Introduction

Viral myocarditis is characterized as localized or diffuse disease of the myocardial parenchyma or interstitium caused by virus infection. After coxsackievirus B3 (CVB3) was first used to induce myocarditis in mice in 1974 by Woodruff, most of the subsequent experimental models of viral myocarditis were induced by CVB3 infection [21]. It showed that an overwhelming level of lipid ROS production was at early infection (3 h postinfection) in the CVB3 group (Fig. 2V, W). Taken together, these results suggested that ferroptosis was activated by CVB3 infection.

In order to determine when ferroptosis occur during CVB3 infection in vitro, we detected the upstream of ferroptosis pathway System XC−. SLC7A11 is a main composition of System XC− [22]. In our study, we found that SLC7A11 expression was decreased at early CVB3 infection but increased obviously at 36 h postinfection (Fig. 2L, M), which was consistent with the level of GSH. However, at 48 h postinfection, the SLC7A11 expresssion declined, another important GSH synthesis enzyme GPX4 was also downregulated (Fig. 2J). To further explore the reason, we measured the change of core regulatory genes in ferroptosis. As shown in Fig. 1H, I, J, the levels of pro-ferroptotic genes ACSL4 and NCOA4 were significantly upregulated at 48 h postinfection, while the negative regulator GPX4 level was downregulated at 48 h (Fig. 2N, O, P). Similar to WB results, the mRNA of ACSL4 and NCOA4 were increased at 48 h postinfection (Fig. 2R, S). Meanwhile, GPX4 mRNA level was reduced (Fig. 2T). Hence, our results has suggested that ferroptosis is occurred mainly at 48 h CVB3 infection in vitro.

The effect of inhibitors on ferroptosis in CVB3 infection

To further explore the role of iron in ferroptosis induced by CVB3 infection, we used two classical ferroptosis inhibitors, ferrostatin-1 (Fer-1) and deferoxamine (DFO). We first used the inducer erastin to induce ferroptosis at 6 μM (Supplementary Fig. 2A), then rescue experiments were performed by treating cells with ferroptosis inhibitors Fer-1 (at 2 μM, Supplementary Fig. 2B) or DFO (at 5 μM, Supplementary Fig. 3C) after induction by erastin. As shown in Fig. 3A, both Fer-1 and DFO could rescue the cell viability decrease induced by CVB3 infection, compared to the non-drug intervention CVB3 group (Fer-1 p < 0.001, DFO p < 0.01).

Fig. 3: The effect of drugs on ferroptosis in CVB3-infected cells.
figure 3

A Cell viability of HeLa cells in different drug treatment groups measured by CCK-8 assays. Fer-1 pretreated at 2 μM, 24 h before CVB3 infection. DFO pretreated at 5 μM 24 h before CVB3 infection. DMSO was used as a solvent control. The results showed that both Fer-1 and DFO could prevent the decrease in cell viability caused by CVB3 infection. B Area of CVB3-positive fluorescence measured with ImageJ, which corresponds to figure E. C MDA detection under different ferroptosis drug interventions. It showed that both Fer-1 and DFO drugs can reduce MDA production, especially DFO (p < 0.01). D Iron concentrations were measured after pretreatment of CVB3-infected HeLa cells with the ferroptosis agonist Erastin (6 μM, 12 h) and inhibitors Fer-1 and DFO. E CVB3 fluorescence (green) determined by IFA in HeLa cells in different drug intervention groups. Cell nuclei were stained with DAPI (blue) (scale bar = 50 μm). F Fluorescence of lipid ROS in HeLa cells pretreated with Fer-1 or DFO and incubated with the BODIPY581/591C11 probe (scale bar = 100 μm). G Percentage of oxidized/non-oxidized cells were calculated by ImageJ software according to figure F. It suggested that both DFO and Fer-1 could decrease the oxidized lipid ROS significantly (p < 0.001). H Flow cytometry was used to detect lipid ROS in different drug pretreatment groups during CVB3 infection. I, J The protein level of ACSL4 in drug groups were measured by western-blot, the gray values of strips were calculated by ImageJ. K, L The protein level of GPX4 in different drug groups were measured by western-blot. Measurement of relative gray values indicating gene protein levels were determined using ImageJ. M, N The mRNA level of ACSL4 and GPX4 in the drug-pretreated groups were detected via qPCR. All results are expressed as the mean ± SD. *p < 0.05 vs. the mock group, **p < 0.01, ***p < 0.001. #p < 0.05 vs. the CVB3 group, ##p < 0.01, ###p < 0.001. n = 3.

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To compare the effects of the two inhibitors on ferroptosis induced by CVB3 infection, we measured iron content, the MDA level, and lipid ROS levels. The results showed that both Fer-1 and DFO can relive the CVB3 virus replication at 24 h of infection (DFO p < 0.001, Fer-1 p < 0.01) (Fig. 3B, E). The iron chelator DFO had the most obvious effect on decreasing MDA and iron content (DFO p < 0.001, Fer-1 p < 0.05). (Fig. 3C, D). Fer-1 had a more obvious effect than DFO on reducing the accumulation of lipid ROS by IFA (Fig. 3F, G) and by flow cytometry (Fig. 3H). Moreover, We detected the changes in the representative ferroptosis genes ACSL4 and GPX4 via qPCR and western-blotting analyses. Both Fer-1 and DFO rescued the mRNA and protein expression of ACSL4 in the CVB3-infected groups (Fig. 3I, J, M), but had no evident effect in non-infected groups, and Fer-1, DFO could also prevent the reducing of GPX4 mRNA and protein levels by CVB3 infection (Fig. 3K, L, N). Taken together, these results indicate that the ferroptosis inhibitors Fer-1 and DFO can both inhibit ferroptosis induced by CVB3 infection.

Downregulation of TFRC inhibited ferroptosis in CVB3-infected HeLa cells

TFRC gene encoded the protein of TFR1, which is a type 2 membrane protein expressed as a homodimer in the cell membrane that constitute the main component of transferrin (Tf) [23]. In light of the TFR1 level has been demonstrated significantly increased in acute myocarditis patients and associated with augmented inflammation (CRP, IL-6) [11], we hypothesized that TFRC might be a key effector gene of pro-ferroptosis in CVB3 infection. To confirm the hypothesis, we searched in KEGG database and screened some candidate genes involved in iron overload-induced ferroptosis, then we analyzed these genes mRNA expression by qPCR (Supplementary Fig. 3A). The analysis identified the TFRC gene, which was upregulated more than fourfold in the CVB3-infected group compared to the mock group at 24 h postinfection (Fig. 4A). Consistently, western-blot analysis showed that TFRC protein levels were also increased after CVB3 infection (Fig. 4B, C). To evaluate the role of TFRC in iron overload, we treated HeLa cells with Fer-1 and DFO. As expected, these two drugs decreased TFRC expression detected by western-blot analysis (Fig. 4F, G). Of note, via immunofluorescence, we found that the subcellular localization of TFRC after CVB3 infection at different time points (Fig. 4E). TFRC began to be observed in the cell membrane. After 24 h of CVB3 infection, it gradually migrated around the nucleus, which was most obvious at 48 h postinfection. To verify this finding, we also did subcellular fractionation analysis using different internal references for nuclear membrane and cytoplasm in western-blot, the results were demonstrated nuclear translocation of TFRC in response to CVB infection in Fig. 4L, V. Thus, it could be inferred that TFRC may play an important role in CVB3-induced infection.

Fig. 4: TFR1 is the key gene leading to ferroptosis in CVB3-infected HeLa cells.
figure 4

A The mRNA level of TFR1 at different CVB3 infection times was detected via qPCR. The TFR1 mRNA increased obviously at 24 h of CVB3 infection (p < 0.001). B, C The protein level of TFR1 and relative gray values at different CVB3 infection times were measured via western-blotting. D TFR1 knockdown effects on three different sequences at the mRNA level via qPCR. E Determination of nuclear localization of TFR1 by immunofluorescence with CVB3 infection. The results showed that the TFR1 was translocated from the cell membrane to the nucleus with CVB3 infection. Scale bar = 10 μm. F, G The effect of ferroptosis drugs on TFR1 protein expression at 48 h post-CVB3 infection determined by western-blotting. H, I The image and gray values of TFR1 knockdown effects on three different sequences at the protein level via western-blotting. J Lipid ROS levels in siNC and siTFR1 groups were detected via immunofluorescence with BODIPY581/591C11 (scale bar = 100 μm). K The statistical results showed that the lipid ROS decreased significantly in siTFR1 group compared to siNC group (p < 0.001). L Subcellular fractionation analysis of TFR1 expressions in cell membrane, nuclear, cytoplasm using different cell component extraction kits and measured by western-blot. Na,K-ATPase, lamin b1 and β-actin were as internal references for cell membrane, nucleus, and cytoplasm in western-blot, respectively. M Cell viability in the siTFR1 group was obviously improved at 48 h post-CVB3 infection, measured by CCK-8 assays. N MDA levels were measured by MDA Assay Kit at a wavelength of 532 nm. It showed knockdown of TFR1 could reduce MDA levels compared to the wide-type group (p < 0.001). O The iron level in siTFR1 and NC groups were measured using an iron assay. It indicated that TFR1 knockdown could decrease cellular iron levels obviously. P, Q Effect of TFR1 knockdown on CVB3 fluorescence detected by IF. Cell nuclei were stained with DAPI (blue) (scale bar = 50 μm). R–T The mRNA levels of ferroptosis genes ACSL4, GPX4 and NCOA4 of TFR1 knockdown via qPCR. U, W The protein levels of major genes in the siTFR1 groups were detected via western-blotting. Corresponding of relative gray values of proteins were measured using ImageJ. V Relative gray values of subcellular fractionation analysis of western-blot strips in figure L measured by ImageJ. It suggested that membrane protein TFR1 was mainly located in membrane in mock group, however, it was partially transfered to nuclear induced by CVB3 infection, especially at 48 h postinfection.

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To confirm the role of TFRC in CVB3 infection, we generated TFRC-knockdown (TFRC-KD) HeLa cells using siRNA. Of the three pairs of siRNAs tested, siTFR1 was the most efficient in knock down of TFRC expression, as verified by qPCR and western-blotting analyses (Fig. 4D, H, I). Phenotypic detection of ferroptosis showed that, the siTFR1 group significantly decreased the lipid ROS compared to NC group (Fig. 4J, K). Additionally, the cell viability was improved by siTFR1 when CVB3 infected 48h, which detected by CCK-8 assay (Fig. 4M). MDA was reversed by TFR1 knockdown after CVB3 infection (Fig. 4N). Cellular iron levels was also lower in the siTFR1 group than siNC group (Fig. 4O). Moreover, we assessed the role of TFRC knockdown on CVB3 virus replication via IFA, the result revealed that the number of virus-infected cells in the siTFR1 group was significantly lower than that in the NC group at 24 h CVB3 postinfection (p < 0.001, Fig. 4P, Q). Next, we examined whether downregulation of TFRC affected typical ferroptosis genes through q-PCR and western-blot. As shown in Fig. 4R, U, W, siTFR1 decreased ACSL4 both at the mRNA and protein level, enhanced GPX4 mRNA and protein levels in CVB3-infected group compared to wild-type group (Fig. 4S, U, W). In non-infected HeLa cells, siTFR1 lowered NCOA4 expression but not ACSL4 and GPX4 mRNA expression compared to mock group (Fig. 4T, U, W). SLC7A11 is a negative regulator of ferroptosis and was upregulated at the protein level in the siTFR1 group (Fig. 4U, W). Taken these together, our results has demonstrated that TFRC is a key gene in ferroptosis and inhibition of TFRC can downregulate ferroptosis in CVB3-infected HeLa cells. Moreover, we found TFRC expression was translocated to nuclear with CVB3 infection by IFA and subcellular analysis.

Sp1 regulates TFRC transcription in HeLa cells by binding to the TFRC promoter region

As shown previously in Fig. 4A, the expression of TFRC mRNA was remarkably upregulated, and a change in TFRC sub-localization to the nucleus was induced by CVB3 (Fig. 4E, L), it inferred that the TFRC was activated at the transcriptional level. To verify our hypothesis, we first searched for the TFRC promoter region in the UCSC Genome database, and a segment −2000 to +200 bp from the transcription start site (TSS) was identified (Fig. 5A). Then, to determine the function of this region, we generated a plasmid containing the −2000 to +200 fragment fused to a luciferase reporter vector as a TFRCp luciferase reporter gene (Fig. 5B, C). As shown in Fig. 5D, according to a dual-luciferase reporter assay, the activity of the TFRC promoter was more than twofold that in the CVB3-infected group compared to the mock group (p < 0.001).

Fig. 5: TFRC transcriptional regulation.
figure 5

A Map of the promoter location in the TFRC gene. B Construction map of the TFRC promoter plasmid. C Construction map of the firefly luciferase reporter plasmid. D The activity of the TFRC promoter in the CVB3 group was significantly higher than that in the mock group based on luciferase reporter assay results. E mRNA level of Sp1 was consistent with that of TFRC at various CVB3 infection times by qPCR. F Effect of Sp1 knockdown and overexpression at the mRNA level determined by qPCR. G The influence of Sp1 upregulation and downregulation on TFRC mRNA expression via qPCR. H, I The effect of Sp1 upregulation and downregulation on TFRC protein expression. J Downregulation of Sp1 reduced the activity of the TFRC promoter by Luciferase Reporter Assay. K Upregulation of Sp1 enhanced the activity of TFRC promoter by Luciferase Reporter Assay. L Chromatin from HeLa cell lysis buffer was subjected to agarose electrophoresis before and after sonication in the ChIP experiment. M The three potential binding sites (BS) for Sp1 within the promoter region of TFRC predicted by JASPAR (http://jaspar.genereg.net/). N, O The binding sites for the transcription factor Sp1 in the TFRC promoter were verified via ChIP-PCR agarose gel electrophoresis (white arrows—indicate binding sites). The input group included total chromatin samples, and IgG served as the negative control. In the IP- Sp1group, the results showed that objective bends (150 bp) only emerged at BS1. All results are expressed as the mean ± SD. *p < 0.05 vs. the mock group, ** p < 0.01, ***p < 0.001. n = 3.

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To further explore which transcription factors interact with the TFRC promoter, we used the JASPAR database (http://jaspar.genereg.net/) for prediction. The TFRC promoter region harbor abundant GC boxes. The transcription factor Sp1 was selected for further verification due to its high expression in mRNA levels measured by qPCR in high binding score transcription factors predicted by JASPAR database (Supplementary Fig. 4A). As shown in Fig. 5E, the expression of Sp1 mRNA was increased along with the increase in TFRC mRNA expression. The data verified that Sp1 overexpression increased the mRNA and protein levels of TFRC; in contrast, Sp1 siRNA decreased TFRC mRNA and protein levels (Fig. 5F, G, H). These results demonstrate that Sp1 regulates TFRC expression at the transcription level.

To address whether Sp1 affects the promoter activity of TFRC, we performed a luciferase reporter assay. Compared to the empty vector, cotransfection with TFRC-Luc and the Sp1 overexpression plasmid resulted in an increase in promoter activity (Fig. 5K), and Sp1 siRNA lowered TFRC promoter activity (Fig. 5J). To confirm the exact position of the Sp1-binding site (BS) in the TFRC promoter, we assessed the three GC box motifs (BS1, BS2, BS3, Fig. 5M) using a ChIP assay. The sheared DNA lengths (200–2000 bp) were confirmed by agarose gel electrophoresis (Fig. 5L). As shown in Fig. 5N, O, based on the ChIP- PCR results, in both non-infected and CVB3-infected cells, Sp1 bound to the BS1 region (tgggcatggt) located at −229 to −238 bp but not to BS2 and BS3 on the TFRC promoter. These results suggest that Sp1 directly activates TFRC transcription by binding to a specific locus on the TFRC promoter.

The protective effect of downregulation of the Sp1/TFRC/Fe axis on ferroptosis

Following exposure to CVB3 infection, compared to mock group, the mRNA level of Sp1 increased from 24 h postinfection (Fig. 6A), while the Sp1 protein level increased from 36 h postinfection (Fig. 6B, C). To confirm that CVB3-induced ferroptosis as mediated by Sp1, HeLa cells were transfected with Sp1 siRNA and overexpression vectors and then exposed to CVB3. Similar to the effect of siTFRC, Sp1 downregulation had a protective effect against CVB3-induced ferroptosis, which was reflected by the increase in cell viability (Fig. 6E), reduced MDA levels (Fig. 6G), inhibition of lipid ROS accumulation (Fig. 6J, K) and decreased the iron level (Fig. 6H). Conversely, Sp1 overexpression exhibited a pro-ferroptotic effect, which was evidenced by decreased cell viability (Fig. 6F) and increased levels of MDA (Fig. 6G), elevated cellular iron level (Fig. 6I). Unexpectedly, siSP1 had no obvious reducing effect on virus replication, but in the Sp1 overexpression group, the underlying mechanism deserves further study (Fig. 6D). Notably, we measured the landmark of ferroptosis molecular ACSL4 and GPX4 in the siSP1group at 48 h post CVB3 infection. In line with the effect of siTFR1, Sp1 silencing lowered ACSL4 mRNA and protein levels and increased the GPX4 levels (Fig. 6M, N, O). Conversely, over-expressing increased ACSL4 mRNA and protein compared to NC group (Fig. 6L, P, Q). Taken together, the results demonstrate that transcription factor SP1 is increased in CVB3 infection. It works by increasing the transcription of TFRC and associate with ferroptosis. Reduction of SP1 exerting beneficial effects on ferroptosis in CVB3 infection.

Fig. 6: Effect of the transcription factor Sp1 on ferroptosis during CVB3 infection.
figure 6

A The mRNA level of Sp1 at different CVB3 infection times was detected via qPCR. B The protein level of Sp1 at different CVB3 infection times was recorded via western-blotting. C Measurement of the relative gray values of Sp1 in the WB analysis was conducted using ImageJ. D CVB3 fluorescence in the SP1 knockdown and overexpression groups was determined via IFA. Cell nuclei were stained with DAPI (blue) (scale bar = 50 μm). E Cell viability in the SP1 knockdown group was obviously increased at 48 h postinfection, based on CCK-8 assay results. F Cell viability in the Sp1 overexpression group was also decreased at 48 h postinfection. G MDA concentrations were determined with Lipid Peroxidation MDA Assay Kit in the Sp1 knockdown and overexpression groups. H, I Iron concentrations were measured in the SP1 knockdown and overexpression groups using an Iron Assay Kit. J The fluorescescence of lipid ROS in the SP1 knockdown group was detected using the BODIPY581/591C11 probe (scale bar = 100 μm). K Percentage of oxidized/non-oxidized cells were calculated by ImageJ software according to figure J. It suggested that siSP1 could decreased the oxidized lipid ROS significantly (p < 0.001). L, M The effect of Sp1 overexpression and knockdown on ACSL4 and GPX4 mRNA expression was determined by qPCR. N, O Relative gray value and images of western-blotting in the effect of knockdown Sp1 on ACSL4 and GPX4 protein expression. P, Q Images of western-blotting and relative gray values in the effect of overexpression Sp1 on ACSL4 and GPX4 protein expression. R Schematic illustrating the mechanism by which the Sp1/TFRC/Fe2+ cascade facilitates ferroptosis in CVB3-infected HeLa cells. All results are expressed as the mean ± SD. ANOVA was used for comparison between groups. *p < 0.05, **p < 0.01, ***p < 0.001.

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In summary, through the Sp1/TFRC/Fe axis (Fig. 6R), CVB3 infection activates the transcription factor Sp1, which targets the TFRC, leading to an increase in TFRC transcription, enhancement of TFR1 protein expression and an increase in iron pumped into cells. Finally, oxygen-free radicals and lipid peroxide levels are greatly increased, and ferroptosis occurs.

Discussion

CVB3 is the predominant pathogen of viral myocarditis in children. Viral replication induces direct cell injury including cytoskeletal disruption and triggers uncontrolled inflammation even after viral clearance [24]. Therefore, an effective therapeutic strategy must target both host cells and the virus simultaneously. A soaring number of studies on ferroptosis and metabolism have revealed exciting insights into the molecular and metabolic mechanisms of cell death [25]. Thus, we hypothesized that CVB3 infection may be an important process facilitating the occurrence of ferroptosis. To validate this hypothesis, in this study, we established a CVB3 infection model in vivo and in vitro, measured the levels of ferroptosis markers: cellular iron, lipid peroxide (lipid ROS or MDA), and examined the expression levels of core ferroptosis genes. Notably, in vitro, the time of cell viability decline was consistent with the peak time of virus infection. We found the level of ferrous iron, lipid peroxide were all elevated by CVB3 infection. Acyl-CoA synthetase long-chain family member 4 (ACSL4) and glutathione peroxidase 4(GPX4) are two main mark genes in ferroptosis. ACSL4 dictates ferroptosis sensitivity by sha** cellular lipid composition and is a sensitive pro-ferroptotic gene. The GPX4 utilizes reduced glutathione (GSH) to reduce lipid hydroperoxides to lipid alcohols, protect cells against membrane lipid peroxidation and inhibit ferropotsis [26]. In our study, the pro-ferroptotic genes ACSL4 increased and anti-ferroptotic genes GPX4 decreased (Figs. 1 and 2). These suggested that ferroptosis is activated during CVB3 infection.

Next, we used classical ferroptosis inhibitors ferrostatin-1 (Fer-1) and deferoxamine (DFO), we found the increased iron level, upregulated lipid peroxide and ACSL4 could all be rescued by Fer-1 and DFO in the ferroptosis induced by CVB3 infection. The mechanism by which Fer-1 prevent cell death has been ascribed to its ability to inhibit lipid peroxidation directly by trap** chain-carrying radicals [27]. During the ferroptosis pathway, Fe, could contribute to the ROS pool in the cell through Fenton reaction in which Fe catalyzes the breakdown of H2O2 to yield hydroxyl radicals. Hence, Fer-1 as a radical-trap** antixodiandts, could inhibit the last step of ferroptosis pathway to slow accumulation of lipid hydroperoxides caused by iron-overload (Fig. 3C, D, F). During the intrinsic pathway of ferroptosis, the expression or activity of intracellular antioxidant enzymes GPX4 is blocked [28]. In our study, we found two ferroptosis inhibitors especially Fer-1 could increase the GPX4 expression, which was consistent with the reports in literature (Fig. 3K, L). Li et al. [29] reported that in diabetic nephropathy mice, iron overload and decreased GPX4 expression were obviously rescued by Fer-1. Liu et al. [30] also found Fer-1 could rescue the downregulated GPX4 in a dose-dependent manner in LPS-induced acute lung injury model in vitro and in vivo. It is mainly dependent on the Fer-1 scavenging of initiating alkoxyl radicals, and the inhibition of the GPX4 activity was relieved. The iron chelator DFO has a significant improvement in life expectancy of thalassemic patients with transfusional iron overload clinically [31]. As results in other experiments [32], we found DFO markedly inhibited increase of ferrous iron, thus rescue the ferroptosis (Fig. 3D).

To further analysis the changes in the metabolites that regulation in ferroptosis with CVB3 infection, we measured the anti-toxisis GSH with infected time points. GSH acts as a regulator of cellular redox state to protect cells from damage caused by lipid peroxides, reactive oxygen and xenobiotics [33]. It is interesting to find that the cell death number was declined at 36 h of infection (Fig. 2C), the lipid peroxide MDA (Fig. 2D), ferrous iron (Fig. 2F), and ACSL4 (Fig. 2H) were all attenuated at 36 h postinfection in accordance with GSH peak level. These indicated that when CVB3 invades and releases harmful substances like lipid peroxide in cellular ferroptosis, host cell initiate GSH as a negative feedback to regulate. However, with imbalance of GSH regulation, the promote ferropotsis factors were increased by CVB3 virus release. At 48 h after CVB3 infection, intracellular free iron reached a peak (Fig. 2F).

As a pivotal indicator and regulator of iron, TFR1 which encoded by TFRC as a critical determinant of ferroptosis sensitivity by modulating cellular iron uptake. TFR1 imports iron from the extracellular environment into cells, contributing to the cellular iron pool required for ferroptosis [34]. In our study, we found TFRC mRNA and protein expression were increased in CVB3 infection model in vivo and in vitro (Figs. 1I, J and 4A, B). To further confirm the function of TFRC in CVB3 infection, we designed siRNA to genetically inhibit TFRC in vitro. TFRC knockdown prevented CVB3-induced ferroptosis in HeLa cells(decreased iron overload, reduced lipid peroxidation, ACSL4 and NCOA4 downregulation, and GPX4 upregulation). Much evidence in reports has shown that TFRC plays an important role in ferroptosis. In an SD heart ischemia/reperfusion (I/R) model, knockdown of TFRC inhibited H/R-induced ferroptosis without p53 deubiquitination [35]. Some researchers have also demonstrated that TFRC-deleted mice exhibit attenuated renal fibrosis, along with reduced renal expression of ferritin and 4-hydroxynonenal, compared with WT mice [36]. During malaria infection, TFRC knockdown decreased parasite-localized lipid peroxides, and led to a substantial decrease in the efficacy of erastin in liver-stage parasite infection [37].

In present study, our team first demonstrated the TFRC was translocation to nuclear with CVB3 infection by IFA and subcellular analysis in western-blot (Fig. 4E, L). Large evidence showed that TFRC can act as a target for infectious diseases because various pathogens use TFRC to enter cells [38, 39]. For example, some New World arenaviruses, which infect humans and cause life-threatening hemorrhagic fever, use TFRC for entry into host cells [40]. Hepatitis C virus also utilizes TFRC for entry in human hepatocytes, and knockdown of TFRC inhibits hepatitis C virus infection in human hepatoma cells [41]. A recent study used erastin to induce lymphoma cell ferroptosis and found that TFRC staining was localized to the plasma membrane and to a perinuclear region associated with the Golgi and the endosomal recycling compartment (ERC) [9]. These all indicate that TFRC could translocation to cytoplasm or nuclear. In our experiment, we further found TFRC recruited to nuclear by a nuclear transcription factor Sp1. Sp1 is a member of the zinc finger family, which bind to specific DNA sequences located in core gene promoter, and interact with the RNA Pol II complex to enhance gene transcription [42]. It is implicated in a variety of essential biological processes, such as cell growth, differentiation, carcinogenesis and apoptosis [43]. In our study, we verified that Sp1 can regulate TFRC transcription and validated that the binding site was located at −229 to −238 bp relative to the TSS using a ChIP assay (Fig. 5D, O). We found that SP1 overexpression upregulated TFRC expression, raised cellular iron levels, increased lipid peroxidation products, and aggravated ferroptosis (Fig. 6F, G, I). Downregulating Sp1 (siSP1) decreased TFRC expression, and rescued the ferroptosis induced by CVB3 infection (Fig. 6E, G, H), which was similar to the effects of siTFRC. This suggests that Sp1/TFRC/Fe forms a functional axis that regulates ferroptosis during CVB3 infection.

Conclusion

Our study revealed that ferroptosis has an important role in CVB3 infection. Inhibition of ferroptosis by Fer-1, DFO or genetic interference could rescue the cell death and reduce virus replication. Mechanistically, the key iron regulation gene TFRC was enhanced by CVB3 infection, leading to the release of large quantities of free iron, which promoted lipid peroxidation and ferroptosis. TFRC expression was activated by the transcription factor Sp1, which bound to a specific site in the TFRC promoter and increased TFRC transcription. Based on these data, we hypothesize that targeting the Sp1/TFRC/Fe pathway may be a potential therapeutic strategy against CVB3-induced injury in the future.