1; melatonin; mitochondrion; mitophagy; parkin.

© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Figures

Figure 1

Melatonin enhances impaired mitochondria elimination in diabetic cardiomyopathy mice A, Representative TEM images of the longitudinal left ventricular wall. B, Mitochondrial number obtained by manual counting per photograph (Magnification, ×9900) per group. C, The percentage of mitochondrial areas per image (Magnification, ×9900). The columns and error bars represent the means and standard deviations (SD) (n = 12) *P < 0.05 vs WT and †P < 0.05 vs DM. D, Representative flow cytometric curves of the fluorescence intensity (MitoTracker Green) in adult cardiomyocytes. E, Mean fluorescence intensity (MitoTracker Green) of flow cytometric analysis. F, Protein expression with representative gel blots of PGC‐1α, NRF‐1, TFAM, and GAPDH (loading control). G, Relative levels of PGC‐1α. H, Relative levels of NRF‐1. I, Relative levels of TFAM. The columns and error bars represent the means and standard deviation (SD) (n = 4) *P < 0.05 vs WT and †P < 0.05 vs DM

Figure 2

Melatonin regulation of mitophagy activity in the diabetic myocardium A, Protein expression with representative gel blots of LC3 I/II, GAPDH (loading control). B, Relative level of LC3 II. The columns and error bars represent the means and standard deviations (SD) (n = 4) *P < 0.05 vs Con; †P < 0.05 vs Mel; ‡P < 0.05 vs HG; §P < 0.05 vs HG + Mel; ||P < 0.05 vs HG + Mel + 3‐MA. C, Representative colocalization images of lysosomes (LysoTracker Green) and mitochondria (MitoTracker Red) (Scale bar: 20 μm). D, Quantitative analysis of LysoTracker/MitoTracker colocalization (percentage of whole cell). The columns and error bars represent the means and standard deviation (SD) (n = 30 cells) *P < 0.05 vs Con; †P < 0.05 vs HG; ‡P < 0.05 vs HG + Mel. E, Representative typical autophagosome engulfing an impaired mitochondrion (Scale bars: 400 nm) in cardiac tissue

Figure 3

Melatonin promotes Parkin‐mediated mitophagy in diabetic cardiomyopathy A, Protein expression with representative gel blots of PINK1, Parkin, GAPDH (loading control). B, Relative levels of PINK1. C, Relative levels of Parkin. D, Protein expression with representative gel blots of Cyto‐Parkin, Mito‐Parkin, Mito‐p62, GAPDH (loading control for Cyto‐Parkin), COX4 (loading control for Mito‐Parkin, Mito‐p62). E, Relative levels of Cyto‐Parkin. F, Relative levels of Mito‐Parkin. G, Relative level of Mito‐p62. The columns and error bars represent the means and standard deviations (SD) (n = 3) *P < 0.05 vs Con; †P < 0.05 vs Mel; ‡P < 0.05 vs DM. H, Representative colocalization images of Parkin (Green) and mitochondria (MitoTracker Red) (Scale bars: 20 μm). I, Relative level of Parkin in confocal images. J, Quantitative analysis of Parkin/MitoTracker Red colocalization (percentage of whole cells). The columns and error bars represent the means and standard deviation (SD) (n = 30 cells). *P < 0.05 vs Con; †P < 0.05 vs Mel; ‡P < 0.05 vs HG

Figure 4

Parkin inhibition counteracts the effects of melatonin on mitophagy. A, Protein expression with representative gel blots of Parkin, Mito‐p62, COX4, p62, LC3, GAPDH (loading control for Parkin, p62, LC3). B, Relative level of Parkin. C, Relative level of p62. D, Relative level of Mito‐p62. E, Relative level of LC3 II. (n = 4). *P < 0.05 vs Con; †P < 0.05 vs HG; ‡P < 0.05 vs HG + Ad‐LacZ; §P < 0.05 vs HG + Ad‐sh‐Parkin; and ||P < 0.05 vs HG + Mel. F, Representative cardiomyocyte images of transmission electron microscopy (TEM) (Scale bars: upper panel 1 μm, and lower panel 400 nm). G, Representative colocalization images of GFP‐LC3 (Green) and mitochondria (MitoTracker Red) (Scale bars: 20 μm). H, Quantitative analysis of GFP‐LC3 punctae per cell. I, Percentage of cells with LC3 and mitochondria (MitoTracker Red, MTR) colocalization. The columns and error bars represent the means and standard deviations (SD) (n = 30 cells). *P < 0.05 vs Con; †P < 0.05 vs HG; ‡P < 0.05 vs HG + Ad‐LacZ; §P < 0.05 vs HG + Ad‐sh‐Parkin; and ||P < 0.05 vs HG + Mel

Figure 5

Melatonin ameliorates DCM phenotypes via Parkin. A, Representative images of echocardiography. B, Measurements of LVEF (%). C, Measurements of LVFS (%). D, Measurements of LVESD (mm). E, Measurements of LVEDD (mm). The columns and error bars represent the means and standard deviations (SD) (n = 12). *P < 0.05 vs WT; †P < 0.05 vs DM; and ‡P < 0.05 vs DM + Mel. F, Representative images of TUNEL staining (Scale bar: 40 μm). G, Quantitative analysis of the apoptotic index (percentage of TUNEL‐positive nuclei, %). The columns and error bars represent the means and standard deviations (SD) (n = 20) *P < 0.05 vs Con; †P < 0.05 vs HG; ‡P < 0.05 vs HG + Ad‐LacZ; §P < 0.05 vs HG + Ad‐sh‐Parkin; and ||P < 0.05 vs HG + Mel. H, Relative ROS level. I, Representative images of mitochondrial morphology (Scale bars: 1 μm). J, Myocardial ATP content. K, Citrate synthase (CS) activity in cardiac tissues. The columns and error bars represent the means and standard deviation (SD). *P < 0.05 vs WT; †P < 0.05 vs DM; and ‡P < 0.05 vs DM + Mel

Figure 6

Mst1 as a negative regulator amongst melatonin‐regulated Parkin signaling. A, Representative colocalization images of GFP‐LC3 and mitochondria (MitoTracker Red) (Scale bars: 20 μm). B, Quantitative analysis of GFP‐LC3 punctae per cell. C, Percentage of cells with LC3 and mitochondria (MitoTracker Red, MTR) colocalization. D, Quantitative analysis of GFP‐LC3 puncta colocalization with mitochondria (MitoTracker Red) per cell. The columns and error bars represent the means and standard deviations (SD) (n = 30 cells). *P < 0.05 vs HG; †P < 0.05 vs HG + Mel; ‡P < 0.05 vs HG + Ad‐sh‐Mst1; §P < 0.05 vs HG + Ad‐Mst1; and ||P < 0.05 vs HG + Ad‐sh‐Mst1 + Mel. E, Representative gel blots of Mst1, Parkin, p‐Parkin (Ser65), Mito‐Parkin, COX4 (loading control for mitochondrial protein) and GAPDH (loading control for whole protein). F, Relative levels of Mst1. G, Relative levels of Parkin. H, Relative levels of p‐Parkin. I, Relative levels of Mito‐Parkin. The columns and error bars represent the means and standard deviations (SD) (n = 4). *P < 0.05 vs DM; †P < .05 vs DM + Mel; ‡P < 0.05 vs DM + Mst1−/−; §P < 0.05 vs DM + Mst1 Tg; and ||P < 0.05 vs DM + Mst1−/− + Mel