eEF2K alleviates doxorubicin-induced cardiotoxicity by inhibiting GSK3β and improving autophagy dysfunction

Figures

• 

  Fig. 1. eEF2K was decreased in DIC. A The intersection of three datasets was shown by the Venn diagram. Data details could be found in Supplementary Material Table S5-S6. B The heatmap of the expression changes of 10 genes in three datasets was displayed. C The mRNA level of eEF2K in animal models was detected through RT-qPCR analysis on the 3rd, 5th, and 7th days after DOX intervention (n = 4). D The expression of eEF2K in mouse heart tissue was shown by immunofluorescence staining. Green signal was used to represent eEF2K, red signal represented cTnT-labeled cardiomyocytes, and blue signal represented Hoechst 33,342-labeled nuclei. Scale bar: 50 μm. E–F The protein level of eEF2K in mouse heart was detected through western blot analysis, with DOX intervention as before (n = 4). G The expression and distribution of eEF2K in cell DIC models were shown by immunofluorescence. Green signal was used to represent eEF2K, cTnT specifically labeled cardiomyocytes, and blue signal represented Hoechst 33,342-labeled nuclei. Scale bar: 50 μm. H The mRNA level of eEF2K in NRCMs was detected through a RT-qPCR analysis (n = 5). I-L The protein level of eEF2K in different concentration–time gradients…

• 

  Fig. 2. eEF2K attenuated doxorubicin-induced cardiotoxicity in NRCMs. A-B The levels of eEF2K protein in NRCMs by western blot analysis (n = 5). C-D PI/Hoechst 33,342 staining indicated the cellular injury (n = 5). The ratio of PI-positive cells to Hoechst 33,342-positive cells was calculated by ImageJ software to indicate the cellular injury. The scale bar represents 250 μm. E–F The cardiomyocyte outline was determined by phalloidin staining. Cardiomyocyte size was quantified by ImageJ software. Hoechst 33,342 staining marks the nuclei, scale bar: 50 μm. G Myocardial cell injury was assessed by LDH detection (n = 5). H-I The levels of eEF2K protein in NRCMs by western blot analysis (n = 5). J-K PI/Hoechst 33,342 staining indicated the cellular injury after eEF2K knockdown (n = 5), scale bar: 250 μm. L Myocardial cell injury was assessed by LDH detection (n = 5). All data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001 indicate statistical differences between groups

• 

  Fig. 3. eEF2K attenuated doxorubicin-induced cardiotoxicity in mice. A Immunofluorescence analysis showed eEF2K localization in cardiac tissue. The green signal represented eEF2K, the red signal denoted cTnT-labeled cardiomyocytes, and the blue represented Hoechst 33,342-labeled nuclei. Scale bar: 50 μm. B-C eEF2K were detected by western blot analysis in murine cardiac tissue (n = 6). D-F LVEF and LVFS were measured by short-axis M-mode echocardiography (n = 6). G-H Serum cTnT and CK-MB levels were quantified by ELISA (n = 6). I-J The gross heart pictures and the HW/TL ratio of mice (n = 6). K-L Representative images of gross cardiac sections stained by HE stains and the cross-section area of hearts were measured (n = 6). M–N Representative images stained with WGA, depicting the cross-sectional area of cardiomyocytes (n = 6). All data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001 indicate statistical differences between groups

• 

  Fig. 4. eEF2K attenuated autophagy dysfunction in NRCMs induced by DOX. A-B Representative fluorescence images of NRCMs infected with mRFP-GFP-LC3 adenovirus (n = 15). The mRFP (red) is stable in the acid organelles while the GFP (green) is not. Therefore, the green fluorescence of GFP quenched when the autophagosome fused with the lysosome to become autolysosome. The yellow dots merged by green dots and red dots represented autophagosomes and the red dots overlay no green dots indicated autolysosomes. The nuclei were stained with Hoechst 33,342 (blue). Scale bar: 20 μm. C-E LC3 was detected by western blot analysis in NRCMs (n = 5). F-I LC3 was detected by western blot analysis following eEF2K overexpression, DOX or chloroquine intervention, reflecting the alterations of autophagy flux (n = 5). J-M LC3 was detected by western blot in NRCMs (n = 5). N–O Representative fluorescence images of lysosomes using LysoSensor Yellow/Blue probes (n = 15). The blue dots with emission peak at 440 nm representing less acidic or neutral lysosomes (impaired lysosomes) as well as the yellow dots with emission peak at 540 nm indicating acid lysosomes (functional lysosomes). The ratio of I440/I540 were…

• 

  Fig. 5. eEF2K improved DOX-induced autophagy blockade in mice heart. A-C LC3 was detected by western blot analysis, reflecting the impact of DOX on autophagy in mouse hearts (n = 4). D-F Western blot analysis evaluated that overexpression of eEF2K attenuated the accumulation of LC3 induced by DOX (n = 6). G-H Representative transmission electron microscope images of autophagic vacuoles in mouse heart (n = 5). Autophagic vacuoles were labeled with red arrows. Scale bar: 2 μm. All data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001 indicate statistical differences between groups

• 

  Fig. 6. eEF2K promoted GSK3β phosphorylation in DIC. A-C The phosphorylation level of GSK3β at Ser9 was detected by western blot analysis in mouse hearts (n = 6). D-F The phosphorylation level of GSK3β at Ser9 was detected in NRCMs following eEF2K overexpression (n = 5). G-I The phosphorylation level of GSK3β at Ser9 was detected following eEF2K knockdown (n = 5). All data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001 indicate statistical differences between groups

• 

  Fig. 7. Inhibition of GSK3β reversed the effects of eEF2K knockdown on aggravating autophagy blockade in DIC. A-B Hoechst 33,342/PI staining indicated the cellular injury (n = 5). The ratio of PI-positive cells to Hoechst 33,342-positive cells was calculated by ImageJ software to indicate the cellular injury. The scale bar represents 250 μm. C Myocardial cell injury was assessed by LDH detection (n = 5). D-E The cardiomyocyte outline was determined by phalloidin staining. Cardiomyocyte size was quantified by ImageJ software. Hoechst 33,342 staining marks the nuclei, scale bar: 50 μm. F-G Representative images of NRCMs infected with mRFP-GFP-LC3 adenovirus (n = 15). The GFP (green) is unstable in the acid organelles like lysosome while the mRFP (red) is not. Therefore, the green fluorescence of GFP quenched when the autophagosome fused with the lysosome to become autolysosome. The yellow dots merged by green dots and red dots represented autophagosomes and the red dots overlay no green dots indicated autolysosomes. The nuclei were stained with Hoechst 33,342 (blue). Scale bar: 20 μm. H-J LC3 were detected by western blot analysis in NRCMs (n = 5). K-N LC3 were detected by western blot…