Application of PPIA-BSG pathway inhibitor in preparation of medicine for resisting myocardial hypertrophy after xenogenous heart transplantation

Abstract

The invention provides application of a PPIA-BSG pathway inhibitor in preparation of a medicine for resisting myocardial hypertrophy after xenogenous heart transplantation, and belongs to the technical field of biological medicine. The PPIA-BSG axis is a key pathway for mediating myocardial hypertrophy after xenogenous heart transplantation. The PPIA inhibitor is utilized to target the pathway, so that the myocardial cell hypertrophy can be remarkably reduced, and a potential treatment strategy is provided for relieving malignant cardiac hypertrophy after xenotransplantation. In addition, extracellular PPIA can be inhibited by using a PPIA-BSG pathway related inhibitor, such as a non-immunosuppressor NIM811, and a remarkable curative effect is shown in the aspects of relieving myocardial hypertrophy and cell apoptosis. Therefore, the PPIA-BSG pathway inhibitor has the effect of resisting cardiac hypertrophy after xenogenic heart transplantation, and provides a treatment strategy and drug target information for cardiac hypertrophy after xenogenic heart transplantation.

Description

Technical Field

[0002] This invention belongs to the field of biomedical technology, and in particular relates to the application of PPIA-BSG pathway inhibitors in the preparation of drugs for treating myocardial hypertrophy after xenotransplantation. Background Technology

[0003] Currently, graft overgrowth after xenotransplantation (xenoHTX) poses a significant challenge, leading to early graft failure and limiting long-term survival. Reported clinical trials of transgenic pig-human xenoHTX have demonstrated significant myocardial hypertrophy and diastolic dysfunction in xenotransplantation. Although current technologies have made some progress in suppressing myocardial hypertrophy after xenotransplantation, the underlying mechanisms driving graft overgrowth remain unclear, thus failing to effectively alleviate malignant cardiac hypertrophy after xenotransplantation. Summary of the Invention

[0004] The purpose of this invention is to provide the application of PPIA-BSG pathway inhibitors in the preparation of drugs for treating myocardial hypertrophy after xenograft heart transplantation, which can effectively alleviate malignant myocardial hypertrophy after xenograft transplantation.

[0005] This invention provides the application of PPIA-BSG pathway inhibitors in the preparation of drugs for treating myocardial hypertrophy after xenotransplantation.

[0006] This invention also provides the application of PPIA-BSG pathway inhibitors in combination with conventional immunosuppressants in the preparation of drugs for treating myocardial hypertrophy after xenograft heart transplantation.

[0007] This invention also provides the use of PPIA-BSG pathway inhibitors in the preparation of drugs to combat inflammatory responses and / or myocardial remodeling after xenograft heart transplantation.

[0008] This invention also provides the application of PPIA-BSG pathway inhibitors in combination with conventional immunosuppressants in the preparation of drugs to combat inflammatory responses and / or myocardial remodeling after xenograft heart transplantation.

[0009] Preferably, the PPIA-BSG pathway inhibitor includes a PPIA inhibitor and / or a BSG inhibitor; the PPIA-BSG pathway inhibitor includes at least one of NIM811, cyclosporine A, a neutralizing anti-CyPA monoclonal antibody, arapovir, NV651, Sanglifehrin A, a CD147 monoclonal antibody meplazumab, AC-73, and licartin.

[0010] Preferably, the PPIA-BSG pathway inhibitor is used to inhibit extracellular PPIA.

[0011] Preferably, the conventional immunosuppressant includes tacrolimus or rituximab.

[0012] The present invention also provides PPIA Genes and / or BSG The application of genes as therapeutic targets in the preparation or screening of drugs for myocardial hypertrophy after xenotransplantation.

[0013] The present invention also provides PPIA Genes and / or BSG The application of genes as therapeutic targets in the preparation or screening of drugs to combat inflammatory responses and / or myocardial remodeling after xenotransplantation.

[0014] Preferably, the anti-xenograft myocardial hypertrophy includes at least one of the following: 1) improving cardiomyocyte hypertrophy; 2) reducing cardiomyocyte damage; 3) inhibiting immune activation and T cell infiltration; 4) maintaining the integrity of myocardial structure; and 5) improving cardiomyocyte apoptosis.

[0015] This invention provides the application of PPIA-BSG pathway inhibitors in the preparation of drugs to combat myocardial hypertrophy after xenotransplantation. This invention utilizes combined single-cell and single-nuclear sequencing analysis to reveal the interaction between immunity and cardiomyocytes, identifying the PPIA-BSG axis as a key pathway mediating myocardial hypertrophy after xenotransplantation. Targeting this pathway with PPIA inhibitors significantly reduces cardiomyocyte hypertrophy, providing a potential therapeutic strategy for alleviating malignant myocardial hypertrophy after xenotransplantation. Furthermore, using PPIA-BSG pathway-related inhibitors, such as the non-immunological inhibitor NIM811, can inhibit extracellular PPIA, showing significant efficacy in reducing myocardial hypertrophy and apoptosis. Therefore, PPIA-BSG pathway inhibitors have an anti-xenotransplantation effect on myocardial hypertrophy, providing therapeutic strategies and drug target information for myocardial hypertrophy after xenotransplantation. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a flowchart of the experiment. Figure 2 To obtain UMAP maps of cell clustering in pigs and monkeys; Figure 3 UMAP plot for clustering single-cell or single-nucleus sequencing data; Figure 4 Cell cluster diagram for cell type clustering identification; Figure 5 This is a distribution map of highly expressed genes in cell clusters; Figure 6 A graph showing the distribution percentage of different cell clusters after xenografting and in non-xenograft samples; Figure 7 This is a graph showing the distribution of the main cell cluster types in pigs and monkeys. Figure 8 Cell clustering distribution map of marker genes; Figure 9 Figure showing enrichment analysis of cardiomyocyte and immune cell types; Figure 10 This is a cluster distribution diagram of the three subtypes of cardiomyocytes; Figure 11 This is a distribution diagram of the proportion of cardiomyocytes in the experimental group; Figure 12 This is a graph showing the changes in functional expression of cardiomyocytes in the experimental group; Figure 13 This is a distribution map of highly expressed genes in porcine cardiomyocytes of the normal control group. Figure 14 Distribution of highly expressed genes in cardiomyocytes during porcine xenotransplantation in the GTKO group; Figure 15 Distribution of highly expressed genes in cardiomyocytes during porcine xenotransplantation in the GTKO+IS group; Figure 16 Figure showing differential gene expression analysis between PTGER3+CMs and normal cardiomyocytes; Figure 17 Figure showing differential gene expression analysis between MYH7B+ CMs and normal cardiomyocytes; Figure 18 This is a diagram showing the expression of inflammatory factors and receptor-related genes in CM subtypes. Figure 19 This is a pseudo-time differentiation trajectory diagram of cardiomyocyte subtypes; Figure 20 This is a pseudo-time differentiation trajectory diagram after cell state clustering; Figure 21 The differentiation trajectory of cell state transition; Figure 22 This is a cluster analysis diagram of differentially expressed genes in time series analysis; Figure 23 UMAP diagram showing subtype clustering of lymphocyte immune cells; Figure 24 A graph showing the distribution percentage of lymphocyte immune cell subtypes in different experimental groups; Figure 25 A diagram showing the differential gene expression of CCR7+CD4+ TC cell populations in the experimental group and the immunosuppressive therapy group; Figure 26 A diagram showing the differential gene expression of XCL1+CD8+ TC cell populations in the experimental group and the immunosuppressive therapy group; Figure 27 UMAP diagram showing subtype clustering of myeloid immune cells; Figure 28 Distribution map of genes highly expressed in myeloid immune cell subtypes; Figure 29 A graph showing the distribution percentage of myeloid immune cell subtypes in different experimental groups; Figure 30 Veen analysis diagram of C1QC+ MC tissue-resident macrophages; Figure 31 A graph showing GO enrichment analysis of C1QC+ MC tissue-resident macrophages; Figure 32 Graph showing GO enrichment analysis of IL1B+ MC in different experimental groups; Figure 33 Graph showing GO enrichment analysis of TIMP1+ MC in different experimental groups; Figure 34 The diagram shows the interaction analysis between cardiomyocytes and other major cell types; where A represents the major ligand-receptor pairs in the heart of a pig without transplantation; B represents the major ligand-receptor pairs in the heart of a pig in the GTKO xenograft group; and C represents the major ligand-receptor pairs in the heart of a pig in the GTKO+IS xenograft+immunosuppressive therapy group. Figure 35 Immunofluorescence analysis of PPIA-BSG in xenotransplantation; Figure 36 Multilabel immunohistochemical analysis of the SD-Balb / c heterotopic heart xenograft model; Figure 37 H&E staining histological analysis of drug administration in the SD-to-Balb / c heterotopic heart xenograft model; Figure 38 WGA staining analysis of drug administration in the SD-to-Balb / c heterotopic heart xenograft model; Figure 39 Immunohistochemical analysis of drug administration in the SD-to-Balb / c heterotopic heart xenotransplantation model; Figure 40 TUNEL staining analysis of apoptosis in cells treated with the SD-to-Balb / c heterotopic heart xenograft model. Detailed Implementation

[0018] This invention provides the application of PPIA-BSG pathway inhibitors in the preparation of drugs for treating myocardial hypertrophy after xenotransplantation.

[0019] This invention utilizes combined single-cell and single-nuclear sequencing analysis to reveal the interaction between the immune system and cardiomyocytes, identifying the PPIA-BSG axis as a key pathway mediating myocardial hypertrophy after xenograft heart transplantation. Targeting this pathway with PPIA and / or BSG inhibitors significantly reduces cardiomyocyte hypertrophy, providing a potential therapeutic strategy for alleviating malignant cardiac hypertrophy after xenograft transplantation. Furthermore, PPIA-BSG may exert its effects through direct crosstalk between cardiomyocytes and immune cells. Extracellular cyclic serine protease A (PPIA) and its receptor, extracellular matrix metalloproteinase inducer (EMMPRIN, BSG), regulate inflammatory processes beyond MMP activity.

[0020] This invention also provides the application of PPIA-BSG pathway inhibitors in combination with conventional immunosuppressants in the preparation of drugs for treating myocardial hypertrophy after xenograft heart transplantation.

[0021] This invention also provides the use of PPIA-BSG pathway inhibitors in the preparation of drugs to combat inflammatory responses and / or myocardial remodeling after xenograft heart transplantation.

[0022] In this invention, xenograft heart transplantation resulted in significant cellular remodeling, characterized by cardiomyocyte dedifferentiation and immune dominance. Single-cell analysis revealed a shift in cardiomyocyte subtypes from contractile (MYOM2+) to stress-responsive (PTGER3+ / MYH7B+), accompanied by downregulation of sarcomere genes (TNNI3, RYR2) and upregulation of fetal genes (NPPB, ANKRD1). Pseudo-temporal analysis further indicated that the dedifferentiation trajectory involved pro-proliferative (LPP, CDK14) and pro-inflammatory (IL6R, STAT3) pathways. This invention also provides the application of PPIA-BSG pathway inhibitors in combination with conventional immunosuppressants in the preparation of drugs to combat inflammatory responses and / or myocardial remodeling after xenograft heart transplantation.

[0023] In one implementation, the PPIA-BSG pathway inhibitor includes a PPIA inhibitor and / or a BSG inhibitor; the PPIA-BSG pathway inhibitor includes at least one of NIM811, cyclosporine A (CsA), a neutralizing anti-CyPA monoclonal antibody (specific monoclonal antibody), arapovir (Debio-025), NV651, Sanglifehrin A (SfA), CD147 monoclonal antibody meplazumab, AC-73, and licartin (metuximab), further comprising NIM811; the PPIA-BSG pathway inhibitor can supplement conventional immunosuppressive therapy regimens, providing a solution for delaying abnormal cardiac hypertrophy after xenograft heart transplantation.

[0024] In this invention, NIM811 can inhibit PPIA activity, thereby blocking the binding of PPIA and CD147. NIM811 has shown significant efficacy in reducing myocardial hypertrophy and apoptosis after xenograft heart transplantation by inhibiting extracellular PPIA.

[0025] As one implementation method, the PPIA-BSG pathway inhibitor is used to inhibit extracellular PPIA.

[0026] As one implementation, the conventional immunosuppressant includes tacrolimus or rituximab; the conventional immunosuppressant can effectively inhibit early immune activation after xenotransplantation (xenoHTX).

[0027] The present invention also provides PPIA Genes and / or BSG The application of genes as therapeutic targets in the preparation or screening of drugs for myocardial hypertrophy after xenotransplantation.

[0028] The present invention also provides PPIA Genes and / or BSG The application of genes as therapeutic targets in the preparation or screening of drugs to combat inflammatory responses and / or myocardial remodeling after xenotransplantation.

[0029] As one implementation, the anti-xenograft myocardial hypertrophy includes at least one of the following: 1) Improve cardiomyocyte hypertrophy; 2) Reduce cardiomyocyte damage; 3) Inhibit immune activation and T cell infiltration; 4) Maintain the integrity of myocardial structure; 5) Improve cardiomyocyte apoptosis.

[0030] In one implementation, the xenotransplantation includes xenotransplantation from a transgenic pig to a non-human primate; the non-human primate includes chimpanzees or monkeys; the monkey includes rhesus monkeys or cynomolgus monkeys.

[0031] To further illustrate the present invention, the application of the PPIA-BSG pathway inhibitor provided by the present invention in the preparation of drugs for treating myocardial hypertrophy after xenograft heart transplantation is described in detail below with reference to the accompanying drawings and embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0032] Experimental Example 1 1. Animal models: All animals were approved by the Animal Ethics Committee of Fuwai Hospital.

[0033] 1.1 Cardiac Xenotransplantation Model: Male GTKO pigs weighing 5-8 kg and 2 months old were used as donors, and rhesus monkeys weighing 14-16 kg were used as recipients to perform a peritoneal xenotransplantation model. (Reference Procedure) Figure 1 .

[0034] 1.2 SD-Balb / c heterotopic heart xenotransplantation model: Sprague Dawley SD rats, aged 7-10 days and weighing 25g, were selected as heart donors and purchased from Vital River Pharmaceuticals in Beijing. Ten-week-old BALB / c rats, weighing 25g, were selected as recipients and purchased from Vital River Pharmaceuticals in Beijing.

[0035] 2. Heart xenotransplantation experiment: intraperitoneal xenoHTX transplantation.

[0036] 3. Dosing regimen: 3.1 Immunosuppressive Protocol for the GTKO Pig-to-Rhesus Monkey Xenotransplantation Model: The pig-to-rhesus monkey xenotransplantation immunosuppressive treatment group received conventional immunosuppressive therapy, while the xenotransplantation control group received no treatment. A list of immunosuppressive drugs used is provided in Table 1.

[0037] Table 1. List of Immunosuppressive Drugs Used

[0038] 3.2 SD-Balb / c heterotopic heart xenotransplantation model: The study was divided into a blank control group and an experimental group. The experimental group was further divided into an FK506 monotherapy group, a NIM811 monotherapy group, and a combination of FK506 and NIM811 therapy group. NIM811 is a specific inhibitor of the PPIA-BSG pathway associated with xenograft myocardial hypertrophy. This inhibitor inhibits PPIA activity, thereby blocking the binding of PPIA and CD147.

[0039] FK506 monotherapy group: In the SD-Balb / c heterotopic heart xenograft model, rats were given the classic immunosuppressant FK506 via intraperitoneal injection for heart transplantation to mice. NIM811 monotherapy group: NIM811 was administered intraperitoneally to rats in the SD-Balb / c heterotopic heart xenograft model before transplantation to mice. FK506 and NIM811 combined treatment group: FK506 and NIM811 were used in combination for rat-to-mouse heart transplantation in the SD-Balb / c heterotopic heart xenograft model.

[0040] The dosage for monotherapy is 10 mg / kg / day, and the dosage for combination therapy is 10 mg / kg / day for each individual drug. The duration of administration is 4 days. The blank control group received no drug treatment.

[0041] 4. Tissue Sampling: Heart tissue samples were taken from the blank control group and the experimental group of GTKO pig-to-rhesus monkey heart xenograft after the xenograft stopped beating. The SD-Balb / c heterotopic heart xenograft model underwent surgical resection on the 4th postoperative day.

[0042] 5. Single-cell extraction and library construction 5.1 Single-cell RNA sequencing: After surgical removal of rhesus monkey donor heart tissue, the heart tissue was dissociated into single cells using 400 U / mL type II collagenase (Worthington, LS004176). Flow cytometry and fluorescence-activated cell sorting (FUC) were used to select 7AAD+ live single cells for capturing non-cardiac cell types. The prepared cell suspension was processed using the Chromium Single-Cell platform at the cell concentration recommended by the manufacturer's instructions. 5' RNA libraries were constructed using the Chromium™ Single-Cell 5' library and Gel Bead Kit v1 (10x Genomics) according to the manufacturer's instructions. After library quality control, sequencing was performed using a next-generation sequencer. The workflow is as follows: Figure 1 As shown. Sequencing data were obtained using Cell RangerSingle-Cell software (10X Genomics, version 8.0.1).

[0043] 5.2 Single-cell RNA sequencing: Approximately 300 mg of tissue was collected from a single sample for myocardial single-cell nucleus extraction. Heart tissue was thawed on ice, washed with cold PBS, and cut into small pieces in lysis buffer. The tissue was homogenized using an Ultra-Turrax probehomogenizer (IKA). The homogenate was then ground in a glass homogenizer to release the nuclei. The homogenate was then filtered sequentially through 100 µm and 70 µm filters. After centrifugation, the supernatant was discarded, and the precipitate was resuspended in freshly prepared sucrose buffer and centrifuged for 1 h. After centrifugation, the supernatant was discarded, and the nucleus pellet was dissolved in 1 mL of 1% BSA (containing 200 U / mL RI). The nucleus count was adjusted to 700–1000 nuclei / µL using trypan blue staining. The prepared nuclear suspension was processed using the Chromium Single-Cell platform. 5' RNA library construction was performed using the Chromium™ Single-Cell 5' Library and Gel Bead Kit v3.1 (10x Genomics) according to the manufacturer's instructions. After library quality control, sequencing was performed using a next-generation sequencer. The workflow is as follows: Figure 1As shown. Sequencing data were obtained using Cell Ranger Single-Cell Software (10X Genomics, version 8.0.1).

[0044] 6. Single-cell transcriptome data analysis 6.1 Single-cell transcriptome data acquisition and format conversion: For xenograft samples, reference genomes and GTF annotation files were downloaded from NCBI, specifically GCF-003339765.1_Mmul10 and GCF_000003025.6_Sscrofa11.1. A combined reference genome was constructed according to the multi-species reference genome guide provided by 10x Genomics. Non-xenograft samples were analyzed using the Sscrofa11.1 reference genome. Fastq files generated from 10xGenomics single-cell (scRNA-seq) and single-nuclear (snRNA-seq) RNA sequencing libraries were processed using the CellRanger Count workflow (v8.0.1).

[0045] 6.2 Quality Control and Filtering: For scRNA-seq samples, the workflow was run using default parameters; for snRNA-seq samples, the additional parameter `--include-introns=True` was applied to merge intron reads, thereby capturing abundant pre-mRNA transcripts in the cell nucleus. The generated matrix was imported into Seurat v4.3.0 for downstream analysis. The potential duplex score for each cell was calculated using Scrublet. The cell screening criteria for scRNA-seq and snRNA-seq samples differed to reflect their specific data characteristics: • scRNA-seq samples: Cells were retained based on the following thresholds: total gene count less than 15,000, more than 200 genes detected, mitochondrial gene content less than 5%, ribosomal gene content less than 50%, and Scrublet duplex score less than 0.3.

[0046] • snRNA-seq samples: Cells were screened using stricter ribosomal gene content thresholds (applicable to nuclear data): total count less than 15,000, more than 200 genes detected, mitochondrial gene content less than 5%, ribosomal gene content less than 5%, and Scrublet bimodal score less than 0.3.

[0047] 6.3 Batch Calibration and Integration: A matrix containing 16,245 overlapping genes between Mmul10 and Sscrofa11.1 was extracted, and the top 2,000 variant features were selected for analysis. The dataset was log-normalized, scaled, and subjected to principal component analysis (PCA). The top 30 PCs were clustered using the Leiden algorithm. Batch calibration was performed using BBKNN. The integrated data was visualized using UMAP.

[0048] 6.4 Cell Clustering and Annotation: The FindAllMarkers function in Seurat v4, combined with the Wilcoxon rank-sum test, was used to identify differentially expressed genes in each cluster. Cells were annotated by marker gene expression, and lymphocyte and myeloid cell clusters were further analyzed through computational separation. Subset analysis followed a similar approach: first, the features of the first 2,000 variables were calculated, then scaling was performed, principal component analysis (PCA) was recalculated, Leiden clustering was performed, and finally, UMAP was used for visualization.

[0049] 6.5 Differentially Expressed Gene (DEG) Identification and Functional Enrichment: Differential gene expression was detected using the FindMarkers function in Seurat, with the default parameter "test.use=wilcox". The Benjamini-Hochberg method was used to estimate the false discovery rate (FDR). DEGs were screened using a minimum log2 (fold change) of 0.25 and a maximum FDR of 0.01. Functional enrichment analysis of the DEGs was performed using the clusterProfiler package in R.

[0050] 6.6 Spatiotemporal Dynamic Pseudo-Time Series Analysis: Pseudo-time series inference was performed using Monocle version 2.30.1. The raw count data in the Seurat object was converted into a CellDataSet object using the `import CDS` function in Monocle. Genes with differential expression between subsets were set as sorted genes. The minimum spanning tree was constructed using the `reduceDimensions` function (reduction_method="DDRTree", num_dim=10, norm_method="log", residualModelFormula="~Celltype", relative_expr=TRUE, scaling=TRUE).

[0051] 6.7 Cell-to-cell communication analysis: Cell-to-cell communication in the dataset was evaluated using the Cellphone database (v5.0.0) with known receptor-ligand pairs. Genes in Seurat objects were renamed with human gene names and then reformatted to the input format described on the CellphoneDB website. Cells were fed into the CellphoneDB computation program for 50 iterations with a precision of 3 and a cluster size of 0.1 cells expressing a particular gene. Interactions were then pruned based on significant sites with p < 0.05. Visualization was performed using the R package CCPlotR (v1.0.0).

[0052] Pathological staining validation analysis of 7 xenograft animal models and SD-Balb / c animal model 7.1 Histological staining - WGA staining analysis Heart tissue sections were baked at 68°C for 45 min using a slide oven. Subsequently, slides were sequentially immersed in xylene I, II, and different concentrations of ethanol (100%, 95%, 80%, 75%, 50%) for 5 min each, followed by washing with distilled water for 5 min to dewax and hydrate. A 10 μg / mL WGA working solution was dropped onto the heart tissue surface and incubated at room temperature in the dark for 1 h. The slides were washed three times with PBS and mounted with DAPI mounting medium.

[0053] 7.2 Histological staining - H&E staining analysis Heart tissue sections were baked at 65°C for 45 minutes using a slide oven. The same procedures as for WGA dewaxing and hydration were followed. Sections were stained in hematoxylin for approximately 5-10 minutes, then rinsed with tap water. The sections were then immersed in 1% hydrochloric acid-ethanol solution to decolorize and turn red. The sections were then rinsed again with running tap water for 3 minutes to restore their blue color, stained with eosin for 0.5 seconds, dehydrated, mounted with neutral resin, and air-dried.

[0054] 7.3 Immunofluorescence staining analysis (CD3+) Heart tissue sections were baked at 65°C for 45 min in a slide oven to prevent detachment. After dewaxing and hydration, the heart tissue sections underwent acid-temperature retrieval and were blocked with serum for 1 h. After washing, they were incubated overnight at 4°C with primary antibody, then washed again and incubated with secondary antibody in the dark for 60 min. After washing, they were mounted with DAPI mounting medium.

[0055] Secondary antibody used: Alexa Fluor 594 is a commonly used, very bright red fluorescent probe.

[0056] 7.4 Multilabel immunohistochemical staining analysis (PPIA, CD147 and cardiomyocyte colocalization) Baking: 68℃ for 1 h; after dewaxing, alkaline repair is performed, followed by containment to remove endogenous catalase, and then blocking with Blocking / Ab Diluent at room temperature for 10 min; after washing, incubate with primary antibody overnight at 4℃, then wash again and incubate with secondary antibody at room temperature for 20 min; after secondary antibody washing, use different labeled fluorescent staining. Depending on the number of antibodies added, different labeled fluorescent staining is selected, and the repair, blocking, primary and secondary antibody incubation, and fluorescent staining steps are repeated. Finally, DAPI mounting and scanning are performed.

[0057] See Table 2 for a list of antibodies / dyes used.

[0058] Table 2 List of Antibodies / Dyes

[0059] Experimental results: I. Cardiac cell atlas analysis of the control and experimental groups: After data was filtered according to threshold quality control, a total of 107,596 cells were finally obtained (including porcine cells and rhesus monkey immune cells, of which 98,655 were from pigs and 8,941 were from rhesus monkeys; scRNA-seq captured 51,866 cells, while snRNA-seq captured 55,730 cells). Figures 2-3 ).

[0060] Cell types were annotated based on classical marker genes and their biological functions, and cell cluster analysis identified 13 major cell clusters ( Figure 4 These cell clusters included three cardiomyocyte types, all characterized by high expression of TTN and RYR2; one endothelial cell type characterized by PLVAP and VWF expression; one epicardial cell type expressing PTX3; and one smooth muscle cell type with elevated expression of MYH11 and ACTA2. In addition, two distinct fibroblast subtypes were identified, characterized by high expression of SPON1 and DCN, respectively. Immune cells were divided into myeloid (highly expressing LYZ) and lymphoid (highly expressing CXCR4) lineages. Other identified cell types included lymphatic endothelial cells (LYVE1), neurons (NRXN1), and erythrocytes (HBB). Figure 5 The distribution of typical cell markers validated the accuracy of the cell definition. Figure 8 ).

[0061] Compared with the single-cell composition of pig heart tissue after xenotransplantation, the number of cardiomyocytes decreased, while the proportion of immune cells increased. Figure 6 Cells derived from rhesus monkeys are mainly lymphocytes and myeloid immune cells. Figure 7Enrichment analysis showed that genes highly expressed in myeloid immune cells were associated with responses to other organisms and inflammatory processes, while genes highly expressed in lymphoid immune cells were associated with T cell differentiation and B cell activation. Figure 9 This further suggests that in cardiac xenotransplantation comparisons, there is a close relationship between the significant changes in cardiomyocyte and immune cell types (myeloid and lymphoid) and the dynamic changes in tissues during xenotransplantation, which requires further in-depth analysis.

[0062] II. Cardiac cell subtype analysis Cardiomyocytes were classified into three subtypes. In the non-GTKO pig heart, MYOM2+ cardiomyocytes were dominant; in the GTKO group, PTGER3+ cardiomyocytes were dominant; and in the GTKO+IS group, both PTGER3+ and MYH7B+ cardiomyocytes were present. Figure 10 and Figure 11 To further investigate whether cardiomyocyte function was normal or its changes in the three experimental groups, the expression of genes related to contractile function (TNNI3, PLN, ACTC1, TNNC1, RYR2, KCNQ1, SCN5A, and MYL3) and heart failure-related genes associated with cardiomyocyte lesions (NPPB, NPPA, and ATP2A2) in the three groups of cardiomyocytes were analyzed. The results showed that the expression of cardiomyocyte contraction-related genes was decreased and NPPB expression was increased in the GTKO group. Although the expression of cardiomyocyte contraction-related genes partially recovered after immunosuppressant treatment in the GTKO+IS group, it was still lower than that in pig hearts not transplanted with GTKO. Figure 12 ).

[0063] The 50 most highly expressed genes in non-GTKO-transplanted porcine cardiomyocytes were identified. These genes are mainly associated with cardiac contraction and calcium ion activity, including TTN, CAMK2D, LMOD2, and TNNI3. Figure 13 In contrast, the 50 most expressed genes in GTKO cardiomyocytes include SLIT2 (PMID: 32564729), a gene regulating cardiac morphology and protecting against myocardial injury; SPIDR (PMID: 39237506), a DNA damage repair gene; and IGF2BP2 (PMID: 38052926), a gene for cardiac stress response. Figure 14 For the GTKO+IS group, ANKRD1 is one of the most highly expressed genes and is known to be involved in the formation of cardiac hypertrophy and the reactivation of fetal cardiac genetic programs (PMID: 25770146). Figure 15 ).

[0064] Further comparison of differentially expressed genes between xenografted cardiomyocytes and normal cardiomyocytes was conducted. The results showed that MYOM2+ cardiomyocytes expressed higher levels of sarcomere and ion channel-related genes, including TTN, TNNI3, LMOD2, RYR2, and KCNJ3, indicating impaired contractile function of cardiomyocytes after xenograft transplantation. In contrast, PTGER3+ cardiomyocytes exhibited higher expression of IL6R, IL4R, and inflammation-related genes (such as STAT3), indicating responsiveness to inflammatory cytokines. Figure 16 Similarly, the expression levels of sarcomere-related genes (TNNT2, TNNI3) in MYOM2+CMs were higher than those in MYH7B+CMs. Figure 17 However, elevated expression of IL6, IL4R, STAT3, and ATF3 was observed in PTGER3+CMs, indicating that inflammation-induced cardiomyocyte effects persisted despite conventional immunosuppressive therapy. Further investigation revealed the expression of inflammatory factors and receptors in the three CM subtypes. Results showed that, in addition to IL4R and IL6R, PTGER3+CMs in the GTKO+IS group also expressed high levels of IL17B, which was associated with the inflammatory response following cardiac xenograft transplantation. Figure 18 Differential gene expression analysis showed that after xenografting, the contractile and calcium ion channel regulation functions of cardiomyocytes were impaired, the expression levels of some genes involved in the inflammatory response were increased, and there was a response to inflammatory cytokines. Although conventional immunosuppressive therapy was given, the inflammatory response still existed.

[0065] The differentiation trajectory of cardiomyocytes from MYOM2+CMs (never transplanted from GTKO pigs) to the predominantly PTGER3+CMs after xenograft transplantation was analyzed using Monocle 2 pseudo-time series analysis. The results showed that normal cardiomyocytes (MYOM2+CMs) were mainly located at one end of the pseudo-time series, while xenografted cardiomyocytes were distributed at both ends. Figure 19 Pseudo-temporal analysis identified five different cardiomyocyte states, with state 4 primarily composed of normal cardiomyocytes. Figure 20 These results highlight the significant differences in gene expression profiles between normal cardiomyocytes and xenograft cardiomyocytes.

[0066] Using state 4 as the root, the differentiation trajectory of normal cardiomyocytes transforming into xenograft cardiomyocytes was traced. In Fate 1, progressively upregulated genes were associated with heart failure, including NPPB and IGF2BP2. Meanwhile, genes upregulated in Fate 2 were associated with cell proliferation, such as LPP, CDK14, BACH2, and RAPGEF5 (PMID: 29290587, PMID: 38586957). Figure 21 and Figure 22Notably, LPP is a LIM protein highly expressed in smooth muscle (PMID: 12760907). These findings indicate that cardiomyocyte types undergo significant changes after xenograft transplantation, corresponding to cell types obtained from cell clustering and different experimental groups. Dedifferentiation of cardiomyocytes due to inflammatory and heart failure responses is a major cause of cardiac dysfunction and hypertrophy.

[0067] III. Subtype Analysis of Lymphocytes Lymphocyte immune cell subtype analysis identified 7 different subtypes ( Figure 23 In non-donor pig hearts, the majority of lymphocytes are CXCR4+CD8+ T cells (TCs), accounting for 90% of the total lymphocytes. After xenografting, significant infiltration of CCR7+CD4+TCs, XCL1+CD8+TCs, and CD48+CD8+TCs from rhesus monkeys was observed in xenograft tumors, and their proportions did not decrease even with immunosuppressants. However, rituximab treatment significantly inhibited the infiltration of CXCR5+ B cells in xenograft tumors. Figure 24 ).

[0068] Further investigation was conducted into the effects of conventional immunosuppressants on T cell gene expression. In CCR7+CD4+TC, immunosuppressive therapy significantly downregulated the expression of immune-activating genes such as IFI27, ISG15, LAG3, IL18, and TIGIT. Figure 25 Similarly, activation of XCL1+CD8+TC was also suppressed, as evidenced by decreased expression of XCL1, IL2RB, IL2RA, ENO1, STAT3, and JAK1. Figure 26 These results indicate that conventional immunosuppressants can effectively inhibit T cell activation, but are insufficient to reduce or eliminate the immune-related responses to xenografts in already activated T cells.

[0069] IV. Subtype Analysis of Myeloid Immune Cells Myeloid immune cell subtype analysis identified 7 different subtypes ( Figure 27 and Figure 28 In non-donor pig hearts, the dominant myeloid immune cell subtypes were MRC1+ macrophages (MCs), C1QC+ macrophages, and ISG15+ macrophages. In the GTKO group, IL1B+ MCs were predominant. Figure 29 These IL1B+MCs are characterized by high expression of PLAC8, CCL20, IL1B, and GAPDH. Figure 28PLAC8 expression indicates that these macrophages were recruited from peripheral blood. Furthermore, high expression of CCL20 and IL1B suggests they play a role in exacerbating the inflammatory response and recruiting more immune cells to infiltrate the graft. In the GTKO+IS group, TIMP1+MC was more abundant and showed high expression of CD52 and CFD. Notably, cleavage of CFD catalytic factor B was observed, a rate-limiting step in the alternative complement activation pathway.

[0070] C1QC+MC, or tissue-resident macrophages, were present in all three groups. Venn analysis identified 538 uniquely upregulated genes in C1QC+MC of the GTKO+IS group. Figure 30 GO enrichment analysis showed that these genes were associated with responses to external biological stimuli, innate immune responses, and defense responses, indicating that conventional immunosuppressive drugs could not suppress the activation of the innate immune system in donor pigs. Figure 31 ).

[0071] The changes in rhesus macaque-derived macrophages (MCs) following conventional xenograft immunosuppressive therapy were further evaluated. Results showed that genes related to humoral immune responses, positive regulation of cytokine production, and complement activation were significantly downregulated in IL1B+ MCs after conventional immunosuppressive therapy. Figure 32 A similar trend was observed in TIMP1+MC; compared to the GTKO+IS group, genes highly expressed in the GTKO group were associated with positive regulation of defense responses, positive regulation of cytokine production, and classical NF-κB signaling transduction. Figure 33 These results indicate that conventional immunosuppressive drugs can effectively inhibit macrophages derived from recipient rhesus monkeys, but have a poor inhibitory effect on macrophages derived from donor pigs.

[0072] V. Analysis of intercellular interactions To further investigate the interactions between cardiomyocytes and non-cardiomyocytes, CellphoneDB was used to analyze the interactions between cardiomyocytes and other major cell types. The results showed that the main ligand-receptor pairs in the non-transplanted donor pig heart were IGF1-IGF1R and IGF2-IGF1R, indicating that the 2-month-old pig heart has strong growth potential. Furthermore, after xenografting, IGF1R signaling was present in both the GTKO and GTKO+IS groups, suggesting that IGF1R may be a key mediator of pig heart growth. Figure 34 (A) Notably, a novel PPIA-BSG signaling pathway was observed in multiple cell types and cardiomyocyte communication in the GTKO group. Figure 34(B in the original text). Previous studies have shown that PPIA binds to its extracellular receptor BSG (CD147), triggering a series of inflammatory responses. Recent evidence suggests that both extracellular and intracellular CyPA (encoded by PPIA) significantly promote cardiovascular inflammation, myocardial ischemia and reperfusion injury, and myocardial remodeling (PMID: 24518139). Moreover, this ligand-receptor pair is also present in the GTKO+IS group ( Figure 34 (C in the text). Based on these findings, it is hypothesized that the PPIA-BSG signaling pathway may be involved in initiating the inflammatory cascade and hypertrophic response in cardiomyocytes.

[0073] To verify this hypothesis, immunofluorescence was first used to examine the expression of PPIA-BSG in xenograft. The results showed that PPIA and CD147 were significantly deposited on the cardiomyocyte membrane in the GTKO group after xenograft (specifically detected by cTNT staining of cardiomyocytes). However, this phenomenon was not observed in pig hearts that did not receive GTKO transplantation. Furthermore, in the GTKO+IS group receiving conventional immunosuppressive drugs, the deposition of PPIA and CD147 on the cardiomyocyte membrane was not completely eliminated, indicating that conventional immunosuppressants cannot completely eliminate PPIA-BSG communication. Figure 35 ).

[0074] To explore whether this phenomenon represents a universal characteristic of xenotransplantation, a rat-to-mouse SD-Balb / c heterotopic heart xenotransplantation model was established. On the 4th day after heart transplantation, the expression of the PPIA-BSG pathway in the heart tissue of this model was detected by multi-label immunohistochemistry. Significant deposition of PPIA and CD147 was also found on the surface of TTN-stained cardiomyocytes. Figure 36 This result indicates that PPIA-BSG pathway expression is also present in SD-Balb / c xenograft heart transplantation, and this pathway is prevalent in xenograft heart transplantation. Therefore, similar research results using the SD-Balb / c animal model are reliable. The pathological staining results and transcriptomic data analysis of the intercellular ligand-receptor communication network in both models are consistent, further demonstrating that PPIA-BSG mediates some inflammatory responses, myocardial remodeling, and myocardial hypertrophy after xenograft transplantation.

[0075] VI. Inhibition of extracellular PPIA can improve cardiomyocyte hypertrophy and damage after cardiac xenotransplantation. In xenograft models, conventional immunosuppressants cannot effectively inhibit the communication link between extracellular PPIA and BSG. Therefore, specific inhibitors targeting the PPIA-BSG pathway are needed to help eliminate or alleviate myocardial remodeling, heart failure, and hypertrophy caused by cardiac xenograft. To demonstrate that blocking PPIA-BSG interaction can reduce cardiomyocyte damage and hypertrophy, NIM811, a non-immunosuppressive inhibitor targeting extracellular PPIA, was administered as monotherapy (10 mg / kg / day) in an SD-to-Balb / c cardiac xenograft model, and its efficacy as monotherapy versus combination therapy with the conventional immunosuppressant tacrolimus was evaluated. The heart graft was harvested on day 4 post-surgery for pathological evaluation.

[0076] H&E staining results showed that in the control group of the SD-to-Balb / c cardiac xenograft model without drug intervention, significant changes occurred in cell morphology and tissue structure, including disruption of cell edge structures, increased cell volume, enlarged nuclei, and obvious myocardial hypertrophy. After intervention with the conventional immunosuppressant tacrolimus (FK506), cell morphology and structure were clearer, and cell arrangement was more orderly, but cardiomyocyte volume was still relatively large. After intervention with the PPIA (CyPA) inhibitor NIM811, cell volume was significantly smaller than that of FK506 cells, with complete and clear cell outlines. The combined use of FK506 and NIM811 further reduced cell volume compared to FK506 alone. Figure 37 ).

[0077] WGA staining was used to label the cell membrane to aid in the identification of cardiomyocyte boundaries and morphology. Statistical analysis of cell size was performed to assess cardiomyocyte hypertrophy. The staining results combined with statistical analysis showed that the control cells (without drug intervention) had significantly larger cell membrane boundaries. FK506 cells had more orderly cell boundaries and were larger. Cardiomyocytes in the NIM811 monotherapy and combination therapy groups were significantly smaller. This result indicates that NIM81 is the primary inhibitor of cardiomyocyte hypertrophy, and its anti-hypertrophy effect is significantly stronger than tacrolimus. However, the combination therapy group did not further reduce cardiomyocyte hypertrophy compared to the NIM811 monotherapy group. Figure 38 ).

[0078] In an immunohistochemical study assessing T cell infiltration using CD3, the inflammatory response showed significant immune infiltration in the control group. Drug intervention reduced cardiomyocyte damage, but interstitial hemorrhage and inflammatory cell infiltration persisted. Tacrolimus significantly inhibited immune cell infiltration, while combination therapy not only better maintained the integrity of myocardial structure but also significantly reduced immune cell infiltration. Figure 39 ).

[0079] To further evaluate cardiomyocyte apoptosis, TUNEL staining was performed. Results showed that significant apoptosis was observed in cardiomyocytes in the control group, while only a few apoptotic cells were observed in the FK506 intervention group. In the combination therapy group, apoptotic cells were mainly concentrated in non-cardiomyocyte cells in the intercellular spaces, such as immune cells and cardiac endothelial cells. Therefore, the results indicate that compared to FK506-tacrolimus monotherapy, NIM811 combined with tacrolimus treatment further improved cardiomyocyte apoptosis. Figure 40 ).

[0080] These findings suggest that NIM811, which blocks extracellular PPIA, has a potent anti-hypertrophy effect and indirectly inhibits immune activation and T cell infiltration by reducing cardiomyocyte apoptosis.

[0081] In summary, this study investigated the role of inflammation in cardiomyocyte hypertrophy and remodeling using a conventional immunosuppressive regimen and a control group without immunosuppression. Single-cell and single-nuclear sequencing revealed the interaction between immunity and cardiomyocytes and identified the PPIA-BSG axis as a key pathway mediating myocardial hypertrophy after xenograft heart transplantation. Targeting this pathway with PPIA inhibitors significantly reduced cardiomyocyte hypertrophy, providing a potential therapeutic strategy for alleviating malignant cardiac hypertrophy after xenograft transplantation. Furthermore, the use of PPIA-BSG pathway-related inhibitors, such as the non-immunosuppressant NIM811, inhibited extracellular PPIA, showing significant efficacy in reducing myocardial hypertrophy and apoptosis. Therefore, NIM811 has an anti-xenograft effect on cardiac hypertrophy after xenograft heart transplantation, providing therapeutic strategies and drug target information for myocardial hypertrophy after xenograft heart transplantation.

[0082] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a PPIA-BSG pathway inhibitor in the preparation of a drug for resisting myocardial hypertrophy after heterotopic heart transplantation.

2. Use of a PPIA-BSG pathway inhibitor combined with a conventional immunosuppressant in the preparation of a drug for resisting myocardial hypertrophy after heterotopic heart transplantation.

3. Use of a PPIA-BSG pathway inhibitor in the preparation of a drug for resisting inflammatory response and / or myocardial remodeling after heterotopic heart transplantation.

4. Use of a PPIA-BSG pathway inhibitor combined with a conventional immunosuppressant in the preparation of a drug for resisting inflammatory response and / or myocardial remodeling after heterotopic heart transplantation.

5. The use according to any one of claims 1 to 4, characterized in that, The PPIA-BSG pathway inhibitor includes a PPIA inhibitor and / or a BSG inhibitor; the PPIA-BSG pathway inhibitor includes at least one of NIM811, cyclosporine A, a neutralizing anti-CyPA monoclonal antibody, alisporivir, NV651, Sanglifehrin A, a CD147 monoclonal antibody Meplazumab, AC-73, and licarin.

6. The use according to any one of claims 1 to 4, characterized in that, The PPIA-BSG pathway inhibitor is used to inhibit extracellular PPIA.

7. Use according to claim 2 or 4, characterized in that, The conventional immunosuppressant includes tacrolimus or rituximab.

8. PPIA Genes and / or BSG Use of genes as therapeutic targets in the preparation or screening of drugs against myocardial hypertrophy after heterologous heart transplantation.

9. PPIA Genes and / or BSG Use of genes as therapeutic targets in the preparation or screening of drugs against inflammatory response and / or myocardial remodeling after heterologous heart transplantation.

10. Use according to any one of claims 1, 2 and 8, characterized in that, The anti-myocardial hypertrophy after heterotopic heart transplantation includes at least one of the following: 1) improving myocardial cell hypertrophy; 2) reducing myocardial cell damage; 3) inhibiting immune activation and T cell infiltration; 4) maintaining the integrity of myocardial structure; 5) improving myocardial cell apoptosis.

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