Abstract Background Ischemia/reperfusion (I/R) is an inevitable pathophysiological process during heart transplantation, and ferroptosis is an important pathogenic mechanism. Unlike other modes of cell death, ferroptosis depends on the accumulation of iron within the cell and the oxidative degradation of polyunsaturated fatty acids. Dysregulation of this pathway has been linked to the progression of multiple pathological conditions, making it an attractive target for therapeutic intervention. Therefore, this study aims to explore the effect of ferroptosis on I/R during heart transplantation. Methods GEO2R was applied to identify differentially expressed genes (DEGs) obtained from [42]GSE50884 data, which was involved in I/R and heart transplantation. And ferroptosis-related DEGs (FRDEGs) were screened by venn diagram with ferroptosis-related genes downloaded from FerDb database. FRDEGs was enriched and analyzed by GO and KEGG, and hub genes related to ferroptosis were screened by Cytoscape software and database STRING. Additionally, considering the relationship between ferroptosis and immunity, CIBERSORTx was to analyze the infiltration of 22 kinds of immune cells in I/R during heart transplantation, and the correlation between each immune cell and the expression of FRDEGs was also discussed. Finally, the mouse model of heart transplantation with I/R was constructed, and the hub genes was verified by RT-qPCR and western blot. Results 12 FRDEGs were identified out of 327 DEGs in [43]GSE50844, which were mainly involved in ferroptosis and other pathways. Three hub genes (SLC7A11, PSAT1, ASNS) were obtained by the degree algorithm of cytohubba plug-in. Immunoinfiltration analysis showed that 16 of 22 immune cells changed, and the immune score of heart transplantation with I/R was higher than that without I/R. In addition, hub genes exhibited significant correlation with Eosinophils, NK cells resting, Dendritic cells resting, NK cells activated and T cells CD4 memory activated. We verified the expression of SLC7A11, PSAT1 and ASNS was higher than that in normal tissues using RT-qPCR and western blot in mouse models of heart transplantation with I/R, companied by ferroptosis aggravated is involved. Conclusions In short, ferroptosis is involved in I/R injury during heart transplantation, which is related to immune cell infiltration. Three hub genes (SLC7A11, PSAT1 and ASNS) identified in this study provide therapeutic targets for ameliorating I/R injury in heart transplantation. Supplementary Information The online version contains supplementary material available at 10.1186/s12872-024-04462-1. Keywords: Heart transplantation, Ischemia/reperfusion, Ferroptosis, Immune cells, Hub genes Introduction Since the first heart transplantation was successfully performed in 1967 by Dr. Christiaan Barnard in South Africa, heart transplantation has emerged as the most effective treatment for end-stage heart disease [[44]1]. With the extensive research and exploration in the field of immunosuppressant related drugs, huge progress has been made in mitigating the failure rate caused by transplant rejection [[45]2]. However, the inevitable problem is gradually being discovered in the field of heart transplantation with cold I/R injury. After preserved in a cold solution, the donor heart undergoes a complex process of reconstruction and reperfusion when transplanted into the recipient’s body, while the sudden influx of oxygen-rich blood can trigger a cascade of inflammatory reactions and oxidative stress, further compromising the transplanted heart [[46]3, [47]4]. Ferroptosis, a recently discovered pattern of programmed cell death, has been shown a significant impact on the occurrence and progression of athophysiological processes of I/R injury in different organs, such as stroke, heart disease and renal degeneration [[48]5]. Owing to the unique roles of iron overload and lipid peroxidation in myocardial I/R injury, ferroptosis may be a therapeutic mechanism and potential cause for such ischemic diseases [[49]6]. Previous studies have shown that ferroptosis inhibitors and iron chelators can reduce infarct size and alleviate myocardial fibrosis after myocardial I/R [[50]7]. In the process, iron accumulation, glutathione metabolism and lipid peroxidation are involved in the molecular mechanism of ferroptosis in cardiomyocytes [[51]8]. Studies have conducted experiments on animal models of I/R [[52]9] as well as analyzed heart tissue samples obtained from human patients with ischemic cardiomyopathy [[53]10], which indicate that increased production of ferroptosis-associated reactive oxygen species worsens myocardial damage [[54]11]. Inhibition of ferroptosis can protect myocardial I/R injury and restore cardiac function [[55]12]. Additionally, immune response is of great significance in the initiation and progression of various cardiovascular diseases [[56]13]. Currently, all major immune cell subtypes, including lymphocytes and myeloid populations, are found in cardiovascular tissue [[57]14]. Following ischemia injury, both innate and adaptive immunity critically regulate the clearance of injured cells and repair responses [[58]15]. A prospective study has found that diseases of the immune system may affect cardiovascular health [[59]16]. During heart transplantation and myocardial I/R, ferroptotic cardiomyocytes release damage-associated molecular patterns that recruit more neutrophils through the involvement of innate immune receptors (such as TLR), triggering aseptic inflammation of damaged myocardial tissue [[60]17]. A study in nonalcoholic steatohepatitis suggests that ferroptosis, rather than necrotic apoptosis, is the primary trigger of inflammation, and that inhibiting ferroptosis can reduce subsequent immune cell invasion and inflammatory response [[61]18]. Besides, follicular helper T (TFH) cells, a specific subset of CD4^+ T cells, which are sensitive to ferroptosis, and selenium supplementation enhances GPX4 expression, increases TFH cell count and promotes antibody response in immunized mice and young adults after influenza vaccination [[62]19]. In summary, understanding the intricate relationship between ferroptosis, immune response, and tissue repair mechanisms can potentially lead to the development of innovative therapeutic strategies for improving outcomes in patients with heart transplantation. Owing to the internal mechanism of ferroptosis in cold I/R injury of heart transplantation has not been clarified, it is necessary to analyze the differentially expressed genes (DEGs) and immune cell infiltration related to ferroptosis in the context of cold I/R injury of heart transplantation, so as to provide new therapeutic ideas for reducing I/R injury of heart transplantation. Materials and methods Data collection As shown in Fig. [63]1, the Gene Expression Omnibus (GEO, [64]https://www.ncbi.nlm.nih.gov/geo/) database was used to search gene microarray date of heart transplantation samples. The [65]GSE50884 dataset, containing 2 without cold ischemia (control group) and 3 with cold ischemia (I/R group) heart tissue sample were acquired. The final 564 ferroptosis-related genes, including ferroptosis markers, drivers, suppressors and unclassified genes, were retrieved from FerrDb online database ([66]http://www.Zhounan.org/ferrdb/current/). Fig. 1. [67]Fig. 1 [68]Open in a new tab Flow diagram. DEGs: differentially expressed genes; ferroptosis-related DEGs: differentially expressed genes; FRDEGs: GEO: Gene Expression Omnibus; GO: gene ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes Identification of ferroptosis-related differentially expressed genes (FRDEGs) The gene expression matrix of [69]GSE50884 dataset were screened and identified according to GEO2R ([70]https://www.ncbi.nlm.nih.gov/geo/geo2r) program. DEGs is defined as |log2FoldChange|>=1 and P < 0.05. FRDEGs is defined as DEGs overlapped with ferroptosis-related genes in [71]GSE50884 and visualized by venn diagram ([72]https://bioinformatics.psb.ugent.be/webtools/Venn/). The ggplot2 package ([73]https://ggplot2.tidyverse.org) and pheatmap package ([74]https://CRAN.R-project.org/package=pheatmap) in R software are applied to generate volcano plots and heatmaps of DEGs and FRDEGs. Functional enrichment analysis of FRDEGs FRDEGs are applied for gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. GO is composed of three categories: biological process, cellular component and molecular function, which is carried out by the R package “clusterProfiler” based on the GO annotation in the R package “org. Mm. egg. db”. KEGG analysis was applied to determine the related signal pathway of FRDEGs, which was carried out by R package “clusterProfiler”. P < 0.05 is statistically significant. R package “ggplot2” draws visual graphics. Protein–protein interaction (PPI) network construction The Protein-Protein Interaction Network of FRDEG was visualized by the STRING database ([75]https://string-db.org/) in order to obtain the diagram. The results obtained in the online database of STRING were imported into Cytoscape v 3.9.1 software ([76]http://cytoscape.org/), and then the overlap of the top genes was identified by CytoHubba plug-in, and the top three significant connection nodes were selected as ferroptosis-related hub genes according to the degree algorithm for further analysis. The degree algorithm is based on the degree of nodes, which refers to the number of connections each node has. The higher the degree, the more connections a node has, making it more important in the network. Correlation analysis of ferroptosis-related hub genes The interaction of three hub genes related to ferroptosis was studied by employing “ggstatsplot” package on R software. Immune infiltration and its score CIBERSORTx ([77]https://cibersort.stanford.edu/) deconvolution algorithm can be calculated according to the RNA matrix in proportion to the body’s immune cells. Immune score was calculated by ESTIMATE algorithm to predict immune cell level. The above results are visualized in R software through box diagram, bar diagram and heat diagram. More importantly, spearman correlation analysis was employed to analyze the relationship between hub genes and immune cell infiltration. Experimental animal and heart transplantation C57BL/6J mice (6-8weeks) were purchased from Hunan An Sheng Mei Pharmaceutical Research Institute Co.,Ltd (Hunan, China) and housed in the Animal Experiment Center of Renmin Hospital of Wuhan University. All experimental protocols were approved by the Medical Faculty Ethics Committee of Wuhan University (Permit Number:20230411B). Heterotopic heart transplantation was performed using a modified non-suture cuff technique [[78]20, [79]21]. Briefly, the donor was given heparin (1000 U/kg) and induced cardiac arrest by the injection of ice-cold, high-potassium cardioplegia solution into the aortic root. The mice (n = 6 per group) were transplanted immediately as control group, or stored in the same University of Wisconsin (UW) solution for 18 h according to the I/R groups [[80]22]. The donor heart was heterotrophically transplanted to the neck vessels of the recipient, and the reperfusion continued for 24 h after the opening of blood flow. After successfully establishing the heart transplantation model, the mice were continuously fed for 12 weeks, then anesthetized and sacrificed with 40 mg/kg body weight sodium phenobarbital (i.p.). The ferroptosis inhibitor, Ferrostatin-1 (Fer-1, Med Chem Express, cas no.347174–05 − 4) was administered intraperitoneally to mice to limit the development of ferroptosis. Beating score At the end of 24 h reperfusion, we using the Stanford cardiac surgery laboratory graft scoring system (0: No contraction, 1: Contraction barely visible or palpable, 2: Obvious decrease in contraction strength, but still contracting in a coordinated manner, 3: Strong coordinated beating but noticeable decrease in strength or rate, 4: Strong contraction of both ventricles, regular rate). Measurement of MDA and SOD levels After successful modeling, myocardial samples were collected. Kits for detecting SOD activities and MDA content were purchased from the Institute of Nanjing Jian Cheng BioEngineering Institute. These myocardial biomarkers were detected with an automated analyzer (Chemray 240/800, Shenzhen, China). Hematoxylin and eosin staining For histological analyses, the donor heart was isolated from the receptor’s neck 24 h after reperfusion and rinsed with phosphate-buffered saline. Then, the cardiac tissues were fixed with 4% buffered paraformaldehyde and embedded in paraffin. These tissues were subsequently sectioned into 4–6 μm slices, dewaxed in xylene, rehydrated with graded alcohol, and stained with hematoxylin and eosin (HE) according to the standard protocol. The morphological structure of each sample was examined under a light microscope, and photomicrographs were taken at 400×magnification (Olympus IX51, Japan). Immunohistochemistry staining Dewaxed paraffin sections were placed in 0.01 M citrate buffer (pH 6.0) and heated for antigen repair. The tissues were incubated at 4 ℃ overnight with the GPX4 (1: 200, A1933, Abclonal) primary antibody. After 40 min of incubation with the secondary antibody (1:100, AS-1109, Aspen, Californian, USA) at 37 ℃ in the dark, the myocardial tissues were stained with DAPI for 20–30 min at room temperature in the dark. Four non-overlapping fields of view in the peri-infarct region of each mouse were randomly selected under a microscope. The magnification light microscope (Olympus, Tokyo, Japan) was used to take images at ×200 fields. RNA extraction and quantitative real-time PCR (RT-qPCR) Total RNA was extracted from the myocardial samples and cell using Trizol reagent. Two micrograms of RNA from each sample were then reversed transcribed into cDNA according to the Prime-Script RT reagent kit instruction (Servicebio, Hubei, China). The RT-qPCR was performed using a SYBR Green qPCR Reagent Kit (Servicebio, Hubei, China) by Bio-Rad CFX Connect Real-Time PCR Detection System (Bio-Rad, USA). The used primers are listed in Table [81]1. The mRNA levels were normalized to β-actin mRNA level. The expression of genes was analyzed by using the 2^−ΔΔCT method. Table 1. Primers for real-time qPCR Gene Forward primer (5′-3′) Reverse primer (5′-3′) Slc7a11 GGTTGTCTCCTGCGACTTCA GGTGGTCCAGGGTTTCTTACTC Tfrc GTTTCTGCCAGCCCCTTATTAT GCAAGGAAAGGATATGCAGCA Psat1 CGGTTTCTTGACAAGGCGGTAG TCTTCGGTTGTGACAGCGTTATAC Asns CAAAGTTGCGTCTGTGGAAATGG GATTGTTCTTCACGGTCTCTAGGTC Gpx4 GCCTGGATAAGTACAGGGGTT CATGCAGATCGACTAGCTGAG Acsl4 CTCACCATTATATTGCTGCCTGT TCTCTTTGCCATAGCGTTTTTCT β-actin TATGCTCTCCCTCACGCCATCC GTCACGCACGATTTCCCTCTCAG [82]Open in a new tab Western blot and band quantitation After treated with different stimulus, cardiac tissues were lysed in ice-cold radio immunoprecipitation assay buffer containing protease inhibitor phenylmethylsulfonyl fluoride and phosphatase inhibitor cocktail (Beyotime, Jiangsu, China) for 20 min, and then centrifuged at 12,000 rpm at 4 °C for 15 min to obtain supernatants. A Pierce BCA Protein Assay Kit (ThermoFisher Scientific) was used to measure the concentration of proteins. Equal amount of protein lysates was loaded into a 5-15% SDS-PAGE geland transferred to polyvinylidene difluoride membrane. Next, the membranes were incubated in protein free rapid blocking buffer (Epizyme Biomedical Technology, Shanghai, China). Primary antibodies were incubated with the membranes overnight at 4 °C, including β-actin (1:1000, GB15001, Abclonal), SLC7A11 (1:1000, A2413, Abclonal), ASNS (1:1000, A1030, Abclonal), PSAT1 (1:1000, 10501-1-AP, Proteintech), GPX4(1:1000, A1933, Abclonal). After 3 cycles of 10-minute washing in TBS/0.1% Tween-20 (0.1% TBST), the membranes were incubated with fluorescent secondary antibody (1:5000, SA00001-2, Proteintech) for 1 h at room temperature. Then, the membranes were washed again with TBST for 3 times, 5 min of each time. The biological image analysis system was used for the final analysis (Bio-Rad, USA). Each sample’s β-actin levels were used to determine the relative expression levels of the target proteins. Statistical analysis All results were analyzed using Graphpad Prism 9.0 software (GraphPad Software, USA) and presented as means ± standard error of mean (SEM). Statistical analysis was carried out using one-way analysis of variance and P < 0.05 was deemed significant. Results Identification of cross-talk genes in DEGs and FRDEGs After standardization of microarray results, our study found a total of 327 DEGs were identified between heart transplantation with or without cold I/R in [83]GSE50884, of which 113 genes were up-regulated and 214 genes were down-regulated. As shown in Fig. [84]2A, the genes |log2FoldChange| ≥ 1 and P < 0.05 are identified as DEGs, whose heat map is shown in Fig. [85]2B. To explore FRDEGs differentially expressed in heart transplantation, 564 ferroptosis-related genes downloaded from FerrDb, a database of regulators, markers, and diseases involved in ferroptosis. A total of 14 FRDEGs were identified after taking the crossover of DEGs and FerrDb according to venn diagram (Fig. [86]2C). The heat map of 14 FRDEGs are shown in Fig. [87]2D and they are divided into three categories: driver, supressor and unclassified (Fig. [88]2E). Fig. 2. [89]Fig. 2 [90]Open in a new tab FRDEGs in ischemia/reperfusion (I/R) during heart transplantation. (A) Volcano plot of [91]GSE50884. (B) Heat map of DEGs. (C) Venn diagram of DEGs and ferroptosis-related genes. (D) Heat map of FRDEGs. (E) Classification of FRDEGs PPI network and identification of ferroptosis-related hub genes The PPI network of FRDEGs, which consists of 14 nodes and 12 edges, is obtained on the online website of STRING (Fig. [92]3A). According to the degree algorithm, the top three FRDEGs (SLC7A11, PSAT1, ASNS) were selected as hub genes (Fig. [93]3B and C). The chord diagram of correlation analysis among ferroptosis-related hub genes is shown in Fig. [94]3C. As shown in Fig. [95]3D-F, the correlation coefficients of PSAT1- SLC7A11, ASNS - SLC7A11 and PSAT1- ASNS are 0.76, 0.87 and 0.97, respectively. However, only PSAT1-ASNS had statistical significance (P < 0.05) and its correlation coefficient greater than 0.7, indicating a close correlation. Fig. 3. [96]Fig. 3 [97]Open in a new tab Identification of ferroptosis-related hub Genes. (A) PPI network of FRDEGs. (B) Hub gene obtained by CytoHubba. The redder the color, the higher the degree score. (C) Calculation results of CytoHubba plugin. Correlation analysis of (D) PSAT1- SLC7A11, (E) ASNS - SLC7A11 and (F) PSAT1- ASNS Functional and pathway enrichment analysis of cross-talk genes In order to explore the function and related ways of 14 FRDEGs as shown in Fig. [98]4A, we carried out GO and KEGG enrichment analysis. As shown in Fig. [99]4B (P < 0.05), biological processes include alpha − amino acid metabolic process, response to oxidative stress, oligopeptide transport, etc. As shown in Fig. [100]4C (P < 0.05), cellular component includes mitochondrial outer membrane, organelle outer membrane, peroxisomal membrane, etc. As shown in Fig. [101]4D (P < 0.05), molecular functions include antioxidant activity, L-amino acid transmembrane transporter activity, organic acid transmembrane transporter activity and so on. Most importantly, the KEGG pathway with statistical differences included only ferroptosis (Fig. [102]4E; P < 0.05). Fig. 4. [103]Fig. 4 [104]Open in a new tab GO and KEGG enrichment analysis of FRDEGs. (A) Statistical parameters (logFC and P.value) for 14 FRDEGs. (B) Biological Process. (C) Cellular component. (D) Molecular function. (E) KEGG pathways Analysis of immune cell infiltration In this study, the properties of 22 immune cell types between heart transplantation with or without I/R were obtained by identifying immune cell types via CIBERSORTx (Fig. [105]5A). Immune cells with obvious changes include B cells memory, T cells CD8, T cells CD4 naïve, T cells CD4 memory activated, T cells follicular helper, NK cells resting, NK cells activated, Macrophages M0, Macrophages M1, Macrophages M2, Dendritic cells resting, Dendritic cells activated, Mast cells resting, Eosinophils, Neutrophils, Monocytes (Fig. [106]5B). The correlation heatmap of each immune cell is shown in Fig. [107]5C. The closer the absolute value of the correlation coefficient is to 1, the more relevant the immune cells are. In addition, heart transplantation with I/R has a higher immune score than that without I/R (Fig. [108]5D). Fig. 5. [109]Fig. 5 [110]Open in a new tab Immune cell landscape in I/R during heart transplantation. (A) Stacked bar chart of 22 kinds of immune cells. (B) Box diagram of immune cells. (C) Correlation heatmap of immune cells. (D) ESTIMATE Immune Score Relationships between ferroptosis-related hub genes and immune infiltration In this study, the correlation efficiency between immune cells and ferroptosis-related hub genes in heart transplantation with or without I/R was estimated to study their relationship and potential interaction. SLC7A11 is negatively and positively correlated with NK cells resting or NK cells activated, respectively (Fig. [111]6A and B; P < 0.05). PSAT1 and ASNS are both positively correlated with Eosinophils or Dendritic cells resting (Fig. [112]6D-E and J-I; P < 0.05). The absolute values of their correlation coefficients are all greater than 0.7, which means that the correlation is good. In addition, SLC7A11, PSAT1 and ASNS were negatively correlated with T cells CD4 memory activated, and the correlation coefficients were all − 0.894 (Fig. [113]6C, G and K; P < 0.05). Therefore, these genes were strongly correlated with immune-infiltrated cells in heart transplantation. Fig. 6. [114]Fig. 6 [115]Open in a new tab Correlation analysis between ferroptosis-related hub gene and immune cells. (A) SLC7A11 and NK cells resting. (B) SLC7A11 and NK cells activated. (C) SLC7A11 and T cells CD4 memory activated. (D) ASNS and Eosinophils. (E) ASNS and Dendritic cells resting. (F) ASNS and T cells CD4 memory activated. (G) PSAT1 and Eosinophils. (I) PAST1 and Dendritic cells resting. (J) PAST1 and T cells CD4 memory activated Inhibition of ferroptosis attenuated myocardial cold I/R injury following heterotopic heart transplantation The myocardial injury and cardiac function of the donor heart were evaluated by beating score and pathological HE staining. The results were observed that the myocardial myofibrillar loss, myocardial cell necrosis, increased vacuolation, and abnormal structure in I/R group compared with the control group (Fig. [116]7C), and the donor heart graft beating score was decreased (Fig. [117]7E). In the I/R + Fer-1 group, the beating score was increased and the myocardial myofibrillator, the myocardial cell necrosis was alleviated, the structure was recovered than in I/R group. Furthermore, the expression of ferroptosis-related protein, GPX4 (Fig. [118]7D), exhibited a marked reduction in myocardial tissue. However, the administration of Fer-1 notably alleviated these alterations. Fig. 7. [119]Fig. 7 [120]Open in a new tab Ferroptosis in heart transplantation mouse model. (A) Timeline for the experimental showing drug administration, heart transplantation surgery, and killing of the mice. (B) Schematic illustration of mice heterotopic heart transplantation. (C) HE staining representative mouse. (D) Representative immunohistochemical staining among the 3 groups. (E) The statistic of GPX4-positive area %. (F and G) The GPX4 protein level. (H) Beating Score. (I and J) The concentrations of MDA and SOD were measured in the myocardium. **P < 0.01 vs. control group; ^##P < 0.01 vs. I/R group. Data are shown as means ± SEM We also examined the changes in markers reflecting oxidative stress by determining the levels of SOD and MDA in the cardiac tissue of mice treated with or without Fer-1 after heart transplantation. The results showed that pretreatment with Fer-1 significantly decreased in MDA (Fig. [121]7F) and increased in SOD (Fig. [122]7G) in the ischemic heart tissue of mice than in I/R group. Validation of hub genes and ferroptosis indexes by RT-qPCR in the model of heart transplantation in mice GPX4, as an important component in the antioxidant stress system, began to decrease during I/R injury and increasing sharply after Fer-1 treatment (Fig. [123]8A). Compared with the control group, the mRNA levels of TRFC and ACSL4 after cold I/R were dramatically upregulated, and then declined in I/R + Fer-1 group (Fig. [124]8B and C). Fig. 8. [125]Fig. 8 [126]Open in a new tab mRNA levels of hub genes and ferroptosis indexes in heart transplantation mouse model. [GPX4 (A), ACSL4 (B), TRFC (C), SLC7A11 (D), PSAT1 (E), and ASNS (F)] among control, I/R and I/R + Fer-1 groups. n = 5. ^**P < 0.01 vs. control group; ^##P < 0.01 vs. I/R group. Data are shown as means ± SEM Next, levels of three ferroptosis-associated hub genes were further validated in mice with cold I/R by RT-qPCR. As shown in Fig. [127]8D-F, SLC7A11, PSAT1 and ASNS in heart transplantation with cold I/R are significantly higher than control group, and then decreased significantly after Fer-1 treatment. Validation of hub genes by western blot in heart transplantation model Three ferroptosis-related hub genes (SLC7A11, PSAT1 and ASNS) were validated at the protein expression level using a mouse model of cold I/R following heart transplantation. As shown in Fig. [128]9, the protein levels of SLC7A11, PSAT1 and ASNS in I/R group were significantly higher than those in the control group, while Fer-1 reversed these changes compared with the I/R group. Fig. 9. [129]Fig. 9 [130]Open in a new tab The protein expression levels of ferroptosis-related hub genes in a mouse model of heart transplantation. The western blotting results of the protein expression levels and semiquantitative analysis of immunoblotting results of PSAT1 (A, B), SLC7A11 (A, C), ASNS (A, D) among control, I/R and I/R + Fer-1 groups. n = 5. ^**P < 0.01 vs. control group; ^##P < 0.01 vs. I/R group. Data are shown as means ± SEM Discussion Heart transplantation is the ultimate treatment for patients with end stage heart disease [[131]23]. However, cold I/R injury of heart transplantation leads to early heart failure and even death following heart transplantation, which greatly affects the prognosis of heart transplant patients. Therefore, it is important to study the key gene of myocardial I/R injury and make intervention. In our study, hub genes (SLC7A11, PSAT1 and ASNS) related to ferroptosis were screened from the mouse transplanted heart obtained from GEO database and verified in the mouse cervical heterotopic heart transplantation model. These genes play an important role in different disease models and may be diagnostic biomarkers and therapeutic targets for cold I/R injury in heart transplantation. Functional enrichment analysis of GO terms showed that FRDEGs were mainly enriched in α-amino acid metabolic process and response to oxidative stress. KEGG pathway analysis indicated that the FRDEGs were mainly enriched in ferroptosis-related pathway. SLC7A11 (Solute Carrier Family 7 Member 11) and SLC3A2 function together as components of the system Xc⁻ cystine transporter, facilitating the import of cystine into cells. This transport system plays a pivotal role in maintaining intracellular glutathione levels and protecting cells from oxidative stress-induced damage [[132]24]. Inhibition of SLC7A11 expression has been shown to significantly exacerbate ferroptosis and reactive oxygen species (ROS) production, thereby impairing cardiac function in I/R injury mice [[133]25]. Conversely, activation of SLC7A11 expression can attenuate ferroptosis and reduce oxidative stress, thus mitigating myocardial injury resulting from I/R injury [[134]26]. However, excessive overexpression of SLC7A11 may deplete NADPH, heighten cellular susceptibility to oxidative stress, and ultimately promote cell death [[135]27]. Therefore, the specific mechanism of SLC7A11 in I/R of heart transplantation needs to be further explored. In terms of phosphoserine aminotransferase 1 (PSAT1), it is a member of the class-V pyridoxal-phosphate-dependent aminotransferase family. Numerous research studies have indicated that PSAT1 is instrumental in inhibiting serine metabolism, which subsequently hinders the occurrence and progression of various types of tumors [[136]28, [137]29]. The molecular mechanisms behind this inhibition involve the suppression of the ROS-dependent JNK/c-Jun signaling pathway, thereby effectively reducing apoptosis [[138]30]. Furthermore, PSAT1 has demonstrated its efficacy in mitigating pulmonary fibrosis in different disease models [[139]31]. Recent investigations have also uncovered its ability to provide protection against hepatic I/R injury. This protective effect is achieved through the alleviation of DNA damage and apoptosis [[140]32]. However, the precise mechanism by which PSAT1 alleviates myocardial I/R injury remains to be thoroughly investigated and understood. As for asparagine synthetase (ASNS), which plays a key role in cancer, such as liver cancer [[141]33], bladder cancer, colon cancer and esophageal cancer. Furthermore, it has been found that ASNS can promote cardiac regeneration in neonatal mice, as well as myocardial cell survival and cell cycle re-entry during myocardial cell dedifferentiation in adult mice [[142]34]. At the same time, there was a study focused on the development and validation of genes associated with ferroptosis in myocardial I/R injury, and ASNS significantly elevated and proposed as a potential protective agent against ferroptosis [[143]35]. Genetic association studies have determined multiple mechanisms regarding the pathogenesis of heart transplantation, which showed that these heart transplantation genes are associated with ferroptosis. We also found that hub genes (SLC7A11, PSAT1 and ASNS) were associated with infiltration of different immune cells in heart transplantation. SLC7A11 partially mediated glutathione production limits the accumulation of reactive oxygen species and supports the activity of mTOR and nuclear factors in activated T cells to drive glycolysis and glutamine breakdown in activated T cells [[144]36]. Inhibition of SLC7A11 can reduce the infiltration of T cells into the central nervous system, reduce the excitotoxic death of mature myelinated oligodendrocytes, and improve the condition of experimental autoimmune encephalomyelitis mice [[145]37]. In patients with T cell acute lymphoblastic leukemia, normal hematopoietic cells can survive due to ASNS activity [[146]38]. However, there is a lack of research between PAST1 and the immune response caused by eosinophils, dendritic cells and T cells, which needs to be further explored and verified. More researches showed that the activity and function of cytotoxic T cells (CD4^+) were regulated by lipid peroxidation and ferroptosis [[147]39]. Therefore, there is a complex relationship between ferroptosis and immune cells, providing a new mechanism for the treatment of I/R injury. Conclusions In this study, we screened three hub genes related to ferroptosis and developed a predictive model for heart transplantation, including SLC7A11, PSAT1 and ASNS, and explored their interaction and relationship with immune cells. However, our study has several limitations. Due to the particularity of heart transplantation model and materials, only one GEO database was found, but the mouse heart transplantation model was adopted during verification to ensure consistency of identification and verification. In addition, we did not use inhibitors of the hub genes to do the rescue experiments and do co-staining of hub genes and specific immune cells in the mouse hearts after heart transplantation with I/R to confirm their relationship, but we will take this part of verification as the next experimental plan. In conclusion, ferroptosis can facilitate new treatment avenues for cold I/R injury. In the subsequent study, we can further analyze the specific molecular mechanisms of hub genes in I/R during heart transplantation. Electronic supplementary material Below is the link to the electronic supplementary material. [148]Supplementary Material 1^ (2.5MB, zip) Abbreviations ASNS Asparagine synthetase TFH Follicular helper T GEO Gene Expression Omnibus; GO: gene ontology KEGG Kyoto Encyclopedia of Genes and Genomes PPI Protein-Protein Interaction Fer-1 Ferrostatin-1 HE Hematoxylin and eosin RT-qPCR Quantitative Real-time PCR SLC7A11 Solute Carrier Family 7 Member 11 PSAT1 Phosphoserine aminotransferase 1 Author contributions XZY and SQ conceived the idea of the study and contributed to the design of the study. QZ and LSQ contributed to the design of the animal study. XR, LYN, XH, and CJH performed the experiments and analyzed the data. TQ and ZYX wrote the manuscript draft. All authors revised the manuscript draft. All authors read and approved the final manuscript. Funding This work is supported by the National Natural Science Foundation of China (82372188 and 82072140), Key research and development Plan of Hubei Province(Grant No.2022BCE007) and Hubei Province Natural Science Foundation of China (No. 2023AFB821). Data availability No datasets were generated or analysed during the current study. Declarations Ethics approval and consent to participate All animal experiments were approved by the Ethics Committee of Renmin Hospital of Wuhan University and were conducted in accordance with the regulations. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Yuxi Zhang and Qiao Tang contributed equally to this work. Contributor Information Qian Sun, Email: queenie_sun@whu.edu.cn. Zhongyuan Xia, Email: xiazhongyuan2005@aliyun.com. References