Cardiomyocyte-derived GPX4 stabilizes BNIP3 to facilitate mitophagy and mitigate myocardial ischemia/reperfusion injury

Domain: biology
Published: 2026-03-29
Editorial outcome: major_revision

Abstract
GPX4 is a crucial regulator of ferroptosis, yet its role in mitochondrial dysfunction during myocardial ischemia/reperfusion injury (MI/RI) is unclear. This study aims to clarify the effect and molecular mechanisms of GPX4 in MI/RI. We analyzed the spatiotemporal dynamics of GPX4 during MI/RI and observed high expression levels in border and normal areas but significant reduction in the ischemic region utilizing spatial transcriptomics, spatial proteomics, and single-cell sequencing. Cardiomyocyte-derived GPX4 notably reduces myocardial damage and mitochondrial dysfunction in MI/RI while also alleviating long-term ventricular remodeling. Mechanistically, our findings reveal that GPX4, through its critical U46 active site, enhances the interaction between BNIP3 and USP20, decreasing ubiquitination at K131 of BNIP3. This process stabilizes BNIP3, promotes mitophagy, improves mitochondrial function, and ultimately preserves cardiac function. Our research defines the role of the GPX4/BNIP3/USP20 complex in MI/RI and uncovers a mechanism linking GPX4 to ferroptosis-related mitochondrial damage, providing valuable insights for advancing ferroptosis studies.

Introduction
Acute myocardial infarction (AMI) continues to be a leading cause of mortality globally1, 2. The Current standard treatment for AMI involves swift and effective myocardial reperfusion, achieved through either thrombolytic therapy or primary percutaneous coronary intervention (PCI), which is essential in alleviating ischemic injury and minimizing infarct size3. Despite the associated benefits, reperfusion can paradoxically induce addtional myocardial injury, a phenomenon known as myocardial ischemia/reperfusion injury (MI/RI)4, 5. This reperfusion injury can initiate various forms of cardiomyocyte death, including apoptosis, autophagy, pyroptosis and ferroptosis6, 7. Given the terminal differentiation and limited regenerative capacity of cardiomyocytes, strategies aimed at suppressing cardiomyocyte death are critical for preserving cardiac function following reperfusion8.

Ischemia followed by reperfusion inflicts significant detrimental effects on cardiomyocytes, particularly by disrupting intracellular glutathione (GSH) metabolism9, 10. This disruption culminates in the excessive accumulation of reac tive oxygen species (ROS), which is closely associated with ferroptosis11. Ferroptosis, an emerging form of programmed cell death characterized by the accumulation of iron- dependent lipid peroxides, plays a pivotal role in the pathogenesis and progression of various cardiovascular diseases, including MI/RI12, 13. This non-apoptotic form of cell death is primarily driven by the excessive accumulation of intracellular ROS resulting from impaired ROS-scavenging systems, such as disturbances in GSH biosynthesis and glutathione peroxidase 4 (GPX4) homeostasis14. GPX4, serves as a vital inhibitor of ROS-induced phospholipid peroxidation and has been recognized as a key regulator of ferroptosis15, 16. Researches have demonstrated that MI/RI correlates with the downregulation of GPX4, leading to ferroptosis processes17, 18. Conversely, upregulation of GPX4 has shown promise in mitigaing myocardial injury and improving cardiac function19, 20. Therefore, elucidating mechanisms through which GPX4 enhances outcomes following MI/RI will significantly bolster its therapeutic potential.

An imbalance in mitochondrial morphology disrupts energy production, enhances mitochondrial ROS generation, and destabilizes calcium homeostasis. Collectively, these factors contribute to cardiomyocyte death following MI/RI in the context of AMI21, 22. Therefore, therapeutic strategies aimed at preserving mitochondrial morphology and functionality may provide a crucial approach for cardioprotection. Mitophagy, which maintains mitochondrial quality by selectively degrading damaged mitochondria and preserving a healthy mitochondrial population, plays a crucial role in cardiomyocytes survival and cardiac function stabilization23, 24, 25. BNIP3 (BCL2/adenovirus e1B 19 kDa protein interacting protein 3) serves as a mitochondrial autophagy receptor essential for maintaining mitochondrial quality control and has significant implications in cardiovascular diseases26, 27, 28. Recent studies have demonstrated that BNIP3-related mitophagy can mitigate ferroptosis through the reduction of mitochondrial ROS levels29. However, there remains limited understanding regarding the direct correlation between GPX4 and BNIP3-mediated mitophagy.

In this study, we present the direct evidence of a link between GPX4 and BNIP3-mediated mitophagy. We initially elucidate the functional significance of a tripartite complex comprising GPX4, BNIP3 and deubiquitinating enzyme USP20 in the context of MI/RI. GPX4 facilitates the interaction between USP20 and BNIP3, thereby stabilizing BNIP3 protein and enhancing its role in mitophagy. This mechanism helps maintain mitochondrial homeostasis, improves mitochondrial function, and ultimately mitigates MI/RI. Consequently, our findings contribute to a deeper molecular understanding of GPX4’s involvement in MI/RI while providing valuable theoretical insights into mitochondrial impairment associated with ferroptosis.

Results
Spatiotemporal Distribution of GPX4 in MI/RI

MI/RI triggers multiple forms of programm ed cell death, but the timing and interplay of these processes remain unclear, prompting us to investigate the role of ferroptosis in its early stages. To explore this hypothesis, we administered various inhibitors targeting cell death via intraperitoneal injection in mice two hours prioIr the I/R procedure. The mice subsequently underwent surgery to surgical induction of 45 minutes of myocardial ischemia followed by 24 hours of reperfusion (Supplementary Fig. S1a). The results from triphenyltetrazolium chloride/Evans Blue (TTC/Evans Blue) staining suggested that emricasan (Emr), an inhibitor of apoptosis, and ferrostatin-1 (Fer), an inhibitor of ferroptosis, reduced myocardial infarct size after 24 hours. In contrast, neither 3-Methyladenine (3MA), an autophagy inhibitor, nor necrostatin-1 (Nec-1), a necroptosis inhibitor, exhibited similar effects during this time frame (Supplementary Fig. S1b-d). Furthermore, treatments with Emr and Fer-1 inhibited the elevation of I/R-induced cardiac injury biomarkers, such as serum cTnT, CK-MB and LDH. 3MA mildly alleviated the elevation of cTnT and CK-MB following myocardial injury but had no significant effect on LDH levels. Notably, Fer-1 demonstrated superior efficacy among them (Supplementary Fig. S1e-g). Consistent with these findings, Fer-1 significantly improved cardiomyocyte viability while reduced cell injury (Supplementary Fig. S1h-i). In conclusion, our study provides substantial evidence indicating that ferroptosis is critically involved in the acute phase of MI/RI.

Given the significant therapeutic efficacy of Fer-1, we conducted a Gene Ontology (GO) analysis on differentially expressed genes in cardiomyocytes treated with either the vehicle (HR + Veh) or Fer-1 (HR + Fer-1). Notably, lipid-related signaling pathways were significantly enriched (Fig. 1a). Among ferroptosis-related genes, GPX4, an enzyme responsible for scavenging lipid peroxides (LPs), plays a critical role in ferroptosis associated with MI/RI (Fig. 1b). To delineate the spatial distribution of GPX4 in myocardium affected by MI/RI, we analyzed the published data to investigate its spatial expression pattern. In the previous study, myocardial tissues from patients with myocardial infarction were categorized into necrotic regions (ischemic zones and border zones) and unaffected left ventricular myocardium (normal zones)30 (Fig. 1c). We observed that GPX4 was highly expressed in both the border and normal zones, whereas its expression was significantly diminished in the ischemic zone (Fig. 1d). Subsequently, we examined the expression levels of GPX4 in myocardial tissues from patients suffering from ischemic cardiomyopathy. Utilizing non-transplanted donor hearts as controls, we found that both mRNA and protein levels of GPX4 were significantly reduced in ischemic myocardial tissues (Fig. 1e-g). To further enhance clinical relevance, we subsequently assessed circulating GPX4 levels in patients with acute myocardial infarction who underwent percutaneous coronary intervention (PCI). Interestingly, when applying the widely used Global Registry of Acute Coronary Events (GRACE) scoring system to assess risk stratification and prognostic outcomes31, we observed that lower serum GPX4 levels were strongly associated with higher GRACE scores (Fig. 1h). These findings suggest that reduced GPX4 expression not only characterizes ischemic myocardial injury but also correlates with adverse prognosis in patients with myocardial infarction.

To further validate our findings in mouse myocardial tissues subjected to MI/RI, we employed spatial proteomics to investigate the spatial expression profile of GPX4 under these conditions. Similarly, we categorized the mouse myocardial tissue into three distinct regions: normal, border, and ischemic areas (Supplementary Fig. S2a-b). Consistent with prior observations12, 32, GPX4 protein levels showed a declining trend in the ischemic area. Conversely, levels in the border area were comparable to those found in the normal area (Supplementary Fig. S2c).

Immunofluorescence staining for GPX4 in mouse heart tissues subjected to MI/RI demonstrated a similar phenomenon (Supplementary Fig. 2d). Additionally, our results revealed that both protein and mRNA levels of Gpx4 decreased in a time-dependent manner in I/R-induced myocardium tissues (Supplementary Fig. 2e-g).

To identify the cellular source of downregulated GPX4 in ischemic myocardium, we performed single-cell RNA sequencing (scRNA-Seq) on approximately 19192 single cells isolated from the MI/RI-treated hearts tissues. Based on the expression of specific marker genes, we classified the cells into seven distinct types: cardiomyocytes (CMs), endothelial cells (ECs), fibroblasts (FBs), macrophages (MPs), smooth muscle cell (SMC) and T cells. (Supplementary Fig. 3a). Interestingly, Gpx4 was broadly expressed across various cell populations in the heart, with the highest proportion observed in cardiomyocytes (Fig. 1i, Supplementary Fig. 3b). Besides, scRNA-seq data showed that Gpx4 expression was highest in cardiomyocytes under baseline conditions (39.31% in sham) and accounted for 27.05% after I/R (Fig. 1j, Supplementary Fig. S3c). Consistently, isolated cardiomyocytes from adult mice after I/R also exhibited markedly decreased GPX4 expression (Fig. 1k, Supplementary Fig. S3d). Based on canonical markers, cardiomyocytes were further subdivided into ischemic, damaged, and remodeling clusters (Supplementary Fig. S3e). Pseudotime trajectory analysis revealed that Tnnt2 decreased and then recovered, Myh7 progressively increased during remodeling, while Gpx4 declined sharply during the ischemic phase and showed no substantial rebound thereafter (Supplementary Fig. S3f-h). These findings highlight cardiomyocytes as the predominant source of GPX4 and indicate that its loss is closely linked to ischemic injury and impaired recovery. Although the role of GPX4 expressed in other cell types in ferroptosis cannot be overlooked33, 34, we focused on cardiomyocyte-derived GPX4 for the subsequent studies. Consistency with our in vivo results, H/R induction triggered a decrease in GPX4 protein and mRNA levels of neonata l rat cardiomyocytes (NRCMs) (Fig. 1l-m, Supplementary Fig. S3i). Altogether, our findings delineate the spatiotemporal distribution of GPX4 in MI/RI, demonstrating a time- dependent reduction in ischemic cardiomyocytes. Importantly, this experimental evidence is supported by clinical observations, where decreased circulating GPX4 levels in acute myocardial infarction patients were associated with adverse prognostic indicators.

Cardiomyocyte-derived GPX4 was identified as a protective factor in MI/RI

We constructed a cardiomyocyte-targeting adeno-associated virus serotype 9 (AAV9) vector that encoding Gpx4 (AAV9-cTnT-Gpx4oe) or an empty vector (AAV9-cTnT-EV). WT (wild-type) mice received tail vein injections of either AAV9-cTnT-Gpx4oe or AAV9-cTnT-EV, resulting in confirmed overexpression of GPX4 in myocardial tissue. After a four-week period that ensured stable GPX4 expression (Supplementary Fig. 4a-d), the mice were subjected to MI/RI surgery, involving 45 minutes of ischemia followed by 24 hours of reperfusion, or underwent a sham operation (Fig. 2a). Infarct size was evaluated through Evans blue/TTC staining. Our findings revealed that cardiomyocyte-specific overexpression of GPX4 significantly reduced the myocardial infarct area induced by MI/RI (Fig. 2b-d). Notably, similar trends were observed in cardiac injury biomarks, including serum cTnT, serum CK-MB and serum LDH levels (Fig. 2e-g). Furthermore, consistent with the aforementioned results, I/R-induced death of cardiomyocyte in the infracted area, as indicated by TUNEL-positive cells, was also reduced by GPX4 overexpression (Fig. 2h-i).

To further investigate the relationship between GPX4 and MI/RI, WT mice were administered RSL3, a specific inhibitor of GPX4, at a dosage of 30 mg/kg via oral gavage daily for 7 consecutive days prior to the onset of ischemia, Subsequently, a model of MI/RI was established (Fig. 2j). Treatment with RSL3 results in an exacerbation of the myocardial infarct area induced by I/R (Fig. 2k-m). The levels of cardiac injury markers, including serum CK-MB, LDH, and cTnT, exhibited similar trends indicative of increased damage (Fig. 2n-p). Additionally, the number of TUNEL-positive cells in myocardial tissues elevated under I/R conditions and was significantly aggravated in mice treated with RSL3 (Fig. 2q-r). Our findings indicate that cardiomyocyte-derived GPX4 plays a protective role in the progression of MI/RI, mitigating myocardial tissue damage.

Cardiomyocyte-derived GPX4 mitigates ventricular remodeling induced by MI/RI and MI

MI/RI can lead to ventricular remodeling and subsequent heart failure if not treated promptly35. Therefore, we assessed whether overexpression of GPX4 in cardiomyocytes could mitigate ventricular remodeling induced by chronic MI/RI, characterized by 45 minutes of ischemia followed by 4 weeks of reperfusion (Fig. 3a). Non -invasive echocardiography demonstrated that cardiomyocyte-specific GPX4 overexpression markedly improved cardiac function, as evidenced by significant increases in ejection fraction (EF) and fractional shortening (FS) (Fig. 3b-d and Supplementary Table 2). Correspondingly, serum ANP levels, a biomarker of heart failure, were significantly reduced in GPX4-overexpressing mice (Fig. 3e). Histological analysis further revealed that GPX4 overexpression attenuated adverse ventricular remodeling, with a pronounced reduction in interstitial fibrosis when compared to that observed in mice treated with AAV9-cTnT-EV (Fig. 3f-g). Consistently, the overexpression of GPX4 significantly mitigated the levels of fibrotic markers, including Col-1a1 and Tgf-β, in the infarcted regions of mouse myocardial tissues subjected to MI/RI compared to the EV group (Fig. 3h). Collectively, these results indicate that GPX4 mitigates pathological ventricular remodeling following MI/RI.

To further confirm the long-term protective role of GPX4, we employed a chronic myocardial infarction (MI) model with 4 weeks of follow-up, which reproduces advanced heart failure and ventricular remodeling36 (Fig. 3i). In this model, cardiomyocyte-specific GPX4 overexpression markedly preserved cardiac function (Fig. 3j-l and Supplementary Table 3), accompanied by reduced serum ANP levels (Fig. 3m), alleviated myocardial fibrosis, and attenuated maladaptive remodeling (Fig. 3n-p). These findings demonstrate that GPX4 confers sustained protection against chronic ventricular remodeling and progression to heart failure. In contrast, mice treated with RSL3 exhibited exacerbated cardiac dysfunction and ventricular remodeling compared to those receiving Veh treatment (Supplementary Fig. S5a-k and Supplementary Table 4).

Confirming the effects of cardiomyocyte-derived GPX4 in vivo, we further explored its function in NRCMs utilizing a H/R model. We initially demonstrated that increasing levels of GPX4 protein significantly reduce H/R-induced cardiomyocyte death (Supplementary Fig. S6a-b). Additionally, iron levels and markers of ROS, evidenced by the decreases in SOD activity, elevations in MDA content, and reduction in GSH levels, indicating lipid peroxidation were diminished in the GPX4oe + H/R group compared to the EV + H/R group (Supplementary Fig. S6c-f). Immunohistochemical staining for 4-HNE, a recognized molecular marker of ferroptosis, revealed that GPX4 overexpression significantly mitigates myocardial ferroptosis following I/R-induced cardiac tissues (Supplementary Fig. S6g). When GPX4 function was inhibited by RSL3, there was a notable increase in cardiomyocytes injury (Supplementary Fig. S6h-i), alongside elevated ROS and ferroptosis levels (Supplementary Fig. S6j-n). In summary, cardiomyocyte-derived GPX4 effectively inhibits ferroptosis and ameliorates MI/RI both in vitro and in vivo.

GPX4 serves as a crucial regulator of mitochondrial function in the progression of MI/RI

Previous studies have established a close association between GPX4 and mitochondrial dysfunction in ferroptosis37. However, the specific regulatory molecular mechanisms involved remain unclear. Mitochondrial dysfunction, recognized as a principal theory underlying MI/RI, plays a pivotal role in mediating the associated pathophysiological processes38, 39. We initially analyzed transcriptome sequencing data from NRCMs subjected to H/R with or without GPX4 overexpression. The Go analysis of this transcriptome revealed numerous differentially regulated mitochondrial signaling pathways (Fig. 4a). Consequently, we proceeded to measure the oxygen consumption rate (OCR) in NRCMs and found that GPX4 overexpression restored both basal, ATP production and maximal oxygen consumption rate (Fig. 4b-c). As shown in Fig. 4d-g, exogenous supplementation of GPX4 significantly ameliorated mitochondrial dysfunction induced by either H/R or I/R injury, as evidenced by elevated ATP level (Fig. 4d), increased complex I activity (Fig. 4e), and improved mitochondrial membrane potential (Fig. 4f-g ). Further ultrastructural analysis using transmission electron microscopy (TEM) revealed that MI/RI induced severe mitochondrial cristae disruption, manifested as fragmented and broken cristae with reduced cristae density. Notably, GPX4 overexpression markedly decreased the proportion of mitochondria exhibiting such cristae damage in MI/RI mice (Fig. 4h-i). Consistently, H/R-induced NRCMs displayed pronounced cristae breakage and density loss, whereas GPX4 overexpression partially preserved cristae structure (Fig. 4j-k). Importantly, under normoxia conditions, no significant differences in mitochondrial function were observed between EV- and GPX4oe-transduced cells, indicating that the protective effect of GPX4 is stress-dependent rather than baseline altering.

In contrast, compared to NRCMs subjected to H/R with vehicle treatment, those treated with RSL3 exhibited pronounced mitochondrial respiratory dysfunction as assessed by marked decreases in basal oxygen consumption rates, ATP production levels, and maximum oxygen consumption capacity (Supplementary Fig. S7a-b). Consistently, the levels of ATP (Supplementary Fig. S7c-d), complex I activity (Supplementary Fig. S7e-f), and mitochondrial membrane potential (Supplementary Fig. S7g-h) exhibited a similar trend. Furthermore, both in vitro and in vivo analyses of mitochondrial ultrastructural revealed more severe mitochondrial damage following RSL3 treatment (Supplementary Fig. S7i-l). Interestingly, we found that RSL3 itself induces mitochondrial dysfunction in cardiomyocytes, and this effect occurs independently of I/R or H/R injury.

The aforementioned findings suggested that GPX4 plays a pivotal role in maintaining mitochondrial integrity during the progression of MI/RI, acting as a key modulator of mitochondrial damage. Notably, we observed that GPX4 overexpression in H/R-treated NRCMs led to a reduction in Tomm20 levels, a marker of mitochondrial mass, accompanied by increased expression of p-Drp1/Drp1 and Fis1, both of which are key regulators of mitochondrial fission (Supplementary Fig. S8a-b). In contrast, treatment with RSL3 resulted in elevated Tomm20 expression, while p-Drp1/Drp1 and Fis1 levels were reduced (Supplementary Fig. S8c-d). These results imply that GPX4 may influence the process of mitochondrial degradation, thereby altering overall mitochondrial quality.

GPX4 directly binds with BNIP3 and promotes BNIP3-dependent-mitophagy in cardiomyocytes

To elucidate the molecular mechanisms by which GPX4 regulates mitochondrial function, we overexpressed GPX4 in cardiomyocytes and subjected them to hypoxia-reoxygenation (H/R) modeling. Co-immunoprecipitation (Co-IP) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to identify potential partners of GPX4. Among the 612 proteins identified by LC-MS/MS, 89 substrate proteins exhibiting a greater than 2-fold change were selected as candidate proteins (Fig. 5a, Dataset I). Notably, Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3), BCL2/adenovirus E1B interacting protein 3-like (BNIP3L), NOD-like receptor X1 (NLRX1), Dynamin-related protein 1 (DRP1), Yes-associated protein 1 (YAP1), and Mothers Against Decapentaplegic Homolog 1 (SMAD1) (Fig. 5a, Dataset II) have been previously implicated in mitochondrial injury. By cross-referencing these two datasets, we identified BNIP3 as a potential binding partner of GPX4 (Fig. 5a). As demonstratedin Fig. 5b, co-transfection of GPX4 and BNIP3 plasmids into NIH/3T3 cells confirmed the interaction between those two proteins. Additionally, we validated the endogenous interaction between GPX4 and BNIP3 in MI/RI-induced infarcted heart tissues (Fig. 5c) and H/R-induced NRCMs (Fig. 5d). We further employed surface plasmon resonance (SPR) to confirm the direct interaction between recombinant mouse GPX4 and recombinant mouse BNIP3 proteins, yielding a dissociation constant (KD) of 3.33e-08M (Fig. 5e). Collectively, these findings substantiate that GPX4 directly interacts with BNIP3 in cardiomyocytes.

It is widely accepted that mitophagy can be initiated by BNIP3, critical regulators of mitochondrial quality and function26, 40. To investigate whether the binding of GPX4 to BNIP3 influences BNIP3-mediated mitophagy, we performed co-staining utilizing a mitophagy dye and mitoTracker to assess the intensity of mitophagy in NRCMs. Our data indicate that, compared with the EV + H/R group, the cardiomyocytes in GPX4oe + H/R group demonstrated significantly elevated mitophagy levels (Fig. 5f-g, and Supplementary Fig. S9a). Furthermore, immunofluorescence staining revealed an increased expression of LAMP in H/R-treated NRCMs with GPX4 overexpression, with clear colocalization with the mitochondrial marker TOMM20 (yellow dots, Supplementary Fig. S9b-c). To clarify the role of mitophagy in regulating mitochondrial quantity, we evaluated mitochondrial numbers by measuring mtDNA copy number measurement. As shown in Supplementary Fig. S9d, GPX4 amplified the observed decline trend regarding mitochondrial numbers. In contrast, inhibition of GPX4 with RSL3 led to a reduction in mitophagy levels (Supplementary Fig. S10a-b) and was accompanied by decreased LAMP1 fluorescence signal (Supplementary Fig. S10c-d). Mitochondrial proteins were subsequently extracted. Our results revealed that GPX4 overexpression led to significantly elevated levels of mitochondrial BNIP3 protein and LC3B-II in H/R-treated NRCMs (Fig. 5h-i). These findings suggest that GPX4 may promote mitophagy primarily by upregulating BNIP3 protein expression. To test this hypothesis, we employed siRNA-mediated Bnip3 knockdown to evaluate whether GPX4 could still enhance mitophagy in the absence of BNIP3. Interestingly, compared with the si-NC + EV + H/R group, BNIP3 silencing reduced mitophagy levels, and the loss of BNIP3 also abolished the GPX4 overexpression–induced increase in mitophagy in NRCMs (Fig. 5j–k, Supplementary Fig. S10e). Collectively, these findings demonstrate that GPX4 directly interacts with BNIP3 and enhances BNIP3-dependent mitophagy, thereby contributing to the maintenance of mitochondrial quality and function under ischemic stress.

GPX4 prevents proteasomal degradation of BNIP3

To clarify how GPX4 increases BNIP3 protein levels, we treated NRCMs with the GPX4 inhibitor RSL3 and applied either autophagy or proteasome inhibitors to determine whether BNIP3 degradation occurs via the autophagy-lysosome pathway or the proteasome pathway. We found that the proteasome inhibitor MG132 effectively prevented BNIP3 degradation, whereas autophagy inhibitors, including 3-methyladenine (3-MA), chloroquine (CQ), and bafilomycin A1 (Baf A1), had no effect on GPX4-mediated BNIP3 regulation (Fig. 6a-b). These results indicate that BNIP3 degradation primarily depends on the proteasome pathway. Interestingly, existing researches indicates that the degradation of BNIP3 is regulated by the ubiquitin-proteasome pathway41. Through Co-IP experiments, we further confirmed that BNIP3 undergoes ubiquitination (Fig. 6c). Moreover, using the cycloheximide to inhibit protein synthesis, we demonstrated that inhibition of proteasome by MG132 activity effectively prevents BNIP3 degradation in NIH/3T3 (Fig. 6d-e). Subsequently, to further validate this hypothesis, we overexpressed GPX4 in cardiomyocytes and co-treated them with MG132 and cycloheximide and confirmed that overexpressing GPX4 plasmids in NIH/3T3 cells significantly inhibited BNIP3 degradation (Fig. 6f-g). Finally, we co-overexpressed GPX4, BNIP3, and UB in NIH/3T3 cells and conducted Co-IP experiments. The obtained results demonstrated a significant reduction in ubiquitination levels of BNIP3 upon GPX4 overexpression (Fig. 6h). This effect was consistently observed in both HR-induced NRCMs (Fig. 6i) and MI/RI-treated myocardial tissues (Fig. 6j). These findings indicated that GPX4 directly interacts wtih BNIP3, thereby inhibiting its degradation via the ubiquitin-proteasome pathway.

USP20 acts as a crucial factor in enhancing the stability of BNIP3 protein through its interaction with GPX4

GPX4 is a critical antioxidant enzyme that plays a vital role in protecting cells from oxidative damage by reducing lipid peroxides15, 16. However, its potential involvement in regulating protein ubiquitination has not been reported. Deubiquitinating enzymes (DUBs) counteract the ubiquitination of substrate proteins, often preventing their targeting for degradation by the proteasome42. Therefore, we hypothesized that DUBs may be involved in the GPX4-BNIP3 interaction system. To investigate this, we utilized Co-IP and LC-MS/MS to screen for potential DUBs functioning within the GPX4-BNIP3 complex. We transfected HL-1 cells with plasmids encoding either GPX4 or BNIP3 for 24 hours, followed by H/R treatment. Subsequently, Co-IP and LC-MS/MS were conducted (Fig. 7a). Notably, we identified 105 proteins that physically interact with GPX4 (Fig. 7b, Dataset I) and 58 proteins binding to BNIP3 (Fig. 7c, Dataset II). We identified USP20 as a potential DUB involved in the GPX4-BNIP3 interaction via comparing these two interactomes (Fig. 7d). Subsequently, we co-transfected NIH/3T3 cells with plasmids encoding GPX4, BNIP3, and USP20 to validate their interaction among each other. The results confirmed that GPX4, BNIP3, and USP20 form a protein complex (Fig. 7e). The endogenous tripartite complex comprising GPX4-BNIP3-USP20 was also confirmed in NRCMs subjected to H/R stimulation (Supplementary Fig. S11a) and myocardial tissues induced by MI/RI (Supplementary Fig. S11b).

To verify our hypothesis that GPX4 influences BNIP3 degradation by removal of its ubiquitin chains through USP20 activity, we employed CRISPR-Cas9 technology to generate Usp20-knockout NIH/3T3 cells (gUsp20-NIH/3T3). Following transfection of these knockout cells with plasmids encoding either empty vector or GPX4, cells were treated with cycloheximide. In gCtrl cells, GPX4 overexpression delayed BNIP3 degradation, whereas in the absence of USP20, the degradation rate of BNIP3 was markedly higher than in gCtrl. Notably, under USP20-deficient conditions, GPX4 overexpression failed to attenuate BNIP3 degradation, suggesting that the regulation of BNIP3 stability by GPX4 is dependent on the involvement of USP20 (Fig. 7f-g). Furthermore, co-transfecting plasmids encoding either GPX4, BNIP3, and UB into gUsp20-NIH/3T3 cells, we observed that GPX4 no longer influenced the ubiquitination level of BNIP3 in the absence of USP20 (Fig. 7h).

We next performed Co-IP experiments in H/R-stimulated cardiomyocytes with or without GPX4 transfection, which demonstrated that GPX4 markedly enhanced the interaction between USP20 and BNIP3 (Supplementary Fig. S12a-b). To further evaluate the molecular basis of this interaction, we employed AlphaFold3-based virtual docking combined with molecular dynamics (MD) simulations. The BNIP3-USP20 dimer exhibited considerable fluctuations, showing only transient stability between 20 ns and 60 ns before returning to an unstable state (Supplementary Fig. S12c). In contrast, the BNIP3-USP20-GPX4 trimer displayed only minor fluctuations around 40 ns and reached a more stable conformation after 60 ns (Supplementary Fig. S12d). Consistently, the trimer showed substantially reduced RMSD values compared with the dimer, indicating improved structural stability (Supplementary Fig. S12e). Complementary surface plasmon resonance (SPR) analysis revealed a dissociation constant (KD) of 4.29e-07 M for the BNIP3-USP20 interaction (Fig. 7i), which decreased to 1.61e-8 M upon GPX4 inclusion (Fig. 7j). Given that KD values are inversely proportional to binding affinity, these results indicate that GPX4 significantly strengthens the interaction between BNIP3 and USP20. In addition, co-IP experiments confirmed that USP20 binds to BNIP3 and deubiquitinates it, while the presence of GPX4 further potentiates the USP20-BNIP3 interaction, thereby enhancing the removal of ubiquitin chains from BNIP3 (Fig. 7k and Supplementary Fig. S12f). Taken together, the results from Co-IP, MD simulations, and SPR consistently demonstrate that GPX4 stabilizes the BNIP3-USP20 complex.

The U46 residue of GPX4 enhances the deubiquitination effect of USP20 on the K131 residue of BNIP3

The U46 residue represents the critical active site of GPX4, and mutation at this site leads to complete loss of enzymatic activity43, 44. Interestingly, AlphaFold3-based structural modeling revealed that the U46 site is located within the binding interface of the GPX4-BNIP3-USP20 complex, suggesting a potential role of U46 in facilitating the interaction between USP20 and BNIP3 (Fig. 8a). Since RSL3 acts by inhibiting the U46 site of GPX443, 45, we first performed IP assays using myocardial tissues subjected to I/R injury with RSL3 treatment and found that BNIP3-USP20 interactions were attenuated (Supplementary Fig. S13a-b). Consistently, in NIH/3T3 cells co-transfected with GPX4, BNIP3, and USP20, GPX4-enhanced BNIP3-USP20 binding was markedly reduced upon RSL3 treatment (Supplementary Fig. S13c-d). To further validate this hypothesis, we generated a GPX4 U46A mutant plasmid (lead to loss of activity) and conducted Co-IP assays in NIH/3T3 cells. The results demonstrated that the U46A mutation abolished the ability of GPX4 to promote BNIP3-USP20 interaction. Moreover, in the U46 mutant background, RSL3 treatment no longer exerted additional inhibitory effects (Fig.8b and Supplementary Fig. S13e). Taken together, these findings indicate that the U46 residue of GPX4 is the critical active site required for promoting the interaction between USP20 and BNIP3.

To accurately identify the deubiquitination site of BNIP3 mediated by USP20 in the presence of GPX4, we conducted an affinity-based ubiquitinated peptide enrichment ubiquitylome analysis in HL-1 cells expressing Flag-GPX4 (Fig.8c). Through a comprehensive analysis of BNIP3 ubiquitination sites, we identified four potential ubiquitination lysine residues susceptible to ubiquitination within the BNIP3 protein: K46, K83, K128, and K131 (Fig.8d). Consequently, we selected these sites and constructed corresponding BNIP3 mutants plasmids by substituting lysine residue with arginine to mimic the deubiquitinated state of BNIP3. Subsequently, HL-1 cells were transfected with plasmids encoding wild-type BNIP3 (WT) plasmids as well as the respective mutant plasmids, followed by H/R modeling and assessment of cell viability. The results indicated that mutation at the K131 site significantly enhanced cell viability (Fig. 8e). Additionally, we observed that the polyubiquitination levels of BNIP3-K131R was significantly lower than that of BNIP3-WT and did not exhibit any further reduction by overexpressing GPX4 both in NIH/3T3 and H/R-stimulated HL-1 (Fig. 8f and Supplementary Fig. S13f). This observation suggests that lysine residue K131 in BNIP3 is crucial for GPX4-mediated USP20-BNIP3 deubiquitination system (Fig. 8g).

To assess the functional significance of K131 ubiquitination, cardiomyocytes were subjected to H/R treatment under different conditions of BNIP3 and USP20 expression. As shown in Supplementary Fig. S13g, USP20 deficiency markedly reduced cell viability, while restoration of BNIP3 expression—particularly the K131R mutant—significantly improved cardiomyocyte survival. Consistent results were obtained for LDH release (Supplementary Fig. S13h). These findings indicate that K131 ubiquitination is essential for BNIP3-mediated protection and that the K131R mutant can partially compensate for USP20 deficiency.

USP20 is essential for GPX4-targeted regulation of BNIP3 in MI/RI

To elucidate the role of the GPX4-BNIP3-USP20 tripartite complex in MI/RI, we generated cardiomyocyte-specific USP20 knockout mice (USP20 CKO) by crossing USP20fl/fl mice with Myh6-Cre mice (Supplementary Fig. 14a-b). USP20fl/fl and USP20 CKO mice subsequently underwent MI/RI surgery following a four-week period of cardiomyocyte-specific overexpression of either EV or GPX4 (Fig. 9a).

In USP20fl/fl mice, GPX4 overexpression significantly reduced infarct size and acute myocardial injury, as evidenced by decreased cardiac injury markers and fewer TUNEL-positive cells following MI/RI). In contrast, USP20 CKO mice displayed larger infarct size and aggravated acute injury, and GPX4 overexpression failed to provide protection (Fig. 9b-h). Besides, we isolated cardiac mitochondria and oxygen consumption rate (OCR) was measured using sequential substrate and inhibitor titrations. Our results showed that GPX4 overexpression significantly promoted the complex I OXPHOS capacity, enhanced the complex II OCR, and increased the respiration of complex IV in USP20fl/fl mice . However, when comparing the EV-IR groups, USP20 CKO mice displayed a marked reduction in multiple OCR parameters relative to USP20fl/fl mice, indicating impaired mitochondrial function. Notably, GPX4 overexpression in USP20 CKO mice did not reverse these defects, suggesting that during the acute phase of I/R, the protective effect of GPX4 on mitochondrial respiration is dependent on the presence of USP20 (Supplementary Fig. S14c-d).

At the chronic stage (4 weeks after MI/RI) (Fig. 9j), GPX4 overexpression improved cardiac function in USP20fl/fl mice, as evidenced by higher EF and FS values, whereas USP20 CKO mice exhibited worse cardiac function compared with their USP20fl/fl counterparts. Interestingly, GPX4 overexpression partially restored cardiac function in USP20 CKO mice but failed to reach the level observed in USP20fl/fl + GPX4oe mice (Fig. 9k-m and Supplementary Table 5). Consistent results were obtained for serum ANP (Fig. 9n) and myocardial fibrosis. Histological staining and qPCR analysis of fibrosis-related genes demonstrated reduced fibrotic remodeling in USP20fl/fl + GPX4oe mice, whereas USP20 CKO mice showed more severe fibrosis, and the protective effect of GPX4 was insufficient to fully reverse the damage caused by USP20 deficiency (Fig. 9o-q).

These findings indicated that USP20 is crucial for GPX4-mediated regulation of BNIP3, and contributes to mitigating MI/RI. More importantly, targeting the GPX4-BNIP3-USP20 axis emerges as an innovative therapeutic strategy for ameliorating MI/RI and unveils a molecular mechanism through which GPX4 modulates GPX4-associated mitochondrial damage (Fig. 10).

Methods
Animals

All animal experiments were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23P, revised 1996) and were approved by the Laboratory Animal Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (approval document No. WYYYI-AEC-YS-2023-0097). Wild-type mice, α-myosin heavy chain promoter-driven heterozygous Cre (T055034, C57BL/6J background, Myh6-Cre+/-) mice and USP20 floxed mice (T0521L23, C57BL/6J background, USP20fl/fl) mice were obtained from GemPharmatech (Nanjing, China). Cardiac-specific deletion of USP20 mice (USP20flox/flox Myh6-Cre, USP20 CKO) was generated by crossing the floxed USP20 mice with Myh6-Cre+/- mice. Mice were housed in specific pathogen-free (SPF) conditions with free access to food and water under a 12 h light/dark cycle.

The myocardial ischemia and reperfusion injury (MI/RI) model was established as previous described48. Mice were anesthetized with 2% isoflurane and ventilated using a MiniVent system. A left thoracotomy was performed, and the left anterior descending coronary artery (LAD) was occluded with a 7-0 silk suture slipknot. Myocardial blanching confirmed complete occlusion. After 45 minutes, the slipknot was released to initiate reperfusion (24 hours for acute I/R injury or two or four weeks for chronic I/R injury). Sham-operated mice underwent identical procedures without tying the suture. Mice received supplemental heat for 30 minutes postoperatively and continued overnight recovery.

Hypoxia-reoxygenation (H/R) injury treatment of cardiomyocytes

Hypoxia was induced by replacing ambient air with 94% N 1% O and 5% CO in a controlled incubator, and using DMEM lacking FBS and glucose for 4 hours. Afterward, cells were reoxygenated by replacing the medium with normal DMEM and incubating under normoxic conditions (21% O , 74% N , 5% CO ) for 6 hours. Control groups were kept under normoxia with glucose and FBS-deprived DMEM for the same duration.

Co-IP combined with LC-MS/MS analysis

For co-immunoprecipitation (Co-IP), antibodies and protein A+G beads were used. For co-immunoprecipitation (Co-IP) experiments, protein complexes were isolated using specific antibodies and protein A/G magnetic beads. Proteins were extracted in SDT lysis buffer and digested using the filter-aided sample preparation (FASP) method. Peptides were analyzed by LC–MS/MS at PTM Bio Co., Ltd. (Zhejiang, China). Peptide separation was performed on a C18 column (1.8 µm, 0.15 × 100 mm) using an EASY-nLC 1000 system (Thermo Fisher Scientific) with a 98-min gradient at 600 nL/min. The mobile phases consisted of 0.1% formic acid (A) and acetonitrile (B). MS/MS analysis was conducted on an LETQ Orbitrap ETD mass spectrometer (Thermo Fisher Scientific). Raw data were processed using Proteome Discoverer (v1.4, Thermo Fisher Scientific) for database searching and protein identification. Candidate substrates were identified based on relative protein intensities. In total, four independent samples were analyzed by LC–MS/MS (n=1 per group).

Spatial proteomics and spatial transcriptomics analysis

For spatial proteomics, heart samples from I/R-treated mice were washed with PBS, immersed in liquid nitrogen-precooled isopentane for 1 minute, and then frozen in O.C.T. Compound. Tissue sections (10 μm) were cut using a CryoStar NX60 cryostat and stained with H&E. The normal, border, and ischemic zones were identified and marked. These regions underwent laser capture microdissection (LCM) and spatial proteomics analysis. Protein extraction, trypsin digestion, and spatial proteomics were performed by PTM Bio Co., Ltd. (Zhejiang, China).

Spatial transcriptome raw data was form the previous published study30. All data analysis was conducted by LC Bio-Technology Co., Ltd. (Hangzhou, China).

Statistical analysis

The results are presented as the mean ± standard error of the mean (SD). For in vitro data, the assumption of normal distribution was based on the central limit theorem, as each experimental dataset represents an average of a large number of cultured cells. For in vivo data, with a sample size of ≥ 6, the normality of each dataset was assessed using the Kolmogorov-Smirnov test. A P-value > 0.05 indicated that the data were approximately normally distributed. Statistical analyses were conducted by using GraphPad Prism 10.0 (GraphPad, San Diego, CA). Depending on the analysis, either a Student’s t-test or one-way ANOVA or two-way ANOVA followed by post hoc multiple comparisons was performed. A P-value < 0.05 was considered statistically significant.

An extended Materials and Methods section is available in the Supplementary Information.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors investigated the role of cardiomyocyte-derived GPX4 in mitigating myocardial ischemia/reperfusion injury (MI/RI) by stabilizing BNIP3 via USP20-mediated deubiquitination, thereby enhancing mitophagy and mitochondrial function. They applied several advanced techniques to determine GPX4 function after heart injury, including spatial transcriptomics, proteomics, and single-cell RNA sequencing, as well as heart injury models. Although the literature has reported GPX4 function in the heart, this work gains novel insights into the role of GPX4/BNIP3/USP20 axis after MI/RI. With that said, here are some specific concerns we have:\n1.In Fig. 1h, the authors classified the heart into three parts: the normal area, the border area, and the ischemic area. However, they showed that the myocardium in these three area was comparable, why?\n2.It is noted that, in this work, GPX4 expression decreased in the infarction region. Can the authors provide data how GPX4 was regulated after IR or MI?\n3.The authors emphasized that GPX4 expression was specifically down-regulated in the ischemic region. Given that cardiomyocytes in this area underwent apoptosis and were progressively replaced by fibrotic tissues, could the reduced GPX4 expression attribute to the loss of viable cardiomyocytes?\n4.The size of the BNIP3 proteins varied inconsistently from different Western blot experiments. For example, in Fig. 6c, the main band appeared ~35 kDa, while in Fig. 6d, it was close to 25 kDa. Overall, the quality of co-IP experiments was low, which need to be improved.\n5.The authors proposed that GPX4 facilitates the interaction between BNIP3 and USP20, thus promoting deubiquitination at the K131 site of BNIP3. However, it will be much improved if the authors can provide data on addressing how GPX4 interacts with BNIP3 and USP20 (since GPX4 function in the heart was reported in the literature).\n6.The author made several spelling errors in the text. For example, in line 65, \"addtional\" should be \"additional\"; in line 104, \"initialy\" should be \"initially\"; in line 209, \"theated\" should be \"treated\"; in line 223, \"specfic\" should be \"specific\"; and in line 463, \"fingding\" should be \"finding.\". It is important to have the errors corrected throughout the manuscript.\n7.According to the figure legends, the y-axis in Fig. 3n should be labeled as FS (%).", "reviewer_2": "This manuscript explored the role and molecular mechanisms of GPX4 in MI/RI. The authors identified that GPX4 stabilized BNIP3 via USP20-mediated deubiquitination, thereby facilitating mitophagy and diminishing myocardial injury resulting from MI/RI. The study was commendably innovative and comprehensive. The current data supported the hypothesis that GPX4 enhanced BNIP3 deubiquitination through USP20, leading to reduced myocardial injury from MI/RI. However, several key data gaps and insufficient evidence limited the overall strength of the conclusions. I would suggest the authors to provide additional experimental data or further data analyses to draw firm conclusions. I have the following comments to improve the manuscript.\n1) The data showed that overexpression of GPX4 in USP20 CKO mice did not significantly reduce the infarct area following MI/RI surgery, suggesting a key role of BNIP3 stability. However, the TTC staining in Fig. 8c without control group data made it impossible to confirm whether GPX4 overexpression reduced the infarct size in the control group. As a result, the protective effect of GPX4 cannot be clearly defined. Could the authors provide control group data for comparison in Fig. 8c? It is essential to include control group data to validate the observed effects and ensure that the protective effects attributed to GPX4 overexpression are not confounded by other variables.\n2) The article proposed that GPX4 stabilized BNIP3 through USP20-mediated deubiquitination, but direct evidence for the stability of BNIP3 protein was lacking. Especially in USP20 CKO mice, was BNIP3 unstable? Could the authors provide direct evidence for BNIP3 stability, such as Western blot or ubiquitination assays, to show whether USP20 depletion affected BNIP3 stability? It is also recommended that the authors provided half-life data for BNIP3 to validate the claim that USP20 directly stabilized BNIP3 through deubiquitination.\n3) While the manuscript suggested that GPX4 regulated mitochondrial function through the GPX4-BNIP3-USP20 axis, the direct evidence supporting this conclusion was insufficient. Could the authors provide mitochondrial function data (e.g., Seahorse analysis) to assess mitochondrial respiration in the USP20 CKO mice? And echocardiography data is necessary to assess whether overexpression of GPX4 was completely ineffective in the USP20 CKO group. How can the authors confirm that the observed effect was specifically due to GPX4 overexpression rather than other factors associated with the experimental model? If overexpression of GPX4 still showed a protective effect in USP20 CKO mice, were there other protective mechanisms? Whether this process was associated with ferroptosis?\n4) The experiments in the article mainly focused on the 24 hours and 2 weeks after MI/RI, but there is little data on the long-term impact on cardiac function. Could the authors extend the experimental timeline to 4 weeks or more to assess long-term cardiac function, particularly in relation to fibrosis and heart failure indicators? Has the cardiac protective effects of GPX4 been verified in other animal models, such as TAC?\n5) The article provided some data on the expression of GPX4 in human myocardial tissues, but lacked more clinical data on the relevance of these findings in patients. Could the author provide the correlation between GPX4 levels in the serum or myocardial tissues in myocardial infarction patients and clinical outcomes such as cardiac function, prognosis? This would greatly enhance the clinical relevance of the study.\n6) The manuscript primarily uses scRNA-seq to demonstrate the high expression of GPX4 in cardiomyocytes, but the analyses were relatively superficial. Could the authors use of the scRNA-seq data to explore GPX4 expression across different cell types and its temporal dynamics during MI/RI? Additionally, spatial transcriptomics and proteomics data were mentioned but not fully explored—could these data be more fully utilized to gain a comprehensive understanding of how GPX4 regulated cellular processes at a global level?\n7) The manuscript had several formatting issues that hindered clarity and the effective presentation of data. Some immunofluorescence images only showed the Merged images, without providing single-channel data, which made the co-localization analysis of signals less intuitive. It is recommended to provide single-channel images and images with high-magnification, and to display cell localization. The fluorescence intensity should be quantitatively analyzed. Some statistical data lack description of the statistical methods used. Could the authors provide more detailed explanations of the statistical methods used and n number for each dataset? Furthermore, it is suggested to perform quantitative analysis on WB and IP-WB protein bands to provide more accurate data on protein and ubiquitination changes.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "GPX4 is a key regulator of ferroptosis; however, its role in mitochondrial dysfunction remains incompletely understood. This study aimed to elucidate the mechanistic link between GPX4 and BNIP3-mediated mitophagy in the context of myocardial ischemia/reperfusion injury (MI/RI). While ferroptosis inhibition has been shown to attenuate myocardial injury, the authors observed that cardiomyocyte-specific GPX4 overexpression mitigates mitochondrial dysfunction and myocardial damage through BNIP3-dependent mitophagy. Furthermore, GPX4 was found to enhance the interaction between BNIP3 and USP20, reducing BNIP3 ubiquitination at K131, thereby stabilizing BNIP3 protein levels. While the manuscript is well-written and provides extensive data, it lacks essential controls and provides only incremental conceptual advancement. The precise mechanistic interplay between GPX4, ferroptosis, USP20, and BNIP3-mediated mitophagy in the regulation of MI/RI remains insufficiently defined.\nMajor concerns:\nThe role of GPX4 in ferroptosis is well established. GPX4 downregulation has been observed in myocardial injury, whereas its upregulation has been shown to improve cardiac function, primarily by inhibiting ferroptosis. This study introduces mitochondrial quality control as an additional process regulated by GPX4. However, the manuscript does not address a critical question: whether GPX4 modulates BNIP3-mediated mitophagy independently of its role in ferroptosis. The mechanism by which GPX4 facilitates the interaction between USP20 and BNIP3 remains unclear. Additionally, whether this pathway plays a definitive role in regulating cardiac function in response to MI/RI remains inconclusive.\nLine 124: How were the inhibitor doses determined? What biomarkers were used to confirm their efficacy in the experiments? Appropriate controls should be included.\nLine 144: “Notably, lipid-related signaling pathways were significantly enriched.” In Figure 1a, many of the processes have p-values >0.05. The authors should highlight and list all the statistically significant pathways. Are lipid-related signaling pathways the most significantly enriched?\nFigure 1: “Both mRNA and protein levels of GPX4 were significantly reduced in ischemic myocardial tissues.” What is the transcriptional mechanism regulating GPX4 expression in this context?\nLine 170: The scRNA-Seq analysis, in its current form, does not provide substantial value to the study. A more informative approach would involve comparing control and diseased hearts to elucidate changes in the proportion of GPX4-expressing cells across different cell types.\nLine 190: “Resulting in confirmed overexpression of GPX4 in myocardial tissue.” GPX4 protein levels should be assessed beyond Flag-tag detection to confirm overexpression.\nFigures 2, 3, and 4: The use of RSL3 raises concerns. How was the dosage determined? Given that RSL3 induces ferroptosis and affects multiple pathways, defining the appropriate dose is critical. Essential controls, including sham groups, are missing. Furthermore, RSL3 itself may modulate GPX4 levels, which must be examined. The authors should include data assessing GPX4 levels and ferroptosis markers in the RSL3 experiments to determine whether the observed effects are GPX4- or ferroptosis-dependent. A GPX4 cKO model would provide stronger mechanistic insight.\nFigure 5: Critical controls are missing across multiple experiments.\nLine 261: “Further ultrastructural analysis using transmission electron microscopy (TEM) revealed that MI/RI induced mitochondrial cristae damage, characterized by both loss and swelling of these structures.” How were “loss and swelling” evaluated without an appropriate control? Additionally, does RSL3 itself induce mitochondrial damage independent of H/R stress?\nLine 280: TOM20 levels are the sole indicator of mitophagy in this part. The authors should provide additional data on mitochondrial markers and autophagy induction, including appropriate control conditions.\nMitophagy Assays: “The cardiomyocytes in the GPX4oe + H/R group demonstrated significantly elevated mitophagy levels.” What is the baseline mitophagy level in NRCMs? Does H/R alone alter mitophagy levels? Figure 6e: The panel only shows one representative cell per condition. At least 200 cells should be quantified to ensure statistical robustness. LC3 Staining: Imaging quality needs improvement, and LC3 RFP/GFP quantification should be provided. Figure 6g: If mitophagy is as elevated as shown in Figures 6e and 6f, the authors need to reconcile this finding with the low LAMP1 levels observed in Figure 1g. Does H/R fail to activate mitophagy in WT cells?\nFigure 6i: Critical controls, including siRNA controls and baseline conditions, should be included.\nLine 334: “GPX4 overexpression led to significantly elevated levels of mitochondrial BNIP3 protein and LC3B-II in H/R-treated NRCMs.” If GPX4 stabilizes BNIP3, an increase in BNIP3 levels should also be detectable under baseline conditions.\nHow does RSL3 regulate BNIP3 levels? The authors propose that RSL3 targets GPX4, thereby altering BNIP3 stability. Is this effect ferroptosis-dependent? Does RSL3 affect GPX4 protein levels or enzymatic activity?\nLine 341: “We found that the proteasome inhibitor MG132 effectively inhibited BNIP3 degradation, whereas autophagy inhibitors, such as 3-methyladenine (3-MA), chloroquine (CQ), and bafilomycin A1 (Baf A1), had no effect on GPX4-mediated BNIP3 degradation.” The authors should provide experimental data confirming the effectiveness of these inhibitors.\nFigure 7f: Mitochondrial protein controls and a gCtrl + Flag-GPX4 condition should be included. Additionally, does USP20 regulate HIF-1 levels?\nThe mechanism by which GPX4 mediates the interaction between USP20 and BNIP3 remains unclear. Is this interaction ferroptosis-dependent or directly mediated by GPX4? Is GPX4 enzyme activity required?\nUSP20 knockout should be validated in USP20-cKO cardiac tissue.\nFigure 8: Baseline cardiac function in USP20 cKO mice should be assessed, both under normal conditions and following injury. Additionally, at least a WT + cTnT-GPX4oe + I/R group should be included for comparison.\nHypoxia was set at 0% oxygen, which is extremely low. Is this level necessary to model physiological ischemia, or would a less severe hypoxic condition better reflect in vivo pathophysiology? The authors should justify this choice and provide data confirming hypoxic stress under these conditions.\nMinor:\n• Line 146: Should reference Fig 1b."}, "author_response": "Response to Reviewers\nCardiomyocyte-derived GPX4 stabilizes BNIP3 to facilitate mitophagy and\nmitigate myocardial ischemia/reperfusion injury\nby Zhong et al.\nThe concerns raised by the reviewers have been comprehensively addressed and the\nRevised components are highlighted in red color.\nOverall, we added a substantial amount of new data which was incorporated into the\nfigures. We have summarized the modified figures below in list form for clarity:\n1. Fig. 1a, h-l\n2. Fig. 3a-p\n3. Fig. 4b-k\n4. Fig. 5c-k\n5. Fig. 7f-g, i-k\n6. Fig. 8a-b\n7. Fig. 9a-q\n8. Supplementary Fig. S1b-g\n9. Supplementary Fig. S3a-i\n10. Supplementary Fig. S4b-d\n11. Supplementary Fig. S7a-l\n12. Supplementary Fig. S8a-d\n13. Supplementary Fig. S9a-c\n14. Supplementary Fig. S10a-e\n15. Supplementary Fig. S12b-f\n16. Supplementary Fig. S13a-e\n17. Supplementary Fig. S14b-d\nThese extensive revisions have further supported the key findings from our original\nobservation with more rigor.\nThe specific point-by-point responses are listed below with our responses highlighted\nin blue.\nReviewer #1 (Overview):\nThe authors investigated the role of cardiomyocyte-derived GPX4 in mitigating\nmyocardial ischemia/reperfusion injury (MI/RI) by stabilizing BNIP3 via USP20-\nmediated deubiquitination, thereby enhancing mitophagy and mitochondrial function.\nThey applied several advanced techniques to determine GPX4 function after heart\ninjury, including spatial transcriptomics, proteomics, and single-cell RNA sequencing,\nas well as heart injury models. Although the literature has reported GPX4 function in\nthe heart, this work gains novel insights into the role of GPX4/BNIP3/USP20 axis\nafter MI/RI. With that said, here are some specific concerns we have:\nResponse:\nWe thank the reviewer for the comments. The specific elements of the critique are\naddressed below.\nReviewer #1, Comment 1:\nIn Fig. 1h, the authors classified the heart into three parts: the normal area, the border\narea, and the ischemic area. However, they showed that the myocardium in these three\narea was comparable, why?\nResponse:\nWe thank the reviewer for this thoughtful comment. In the LAD ligation model, the\nischemic area was defined as the apical and anterior wall myocardium distal to the\nligation site (LAD-supplied region, as illustrated by the red boxes in Revised\nSupplementary Fig. S2a), the border zone was defined as the transitional region at\nthe interface between the infarct core and the normally perfused myocardium (as\nillustrated by the orange boxes in Revised Supplementary Fig. S2a), and the normal\narea was defined as tissue remote from the LAD territory (e.g., right ventricle or\nposterior wall, as illustrated by the green boxes in Revised Supplementary Fig. S2a).\nThese definitions are widely used in myocardial ischemia/reperfusion studies and are\nsupported by our long-term experience with this model.\nReviewer #1, Comment 2:\nIt is noted that, in this work, GPX4 expression decreased in the infarction region. Can\nthe authors provide data how GPX4 was regulated after IR or MI?\nResponse:\nWe appreciate the reviewer’s insightful comment. Indeed, ischemic regions are\ncharacterized by cardiomyocyte apoptosis and progressive fibrotic replacement,\nwhich could theoretically contribute to reduced GPX4 levels. To address this, we\nperformed additional analyses:\nTo determine whether GPX4 down-regulation involves active transcriptional\nrepression, we performed a systematic analysis of transcriptional regulation\n(Response Fig. R1-2a). First, we screened potential transcription factors (TFs) from\nthe ENCODE database, filtered them by I/R-related literature, and confirmed their\nexpression in cardiomyocytes using single-cell RNA-seq, which highlighted Atf4,\nSmad4, Sp1, Sp3 as relatively abundant candidates (Response Fig. R1-2b). We next\nused ENCODE ChIP-seq data to assess the binding of these transcription factors\n(TFs) to the GPX4 promoter. The results revealed binding peaks for all of the TFs,\nindicating their direct interaction with the GPX4 promoter (Response Fig. R1-2c).\nAmong these, we focused on SP1 and SP3, as both are critical transcription factors in\nthe heart, with knockout of either SP1 or SP3 leading to spontaneous hypertrophic\ncardiomyopathy1 or embryonic lethality2. To investigate their role in regulating\nGPX4, we silenced Sp1 and Sp3 using siRNA. Knockdown of Sp1 and Sp3\nsignificantly reduced GPX4 mRNA levels (Response Fig. R1-2d). These findings\nsuggest that SP1 and SP3 may play crucial roles in the transcriptional regulation of\nGPX4 in the context of myocardial ischemia/reperfusion injury.\nTaken together, these results indicate that reduced GPX4 expression in ischemic\nregions is not merely a reflection of cardiomyocyte loss, but is also mediated by\ntranscriptional regulation in surviving cardiomyocytes.\nReviewer #1, Comment 3:\nThe authors emphasized that GPX4 expression was specifically down-regulated in the\nischemic region. Given that cardiomyocytes in this area underwent apoptosis and\nwere progressively replaced by fibrotic tissues, could the reduced GPX4 expression\nattribute to the loss of viable cardiomyocytes?\nResponse:\nWe sincerely thank the reviewer for raising this important point. To clarify\nwhether the reduction of GPX4 is merely due to the loss of viable cardiomyocytes, we\nperformed additional analyses.\nFirst, we re-analyzed the scRNA-seq dataset and quantified GPX4 expression\nacross major cardiac cell types, including cardiomyocytes (CMs), endothelial cells\n(ECs), fibroblasts (FBs), macrophages (MPs), smooth muscle cells (SMCs), and T\ncells. Under baseline conditions, GPX4 expression was predominantly detected in\ncardiomyocytes (39.31% in sham). In the I/R group, GPX4 expression was mainly\nobserved in fibroblasts (32.73% in I/R) and cardiomyocytes (27.05% in I/R). These\nfindings suggest that I/R treatment may markedly alter the GPX4 expression\nproportion in cardiomyocytes (Revised Fig. 1k and Revised Supplementary Fig.\nS3c).\nAs you mentioned, these changes in expression may be related to the proportion\nof cardiomyocytes. Therefore, we isolated adult cardiomyocytes from I/R hearts and\nnormalized GPX4 expression to Tnnt2. The results confirmed that GPX4 levels were\nsignificantly decreased after I/R (Revised Fig.1l and Revised Supplementary Fig.\nS3d). In addition, data from our original manuscript showed that, in an in vitro model\nof H/R-induced injury in NRCMs, Gpx4 expression exhibited a progressive decrease\nas the severity of H/R injury increased, after normalization to β-actin (Revised\nFig.1m-n).\nTogether, these results demonstrate that the down-regulation of GPX4 in the\nischemic region cannot be simply explained by cardiomyocyte loss, but rather reflects\na true decrease in GPX4 expression within surviving cardiomyocytes.\nReviewer #1, Comment 4:\nThe size of the BNIP3 proteins varied inconsistently from different Western blot\nexperiments. For example, in Fig. 6c, the main band appeared ~35 kDa, while in Fig.\n6d, it was close to 25 kDa. Overall, the quality of co-IP experiments was low, which\nneed to be improved.\nResponse:\nWe sincerely apologize for the inconsistency observed in our initial BNIP3 co-IP\nresults and appreciate the reviewer’s careful observation. To address this concern, we\noptimized the experimental conditions, including antibody selection and IP\nprocedures, which improved the specificity and quality of the immunoprecipitation.\nWith these refinements, the BNIP3 bands appeared more consistent across\nexperiments. We have repeated the co-IP assays accordingly and replaced the previous\ndata with the updated results in the Revised Fig.5c and 5d.\nReviewer #1, Comment 5:\nThe authors proposed that GPX4 facilitates the interaction between BNIP3 and\nUSP20, thus promoting deubiquitination at the K131 site of BNIP3. However, it will\nbe much improved if the authors can provide data on addressing how GPX4 interacts\nwith BNIP3 and USP20 (since GPX4 function in the heart was reported in the\nliterature).\nResponse:\nWe thank the reviewer for this constructive comment.\nIn our original manuscript, we demonstrated that GPX4 promotes the interaction\nbetween USP20 and BNIP3 (Revised Supplementary Fig. S12a-b).\nTo further confirm this finding, we generated purified recombinant GPX4,\nBNIP3, and USP20 proteins, and performed surface plasmon resonance (SPR) assays to\nquantitatively assess their binding affinities. Specifically, BNIP3 was immobilized on\nthe sensor chip, and USP20 was injected at increasing concentrations (3.125–100\nnM). The measured dissociation constant (KD) was 4.29 × 10⁻⁷ M (Revised Fig. 7i).\nWhen GPX4 was included in the reaction, the KD between USP20 and BNIP3\ndecreased to 1.61 × 10⁻⁸ M, indicating a markedly stronger binding (Revised Fig. 7j).\nSince the KD value is inversely proportional to the binding affinity (i.e., the smaller\nthe KD, the stronger the interaction between two proteins), these results demonstrate\nthat GPX4 significantly enhances the binding between USP20 and BNIP3.\nCollectively, these SPR data corroborate our co-IP results and provide direct\nbiophysical evidence that GPX4 facilitates the formation of the USP20–BNIP3\ncomplex.\nThen, we employed AlphaFold3-based virtual docking combined with molecular\ndynamics (MD) simulations to evaluate the binding of the three proteins. The BNIP3–\nUSP20 dimer exhibited considerable fluctuations, showing a transiently stable phase\nbetween 20 ns and 60 ns but eventually returning to an unstable state (Revised\nSupplementary Fig. S12c). In contrast, the BNIP3–USP20–GPX4 trimer displayed\nonly minor fluctuations around 40 ns and reached a more stable conformation after 60\nns (Revised Supplementary Fig. S12d). Notably, the RMSD values of the trimer\nwere markedly lower compared with the dimer, indicating improved structural\nstability (Revised Supplementary Fig. S12e). These results suggest that GPX4\nstabilizes the interaction between USP20 and BNIP3.\nSubsequently, we co-transfected NIH/3T3 cells with HA-UB, Flag-USP20, His-\nBNIP3, and Flag-GPX4. Co-immunoprecipitation assays confirmed that GPX4 indeed\nenhances the interaction between USP20 and BNIP3, and further reduces the\nubiquitination level of BNIP3 (Revised Fig. 7k).\nTo further elucidate the molecular mechanism by which GPX4 regulates the\nBNIP3–USP20 complex, we employed AlphaFold3 to evaluate the three-\ndimensional conformation of the ternary complex. The predicted binding interface\nincluded the U46 site of GPX4, suggesting a potential role of U46 in facilitating the\ninteraction between USP20 and BNIP3 (Revised Fig. 8a). The U46 residue\nrepresents the critical active site of GPX4, and mutation at this site leads to complete\nloss of enzymatic activity3, 4. Since RSL3 acts by inhibiting the U46 site of GPX44, 5,\nwe first performed IP assays using myocardial tissues subjected to I/R injury with\nRSL3 treatment and found that BNIP3-USP20 interactions were attenuated (Revised\nSupplementary Fig. S13a). Consistently, in NIH/3T3 cells co-transfected with\nGPX4, BNIP3, and USP20, GPX4-enhanced BNIP3-USP20 binding was markedly\nreduced upon RSL3 treatment (Revised Supplementary Fig. S13c). To further\nvalidate this hypothesis, we generated a GPX4 U46A mutant plasmid (lead to loss of\nactivity) and conducted co-IP assays in NIH/3T3 cells. The results demonstrated that\nthe U46A mutation abolished the ability of GPX4 to promote BNIP3-USP20\ninteraction. Moreover, in the U46 mutant background, RSL3 treatment no longer\nexerted additional inhibitory effects (Revised Fig.8b).\nTaken together, these results provide consistent biochemical, computational, and\ncellular evidence that GPX4 functions as a facilitator of the USP20–BNIP3\ninteraction through its U46 site, thereby promoting the deubiquitination of BNIP3 at\nK131.\nReviewer #1, Comment 6:\nThe author made several spelling errors in the text. For example, in line 65,\n\"addtional\" should be \"additional\"; in line 104, \"initialy\" should be \"initially\"; in line\n209, \"theated\" should be \"treated\"; in line 223, \"specfic\" should be \"specific\"; and in\nline 463, \"fingding\" should be \"finding.\". It is important to have the errors corrected\nthroughout the manuscript.\nResponse:\nWe sincerely thank the reviewer for carefully pointing out these spelling errors. We\nhave carefully proofread the entire manuscript and corrected all mistakes mentioned\n(e.g., “addtional” → “additional”, “initialy” → “initially”, “theated” → “treated”,\n“specfic” → “specific”, “fingding” → “finding”), as well as other minor errors\nthroughout the text. The Revised manuscript has been polished to ensure accuracy and\nconsistency.\nReviewer #1, Comment 7:\nAccording to the figure legends, the y-axis in Fig. 3n should be labeled as FS (%).\nResponse:\nWe sincerely thank the reviewer for pointing out this labeling error. In the Revised\nmanuscript, we have corrected the y-axis in Fig. 3n to “FS (%)” so that it is consistent\nwith the figure legend. In addition, we carefully re-examined all figures and legends\nthroughout the manuscript to ensure that axis labels, units, and descriptions are\naccurate and consistent. We believe these corrections improve the overall clarity and\npresentation quality of the figures.\nReviewer #2 (Overview):\nThis manuscript explored the role and molecular mechanisms of GPX4 in MI/RI. The\nauthors identified that GPX4 stabilized BNIP3 via USP20-mediated deubiquitination,\nthereby facilitating mitophagy and diminishing myocardial injury resulting from\nMI/RI. The study was commendably innovative and comprehensive. The current data\nsupported the hypothesis that GPX4 enhanced BNIP3 deubiquitination through\nUSP20, leading to reduced myocardial injury from MI/RI. However, several key data\ngaps and insufficient evidence limited the overall strength of the conclusions. I would\nsuggest the authors to provide additional experimental data or further data analyses to\ndraw firm conclusions. I have the following comments to improve the manuscript.\nResponse:\nWe sincerely thank the reviewer for the positive evaluation of our work and for\nrecognizing its novelty and comprehensiveness. We have carefully considered the\nreviewer’s valuable comments and made systematic revisions and additions\naccordingly. We hope that the Revised version addresses the concerns raised and\nmeets the reviewer’s expectations.\nReviewer #2, Comment 1:\nThe data showed that overexpression of GPX4 in USP20 CKO mice did not\nsignificantly reduce the infarct area following MI/RI surgery, suggesting a key role of\nBNIP3 stability. However, the TTC staining in Fig. 8c without control group data\nmade it impossible to confirm whether GPX4 overexpression reduced the infarct size\nin the control group. As a result, the protective effect of GPX4 cannot be clearly\ndefined. Could the authors provide control group data for comparison in Fig. 8c? It is\nessential to include control group data to validate the observed effects and ensure that\nthe protective effects attributed to GPX4 overexpression are not confounded by other\nvariables.\nResponse:\nWe sincerely thank the reviewer for the valuable suggestion. We fully acknowledge\nthe importance of control data to clarify the protective effect of GPX4 overexpression.\nTo address this concern, we performed additional experiments by including the\ncorresponding control groups.\nSpecifically, we injected AAV9 vectors carrying either EV or GPX4 under a\ncardiac-specific promoter into USP20fl/fl and USP20 CKO mice. Four weeks after\nviral injection, we subjected the mice to MI/RI surgery (45 min ischemia, 24h\nreperfusion) and assessed infarct size and cardiac injury (Revised Fig. 9a). In\nUSP20fl/fl mice, GPX4 overexpression significantly reduced the infarct size compared\nwith the EV group, confirming the protective effect of GPX4 under normal\nconditions. Consistently, GPX4 overexpression also decreased cardiac injury markers\nand TUNEL-positive cell death. However, in USP20 CKO mice, the infarct size was\nmarkedly increased, and GPX4 overexpression failed to rescue the injury, indicating\nthat the protective effect of GPX4 is abolished in the absence of USP20. Similar\ntrends were observed in serum biomarkers and TUNEL staining (Revised Fig. 9b-h).\nTo further strengthen these findings, we established a long-term MI/RI model (45\nmin ischemia, 4 weeks reperfusion) (Revised Fig. 9j). GPX4 overexpression\nimproved cardiac function in USP20fl/fl mice, as evidenced by higher EF and FS\nvalues, whereas USP20 CKO mice exhibited worse cardiac function compared with\ntheir USP20fl/fl counterparts. Interestingly, GPX4 overexpression partially restored\ncardiac function in USP20 CKO mice but failed to reach the level observed in\nUSP20fl/fl + GPX4oe mice (Revised Fig. 9k-m and Revised Supplementary Table\n5). Consistent results were obtained for serum ANP (Revised Fig. 9n) and myocardial\nfibrosis. Histological staining and qPCR analysis of fibrosis-related genes\ndemonstrated reduced fibrotic remodeling in USP20fl/fl + GPX4oe mice, whereas\nUSP20 CKO mice showed more severe fibrosis, and the protective effect of GPX4\nwas insufficient to fully reverse the damage caused by USP20 deficiency (Revised\nFig. 9o-q).\nTaken together, these findings indicated that USP20 is crucial for GPX4-\nmediated regulation of BNIP3, and contributes to mitigating MI/RI. However, we\nobserved a divergence between the acute and chronic phases of MI/RI in USP20-\ndeficient mice. During the acute stage, GPX4 overexpression failed to confer\nprotection against infarct size and cardiomyocyte death in USP20 cKO hearts,\nhighlighting the essential requirement of USP20-mediated BNIP3 stabilization for\nrapid mitophagy and early cardiomyocyte survival. However, at later time points,\nGPX4 overexpression partially ameliorated cardiac dysfunction and adverse\nremodeling despite USP20 deficiency. This discrepancy suggests that GPX4 may\nexert protective effects through USP20-independent mechanisms in the chronic phase,\nsuch as suppression of lipid peroxidation, attenuation of inflammation, or modulation\nof fibrotic remodeling. These findings underscore the phase-specific roles of GPX4,\nwith its acute cardioprotective effects critically dependent on USP20, while its long-\nterm benefits may also involve additional pathways beyond the USP20–BNIP3 axis.\nFuture work should aim to dissect these temporal dynamics and identify\ncomplementary mechanisms that sustain GPX4-mediated protection during post-\nischemic remodeling.\nReviewer #2, Comment 2:\nThe article proposed that GPX4 stabilized BNIP3 through USP20-mediated\ndeubiquitination, but direct evidence for the stability of BNIP3 protein was lacking.\nEspecially in USP20 CKO mice, was BNIP3 unstable? Could the authors provide\ndirect evidence for BNIP3 stability, such as Western blot or ubiquitination assays, to\nshow whether USP20 depletion affected BNIP3 stability? It is also recommended that\nthe authors provided half-life data for BNIP3 to validate the claim that USP20 directly\nstabilized BNIP3 through deubiquitination.\nResponse:\nWe sincerely thank the reviewer for this valuable suggestion. To address this point, we\nperformed additional experiments to directly assess BNIP3 stability.\nIn vivo: We examined BNIP3 protein expression in cardiac tissues from USP20\nCKO and USP20fl/fl mice subjected to I/R injury. The results showed that BNIP3\nprotein levels were significantly reduced in USP20 CKO hearts compared with\nUSP20fl/fl controls, indicating decreased BNIP3 stability upon USP20 deletion\n(Response Fig. R2-2a-b).\nIn vitro half-life assay: Using USP20 knockout (gUsp20) cells and control\n(gCtrl) cells, we treated cultures with cycloheximide (CHX) to block new protein\nsynthesis. BNIP3 protein degradation was markedly accelerated in gUsp20 cells\ncompared with gCtrl cells, demonstrating that USP20 prolongs BNIP3 half-life\n(Response Fig. R2-2c-d).\nTogether, these data provide direct evidence that USP20 depletion decreases BNIP3\nprotein stability both in vivo and in vitro, thereby supporting our conclusion that\nUSP20 stabilizes BNIP3 through deubiquitination.\nReviewer #2, Comment 3:\nWhile the manuscript suggested that GPX4 regulated mitochondrial function through\nthe GPX4-BNIP3-USP20 axis, the direct evidence supporting this conclusion was\ninsufficient. Could the authors provide mitochondrial function data (e.g., Seahorse\nanalysis) to assess mitochondrial respiration in the USP20 CKO mice? And\nechocardiography data is necessary to assess whether overexpression of GPX4 was\ncompletely ineffective in the USP20 CKO group. How can the authors confirm that\nthe observed effect was specifically due to GPX4 overexpression rather than other\nfactors associated with the experimental model? If overexpression of GPX4 still\nshowed a protective effect in USP20 CKO mice, were there other protective\nmechanisms? Whether this process was associated with ferroptosis?\nResponse:\nWe sincerely thank the reviewer for the insightful comments. To address these\nconcerns, we first isolated mitochondria from mouse hearts and performed oxygen\nconsumption rate (OCR) assays to evaluate the impact of GPX4 on mitochondrial\nrespiration in USP20 CKO mice. In addition, we established a 4-week I/R model to\nassess long-term changes in cardiac function by echocardiography.\nFirst, we delivered AAV9 vectors encoding either EV or GPX4 driven by a\ncardiac-specific promoter into USP20fl/fl and USP20 CKO mice. Four weeks later, the\nmice were subjected to MI/RI surgery (45 min ischemia followed by 24 h\nreperfusion). Then, we isolated cardiac mitochondria and oxygen consumption rate\n(OCR) was measured using sequential substrate and inhibitor titrations. Complex I–\nlinked respiration was assessed with pyruvate, malate, and glutamate (PMG),\nfollowed by rotenone to inhibit complex I. Complex II activity was determined by the\naddition of succinate, and complex IV respiration was measured using\nTMPD/ascorbate, followed by sodium azide to block complex IV. As the results\nshown in Revised Supplementary Fig. S14c-d, GPX4 overexpression significantly\npromoted the complex I OXPHOS capacity, enhanced the complex II OCR, and\nincreased the respiration of complex IV (a-b) in USP20fl/fl mice. However, when\ncomparing the EV-IR groups, USP20 CKO mice displayed a marked reduction in\nmultiple OCR parameters relative to USP20fl/fl mice, indicating impaired\nmitochondrial function. Notably, GPX4 overexpression in USP20 CKO mice did not\nreverse these defects, suggesting that the protective effect of GPX4 on mitochondrial\nrespiration depends on the presence of USP20.\nAs consistent with our response to Comment 1, we found that GPX4\noverexpression improved cardiac function in USP20fl/fl mice, as evidenced by higher\nEF and FS values, whereas USP20 CKO mice exhibited worse cardiac function\ncompared with their USP20fl/fl counterparts. Interestingly, GPX4 overexpression\npartially restored cardiac function in USP20 CKO mice but failed to reach the level\nobserved in USP20fl/fl + GPX4oe mice (Revised Fig. 9k-m and Revised\nSupplementary Table 5). These findings demonstrate that USP20 is indispensable\nfor the cardioprotective effects of GPX4 at both the mitochondrial and organ levels.\nAfter adding appropriate control groups, our results showed that in USP20fl/fl mice,\nGPX4 overexpression provided significant protection against both acute and chronic\ninjury compared with EV controls. In contrast, in USP20 CKO mice, no obvious\ndifferences were observed between EV and GPX4oe groups in the setting of acute\ninjury, whereas in the chronic phase, GPX4 overexpression conferred partial\ntherapeutic benefit. First of all, we would like to explain that in all experimental\ngroups, AAV9-cTnT-EV was included as the control to rigorously account for\npotential effects of viral delivery. Therefore, the differences observed between EV and\nGPX4oe groups can be attributed specifically to GPX4 overexpression rather than to\nnonspecific effects of the experimental model.\nWith regard to the observed differences between the acute and chronic phases of\nMI/RI in USP20-deficient mice, our interpretation is as follows: In the acute stage,\nGPX4 overexpression failed to protect against infarct size and cardiomyocyte death in\nUSP20 conditional knockout (CKO) hearts, underscoring the necessity of USP20-\nmediated BNIP3 stabilization for rapid mitophagy and early cardiomyocyte survival.\nAt later time points, however, GPX4 overexpression partially improved cardiac\nfunction and attenuated adverse remodeling despite the absence of USP20. This\ndiscrepancy suggests that GPX4 may exert protective effects through USP20-\nindependent mechanisms during the chronic phase, such as suppression of lipid\nperoxidation, mitigating inflammation, or modulating fibrotic remodeling. In this\ncontext, GPX4-dependent anti-ferroptotic activity likely plays a particularly important\nrole. Collectively, these findings highlight the phase-specific actions of GPX4: its\nacute cardioprotective effects rely heavily on the GPX4-BNIP3-USP20 axis to\nmaintain mitochondrial quality control, whereas its long-term benefits may\nadditionally involve ferroptosis-related pathways beyond this axis.\nReviewer #2, Comment 4:\nThe experiments in the article mainly focused on the 24 hours and 2 weeks after\nMI/RI, but there is little data on the long-term impact on cardiac function. Could the\nauthors extend the experimental timeline to 4 weeks or more to assess long-term\ncardiac function, particularly in relation to fibrosis and heart failure indicators? Has\nthe cardiac protective effects of GPX4 been verified in other animal models, such as\nTAC?\nResponse:\nWe sincerely thank the reviewer for this insightful suggestion.\nTo address this concern, we extended our studies to a long-term\nischemia/reperfusion (I/R) model with 4 weeks of reperfusion. In this setting, mice\nwere pretreated with AAV9 carrying a cardiomyocyte-specific GPX4 overexpression\nconstruct via tail vein injection, followed by MI/RI surgery 4 weeks later (Revised\nFig. 3a). Echocardiographic assessment demonstrated that GPX4 overexpression\nsignificantly preserved cardiac function at 4 weeks post-I/R (Revised Fig. 3b-d) , and\nits overexpression reduced circulating biomarkers (serum ANP) of heart failure\n(Revised Fig. 3e). Consistently, histopathological analysis together with qPCR\nassessment of cardiac fibrosis markers revealed that GPX4 overexpression markedly\nreduced myocardial fibrosis (Revised Fig. 3f-h).\nTo further confirm the long-term protective role of GPX4, we employed a\nchronic myocardial infarction (MI) model with 4 weeks of follow-up, which\nreproduces advanced heart failure and ventricular remodeling, thereby serving as an\nalternative to TAC (Revised Fig. 3i). In this model, cardiomyocyte-specific GPX4\noverexpression significantly improved cardiac performance, attenuated myocardial\nfibrosis, and mitigated maladaptive remodeling (Revised Fig. 3j-p).\nTogether, these additional data provide compelling evidence that GPX4 exerts\nsustained cardioprotective effects during the chronic phase of MI/RI injury. Its\noverexpression not only alleviates cardiac dysfunction in the long term but also\nreduces heart failure progression and structural remodeling following myocardial\ninfarction\nReviewer #2, Comment 5:\nThe article provided some data on the expression of GPX4 in human myocardial\ntissues, but lacked more clinical data on the relevance of these findings in patients.\nCould the author provide the correlation between GPX4 levels in the serum or\nmyocardial tissues in myocardial infarction patients and clinical outcomes such as\ncardiac function, prognosis? This would greatly enhance the clinical relevance of the\nstudy.\nResponse:\nWe thank the reviewer for this important suggestion to strengthen the clinical\nrelevance of our study. In response, we measured circulating GPX4 levels in patients\nwith acute myocardial infarction undergoing PCI (n=70) and compared them with\nindividuals confirmed to be free of coronary artery disease after coronary angiography\n(n=29). The results demonstrated that serum GPX4 levels were significantly reduced\nin myocardial infarction patients (Revised Fig. 1h). Moreover, correlation analysis\nrevealed that lower GPX4 levels were associated with higher GRACE scores,\nsuggesting a link between reduced GPX4 expression and adverse prognosis (Revised\nFig. 1i). While the current analysis was based on a relatively small cohort, these\nfindings provide initial clinical evidence supporting the translational relevance of our\nmechanistic data. We plan to expand patient sample size and integrate cardiac\nfunction parameters and long-term outcomes in future studies to further validate this\nassociation.\nReviewer #2, Comment 6:\nThe manuscript primarily uses scRNA-seq to demonstrate the high expression of\nGPX4 in cardiomyocytes, but the analyses were relatively superficial. Could the\nauthors use of the scRNA-seq data to explore GPX4 expression across different cell\ntypes and its temporal dynamics during MI/RI? Additionally, spatial transcriptomics\nand proteomics data were mentioned but not fully explored—could these data be more\nfully utilized to gain a comprehensive understanding of how GPX4 regulated cellular\nprocesses at a global level?\nResponse:\nWe sincerely thank the reviewer for this valuable suggestion. We agree that the initial\nanalyses were limited, and we have now performed additional in-depth analyses to\naddress these points.\nFirst, we re-analyzed the scRNA-seq dataset and quantified GPX4 expression\ndistribution in major cardiac cell populations, including CMs (cardiomyocytes), ECs\n(endothelial cells) , FBs (fibroblasts), MPs (macrophages), SMCs (smooth muscle\ncell) and T cells. The results confirmed that under baseline conditions, GPX4\nexpression was predominantly detected in cardiomyocytes (39.31% in sham). In the\nI/R group, GPX4 expression was mainly observed in fibroblasts (32.73% in I/R) and\ncardiomyocytes (27.05% in I/R). These findings suggest that I/R treatment may\nmarkedly alter the GPX4 expression proportion in cardiomyocytes (Revised Fig. 1k\nand Revised Supplementary Fig. S3c).\nBased on canonical markers of ischemia, damaged, and remodeling, we first re-\nclassified cardiomyocyte populations from sham and I/R groups (Revised\nSupplementary Fig. S3e). We then performed pseudotime trajectory analysis using\nthe ischemic cluster as the starting point, which generated a branched timeline and\nassociated gene expression maps (Revised Supplementary Fig. S3f-g). Along this\nischemia–damaged–remodeling continuum, Tnnt2, a marker of cardiomyocyte injury,\ninitially decreased and subsequently recovered, whereas Myh7, a marker of\npathological remodeling, progressively increased and peaked during the remodeling\nstage. In contrast, Gpx4 expression was markedly reduced during the ischemic phase\nand did not show a substantial rebound during either the injury or remodeling phases\n(Revised Supplementary Fig. S3h).\nAs for the spatial transcriptomics and proteomics data, we sincerely thank the\nreviewer for this thoughtful suggestion. In our study, the primary purpose of\nincorporating publicly available spatial transcriptomic and proteomic datasets was to\nvalidate the spatial distribution pattern of Gpx4 in myocardial ischemia/reperfusion\ninjury (eg. Revised Fig. 1c-d and Revised Supplementary Fig. S2a-c). This analysis\nconfirmed a clear regional difference in Gpx4 expression, with the lowest levels in\nischemic cores and higher expression in border and remote areas. We agree that\ndeeper expression profiling and integrative analyses would be valuable. However,\ngiven the complexity of these datasets and technical limitations, we were unable to\nfully expand such analyses at this stage. We have now clarified this point in the\nresponse letter and plan to pursue more comprehensive spatial analyses in future\nstudies.\nTogether, these integrated single-cell analyses provide a more comprehensive view of\nGPX4 regulation during the progression of I/R in cardiomyocytes. The new analyses\nand corresponding figures have been incorporated into the Revised manuscript (see\nRevised Fig. 1k, Supplementary Fig. S3c and Supplementary Fig. S3e-h).\nReviewer #2, Comment 7:\nThe manuscript had several formatting issues that hindered clarity and the effective\npresentation of data. Some immunofluorescence images only showed the Merged\nimages, without providing single-channel data, which made the co-localization\nanalysis of signals less intuitive. It is recommended to provide single-channel images\nand images with high-magnification, and to display cell localization. The fluorescence\nintensity should be quantitatively analyzed. Some statistical data lack description of\nthe statistical methods used. Could the authors provide more detailed explanations of\nthe statistical methods used and n number for each dataset? Furthermore, it is\nsuggested to perform quantitative analysis on WB and IP-WB protein bands to\nprovide more accurate data on protein and ubiquitination changes.\nResponse:\nWe sincerely thank the reviewer for these constructive suggestions and apologize for\nthe lack of clarity in the initial submission. We have carefully Revised the manuscript\nand addressed each point as follows:\n1. We have added the corresponding single-channel images, higher-magnification\nviews, and cell localization panels to improve the clarity of signal co-localization.\nIn addition, we have performed fluorescence intensity quantification, and the results are\nnow included in the Revised figures (see Revised Fig. 5f-g; Revised Fig. 5j-k;\nRevised Supplementary Fig. S9a-c; Revised Supplementary Fig. S10a-e).\n2. We have updated the figure legends and Methods section to clearly describe the\nstatistical tests used (e.g., Student’s t-test, one-way or two-way ANOVA), and we\nhave specified the sample size (n) for each dataset.\n3. We have performed densitometric quantification of WB and IP-WB protein bands,\nnormalized to appropriate loading controls, to provide more accurate measurements of\nprotein abundance and ubiquitination. These quantitative data are now incorporated\ninto the Revised manuscript (see Revised Supplementary Fig. S12b,f; and Revised\nSupplementary Fig. S13b,d,e). We also have clarified that all key co-\nimmunoprecipitation experiments were independently replicated at least three times to\nensure reproducibility. The corresponding revisions have been incorporated into the\nfigure legends, with the changes highlighted in red for clarity. However, we\nacknowledge that quantitative analysis of UB or HA-UB bands in the IP experiments\npresents technical limitations, as the diffuse nature of these signals precludes accurate\nmeasurement with ImageJ. Nevertheless, the presented results already allow clear\nvisualization of the differences in UB or HA-UB expression between groups, which\nwe believe adequately support our conclusions. Therefore, quantitative densitometry\nof UB or HA-UB was not performed.\nWe believe these revisions significantly improve the rigor, transparency, and clarity of\ndata presentation in the manuscript.\nReviewer #3 (Overview):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nResponse:\nWe sincerely thank the reviewer for the clarification and for their contribution to the\npeer-review process. We greatly appreciate the time and effort dedicated to evaluating\nour manuscript and providing valuable feedback, which has significantly improved\nthe quality of our work.\nReviewer #4 (Overview):\nGPX4 is a key regulator of ferroptosis; however, its role in mitochondrial dysfunction\nremains incompletely understood. This study aimed to elucidate the mechanistic link\nbetween GPX4 and BNIP3-mediated mitophagy in the context of myocardial\nischemia/reperfusion injury (MI/RI). While ferroptosis inhibition has been shown to\nattenuate myocardial injury, the authors observed that cardiomyocyte-specific GPX4\noverexpression mitigates mitochondrial dysfunction and myocardial damage through\nBNIP3-dependent mitophagy. Furthermore, GPX4 was found to enhance the\ninteraction between BNIP3 and USP20, reducing BNIP3 ubiquitination at K131,\nthereby stabilizing BNIP3 protein levels. While the manuscript is well-written and\nprovides extensive data, it lacks essential controls and provides only incremental\nconceptual advancement. The precise mechanistic interplay between GPX4,\nferroptosis, USP20, and BNIP3-mediated mitophagy in the regulation of MI/RI\nremains insufficiently defined.\nResponse:\nWe thank the reviewer for the comments. The specific elements of the critique are\naddressed below.\nMajor concerns:\nReviewer #4, Comment 1:\nThe role of GPX4 in ferroptosis is well established. GPX4 downregulation has been\nobserved in myocardial injury, whereas its upregulation has been shown to improve\ncardiac function, primarily by inhibiting ferroptosis. This study introduces\nmitochondrial quality control as an additional process regulated by GPX4. However,\nthe manuscript does not address a critical question: whether GPX4 modulates BNIP3-\nmediated mitophagy independently of its role in ferroptosis. The mechanism by which\nGPX4 facilitates the interaction between USP20 and BNIP3 remains unclear.\nAdditionally, whether this pathway plays a definitive role in regulating cardiac\nfunction in response to MI/RI remains inconclusive.\nResponse:\nWe sincerely thank the reviewer for these three critical comments. Below, we provide\ndetailed responses to each of these points individually.\n1. whether GPX4 modulates BNIP3-mediated mitophagy independently of its role in\nferroptosis.\nResponse:\nWe thank the reviewer for raising this important question. To distinguish ferroptosis-\ndependent from ferroptosis-independent effects, we first inhibited ferroptosis and then\nexamined the impact of GPX4 on BNIP3-mediated mitophagy. Specifically, we used\nliproxstatin-1 (Lip-1), a well-characterized ferroptosis inhibitor that functions as a\nradical-trapping antioxidant, suppressing lipid peroxidation by scavenging lipid\nperoxyl radicals independently of GPX4 enzymatic activity6, 7. Subsequently, we\noverexpressed GPX4 in cardiomyocytes and assessed cell viability as well as LDH\nrelease following hypoxia/reoxygenation injury. These experiments allowed us to\ndetermine whether GPX4 supplementation could further improve cardiomyocyte\ninjury even under ferroptosis-suppressed conditions.\nThe results showed that either GPX4 overexpression or Lip-1 treatment improved\ncardiomyocyte viability and reduced LDH release associated with cell death.\nImportantly, when GPX4 was overexpressed in the presence of Lip-1, cardiomyocyte\ninjury induced by hypoxia/reoxygenation was further attenuated (Response Fig. R4-\n1-1a-b),\nindicating that GPX4 confers additional protection beyond ferroptosis\ninhibition.\nTogether, these findings support that GPX4 promotes BNIP3-mediated mitophagy in a\nmanner that is at least partly independent of its ferroptosis-regulatory activity.\n2. The mechanism by which GPX4 facilitates the interaction between USP20 and\nBNIP3 remains unclear.\nResponse:\nWe thank the reviewer for this constructive comment.\nIn our original manuscript, we demonstrated that GPX4 promotes the interaction\nbetween USP20 and BNIP3 (Revised Supplementary Fig. S12a-b).\nTo further confirm this finding, we generated purified recombinant GPX4,\nBNIP3, and USP20 proteins, and performed surface plasmon resonance (SPR) assays\nto quantitatively assess their binding affinities. Specifically, BNIP3 was immobilized\non the sensor chip, and USP20 was injected at increasing concentrations (3.125-100\nnM). The measured dissociation constant (KD) was 4.29 × 10-⁷ M (Revised Fig. 7i).\nWhen GPX4 was included in the reaction, the KD between USP20 and BNIP3\ndecreased to 1.61 × 10-⁸ M, indicating a markedly stronger binding (Revised Fig. 7j).\nSince the KD value is inversely proportional to the binding affinity (i.e., the smaller\nthe KD, the stronger the interaction between two proteins), these results demonstrate\nthat GPX4 significantly enhances the binding between USP20 and BNIP3.\nCollectively, these SPR data corroborate our co-IP results and provide direct\nbiophysical evidence that GPX4 facilitates the formation of the USP20-BNIP3\ncomplex.\nSubsequently, we employed AlphaFold3-based virtual docking combined with\nmolecular dynamics (MD) simulations to evaluate the binding of the three proteins.\nThe BNIP3-USP20 dimer exhibited considerable fluctuations, showing a transiently\nstable phase between 20 ns and 60 ns but eventually returning to an unstable state\n(Revised Supplementary Fig. S12c). In contrast, the BNIP3-USP20-GPX4 trimer\ndisplayed only minor fluctuations around 40 ns and reached a more stable\nconformation after 60 ns (Revised Supplementary Fig. S12d). Notably, the RMSD\nvalues of the trimer were markedly lower compared with the dimer, indicating\nimproved structural stability (Revised Supplementary Fig. S12e). These results\nsuggest that GPX4 stabilizes the interaction between USP20 and BNIP3.\nSubsequently, we co-transfected NIH/3T3 cells with HA-UB, Flag-USP20,\nHis-BNIP3, and Flag-GPX4. Co-immunoprecipitation assays confirmed that GPX4 indeed\nenhances the interaction between USP20 and BNIP3, and further reduces the\nubiquitination level of BNIP3 (Revised Fig. 7k).\nTo further elucidate the molecular mechanism by which GPX4 regulates the\nBNIP3–USP20 complex, we employed AlphaFold3 to evaluate the three-\ndimensional conformation of the ternary complex. The predicted binding interface\nincluded the U46 site of GPX4, suggesting a potential role of U46 in facilitating the\ninteraction between USP20 and BNIP3 (Revised Fig. 8a). The U46 residue\nrepresents the critical active site of GPX4, and mutation at this site leads to complete\nloss of enzymatic activity3, 4. Since RSL3 acts by inhibiting the U46 site of GPX44, 5,\nwe first performed IP assays using myocardial tissues subjected to I/R injury with\nRSL3 treatment and found that BNIP3-USP20 interactions were attenuated (Revised\nSupplementary Fig. S13a). Consistently, in NIH/3T3 cells co-transfected with\nGPX4, BNIP3, and USP20, GPX4-enhanced BNIP3-USP20 binding was markedly\nreduced upon RSL3 treatment (Revised Supplementary Fig. S13c). To further\nvalidate this hypothesis, we generated a GPX4 U46A mutant plasmid (lead to loss of\nactivity) and conducted co-IP assays in NIH/3T3 cells. The results demonstrated that\nthe U46A mutation abolished the ability of GPX4 to promote BNIP3-USP20\ninteraction. Moreover, in the U46 mutant background, RSL3 treatment no longer\nexerted additional inhibitory effects (Revised Fig.8b).\nTaken together, these results provide consistent biochemical, computational, and\ncellular evidence that GPX4 functions as a facilitator of the USP20-BNIP3 interaction\nthrough its U46 site, thereby promoting the deubiquitination of BNIP3 at K131.\n3. Additionally, whether this pathway plays a definitive role in regulating cardiac\nfunction in response to MI/RI remains inconclusive.\nResponse:\nWe sincerely thank the reviewer for the valuable suggestion. As noted in your\nComment 19, we have added USP20fl/fl mice with EV and GPX4oe as control groups\nto the experiments involving the USP20 CKO model in the original manuscript\n(Revised Fig. 9). In this Revised analysis, we obtained the following findings:\nSpecifically, we injected AAV9 vectors carrying either EV or GPX4 under a\ncardiac-specific promoter into USP20fl/fl and USP20 CKO mice. Four weeks after\nviral delivery, the mice were subjected to MI/RI surgery (45 min ischemia, 24 h\nreperfusion) and evaluated for infarct size and cardiac injury (Revised Fig. 9a).\nIn USP20fl/fl mice, GPX4 overexpression significantly reduced infarct size compared\nwith the EV group, confirming its protective effect under normal conditions.\nConsistently, GPX4 overexpression also lowered cardiac injury markers and TUNEL-\npositive cell death. By contrast, in USP20 CKO mice, infarct size was markedly\nincreased, and GPX4 overexpression failed to provide protection, indicating that the\ncardioprotective effect of GPX4 is abolished in the absence of USP20. Similar\npatterns were observed for serum biomarkers and TUNEL staining (Revised Fig. 9b-\nh).\nTo further substantiate these findings, we employed a long-term MI/RI model\n(45 min ischemia, 4 weeks reperfusion) (Revised Fig. 9j). In USP20fl/fl mice, GPX4\noverexpression markedly improved cardiac function, as reflected by higher EF and FS\nvalues. In contrast, USP20 CKO mice exhibited more severe cardiac dysfunction than\ntheir USP20fl/fl counterparts. Interestingly, GPX4 overexpression partially restored\ncardiac function in USP20 CKO mice, although the improvement did not reach the\nlevel observed in USP20fl/fl + GPX4oe mice (Revised Fig. 9k–m and Revised\nSupplementary Table 5). Consistent trends were seen for serum ANP (Revised Fig.\n9n) and myocardial fibrosis. Histological staining and qPCR of fibrosis-related genes\nfurther demonstrated reduced fibrotic remodeling in USP20fl/fl + GPX4oe mice,\nwhereas USP20 CKO mice displayed more extensive fibrosis, and the protective\neffect of GPX4 was insufficient to fully counteract the detrimental impact of USP20\ndeficiency (Revised Fig. 9o–q).\nIn our Revised study (Revised Fig. 9), we analyzed both the acute (24 h\nreperfusion) and chronic (4 weeks reperfusion) phases of MI/RI in USP20fl/fl and\nUSP20 CKO mice with or without GPX4 overexpression. Taken together, our results\nrevealed a clear divergence between the acute and chronic settings. During the acute\nphase, GPX4 overexpression failed to reduce infarct size or cardiomyocyte death in\nUSP20 CKO hearts, highlighting the essential requirement of USP20-mediated\nBNIP3 stabilization for rapid mitophagy and early cell survival. However, at later\nstages, GPX4 overexpression partially improved cardiac function and attenuated\nadverse remodeling despite the absence of USP20. These findings suggest that while\nthe acute cardioprotective effect of GPX4 is critically dependent on the USP20–\nBNIP3 axis, GPX4 may also exert USP20-independent effects in the chronic phase,\nlikely through suppression of lipid peroxidation, reduction of inflammation, or\nmodulation of fibrotic remodeling.\nTherefore, in conjunction with the results shown in Response Fig. R4-1, our\ndata demonstrate that GPX4 exerts functions independent of the ferroptosis pathway,\nand that the GPX4-USP20-BNIP3 axis plays an indispensable role in preserving\ncardiac function both during the acute injury response and throughout the chronic\nremodeling phase.\nIn conclusion, we here present a summary in response to the three key\nquestions outlined above：\nOur additional results not only extend the canonical understanding of GPX4 as a\nlipid peroxidation scavenger within the ferroptosis pathway but also uncover an\nunexpected role in regulating protein–protein interactions. Specifically, we identified\nthat GPX4 engages its enzymatic active site (U46) to influence the USP20–BNIP3\ncomplex, thereby linking GPX4 activity to mitochondrial quality control. This dual\nfunctionality highlights GPX4 as both a critical suppressor of ferroptotic stress and a\nmodulator of mitophagy signaling. Collectively, these findings establish a broader\nmolecular framework in which GPX4 exerts cardioprotective effects through\nferroptosis-dependent and -independent mechanisms, underscoring its indispensable\nrole in preserving cardiac function during both the acute injury response and the\nchronic remodeling phase of MI/RI. Future studies will further dissect how these\nparallel pathways are integrated and how they may be leveraged for therapeutic\nintervention.\nReviewer #4, Comment 2:\nLine 124: How were the inhibitor doses determined? What biomarkers were used to\nconfirm their efficacy in the experiments? Appropriate controls should be included.\nResponse:\nWe thank the reviewer for raising this important point.\nIn our study, the doses of the inhibitors (3-Methyladenine [3-MA, 20 mg/kg],\nNecrostatin-1 [Nec-1, 1mg/kg], Emricasan [Emr, 2.5mg/kg], and Ferrostatin-1 [Fer-1,\n1mg/kg]) were determined based on previously published literature in cardiac\nischemia/reperfusion models8. To evaluate their efficacy, we assessed classical\nbiomarkers associated with the respective cell death pathways, including LC3B-I/II\nand P62 for autophagy (3-MA), cleaved caspase-3, Bax, Bcl-2 for apoptosis (z-VAD-\nFMK), p-RIPK1, p-RIPK3 and p-MLKL activation for necroptosis (Nec-1), and\nGPX4 and SLC7A11 for ferroptosis (Fer-1). Both in vivo (cardiac tissue) and in vitro\n(cardiomyocyte) Western blot analyses confirmed that these biomarkers were\neffectively suppressed by the corresponding inhibitors, demonstrating that all four\nforms of cell death were successfully attenuated under the respective treatments\n(Response Fig. R4-2 a-d for in vivo, e-h for in vitro).\nIn the animal experiments, sham-operated mice treated with vehicle were used as\ncontrols to ensure rigorous experimental design. Consistent with the conclusions of\nthe original manuscript, we found that among the four inhibitors tested, Fer-1 exerted\nthe most pronounced protective effect against myocardial ischemia/reperfusion injury\nboth in vivo and in vitro. The corrected data have now been included in the Revised\nmanuscript, replacing the previous version (see Revised Supplementary Fig. S1a-g).\nReviewer #4, Comment 3:\nLine 144: “Notably, lipid-related signaling pathways were significantly enriched.” In\nFigure 1a, many of the processes have p-values >0.05. The authors should highlight\nand list all the statistically significant pathways. Are lipid-related signaling pathways\nthe most significantly enriched?\nResponse:\nWe sincerely thank the reviewer for this important comment and apologize for our\noversight in the initial submission. To address this issue, we re-analyzed the\ntranscriptomic sequencing data and now provide the top 30 enriched pathways, as\nshown in Response Fig. R4-3a. Among these, nine pathways were lipid-related and\none pathway was associated with the inhibition of ferroptosis, as illustrated in\nRevised Fig. 1a. The Revised analysis replace the previous version in the manuscript,\nensuring that only statistically significant pathways are highlighted and properly\nreported.\nReviewer #4, Comment 4:\nFigure 1: “Both mRNA and protein levels of GPX4 were significantly reduced in\nischemic myocardial tissues.” What is the transcriptional mechanism regulating GPX4\nexpression in this context?\nResponse:\nWe appreciate the reviewer’s insightful comment. Indeed, ischemic regions are\ncharacterized by cardiomyocyte apoptosis and progressive fibrotic replacement,\nwhich could theoretically contribute to reduced GPX4 levels. To address this, we\nperformed additional analyses:\nTo determine whether GPX4 down-regulation involves active transcriptional\nrepression, we performed a systematic analysis of transcriptional regulation\n(Response Fig. R1-2a). First, we screened potential transcription factors (TFs) from\nthe ENCODE database, filtered them by I/R-related literature, and confirmed their\nexpression in cardiomyocytes using single-cell RNA-seq, which highlighted Atf4,\nSmad4, Sp1, Sp3 as relatively abundant candidates (Response Fig. R1-2b). We next\nused ENCODE ChIP-seq data to assess the binding of these transcription factors\n(TFs) to the GPX4 promoter. The results revealed binding peaks for all of the TFs,\nindicating their direct interaction with the GPX4 promoter (Response Fig. R1-2c).\nAmong these, we focused on SP1 and SP3, as both are critical transcription factors in\nthe heart, with knockout of either SP1 or SP3 leading to spontaneous hypertrophic\ncardiomyopathy1 or embryonic lethality2. To investigate their role in regulating\nGPX4, we silenced Sp1 and Sp3 using siRNA. Knockdown of Sp1 and Sp3\nsignificantly reduced GPX4 mRNA levels (Response Fig. R1-2d). These findings\nsuggest that SP1 and SP3 may play crucial roles in the transcriptional regulation of\nGPX4 in the context of myocardial ischemia/reperfusion injury.\nTaken together, these results indicate that reduced GPX4 expression in ischemic\nregions is not merely a reflection of cardiomyocyte loss, but is also mediated by\ntranscriptional regulation in surviving cardiomyocytes.\nReviewer #4, Comment 5:\nLine 170: The scRNA-Seq analysis, in its current form, does not provide substantial\nvalue to the study. A more informative approach would involve comparing control and\ndiseased hearts to elucidate changes in the proportion of GPX4-expressing cells\nacross different cell types.\nResponse:\nWe sincerely thank the reviewer for this valuable suggestion. We agree that the initial\nanalyses were limited, and we have now conducted more comprehensive\ninvestigations to address your concerns. Specifically, we re-analyzed the scRNA-seq\ndataset and quantified GPX4 expression distribution in major cardiac cell populations,\nincluding CMs (cardiomyocytes), ECs (endothelial cells) , FBs (fibroblasts),\nMPs (macrophages), SMCs (smooth muscle cell) and T cells. The results confirmed that\nunder baseline conditions, GPX4 expression was predominantly detected in\ncardiomyocytes (39.31% in sham). In the I/R group, GPX4 expression was mainly\nobserved in fibroblasts (32.73% in I/R) and cardiomyocytes (27.05% in I/R). These\nfindings suggest that I/R treatment may markedly alter the GPX4 expression\nproportion in cardiomyocytes (Revised Fig. 1k and Revised Supplementary Fig.\nS3c).\nReviewer #4, Comment 6:\nLine 190: “Resulting in confirmed overexpression of GPX4 in myocardial tissue.”\nGPX4 protein levels should be assessed beyond Flag-tag detection to confirm\noverexpression.\nResponse:\nWe sincerely thank the reviewer for this helpful suggestion. In addition to Flag-tag\ndetection, we have now directly assessed GPX4 protein expression in myocardial\ntissue by Western blot (Revised Supplementary Fig. S4c-d). The results confirmed\nsuccessful overexpression of GPX4, and the corresponding protein bands have been\nincluded in the Revised manuscript (see Revised Supplementary Fig. S4c-d).\nReviewer #4, Comment 7:\nFigures 2, 3, and 4: The use of RSL3 raises concerns. How was the dosage\ndetermined? Given that RSL3 induces ferroptosis and affects multiple pathways,\ndefining the appropriate dose is critical. Essential controls, including sham groups, are\nmissing. Furthermore, RSL3 itself may modulate GPX4 levels, which must be\nexamined. The authors should include data assessing GPX4 levels and ferroptosis\nmarkers in the RSL3 experiments to determine whether the observed effects are\nGPX4- or ferroptosis-dependent. A GPX4 cKO model would provide stronger\nmechanistic insight.\nResponse:\nWe thank the reviewer for raising this important concern.\nThe concentration of RSL3 used in our study was selected based on previously\npublished reports (30mg/kg in vivo9, 10 and 100nM in vitro11, 12), and in our hands this\ndose consistently produced the expected biological effects, as reflected in the data\npresented.\nIn vivo, mice treated with RSL3 alone showed reduced GPX4 protein levels in cardiac\ntissues, as confirmed by Western blot analysis4, 13, 14.\nImportantly, our key in vivo findings (Revised Fig. 9, also detailed in our response to\nComment 1) demonstrated that the GPX4-BNIP3-USP20 axis protects cardiac\nfunction through the regulation of mitophagy in a manner that is independent of\nferroptosis. In line with this, our mechanistic studies further showed that RSL3\ndirectly targets the catalytic U46 residue of GPX4, thereby disrupting the GPX4-\nBNIP3-USP20 interaction. These results indicate that the inhibitory effect of RSL3 on\nmitophagy cannot be solely attributed to ferroptosis induction, but rather reflects a\ndirect interference with the GPX4-USP20-BNIP3 complex. Together, these data\nclarify that while RSL3 is classically recognized as a ferroptosis inducer, its impact in\nour study highlights an additional, ferroptosis-independent mechanism, namely the\ndisruption of GPX4-mediated stabilization of BNIP3 by USP20.\nWe fully agree that a cardiomyocyte-specific GPX4 CKO model would provide\nstronger mechanistic evidence. However, global GPX4 knockout is known to cause\nembryonic lethality, as previously reported15, and our own prior attempts were also\nunsuccessful due to early lethality. Establishing a cardiomyocyte-specific GPX4 CKO\nmodel would require substantial time and resources that are currently beyond the\nscope of this study. We sincerely apologize for this limitation. Nevertheless, to\naddress this question within the feasibility of our current system, we employed\ncomplementary approaches, including cardiomyocyte-specific GPX4 overexpression\nvia AAV9-cTnT vectors and the use of the GPX4 inhibitor RSL3. Despite the absence\nof genetic knockout data, our in vivo and in vitro results consistently demonstrated\nthat GPX4 confers protection against myocardial I/R injury, and further revealed that\nthe GPX4-BNIP3-USP20 complex plays an important role in maintaining\nmitochondrial integrity under these conditions. In future studies, we plan to establish\ncardiomyocyte-specific GPX4 CKO models, which will allow us to further validate\nand strengthen the mechanistic conclusions.\nReviewer #4, Comment 8:\nFigure 5: Critical controls are missing across multiple experiments.\nResponse:\nWe sincerely thank the reviewer for raising this critical point. We agree that the\nabsence of appropriate controls limited the objective evaluation of GPX4\noverexpression and RSL3 treatment on mitochondrial function in our initial\nsubmission. To address this concern, we have now included the missing control\ngroups and repeated the experiments. The newly generated data are provided in the\nRevised manuscript (Revised Fig. 4a-k and Revised Supplementary Fig. S7a-l).\nComprehensive assessments—including OCR measurements, ultrastructural\nevaluation by electron microscopy, ATP production, mitochondrial complex I activity,\nas well as JC-1 and DHE assays—demonstrated that GPX4 overexpression\nsignificantly ameliorated mitochondrial structural damage and functional impairment\ninduced by myocardial I/R injury, both in vivo and in vitro, consistent with the\nconclusions of our original manuscript. Furthermore, RSL3 treatment aggravated\nmitochondrial dysfunction and ultrastructural abnormalities following I/R injury.\nInterestingly, we also observed that RSL3 alone, even in the absence of I/R, was\nsufficient to induce mitochondrial dysfunction and structural damage.\nReviewer #4, Comment 9:\nLine 261: “Further ultrastructural analysis using transmission electron microscopy\n(TEM) revealed that MI/RI induced mitochondrial cristae damage, characterized by\nboth loss and swelling of these structures.” How were “loss and swelling” evaluated\nwithout an appropriate control? Additionally, does RSL3 itself induce mitochondrial\ndamage independent of H/R stress?\nResponse:\nWe thank the reviewer for this insightful comment. In our original submission, the\ndescription of mitochondrial damage assessment was not sufficiently detailed. In the\nRevised manuscript, we have included appropriate sham-operated controls for\ncomparison. Based on the approach described by Lai et al., mitochondrial\nultrastructural integrity was evaluated by quantifying cristae length per mitochondrion\non TEM images using ImageJ. At least 20 mitochondria per sample were analyzed to\nensure statistical robustness. These new data and detailed methods have been\nincorporated into the Revised manuscript (see Revised Fig. 4h-k and Revised\nSupplementary Fig. S7i-l).\nRegarding the effect of RSL3 itself on cardiomyocyte mitochondria, we performed\nadditional experiments to evaluate mitochondrial morphology and function both in\nvivo and in vitro. TEM analysis revealed that RSL3 treatment alone induced\nmitochondrial structural abnormalities, while functional assays (ATP production,\nOCR, and mitochondrial membrane potential) confirmed impaired mitochondrial\nperformance. These results demonstrate that RSL3 itself can affect mitochondrial\nintegrity independent of I/R stress. These new data have been incorporated into the\nRevised manuscript (Revised Supplementary Fig. 7a-l).\nReviewer #4, Comment 10:\nLine 280: TOM20 levels are the sole indicator of mitophagy in this part. The authors\nshould provide additional data on mitochondrial markers and autophagy induction,\nincluding appropriate control conditions.\nResponse:\nWe sincerely thank the reviewer for this helpful suggestion. To address this concern,\nwe have now included appropriate control groups (EV+Normaxia, GPX4oe+Normaxia\nand Veh+Normaxia, RSL3+Normaxia) and expanded our assessment beyond TOM20\nalone. In addition to TOM20 degradation, we examined mitochondrial dynamics-\nrelated proteins (FIS1, DRP1, and p-DRP1) to provide supporting evidence for\nmitochondrial turnover. While these markers primarily reflect mitochondrial fission,\ntheir changes are closely associated with mitophagy initiation. The increased\nexpression of p-Drp1/Drp1 and Fis1 suggests enhanced mitochondrial fission. Since\nmitochondrial fission is generally considered a prerequisite for mitophagy, these\nmarkers indirectly indicate an increase in mitophagic activity16. Furthermore, we also\nconfirmed the efficiency of GPX4 overexpression and the inhibitory effect of RSL3\non GPX4 in cardiomyocytes. These new data have been incorporated into the Revised\nmanuscript (Revised Supplementary Fig. S8a-d).\nReviewer #4, Comment 11:\nMitophagy Assays: “The cardiomyocytes in the GPX4oe + H/R group demonstrated\nsignificantly elevated mitophagy levels.” What is the baseline mitophagy level in\nNRCMs? Does H/R alone alter mitophagy levels? Figure 6e: The panel only shows\none representative cell per condition. At least 200 cells should be quantified to ensure\nstatistical robustness. LC3 Staining: Imaging quality needs improvement, and LC3\nRFP/GFP quantification should be provided. Figure 6g: If mitophagy is as elevated as\nshown in Figures 6e and 6f, the authors need to reconcile this finding with the low\nLAMP1 levels observed in Figure 1g. Does H/R fail to activate mitophagy in WT\ncells?\nResponse:\nWe sincerely thank the reviewer for these constructive comments. To address these\nconcerns:\nBaseline and H/R-induced mitophagy: We have now included normoxia controls. At\nbaseline, NRCMs exhibited a low but detectable level of mitophagy. H/R treatment\nalone modestly increased mitophagy compared with sham conditions, whereas GPX4\noverexpression further enhanced mitophagy flux under H/R, consistent with its\nprotective role. In contrast, RSL3 treatment partially suppressed mitophagy (Revised\nFig. 5f, 5j; Revised Supplementary Fig. S10a).\nImage presentation: To improve transparency, we now provide images of all\nindividual channels in addition to merged panels for more straightforward evaluation\n(Revised Supplementary Fig. S9a; Revised Supplementary Fig. S10e).\nCell quantification: For quantification, we analyzed 50 cells per group and found that\nstatistical significance was already clearly established at this level, ensuring the\nreliability of our conclusions (Revised Fig. 5f, 5j and Revised Supplementary Fig.\nS10b).\nLC3 Staining: While LC3B-RFP/GFP is a widely used autophagy reporter, it does not\nspecifically distinguish mitophagy. We therefore placed greater emphasis on\nLAMP1/TOMM20 colocalization, which directly reflects mitochondrial\nautophagosome–lysosome fusion and provides a more accurate assessment of\nmitophagy. For this reason, we decided not to include LC3B-RFP/GFP quantification\nin the Revised manuscript.\nLAMP1/TOMM20 Staining: In the Revised manuscript, we have optimized the\nstaining conditions and now provide higher-quality images of LAMP1 and TOMM20\nfor clearer evaluation. The new data demonstrate that H/R indeed activates mitophagy\nin cardiomyocytes, as evidenced by increased LAMP1/TOMM20 colocalization.\nMoreover, GPX4 overexpression enhanced LAMP1 expression and further increased\nLAMP1–TOMM20 colocalization, whereas RSL3 treatment showed the opposite\neffect. To ensure the robustness of these findings, we also quantified fluorescence\nsignals from 20 cells per group, and the results confirmed the trends observed in the\nrepresentative images (Revised Supplementary Fig. S9b-c; Revised\nSupplementary Fig. S9c-d).\nTogether, these revisions clarify baseline and H/R-induced mitophagy, provide\nappropriate controls and quantification, and reconcile our findings across different\nassays.\nReviewer #4, Comment 12:\nFigure 6i: Critical controls, including siRNA controls and baseline conditions, should\nbe included.\nResponse:\nWe thank the reviewer for this valuable suggestion. In the Revised manuscript, we\nhave included the appropriate negative control siRNAs and baseline (normoxia)\nconditions to ensure the specificity and reliability of our findings. The experimental\ngroups were designed as follows: si-NC + EV + Normoxia, si-Bnip3 + EV +\nNormoxia, si-NC + EV + H/R, si-Bnip3 + EV + H/R, and si-Bnip3 + GPX4oe + H/R.\nUnder optimized experimental conditions, we also provide higher-quality images,\nincluding single-channel images for clearer evaluation. In addition, mitophagy was\nquantified in 50 cells per group to ensure statistical robustness (see Revised Fig. 5j-k\nand Revised Supplementary Fig. S10e).\nThe results demonstrated that mitophagy levels remained low under normoxia.\nCompared with the si-NC + EV + H/R group, BNIP3 silencing markedly reduced\nmitophagy, and importantly, BNIP3 depletion abolished the GPX4 overexpression-\ninduced increase in mitophagy in NRCMs. These new data further support our\nconclusion that BNIP3 is required for GPX4-mediated regulation of mitophagy under\nH/R stress.\nReviewer #4, Comment 13:\nLine 334: “GPX4 overexpression led to significantly elevated levels of mitochondrial\nBNIP3 protein and LC3B-II in H/R-treated NRCMs.” If GPX4 stabilizes BNIP3, an\nincrease in BNIP3 levels should also be detectable under baseline conditions.\nResponse:\nWe appreciate the reviewer’s insightful comment. To address this, we added\nnormoxia control groups (EV + Normaxia and GPX4oe + Normaxia) and performed\nWestern blot analysis of mitochondrial fractions across all conditions. The results\nshowed that GPX4 overexpression increased BNIP3 levels under H/R conditions, but\nno such increase was observed under normoxia (Revised Fig. 5h-i). This finding is\nconsistent with the fact that BNIP3 expression is intrinsically very low under baseline\nconditions and is markedly upregulated in response to hypoxic or ischemic stress.\nTherefore, the stabilizing effect of GPX4 on BNIP3 becomes apparent only when\nBNIP3 is induced under H/R, where USP20-mediated deubiquitination prevents its\ndegradation.\nReviewer #4, Comment 14:\nHow does RSL3 regulate BNIP3 levels? The authors propose that RSL3 targets\nGPX4, thereby altering BNIP3 stability. Is this effect ferroptosis-dependent? Does\nRSL3 affect GPX4 protein levels or enzymatic activity?\nResponse:\nWe thank the reviewer for these important questions. Below, we provide detailed\nresponses to each of these points individually.\n1. How does RSL3 regulate BNIP3 levels?\nResponse: As shown by the comparison between lanes 1 and 2 in the Western blot\nresults of Fig. 6a, the presence of RSL3 promotes BNIP3 degradation (Revised Fig.\n6a-b).\n2. Is this effect ferroptosis-dependent? Does RSL3 affect GPX4 protein levels or\nenzymatic activity?\nResponse: As the data shown in the Response Fig. R4-14 a-b that, both GPX4\noverexpression and Lip-1 treatment improved cardiomyocyte viability and reduced\nLDH release under H/R conditions. Notably, GPX4 overexpression in the presence of\nLip-1 provided additional protection, indicating that GPX4 acts beyond ferroptosis\ninhibition. These findings support the conclusion that GPX4 promotes BNIP3-USP20\nmediated mitophagy at least partly independently of its role in ferroptosis regulation.\nRSL3 is recognized as an irreversible covalent inhibitor that alkylates the catalytic\nU46 residue of GPX4 via its chloroacetamide electrophile. This modification blocks\nthe GSH-dependent reduction cycle, leading to the accumulation of phospholipid\nhydroperoxides and rapid induction of ferroptosis4, 5. In MI/RI myocardial tissues\ntreated with RSL3, we observed that RSL3 suppressed the interaction between USP20\nand BNIP3 (Revised Supplementary Fig. S13a). Consistently, in 3T3 cells, RSL3\nalso inhibited the interaction among GPX4, USP20, and BNIP3 (Revised\nSupplementary Fig. S13c). To further confirm the role of GPX4-U46, we generated a\nGPX4 U46 mutant plasmid that disrupts the RSL3 binding site. Co-IP results showed\nthat RSL3 reduced the USP20–BNIP3 interaction mediated by wild-type GPX4 (lane\n2 vs. lane 4 in Revised Fig. 8b), whereas this inhibitory effect was abolished in the\nGPX4-U46 mutant (lane 3 vs. lane 5 in Revised Fig. 8b). Together, these findings\ndemonstrate that the U46 residue of GPX4 is a critical site for regulating the USP20–\nBNIP3 interaction, and that RSL3 disrupts the GPX4–BNIP3–USP20 complex\nthrough targeting this catalytic site.\nReviewer #4, Comment 15:\nLine 341: “We found that the proteasome inhibitor MG132 effectively inhibited\nBNIP3 degradation, whereas autophagy inhibitors, such as 3-methyladenine (3-MA),\nchloroquine (CQ), and bafilomycin A1 (Baf A1), had no effect on GPX4-mediated\nBNIP3 degradation.” The authors should provide experimental data confirming the\neffectiveness of these inhibitors.\nResponse:\nWe thank the reviewer for this helpful suggestion. To confirm the effectiveness of the\nautophagy inhibitors, we applied 3-MA, CQ, and Baf A1 at the concentrations used in\nour experiments and evaluated their activity by monitoring LC3B-I/II conversion\nusing Western blot. The results demonstrated that all three inhibitors exerted their\nexpected effects on autophagy under these conditions, confirming that the lack of\nimpact on GPX4-mediated BNIP3 degradation was not due to ineffective inhibitor\nactivity (Response Fig. 4-15a-b).\nReviewer #4, Comment 16:\nFigure 7f: Mitochondrial protein controls and a gCtrl + Flag-GPX4 condition should\nbe included. Additionally, does USP20 regulate HIF-1 levels?\nResponse:\nWe thank the reviewer for these insightful comments. In the Revised manuscript, we\nhave added the appropriate control groups. Consistent with the results of the original\nmanuscript, in the absence of USP20, GPX4 overexpression was no longer able to\nenhance BNIP3 stability, supporting the conclusion that GPX4 exerts its stabilizing\neffect on BNIP3 in a USP20-dependent manner (Revised Fig. 7a-b). We would like\nto note that the primary objective of this experiment was to demonstrate the\nrequirement of USP20 in mediating the GPX4-BNIP3 interaction; therefore, inclusion\nof mitochondrial protein controls is of limited relevance in this context.\nAdditionally, to address the reviewer’s concern regarding the mitochondrial protein\ncontrols,we included additional groups (gCtrl + EV, gCtrl + GPX4oe, gUsp20 + EV,\nand gUsp20 + GPX4oe) to examine whether GPX4 overexpression affects TOMM20\nprotein levels in 3T3 cells. The results showed that GPX4 overexpression in gCtrl\ncells led to a reduction in TOMM20 levels, consistent with activation of mitophagy\nand loss of mitochondrial mass. In contrast, in gUsp20 cells lacking USP20,\nGPX4 overexpression did not further reduce TOMM20 expression, indicating that\nUSP20 is required for GPX4-mediated activation of mitophagy (Response Fig. R4-\n16a-b).\nThese findings support our conclusion that GPX4 promotes mitophagy through the\nUSP20-BNIP3 axis.\nRegarding HIF-1α, we further overexpressed USP20 in cardiomyocytes and examined\nHIF-1α protein levels by Western blot. The results showed that USP20 increased HIF-\n1α expression under both normoxic and H/R conditions (Response Fig. R4-16c).\nThese findings suggest that USP20 may regulate HIF-1α levels, although the precise\nmechanism remains to be elucidated and will be explored in future studies.\nReviewer #4, Comment 17:\nThe mechanism by which GPX4 mediates the interaction between USP20 and BNIP3\nremains unclear. Is this interaction ferroptosis-dependent or directly mediated by\nGPX4? Is GPX4 enzyme activity required?\nResponse:\nWe appreciate the reviewer’s insightful question. As described in detail in our\nresponse to your Comment 1, we provide here a concise summary of the findings.\nOur data indicate that the interaction between USP20 and BNIP3 is directly facilitated\nby GPX4 and is not solely dependent on ferroptosis inhibition. Specifically, GPX4\noverexpression enhanced USP20–BNIP3 binding even when ferroptosis was\npharmacologically suppressed (Lip-1 treatment), suggesting a ferroptosis-independent\nmechanism (Response Fig. R4-1-1).\nMoreover, mutation of the U46 catalytic residue in GPX4 abolished its ability to\npromote USP20–BNIP3 interaction, indicating that GPX4 enzymatic activity is\nrequired for this regulatory function.\nReviewer #4, Comment 18:\nUSP20 knockout should be validated in USP20-cKO cardiac tissue.\nResponse:\nWe thank the reviewer for this valuable suggestion. To validate USP20 knockout in\ncardiac tissues, we isolated adult cardiomyocytes from USP20fl/fl and USP20 CKO\nmice. Western blot analysis, normalized to Tnnt2, confirmed that USP20 protein\nexpression was successfully deleted in USP20 CKO cardiomyocytes, demonstrating\nefficient knockout (Response Fig. R4-18).\nReviewer #4, Comment 19:\nFigure 8: Baseline cardiac function in USP20 cKO mice should be assessed, both\nunder normal conditions and following injury. Additionally, at least a WT + cTnT-\nGPX4oe + I/R group should be included for comparison.\nResponse:\nWe sincerely thank the reviewer for this constructive suggestion and apologize for our\noversight in not including these critical control groups in the original submission. We\nfully acknowledge the importance of incorporating appropriate controls, and in future\nexperimental designs we will pay closer attention to this aspect.\nTo address the reviewer’s concern, we first assessed baseline cardiac function in\nUSP20 CKO mice compared with USP20fl/fl controls. Echocardiography revealed that\nbaseline cardiac function was comparable between the two groups. However, after 4\nweeks of MI/RI (45 min ischemia, 4 weeks reperfusion), USP20 CKO mice exhibited\nmarkedly worse cardiac function compared with USP20fl/fl controls, indicating that\nUSP20 deletion exacerbates long-term post-injury remodeling. (Response Fig. R4-\n19).\nWe then performed additional experiments by introducing cardiomyocyte-specific\nGPX4 overexpression (AAV9-cTnT-GPX4) into both USP20fl/fl and USP20 CKO\nmice. In USP20fl/fl mice, GPX4 overexpression significantly reduced infarct size and\nacute myocardial injury, as evidenced by decreased cardiac injury markers and fewer\nTUNEL-positive cells following MI/RI (45 min ischemia, 24h reperfusion). In\ncontrast, USP20 CKO mice displayed larger infarct size and aggravated acute injury,\nand GPX4 overexpression failed to provide protection. In the long-term (4 weeks after\nMI/RI), GPX4 overexpression significantly improved cardiac function in USP20fl/fl\nmice, as reflected by higher EF and FS values. In USP20 CKO mice, GPX4\noverexpression conferred partial improvement in cardiac function; however, the effect\nwas insufficient to fully reverse the deficits caused by USP20 deficiency. Consistent\ntrends were observed in myocardial fibrosis, where histological staining and qPCR\nanalysis of fibrosis-related genes revealed reduced fibrotic remodeling in USP20fl/fl +\nGPX4oe mice, while USP20 CKO + GPX4oe mice showed only modest attenuation of\nfibrosis without complete normalization (Revised Fig. 9).\nReviewer #4, Comment 20:\nHypoxia was set at 0% oxygen, which is extremely low. Is this level necessary to\nmodel physiological ischemia, or would a less severe hypoxic condition better reflect\nin vivo pathophysiology? The authors should justify this choice and provide data\nconfirming hypoxic stress under these conditions.\nResponse:\nWe sincerely thank the reviewer for pointing out this issue. This was a typographical\nerror in the original manuscript. The hypoxic condition we used was 1.0% O₂, not 0%.\nThis level is widely adopted in in vitro studies to model ischemic stress in\ncardiomyocytes and has been demonstrated to induce robust hypoxia-related\nresponses17, 18. We have corrected this error in the Revised manuscript and clarified\nthe description in the Methods section. We apologize for the oversight and any\nconfusion it may have caused.\nMinor concerns:\nReviewer #4, Comment 1:\nLine 146: Should reference Fig 1b.\nResponse:\nWe thank the reviewer for noting this oversight. We have corrected the text at line 146\nto reference Fig. 1b in the Revised manuscript. In addition, we carefully re-examined\nthe entire manuscript and corrected similar minor errors to ensure consistency\nbetween the text and figure references.\nReference:\n1. Zhang F, et al. Deficiency of Transcription Factor Sp1 Contributes to Hypertrophic\nCardiomyopathy. Circ Res 134, 290-306 (2024).\n2. van Loo PF, et al. Transcription factor Sp3 knockout mice display serious cardiac\nmalformations. Mol Cell Biol 27, 8571-8582 (2007).\n3. Liu J, Tang D, Kang R. Targeting GPX4 in ferroptosis and cancer: chemical strategies\nand challenges. Trends Pharmacol Sci 45, 666-670 (2024).\n4. Liu H, et al. Small-molecule allosteric inhibitors of GPX4. Cell Chem Biol 29, 1680-\n1693.e1689 (2022).\n5. Yang WS, Kim KJ, Gaschler MM, Patel M, Shchepinov MS, Stockwell BR.\nPeroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis. Proc\nNatl Acad Sci U S A 113, E4966-4975 (2016).\n6. Zilka O, et al. On the Mechanism of Cytoprotection by Ferrostatin-1 and\nLiproxstatin-1 and the Role of Lipid Peroxidation in Ferroptotic Cell Death. ACS\nCent Sci 3, 232-243 (2017).\n7. Friedmann Angeli JP, et al. Inactivation of the ferroptosis regulator Gpx4 triggers\nacute renal failure in mice. Nat Cell Biol 16, 1180-1191 (2014).\n8. Cai W, et al. Alox15/15-HpETE Aggravates Myocardial Ischemia-Reperfusion Injury\nby Promoting Cardiomyocyte Ferroptosis. Circulation 147, 1444-1460 (2023).\n9. Park SY, et al. Irreversible HER2 inhibitors overcome resistance to the RSL3\nferroptosis inducer in non-HER2 amplified luminal breast cancer. Cell Death Dis 14,\n532 (2023).\n10. Xie X, et al. BACH1-induced ferroptosis drives lymphatic metastasis by repressing\nthe biosynthesis of monounsaturated fatty acids. Cell Death Dis 14, 48 (2023).\n11. Park TJ, et al. Quantitative proteomic analyses reveal that GPX4 downregulation\nduring myocardial infarction contributes to ferroptosis in cardiomyocytes. Cell Death\nDis 10, 835 (2019).\n12. Sun S, Shen J, Jiang J, Wang F, Min J. Targeting ferroptosis opens new avenues for\nthe development of novel therapeutics. Signal Transduct Target Ther 8, 372 (2023).\n13. Cheff DM, et al. The ferroptosis inducing compounds RSL3 and ML162 are not\ndirect inhibitors of GPX4 but of TXNRD1. Redox Biol 62, 102703 (2023).\n14. Zheng C, et al. Structure-activity relationship study of RSL3-based GPX4 degraders\nand its potential noncovalent optimization. Eur J Med Chem 255, 115393 (2023).\n15. Zheng J, Conrad M. The Metabolic Underpinnings of Ferroptosis. Cell Metab 32,\n920-937 (2020).\n16. Tong M, et al. Distinct Roles of DRP1 in Conventional and Alternative Mitophagy in\nObesity Cardiomyopathy. Circ Res 133, 6-21 (2023).\n17. Hu X, et al. Stromal cell derived factor-1 alpha confers protection against myocardial\nischemia/reperfusion injury: role of the cardiac stromal cell derived factor-1 alpha\nCXCR4 axis. Circulation 116, 654-663 (2007).\n18. Zhang X, et al. Propofol inhibits myocardial injury induced by microvesicles derived\nfrom hypoxia-reoxygenated endothelial cells via lncCCT4-2/CCT4 signaling. Biol\nRes 56, 20 (2023)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all major concerns that i had, so I have no further suggestions.", "reviewer_2": "The revised manuscript represents a substantial improvement over the previous version, offering a coherent mechanistic framework that links GPX4 to USP20–BNIP3-mediated mitophagy. The authors have strengthened the study through the inclusion of new biochemical and functional assays, long-term in vivo validation, and clinical correlation analyses. I recommend acceptance of the manuscript pending minor revisions. Addressing the following points will further strengthen the causal evidence and enhance the mechanistic depth of the study.\nMajor Comments:\nThe identification of K131 as a critical ubiquitination site is a valuable addition. However, the current evidence remains largely biochemical. To firmly establish functional relevance, it is essential to test whether the BNIP3-K131R mutant can rescue the phenotype in USP20-deficient or GPX4-silenced backgrounds.\nIn the chronic phase of I/R injury, GPX4 overexpression partially restores cardiac function even under USP20-deficient conditions. The authors attribute this “partial recovery” to USP20-independent antioxidant or anti-fibrotic effects of GPX4; however, no direct evidence—such as measurements of ROS or lipid peroxidation—is provided. If such data cannot be included at this stage, this point should be clearly framed as a hypothesis in the Discussion, accompanied by specific proposals for future experimental validation.\nMinor Comments:\n1）Please specify in each Co-IP figure legend whether IgG negative controls were included to rule out nonspecific binding.\nIf not shown, please add representative IgG control blots to the Supplementary Materials.\n2）Please correct the typographical error in the Fig. 9d X-axis label from “AAR/LV” to “IF/AAR.”\n3）Masson staining images and Fig. 4h currently lack visible scale bars, although these are mentioned in the legends.\nClearly visible scale bars should be added to all histological and immunofluorescence images.\n4）Several figure citations are inaccurate and require correction:\nLine 306 should refer to Fig. 4h–i\nLine 308 should refer to Fig. 4j–k\nLine 356 should refer to Fig. 5e", "reviewer_4": "My comments have been addressed."}, "author_response": "Response to Reviewers\nCardiomyocyte-derived GPX4 stabilizes BNIP3 to facilitate mitophagy and\nmitigate myocardial ischemia/reperfusion injury\nby Zhong et al.\nThe concerns raised by the reviewers have been comprehensively addressed and the\nRevised components are highlighted in red color.\nOverall, we added a substantial amount of new data which was incorporated into the\nfigures. We have summarized the modified figures below in list form for clarity:\n1. Supplementary Fig. S13g\n2. Supplementary Fig. S13h\nThese extensive revisions have further supported the key findings from our original\nobservation with more rigor.\nThe specific point-by-point responses are listed below with our responses highlighted\nin blue.\nReviewer #1 (Overview):\nThe authors have addressed all major concerns that i had, so I have no further\nsuggestions.\nResponse:\nWe sincerely thank the reviewer for the positive evaluation of our revised manuscript.\nWe are grateful for the constructive comments provided during the previous round,\nwhich significantly helped us strengthen the mechanistic framework and overall\nquality of the study. We appreciate the reviewer’s time and effort in reassessing the\nrevised version and are pleased that all major concerns have been fully addressed.\nThank you again for your thorough review and supportive remarks.\nReviewer #2 (Overview):\nThe revised manuscript represents a substantial improvement over the previous\nversion, offering a coherent mechanistic framework that links GPX4 to USP20-\nBNIP3-mediated mitophagy. The authors have strengthened the study through the\ninclusion of new biochemical and functional assays, long-term in vivo validation, and\nclinical correlation analyses. I recommend acceptance of the manuscript pending\nminor revisions. Addressing the following points will further strengthen the causal\nevidence and enhance the mechanistic depth of the study.\nResponse:\nWe sincerely thank the reviewer for the positive evaluation of our revised manuscript\nand for acknowledging the substantial improvements made in mechanistic clarity,\nbiochemical validation, and in vivo relevance. We greatly appreciate the reviewer’s\nrecognition of the strengthened GPX4-USP20-BNIP3 mechanistic framework and the\nadded functional and clinical evidence.\nWe also thank the reviewer for outlining the remaining minor points that could further\nreinforce the causal relationship and mechanistic depth. We have carefully considered\neach comment and have fully addressed all points in the revised manuscript. Detailed\npoint-by-point responses are provided below.\nWe are grateful for the reviewer’s constructive feedback and supportive\nrecommendation for acceptance pending minor revisions.\nMajor Comments:\nReviewer #2, Comment 1:\nThe identification of K131 as a critical ubiquitination site is a valuable addition.\nHowever, the current evidence remains largely biochemical. To firmly establish\nfunctional relevance, it is essential to test whether the BNIP3-K131R mutant can\nrescue the phenotype in USP20-deficient or GPX4-silenced backgrounds.\nResponse:\nWe appreciate the reviewer’s insightful comment and fully agree that functional\nvalidation is essential to establish the relevance of K131 ubiquitination in BNIP3\nregulation. To address this point, we conducted additional experiments using an in\nvitro hypoxia/reoxygenation (H/R) model of cardiomyocytes (HL-1).\nFour experimental groups were included: si-NC + H/R, si-USP20 + H/R, si-USP20\n+ BNIP3-WToe+ H/R, and si-USP20 + BNIP3-K131Roe+ H/R. As shown in the\nRevised Supplementary Fig. S13g, cell viability was markedly reduced in the si-\nUSP20 + H/R group compared with control cells. Overexpression of BNIP3-WT\nslightly improved cell viability, whereas BNIP3-K131Roe led to a significant\nrestoration of cardiomyocyte viability. Consistent with these results, LDH release\nshowed a similar trend, indicating reduced cellular injury in the BNIP3-K131Roe\ngroup (Revised Supplementary Fig. S13h).\nThese results provide functional evidence that ubiquitination at K131 is critical for\nBNIP3-mediated protection, and that the BNIP3-K131R mutant can partially rescue\nthe phenotype caused by USP20 deficiency under H/R injury conditions.\nReviewer #2, Comment 2:\nIn the chronic phase of I/R injury, GPX4 overexpression partially restores cardiac\nfunction even under USP20-deficient conditions. The authors attribute this “partial\nrecovery” to USP20-independent antioxidant or anti-fibrotic effects of GPX4;\nhowever, no direct evidence-such as measurements of ROS or lipid peroxidation-is\nprovided. If such data cannot be included at this stage, this point should be clearly\nframed as a hypothesis in the Discussion, accompanied by specific proposals for\nfuture experimental validation.\nResponse:\nWe thank the reviewer for this valuable and insightful comment. In the revised\nmanuscript, we have carefully clarified this point in the Discussion section.\nSpecifically, we acknowledge that although GPX4 overexpression partially restored\ncardiac function in USP20-deficient mice during the chronic phase of I/R injury, the\ncurrent study does not provide direct biochemical evidence to confirm USP20-\nindependent antioxidant or anti-fibrotic effects.\nWe have therefore reframed this interpretation as a hypothesis, suggesting that\nGPX4 may confer partial protection through additional mechanisms beyond the\nUSP20-BNIP3 axis, such as suppression of lipid peroxidation, attenuation of\ninflammation, or modulation of fibrotic remodeling. Furthermore, we have added a\nstatement outlining future experimental directions, including direct assessment of\noxidative stress (ROS and lipid peroxidation levels) and fibrosis-related signaling in\nUSP20-deficient models, to validate this hypothesis.\nThe revised sentences can be found in the Discussion of the revised manuscript.\n“Although GPX4 overexpression partially restored cardiac function in USP20-\ndeficient mice during the chronic phase, the underlying mechanisms remain to be\nelucidated. We hypothesize that GPX4 may exert USP20-independent protective\neffects by suppressing lipid peroxidation, reducing inflammation, or modulating\nfibrotic remodeling. Future work will focus on directly evaluating oxidative stress and\nlipid peroxidation to test this hypothesis.”\nMinor Comments:\nReviewer #2, Comment 1:\nPlease specify in each Co-IP figure legend whether IgG negative controls were\nincluded to rule out nonspecific binding. If not shown, please add representative IgG\ncontrol blots to the Supplementary Materials.\nResponse:\nWe thank the reviewer for this helpful suggestion. We have now specified this\ninformation in each relevant figure legend. In addition, representative IgG control\nblots have been provided below for clarity.\nReviewer #2, Comment 2:\nPlease correct the typographical error in the Fig. 9d X-axis label from “AAR/LV” to\n“IF/AAR.”\nResponse:\nWe thank the reviewer for carefully identifying this typographical error. The X-axis\nlabel in Fig. 9d has been corrected from “AAR/LV” to “INF/AAR” in the revised\nfigure. We appreciate the reviewer’s attention to detail.\nReviewer #2, Comment 3:\nMasson staining images and Fig. 4h currently lack visible scale bars, although these\nare mentioned in the legends. Clearly visible scale bars should be added to all\nhistological and immunofluorescence images.\nResponse:\nWe thank the reviewer for pointing out this issue. We have now added clearly visible\nscale bars to all histological and immunofluorescence images, including the Masson\nstaining panels and Fig. 4h. The corresponding figure legends have been updated to\nensure consistency. We appreciate the reviewer’s attention to detail, which has helped\nimprove the clarity of the figures.\nReviewer #2, Comment 4:\nSeveral figure citations are inaccurate and require correction:\nLine 306 should refer to Fig. 4h–i\nLine 308 should refer to Fig. 4j–k\nLine 356 should refer to Fig. 5e\nResponse:\nWe appreciate the reviewer’s attention to detail, which has helped improve the\naccuracy and clarity of the manuscript.\nReviewer #4 (Overview):\nMy comments have been addressed.\nResponse:\nWe sincerely thank the reviewer for the careful evaluation of our revised manuscript.\nWe are pleased that all previous comments have been fully addressed to your\nsatisfaction. We greatly appreciate the time and constructive feedback provided\nthroughout the review process, which has helped us improve the clarity and rigor of\nthe study.\nThank you again for your supportive remarks."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors addressed all my questions, I have no further comments."}, "author_response": ""}]

Decision letter