Oxidative stress induces mitochondrial iron overload and ferroptotic cell death

Oxidative stress has been shown to induce cell death in a wide range of human diseases including cardiac ischemia/reperfusion injury, drug induced cardiotoxicity, and heart failure. However, the mechanism of cell death induced by oxidative stress remains incompletely understood. Here we provide new evidence that oxidative stress primarily induces ferroptosis, but not apoptosis, necroptosis, or mitochondria-mediated necrosis, in cardiomyocytes. Intriguingly, oxidative stress induced by organic oxidants such as tert-butyl hydroperoxide (tBHP) and cumene hydroperoxide (CHP), but not hydrogen peroxide (H2O2), promoted glutathione depletion and glutathione peroxidase 4 (GPX4) degradation in cardiomyocytes, leading to increased lipid peroxidation. Moreover, elevated oxidative stress is also linked to labile iron overload through downregulation of the transcription suppressor BTB and CNC homology 1 (Bach1), upregulation of heme oxygenase 1 (HO-1) expression, and enhanced iron release via heme degradation. Strikingly, oxidative stress also promoted HO-1 translocation to mitochondria, leading to mitochondrial iron overload and lipid reactive oxygen species (ROS) accumulation. Targeted inhibition of mitochondrial iron overload or ROS accumulation, by overexpressing mitochondrial ferritin (FTMT) or mitochondrial catalase (mCAT), respectively, markedly inhibited oxidative stress-induced ferroptosis. The levels of mitochondrial iron and lipid peroxides were also markedly increased in cardiomyocytes subjected to simulated ischemia and reperfusion (sI/R) or the chemotherapeutic agent doxorubicin (DOX). Overexpressing FTMT or mCAT effectively prevented cardiomyocyte death induced by sI/R or DOX. Taken together, oxidative stress induced by organic oxidants but not H2O2 primarily triggers ferroptotic cell death in cardiomyocyte through GPX4 and Bach1/HO-1 dependent mechanisms. Our results also reveal mitochondrial iron overload via HO-1 mitochondrial translocation as a key mechanism as well as a potential molecular target for oxidative stress-induced ferroptosis in cardiomyocytes.

Loss of cardiomyocytes by apoptotic and/or necrotic cell death contributes to the pathogenesis of multiple forms of heart disease such as ischemia/reperfusion injury (I/R), myocarditis, cardiomyopathy, drug induced cardiotoxicity, and heart failure of diverse etiologies 1, 2 .Apoptosis has been well established as a form of regulated cell death, which is tightly regulated by death receptor-or mitochondria-mediated signaling pathways 1 .In contrast, necrosis had long been regarded as an unregulated and passive process, characterized by cellular swelling, plasma membrane rupture, and cell lyses 3 .However, recent studies have overturned this notion and revealed that necrosis can also occur in a highly regulated and genetically controlled manner, termed "regulated necrosis" 1 .Indeed, several regulated necrosis pathways have recently been identified, including necroptosis, mitochondria-mediated necrosis, ferroptosis, pyroptosis, and other regulated necrotic processes 1 .
Necroptosis is a form of regulated necrosis mediated by death receptors and executed through the induction of receptor-interacting protein kinase 1 and 3 (RIPK1-RIPK3) necrosome, phosphorylation and oligomerization of mixed lineage kinase domain-like protein (MLKL), and plasma membrane disruption [4][5][6] .In contrast to necroptosis, the defining event in the mitochondria-mediated necrosis is the opening of the mitochondrial permeability transition pore (mPTP) on the inner mitochondrial membrane regulated by cyclophilin D (CypD) and/ or increased outer mitochondrial membrane permeability mediated by Bax and Bak 7,8 .Ferroptosis has recently been identified as a new form of regulated necrosis, which is characterized by iron-dependent lipid peroxidation, irreparable lipid damage, membrane disruption, and necrotic cell death 9,10 .Glutathione peroxidase 4 (GPX4) is Cell culture.All experiments involving animals were approved by the Institutional Animal Care and Use Committees of the University of Washington, and all studies were performed in accordance with relevant guidelines and regulations.All methods were reported in accordance with ARRIVE guidelines.Neonatal rat cardiomyocytes were isolated from hearts of 1-to 2-day-old Sprague-Dawley rat pups as previously described 28 .Briefly, neonatal hearts were collected, the atria were removed, and the ventricles were minced in HBSS prior to enzymatic digestion.The ventricular tissue was subjected to 5 rounds of enzymatic digestion using 0.05% pancreatin (Sigma-Aldrich) and 84 U/ml collagenase (Worthington).Cells were collected by centrifugation at 500g for 5 min at 4 °C and resuspended in M199 medium.After separation from fibroblasts, enriched cardiomyocytes were plated on culture dishes coated with 1% gelatin.Cells were grown in M199 medium supplemented with 2% bovine growth serum (Thermo Fisher Scientific), 100 U/ml penicillin-streptomycin, and 2 mM L-glutamine.For simulated ischemia/reperfusion (sI/R), ischemia was imposed by a buffer exchange to ischemic solution (20 mM HEPES, pH 6.6, 20 mM deoxyglucose, 125 mM NaCl, 8 mM KCl, 1.2 mM KH 2 PO 4 , 1.25 mM MgSO 4 , 1.2 mM CaCl 2 , 6.25 mM NaHCO 3 , 5 mM sodium lactate) and placing in a humidified chamber equilibrated with 95% N2 and 5% CO2.After 6 h of simulated ischemia, reperfusion was initiated by buffer exchange to normal culture medium in 95% room air and 5% CO2 for 12 h.
Cell death assays.Cell death was assessed using a Cell Meter Apoptotic and Necrotic Detection kit (ATT Bioquest) as we previously described 30 .Briefly, cells were incubated at 37 °C for 30 min with Apopxin Green Measurement of lipid peroxidation.Lipid peroxidation was measured using C11-BODIPY 581/591 (Invitrogen) according to the manufacturer's instructions.Cells were incubated in 10 μM C11-BODIPY581/591 for 30 min at 37 °C.Fluorescence measurements were performed using a BioTek Synergy 2 microplate reader with excitation wavelength of 581 nm and an emission wavelength of 591 nm.

Labile iron levels.
The labile iron levels in cardiomyocytes were measured using the calcein-AM method (Yoshida M 3145).Briefly, cells were incubated with 1 μM calcein-AM at 37 °C for 10 min followed by washing with PBS.Fluorescence intensity was measured using a BioTek Synergy 2 fluorescence microplate reader.Cells were then treated with 100 μM 2′,2′-bipyridine (BIP) at 37 °C for 10 min and the fluorescence was measured again.The changes in fluorescence upon BIP treatment was used to determine the labile iron pool.
Cytosolic and mitochondrial fractions.Cytosolic and mitochondrial fractions were prepared based on the method by Frezza et al. with some modifications 31 .Briefly, cells were suspended in sucrose-mannitol buffer (20 mM HEPES, pH 7.5, 2 mM EDTA, 70 mM sucrose, 220 mM mannitol, 5 mM NaF, protease inhibitor cocktail [Roche]) and homogenized using a Teflon homogenizer.The homogenates were centrifuged at 600g for 10 min at 4 °C.The supernatant was re-centrifuged at 10,000×g for 15 min at 4 °C to collect the supernatant (cytosolic fraction) and pellet (mitochondrion fraction).The purity of cytosolic and mitochondrial fractions was validated by Western blotting using anti-GAPDH and anti-VDAC antibodies, respectively.

Mitochondrial iron.
Mitochondrial iron was measured using mito-FerroGreen (Dojindo), a fluorescence probe for mitochondrial ferrous ion (Fe 2+ ).Cells were incubated in 5 μM mito-FerroGreen for 30 min at 37 °C.After three washes in PBS, 100 μM ammonium iron sulfate was then added to the cells.Mitochondrial iron was fluorometrically measured using a BioTek Synergy 2 microplate reader at an excitation wavelength of 505 nm and an emission wavelength of 535 nm or visualized using an EVOS FL fluorescence microscope.

Mitochondrial lipid peroxidation.
Mitochondrial lipid peroxidation was assessed using mitoPeDPP (Dojindo), a fluorescence probe that specifically detects lipid peroxides in the mitochondrial inner membrane.Cells were incubated with 0.5 μM mitoPeDPP solution for 30 min at 37 °C.After three washes with PBS, mitochondrial lipid peroxidation was fluorometrically measured using a BioTek Synergy 2 microplate reader at an excitation wavelength of 452 nm and an emission wavelength of 470 nm.
Measurement of mitochondrial ROS.MitoSOX Red (Invitrogen) was used for analyzing mitochondrial ROS.Cells were loaded with MitoSOX at 5 µM concentration for 30 min at 37 °C.After washing three times with PBS, fluorescence was detected by an EVOS FL digital fluorescence microscope (AMG) and quantified using ImageJ software.Data were collected from at least 3000 cells.

Statistics.
Results are presented as mean ± SEM.Statistical analysis was performed using GraphPad Prism 9 (GraphPad).Statistical analysis was performed using the Student's two-tailed t test for comparison between 2 groups.Comparisons between multiple groups were made using one-way analysis of variance (ANOVA) with Tukey's post hoc test.Comparison of multiple groups with multiple conditions was performed using 2-way ANOVA with Tukey's multiple-comparison test.P < 0.05 was considered significant.

Organic oxidants induce ferroptosis in cardiomyocytes.
Oxidative stress primarily induces nonapoptotic cell death, but the cell death mechanism remains unclear 7,22 .Here, we examined whether and how oxidative stress induces ferroptosis in cardiomyocytes.tert-butyl hydroperoxide (tBHP), an organic peroxide, was used to generate oxidative stress, which induced necrotic cell death in cardiomyocytes, as indicated by increased propidium iodide (PI) uptake, an indicator of impaired plasma membrane integrity (Fig. 1A,B).Moreover, tBHP also promoted the release of high mobility group box 1 (HMGB1) into the culture supernatant, another marker of necrotic cell death 30 (Fig. 1C).Importantly, tBHP-induced cell death and HMGB1 release were largely inhibited by classic ferroptosis inhibitors such as ferrostatin-1 (Fer-1) and deferoxamine (DFO), indicating the induction of ferroptosis (Fig. 1A-C).In contrast, tBHP-induced cell death was not affected by necrostatin-1 s (Nec-1 s) or cyclosporine A (CsA), suggesting a mechanism distinct from necroptosis or mitochondria-mediated necrosis (Fig. 1A-C).Consistent with this observation, genetic deletion of Ripk1, Ripk3, or Ppif (CyPD) also had minimal effects on tBHP-induced cell death in mouse embryonic fibroblasts (MEFs) (Fig. 1D).These results indicate that tBHP primarily induces ferroptosis, but not necroptosis or mitochondria-dependent necrosis, in cardiomyocytes.
The effect of oxidative stress-induced cell death was further assessed using cumene hydroperoxide (CHP) and H 2 O 2 as the oxidizing agents.Like tBHP, CHP markedly induced cell death and HMGB1 release in cardiomyocytes, which was effectively inhibited by ferroptosis inhibitors such as Fer-1, DFO, and liproxstatin-1 (Lip-1) (Fig. 1E,F).Intriguingly, H 2 O 2 also induced cardiomyocyte death and HMGB1 release, but these effects were resistant to ferroptosis inhibition (Fig. 1G,H).These results reveal that ferroptosis is selectively induced by organic oxidants but not H 2 O 2 .

Organic oxidants promote glutathione depletion, GPX4 downregulation, and enhanced lipid peroxidation in cardiomyocytes.
Here, we examined whether organic oxidants promote lipid peroxidation, a key feature of ferroptosis.Indeed, both tBHP and CHP greatly induced lipid peroxidation in cardiomyocytes, which was largely blocked by Fer-1, Lip-1, or DFO (Fig. 2A).In contrast, H 2 O 2 only moderately increased lipid peroxidation, which was not affected by treatment with Fer-1, Lip-1, or DFO (Fig. 2A).These results are consistent with our finding that tBHP and CHP, but not H 2 O 2 , induced ferroptosis in cardiomyocytes.
To understand the mechanism of organic oxidants -induced ferroptosis, we assessed the effect of tBHP on GSH-GPX4 signaling.We found that tBHP induced GSH depletion in cardiomyocytes in a time-dependent manner (Fig. 2B).Pretreatment with the reduced GSH, but not the oxidized GSSG, inhibited tBHP-induced cell death (Fig. 2C).Moreover, GPX4 was also markedly downregulated upon tBHP stimulation (Fig. 2D).To further determine the role of GPX4 in tBHP-induced ferroptosis, we examined the rates of cell death in cardiomyocytes transduced with adenoviral vectors encoding GPX4 shRNA or wild-type GPX4.GPX4 silencing further promoted tBHP-induced cell death and HMGB1 release, whereas GPX4 overexpression showed the opposite effects (Fig. 2E-H), suggesting that GPX4 confers cell death resistance to oxidative stress.These results indicate that organic oxidants induce cardiomyocyte ferroptosis through GSH depletion and GPX4 downregulation.

Organic oxidants promote labile iron accumulation via the Bach1-HO-1 pathway. Next, we
examined whether oxidative stress induces iron overload, which is a defining feature of ferroptosis.Indeed, both tBHP and CHP greatly increased labile iron levels in cardiomyocytes as assessed by calcein-acetoxymethyl ester assay 32 (Fig. 3A).Moreover, tBHP-induced iron overload correlates with a marked upregulation of heme oxygenase-1 (HO-1), an enzyme that drives iron release via heme degradation 33 .Conversely, the transcription factor BTB and CNC homology 1 (Bach1), a transcriptional suppressor for HO-1 34 , was downregulated by tBHP and CHP (Fig. 3B).Deletion of HO-1 markedly reduced labile iron levels induced by tBHP or CHP, further confirming the role of HO-1 in oxidative stress-induced iron overload (Fig. 3C).Moreover, deletion of HO-1 also inhibited tBHP-or CHP-induced cardiomyocyte death (Fig. 3D).Similar effect was obtained in cardiomyocytes treated with zinc protoporphyrin IX (ZnPP), an HO-1 inhibitor (Supplemental Fig. 1).Conversely, HO-1 overexpression further promoted tBHP-induced cell death (Supplemental Fig. 2A).To determine the mechanism by which oxidative stress promotes HO-1 expression, we showed that tBHP or CHP promoted the degradation of Bach1, which was blocked by pretreatment with the proteasome inhibitor MG-132 (Fig. 3E).Moreover, deletion of Bach1 with an adenoviral vector encoding Bach1 shRNA was sufficient to promote HO-1 expression in cardiomyocytes (Supplemental Fig. 2B), suggesting that oxidative stress induces HO-1 expression through Bach1 degradation.Moreover, overexpression of Bach1 inhibited cell death tBHP-or CHP-induced cell death (Fig. 3F).Together, these results reveal a key role for the Bach1-HO-1 signaling pathway in organic oxidants-induced iron overload and ferroptosis in cardiomyocytes.

Organic oxidants promote mitochondrial iron overload. The role of mitochondria in ferroptosis has
been controversial, possibly depending on cell types and cellular contexts [35][36][37][38] .Here, we identified a critical role of mitochondria in mediating oxidative stress-induced ferroptosis in cardiomyocytes.Intriguingly, a marked increase in mitochondrial ferrous iron (Fe 2+ ) was detected in cardiomyocytes after tBHP treatment (Fig. 4A).Strikingly, this effect was associated with a significant translocation of HO-1 from the cytosol to mitochondria (Fig. 4B).Moreover, tBHP induced mitochondrial iron accumulation was largely abrogated by HO-1 deletion, revealing a key role for HO-1 in mediating mitochondrial iron overload (Fig. 4C).Importantly, overexpressing mitochondrial ferritin (FTMT), a mitochondrial matrix protein that chelates iron, effectively inhibited tBHPinduced cell death in cardiomyocytes (Fig. 4D), further suggesting that mitochondrial iron overload plays a key role in tBHP-induced ferroptosis.Conversely, deletion of FTMT further promoted tBHP-induced cell death in cardiomyocytes (Fig. 4E).In contrast to FTMT, overexpression of ferritin heavy chain 1 (FTH1), which chelates cytosolic iron, moderately inhibited ferroptosis (Supplemental Fig. 3).Together, these data identified mitochondrial iron overload as a key mediator of organic oxidants-induced ferroptosis in cardiomyocytes.

Mitochondrial iron and ROS accumulation mediates cardiomyocyte death induced by simulated ischemia/reperfusion or doxorubicin.
To further investigate the role of mitochondrial iron and ROS in pathological conditions associated with elevated oxidative stress 17,19 , cardiomyocytes were subjected to simulated ischemia and reperfusion (sI/R) or the chemotherapeutic agent doxorubicin (DOX).Mitochondrial iron levels were markedly elevated in cardiomyocytes subjected to sI/R or DOX (Fig. 6A).Elevated mitochondrial lipid peroxidation was also detected under these conditions (Fig. 6B).To examine the role of mitochondrial iron and ROS in sI/R-or DOX-induced cell death, cardiomyocytes were transduced with Ad-FTMT or Ad-mCAT to prevent mitochondrial iron or ROS accumulation.Overexpressing FTMT or mCAT diminished mitochondrial lipid peroxidation and cell death induced by sI/R, associated with reduced HMGB1 release (Fig. 6C-E).Similar effects were obtained in cardiomyocytes treated with DOX (Fig. 6F-H).These results suggest that targeted inhibition of mitochondrial iron or ROS accumulation prevents cell death in pathological conditions associated with oxidative stress.

Discussion
In the present study, we provide new evidence that oxidative stress induced by organic oxidants primarily causes ferroptotic cell death in cardiomyocytes, highlighting the significance of this new cell death modality in heart disease driven by elevated oxidative stress.Mechanistically, oxidative stress promotes GSH depletion and GPX4 downregulation, leading to enhanced lipid peroxidation (Fig. 7).Moreover, we provide mechanistic evidence linking elevated oxidative stress to labile iron overload through the Bach1-HO-1 signaling (Fig. 7).Our data also reveal the interdependence of lipid peroxidation and iron overload in ferroptosis signaling, as blockade of either pathway prevented oxidative stress-induced ferroptosis in cardiomyocytes.Importantly, we further identified HO-1 mitochondrial translocation as a previously undescribed mechanism that mediates iron overload and lipid ROS accumulation within the mitochondria (Fig. 7).Strikingly, targeted inhibition of mitochondrial iron overload and ROS accumulation, by overexpressing FTMT or mCAT, respectively, markedly inhibited oxidative stress-induced ferroptosis.These results suggest that mitochondrial iron overload and lipid ROS accumulation may represent potential therapeutic targets in oxidative stress-induced pathological conditions.
Here we showed that organic oxidants such as tBHP and CHP, but not H 2 O 2 , primarily induced ferroptosis in cardiomyocytes.Consistent with this observation, tBHP and CHP greatly enhanced lipid peroxidation in cardiomyocytes, whereas H 2 O 2 only had moderate effect.These results reveal distinct effects of different oxidative stress inducers, possibly depending on their chemical properties.Indeed, it has been shown that tBHP can be metabolized to produce peroxyl and alkoxyl radicals 40 , which can initiate lipid peroxidation of membrane phospholipids.Mechanistically, we further show that tBHP induces GSH depletion as well as GPX4 downregulation in cardiomyocytes, leading to GPX4 inactivation and elevated lipid peroxidation.Notably, GSH depletion and/or GPX4 downregulation have also been linked to pathological oxidative stress in vivo, such as ischemia/ reperfusion cardiac injury and doxorubicin induced cardiomyopathy 41,42 .
It is well established that iron overload promotes ROS accumulation via the Fenton reaction 43 , but whether the reverse is also true has not been directly investigated.Here we show that elevated oxidative stress can also  induce iron overload in cardiomyocytes.Mechanistically, we found that oxidative stress promoted the expression of HO-1, an enzyme that drives labile iron release through heme degradation.Notably, ablation of HO-1 abolished, whereas overexpression of HO-1 exacerbated, oxidative stress-induced labile iron overload and ferroptosis.These data suggest that oxidative stress-induced iron overload is mainly originated intracellularly through HO-1 mediated iron release.It is possible that iron overload can also occur through additional mechanisms, such as nuclear receptor coactivator 4 (NCOA4) mediated ferritinophagy and lysosomal iron release 44 , which warrant further investigation.Consistent with our observations, HO-1 have also been shown to promote iron overload in beta-thalassemia, sickle cell disease, and anthracycline cardiotoxicity [45][46][47] .Moreover, transgenic overexpression of HO-1 promoted iron accumulation in the brain 48 .Of note, although HO-1 is commonly regarded as a cytoprotective enzyme 49 , recent studies indicate that HO-1 can also exert detrimental effects 50 .Moreover, both pro-and anti-ferroptotic roles of HO-1 have been observed depending on cell types and pathological conditions 50 .To explain this discrepancy, accumulating evidence suggests that moderate activation of HO-1 elicits a cytoprotective effect whereas excessive and/or prolonged activation of HO-1 increases labile Fe 2+ , leading to ferroptotic cell death 50 .Importantly, a recent study by Miyamoto et al. showed that HO-1 silencing prevented sI/R-induced ferroptosis in cardiomyocytes 51 .Moreover, inactivation of HO-1 with ZnPP also attenuated doxorubicin-induced cardiac ferroptosis and cardiotoxicity 47 .Therefore, these findings support a detrimental role of HO-1 activation in the settings of sI/R and doxorubicin insults, where HO-1 is markedly upregulated, by promoting ferroptosis of cardiomyocytes.Intriguingly, the upregulation of HO-1 in cardiomyocytes in response to oxidative stress was associated with the downregulation of Bach1, a transcriptional repressor of HO-1 34 .HO-1 is positively regulated by the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), but negatively regulated by Bach1 52 .Notably, inactivation of Bach1 is a prerequisite for HO-1 induction, which is dominant over NRF2-mediated HO-1 transcription 53 .Moreover, Bach1 inactivation can induce HO-1 expression without NRF2 nuclear accumulation 53 .Consistent with this notion, our results indicate that the primary event leading to HO-1 induction in response to oxidative stress is Bach1 downregulation.Importantly, deletion of Bach1 was sufficient to induce HO-1 expression in cardiomyocytes.Moreover, forced overexpression of Bach1 inhibited oxidative stress-induced ferroptosis.Together, these data suggest that Bach1-HO-1 signaling critically regulates oxidative stress-induced ferroptosis.
This study also reveals that mitochondria play a key role in oxidative stress-induced ferroptosis in cardiomyocytes.The role of mitochondria in ferroptosis has been controversial [35][36][37][38] .For instance, it has been shown that depletion of mitochondria had no effect on RLS3-induce ferroptosis in HT-1080 cells 35 .In contrast, subsequent studies found that mitochondrial DNA depletion or mitochondrial ROS quenching inhibited RSL3-induced ferroptosis 37,38 .Moreover, mitochondria depletion prevented ferroptosis induced by cysteine-deprivation or Erastin 36 .Here we showed that mitochondrial free iron levels as well as lipid peroxidation were markedly elevated following oxidative stress.Moreover, targeted inhibition of mitochondrial iron overload by overexpressing FTMT markedly inhibited oxidative stress-induced mitochondrial lipid peroxidation and ferroptosis.Targeted inhibition of mitochondrial ROS, using mitoQ, SKQ1, or mCAT, also largely inhibited ferroptosis.These results suggest that mitochondrial iron overload and lipid ROS accumulation play an important role in oxidative stress-induced ferroptosis in cardiomyocytes.Of note, mitochondrial permeability transition pore (mPTP) has been implicated in oxidative stress-induced necrosis as well as ischemia/reperfusion injury 7 .However, Dixon et al. demonstrated that ferroptosis is independent of mPTP, since ferroptosis is not affected by inactivation of CypD, a key component of mPTP 9 .Consistent with this, we found that ablation of Ppif (gene encoding CypD) had no effect on tBHP-induced cell death in MEFs.Moreover, inactivation of CypD with CsA also failed to inhibit tBHP-induced cell death in cardiomyocytes.Similar effects were obtained in H9c2 cardiomyoblasts 54 .In contrast, another study showed that Ppif deletion prevented tBHP-induced cell toxicity in a human pancreatic cell line 55 .These results suggest that tBHP may induce cell death though mPTP-dependent or -independent mechanisms depending on cell types and cellular contexts.
Importantly, we further identified HO-1 mitochondrial translocation as a key mechanism for oxidative stressinduced mitochondrial iron overload and ROS accumulation.Our data suggest that oxidative stress promotes HO-1 translocation from the cytosol into mitochondria where it catalyzes heme degradation and iron release.In support of this notion, deletion of HO-1 abrogated oxidative stress-induced mitochondrial iron overload.Notably, HO-1 mitochondrial translocation has also been detected in several cell types in response to hypoxia, leading to mitochondrial ROS accumulation and dysfunction 56 .The precise mitochondria targeting sequence of HO-1 has not been identified 56 .It is possible that a cryptic mitochondria-targeting signal may exit which might be activated by oxidative stress 56 .Therefore, the mechanism of HO-1 mitochondrial translocation warrants further investigation.Importantly, overexpressing FTMT, a mitochondrial iron chelating protein, largely inhibited oxidative stress-induced ferroptosis, further linking mitochondria iron overload to ferroptosis.Consistent with our findings, a recent study on the dihydroorotate dehydrogenase (DHODH)-mediated mitochondria ferroptosis defense system also points to the role of mitochondrial iron in ferroptosis 57 .Moreover, mice with heart-specific overexpression of ABCB8, which exports iron out of the mitochondria, were more resistant to DOX cardiotoxicity, although the role of ABCB8 in ferroptosis has not been directly investigated 58 .Of note, other potential mechanisms may also contribute to mitochondria iron overload in ferroptosis.For example, increased mitochondrial iron uptake through iron transporters, such as mitoferrin-2, can mediate mitochondrial iron overload 59,60 .Moreover, in photodynamic therapy-induced ferroptosis, cytosolic iron is translocated into mitochondria via the mitochondrial Ca 2+ and Fe 2+ uniporter (MCU), leading to mitochondrial iron overload.Nonetheless, these results highlight a key role of mitochondrial iron in mediating ferroptosis, suggesting that targeting mitochondrial iron overload may represent a new strategy for preventing ferroptosis.Whether mitochondrial iron overload mediates oxidative stress-induced ferroptosis in the heart in vivo warrants further investigation.Indeed, it has been shown that mitochondrial iron levels are elevated in the heart subjected to ischemia/reperfusion injury and pressure overload 61,62 .Moreover, HO-1 is also upregulated in the heart under these pathological conditions.It will be important to further investigate the role of HO-1 mediated mitochondrial iron overload in cardiac ferroptosis in vivo and the physiological implications of this mechanism in the pathogenesis of oxidative stress-induced heart disease.
In summary, the present study identified ferroptosis as the major form of cardiomyocyte death triggered by organic oxidants-induced oxidative stress, in contrast to previous studies implicating other forms of cell death in this process, such as apoptosis and necroptosis.We also provide mechanistic evidence that oxidative stress induces cardiomyocyte ferroptosis by promoting lipid peroxidation via GSH depletion and GPX4 inactivation as well as iron overload through Bach1-HO-1 signaling.Moreover, we identified HO-1 mitochondrial translocation as a novel mechanism mediating mitochondrial iron overload and ROS accumulation.Targeted inhibition of mitochondrial iron overload or ROS accumulation effectively inhibited oxidative stress-induced ferroptosis.Therefore, targeting Bach1-HO-1 signaling and mitochondrial iron overload may serve as potential cytoprotective strategies in pathological conditions associated with oxidative stress.

Figure 6 .
Figure 6.Inhibition of mitochondrial iron or ROS accumulation prevented simulated ischemia/reperfusion or doxorubicin induced cell death in cardiomyocytes.(A) Mitochondrial Fe 2+ levels in cardiomyocytes subjected to 6 h simulated ischemia and 12 h reperfusion (sI/R) or control condition (left panel).Mitochondrial Fe 2+ levels were also measured in cells treated with 10 μM DOX or vehicle control for 12 h (right panel).*P < 0.05 vs Control or Veh.n = 4. (B) Mitochondrial lipid peroxidation assessed with MitoPeDPP in cardiomyocytes subjected to sI/R or DOX as in A. *P < 0.05 vs Control or Veh.n = 4. (C) Mitochondrial lipid peroxidation assessed with MitoPeDPP in cardiomyocytes infected with Ad-βgal, Ad-FTMT, or Ad-mCAT and subjected to sI/R or control condition.*P < 0.05 vs Control.#P < 0.05 vs Ad-βgal sI/R.n = 3. (D) Quantification of cell death from cardiomyocytes infected with Ad-βgal, Ad-FTMT, or Ad-mCAT and subjected to sI/R or control condition.*P < 0.05 vs Control.#P < 0.05 vs Ad-βgal sI/R.n = 4. (E) Western blotting for the indicated proteins from cells treated as in D. (F) Mitochondrial lipid peroxidation assessed with MitoPeDPP in cardiomyocytes infected with Ad-βgal, Ad-FTMT, or Ad-mCAT followed by treatment with DOX or vehicle control for 12 h.*P < 0.05 vs Veh.#P < 0.05 vs Ad-βgal DOX.n = 3. (G) Quantification of cell death from cardiomyocytes infected with Ad-βgal, Ad-FTMT, or Ad-mCAT followed by treatment with DOX or vehicle control for 12 h.*P < 0.05 vs Veh.#P < 0.05 vs Ad-βgal DOX.n = 4. (H) Western blotting for the indicated proteins from cells treated as in G.

Figure 7 .
Figure 7. Proposed mechanisms by which oxidative stress induces mitochondrial iron overload and ferroptosis in cardiomyocytes.Oxidative stress induced by organic oxidants promotes GSH depletion and GPX4 downregulation, leading to increased lipid peroxidation.Oxidative stress also increases labile iron levels through downregulation of the transcription suppressor Bach1, upregulation of HO-1 expression, and enhanced iron release via heme degradation.Moreover, oxidative stress also promotes HO-1 translocation to mitochondria, leading to mitochondrial iron overload and lipid ROS accumulation.Targeted inhibition of mitochondrial iron overload and ROS accumulation prevents oxidative stress-induced ferroptosis in cardiomyocytes.ZnPP, zinc protoporphyrin IX, HO-1 inhibitor; FTMT, mitochondrial ferritin; mCAT, mitochondrial catalase.