EBBP-Mediated Integrated Stress Response Attenuates Anthracycline-Induced Cardiotoxicity by Inhibiting the Ferroptosis of Cardiomyocytes

Abstract

Anthracyclines are potent chemotherapeutics, but their clinical application is constrained by dose-dependent cardiotoxicity, in which ferroptosis plays a critical role. Here, EBBP (Estrogen-responsive B Box Protein) is identified as a key cardioprotective regulator in anthracycline-induced cardiotoxicity. Transcriptomic profiling of doxorubicin (DOX)-treated hearts reveals significant EBBP upregulation. Cardiac-specific overexpression of EBBP protects against myocardial injury and dysfunction by reducing DOX-induced ferroptosis. Conversely, EBBP silencing exacerbates DOX-induced cardiac damage, an effect reversed by ferroptosis inhibitor ferrostatin-1 (Fer-1). The molecular targets of EBBP are subsequently identified through bulk RNA sequencing, molecular docking analysis, co-immunoprecipitation experiments, and ubiquitination assays. Mechanistically, EBBP interacts with GRP78 to promote its K63-linked ubiquitination, disrupting the inhibitory GRP78-PERK interaction and activating PERK-mediated integrated stress response (ISR). This signaling cascade ultimately leads to the activation of downstream effectors ATF4 and Nrf2, which coordinately upregulates the SLC7A11/GSH/GPX4 axis and restores iron homeostasis. Importantly, pharmacological inhibition of PERK abolishes the protective effects of EBBP against myocardial injury and ferroptosis. Overall, our findings identify EBBP as a novel suppressor of ferroptosis in anthracycline-induced cardiotoxicity via the PERK-mediated ISR, thereby underscoring its therapeutic potential for preventing anthracycline-induced cardiomyopathy.

Keywords: EBBP; K63‐linked ubiquitination; PERK‐mediated integrated stress response; anthracycline‐induced cardiotoxicity; ferroptosis.

Figure 1

EBBP is significantly upregulated in anthracycline‐induced cardiotoxicity. A) Venn diagram showing differentially expressed genes that are consistently upregulated or downregulated across all three GEO datasets (GSE40289, GSE226116, GSE233644, DOX vs Vehicle). B) The mRNA levels of candidate genes in cardiac tissues of mice treated with DOX (n = 6). C) Immunohistochemistry and statistical analysis of EBBP expression in the myocardium of DOX‐treated mice (scale bar, 100 µm). D) Immunoblots and statistical analysis of EBBP protein level in the heart of mice treated with DOX (n = 6). E) Immunoblots and statistical analysis of EBBP expression in NRCMs treated with different concentrations of doxorubicin for 24 h (n = 3). F) Representative immunofluorescence staining of EBBP (red) and cTnT (green) in mouse heart tissue. Nuclei were stained with DAPI (blue). (scale bars, 50 µm; n = 6). Statistical analyses of EBBP mean fluorescence intensity are presented in the panel on the right. G,I) Adenovirus‐mediated EBBP overexpression or depletion in vitro was assessed by western blot analysis. H,J) Following infection with Ad‐EBBP or Ad‐shEBBP, H9c2 was subjected to a DOX (1 µm) treatment for a duration of 24 h. Cell viability was measured by CCK8 (n = 3). Values are presented as the mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 vs Vehicle group; ^## p < 0.01 vs DOX+ Ad‐Null or DOX+ Ad‐shNC.

Figure 2

EBBP overexpression ameliorates anthracycline‐induced cardiac injury. AAV‐NC or AAV‐EBBP was administered via tail vein injection, and the DoIC model was established 14 days thereafter. A) Representative images of transthoracic echocardiography. B) Representative images of Masson's trichrome staining (scale bars, 100 µm). C) Representative images of TUNEL staining (scale bars, 50 µm). D) Changes in body weight of mice after DOX injection (n = 6). E) Changes in HW/TL (n = 6). F,G) Statistical analysis of LVEF and FS (n = 6). H–J) The mRNA levels of Anp, Bnp, and Myh7 in cardiac tissues (n = 6). K–M) Statistical analysis of serum AST, CK‐MB, and LDH levels (n = 6). N) Statistical analysis of fibrosis area (n = 6). O) Quantitative analysis of TUNEL‐positive cells via immunofluorescence staining (n = 6). Values are presented as the mean ± SD. ***p < 0.001 vs Vehicle + AAV‐NC; ^## p < 0.01 and ^### p < 0.001 vs DOX + AAV‐NC group.

Figure 3

EBBP deficiency aggravates anthracycline‐induced cardiac injury. AAV‐shNC or AAV‐shEBBP was administered via tail vein injection, and the DoIC model was established 14 days thereafter. A) Representative images of transthoracic echocardiography. B) Representative images of Masson's trichrome staining (scale bars, 100 µm). C) Representative images of TUNEL staining (scale bars, 50 µm). D) Changes in body weight of mice after DOX injection (n = 6). E) Changes in HW/TL (n = 6). F,G) Statistical analysis of LVEF and FS (n = 6). H–J) The mRNA levels of Anp, Bnp, and Myh7 in cardiac tissues (n = 6). K–M) Statistical analysis of serum AST, CK‐MB, and LDH levels (n = 6). N) Statistical analysis of fibrosis area (n = 6). O) Quantitative analysis of TUNEL‐positive cells via immunofluorescence staining (n = 6). Values are presented as the mean ± SD. ***p < 0.001 vs Vehicle + AAV‐shNC; ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 vs DOX + AAV‐ shNC group.

Figure 4

EBBP deficiency aggravates anthracycline‐induced cardiomyocyte ferroptosis in vitro. A) KEGG pathway enrichment analysis of the GEO dataset (GSE233644) comparing DOX‐treated murine hearts with control murine hearts. B) The effects of various cell death inhibitors on the viability of H9c2 cells treated with DOX (1 µm) (n = 3). C) The effects of various cell death inhibitors on the viability of H9c2 cells transduced with Ad‐shEBBP and treated with DOX (1 µm) (n=3). D–L) Following infection with Ad‐shNC or Ad‐shEBBP, H9c2 was pretreated with Fer‐1 (2 µm) for 2h and then treated with DOX (1 µm) for 24 h. D) The mRNA level of Ptgs2 was detected by qRT‐PCR (n = 3). E) Representative pictures and statistical analysis of intracellular lipid peroxide by flow cytometry (n = 3). F) MDA level in cells (n = 3). G) Quantification of fluorescent immunohistochemistry staining for DHE in H9c2 cells (n = 3). H) Quantification of intracellular Fe²⁺ levels (FerroOrange staining) in H9c2 cells (n = 3). I) Statisticaof mitochondrial membrane potential (JC‐1 staining) in H9c2 cells (scale bars, 20 µm; n = 3). J–L) Representative images of DHE staining (scale bars, 100 µm), FerroOrange staining (scale bars, 50 µm), and JC‐1 staining (scale bars, 20 µm). Values are presented as the mean ± SD. ***p < 0.001 vs Vehicle +Ad‐shNC; ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 vs DOX+ Ad‐shNC; ^&&& p < 0.001 vs DOX+Ad‐shEBBP group.

Figure 5

EBBP attenuates anthracycline‐induced myocardial ferroptosis in vivo. A–H) AAV‐NC or AAV‐EBBP was administered via tail vein injection, and the DoIC model was established 14 days thereafter. A) Representative images and quantification of fluorescent immunohistochemistry staining for DHE in mouse myocardium (scale bars, 100 µm, n = 6). B) Representative images and quantification of immunohistochemistry for 4‐HNE in mouse myocardium (scale bars, 50 µm, n = 6). C) Representative transmission electron micrographs of cardiac tissues (Scale bar, 500 nm). D) The mRNA levels of Ptgs2 mRNA in cardiac tissues (n = 6). E) Plasma MDA level (n = 6). F) MDA levels in cardiac tissues (n = 6). G,H) Fe²⁺ and Fe³⁺ levels in cardiac tissues (n = 6). I–P) AAV‐shNC or AAV‐shEBBP was administered via tail vein injection, and the DoIC model was established 14 days thereafter. I) Representative images and quantification of fluorescent immunohistochemistry staining for DHE in mouse myocardium (scale bars, 100 µm, n = 6). J) Representative images and quantification of immunohistochemistry for 4‐HNE in mouse myocardium (scale bars, 50 µm, n = 6). K) Representative transmission electron micrographs of cardiac tissues (Scale bar, 500 nm). L) The mRNA levels of Ptgs2 mRNA in cardiac tissues (n = 6). M) Plasma MDA level (n = 6). N) MDA levels in cardiac tissues (n = 6). O‐P) Fe²⁺ and Fe³⁺ levels in cardiac tissues (n = 6). Values are presented as the mean ± SD. ***p < 0.001 vs Vehicle + AAV‐NC or Vehicle + AAV‐shNC; ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 vs DOX + AAV‐NC or DOX + AAV‐ shNC group.

Figure 6

EBBP inhibits anthracycline‐induced cardiomyocyte ferroptosis by regulating SLC7A11/GSH/GPX4 and iron homeostasis. A,B) Immunoblots for EBBP, FTH1, SLC7A11, and GPX4 in cardiac tissues. C,D) Statistical analysis of the protein level of EBBP, FTH1, SLC7A11, GPX4, and GSH/GSSG in cardiac tissues administered with AAV‐NC or AAV‐EBBP (n = 6). E,F) Statistical analysis of the protein level of EBBP, FTH1, SLC7A11, GPX4, and GSH/GSSG in cardiac tissues administered with AAV‐shNC or AAV‐shEBBP (n = 6). G,H) Representative western blots of EBBP, FTH1, SLC7A11, and GPX4 in H9c2 cells. I,J) Statistical analysis of the protein level of EBBP, FTH1, SLC7A11, GPX4, and GSH/GSSG in H9c2 cells infection with Ad‐Null or Ad‐EBBP (n = 3). K,L) Statistical analysis of the protein level of EBBP, FTH1, SLC7A11, GPX4, and GSH/GSSG in H9c2 cells infection with Ad‐shNC or Ad‐shEBBP (n = 3). Values are presented as the mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 vs Vehicle + Ad‐Null or Vehicle+ Ad‐shNC; ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 vs DOX+ Ad‐Null or DOX+ Ad‐shNC.

Figure 7

EBBP promotes the activation of the PERK/ATF4 axis and Nrf2. A) Following infection with Ad‐Null or Ad‐EBBP, H9c2 was subjected to DOX (1 µm) for 24 h. Representative western blots of p‐PERK, PERK, p‐ eIF2α, eIF2α, ATF4, p‐Nrf2, and Nrf2 in H9c2 cells. B) Representative western blots of nuclear p‐Nrf2 in H9c2 cells. C–H) Statistical analysis of the protein levels in (A,B) (n = 3). I) Following infection with Ad‐shNC or Ad‐shEBBP, H9c2 was subjected to DOX (1 µm) for 24 h. Representative western blots of p‐PERK, PERK, p‐eIF2α, eIF2α, ATF4, p‐Nrf2, and Nrf2 in H9c2 cells. J) Representative western blots of nuclear p‐Nrf2 in H9c2 cells. K–P) Statistical analysis of the protein levels in (I,J) (n = 3). Values are presented as the mean ± SD. **p < 0.01 and ***p < 0.001 vs Vehicle + Ad‐Null or Vehicle+ Ad‐shNC; ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 vs DOX+ Ad‐Null or DOX+ Ad‐shNC.

Figure 8

Pharmacological inhibition of PERK attenuates EBBP's cardioprotective effects in anthracycline‐induced cardiac injury. A) Images of transthoracic echocardiography. B) Representative images of Masson's trichrome staining in heart (scale bars, 100 µm). C) Representative images of TUNEL staining in heart (scale bars, 50 µm). D) Changes in body weight of mice after DOX injection (n = 6). E) Changes in HW/TL (n = 6). F‐G) Quantification analysis of LVEF and FS (n = 6). H–J) The mRNA levels of Anp, Bnp, and Myh7 in cardiac tissues (n = 6). K–M) Quantitative analysis of serum AST, CK‐MB, and LDH levels (n = 6). N) Statistical analysis of fibrosis area (n = 6). O) Quantitative analysis of TUNEL‐positive cells via immunofluorescence staining (n = 6). Values are presented as the mean ± SD. ***p < 0.001 vs Vehicle + AAV‐NC; ^## p < 0.01 and ^### p < 0.001 vs DOX + AAV‐NC group; ^&&& p < 0.001 vs DOX + AAV‐EBBP group.

Figure 9

EBBP promotes PERK activation through the non‐degradative ubiquitination of GRP78. A) Molecular docking predicted an interaction between EBBP and GRP78. Red, EBBP. Blue, GRP78. B) Coimmunoprecipitation (co‐IP) experiments of the interaction between EBBP and GRP78 in 293T cells. WCL, whole‐cell lysis. C) In DOX‐treated NRCMs, an endogenous IP study was performed to investigate the interaction between EBBP and GRP78. D) A diagrammatic representation of the domains of EBBP and the shortened mutants is shown. Co‐IP analysis of the interaction domains of EBBP and GRP78. E) NRCMs that have been transfected with Ad‐Null or Ad‐EBBP were subjected to ubiquitination tests in order to determine the level of ubiquitination of endogenous GRP78. F) Ubiquitination assays determine the ubiquitination of HA‐GRP78 in HEK293T cells. G,H) The ubiquitination of HA‐GRP78 in response to Flag‐EBBP transfection was investigated in HEK293T cells transfected with the wild‐type (WT) and mutant Myc‐Ub plasmids to determine possible lysine ubiquitination types. I) Representative western blots of GRP78 and EBBP in NRCMs infected with Ad‐EBBP followed by stimulation with DOX (1 um for 24 h). J) Assays of coimmunoprecipitation (co‐IP) between GRP78 and PERK in 293T cells. K) Endogenous IP analysis of the interaction between GRP78 and PERK in NRCMs treated with DOX was investigated, aiming to determine how EBBP overexpression influences this interaction.