Drug delivery system and application thereof in preparation of drug for treating myocardial ischemia-reperfusion injury

Abstract

The invention discloses a drug delivery system and application thereof in preparation of drugs for treating myocardial ischemia-reperfusion injury, and relates to the technical field of myocardial ischemia-reperfusion injury. The drug delivery system is constructed on the basis of lipidosome, a transmembrane structure domain of a lipidosome membrane layer and a SpyCatcher-SpyTag system, and the protective effect of a small-molecule inhibitor or a copper death inhibitor of Cpeb3 on the cardiac function is further improved through the delivery system. Pharmacological experiments show that the drug delivery system shows an excellent myocardial protection effect in both small and large animal models, can inhibit fibrosis, improve cardiac functions and enhance electrical conduction, and has minimal side effects and good biocompatibility. Therefore, the drug delivery system has important clinical application potential in the aspect of treating MIRI, and is particularly suitable for old patients.

Description

Technical Field

[0001] The present invention relates to the technical field of myocardial ischemia-reperfusion injury, and in particular to a drug delivery system and application thereof in preparing a drug for treating myocardial ischemia-reperfusion injury. Background Art

[0002] Metal ions are important elements for maintaining normal physiological functions, and the lack of metal ions can lead to the interruption of biological activities. For example, zinc deficiency may cause growth retardation, while iron deficiency may lead to anemia. Studies have shown that excess iron ions in myocardial tissue can induce ferroptosis. As another important metal ion, copper, it remains to be explored whether it can cause cardiomyocyte death by inducing mitochondrial dysfunction and excessive production of reactive oxygen species (ROS). During myocardial ischemia-reperfusion injury (IRI), cardiomyocytes undergo a series of stress responses, including nutrient deficiency, ROS release, and secondary inflammatory damage. Single-cell sequencing studies have shown that cardiomyocytes are highly heterogeneous, and different cell types may respond differently to the same stimulus.

[0003] During MIRI, the metabolic pattern of cardiomyocytes changes from fatty acid metabolism to glucose metabolism dominated by glycolysis. The ATP generated by glycolysis not only maintains cell activity, but also supports Na + / K + -ATPase and Ca 2+ -ATPase pump function, which is essential for maintaining the myocardial potential gradient and the propagation of action potential. Hexokinase 2 (HK2) is a key enzyme in the glycolysis pathway, responsible for catalyzing the phosphorylation of glucose to produce glucose-6-phosphate. It is mainly located in the outer membrane of mitochondria and regulates mitochondrial energy supply. Studies have shown that amino acid metabolism and related proteins play an important role in regulating glycolysis, including proteins such as c-Myc, Sirt1 and Akt, which connect glycolysis and amino acid metabolism by affecting HK2. Cpeb (cytoplasmic polyadenylation element binding protein) family RNA binding proteins (including Cpeb1 / 2 / 3 / 4) regulate the stability and translation activity of mRNA by binding to the 3' untranslated region (3'-UTR) of mRNA, especially under stress conditions such as hypoxia or energy deficiency. Cpeb3 affects amino acid metabolism by regulating the mTOR signaling pathway, ATF4-mediated stress response and GCN2 / eIF2α pathway.

[0004] The interaction between RNA-binding proteins and glycolytic proteins may play a key role in metabolic reprogramming. Liquid-liquid phase separation (LLPS) is an emerging intracellular regulatory mechanism that forms a dynamic "droplet" structure through intermolecular interactions, allowing metabolic enzymes to concentrate under stress conditions to optimize cellular responses. Studies have shown that glycolytic enzymes such as PKM2 and PFK1 can undergo LLPS and localize to specific regions to regulate glycolytic flux and efficiency. On the other hand, the role of RNA-binding proteins in regulating RNA modifications has received increasing attention, especially the widespread m6A modification (N6-methyladenosine), which affects mRNA stability, splicing, and translation.

[0005] Copper deficiency can cause disturbances in biological activities, while the role of copper excess in myocardial ischemia-reperfusion injury (MIRI), especially in specific cardiomyocyte subpopulations, remains unclear.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a drug delivery system and its application in preparing drugs for treating myocardial ischemia-reperfusion injury to solve the above technical problems.

[0008] The present invention is achieved in that:

[0009] In a first aspect, the present invention provides a drug delivery system, comprising: a small molecule inhibitor of Cpeb3 or a copper death inhibitor and a liposome; the liposome comprises a membrane layer, the small molecule inhibitor of Cpeb3 or the copper death inhibitor is located in the membrane layer, and the membrane layer further comprises a transmembrane domain; the transmembrane domain is connected to a spy catcher (SpyCatcher);

[0010] The copper death inhibitor is selected from molybdenum tetrasulfide, UK5099 and Antimycin A; the small molecule inhibitor of Cpeb3 is selected from the compound shown in formula I or its salt and the compound shown in formula II or its salt.

[0011] Formula I is: Formula II is:

[0012] In a second aspect, the present invention also provides a method for preparing a drug delivery system, which comprises the following steps: first preparing a membrane layer of a liposome, and then mixing the membrane layer of the liposome with a transmembrane domain connected to a spy catcher to obtain a liposome modified with a transmembrane domain-spy catcher; mixing the liposome modified with a transmembrane domain-spy catcher with a small molecule inhibitor of Cpeb3 or a copper death inhibitor; obtaining a liposome encapsulating a small molecule inhibitor of Cpeb3 or a copper death inhibitor.

[0013] In a third aspect, the present invention also provides an application of a drug delivery system in the preparation of a drug for preventing and / or treating myocardial ischemia-reperfusion injury.

[0014] The present invention has the following beneficial effects:

[0015] The present invention confirms that copper overload exists during myocardial ischemia-reperfusion injury (MIRI) and induces Gpc6 positive cardiomyocytes (Gpc6 + Copper overload promotes the high expression of Cpeb3, which interacts with HK2 through liquid-liquid phase separation (LLPS) and m6A modification, regulates metabolic reprogramming and induces copper death.

[0016] The present invention identifies a small molecule inhibitor of Cpeb3 through high-throughput drug screening, and the small molecule inhibitor of Cpeb3 can significantly reverse the changes in copper death, aerobic respiration and / or glycolysis caused by Cpeb3 activation. In addition, copper death inhibitors such as TTM and other substances have the effects of improving the survival rate of cardiomyocytes, reversing the changes in aerobic respiration and / or glycolysis induced by Cu, and reversing the changes in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle metabolites caused by Cu stimulation.

[0017] The present invention provides a drug delivery system for encapsulating a small molecule inhibitor of Cpeb3 or a copper death inhibitor, which is constructed based on liposomes, transmembrane domains of liposome membrane layers, and a SpyCatcher-SpyTag system, and the delivery system further improves the protective effect of the small molecule inhibitor of Cpeb3 or the copper death inhibitor on cardiac function. Toxicological experiments have shown that the drug delivery system exhibits excellent myocardial protection in small (rat) and large animal (Bama miniature pig) models, can inhibit fibrosis, improve cardiac function and enhance electrical conduction, while having minimal side effects and good biocompatibility. This shows that the drug delivery system has important clinical application potential in the treatment of MIRI, and is particularly suitable for elderly patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 To investigate how copper overload occurs in MIRI and acts on Gpc6 via Cpeb3-mediated metabolic reprogramming +CMs, leading to subsequent copper death experimental results; (A) Peripheral blood was collected from healthy individuals (n = 20) and myocardial infarction patients (24 hours; n = 40), and copper levels and cTnI were measured. (B) Correlation analysis between copper and cTnI was performed. Single-cell sequencing data of myocardial infarction mouse hearts were analyzed

[23] , and clustered cardiomyocytes were displayed using unified manifold approximation and projection (UMAP) maps (snRNA-seq) (right), which were annotated according to spatial localization and gene expression patterns. (C) Quantification of the composition of clustered cardiomyocytes; (D) Violin plots using the specific cardiomyocyte marker Gpc6 were used to display snRNA-seq data in different cardiomyocyte clusters. Gpc6 was isolated by flow cytometry using adult rat primary cardiomyocytes (ARPCs) + CMs, subjected to myocardial infarction (hypoxia and starvation for 1 h): (E) Cell survival after 24 h of treatment with different concentrations of elesclomol-Cu (ratio 1:1, ES_Cu) or disulfiram-Cu (ratio 1:1, DS_Cu) was measured by CCK-8 method; (F) Mitochondrial oxygen consumption rate (OCR) was analyzed by hippocampal model mitochondrial stress test after 24 h of ES_Cu (50 nM) treatment; (G) Extracellular acidification rate (ECAR) was analyzed by hippocampal model mitochondrial stress test; Oligo (oligomycin), FCCP (carbon cyano trifluoromethoxyphenylhydrazine), Rot (rot), Ant (antimycin A), 2-DG (2-deoxyglucose), Rot+Ant represents a mixture of equal concentrations of Rot and Ant. (H) Volcano plot showing Gpc6 24 h after myocardial infarction + Differentially expressed genes (DEGs) between CMs and Gpc6-CMs, threshold of differentially expressed genes: padj < 0.05, blue indicates down-regulation, red indicates up-regulation, and gray indicates no change, Gpc6 + Upregulated Cpeb3 and downregulated HK2 in CMs are marked in green. Gpc6 isolated from ARPCs + CMs were knocked down (shCpeb3) or overexpressed (HK2-OE) Cpeb3 and subjected to myocardial infarction (hypoxia and starvation for 1 h). They were stimulated with ES_Cu (50 nM) for 24 h in the absence or presence of TTM (10 μM; molybdenum tetrasulfide, a copper death inhibitor). (I) Cell survival was measured by CCK-8 assay; (J) OCR was analyzed by hippocampal model mitochondrial stress test; (K) ECAR was analyzed. (L) The proposed model summarizes the role of copper-Cpeb3 signaling in Gpc6 +Role of copper death associated with metabolic reprogramming of CMs. (M) Analysis of single-cell sequencing data of human hearts at different ages to quantify the composition of clustered cardiomyocytes, with clusters 1 and 3 marked by high expression of Gpc6. Data are presented as mean ± standard deviation. A was determined by two-tailed t-test, B was calculated by Pearson correlation, and I was calculated by one-way ANOVA, **p<0.01, ****p<0.001, ns indicates no statistical difference;

[0020] Figure 2 To establish myocardial ischemia-reperfusion injury (MIRI) models in Bama miniature pigs and rats, we investigated the expression of copper ions and Gpc6 in myocardial cells and myocardial tissues. + Results of myocardial cell ratio detection; Myocardial ischemia-reperfusion injury (MIRI) model was established in (C) Bama miniature pigs and (F) rats by ligating the left anterior descending artery, and ST segment elevation in the electrocardiogram confirmed the successful establishment of the model; (A) Detection of copper ion content in myocardial tissue of Bama miniature pigs at different time points after MIRI. (B) Analysis of single-cell sequencing data from mouse hearts after myocardial infarction3 to quantify the composition of clustered myocardial cells. (D, E) Primary cardiomyocytes of adult Bama miniature pigs and (G, H) rats were isolated at different time points after MIRI, and Gpc6 was determined by (E, H) flow cytometry. + Cardiomyocytes (Gpc6 + The ratio of CMs was calculated and statistically analyzed (D, G). Data are expressed as mean ± SD. P values ​​were calculated by one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, ns indicates no statistical difference;

[0021] Figure 3 Gpc6 isolated from adult rat primary cardiomyocytes (ARPCs) + Cardiomyocytes (Gpc6 + CMs) were treated with infarction (IF, hypoxia and starvation for 1 hour), and then stimulated with ES_Cu (50 nM) for 24 hours, or stimulated with TTM (10μM), Z-VAD-FMK (20μM), Nec-1 (20μM) or Fer-1 (10μM) for 24 hours for cell viability test results; (A) CCK-8 method was used to detect cell viability; (B, C) PI (propidium iodide) staining combined with flow cytometry was used to detect cell death, and (C) statistical analysis was performed. Gpc6 isolated from ARPCs +CMs were treated with infarction (hypoxia and starvation for 1 h), (D) stimulated with different concentrations of Elesclomol-Cu (ratio = 1:1, ES_Cu) or Disulfiram-Cu (ratio = 1:1, DS_Cu) for 24 h, and the cell survival rate was measured by CCK-8 method; (E) stimulated with ES_Cu (50 nM) for 24 h, and the cell survival rate was measured by CCK-8 method in the presence or absence of TTM (10 μM), Z-VAD-FMK (20 μM), Nec-1 (20 μM), or Fer-1 (10 μM). Gpc6 isolated from ARPCs by flow cytometry + CMs were treated with infarction (hypoxia and starvation for 1 hour) and then subjected to hippocampal mitochondrial stress test with or without ES_Cu (50 nM) stimulation for 24 hours: (F) mitochondrial oxygen consumption rate (OCR) was detected, and spare respiratory capacity, basal respiration, proton leak and ATP production capacity were calculated; (G) extracellular acidification rate (ECAR) was detected, and glycolytic capacity, glycolytic level and glycolytic reserve capacity were further calculated. Gpc6-CMs isolated from ARPCs were treated with infarction (hypoxia and starvation for 1 hour) and then subjected to hippocampal mitochondrial stress test with or without ES_Cu (50 nM) stimulation for 24 hours: (H) OCR and (I) ECAR were detected, and the relevant indicators were calculated. Data are expressed as mean ± standard deviation (mean ± SD). P values ​​were calculated by one-way ANOVA, *p<0.05, **p<0.01, ***p<0.005, ****p<0.001, ns indicates no statistical difference;

[0022] Figure 4 To establish the MIRI model using Bama miniature pigs, samples were collected from different regions of myocardial tissue for protein extraction or paraffin sectioning, followed by (A) Western blot and (B) immunofluorescence assays to detect the expression levels of Cpeb3 and HK2, scale bar = 50 μm. (C) Gpc6 isolated from adult rat primary cardiomyocytes (ARPCs) + CMs and Gpc6-CMs were subjected to infarction treatment (IF, hypoxia and starvation for 1 h) or not, and then stimulated with ES_Cu (50 nM) for 24 h or not, and the expression levels of Cpeb3 and HK2 were detected by Western blotting. +CMs were stimulated with or without ES_Cu (50 nM) for 24 h under conditions of (D) knockdown of Cpeb3 (shCpeb3) or (E) knockdown of HK2 (shHK2), and Western blot was performed to evaluate the expression levels of (D) HK2 and (E) Cpeb3. Gpc6 was extracted based on published single-cell sequencing data after myocardial infarction in mice. + The whole genome expression data of high-expressed Cpeb3 and low-expressed HK2 in CMs were subjected to KEGG pathway analysis to show the relevant pathways of (F) high expression of Cpeb3 and (G) low expression of HK2. Gene set enrichment analysis (GSEA) showed that: (H) HK2 was negatively correlated with copper death and positively correlated with glycolysis; (I) On the contrary, Cpeb3 was positively correlated with copper death and negatively correlated with glycolysis;

[0023] Figure 5 Gpc6 isolated from adult rat primary cardiomyocytes (ARPCs) + CMs knocked down Cpeb3 (shCpeb3) or overexpressed HK2 (HK2-OE), then underwent infarction treatment (IF, hypoxia and starvation for 1 h), and were stimulated with or without ES_Cu (50 nM) for 24 h in the presence or absence of TTM (10 μM). (A, B) Cell mortality was detected by PI (propidium iodide) staining combined with flow cytometry, (B) and statistical analysis was performed; (C) Mitochondrial ROS levels were detected by flow cytometry. Seahorse mitochondrial stress test was performed to detect (D) mitochondrial oxygen consumption rate (OCR), calculate spare respiratory capacity, basal respiration, proton leak, and ATP production; (E) The extracellular acidification rate (ECAR) was detected, and glycolytic capacity, glycolytic level, and glycolytic reserve were further calculated; the effects of shCpeb3, HK2-OE, or TTM on Gpc6 after infarction and ES_Cu stimulation were analyzed. + Effect of glucose-derived metabolites and percentage of total metabolites in CMs. Cells were incubated with [U-13C]glucose (1.5 mM) for 24 h, and metabolites related to (F) glucose-6-phosphate (Glc-6P), (G) glycolysis, (H) pentose phosphate pathway (PPP), and (I) tricarboxylic acid cycle (TCA) were detected. Data are presented as mean ± standard deviation (mean ± SD). P values ​​were calculated by one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, ns indicates no statistical difference;

[0024] Figure 6To analyze single-cell sequencing data (snRNA-seq) from human hearts, 12-14 images were drawn using the unified manifold approximation and projection (UMAP) method to show: (A) all cells and (B) clustered cardiomyocytes (right), with clusters annotated based on gene expression patterns. Data are from representative samples of different age groups (young group, age ≤ 45; middle-aged group, 45 < age < 60; old group, age ≥ 60). (C) In different clusters of cardiomyocytes, violin plots are used to show the expression of Gpc6, a marker for specific cardiomyocyte subsets, in snRNA-seq data;

[0025] Figure 7 Com2 is an inhibitor of Cpeb3. (A) Flow chart of screening small molecule Cpeb3 inhibitors. IF (1 hour of hypoxia and starvation treatment), Gpc6 isolated from adult rats + CMs (cardiomyocytes) and CMFs (cardiac fibroblasts). Cell viability was detected by CCK-8 and SPR (surface plasmon resonance) analysis. (B) Chemical structure of Com2. (C) Binding of Com2 to rat Cpeb3 recombinant protein was detected by SPR assay. Gpc6 isolated from ARPCs + CMs, Cpeb3 was knocked down (shCpeb3) and subjected to myocardial infarction (hypoxia and starvation for 1 h), stimulated with ES_Cu (50 nM) for 24 h, treated in the presence or absence of Com2 (5 μM), (D) cell survival was detected by CCK-8, (E) OCR (oxygen consumption rate) was analyzed by Seahorse mitochondrial stress test, (F) ECAR (extracellular acidification rate) was analyzed. Data are expressed as mean ± SD. P values ​​were calculated by one-way analysis of variance (ANOVA), ****p<0.001, ns indicates no statistical difference;

[0026] Figure 8 (A) Chemical structure of Com1; (B) SPR determination of the binding of Com1 to rat Cpeb3 recombinant protein. Gpc6 isolated from ARPCs +CMs were subjected to ischemia-reperfusion injury (hypoxia and starvation for 1 h) and stimulated with ES_Cu (50 nM) for 24 h in the absence or presence of Com1 (5 μM) or Com2 (5 μM): (C) Cell survival was detected by CCK-8 method, (D) Cell death was detected by PI (propidium iodide) staining flow cytometry, (E) and statistical analysis was performed, (F) Mitochondrial ROS levels were assessed by flow cytometry. The Seahorse mitochondrial stress test was used to detect: (G) Extracellular acidification rate (ECAR), which was further calculated for glycolytic capacity, glycolysis and glycolytic reserve, (H) Mitochondrial oxygen consumption rate (OCR), which was used to calculate spare respiratory capacity, basal respiration, proton leak and ATP production. Data are expressed as mean ± standard deviation (mean ± SD). P values ​​were calculated by one-way ANOVA, *p<0.05, **p<0.01, ***p<0.005, ****p<0.001, ns indicates no statistical difference;

[0027] Fig. 9 (A) Schematic diagram of the docking interaction between the complex Com2 (blue) and rat Cpeb3 protein (red), solid green lines represent hydrogen bonds, and dashed green lines represent π-π bonds. (B) SPR was used to measure the binding effect of Com2 with wild-type or specific amino acid mutant rat Cpeb3 recombinant proteins. The binding of Com2 to (C) human, (D) pig, and (E) mouse Cpeb3 recombinant proteins was measured by SPR. Gpc6 isolated from ARPCs + CMs were subjected to ischemia-reperfusion injury (hypoxia and starvation for 1 h) and stimulated with ES_Cu (50 nM) for 24 h in the absence or presence of Com2 (5 μM) or Cpeb3 knockdown (shCpeb3): (F) Cell death was detected by PI (propidium iodide) staining flow cytometry, (G) and statistical analysis, (H) Mitochondrial ROS levels were assessed by flow cytometry. The Seahorse mitochondrial stress test was used to detect: (I) mitochondrial oxygen consumption rate (OCR) and (J) extracellular acidification rate (ECAR), and the relevant index was calculated. Data are expressed as mean ± SD. P values ​​were calculated by one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, ns indicates no statistical difference;

[0028] Fig.10 (A) Gpc6 isolated from ARPCs +CMs were subjected to ischemia (hypoxia and starvation for 1 h) after Cpeb3 knockdown (shCpeb3) and then stimulated with ES-Cu (50 nM) for 24 h in the absence or presence of Com2 (5 μM). + Effect of glucose-derived metabolites and percentage of total metabolites in CMs. After ischemia and ES_Cu stimulation, cells were cultured with [U-13C]glucose (1.5 mM) for 24 h, and the following metabolites were detected: (A) Glc-6P, (G) glycolysis, (H) tricarboxylic acid cycle (TCA), (G) pentose phosphate pathway (PPP) related metabolites. Data are expressed as mean ± standard deviation (mean ± SD). P values ​​were calculated by one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, ns indicates no statistical difference;

[0029] Fig.11 To study the interaction between Cpeb3 and HK2 through LLPS and m6A modification. (A) Immunoprecipitation-Western blotting (IP-western blotting) was used to detect the interaction between Cpeb3 and HK2 in rat Gpc6 + Binding in CMs, stimulated by 1 h of hypoxia and starvation, in the presence or absence of ES_Cu (50 nM) treatment.

[0030] (B) Gpc6 in rats with Cpeb3 knockdown (shCpeb3) or HK2 overexpression (HK2-OE) + CMs, subjected to myocardial infarction (hypoxia and starvation for 1 h), were stimulated with ES_Cu (50 nM) for 24 h, and the fluorescence redistribution of the photobleached region of HK2-mCherry aggregates was quantitatively measured in the presence or absence of Com2 (5 μM) (n=10). (C) The affinity of recombinant rat Cpeb3 and HK2 was detected by SPR assay. Rat Gpc6 +CMs were subjected to myocardial infarction (hypoxia and starvation for 1 h) and stimulated with ES_Cu (50 nM) for 24 h in the presence or absence of Com2 (5 μM). (D) The binding of HK2 mRNA to Cpeb3 was measured by RIP assay, (E) The m6A modification of HK2 mRNA was detected by MeRIP qPCR assay, (F) Cpeb3 was knocked down and the binding effect of HK2 mRNA to ALKBH5 / YTHDC1 was tested by RNA pull-down assay. (G) Com2 (5 μM) or not was further added, and new RNA synthesis was blocked with actinomycin D (5 μg / mL), and the stability of HK2 mRNA was tested, which was measured by qRT-PCR with fold change after 0 h. (H) The proposed model summarizes the Cpeb3-HK2 signaling pathway in Gpc6 + Role of cuproptosis in CMs associated with metabolic reprogramming. Data are presented as mean ± SD. P values ​​were calculated by one-way analysis of variance (ANOVA), *p < 0.05, ****p < 0.001, ns indicates no statistical difference;

[0031] Fig.12 (A) Simulation of the binding between Cpeb3 and HK2 across species. (B) Gpc6 isolated from ARPCs + CMs, after Cpeb3 knockdown (shCpeb3), were subjected to ischemia (hypoxia and starvation for 1 h) and then stimulated for 24 h in the absence or presence of Com2 (5 μM) or with ES_Cu (50 nM). The expression of Cpeb3 (green) and HK2 (yellow) and their colocalization with mitochondria (red) and nuclei (blue) were detected by immunofluorescence. Nuclei were stained with Hoechst 33342, scale bar = 20 μm. Gpc6 isolated from ARPCs + CMs were subjected to ischemia (hypoxia and starvation for 1 h) and then stimulated with ES_Cu (50 nM) for 24 h. (C) Time-lapse images of Cpeb3-GFP (left) and HK2-mcherry (right), (D) FRAP analysis of Cpeb3-GFP (top) and HK2-mcherry (bottom) aggregates, (E) FRAP quantitative analysis of bleached areas in Cpeb3-GFP and HK2-mcherry aggregates.

[0032] Fig.13(A) Representative fluorescence images of HK2-mcherry droplets in the presence or absence of Cpeb3 or Com2, Scale bar = 10 μm. The affinity of recombinant rat mutant Cpeb3 to HK2 was determined by (B) SPR assay and (C) fluorescence polarization (FP) assay. (D) Gpc6 isolated from ARPCs + CMs, after Cpeb3 knockdown (shCpeb3) or overexpression (Cpeb3-OE), were subjected to ischemia (hypoxia and starvation for 1 h) and then stimulated with ES_Cu (50 nM) for 24 h in the absence or presence of Com2 (5 μM). FRAP analysis of HK2-mcherry aggregates was performed. Data are presented as mean ± standard deviation (SD). P values ​​were calculated by one-way ANOVA, ***p<0.005, ns indicates no statistical difference;

[0033] Fig.14 (A) Gpc6 isolated from ARPCs + CMs were subjected to ischemia (hypoxia and starvation for 1 h) or not, and then stimulated in the absence or presence of ES_Cu (50 nM) for 24 h, and the expression levels of cytoplasmic Cpeb3 (CytCpeb3) and nuclear Cpeb3 (NucCpeb3) were detected by Western blotting. (B) (a) HK2 mRNA sequence information from different species, including the start position of 5'-UTR, CDS and 3'-UTR; (b) CPE sequence information in HK2 mRNA 3'-UTR of mouse, (c) rat, (d) pig and (e) human; and the m6A site predicted by SRAMP (http: / / www.cuilab.cn / sramp) and its corresponding secondary structure. (C) Rat Gpc6 + CMs were subjected to ischemia (hypoxia and starvation for 1 h) and then stimulated with ES-Cu (50 nM) for 24 h. The binding of HK2 mRNA and Cpeb3 was measured using RIP assay. + In CMs, (D) the binding effect of wild-type and mutant HK2 mRNA 3'-UTR with Cpeb3 protein was evaluated by luciferase reporter gene assay; (E) HK2 mRNA binding region and (F) sequence binding with Cpeb3 protein were detected by RNA pull-down assay. Data are expressed as mean ± standard deviation (SD). P values ​​were calculated by one-way ANOVA, ***p<0.005, ****p<0.001, ns indicates no statistical difference;

[0034] Fig.15 Rat Gpc6 +After CMs were subjected to ischemia (hypoxia and starvation for 1 hour), (B, C, D) Cpeb3 gene knockout (shCpeb3) or (A) non-knockout were performed, followed by ES_Cu (50 nM) stimulation for 24 hours, and the m6A RNA modification of HK2 mRNA was detected by MeRIP assay; proteins bound to Cpeb3 were collected by immunoprecipitation with Cpeb3-coated magnetic beads, and the most significantly changed proteins were evaluated by mass spectrometry (MS), and statistical analysis was performed for (C) ALKBH5 and (D) YTHDC1, two proteins associated with m6A modification, which showed the most significant changes in abundance changes. (E) Rat Gpc6 + CMs were subjected to ischemia (hypoxia and starvation for 1 h) and subjected to Cpeb3 knockout (shCpeb3) or YTHDC1 overexpression (YTHDC1-OE) and stimulated with ES-Cu (50 nM) for 24 h. The nuclear and cytoplasmic fractions of HK2 mRNA were detected by qRT-PCR. (F) Rat Gpc6 + CMs were subjected to ischemia (hypoxia and starvation for 1 hour) and stimulated with ES_Cu (50 nM) for 24 hours. The colocalization of Cpeb3, YTHDC1, and YTHDC1 in the nucleus was detected by confocal microscopy in the absence or presence of Com2 (5 μM). Barcode = 10 μm. Data are expressed as mean ± standard deviation (SD). P values ​​were calculated by one-way ANOVA, **p < 0.01, ****p < 0.001, ns indicates no statistical difference;

[0035] Fig.16Synthesis and characterization of editable targeted liposomes. (A) Schematic diagram of the preparation of editable targeted liposomes, in which the SpyTag-Ligand part is exchangeable, ensuring that the liposomes have editable targeting capabilities. STMD is the transmembrane domain of transmembrane proteins, ensuring that the STMD-SpyCatcher component is stably anchored on the liposome surface. The agent to be delivered is encapsulated within the liposome. (B) Transmission electron microscopy (TEM) images of liposomes (Lp), STMD-SpyCatcher-modified liposomes, i.e., platform Lp (PLp), and SpyTag-Ligand-coupled PLp, i.e., functional PLp (FPLp), scale bar = 50 nm. (C) Fluorescein (Rho) was loaded into PLp, and single-chain variant fragments for heart (Gpc6), liver (HAS), spleen (CD3), lung (SFTPA1), and kidney (Podxl) were synthesized as targeting ligands to form different SpyTag-Ligand parts to target different organs, and finally injected into rats through the tail vein. After 0.5 hours, the targeting ability of isolated organs was detected by fluorescence imaging. (D) Immunofluorescence detection of rat heart slices to evaluate the targeting of Gpc6 + The capabilities of CMs;

[0036] Fig.17 (A) Schematic diagram of the STMD-SpyCatcher complex designed, in which a 6×His tag was inserted in the middle and connected by a flexible connector, and a western blot experiment of STMD-SpyCatcher was performed, prepared by incubation at 15°C for 16 hours using different STMDs (bands). (B) Anti-His immunofluorescence was used to detect and quantify the amount of different STMD-SpyCatcher structural modifications on the liposome membrane to form PLp, scale bar = 5μm. (C) Statistical analysis was then performed. (D) Schematic diagram of the SpyTag-Ligand complex designed, in which a Flag tag was inserted in the middle, and a western blot experiment of SpyTag-Ligand was performed, prepared by incubation at 15°C for 16 hours using different Ligands (bands). (E) Anti-His and anti-Flag immunofluorescence were used to detect and quantify the amount of different SpyTag-Ligand structural modifications on PLp to form FPLp, scale bar = 5μm;

[0037] Fig.18SD rats (180-220 g) were used to establish the MIRI model, with n=10 in each group. The synthetic rat Gpc6 scFv was used as a ligand to form PLp, and loaded with Com2 (PLp(Com2), 23.34±5.22 wt%). After blood supply was restored, FPLp (20 mg / Kg), Com2 (5 mg / Kg) or FPLp(Com2) (20 mg / Kg, Com2 dose is about 5 mg / kg) was injected intravenously at the beginning of reperfusion, once every 8 hours, for a total of 15 days, and the saline group was injected with an equal volume of saline, while the sham group was not ligated. After 24 hours, (A) Evans Blue & TTC (2,3,5-triphenyltetrazolium chloride) staining was used to detect reperfusion injury, (B) IF / AAR, IF (infarct size), AAR (ischemic area); (C) cTnI and (D) CK-MB (creatine kinase-MB) in plasma were detected. After 4 weeks, cardiac ultrasound examination was performed, (E, left) Doppler flow, (E, middle) M mode and (E, right) two-dimensional speckle tracking were performed, (F) E / A (early peak / atrial peak) ratio, (G) LVEF (left ventricular ejection fraction), (H) LVFS (left ventricular ejection fraction), (I) PLAXL (left ventricular parasternal long axis view) strain and (J) HR (heart rate); (K, L) Masson staining was used to detect fibrosis area, (K) and the percentage of left ventricular myocardial fibrosis area was calculated. (M) Action potentials were detected by optical imaging, (N) active time of the action potential phase, and (O) APD90 (action potential duration, 90% repolarization) were used for statistical analysis. Data are presented as mean ± standard deviation. P values ​​were calculated by one-way ANOVA, *p<0.05, **p<0.01, ***p<0.005, ****p<0.001, ns indicates no statistical difference;

[0038] Fig.19The cardioprotective effect of PLp (Com2) in the Bama mini pig MIRI model. (A) The MIRI model was established using Bama mini pigs. At the beginning of reperfusion, PLp (Com2) (20 mg / Kg, the dose of Com2 was about 5 mg / kg) was injected intravenously every 8 hours for 15 days. An equal volume of saline was injected as a saline control group, while the sham operation group was not ligated. (B) Rhodamine (Rho) was loaded into PLp and finally injected into rats via intravenous injection. After 0.5 hours, the targeting ability of the isolated organs of Bama mini pigs was detected by fluorescence imaging. (C) The plasma cTnI level was monitored, and (D) the degree of ST segment elevation in the electrocardiogram was monitored. Four weeks later, echocardiography was performed, including Doppler flow, M-mode, and 2D speckle tracking, and (E) E / A (early peak / diastolic peak) ratio, (F) LVFS (left ventricular ejection fraction), and (G) PLAXL (left ventricular parasternal long axis view) strain were calculated; (H) fibrosis area was measured by tissue sectioning and Masson staining. (I) Action potential was detected by optical imaging, and (J) calcium ion (Ca) was detected. 2+ ) transient duration. Electrocardiography was performed to evaluate ventricular electrophysiological stability and ventricular arrhythmia (VA) susceptibility, and **(K)**VA inducibility and **(L)**VF threshold were measured. Data are expressed as mean ± standard deviation (SD). P values ​​were calculated by one-way analysis of variance (ANOVA), *p<0.05, **p<0.01, ***p<0.005, ****p<0.001, ns indicates no statistical difference;

[0039] Fig. 20SD rats (180-220 g) were used to establish the MIRI model, with n=10 in each group. The synthetic rat Gpc6 scFv was used as a ligand to form PLp, and Com2 was loaded (PLp(Com2), 23.34±5.22wt%). After blood supply was restored, FPLp (20 mg / Kg), Com2 (5 mg / Kg) or FPLp(Com2) (20 mg / Kg, Com2 dose was about 5 mg / kg) was injected intravenously at the beginning of reperfusion, once every 8 hours, for a total of 15 days. The saline group was injected with an equal volume of saline, while the sham group was not ligated. After 4 weeks, (A) the Ca2+ mutation time course was detected by optical imaging, and the (B) active time in the Ca2+ mutation time course stage and (C) CTD90 (calcium transport duration, 90% recovery) were statistically analyzed. Electrocardiograms were evaluated to assess ventricular electrophysiological stability and susceptibility to ventricular arrhythmias (VA), and (D) VA inducibility and (E) VF thresholds were measured. (F) Kaplan-Meier survival curves represent survival rates throughout the entire process. (G) Liver and (H) kidney function were assessed by peripheral blood, including ALB (albumin), γ-GT (γ-glutamyl transpeptidase), AST (aspartate transaminase), ALT (alanine transaminase), ALP (alkaline phosphatase), TP (total protein), Glu (urine glucose), UA (uric acid), UREA (urea nitrogen), and CREA (creatinine). (I) Heart, liver, spleen, lung, and kidney were collected, embedded in paraffin, and stained with HE, magnification 100 μm. Data are expressed as mean ± SD. P values ​​were calculated by one-way ANOVA, *p<0.05, ***p<0.005, ****p<0.001, ns indicates no statistical difference. Survival rate was analyzed by log-rank (Mantel-Cox) test;

[0040] Fig.21A ligand was synthesized using the scFv of porcine Gpc6 to form PLp and loaded with Com2 (PLp(Com2), 23.34±5.22wt%). The MIRI model was established using Bama miniature pigs. PLp(Com2) (20 mg / Kg, Com2 dosage is about 5 mg / kg) was injected intravenously at the beginning of reperfusion, once every 8 hours for a total of 15 days. The saline group was injected with an equal volume of saline, while the sham group was not ligated. After 24 hours, (A) CK-MB (creatine kinase-MB) in plasma and (B) ST segment elevation in electrocardiogram were monitored. After 4 weeks, cardiac ultrasound examinations were performed, including (E, left) Doppler flow, (E, right) M mode and (E, bottom) two-dimensional speckle tracking, and (C) LVEF (left ventricular ejection fraction) and (D) HR (heart rate) were calculated; (F) heart tissue sections and Masson staining were performed to test the fibrosis area. (G, top) Action potential and (G, bottom) Ca2+ mutation time course were detected by optical imaging, and (H) APD90 (action potential duration, 90% repolarization) and (I) CTD90 (calcium transport duration, 90% recovery) were statistically analyzed. Data are expressed as mean ± standard deviation. P values ​​were calculated by one-way ANOVA, *p<0.05, **p<0.01, ***p<0.005, ns indicates no statistical difference;

[0041] Fig. 22 The ligand was synthesized using the scFv of porcine Gpc6 to form PLp and loaded with Com2 (PLp(Com2), 23.34±5.22wt%). The MIRI model was established using Bama miniature pigs, and PLp(Com2) (20 mg / Kg, Com2 dosage was approximately 5 mg / kg) was injected intravenously at the beginning of reperfusion, once every 8 hours for a total of 15 days. The saline group was injected with an equal volume of saline, while the sham group was not ligated. After 4 weeks, (A) kidney and (H) liver function were evaluated by peripheral blood, and the following indicators were detected: ALB (albumin), γ-GT (γ-glutamyl transpeptidase), AST (aspartate aminotransferase), ALT (alanine aminotransferase), ALP (alkaline phosphatase), TP (total protein), Glu (urine glucose), UA (uric acid), UREA (urea nitrogen), CREA (creatinine). Heart, liver, spleen, lung and kidney were collected and embedded in paraffin, followed by HE staining. (C) Tissue section staining image, barcode = 100 μm. Data are expressed as mean ± standard deviation. P value was calculated by one-way ANOVA, ns indicates no statistical difference. DETAILED DESCRIPTION

[0042] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0043] SpyTag / SpyCatcher is derived from the pilin domain that can spontaneously form isopeptide bonds, which are completely based on natural amino acids. The high irreversible reactivity and stable connection of SpyTag / SpyCatcher make it widely used in protein hydrogels, protein topological structures, synthetic drug carriers and synthetic biology fields of Spy networks. The SpyCatcher in the present invention can be derived from any commercially available SpyCatcher structure.

[0044] In a first aspect, the present invention provides a drug delivery system, comprising: a small molecule inhibitor of Cpeb3 or a copper death inhibitor and a liposome; the liposome comprises a membrane layer, the small molecule inhibitor of Cpeb3 or the copper death inhibitor is located in the membrane layer, and the membrane layer further comprises a transmembrane domain; the transmembrane domain is connected to a spy catcher (SpyCatcher);

[0045] The copper death inhibitor is selected from molybdenum tetrasulfide, UK5099 and Antimycin A; the small molecule inhibitor of Cpeb3 is selected from the compound shown in formula I or its salt and the compound shown in formula II or its salt.

[0046] Formula I is: CAS: 152684-55-4, named Com2 in this application; Formula II is: CAS: 1228690-19-4, named as Com1 in this application.

[0047] The present invention utilizes the unique advantages of small molecule compounds and identifies two potential Cpeb3 small molecule inhibitors, Com1 and Com2, through high-throughput drug screening. Com2 has a stronger binding affinity to Cpeb3 (KD = 3.29 μM) and exhibits robust binding ability to Cpeb3 protein in multiple species (such as humans, pigs and mice). Com2 binds to the molecular pocket of Cpeb3 through hydrogen bonds and π-π conjugation, which is composed of TYR-7, ARG-1, LYS-82, THR-83, SER-85 and ASP-86, among which hydrogen bonds and π-π interactions are medium-weak non-covalent interactions. In Gpc6 +In CMs, Com2 significantly inhibited ES_Cu (Elesclomol-Cu (ratio = 1:1))-induced copper death and mitochondrial ROS production, and its effect was better than Com1 and comparable to that of Cpeb3 knockdown. Compared with Com1, Com2 more effectively reversed the changes in aerobic respiration and glycolysis induced by ES_Cu, and its effects were similar to those of Cpeb3 knockdown. In addition, Com2 treatment significantly reversed the changes in glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle metabolites induced by ES_Cu stimulation, with no significant difference from the effects of Cpeb3 knockdown. These results indicate that Com2 is an excellent small molecule inhibitor of Cpeb3, which can effectively counteract the Gpc6-positive cardiomyocytes (Gpc6 + Aerobic respiration and glycolysis in CMs were altered, thereby regulating metabolic reprogramming and inhibiting the occurrence of copper death.

[0048] The present invention provides a drug delivery system for encapsulating a small molecule inhibitor of Cpeb3 or a copper death inhibitor, which is constructed based on liposomes, transmembrane domains of liposome membrane layers, and a SpyCatcher-SpyTag system, and the delivery system further improves the protective effect of the small molecule inhibitor of Cpeb3 or the copper death inhibitor on cardiac function. Toxicological experiments have shown that the drug delivery system exhibits excellent myocardial protection in small (rat) and large animal (Bama miniature pig) models, can inhibit fibrosis, improve cardiac function and enhance electrical conduction, while having minimal side effects and good biocompatibility. This shows that the drug delivery system has important clinical application potential in the treatment of MIRI, and is particularly suitable for elderly patients.

[0049] In a preferred embodiment of the present invention, the transmembrane domain is selected from at least one of the following transmembrane proteins: transmembrane receptor proteins, membrane transport proteins, ion channel proteins, pump proteins and lipid-anchored proteins.

[0050] In a preferred embodiment of the present invention, the transmembrane receptor protein is selected from GPCR or β2AR; the membrane transport protein is CFTR; the ion channel protein is selected from sodium, calcium or potassium ion channel protein;

[0051] The pump protein is selected from Na+ / K+ATPase, Ca2+-ATPase, H+-ATPase or H+-ATP synthase;

[0052] The anchoring protein is integrin.

[0053] Transmembrane proteins can also be divided into extrinsic membrane proteins, intrinsic membrane proteins and lipid-anchored proteins according to their functions. Therefore, they are not limited to transmembrane receptor proteins, membrane transport proteins, ion channel proteins, pump proteins and lipid-anchored proteins.

[0054] "G-protein coupled receptors" or "GPCRs" are polypeptides that share a common structural motif, with an extracellular amino terminus (N-terminus), an intracellular carboxyl terminus (C-terminus), and seven hydrophobic transmembrane spans (seven regions of 22 to 24 hydrophobic amino acids forming seven alpha helices), each spanning the membrane. Each span is identified by a number, i.e., transmembrane-1 (TM1), transmembrane-2 (TM2), etc.

[0055] In a preferred embodiment of the present invention, the sodium ion channel protein is selected from SCN5A, Nav1.7, Nav1.8 or Nav1.9;

[0056] In a preferred embodiment of the present invention, the transmembrane domain is selected from the transmembrane domain of β2AR, and its amino acid sequence is GTVPSDNIDSQGRNCSTNDSLL.

[0057] In a preferred embodiment of the present invention, the raw material of the membrane layer is selected from simulated plasma membrane, synthetic plasma membrane, membrane molecules extracted from biological cells, a mixture of different membrane components, or a combination thereof.

[0058] In a preferred embodiment of the present invention, the membrane layer is prepared by the following method: dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylethanolamine (DOPE) and cholesterol are mixed in a molar ratio of (4:3:3), (5:3:2), (6:3:1) or (7:2:1), and after removing the organic solvent, a lipid membrane is obtained, which is then hydrated and mixed with a transmembrane domain connected to a spy catcher.

[0059] In a preferred embodiment of the present invention, the spy catcher is further connected to a spy tag (SpyTag) and a ligand; the spy catcher and the spy tag are coupled by spontaneously forming an isopeptide bond, and the spy tag is operably connected to the ligand;

[0060] In a preferred embodiment of the present invention, in the drug delivery system, the molar ratio of the transmembrane domain connected with the spy catcher to the conjugate of the spy tag and the ligand is 1:2, 1:1 or 2:1;

[0061] In a preferred embodiment of the present invention, in the drug delivery system, the mass percentage of the small molecule inhibitor of Cpeb3 or the copper death inhibitor in the drug delivery system is (17.11-26.48) wt%.

[0062] In a preferred embodiment of the present invention, the transmembrane domain is located at the C-terminus of the spy catcher;

[0063] In a preferred embodiment of the present invention, the spy catcher is connected to the transmembrane domain via a connecting peptide;

[0064] In a preferred embodiment of the present invention, a first protein tag sequence is further provided between the spy catcher and the transmembrane domain or at the N-terminus of the spy catcher;

[0065] In a preferred embodiment of the present invention, a second protein tag sequence is provided between the ligand tag and the ligand. The protein tag sequence can facilitate the purification of the ligand tag.

[0066] In a preferred embodiment of the present invention, the first protein tag sequence and the second protein tag sequence are each independently selected from any one of the following:

[0067] His, Flag, GST, MBP, HA, Myc, GFP or Fc. The first protein tag sequence and the second protein tag sequence may be the same or different.

[0068] In a preferred embodiment of the present invention, the connecting peptide is a rigid connecting peptide or a flexible connecting peptide;

[0069] In a preferred embodiment of the present invention, the connecting peptide is a flexible connecting peptide.

[0070] In a preferred embodiment of the present invention, the length of the connecting peptide is 6 to 59 amino acids.

[0071] In a preferred embodiment of the present invention, the length of the connecting peptide is 15 to 35 amino acids, for example, 15 to 20 amino acids, 18 to 22 amino acids, 20 to 30 amino acids, or 30 to 35 amino acids.

[0072] In a preferred embodiment of the present invention, the sequence of the connecting peptide is GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS. In other embodiments, the GS connecting peptide is any one of the following: GGS, GGGS, (GS)2, GGSGG, GGGGS, GGSSG, (GGS)2, (GS)3, GGSGGSG, GGSGSGSG, GGGSGGG, (GS)4, GGSGGSGG, (GGGS)2, GSGSGSGSG, (GGS)3, GGSGGSGGSG, (GS)5, (GGGGS)2, (GGSGG)2, (G)3, (G)4, (G)5, (G)6, (G)7, (G)8, (G)9, (G)10.

[0073] In a preferred embodiment of the present invention, the spy catcher is also coupled with a fluorescent dye, and by detecting the fluorescent signal of the fluorescent dye, the subsequent cell counting and cell detection after the targeted cells of the drug delivery system, as well as the drug release effect detection can be achieved quickly and with high sensitivity.

[0074] In an optional embodiment, in order to facilitate the protease cleavage operation, a protease recognition sequence, such as TVMV enzyme and / or TEV protease, can be operably connected to the N-terminus or C-terminus of the spy catcher.

[0075] In a preferred embodiment of the present invention, the ligand is selected from the ligand targeting cardiac Gpc6 protein.

[0076] In a preferred embodiment of the present invention, the ligand is any one of F(ab')2, Fab', Fab, Fv, Fab'-SH and scFv of an antibody against cardiac Gpc6, especially the ligand is scFv of an antibody against cardiac Gpc6.

[0077] In a second aspect, the present invention also provides a method for preparing a drug delivery system, which comprises the following steps: first preparing a membrane layer of a liposome, and then mixing the membrane layer of the liposome with a transmembrane domain connected to a spy catcher to obtain a liposome modified with a transmembrane domain-spy catcher; mixing the liposome modified with a transmembrane domain-spy catcher with a small molecule inhibitor of Cpeb3 or a copper death inhibitor; obtaining a liposome encapsulating a small molecule inhibitor of Cpeb3 or a copper death inhibitor;

[0078] In a preferred embodiment of the present invention, liposomes encapsulating small molecule inhibitors of Cpeb3 or copper death inhibitors are mixed with SpyTag and ligands;

[0079] In a preferred embodiment of the present invention, in the drug delivery system, the molar ratio of the transmembrane domain connected with the spy catcher to the conjugate of the spy tag and the ligand is 1:2, 1:1, 2:1;

[0080] In a preferred embodiment of the present invention, in the drug delivery system, the mass percentage of the small molecule inhibitor of Cpeb3 or the copper death inhibitor in the drug delivery system is (17.11-26.48) wt%.

[0081] In a third aspect, the present invention also provides an application of a drug delivery system in the preparation of a drug for preventing and / or treating myocardial ischemia-reperfusion injury.

[0082] In a preferred embodiment of the present invention, the drug is administered by intravenous injection or oral administration.

[0083] In a preferred embodiment of the present invention, the drug has at least one of the following uses:

[0084] (1) Inhibit Cu-induced copper death and mitochondrial ROS production;

[0085] (2) reversing Cu-induced changes in aerobic respiration and / or glycolysis;

[0086] (3) Reversing the changes in glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle metabolites induced by Cu stimulation;

[0087] (4) Inhibit the binding of Cpeb3 to HK2 and inhibit the formation of LLPS between Cpeb3 and HK2;

[0088] (5) increase the level of m6A modification on HK2 mRNA;

[0089] (6) Promote the recognition of m6A by the reader protein YTHDC1, inhibit the degradation of HK2 mRNA, increase the expression of HK2, and restore mitochondrial function;

[0090] (7) suppressing ST segment elevation in the subject's electrocardiogram;

[0091] (8) Alleviate IRI-induced myocardial damage;

[0092] (9) improving the overall or local diastolic and systolic function of the subject's heart;

[0093] (10) Enhance the deformability of longitudinal myocardial fibers;

[0094] (11) Reduced myocardial fibrosis;

[0095] (12) Reduced scar formation;

[0096] (13) Inhibit the activation of action potential and calcium transients in cardiomyocytes, and inhibit the prolongation of cardiomyocyte repolarization;

[0097] (14) Reduce the susceptibility of subjects to ventricular arrhythmias (VA);

[0098] (15) Increase the threshold for sudden stimulation of ventricular fibrillation (VF) in subjects;

[0099] (16) Reduce the mortality caused by ventricular arrhythmias;

[0100] In a preferred embodiment of the present invention, use (1) refers to: inhibiting Cu-induced copper death of Gpc6-positive cardiomyocytes.

[0101] In a preferred embodiment of the present invention, the drug further comprises a pharmaceutically acceptable carrier, including but not limited to a filler, a lubricant, a disintegrant, a binder, a glidant, and the like.

[0102] In the preferred technical scheme of the present invention, the pharmaceutically acceptable carrier includes but is not limited to polyvinyl pyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methyl cellulose and its derivatives, ethyl cellulose and its derivatives, hydroxypropyl cellulose and its derivatives, starch and its derivatives, polyethylene glycol and its derivatives, lactose, sucrose, mannitol, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, calcium hydrogen phosphate, calcium stearate, sodium stearyl fumarate, silicon dioxide, titanium dioxide, talc, indigo or a combination thereof.

[0103] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0104] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0105] Experimental Materials and Methods

[0106] (A) Patient information and blood samples.

[0107] This study enrolled patients with acute ST-segment elevation myocardial infarction (STEMI) who underwent percutaneous coronary intervention (PCI) in Sichuan Provincial People's Hospital from January 2022 to December 2023 (n = 40). All patients underwent corresponding laboratory tests. Inclusion criteria: (1) clinical diagnosis of acute STEMI; (2) PCI within 12 hours of symptom onset; (3) patients and their families signed the relevant consent forms for inclusion. Exclusion criteria: (1) patients with advanced malignant tumors; (2) patients with multiple organ dysfunction; (3) patients with severe coagulation dysfunction; (4) patients with poor treatment compliance. Peripheral blood was collected 24 hours after PCI to detect copper ion content. Healthy subjects who underwent physical examination in Sichuan Provincial People's Hospital during the same period were selected as the control group (n = 20), and peripheral blood was collected for copper ion detection. All enrolled subjects signed informed consent forms, and this study was approved by the Ethics Committee of Sichuan Provincial People's Hospital.

[0108] (ii) Plasma and tissue copper detection.

[0109] Plasma samples were collected with heparin as an anticoagulant, centrifuged at 1000g for 5 minutes at 4°C, and the supernatant was placed on ice for measurement. The copper ion level in peripheral blood was detected using a serum copper content assay kit (#BC5645, Solarbio, Beijing, China) according to the manufacturer's instructions. About 0.1g of heart tissue was measured and 1mL of distilled water was added. The mixture was then homogenized on ice. The homogenate was then centrifuged at 10000g for 10 minutes at 4°C, and the resulting supernatant was collected. The measurement of copper content was carried out according to the instructions of the tissue copper content assay kit (#BC5565, Solarbio, Beijing, China).

[0110] (3) MIRI model.

[0111] The MIRI model was established according to a previous report. Spf mice (8 weeks), SD rats (180–230 g), and Bama miniature pigs (15–20 kg) were obtained from Chengdu Dashuo Experimental Animal Co., Ltd. (Chengdu, China), and all animal procedures were approved by the Animal Care and Use Committee of Sichuan Provincial People’s Hospital. SD rats or mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg), intubated, and connected to a small animal ventilator to ensure breathing. The degree of myocardial ischemia was evaluated by electrocardiographic monitoring. The heart was exposed by thoracotomy at the fourth intercostal space on the left side, and the left anterior descending coronary artery (LCA) was occluded with 8-0 (mice) or 6-0 (rat) silk sutures, and a slipknot was tied 2–3 mm distal to the origin of the LCA. T wave reversal and ST-segment elevation were observed on the electrocardiogram, indicating ischemia, and the slipknot was released 45 minutes later to allow reperfusion of the affected area. For Bama miniature pigs, fasting for 12 h before surgery was performed to minimize the risk of aspiration. Anesthesia was initiated with isoflurane and maintained with 1-2% isoflurane via endotracheal intubation. Vital signs, including heart rate, respiratory rate, and oxygen saturation, were continuously monitored using an electrocardiogram. The left chest was shaved and disinfected with povidone-iodine solution. A midline thoracotomy was performed to expose the heart, and the LCA coronary artery (second diagonal branch) was preoccluded for 45 minutes before ligation. The chest cavity was closed in layers using absorbable sutures. Antibiotics (cefazolin, 25 mg / kg) and analgesics (lidocaine, 2 mg / kg) were given postoperatively and closely monitored during recovery. Myocardial infarction was confirmed by electrocardiogram immediately after surgery. At the conclusion of the study, the animals were humanely euthanized with an overdose of pentobarbital, and tissues were removed from major organs.

[0112] (IV) Bioinformatics analysis.

[0113] All bioinformatics analyses were performed using the CeleLens Cloud platform provided by Singleron (https: / / www.celelenscloud.cn / # / my_analyzes). Sc / snRNA-seq data of mouse heart after myocardial infarction were obtained from the Gene Expression Omnibus (GSE214611), and Sc / snRNA-seq data of human heart were obtained from the Human Cell Atlas Data Coordination Platform (ERP123138), the European Genome-phenome Archive (EGAS00001006374), and cellxgene (https: / / cellxgene.cziscience.com / collections / 8191c283-0816-424b-9b61-c3e1d6258a77). The Seurat package was used to classify and annotate the data, identify different cell types, and extract marker genes for each subpopulation. Subsequently, DESeq2 was used to perform differential expression analysis between the two groups of cells, and a volcano plot was generated to visualize significantly differentially expressed genes. For the expression levels of CPEB3 and HK2, KEGG analysis was performed to identify their related biological pathways. In addition, GSEA was used to analyze the copper death pathway and glycolysis pathway to evaluate the enrichment of related gene sets. All analyses were performed in the R environment to ensure the reliability of data processing and the reproducibility of results, thereby revealing the potential functions and mechanisms of different cell subpopulations in copper death and metabolic regulation.

[0114] (V) Cell activity and death.

[0115] To simulate the damage of MIRI to cardiomyocytes in vitro, Gpc6+CMs / Gpc6-CMs were isolated by flow cytometry using adult rat primary cardiomyocytes (ARPCs), and all cells were ischemic (hypoxia and starvation for 1 h) to simulate MIRI and then treated with different drugs. CCK8 detection was performed according to the protocol provided by cell Counting Kit-8 (#C0037, Beyotime, Beijing, China) to evaluate cell death. In addition, propidium iodide (#C1062S, Beyotime, Beijing, China) staining was used to identify cells with impaired membrane integrity and analyzed using flow cytometry.

[0116] (vi) Flow cytometry.

[0117] Single cell suspension was obtained from myocardial tissue by enzyme digestion (#abs9482, Absin, Shanghai, China) and washed with PBS buffer to remove residual enzymes. After the cell suspension was adjusted to the appropriate concentration, anti-cTnI antibody (rat: #MG270057, Abmart, Shanghai, China; pig: #ab231064, Abcam, Boston, MA, USA) and Gpc6 (mouse: #PC22025, Abmart, Shanghai, China; pig: Abconal custom antibody) were added and mixed gently. The mixture was incubated at 4°C for 1 hour to ensure that the antibody was fully bound to the target cells. The cells were then washed three times with PBS to remove unbound primary antibody. Fluorescently labeled secondary antibody (FITC: #F-2761, Invit rogen, USA; PE: #12-4739-81, Invitrogen, USA) was added and incubated at 4°C for 30 minutes to ensure that the secondary antibody specifically bound to the primary antibody. The cells were washed three times with PBS to remove the unbound secondary antibody. Subsequently, the cells were analyzed by flow cytometry. In the flow cytometer, the fluorescent dye was excited with a laser, and the forward scatter and side scatter data were collected to determine the size and complexity of the cells. The expression of cTnI and Gpc6 was detected using specific fluorescence channels. Positive cells and negative cells were distinguished according to the fluorescence intensity, and the number of gpc6-positive cardiomyocytes and the proportion of the total number of cells were finally recorded.

[0118] (VII) Seahorse analysis.

[0119] Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of isolated cardiomyocytes were assessed by Seahorse XF analyzer (Agilent, Santa Clara, CA) according to the manufacturer's instructions. Cell-Tak (22 μg / ml, pH 6.5-8, #354241, Corning) was applied to the wells of Seahorse XFe96 spheroid microplates (#102959-100, Agilent) and incubated at 37°C for 20 minutes. Afterwards, the wells were neutralized with deionized water, rinsed twice, dried and stored at 4°C. The isolated cardiomyocytes were then transferred to the wells, placed in culture medium, and incubated overnight at 37°C to allow attachment. The next day, the buffer was changed to low-phosphate DMEM / F12 buffer pre-warmed to 37°C in a CO2-free incubator. The sensor chip was calibrated with the provided Seahorse buffer one day before the test.

[0120] For OCR evaluation, the combination of oligomycin (4 μmol / L), FCCP (4 μmol / L), and rotundone (2 μmol / L) and antimycin A (2 μmol / L) was measured using Seahorse XF Mitochondrial Stress Test Kit (#103015-100, Agilent, Santa Clara, CA). ECAR was measured by Seahorse XF Glycolysis Stress Test Kit (#103020-100, Agilent, Santa Clara, CA) under baseline conditions and after the addition of glucose (20 mmol / L), oligomycin (4 μmol / L), and 2-deoxyglucose (2-DG, 100 mmol / L) in sequence. Each injection port of the sensor plate was filled with 25 μl of test compounds or solvents diluted with DMEM / F12 buffer. After calibration, the cell plate was analyzed in an XF96 instrument, and the measurement protocol was set to 3 minutes of mixing followed by 3 minutes of measurement. Results read on the Seahorse XF96 Analyzer were normalized by the total protein level in each well.

[0121] (VIII) Western blot and co-immunoprecipitation (Co-IP).

[0122] Tissues or cells were lysed with RIPA lysis buffer (#R0010, Solarbio, Beijing, China), and protein concentration was determined by BCA protein quantification kit (#PC0020, Solarbio, Beijing, China). Proteins were separated by SDS-PAGE and transferred to PVDF membrane. The membrane was incubated with primary antibody at 4°C overnight and then incubated with secondary antibody at 37°C for 1 hour. Amersham ImageQuant TM Visualization of the bands was performed using the Cytiva 800 system (Cytiva, Sweden).

[0123] In Co-IP experiments, Pierce TM Classic Magnetic IP / Co-IP Kit (Thermo Scientific TM, USA), after cell lysis, the target protein was captured using specific antibodies, allowing other interacting proteins to co-precipitate. Subsequently, the antibody-protein complex was immobilized using Protein A / G agarose beads and the unbound components were removed. Finally, the bound proteins were eluted and analyzed by Western blot to confirm the interaction between the target protein and other proteins. The antibodies used in the experiment included: Cpeb3 (#PC11340, Abmart, Shanghai, China), HK2 (#TD6176, Abmart, Shanghai, China), ALKBH5 (#M008844, Abmart, Shanghai, China), YTHDC1 (#14392-1-AP, Proteintech, Wuhan, China), LaminB1 (#P60054, Abmart, Shanghai, China), GAPDH (#TA7021, Abmart, Shanghai, China), His-Tag (#M30111, Abmart, Shanghai, China), Flag-Tag (#TT0053, Abmart, Shanghai, China).

[0124] (IX) Immunofluorescence.

[0125] The cells were fixed with 4% paraformaldehyde for 10 to 20 minutes and washed three times with PBS. Subsequently, 0.2%-0.5% Triton X-100 (prepared in PBS) was added, incubated at room temperature for 5 to 10 minutes, and then washed three times with PBS. Nonspecific binding was blocked with 3% BSA for 30 to 60 minutes. A 1:500 dilution of primary antibody was added and incubated overnight at 4°C. Then, the cells were washed three times with PBS, and a 1:1000 dilution of fluorescently labeled secondary antibody was added, incubated for 60 minutes in the dark, and washed again with PBS three times. Finally, the nucleus was stained with DAPI for 5 minutes, and after washing, the slides were sealed with anti-fluorescence attenuation sealing agent for microscopic observation.

[0126] For tissue samples, fresh tissues were first fixed with 4% paraformaldehyde for 24 hours, dehydrated with gradient alcohol, and paraffin-embedded after being transparentized with xylene. The slices were 5 μm thick and mounted on glass slides. After baking at 60°C for 30 minutes, the slides were dewaxed with xylene and alcohol, and then washed with distilled water. The slices were placed in citric acid buffer, incubated at 95°C for 20 minutes, and washed three times with PBS after cooling. Nonspecific binding was blocked with 3% BSA for 30 minutes. Primary antibody was added and incubated overnight at 4°C. Subsequently, the slides were washed three times with PBS, fluorescently labeled secondary antibodies were added, incubated for 1 hour under light-proof conditions, and washed again three times. The nuclei were stained with DAPI for 5 minutes, and after washing, the slides were sealed with sealing agents for microscopic observation.

[0127] The antibodies used in the experiments included: Cpeb3 (#PC11340, Abmart, Shanghai, China), HK2 (#TD6176, #MG680856, Abmart, Shanghai, China), Gpc6, YTHDC1 and ALKBH5 (customized by Abconal), His-Tag (#M30111, Abmart, Shanghai, China) and Flag-Tag (#TT0053, Abmart, Shanghai, China).

[0128] (10) Metabolomics.

[0129] Metabolite detection was analyzed based on methods from previous studies. Two complementary experiments were performed to quantitatively detect metabolites in glycolysis, the pentose phosphate pathway (PPP), and the tricarboxylic acid cycle (TCA). The first experiment used ion chromatography-mass spectrometry (IC-MS) to analyze pyruvate and glucose-6P levels in the culture medium; the second experiment used ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) to separate hexose sugars, including glucose.

[0130] Isolated cardiomyocytes were incubated with 10 mM [U-13C] labeled glucose for 6 h. Metabolites were extracted using 80% methanol and concentrated by Labconco CentriVap. The dried metabolites were resuspended in 50% acetonitrile-water solution and loaded onto a Luna 3 μm NH2 100A column (150 × 2.0 mm, Phenomenex). Chromatographic separation was performed on a VanquishFlex system (Thermo Scientific) at a flow rate of 200 μL / min, using a linear gradient from 15% to 95% mobile phase A (5 mM NH4AcO, pH 9.9) for 18 min, followed by isocratic elution at 95% A for 9 min.

[0131] Metabolites were detected by Q Exactive mass spectrometer (Thermo Scientific) in full scan mode, switching polarity (+3.5kV / -3.5kV), and the mass-to-charge ratio range of 65-975m / z was detected. Quantitative analysis was performed using TraceFinder 4.1 software based on peak area, expected retention time and accurate mass (error <5ppm). Detection values ​​were standardized to cell counts, and the relative amount of metabolites was calculated by summing all isotopologues of each metabolite and correcting for natural C13 abundance.

[0132] (XI) Mitochondrial reactive oxygen species (ROS) detection.

[0133] Use of MitoSOX TMMitochondrial superoxide indicator (#M36005, Invitrogen TM , USA) to measure mitochondrial reactive oxygen species. First, the cell samples were cultured under appropriate conditions, and then the cells were collected. The cells were washed once with PBS to remove the culture medium, and then 1μl of 5mM MitoSOX Green was diluted in 0.5ml PBS to prepare a working solution. 1ml of the working solution was added to the cells and incubated at 37°C for 30 minutes. After incubation, the supernatant was discarded and the cells were washed twice with PBS. The cells were then analyzed using a flow cytometer, and appropriate parameters were set to detect the fluorescence signal of MitoSOX. Through this analysis, the level of mitochondrial ROS in each cell can be quantitatively determined, and the differences between different treatment groups can be compared. The data obtained can be used to evaluate mitochondrial function and oxidative stress status in cells.

[0134] (XII) Detection of cTnI and CK-MB.

[0135] Cardiac troponin I (cTnI) levels were determined using specific ELISA kits for different species: porcine TNNI3 ELISA kit (#ELK5656, ELK Biotech, Wuhan, China), rat cTnI ELISA kit (#D731143, Sanger Biotech, Shanghai, China), and mouse cTnI ELISA kit (#D721149, Sanger Biotech, Shanghai, China) according to the manufacturer's instructions. Similarly, creatine kinase-MB (CK-MB) levels were determined using the relevant kits for each species: porcine kit (#CB10105-Pg, Coibo, Shanghai, China), rat kit (#SEKR-0059, Solarbio, Beijing, China), and mouse kit (#SEKM-0152, Solarbio, Beijing, China). After the model was successfully established for each group, 0.2 mL of venous blood was collected to determine the cTnI concentration.

[0136] (XIII) Evans Blue & TTC staining and Masson staining

[0137] 24 hours after myocardial ischemia-reperfusion injury, the degree of myocardial injury was assessed by double staining technique of Evans Blue (#E8010, Solarbio, Beijing, China) and TTC (diphenyltetrazolium chloride, #T8170, Solarbio, Beijing, China). First, 0.5% Evans Blue solution was perfused for 10 minutes to mark the necrotic myocardium. The heart was then removed, cut into slices of approximately 1 cm thick, and stained with 1% TTC solution in a 37°C water bath for 30 minutes. Healthy myocardial tissue appears red after TTC staining, while the ischemic injury area remains pale. After the experiment, the staining results were quantitatively evaluated using image analysis software, and the proportion of infarct area to total myocardial area was calculated to objectively assess the degree of ischemia-reperfusion injury.

[0138] Four weeks after myocardial ischemia-reperfusion injury, the degree of myocardial fibrosis was assessed using Masson staining (#G1346, Solarbio, Beijing, China). The rat hearts were quickly rinsed with saline and fixed in 10% neutral buffered formaldehyde for 24 hours. After fixation, the hearts were cut into 5 μm thick slices, dehydrated and transparentized. Masson staining solution was then used for staining. The specific steps include: staining with Weigert iron solution (containing 1% potassium ferrocyanide and 1% ferric chloride) at room temperature for 10 minutes, followed by rinsing with distilled water to dye the fiber background blue. Next, acid fuchsin solution was added for staining for 2 minutes, and excess dye was washed off. Finally, the slices were rinsed with 1% acetic acid and sealed with resin. After staining, the degree of myocardial fibrosis was observed under a microscope, and the distribution and proportion of blue collagen fibers in myocardial tissue were analyzed.

[0139] 14. Echocardiography

[0140] Echocardiography of the hearts of rats, mice, and pigs was performed using FUJIFILM Vevo 3100LT and Finno Ultrasound VINNO ULTIMUS 9E. All animals were properly anesthetized and immobilized before the experiment. For rats and mice, isoflurane was used for anesthesia, and the anesthetic concentration was maintained at 2-3% to ensure the depth and stability of anesthesia. After anesthesia, the animals were placed in a warm environment to prevent hypothermia, and vital signs were monitored using a small anesthesia machine. For pigs, Tiletamine-Zolazepam was first used at a dose of 10 mg / kg body weight for induction. After anesthesia induction, pigs were maintained in an appropriate state of anesthesia with 2-3% isoflurane. During anesthesia, the heart rate, respiratory rate, and body temperature of the pigs were continuously monitored to ensure safety and anesthetic efficacy.

[0141] Using Vevo 3100LT, operators acquired cardiac images of rats and mice through high-frequency probes and calculated important cardiac function parameters. The E / A ratio was obtained through the mitral Doppler flow velocity spectrum, and the left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVFS) were calculated by measuring the dynamic changes in the cardiac chamber contour through the left ventricular short axis M-mode. Subsequently, VINNO ULTIMUS9E was used to perform ultrasound examinations on rats, mice, and pigs. The device focuses on obtaining strain data of the left ventricular long axis section (PLAXL) and calculates myocardial strain by dynamically tracking the long axis view. In addition, for pigs, the E / A ratio, LVEF, LVFS, and heart rate (HR) were also recorded to comprehensively evaluate cardiac function. All ultrasound images and parameters were carefully analyzed to ensure the accuracy of the data, providing a reliable basis for cardiac function evaluation for subsequent studies.

[0142] (XV) Electrophysiological integration testing

[0143] Electrophysiological integration indices were detected by an optical mapping system. First, heparin sodium injection (3000U / kg) was intraperitoneally injected, and isoflurane anesthesia was induced after waiting for 15 minutes. The skin was disinfected with alcohol, and the xiphoid process was exposed by incision on the chest skin. The xiphoid process was lifted with forceps, and the ribs were cut along the left and right midline to open the chest cavity and expose the heart. The lung tissue was clamped with forceps, and the heart was quickly removed and placed in a 4°C calcium-free pre-filled oxygenated (95% O2, 5% CO2) modified Krebs buffer (118mmol / L NaCl, 4.2mmol / L KCl, 1.2mmol / L KH2PO4, 1.2mmol / L MgSO4, 23mmol / L NaHCO3, 20mmol / L D-glucose, 2mmol / L sodium pyruvate, 1.8mmol / LCaCl2, pH 7.35-7.45) for flushing. The aorta was quickly inserted into the perfusion needle using a horizontal Langendorff perfusion apparatus and fixed with surgical sutures to observe whether there were any bubbles entering the heart. The heart was fixed in a silicone rubber chamber for retrograde perfusion and ventilation with carbogen gas. After the Langendorff-isolated heart was stabilized for 10 minutes, the heart was allowed to resume normal rhythm and loaded with fluorescent dyes in sequence.

[0144] After 10 minutes of stabilization, all room lights were turned off and continuous perfusion with Krebs buffer was performed. The cardioplegic agent Blebbistatin was injected from the Y-shaped inlet to eliminate the mechanical contraction of the heart, and it stopped after about 1-2 minutes. Part of the perfusate was removed, the oxygen supply was reduced from the circulating reservoir, and Pluronic F127 was added to the remaining perfusate. After 10 minutes of continuous perfusion, the calcium indicator Rhod-2AM was slowly injected from the Y-shaped inlet, and the circulation was continued for 15 minutes. Then, the voltage-sensitive dye RH237 was slowly injected, and the previously removed Krebs buffer was returned to the perfusion reservoir and the circulation continued.

[0145] Transfer the dye-loaded heart to the bath, place ECG electrodes on both sides of the heart, attach the red electrode to the tip of the left ventricle, the blue electrode to the right atrium, and the black electrode to the ground in the bath. Connect to the biosignal acquisition system to record the electrocardiogram (ECG), and adjust the sampling rate to automatically monitor continuously. Using the optical imaging system microscope, focus the excitation light on the heart, adjust the distance between the lens and the heart, select the maximum resolution and frame the appropriate imaging area. Record three action potentials and calcium ion signals in sinus rhythm.

[0146] Insert the pacing electrode approximately 1 mm into the right ventricle and deliver the pacing stimulus. The initial current intensity is 0 mA, the frequency is 6 Hz, the pulse width is 2 ms, and the ECG is closely observed. When the ECG shows a clear 1:1 pacing corresponding to the stimulus, the current intensity is the diastolic threshold current. Set the pacing current to twice the diastolic threshold current, and record the action potential and calcium ion signal at this 6 Hz pacing rate. After eight consecutive S1 stimuli, apply an early additional stimulus S2, the S1S1 interval is reduced by 20 ms compared to the inherent RR interval, and the S1S2 interval is gradually reduced by 5 ms until S2 cannot induce a QRS waveform. At this time, the S1S2 interval is recorded as the effective ventricular reentry period.

[0147] Ventricular arrhythmias were induced using 50 Hz high-frequency pulses, and the intensity of the stimulation current was gradually increased from 5 mA to 10 mA, then to 15 mA, and finally to 20 mA. The interval between each stimulation was 3 minutes, and the occurrence, duration, and fibrillation threshold of ventricular arrhythmias were recorded after each stimulation. ECGs of all hearts were recorded under sinus rhythm, normal pacing, and induced ventricular arrhythmias, and optical measurements of membrane potential and calcium ion signals of dye-loaded rat hearts were observed before and after ischemia-reperfusion, and action potential depolarization, repolarization time, action potential duration, and calcium transients were analyzed.

[0148] (XVI) Protein purification

[0149] Plasmids containing Cpeb3 from different species and its specific amino acid mutants, Cpeb3-GFP and HK2-mcherry were synthesized by Qingke Biotechnology (Beijing, China) using the pGEX-4T-1 vector and subsequently transformed into competent Escherichia coli cells. The transformed bacteria were inoculated on LB agar plates supplemented with antibiotics and cultured overnight to select plasmids containing the ampicillin resistance gene. One positive clone was inoculated into LB liquid medium containing antibiotics and cultured overnight at 37°C with shaking.

[0150] Subsequently, a small amount of overnight culture was transferred to fresh LB medium containing antibiotics and cultured at 37°C with shaking until the optical density at 600nm (OD600) reached 0.6-0.8. At this stage, isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to induce protein expression, and the culture medium was continued to be cultured at 37°C with shaking for 6 hours. After induction, bacterial cells were collected by centrifugation at 6000rpm for 10 minutes. The obtained cell pellet was resuspended in a lysis buffer containing protease inhibitors to prevent protein degradation. The cells were broken by ultrasound to effectively release the target protein into the solution.

[0151] After cell lysis, centrifugation is performed to remove cell debris, and the supernatant containing dissolved proteins is collected for purification. The target protein with a His tag is separated using a Ni-NTA affinity column and eluted with an elution buffer containing different concentrations of imidazole. The individual fractions collected during the elution process are analyzed for protein purity by SDS-PAGE. If the purity is insufficient, the protein is further concentrated by dialysis or ultrafiltration. Finally, the purified protein is stored in a suitable buffer for subsequent experiments.

[0152] (XVII) Virtual Docking

[0153] Virtual docking was completed by AutoDock software. This study aims to explore the binding interaction between rat Cpeb3 and small molecule compounds using molecular docking technology. First, the three-dimensional structure of Cpeb3 was obtained, and the ligand structure of the small molecule compound was prepared. Then the docking grid frame was defined, and the docking simulation was performed. By analyzing the binding affinity and visualizing the interaction, the key hydrogen bonds and hydrophobic interactions were identified, and the distances between the interacting groups were measured. Finally, the binding mechanism of Cpeb3 with small molecule compounds was revealed, providing a theoretical basis for subsequent drug design.

[0154] 18. High-throughput drug screening

[0155] Using the molecular modeling software AutoDock, the three-dimensional structure of Cpeb3 (UniProt number: D4AD99) was constructed and its energy was minimized to ensure the accuracy of the model. Subsequently, compounds with specific chemical properties and biological activity information were screened from the 19 million compounds provided by Topscience to establish a compound database. Using molecular docking technology, each compound was docked with the binding site of Cpeb3 to evaluate its binding affinity. Potential high-affinity compounds were screened by calculating key parameters such as binding energy, number of hydrogen bonds, and hydrophobic interactions. During the virtual screening process, the PLANET model based on image neural network was used to score the compounds to capture the complex relationship between molecular structure and biological activity. Combined with the results of molecular docking, 300 candidate compounds were finally selected.

[0156] Next, primary Gpc6-positive cardiomyocytes (Gpc6 + CMs) and cardiac fibroblasts (CMFs). First, the cardiac tissue was digested with collagenase to extract cells, and then the expression of Gpc6 was detected by flow cytometry. During cell culture, cardiomyocytes used DMEM / F12 medium, and cardiac fibroblasts used DMEM medium. The medium was supplemented with 10% fetal bovine serum and antibiotics, and the cells were cultured at 37°C and 5% CO. In order to simulate the myocardial infarction environment, the cells were placed under hypoxia (1% O) and starvation (serum-free medium) conditions for 1 hour to induce stress response. Subsequently, 50nM ES_Cu and 5μM of different candidate compounds were added to the culture medium and incubated for 24 hours.

[0157] Cell viability was assessed using the CCK8 kit according to the manufacturer's instructions, and appropriate control groups (such as untreated group and ES_Cu-only treated group) were set up to ensure the reliability of the experiment. + In the CMs group, cell viability increased significantly by 1.5 times, indicating that these compounds have good activity in promoting cardiomyocyte survival and functional recovery, which may be related to their regulation of cell metabolism, antioxidant pathways or inhibition of apoptotic signals. In the CMFs group, the increase in cell viability did not exceed 1.1 times, indicating that these compounds had no significant promoting effect on cardiac fibroblasts, highlighting their specificity and selectivity. Based on this result, the Gpc6 + Compounds with cell viability increased by more than 1.5 times in the CMs group and no significant viability increase in the CMFs group were defined as “hits”, demonstrating their superiority in targeting cardiomyocytes.

[0158] For the hit compounds screened, a surface plasmon resonance (SPR) experiment was performed. In the SPR experiment, Cpeb3 protein was prepared by a recombinant expression system and immobilized on a sensor chip. Different concentrations of the screening compounds were injected, and their binding and dissociation processes were monitored in real time. The affinity constant (KD) was calculated by data analysis, and compounds with KD ≤ 50 μM were defined as effective hit compounds.

[0159] (19) Surface Plasmon Resonance (SPR) Experiment

[0160] use SPR experiments were performed using R4 (Sartorius, Germany). The target protein was produced by a recombinant expression system, carrying a histidine tag, and then purified. The protein was immobilized on a pre-treated Ni-NTA sensor chip to ensure uniform binding of the protein, thereby improving the repeatability and reliability of the experiment.

[0161] During the experiment, different concentrations of candidate compounds or proteins are injected into the sensor surface in sequence by flow injection. The instrument continuously monitors the changes in reflected light intensity during the binding process and generates sensor response curves (senorgrams). These response curves show the rate of the binding reaction and the changes during the dissociation process, clearly reflecting the interaction between the mobile phase and the fixed protein.

[0162] In the data analysis stage, dedicated software is used for baseline correction to eliminate background noise and extract the real signal. The equilibrium dissociation constant (KD) is calculated by fitting the binding and dissociation curves at different concentrations.

[0163] (XX) RNA immunoprecipitation (RIP) and methylated RNA immunoprecipitation (MeRIP)

[0164] RIP and MeRIP experiments were performed according to previous literature methods. RIP experiments were performed using EZ-Magna RIP TMRNA Binding Protein Immunoprecipitation Kit (#17-701, Sigma-Aldrich, Billerica, MA, USA) was used according to the manufacturer's instructions. Briefly, RNA binding proteins were extracted by cell lysis using the provided RIP lysis buffer. Target RNA binding proteins were captured by immunoprecipitation using magnetic beads conjugated to anti-Cpeb3 antibody or control IgG. To detect the enrichment of HK2 mRNA in the immunoprecipitated RNA samples, qRT-PCR analysis was performed. In addition, to analyze the m6A modification level in HK2 mRNA, MeRIP experiments were performed based on established methods. In this step, 5 μg of anti-m6A antibody (#A19841, ABclonal, Wuhan, China) or control IgG was conjugated to magnetic beads in the Magna RIP kit (#MAGNARIP01, Merck, Germany). Then, 50 μg of total RNA (extracted from cells by standard RNA isolation techniques) was incubated with anti-m6A antibodies in RIP immunoprecipitation buffer (provided by Magna RIP kit) at 4 ° C overnight to specifically bind to m6A-modified RNA. After incubation, the samples were treated with proteinase K to remove residual proteins to ensure purified RNA samples. Subsequently, RNA was purified using RNeasy Mini Kit (#74104, Qiagen, Germany). The enrichment of HK2 mRNA in the m6A immunoprecipitated RNA fraction was quantitatively detected by qRT-PCR analysis.

[0165] (XXI) RNA Pull-Down Experiment

[0166] Biotin-labeled HK2 mRNA was synthesized by in vitro transcription, using the pcDNA3.1 vector as a template. Biotin RNA Labeling Mix (#11685597910, Merck, Germany) and T7 RNA polymerase (#RPOLT7-RO, Merck, Germany) containing biotin-UTP were added to drive the reaction during transcription. To remove residual DNA that may contaminate RNA, the synthesized product was treated with RNase-free DNase I (#69182, Merck, Germany) at a concentration of 1U / μg RNA at 37°C for 30 minutes and then heated at 75°C for 10 minutes to inactivate. Biotin-labeled RNA was purified by RNeasy Mini Kit (#74104, Qiagen, Germany) and eluted with 30μL of RNase-free water to obtain a pure and concentrated RNA product. Subsequently, the purified biotin-labeled RNA was added to the pre-prepared cell lysate (prepared from cultured cells by RIPA buffer containing protease inhibitors) and incubated at 4°C for 2 hours. This incubation step promotes the interaction between RNA and protein to form RNA-protein complexes. TM M-270 Streptavidin (#65305, Invitrogen, USA) captures biotin-labeled RNA-protein complexes. The magnetic beads were pre-washed with PBS before adding to the mixture and gently rotated at 4°C for 1 hour to achieve binding. After capture, the magnetic beads were washed three times with PBS containing 0.1% Tween-20 to remove unbound proteins. Subsequently, the bound proteins were eluted from the magnetic beads using 2× Laemmli sample buffer and denatured at 95°C for 5 minutes. The eluted samples were analyzed by Western blot to identify proteins that interact with biotin-labeled HK2 mRNA, thereby revealing the functional dynamics of HK2 in the cellular environment.

[0167] (XXII) qRT-PCR experiment

[0168] Total RNA was extracted using RNAsimple Total RNA Kit (#DP419, Tiangen, Beijing, China) and the manufacturer's instructions were strictly followed to ensure high-quality RNA to meet the requirements of subsequent experiments. For cDNA synthesis, 1,000 ng of extracted total RNA was used as a template and PrimeScript TMRT Reagent Kit with gDNA Eraser (#RR047, TaKaRa, Dalian, China) was used to reverse transcribe and synthesize cDNA. The kit contains a gDNA removal step to effectively remove genomic DNA contamination, thereby improving the accuracy and reliability of downstream experimental results. After cDNA synthesis, qPCR amplification was performed using a one-step fluorescent quantitative PCR kit (One-step Reverse Transcription PCR Kit, #FP313-01, Tiangen, Beijing, China) to quantitatively evaluate the expression level of the target gene. GAPDH was selected as an internal reference gene to normalize the expression data to compensate for changes in cDNA input and overall PCR efficiency. Relative gene expression levels were calculated by 2 -(ΔΔCt) Methods were calculated to enable robust comparison of gene expression between samples.

[0169] To study RNA stability, cells were treated with 5 μg / mL of Actinomycin D, a drug that inhibits transcription. Cells were then collected at different time points and RNA was extracted to determine the degradation rate of mRNA. RNA extraction was still performed using the RNAsimpleTotal RNA Kit, and the quality and concentration of the resulting RNA samples were assessed using a Nanodrop spectrophotometer. By comparing expression levels at different time points, the degradation kinetics of the target mRNA under transcription inhibition conditions were analyzed. Primer sequences

[0170] HK2 Forward:5'-CTGCCCTTCTGCTTCTCTG-3';

[0171] HK2 Reverse:5'-GCTGGTGGTGAGGTGATG-3'.

[0172] (XXIII) Luciferase reporter gene experiment

[0173] To identify the binding site of Cpeb3 in the 3′-UTR region of HK2 mRNA, the wild-type and mutant sequences of HK2 3′-UTR were cloned into the pmirGLO-luciferase vector. + CMs cells were plated at 5×10 per well. 3Cells were seeded in 96-well plates at a density of 10 cells / mL, and each condition was performed in triplicate. Cells were co-transfected with pmirGLO-HK2 wild-type 3′-UTR or pmirGLO-HK2 mutant 3′-UTR vectors and shRNA against Cpeb3 to inhibit the expression of Cpeb3. Luciferase activity was measured 48 hours after transfection using a dual-luciferase assay system (#RG029, Beyotime, Beijing, China) according to the manufacturer's instructions. Firefly and Renilla luciferase activities were quantified, and the relative luciferase activity was determined by calculating the ratio of firefly luciferase to Renilla luciferase activity to normalize the transfection efficiency. This approach provides insight into the interaction between Cpeb3 and the 3′-UTR region of HK2 mRNA.

[0174] (XXIV) Fluorescence polarization method

[0175] In order to detect the binding of Cpeb3 protein to HK2 protein, fluorescence polarization technology was used, using SpectraMaxiD5 fluorescence polarization detector (Molecular Devices). First, recombinant HK2 protein and Cpeb3 protein (including wild type and mutant) labeled with green fluorescent protein (GFP) were prepared. In each experimental group, different concentrations of HK2 protein (including control group and HK2 overexpression group) were set. An equal amount of Cpeb3 protein was added to each reaction tube and gently mixed, followed by measurement using a fluorescence polarization detector. In the fluorescence polarization detection, the excitation wavelength was set to 488nm and the emission wavelength was set to 520nm to adapt to the fluorescence characteristics of GFP. The fluorescence polarization values ​​under different conditions were recorded, and the polarization changes of wild-type and mutant GFP-Cpeb3 proteins when binding to HK2 protein were compared to evaluate the binding affinity between the two.

[0176] (XXV) Fluorescence recovery after photobleaching (FRAP) experiment

[0177] Different treatment strategies were applied to cultured cells prior to imaging. Using a ZEISS LSM 980 confocal microscope, regions of interest (ROI) containing GFP or mCherry signals were completely photobleached and treated using a focused 488nm laser beam. After photobleaching, the recovery of fluorescence intensity within the ROI was monitored in real time, and data was captured at predetermined intervals to quantify the recovery kinetics. Fluorescence intensity was measured before bleaching and at the beginning of recovery, allowing a comprehensive analysis of the dynamics of the movement and redistribution of fluorescent proteins after treatment.

[0178] (XXVI) RNA Pulldown / Mass Spectrometry (MS)

[0179] In rat Gpc6 +Cpeb3 protein was knocked down or not in CMs. Nuclear proteins were extracted using a nuclear and cytoplasmic protein extraction kit (#P0027, Beyotime, Beijing, China), and RNA pull-down experiments of HK2 mRNA were performed. After overnight incubation at 4°C, Protein A / G magnetic beads were added to capture the antibody-antigen complex, and non-specifically bound proteins were removed by washing. Subsequently, the bound proteins were enzymatically hydrolyzed, and the resulting peptides were purified using a C18 solid phase extraction column and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). For mass spectrometry data analysis, the obtained mass spectrometry data were processed using MaxQuant software and compared with the protein database. Based on the mass spectra of the peptides, the m6A-modified proteins that interacted with Cpeb3 were identified, with special attention paid to the changes in relative abundance to distinguish the differences between the control group and the Cpeb3 knockdown group.

[0180] (XXVII) Synthesis of STMD-SpyCatcher and SpyTag-Ligand

[0181] The sequences of SpyCatcher and SpyTag were obtained according to previous studies. STMD-SpyCatcher consists of SpyCatcher, His tag, linker peptide and STMD, which were synthesized and purified in the E. coli expression system using the pGEX-4T-1 vector. The manufacturer is Qingke (Beijing, China). The amino acid sequences of STMD of different proteins are as follows:

[0182] Transmembrane receptor β2AR: GTVPSDNIDSQGRNCSTNDSLL

[0183] Ion channel SCN5A: VAISTMVQPIFSYFIMITI

[0184] Transporter CFTR: AAVKILVHSLFNMLIMCTILTNCV

[0185] Pump protein Na+ / K+ATPase: EWVKFCRQLFGGFSILLWIGA

[0186] Anchoring protein Integrin: IIPIVAGVVAGIVLIGLALLLIW

[0187] The amino acid sequence of SpyCatcher is as follows:

[0188] MIELELPAPNGSVVVLKEGTQFTVTVEDLGSKISPFGKIVEIFDIKGTKIFGDDGEEAEEFKHLMMEGVVHGVHEQNFEGVTYRVDNNRIL.

[0189] The amino acid sequence of SpyTag is as follows: AHIVMVDAYKPTK.

[0190] The selected linker peptide was a reported flexible linker sequence: GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS. The purified STMD-SpyCatcher was verified by Western blot experiment using anti-His antibody (#M30111, Abmart, Shanghai, China). SpyTag-Ligand consists of SpyTag, Flag tag and ligand, synthesized by Abconal, and different ligands were designed using scFv (single-chain variable fragment) to target different organs: heart (Gpc6: heavy chain variable region, QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVKQSHGKSLEWIGDINPNSGGTNYNQKFKGRVTMTTDTSTSTVYMELSSLRSEDTAVYYCAR; linker, GGGSGGGGSGGGGS; light chain variable region, DIQLTQSPSSLSASVGDRVTITCRASQGISDWLNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK), liver (HAS: heavy chain variable region, EVQLVESGGGLVQPGGSLR LSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARG; linker, GGGSGGGGSGGGGS; light chain variable region, QQVQLQESGPGLVKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGYIYYSGSTYYNPSLKSRLTISVDTSKNQFSLKLSSVTAADTAVYYCAR), spleen (CD3: heavy chain variable region, QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWIHWVKQRPGQGLEWIGYINPYYGSTYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYYCTRGGYFDYWGQGTLVTVSS; linker, GGGSGGGGSGGGGS;Light chain variable region, QQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWIGDIYPGDGDTKYNEKFKGRVTMTTDTSTSTAYMELSSLRSEDTAVYYCAR), lung (SFTPA1: heavy chain variable region, QVQLVQSGAEVKKPGESNKISYVYWYQKPKGELVSGSGVPSRFSGSGSGTQGTQGELARSYNQKFKGRGSGIGEGSGVPSRFSGSGSGTGELQGAGSQVGQESGQVTFTQKTFSFYKQ; linker, GGGSGGGGSGGGGS; light chain variable region, DIVLTQSPDSLAVVLAGLQKGGSKLTVPSPFNLELNLPFLQGSANVNLVLLLEPGITLAPQLVPK LGTPFLLFQYPYSKLSGLQYKQGGKKLEPLVYFHQPN) and kidney (Podxl: heavy chain variable region, QVQLVQSGAELVKKPGESNKISYVYYWYYTKFQQRGEVSKFNSRVDPNSFSGSGSGTQGTQGGGSLRLSCAQKLLGLLSYLREYNNGAQQHYHAGGLQFQSVEKNSKKYQTCSNNSRFSEMGVLRPLK; linker, GGGSGGGGSGGGGS; light chain variable region, DIVLTQSPDSLAVVLAGLQKGGSKLVPSPQNLELDQPKFLEGSNADYLSLQPNQYYLRGKPQWLPGWGTFGRVVHQNKLDSFNSNRYWNFVRKSNNYYNWFGRLKRWGRVRAKLLTLGAVT). The synthesis results were verified by Western blot experiments using anti-Flag antibody (#TT0053, Abmart, Shanghai, China).

[0191] (XXVIII) Preparation of liposomes (Lp), liposome delivery platforms (PLp) and functionalized liposome delivery platforms (FPLp)

[0192] Liposomes (Lp) were prepared as follows: dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylethanolamine (DOPE), and cholesterol were mixed in an organic solvent at a molar ratio of 700:200:100. After evaporation of the organic solvent using argon purge, the resulting lipid film was dried overnight under vacuum to ensure complete removal of the organic solvent. Subsequently, the lipid film was hydrated at 40°C for 5 minutes, during which STMD-SpyCatcher solution or PBS (depending on the target requirements) was added. The hydrated mixture was sonicated to promote dispersion and stirred at 40°C overnight to form liposomes modified with STMD-SpyCatcher, referred to as PLp. To further functionalize the liposomes, a SpyTag-Ligand solution was prepared and mixed with PLp at a 1:1 molar ratio (final concentration of 50-200 μM). This mixed solution was gently stirred at room temperature for 1 hour to promote the binding of SpyTag-Ligand to PLp. The resulting product is a functionalized liposome, called FPLp. In order to achieve a uniform particle size (about 100 nm), Lp, PLp and FPLp were extruded through an Avanti Mini-Extruder equipped with a 0.1 μm polycarbonate membrane. Free STMD-SpyCatcher and unbound SpyTag-Ligand were then removed by filtration using a 30 kDa molecular weight cutoff membrane, and the purified functionalized liposome preparation was finally obtained.

[0193] (XXIX) Characterization of Lp, PLp and FPLp

[0194] Transmission electron microscopy (TEM) was used to detect the changes in the physical morphology and membrane thickness of different liposomes. PLp was modified with rhodamine as a fluorescent indicator, combined with different SpyTag-Ligands, and encapsulated with rhodamine to form targeted functionalized liposomes (FPLp(Rho)) for different organs.

[0195] FPLp(Rho) was injected intravenously into the animals, and the heart, liver, spleen, lung and kidney were removed respectively. The fluorescence distribution in each tissue was detected using the PerkinElmer IVIS Spectrum fluorescence imaging system to evaluate the targeting effect of different SpyTag-Ligand modifications on the corresponding organs. For cardiac tissue, paraffin sections were prepared and Gpc6 immunofluorescence staining was performed to evaluate the targeting effect of FPLp on Gpc6-positive cardiomyocytes.

[0196] (30) Blood biochemistry and heart section histology analysis

[0197] Peripheral blood was collected from the experimental animals for liver and kidney function tests, which were performed by Lilai Biomedical (Chengdu, China), including albumin (ALB), γ-glutamyl transpeptidase (γ-GT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total protein (TP), glucose (Glu), uric acid (UA), urea nitrogen (UREA), and creatinine (CREA) in urine.

[0198] Four weeks after transplantation, all animals were euthanized, and organs were collected and cut into 5 μm thick paraffin sections. Biotoxicity was tested by HE staining. Part of the heart was cut into 5 mm thick sections, and imaging of the infarct area was performed.

[0199] 31. Statistical analysis

[0200] All data are presented as mean ± standard deviation (mean ± sd). The Student's unpaired t test was used for statistical analysis of normal distribution data, and the nonparametric Mann-Whitney rank sum test was used to compare the differences between samples for non-normal distribution data. One-way ANOVA was used for some experiments, combined with Bonferroni post hoc test for specific analysis.

[0201] Animal survival analysis used the Kaplan-Meier method, and differences were detected by log-rank (Mantel-Cox) test. Linear correlation analysis used the Pearson correlation coefficient, which ranged from -1 to 1, to evaluate the linear relationship between two continuous variables, and 0 indicated no correlation. For non-normally distributed data, the median (25% and 75% quantiles) was used for descriptive statistics, and the Mann-Whitney U test (non-parametric test) was used to compare the two groups of data, and the results were expressed as Z statistics. For the comparison of the percentages between the two groups, a 2×2 contingency table analysis was used, and the Fisher exact test was used to calculate the significant differences. The statistical significance threshold was set at P<0.05. All statistical analyses were performed using GraphPad Prism software.

[0202] Example 1

[0203] Based on the aforementioned experimental methods, the results showed that copper overload induced Gpc6 + Cpeb3-dependent metabolic reprogramming in CMs leads to copper death in MIRI.

[0204] Previous studies have shown that iron overload during MIRI can induce ferroptosis, but it is unclear whether copper can induce a similar phenomenon. Human data and animal experimental results (Table 1) show that copper levels increase after MIRI ( Figure 1 Middle A, Figure 2 A) and is positively correlated with myocardial injury ( Figure 1 (B) Previous studies have analyzed the heterogeneity of cardiomyocytes after myocardial infarction in mice. The present invention re-analyzed the heterogeneity of cardiomyocytes and found that a type of Gpc6-positive cardiomyocyte (Gpc6 + The change in cell number of CMs was negatively correlated with the copper ion concentration ( Figure 1 C,D, Figure 2 Middle B). In Bama miniature pigs ( Figure 2 Middle C, Figure 2 Middle D, Figure 2 Middle E) and rats ( Figure 2 Middle F, Figure 2 Middle G, Figure 2 A similar trend was observed in H), suggesting that Gpc6 + The status of CMs may be related to the copper concentration.

[0205] Table 1. General information of myocardial infarction and healthy subjects M(P 25 ,P 75 )]

[0206]

[0207]

[0208] The experiments showed that ES_Cu and DS_Cu promoted Gpc6 in a dose-dependent manner + Copper death in CMs ( Figure 1 Middle E, Figure 3 Middle B, Figure 3 C), rather than other forms of cell death ( Figure 3 A), and had no significant effect on Gpc6-negative cardiomyocytes (Gpc6-CMs) ( Figure 3 Middle B, Figure 3 Middle C, Figure 3 Middle D, Figure 3 (E) Spatial transcriptomic studies of human myocardial infarction showed that the specificity of disease damage is highly dependent on the heterogeneity of cardiomyocytes, and different cell types have different metabolic characteristics under the same stimulation. Our data further revealed that ES_Cu significantly inhibited Gpc6 + Mitochondrial respiration of CMs ( Figure 1 Middle F, Figure 3 F) and glycolysis ( Figure 1 Middle G, Figure 3 G), but had no significant effect on Gpc6-CMs ( Figure 3 Middle H, Figure 3 Middle I).

[0209] 24 hours after MIRI, Gpc6 + CMs showed high expression of Cpeb3 and low expression of HK2 in the infarct edge area. Figure 1 Middle H, Figure 4 Middle A, Figure 4 B) phenomenon, this expression pattern is induced by ES_Cu ( Figure 4 C), and the expression of HK2 is regulated by Cpeb3 ( Figure 4 Middle D, Figure 4 Middle E). KEGG and GSEA analysis showed that both Cpeb3 and HK2 were involved in copper death and glycolysis ( Figure 4 Middle F, Figure 4 The correlation between the two is significant ( Figure 4 Middle H, Figure 4 (I) The enriched RNA-binding proteins can induce ferroptosis and regulate cellular metabolism, including glycolysis.

[0210] The results confirmed that ES_Cu in Gpc6 + Activated copper death and metabolic changes in CMs are mainly regulated by Cpeb3, while HK2 plays a weaker role ( Figure 1 I, J, K, Figure 5 Middle A, Figure 5 Middle B, Figure 5 Middle C, Figure 5 Middle D, Figure 5 This process affects the production of metabolic byproducts in glycolysis, the tricarboxylic acid cycle (TCA), and the pentose phosphate pathway ( Figure 5 Middle F, Figure 5 Middle G, Figure 5 Middle H, Figure 5 I), leading to metabolic reprogramming and copper death ( Figure 1 Middle L).

[0211] It is well known that myocardial infarction is usually more serious in the elderly. Single-cell sequencing data of human heart in different age groups showed that Gpc6 + The proportion of CMs increases with age ( Figure 1 Medium M, Figure 6 Middle A, Figure 6 Middle B, Figure 6 This may explain the increased susceptibility of myocardial injury in the elderly.

[0212] Example 2

[0213] This example screens and identifies small molecule inhibitors of Cpeb3.

[0214] Taking advantage of the unique advantages of small molecule compounds, high-throughput drug screening ( Figure 7Middle A) Two potential small molecule inhibitors of Cpeb3 were identified, Com1 ( Figure 8 A) and Com2( Figure 7 Middle B). Com2 has a stronger binding affinity to Cpeb3 (KD = 3.29 μM) ( Figure 7 Middle C, Figure 8 B), and showed robust binding ability to Cpeb3 in multiple species ( Fig. 9 Middle C, Fig. 9 Middle D, Fig. 9 Middle E). Com2 binds to the molecular pocket of Cpeb3 through hydrogen bonds and π-π conjugation, which is composed of TYR-7, ARG-1, LYS-82, THR-83, SER-85 and ASP-86 ( Fig. 9 Middle A, Fig. 9 In B), hydrogen bonds and π-π interactions are medium-weak non-covalent interactions. + In CMs, Com2 significantly inhibited ES_Cu-induced copper death and mitochondrial ROS production, which was superior to Com1 and comparable to shCpeb3 (Cpeb3 knockdown) ( Figure 7 Middle D, Figure 8 Middle C, Figure 8 Middle D, Figure 8 Middle E, Figure 8 Middle F, Fig. 9 Middle F, Fig. 9 Middle G, Fig. 9 Compared with Com1, Com2 more effectively reversed the changes in aerobic respiration and glycolysis induced by ES_Cu, and its effects were similar to those of shCpeb3 ( Figure 7 Middle E, 3F, Figure 8 Middle G, Figure 8 Middle H, Fig. 9 Middle I, Fig. 9 In addition, Com2 treatment significantly reversed the changes in glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle metabolites induced by ES_Cu stimulation, with no significant difference from the effect of shCpeb3 ( Fig.10 Middle A, Fig.10 Middle B, Fig.10 Middle C, Fig.10 These results indicate that Com2 is an excellent small molecule inhibitor of Cpeb3 that can effectively counteract the Gpc6 regulation caused by overexpression of Cpeb3. + Aerobic respiration and glycolysis were altered in CMs, thereby regulating metabolic reprogramming and inhibiting the occurrence of copper death.

[0215] Example 3

[0216] Cpeb3 and HK2 regulate the crosstalk between metabolic reprogramming and copper death via liquid-liquid phase separation (LLPS) and m6A modification.

[0217] In Gpc6 + In CMs, Cpeb3 regulates the expression of HK2, which controls energy production during glycolysis by catalyzing glucose phosphorylation by properly localizing in the outer membrane of mitochondria. The prediction results of Haddock2.4 showed that Cpeb3 and HK2 have strong interaction ability between different species (Table 2, Table 3, Table 4, Fig.12 Middle A). ES_Cu significantly enhances the binding between Cpeb3 and HK2 ( Fig.11 A), causing HK2 to shift from the mitochondrial outer membrane, while some Cpeb3 also translocates to the nucleus ( Fig.12 Com2 can inhibit the binding of Cpeb3 to HK2 without affecting the translocation of Cpeb3 to the nucleus ( Fig.12 (B). PKM2 and PFK1, regulators of glycolysis, are also capable of liquid-liquid phase separation (LLPS). Meanwhile, the RNA-binding protein FXR1 activates mRNA translation via LLPS. Our results show that both Cpeb3 and HK2 exhibit liquid-like properties of fusion and cleavage ( Fig.12 C) and recovers rapidly after photobleaching ( Fig.11 Middle B, Fig.12 Middle D, Fig.12 E), which indicates that they have a strong affinity through LLPS ( Fig.11 C, KD = 47.4 nM), and the amino acid binding site was consistent with the prediction of Haddock2.4 ( Fig.13 Middle B, Fig.13 C, Table 4 rats), while Com2 effectively inhibited the LLPS formation between Cpeb3 and HK2 ( Fig.11 Middle B, Fig.13 Middle A, Fig.13 Middle D). LLPS can lead to protein translocation, and Cpeb3 also controls this process by regulating the expression of HK2 ( Fig.13 D), and under the stimulation of ES_Cu, Cpeb3 translocates to the nucleus ( Fig.12 Middle B, Fig.14 (A).

[0218] Table 2. Prediction of the binding ability of Cpeb3 and HK2 in different species using HADDOCK 2.4

[0219]

[0220] Table 3. Interface residues between Cpeb3 and HK2 (human and pig)

[0221]

[0222]

[0223]

[0224] Table 4. Interface residues between Cpeb3 and HK2 (rat, mouse)

[0225]

[0226]

[0227]

[0228] Cpeb3 mainly binds to U-rich cytoplasmic polyadenylation elements (CPEs), including sequences such as UUUUUUAU, which are present in the 3' untranslated region (3'-UTR) of mRNA and regulate polyadenylation and translation processes. The 3'-UTR of HK2 mRNA from different species is rich in CPEs ( Fig.14 In B), Cpeb3 is able to bind to the CPE region on the 3'-UTR of HK2 mRNA, and ES_Cu promotes this binding ( Fig.14 Middle C, Fig.14 Middle D, Fig.14 Middle E, Fig.14 F), while Com2 effectively inhibited their interaction ( Fig.11 (middle D).

[0229] m6A modification is widely present in RNA, and RNA-binding proteins can directly or indirectly participate in the addition, recognition, and removal of m6A. They can help add m6A as part of the "writing" complex, or recognize m6A as a "reading" protein to mediate downstream functions, or affect demethylation through interaction with "erasing" proteins. The SRAMP prediction server (http: / / www.cuilab.cn / sramp) shows that the 3'-UTR of HK2 in different species is rich in m6A modification sites ( Fig.14 B), and ES_Cu inhibited the m6A modification level of HK2 mRNA ( Fig.15 This effect is attributed to the activation of Cpeb3 ( Fig.15 B), while Com2 can prevent the inhibition of m6A modification ( Fig.11 Middle E).

[0230] Mass spectrometry experiments confirmed that the abundance of m6A eraser protein ALKBH5 and reader protein YTHDC1 on HK2 mRNA was significantly regulated by Cpeb3 ( Fig.15 Middle C, Fig.15 Cpeb3 can promote ALKBH5 to bind to the m6A modification site on HK2 mRNA in the nucleus, thereby preventing the recognition of the reader protein YTHDC1 ( Fig.11 Middle F, Fig.15 Middle F). The reader protein YTHDC1 regulates mRNA nuclear export, thereby affecting RNA stability but not Gpc6. + Nuclear export of HK2 mRNA in cardiomyocytes Fig.15 Treatment with actinomycin D showed that Cpeb3 could reduce the stability of HK2 mRNA through m6A-mediated RNA degradation, while Com2 could prevent HK2 mRNA degradation ( Fig.11 These findings suggest that Gpc6 + In cardiomyocytes, under the stimulation of copper ions, Cpeb3 forms a liquid with HK2 in the form of LLPS, causing the HK2 protein to translocate from the outer membrane of the mitochondria to the cytoplasm. The HK2 protein detaches from the outer membrane of the mitochondria where it should be located, resulting in the inhibition of the mitochondrial glycolysis pathway it promotes, thereby inhibiting mitochondrial function. In addition, Cpeb3 can enter the nucleus, promote the binding of ALKBH5 to the m6A modification site on HK2 mRNA, reduce the m6A modification level, inhibit the recognition of m6A by the reader protein YTHDC1, promote the degradation of HK2 mRNA, inhibit the expression of HK2, further inhibit mitochondrial function, and ultimately induce copper death due to metabolic reprogramming ( Fig.11 Middle H).

[0231] Example 4

[0232] Design and characterization of editable targeted liposomes as drug delivery systems.

[0233] Liposomes are widely accepted for their excellent biocompatibility, but their lack of targeting ability limits their application. Membrane proteins can be divided into five types: transmembrane receptors (such as β2AR), ion channels (such as SCN5A), transport proteins (such as CFTR), pump proteins (such as Na+ / K+ATPase), and anchoring proteins (such as integrins). Each molecule of these transmembrane proteins contains a fragment with a strong affinity for the phospholipid bilayer, namely the transmembrane domain (STMD), which can be stably inserted into the bilayer. Based on the interaction between SpyCatcher and SpyTag and the characteristics of STMD, this example designed and synthesized a flexible and editable targeted delivery platform, called PLp ( Fig.16The variable SpyTag-Ligand provides the flexibility to target different tissues or cells. Fig.17 A) The experiment of binding to liposomes showed that β2AR-SpyCatcher has the strongest affinity to liposome membrane ( Fig.17 Middle B, Fig.17 C). We synthesized single-chain variable fragments (scFv) targeting the heart (Gpc6), liver (HAS), spleen (CD3), lung (SFTPA1), and kidney (Podxl) as ligands to form different SpyTag-Ligands. Under the interaction between SpyCatcher and SpyTag, SpyTag-Ligand and STMD-SpyCatcher can bind quickly and efficiently ( Fig.17 E). Liposomes (Lp) only form delivery liposome platforms (PLp) after modification with STMD-SpyCatcher, and further form functionalized targeted delivery liposome platforms (FPLp) after coupling with variable SpyTag-Ligand. Under appropriate conditions, FPLp can be reduced to PLp ( Fig.16 (B). PLp modified with variable SpyTag-Ligand can effectively target specific organs or tissues ( Fig.16 C), or even specific cells ( Fig.16 These results indicate that the synthesized PLp exhibits universal flexibility, and the FPLp formed after modification with different SpyTag-Ligands can effectively target specific organs, tissues, and even cells, showing a wide range of application potential.

[0234] Example 5

[0235] This example provides cardioprotection in vivo by targeting inhibition of Cpeb3 through a drug delivery system.

[0236] After modifying PLp with SpyTag-Gpc6, FPLp was formed, and its good targeting ability to the heart of Bama miniature pigs was further verified by encapsulating rhodamine ( Fig.19 Middle A, Fig.19 Middle B). FPLp encapsulated Com2 was used to treat myocardial ischemia-reperfusion injury (MIRI) in rats and Bama miniature pigs, significantly improving reperfusion injury ( Fig.18 Middle A, Fig.18 B) and relieved the rats ( Fig.18 Middle C, Fig.18 Middle D) and Bama miniature pigs ( Fig.19 Middle C, Fig.21A) myocardial injury, while significantly inhibiting ST segment elevation ( Fig.19 Middle D, Fig.21 (B). The effect of PLp(Com2) was significantly better than that of Com2 alone. Echocardiographic evaluation using Doppler flow mode, M mode, and two-dimensional speckle tracking showed that PLp(Com2) significantly improved the Fig.18 Middle E) and Bama miniature pigs ( Fig.21 Diastolic function ( Fig.18 Middle F; Fig.19 E) and contractile function ( Fig.18 Middle G, Fig.18 Middle H; Fig.19 Middle F, Fig.21 C) and did not affect heart rate ( Fig.18 Middle J; Fig.21 Importantly, the deformability of the longitudinal fibers of the heart was also improved ( Fig.18 middle I; Fig.19 G), indicating that PLp(Com2) protected global and local contractile function. PLp(Com2) also effectively reduced myocardial fibrosis ( Fig.18 Middle K, Fig.18 Middle L; Fig.19 Middle H, Fig.21 These results indicate that PLp(Com2) significantly enhanced cardiac contraction, relaxation, and functional performance, effectively inhibited myocardial fibrosis and reduced scar formation in the long term, and the therapeutic effect was significantly better than that of Com2 alone. In addition, PLp(Com2) significantly inhibited the activation of action potentials and calcium transients and the prolongation of repolarization ( Fig.18 Middle M, Fig.18 Medium N, Fig.18 Middle O, Fig. 20 Middle A, Fig. 20 Middle B, Fig. 20 Middle C; Fig.19 Middle I, Fig.19 Middle J, Fig.21 Middle G, Fig.21 Middle H, Fig.21 Due to the increased susceptibility to premature ventricular contractions (VA) and lowered threshold for ventricular fibrillation (VF) caused by aggravated MIRI, PLp(Com2) treatment significantly alleviated this problem ( Fig. 20 Middle D, Fig. 20 Middle E; Fig.19 Middle K, Fig.19 L), while reducing mortality caused by VA ( Fig. 20 Middle (F). The protective effect of PLp(Com2) on cardiac function is similar to its effect of improving electrical conduction, showing a significantly better effect than Com2 alone.

[0237] Toxicology experiments showed that although PLp(Com2) treatment in rats ( Fig. 20 Middle G, Fig. 20 Middle H) and Bama miniature pigs ( Fig. 22 Middle A, Fig. 22 B) caused some fluctuations in liver and kidney function indicators, but these fluctuations were still within the normal range and had almost no adverse effects on rats ( Fig. 20 Medium I) or Bama miniature pig ( Fig. 22 These results indicate that PLp(Com2) exhibits excellent cardioprotective effects in both small and large animal models, inhibits fibrosis, improves cardiac function and enhances electrical conduction, with minimal side effects and good biocompatibility. This suggests that PLp(Com2) has important clinical application potential in the treatment of MIRI, especially for elderly patients.

[0238] In summary, during myocardial ischemia-reperfusion injury (MIRI), overload of metal ions can trigger specific cell death. In addition to the known ferroptosis, the present invention also confirmed that copper death occurred in specific Gpc6+ cardiomyocytes (CMs). The induction of copper death is associated with the activation and nuclear translocation of Cpeb3, which further regulates the intracellular localization and expression of HK2 through liquid-liquid phase separation (LLPS) and m6A modification, thereby inducing metabolic reprogramming and triggering copper-specific cell death. The small molecule Com2 identified by high-throughput drug screening can significantly reverse copper death caused by Cpeb3 activation. FPLp, an editable targeted delivery platform constructed based on liposomes, transmembrane proteins and SpyCatcher-SpyTag systems, exhibits good cardioprotective effects when used in combination with Com2.

[0239] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A drug delivery system, characterized in that The invention comprises: a small molecule inhibitor of Cpeb3 or a copper death inhibitor and a liposome; the liposome comprises a membrane layer, the small molecule inhibitor of Cpeb3 or the copper death inhibitor is located in the membrane layer, the membrane layer further comprises a transmembrane domain; the transmembrane domain is connected with a spy catcher; The copper death inhibitor is selected from molybdenum tetrasulfide, UK5099 and Antimycin A; the small molecule inhibitor of Cpeb3 is selected from the compound or its salt shown in formula I and the compound or its salt shown in formula II; Formula I is: Formula II is:

2. The drug delivery system according to claim 1, characterized in that The transmembrane domain is selected from at least one of the following transmembrane proteins: transmembrane receptor proteins, membrane transport proteins, ion channel proteins, pump proteins and lipid-anchored proteins; Preferably, the transmembrane receptor protein is selected from GPCR or β2AR; the membrane transport protein is CFTR; the ion channel protein is selected from sodium, calcium or potassium ion channel protein; The pump protein is selected from Na+ / K+ATPase, Ca2+-ATPase, H+-ATPase or H+-ATP synthase; The anchoring protein is integrin; Preferably, the sodium ion channel protein is selected from SCN5A, Nav1.7, Nav1.8 or Nav1.9; Preferably, the transmembrane domain is selected from the transmembrane domain of β2AR, and its amino acid sequence is GTVPSDNIDSQGRNCSTNDSLL.

3. The drug delivery system according to claim 1, characterized in that The raw material of the membrane layer is selected from simulated plasma membrane, synthetic plasma membrane, membrane molecules extracted from biological cells, a mixture of different membrane components, or a combination thereof; Preferably, the membrane layer is prepared by the following method: dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylethanolamine (DOPE) and cholesterol are mixed in a molar ratio of (4:3:3), (5:3:2), (6:3:1) or (7:2:1), and after removing the organic solvent, a lipid membrane is obtained, which is then mixed with a transmembrane domain connected to a spy catcher after hydration.

4. The drug delivery system according to claim 1, characterized in that The spy catcher is also connected to a spy tag (SpyTag) and a ligand; the spy catcher and the spy tag are coupled by spontaneously forming an isopeptide bond, and the spy tag is operably connected to the ligand; Preferably, in the drug delivery system, the molar ratio of the transmembrane domain connected with the spy catcher to the conjugate of the spy tag and the ligand is 2:1, 1:1 or 1:2; Preferably, in the drug delivery system, the mass percentage of the small molecule inhibitor of Cpeb3 or the copper death inhibitor in the drug delivery system is (17.11-26.48) wt%.

5. The drug delivery system according to claim 4, characterized in that The transmembrane domain is located at the C-terminus of the spy catcher; Preferably, the spy catcher is connected to the transmembrane domain via a connecting peptide; Preferably, a first protein tag sequence is further provided between the spy catcher and the transmembrane domain or at the N-terminus of the spy catcher; Preferably, a second protein tag sequence is further provided between the spy tag and the ligand; Preferably, the connecting peptide is a rigid connecting peptide or a flexible connecting peptide; Preferably, the connecting peptide is a flexible connecting peptide; Preferably, the length of the connecting peptide is 6 to 59 amino acids; Preferably, the length of the connecting peptide is 15 to 35 amino acids; Preferably, the first protein tag sequence and the second protein tag sequence are each independently selected from any one of the following: His, Flag, GST, MBP, HA, Myc, GFP or Fc; Preferably, the spy catcher is also coupled with a fluorescent dye; Preferably, the ligand is selected from ligands targeting cardiac Gpc6 protein; Preferably, the ligand is any one of F(ab')2, Fab', Fab, Fv, Fab'-SH and scFv of an anti-cardiac Gpc6 antibody.

6. The method for preparing the drug delivery system according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: first preparing a membrane layer of liposomes, then mixing the membrane layer of liposomes with a transmembrane domain connected with a spy catcher to obtain a liposome modified with a transmembrane domain-spy catcher; mixing the liposome modified with a transmembrane domain-spy catcher with the small molecule inhibitor of Cpeb3 or the copper death inhibitor; obtaining a liposome encapsulating the small molecule inhibitor of Cpeb3 or the copper death inhibitor; Preferably, the liposomes encapsulating the small molecule inhibitor of Cpeb3 or the copper death inhibitor are mixed with the SpyTag and the ligand; Preferably, in the drug delivery system, the molar ratio of the transmembrane domain connected with the spy catcher to the conjugate of the spy tag and the ligand is 2:1, 1:1 or 1:2; Preferably, in the drug delivery system, the mass percentage of the small molecule inhibitor of Cpeb3 or the copper death inhibitor in the drug delivery system is (17.11-26.48) wt%.

7. Use of the drug delivery system according to any one of claims 1 to 5 in the preparation of a drug for preventing and / or treating myocardial ischemia-reperfusion injury.

8. The use according to claim 7, characterized in that: The drug is administered by intravenous injection or oral gavage.

9. The use according to claim 7 or 8, characterized in that: The drug has at least one of the following uses: (1) Inhibit Cu-induced copper death and mitochondrial ROS production; (2) reversing Cu-induced changes in aerobic respiration and / or glycolysis; (3) Reversing the changes in glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle metabolites induced by Cu stimulation; (4) Inhibit the binding of Cpeb3 to HK2 and inhibit the formation of LLPS between Cpeb3 and HK2; (5) increase the level of m6A modification on HK2 mRNA; (6) Promote the recognition of m6A by the reader protein YTHDC1, inhibit the degradation of HK2 mRNA, increase the expression of HK2, and restore mitochondrial function; (7) suppressing ST segment elevation in the subject's electrocardiogram; (8) Alleviate IRI-induced myocardial damage; (9) improving the overall or local diastolic and systolic function of the subject's heart; (10) Enhance the deformability of longitudinal myocardial fibers; (11) Reduced myocardial fibrosis; (12) Reduced scar formation; (13) Inhibit the activation of action potential and calcium transients in cardiomyocytes, and inhibit the prolongation of cardiomyocyte repolarization; (14) Reduce the susceptibility of subjects to ventricular arrhythmias (VA); (15) Increase the threshold for sudden stimulation of ventricular fibrillation (VF) in subjects; (16) Reduce the mortality caused by ventricular arrhythmias; Preferably, the use (1) refers to: inhibiting Cu-induced copper death of Gpc6-positive cardiomyocytes.

10. The use according to claim 7 or 8, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

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