ATRA alleviates doxorubicin-induced cardiomyocyte ferroptosis by suppressing KEAP1-mediated NRF2 degradation

Q: What are the key findings of this study?

All-trans retinoic acid attenuated doxorubicin-induced cardiomyocyte ferroptosis and improved left ventricular ejection fraction in mice by suppressing KEAP1-mediated NRF2 degradation.

Q: What is the clinical evidence from this study?

Population: Doxorubicin-induced cardiotoxicity and cardiomyocyte ferroptosis (n=40). Intervention: All-trans retinoic acid (ATRA) vs. Doxorubicin alone (2 mg/kg intraperitoneally in vivo; 2 µM in vitro), vehicle control, or Doxorubicin + Deferoxamine (50 mg/kg). Primary outcome: Cardiomyocyte ferroptosis and left ventricular cardiac function (LVEF and LVFS) (p=<0.05).

Q: What does this research mean for the field?

All-trans retinoic acid attenuates doxorubicin-induced cardiomyocyte ferroptosis and improves left ventricular function by disrupting KEAP1-mediated NRF2 degradation and activating the NRF2-SLC7A11 pathway. Novelty: ClaimNovelty.NOVEL_FINDING. Consensus alignment: ConsensusAlignment.NEUTRAL.

Q: What question did this study set out to answer?

To determine whether all-trans retinoic acid (ATRA) mitigates doxorubicin-induced cardiomyocyte ferroptosis and to elucidate the underlying molecular mechanism.

Wenjin Xu; Xie Yuxin; Yaxin Guo; Yi Yide; Mengxiang CHEN; Hsiang‐i Tsai; Shengxiang Fu; Haitao Zhu; Liao Xiang

doi:10.21203/rs.3.rs-10690138/v1

October 12, 2026Open Access

ATRA alleviates doxorubicin-induced cardiomyocyte ferroptosis by suppressing KEAP1-mediated NRF2 degradation

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Key result

All-trans retinoic acid preserves LVEF and attenuates doxorubicin-induced cardiomyocyte ferroptosis in mice.

P<0.05
n=40

Why the study?

Doxorubicin use is limited by dose-dependent cardiotoxicity involving cardiomyocyte ferroptosis, yet effective strategies to suppress this process remain limited. Whether all-trans retinoic acid mitigates doxorubicin-induced cardiomyocyte ferroptosis and its underlying mechanism were unknown.

Does all-trans retinoic acid (ATRA) mitigate doxorubicin-induced cardiomyocyte ferroptosis and improve cardiac function in preclinical cellular and mouse models?

Key Points

To determine whether all-trans retinoic acid (ATRA) mitigates doxorubicin-induced cardiomyocyte ferroptosis and to elucidate the underlying molecular mechanism.
Tested the effects of ATRA on doxorubicin-induced ferroptosis in AC16 human cardiomyocytes and 8-week-old male C57BL/6 mice using cell viability, MDA, GSH, and lipid ROS assays.
Employed cycloheximide chase, MG132 proteasome inhibition, co-immunoprecipitation, molecular docking, and point mutagenesis at KEAP1 Arg415 to evaluate the KEAP1–NRF2 interaction.
Assessed cardiac structure and function in vivo through echocardiography measuring left ventricular ejection fraction (LVEF) and fractional shortening (LVFS), alongside cardiac injury markers.
Doxorubicin reduced cell viability (IC50 approximately 2 µM), depleted GSH, elevated MDA, and downregulated SLC7A11, whereas 20 µM ATRA reversed these ferroptotic changes in vitro and in vivo.
ATRA disrupted the KEAP1–NRF2 interaction and inhibited NRF2 proteasomal degradation depending on KEAP1 Arg415, while protection was lost in NRF2-deficient and SLC7A11-deficient cells.
In mice, ATRA restored myocardial NRF2, GPX4, and SLC7A11 levels, reduced ANP, BNP, cTnT, and MYH7 expression, and improved LVEF and LVFS.

Structured PICO

Does all-trans retinoic acid (ATRA) mitigate doxorubicin-induced cardiomyocyte ferroptosis and improve cardiac function in preclinical cellular and mouse models?

P

Population

40 male C57BL/6 mice aged 6-8 weeks in an acute doxorubicin-induced cardiotoxicity model, treated for 14 days alongside in vitro human AC16 cardiomyocytes.

I

Intervention

All-trans retinoic acid (ATRA): 20 µM for 48 hours in AC16 human cardiomyocytes in vitro; 10 mg/kg via intraperitoneal injection every two days for 14 days in C57BL/6 mice in vivo.

C

Comparator

In vitro: untreated control, doxorubicin alone (2 µM), and ferroptosis inhibitors/inducers (ferrostatin-1 2 µM, erastin 5 µM, RSL3 10 µM). In vivo: control group, ATRA alone (10 mg/kg), doxorubicin alone (2 mg/kg), and doxorubicin (2 mg/kg) + deferoxamine (50 mg/kg) administered intraperitoneally every two days for 14 days.

O

Outcome

Cardiomyocyte ferroptosis (cell viability, lipid ROS accumulation, malondialdehyde [MDA], reduced glutathione [GSH], and expression of SLC7A11, GPX4, and NRF2) and in vivo cardiac function (left ventricular ejection fraction [LVEF] and fractional shortening [LVFS] via transthoracic echocardiography at 14 days).surrogate

Main Result

p-value: p=<0.05

Limitations

Mechanistic experiments were mainly performed in AC16 cell lines rather than primary cardiomyocytes, hiPSC-derived cardiomyocytes, or cardiac-specific genetic models.
Direct biophysical evidence for ATRA-KEAP1 binding is lacking beyond molecular docking and mutagenesis.
The animal study used a short-term 14-day acute doxorubicin injury protocol rather than chronic cumulative exposure.
The study did not evaluate whether ATRA alters the antitumour efficacy of doxorubicin in tumour-bearing models.
Findings require further mechanistic and translational validation before clinical application.

Implication

This preclinical study identifies all-trans retinoic acid (ATRA) as a potential cardioprotective agent that attenuates doxorubicin-induced cardiotoxicity and ferroptosis by stabilizing NRF2 via KEAP1 Arg415 and activating the NRF2-SLC7A11 pathway.

Abstract

Objective Doxorubicin (DOX), a first-line anthracycline chemotherapeutic agent, is limited by dose-dependent cardiotoxicity. Ferroptosis is increasingly implicated in DOX-induced cardiomyocyte injury, yet effective strategies to suppress this process remain limited. This study investigated whether all-trans retinoic acid (ATRA) mitigates DOX-induced cardiomyocyte ferroptosis and defined the underlying mechanism. Methods We assessed the effects of ATRA on DOX-induced cardiomyocyte ferroptosis in vitro and in vivo using western blotting, quantitative real-time PCR, malondialdehyde (MDA) assays, glutathione (GSH) measurements and lipid peroxidation analysis. Loss-of-function models, cycloheximide chase assays, proteasome inhibition assays, molecular docking and co-immunoprecipitation were used to determine how the NRF2–SLC7A11 pathway mediates the protective effect of ATRA. Results DOX reduced cell viability, depleted GSH, increased MDA and downregulated SLC7A11 in cardiomyocytes. These effects were reversed by ATRA in vitro and in vivo. The anti-ferroptotic effect of ATRA was lost in SLC7A11-deficient and NRF2-deficient cells, indicating that the NRF2-SLC7A11 axis is required for protection. Mechanistically, ATRA stabilised NRF2 by reducing NRF2-KEAP1 interaction and proteasomal degradation. Co-immunoprecipitation showed that ATRA weakened the interaction between KEAP1 and NRF2. Molecular docking and mutagenesis suggested that KEAP1 Arg415 is functionally required for ATRA-mediated disruption of the KEAP1–NRF2 interaction. In vivo, ATRA increased myocardial NRF2 expression, suppressed DOX-induced ferroptosis and improved cardiac function, as shown by higher left ventricular ejection fraction and fractional shortening. Conclusion ATRA attenuates DOX-induced cardiomyocyte ferroptosis by disrupting KEAP1-mediated NRF2 degradation and activating the NRF2-SLC7A11 antioxidant pathway. These findings identify ATRA as a candidate cardioprotective strategy for anthracycline-related cardiac injury, pending further mechanistic and translational validation.

Discussion

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Cite This Study

Xu et al. (2026) studied Doxorubicin-induced cardiotoxicity and cardiomyocyte ferroptosis (n=40). All-trans retinoic acid (ATRA) vs. Doxorubicin alone (2 mg/kg intraperitoneally in vivo; 2 µM in vitro), vehicle control, or Doxorubicin + Deferoxamine (50 mg/kg) was evaluated on Cardiomyocyte ferroptosis and left ventricular cardiac function (LVEF and LVFS) (p=<0.05). All-trans retinoic acid attenuated doxorubicin-induced cardiomyocyte ferroptosis and improved left ventricular ejection fraction in mice by suppressing KEAP1-mediated NRF2 degradation.

synapsesocial.com/papers/6ae28f42cbadf70daf6c4487 https://doi.org/10.21203/rs.3.rs-10690138/v1

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ATRA alleviates doxorubicin-induced cardiomyocyte ferroptosis by suppressing KEAP1-mediated NRF2 degradation

Key Points

Structured PICO

Main Result

Limitations

Implication

Abstract

Discussion

Cite This Study

ATRA alleviates doxorubicin-induced cardiomyocyte ferroptosis by suppressing KEAP1-mediated NRF2 degradation

Key Points

Structured PICO

Main Result

Limitations

Implication

Abstract

Discussion

Cite This Study

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