Melatonin attenuates diabetic cardiomyopathy by increasing autophagy of cardiomyocytes via regulation of VEGF-B/GRP78/PERK signaling pathway

Figures

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  Fig. 1. Mel alleviated cardiac dysfunction in diabetic mice. A Representative images of echocardiography in mice. B, C Values of EF and FS, n = 6. D CK-MB release in serum, n = 6. E Ratio of heart weight to tibia length, n = 6. F Myocardial tissues were stained with H&E (scale bar = 50 μm), WGA (scale bar = 50 μm), Masson (scale bar = 100 μm) and Sirius red (scale bar = 50 μm). G Size of myocardial cells were assessed by staining with WGA, n = 8. H, I The mRNA expressions of Anf and Myhc, n = 6. J Calcein-AM/PI double staining in NRVMs (scale bar = 200 μm). K Quantification of PI-positive cells, n = 5. Data were expressed as the mean ± SD.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. One-way ANOVA followed by a post hoc Tukey’s test

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  Fig. 2. Mel attenuated DCM through increasing cardiomyocytes autophagy. A Western blot for p62 and LC3 in mice. B Quantification for p62 and LC3-II/LC3-I in mice, n = 3. C Western blot for p62 and LC3 in NRVMs. D Quantification for p62 and LC3-II/LC3-I in NRVMs, n = 3. E Autophagic flux was used to detect autophagy in NRVMs (scale bar = 10 µm). F Western blot for p62 and LC3 in NRVMs. G Quantification for p62 and LC3-II/LC3-I in NRVMs, n = 3. H Autophagic flux in NRVMs (scale bar = 10 µM) I Calcein-AM/PI double staining in NRVMs (scale bar = 200 μm) J Quantification of PI-positive cells, n = 5. Data were expressed as the mean ± SD.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. One-way ANOVA followed by a post hoc Tukey’s test

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  Fig. 3. Mel down-regulated expression of VEGF-B either in vivo or in vitro. A The mRNA expression of Vegfb in mice, n = 6. B Western blot for VEGF-B in mice. C Quantification for VEGF-B in mice, n = 3. D The immunohistochemical analysis for VEGF-B in mice myocardial tissues (scale bar = 100 μm). E The mRNA expression of Vegfb in NRVMs, n = 5. F Western blot for VEGF-B in NRVMs. G Quantification for VEGF-B in NRVMs, n = 3. H The NRVMs were subjected to immunofluorescence analysis with anti-VEGF-B antibody (scale bar = 100 μm). I Quantification for mean fluorescence of VEGF-B in NRVMs, n = 5. Data were expressed as the mean ± SD.*p < 0.05, **p < 0.01, ***p < 0.001. One-way ANOVA followed by a post hoc Tukey’s test

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  Fig. 4. VEGF-B overexpression abolished the cardioprotective effect of Mel on DCM. A Representative images of echocardiography in mice. B Values of EF and FS, n = 6. C CK-MB release in serum, n = 6. D Ratio of heart weight to tibia length, n = 6. E Myocardial tissues were stained with H&E (scale bar = 50 μm), WGA (scale bar = 50 μm), Masson (scale bar = 100 μm) and Sirius red (scale bar = 50 μm). F Size of myocardial cells were assessed by staining with WGA, n = 8. G The mRNA expression of Myhc, n = 6. H Calcein-AM/PI double staining in NRVMs (scale bar = 200 μm). I Quantification of PI-positive cells, n = 5. J Autophagic flux in NRVMs (scale bar = 10 µm). Data were expressed as the mean ± SD.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. One-way ANOVA followed by a post hoc Tukey’s test

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  Fig. 5. VEGF-B interacted with GRP78 in NRVMs. A Co-immunoprecipitation was performed to detect the interaction of VEGF-B and GRP78 in NRVMs and (B) in 293 T cells. C We constructed GST-GRP78-FL plasmid, expressed and purified in Rosetta (DE3). D Co-incubated with Flag-VEGF-B to detect the interaction between GRP78 and VEGF-B. E Western blot for GRP78 and quantification for GRP78 in mice, n = 3. F Western blot for GRP78 and quantification for GRP78 in NRVMs, n = 3. G Western blot for GRP78 and VEGF-B in NRVMs. H Quantification for GRP78 and VEGF-B in NRVMs, n = 3. I Immunofluorescence staining for VEGF-B, GRP78, and their co-localization in NRVMs (scale bar = 50 μm). J Different truncates of GRP78. K We established and purified GST-GRP78-FL, GST-GRP78-NBD and GST-GRP78-SBD in Rosetta (DE3). L VEGF-B interacted with SBD domain, NBD domain and FL of GRP78. Data were expressed as the mean ± SD.*p < 0.05, **p < 0.01. One-way ANOVA followed by a post hoc Tukey’s test

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  Fig. 6. Mel enhanced autophagy and protected against DCM via increasing phosphorylation of PERK. A Co-immunoprecipitation was performed to detect the interaction of PERK and GRP78 in NRVMs. B The experimental protocol in mice. C Representative images of echocardiography in mice. D Values of EF and (E) FS, n = 6. F CK-MB release in serum, n = 6. G Ratio of heart weight to tibia length, n = 6. H, I The mRNA expressions of Myhc and Anf, n = 6. J Myocardial tissues were stained with H&E (scale bar = 50 μm), WGA (scale bar = 50 μm), Masson (scale bar = 100 μm) and Sirius red (scale bar = 50 μm). K Size of myocardial cells were assessed by staining with WGA, n = 8. L Western blot for PERK-related pathways and autophagy proteins mice, n = 3. M Autophagic flux in NRVMs (scale bar = 10 µm) Data were expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.01, ****p < 0.0001. One-way ANOVA followed by a post hoc Tukey’s test

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  Fig. 7. Mel attenuated DCM through VEGF-B/PERK signaling pathway. A Representative images of echocardiography in mice. B Values of EF and (C) FS, n = 6. D CK-MB release in serum, n = 6. E Myocardial tissues were stained with H&E (scale bar = 50 μm), WGA (scale bar = 50 μm), Masson (scale bar = 100 μm) and Sirius red (scale bar = 50 μm). F Size of myocardial cells were assessed by staining with WGA, n = 8. G Ratio of heart weight to tibia length, n = 6. H The mRNA expression of Myhc, n = 6. I Western blot for PERK-related pathways and autophagy proteins in GSK treated mice, n = 3. J Western blot for PERK-related pathways and autophagy proteins in mice, n = 3. K Immunofluorescence staining for ATF4 translocation in NRVMs (scale bar = 10 μm). L Autophagic flux in NRVMs (scale bar = 10 μm). Data were expressed as the mean ± SD.*p < 0.05, **p < 0.01, ***p < 0.01, ****p < 0.0001. One-way ANOVA followed by a post hoc Tukey’s test

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  Fig. 8. The overview diagram of this study. Mel attenuated DCM by increasing autophagy of cardiomyocytes via regulation of VEGF-B/GRP78/PERK signaling pathway