eEF2K was decreased in DIC

DIC dataset was analyzed online on the GEO database. Differential gene expression was assessed across three RNA-seq datasets, identifying 10 differentially genes (Fig. 1 A). Detailed dataset information was provided (supplementary material Table S5-6). Heatmap analysis of these 10 genes revealed a downregulation of eEF2K (Fig. 1 B). Subsequently, an animal model of acute DIC (15 mg/kg) was established. Through RT-qPCR, we confirmed that eEF2K expression was downregulated in mouse heart at the mRNA level on the 3rd, 5th, and 7th days after DOX intervention (Fig. 1 C). Continuous stimulation with dox resulted in decreased expression of eEF2K over time (Fig. 1 E, F). According to previous literature reports (Chen et al. 2022a; Yang et al. 2023), a 5-day DOX intervention protocol was selected for subsequent animal experiments. Immunofluorescence staining also revealed that eEF2K was decreased by DOX in mouse heart (Fig. 1 D). According to RT-qPCR analysis, we found that eEF2K was decreased by DOX at the mRNA level in vitro (Fig. 1 H). The different concentrations of DOX were used, which resulted in a dose-dependent reduction of eEF2K expression (Fig. 1 I, J). Given that DOX concentrations exceeding 2 μM are not clinically relevant (Li et al. 2016), a concentration of 1 μM DOX was employed. Furthermore, the different-times intervention of DOX was used, which suggested that there was a time-dependent decrease of eEF2K at the protein level (Fig. 1 K, L). Immunofluorescence of NRCMs also revealed a reduction of eEF2K induced by DOX (Fig. 1 G).

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 was detected through western blot analysis in NRCMs (n = 5). All data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001 indicate statistical differences between groups

eEF2K attenuated doxorubicin-induced cardiotoxicity in NRCMs

To study the function of eEF2K in DIC, we utilized adenovirus to artificially enhance its expression in vitro (Supplementary Fig. S2 A, B). According to western blot analysis, eEF2K levels were lowered by DOX, yet adenovirus-mediated overexpression reinstated them. (Fig. 2 A, B). PI/Hoechst 33,342 staining demonstrated that DOX induced cardiomyocyte injury and death compared to that in the CTR group, but the overexpression of eEF2K reduced cardiomyocyte damage (Fig. 2 C, D). Similarly, LDH detection also suggested that the overexpression of eEF2K reduced cardiomyocyte injury induced by DOX (Fig. 2 G). Furthermore, Phalloidin staining revealed that myocardial cell area was reduced by DOX, whereas the overexpression of eEF2K attenuated DOX-induced cardiomyocyte atrophy (Fig. 2 E, F).

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

Secondly, eEF2K was knocked down by siRNA in vitro (Supplementary Fig. S3 A, B), and eEF2K was further reduced by si-eEF2K in DOX-treated NRCMs (Fig. 2 H, I). Although PI/Hoechst 33,342 staining revealed that eEF2K knockdown alone did not induce NRCMs injury, eEF2K knockdown exacerbated DOX-induced cardiomyocyte injury (Fig. 2 J, K). LDH detection supported these findings, indicating a exacerbation of DIC upon eEF2K knockdown (Fig. 2 L).

Overexpression of eEF2K attenuated doxorubicin-induced cardiotoxicity in mice

Adeno-associated virus (AAV) was introduced into mice through an injection in the tail vein, which resulted in targeted overexpression of eEF2K specifically in the cardiac tissue (Supplementary Fig. S4 A-B). An equal amount of empty vector virus was administered to the negative control (NC) group. According to immunofluorescence analysis, the DOX group showed a decrease in eEF2K fluorescence intensity, whereas AAV-eEF2K infection led to an increase. (Fig. 3 A). Western blot similarly showed that cardiac eEF2K was reduced in the DOX group, but it was elevated through AAV-mediated overexpression (Fig. 3 B-C).

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

M-mode echocardiography revealed that DOX led to a reduction in both LVEF and LVFS compared to the Sham group. In contrast, AAV-eEF2K mitigated the reduction of LVEF and LVFS caused by DOX (Fig. 3 D-F). Additionally, the DOX group had a thinner left ventricular wall, whereas wall thinning was less pronounced with DOX + AAV-eEF2K than in the DOX + NC group. (Supplementary Fig. S5 A-F). Detection of cTnT and CK-MB was used to assess myocardial injury. According to the results, DOX elevated cTnT and CK-MB levels, while AAV-eEF2K alleviated the myocardial damage induced by DOX. In the DOX + AAV-eEF2K group, cTnT and CK-MB levels were lower than those in the DOX + NC group (Fig. 3 G-H).

Because previous research has established myocardial atrophy as a principal pathological feature of DIC (Chen et al. 2022a; Ferreira de Souza et al. 2018; Lipshultz et al. 1991; Orogo and Gustafsson 2015), we evaluated the heart mass through gross cardiac specimens and the HW/TL ratio. The results suggested that both heart volume and HW/TL ratio were reduced by DOX (Fig. 3 I-J). Concurrently, we observed that there was a decline body weight following DOX intervention (Supplementary Fig. S6 A-C). However, the analysis of HW/BW ratio also indicated that AAV-eEF2K attenuated the heart mass loss induced by DOX (Supplementary Fig. S6 D). We selected heart tissue sections at the papillary muscle level for HE staining. Findings indicated a decrease in the mouse heart's cross-sectional area due to DOX, which AAV-eEF2K could help counteract (Fig. 3 K-L). Furthermore, the average cross-sectional area of cardiomyocytes was found to be smaller in the DOX group than in the Sham group, as indicated by WGA staining, and AAV-eEF2K could attenuate the cardiomyocyte atrophy induced by DOX (Fig. 3 M–N).

eEF2K attenuated autophagy dysfunction induced by DOX

For additional confirmation, NRCMs were transfected with tandem mRFP-GFP-LC3 adenovirus to observe the number of autophagosomes and autolysosomes in vitro. Between the groups, the number of autophagosomes showed no statistical difference. Nonetheless, DOX induced a marked increase in autolysosomes compared to the CTR group. There was no statistical difference among DOX + NC group and DOX group. The overexpression of Ad-eEF2K led to a decrease in autolysosome accumulation caused by DOX (Fig. 4 A-B), which indicated that eEF2K might mitigate the DOX-induced impairment in autolysosome degradation. Western blot also suggested that eEF2K improved the DOX-induced elevation of LC3-II, thereby attenuating autophagic dysfunction (Fig. 4 C-E).

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 measured. Scale bar: 20 μm. All data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001 indicate statistical differences between groups

Chloroquine (CQ), a specific autophagy inhibitor, was employed to further elucidate the autophagy flux. Compared to the CTR group, the CQ group displayed a significant rise in LC3-II, suggesting the preservation of autophagy flux in normal cells. The DOX + CQ group showed a reduction in LC3-II, suggesting a reduction of autophagy flux induced by DOX compared to that in the CQ group (Supplementary Fig. S7 A-D). Additionally, LC3-II levels rose in both the Ad-eEF2K + CQ group and the Ad-eEF2K + DOX + CQ group when compared to those not treated with chloroquine, which indicated that eEF2K could restore the decrease of autophagy flux induced by DOX (Fig. 4 F-I).

Recent research has elucidated that there was an inhibition of lysosomal acidification to inhibit autophagy flux (Li et al. 2016). Therefore, we employed the LysoSensor Yellow/Blue probe to stain lysosomes. The increase of the ratio of I440/I540 suggested that DOX led to a disruption of the lysosomal acidic environment, but Ad-eEF2K decreased the ratio and restored lysosomal acidification dysfunction induced by DOX (Fig. 4 J-M).

Additionally, our findings revealed that eEF2K knockdown elevated LC3-II accumulation induced by DOX, resulting in aggravating autophagy blockade (Fig. 4 N–O). Overall, these results implicated that eEF2K restored autolysosomes accumulation and autophagy flux reduction induced by DOX, thereby attenuating DIC.

Overexpression of eEF2K improved DOX-induced autophagy blockade in mice heart

Mice were grouped based on DOX-intervention durations, which revealed that DOX induced autophagic dysfunction in mouse hearts over time, peaking at 5 days (Fig. 5 A-C). Through Western blot examination, it was found that augmenting eEF2K levels using an adenovirus led to changes in LC3-II concentrations, indicating that eEF2K could alleviate the accumulation of LC3-II instigated by DOX (Fig. 5 D-F). Furthermore, TEM was employed to evaluate the abundance of autophagic vacuoles in cardiac tissue. The results showed a low number of autophagic vacuoles in normal heart tissue, whereas DOX increased their amount. However, overexpression of eEF2K decreased the amount of autophagic vacuoles induced by DOX in cardiac tissue (Fig. 5 G-H). Collectively, these results indicate that DOX caused a blockage in cardiac autophagy, leading to the buildup of autophagic vacuoles and LC3-II, while eEF2K improved DOX-induced autophagy blockade by enhancing the degradation of autophagic vacuoles.

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

eEF2K promoted GSK3β phosphorylation in DIC

Recently, eEF2K has been reported to facilitate GSK3β phosphorylation at the Ser9 site (Chen et al. 2022b). However, whether eEF2K promotes phosphorylation in DIC is still unclear.

We used western blot analysis to detect the phosphorylation level of GSK3β, revealing that DOX reduced p-GSK3β levels in vivo. However, overexpression eEF2K increased the p-GSK3β level (Fig. 6 A-C). In addition, overexpression eEF2K attenuated the reduction of p-GSK3β levels induced by DOX in vitro (Fig. 6 D-F). Similarly, eEF2K knockdown not only led to a decrease of p-GSK3β levels in normal cells, but also further decreased p-GSK3β levels in DIC (Fig. 6 G-I). These findings suggested that eEF2K promoted GSK3β phosphorylation at the Ser9 site in DIC.

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

Inhibition of GSK3β reversed the effects of eEF2K knockdown on aggravating autophagy blockade in DIC

According to studies, DOX reduces the phosphorylation level at Ser9 of GSK3β, resulting in its heightened activation (Li et al. 2020; Wang et al. 2021; Zhang et al. 2020). Like previous studies, the findings indicated that the phosphorylation level of GSK3β was decreased by DOX at different concentrations intervention (Supplementary Fig. S8 D-F). The study showed that knocking down eEF2K worsens the injury to cardiomyocytes induced by DOX. Given the overactivation of GSK3β in the DIC, we used a selective GSK3β inhibitor, AR-A014418 (ARi), to investigate the therapeutic potential of inhibiting GSK3β. We found that inhibiting GSK3β could alleviate DIC (Supplementary Fig. S8 A-C). PI/Hoechst 33,342 staining showed that ARi attenuated the exacerbated cardiomyocyte injury resulting from eEF2K knockdown in DIC (Fig. 7 A-B). Similarly, LDH detection suggested that si-eEF2K notably exacerbated DIC, whereas ARi alleviated the injury (Fig. 7 C). It is worth noting that knockdown eEF2K alone did not cause damage. Furthermore, phalloidin staining demonstrated that si-eEF2K exacerbated DOX-induced cardiomyocyte atrophy, but ARi reversed the atrophic response (Fig. 7 D-E). These findings suggested that GSK3β inhibition by ARi attenuated the exacerbated cardiomyocyte injury resulting from eEF2K knockdown in DIC.

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 analysis following eEF2K knockdown, ARi and chloroquine intervention in DIC, reflecting the alteration of autophagy flux (n = 5). O-P 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 measured. Scale bar: 20 μm. All data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001 indicate statistical differences between groups

Subsequently, we investigated how ARi influences the autophagy blockade caused by DOX. The mRFP-GFP-LC3 fluorescence showed that eEF2K knockdown aggravated the accumulation of autolysosome compared to that in the DOX group, further reducing the autophagy flux. However, ARi attenuated autolysosomes accumulation and restored autophagy flux (Fig. 7 F-G). Moreover, the analysis using western blot demonstrated that eEF2K knockdown resulted in elevated LC3-II levels relative to the DOX group, but LC3-II accumulation was reduced after ARi intervention (Fig. 7 H-J). These results suggested that GSK3β inhibition could attenuate the autophagy blockade induced by eEF2K knockdown in DIC.

Autophagy flux was further detected by chloroquine intervention. Our findings suggested that DOX decreased cardiomyocyte autophagy flux, and eEF2K knockdown further exacerbated this reduction. Nevertheless, the inhibition of GSK3β by ARi partially reversed the reduced autophagy flux resulting from eEF2K knockdown in DIC (Fig. 7 K-N). Additionally, lysosomal acidification function was assessed using the LysoSensor Yellow/Blue probe. Our results indicated that lysosomal acidification dysfunction induced by DOX was exacerbated by eEF2K knockdown, but ARi partially reversed this lysosomal dysfunction (Fig. 7 O-P).

In conclusion, our results suggested that inhibition of GSK3β by ARi could attenuate lysosome dysfunction, alleviate autophagy blockade, and restore autophagy flux, thereby improving DIC.