Application of a small molecule inhibitor of cuproptosis in myocardial ischemia-reperfusion injury

Abstract

The present invention discloses the application of a small molecule inhibitor of cuproptosis in myocardial ischemia-reperfusion injury, which relates to the technical field of myocardial ischemia-reperfusion injury. Specifically reducing or knocking out Cpeb3 expression can significantly promote myocardial repair, improve action potential stability, and reduce mortality. Therefore, Cpeb3 is a specific intervention target for cuproptosis in Gpc6<supgt;+< / supgt> CMs. The small molecule inhibitor of Cpeb3 can significantly reverse the changes in cuproptosis, aerobic respiration, and / or glycolysis triggered by Cpeb3 activation. In addition, the cuproptosis inhibitor can improve the survival rate of cardiomyocytes, reverse the changes in Cu-induced aerobic respiration and / or glycolysis, and reverse the changes in glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle metabolites caused by Cu stimulation. Therefore, the present invention provides a new solution for the prevention and treatment of myocardial ischemia-reperfusion injury and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of myocardial ischemia-reperfusion injury. Specifically, it relates to the application of a small molecule inhibitor of cuproptosis in myocardial ischemia-reperfusion injury. Background Art

[0002] Metal ions are important elements for maintaining normal physiological functions. 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 excessive iron ions in myocardial tissue can induce ferroptosis. As another important metal ion, copper, it is still necessary to explore whether it can trigger myocardial cell death by inducing mitochondrial dysfunction and excessive production of reactive oxygen species (ROS). During myocardial ischemia-reperfusion injury (IRI), myocardial cells undergo a series of stress responses, including nutrient deficiency, ROS release, and secondary inflammatory injury. Single-cell sequencing studies have shown that myocardial cells have a high degree of heterogeneity, and different cell types may respond differently to the same stimulus.

[0003] During the process of MIRI, the metabolic pattern of myocardial cells changes from mainly fatty acid metabolism to glucose metabolism mainly based on glycolysis. The ATP generated by glycolysis not only maintains cell viability but also supports the functions of the Na⁺ / K⁺-ATPase and Ca²⁺-ATPase pumps, which are crucial for maintaining the myocardial potential gradient and the propagation of action potentials. Hexokinase 2 (HK2) is a key enzyme in the glycolysis pathway, responsible for catalyzing the phosphorylation of glucose to produce glucose-6-phosphate, mainly located on the outer mitochondrial membrane, and regulating mitochondrial energy supply. Studies have shown that amino acid metabolism and related proteins play important roles in regulating glycolysis, including proteins such as c-Myc, Sirt1, and Akt, which affect the connection between HK2, glycolysis, and amino acid metabolism. RNA-binding proteins of the Cpeb (cytoplasmic polyadenylation element-binding protein) family (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, the stress response mediated by ATF4, and the 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 dynamic "droplet" structures through intermolecular interactions, enabling 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 disorders in biological activities, while the role of copper excess in myocardial ischemia-reperfusion injury (MIRI), especially in specific subsets of cardiomyocytes, remains unclear.

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

[0007] The object of the present invention is to provide the application of a Cpeb3 gene inhibitor and a ferroptosis inhibitor in the preparation of a drug for treating myocardial ischemia-reperfusion injury to solve the above technical problems.

[0008] The present invention is implemented as follows:

[0009] In a first aspect, the present invention provides the application of a Cpeb3 gene inhibitor and / or an inhibitor of Cpeb3 protein in the preparation of a drug for preventing and / or treating myocardial ischemia-reperfusion injury. The Cpeb3 gene inhibitor is a substance that can inhibit the level of the Cpeb3 gene in cells, tissues, or organs, and the inhibitor of Cpeb3 protein is a substance that can inhibit the level or activity of Cpeb3 protein in cells, tissues, or organs.

[0010] In a second aspect, the present invention also provides the application of a small molecule inhibitor of Cpeb3 or a ferroptosis inhibitor in the preparation of a drug for preventing and / or treating myocardial ischemia-reperfusion injury. The ferroptosis inhibitor is selected from molybdenum tetrasulfide, UK5099, and Antimycin A; the small molecule inhibitor of Cpeb3 is selected from the compounds shown in Formula I or their salts and the compounds shown in Formula II or their salts:

[0011] Formula I is: , CAS: 152684-55-4, named Com2 in this application; Formula II is: , CAS: 1228690-19-4, named Com1 in this application.

[0012] The present invention has the following beneficial effects:

[0013] The present invention confirms that copper overload exists during myocardial ischemia-reperfusion injury (MIRI) and induces cuproptosis in Gpc6-positive cardiomyocytes (Gpc6 + CMs). Copper overload promotes the high expression of Cpeb3, interacts with HK2 through liquid-liquid phase separation (LLPS) and m6A modification, regulates metabolic reprogramming and triggers cuproptosis.

[0014] In the MIRI model, specific knockout of Cpeb3 in cardiomyocytes can significantly alleviate IRI-induced myocardial injury. It is manifested that the cardiac diastolic and systolic functions of the animal model with Cpeb3 knockout are improved, and the deformation ability of longitudinal myocardial fibers is enhanced. It shows that knocking out Cpeb3 can protect the overall and local systolic functions of the heart. In addition, Cpeb3 knockout effectively reduces myocardial fibrosis.

[0015] Cpeb3 knockout significantly inhibits the activation of action potential and calcium transient in cardiomyocytes, inhibits the prolongation of cardiomyocyte repolarization, reduces the susceptibility of the animal model to ventricular arrhythmia (VA); increases the threshold of the animal model for sudden stimulus-induced ventricular fibrillation (VF). Since the increased incidence of VA is associated with higher mortality, specifically reducing or knocking out Cpeb3 expression can significantly promote myocardial repair, improve action potential stability, and reduce mortality. Therefore, Cpeb3 is a specific intervention target for cuproptosis in Gpc6 + CMs.

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

[0017] Therefore, the present invention provides a new solution for the prevention and treatment of myocardial ischemia-reperfusion injury, with good application prospects. 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 will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0019] Figure 1To investigate the occurrence of copper overload in MIRI and its effect on Gpc6 through Cpeb3-mediated metabolic reprogramming + CMs, leading to the experimental result graph of subsequent cuproptosis; (A) Peripheral blood was collected from healthy individuals (n = 20) and patients with myocardial infarction (24 hours; n = 40), and copper levels and cTnI were measured. (B) Correlation analysis was performed between copper and cTnI. Single-cell sequencing data of the hearts of myocardial infarction mice were analyzed, and the Uniform Manifold Approximation and Projection (UMAP) graph (snRNA-seq) was used to show the clustered cardiomyocytes, which were annotated according to spatial localization and gene expression patterns. (C) Quantitative composition of the clustered cardiomyocytes; (D) Violin plot of the specific cardiomyocyte marker Gpc6 was applied to show the snRNA-seq data in different cardiomyocyte clusters. Gpc6 was isolated from adult rat primary cardiomyocytes (ARPCs) by flow cytometry + CMs, subjected to myocardial infarction (hypoxia and starvation for 1 hour): (E) Cell viability was detected by CCK-8 assay after treatment with different concentrations of elesclomol-Cu (ratio 1:1, ES_Cu) or disulfiram-Cu (ratio 1:1, DS_Cu) for 24 hours; (F) Mitochondrial oxygen consumption rate (OCR) was analyzed by Seahorse model mitochondrial stress test within 24 hours of treatment with ES_Cu (50 nM); (G) Extracellular acidification rate (ECAR) was analyzed by Seahorse model mitochondrial stress test; Oligo (oligomycin), FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone), Rot (rotenone), Ant (antimycin A), 2-DG (2-deoxyglucose), Rot+Ant represent a mixture of equal concentrations of Rot and Ant. (H) Volcano plot showing the differentially expressed genes (DEGs) between Gpc6 + CMs and Gpc6-CMs 24 hours after myocardial infarction, threshold for differentially expressed genes: padj < 0.05, Gpc6 isolated from ARPCs + CMs were transfected with Cpeb3 knockdown (shCpeb3) or HK2 overexpression (HK2-OE), and subjected to myocardial infarction (hypoxia and starvation for 1 hour), and stimulated with ES_Cu (50 nM) for 24 hours in the absence or presence of TTM (10 μM; molybdenum disulfide, a cuproptosis inhibitor), (I) Cell viability was detected by CCK-8 assay; (J) OCR was analyzed by Seahorse model mitochondrial stress test; (K) ECAR was analyzed. (L) The proposed model summarizes the copper-Cpeb3 signal in Gpc6 +Role in cuproptosis related to CMs metabolic reprogramming. (M) Analyze single-cell sequencing data of human hearts of different ages, quantify the composition of cardiomyocytes, and clusters 1 and 3 are labeled as high expression of Gpc6. Data are presented as mean ± standard deviation. A is determined by two-tailed t-test, B is calculated by Pearson correlation, I is calculated by one-way ANOVA, **p < 0.01, ****p < 0.001, ns indicates no statistical difference;

[0020] Figure 2 Cpeb3 plays a role as a key regulator in the MIRI mouse model. Conditional knockout mice (cardiomyocyte-specific Cpeb3 gene deletion mice, C57BL / 6J-Cpeb3em1flox) were used, with n = 10 in each group. At 24 hours after MIRI treatment in mice, (A) measure the cTnI level in plasma, (B) detect reperfusion injury by Evans blue and TTC (2,3,5-triphenyltetrazolium chloride) staining, AAR (area at risk), IF (infarct size). After 4 weeks, echocardiography was performed, including Doppler flow, M-mode ultrasound, and two-dimensional speckle tracking, and calculate (C) E / A (early peak / atrial peak) ratio, (D) LVFS (left ventricular ejection fraction), (E) PLAXL (left ventricular parasternal long-axis view) strain; (F) test the fibrotic area by Masson staining;

[0021] (G) Detect action potential by optical mapping, (H) detect the duration of Ca2+ transient changes. Electrocardiogram was performed to evaluate ventricular electrophysiological stability and arrhythmia (VA) susceptibility, and measure (I) VA inducibility, (J) VF threshold. (K) Kaplan-Meier survival curve shows the survival rate of mice throughout the process. Data are presented as mean ± standard deviation. P values are calculated by one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, ns indicates no statistical difference. The survival rate was analyzed by log-rank (Mantel-Cox) test;

[0022] Figure 3 Com2 is an inhibitor of Cpeb3. (A) Flow chart for screening small molecule Cpeb3 inhibitors. IF (hypoxia and starvation treatment for 1 hour), 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) Detect the binding of Com2 to rat Cpeb3 recombinant protein by SPR experiment. Gpc6 isolated from ARPCs +For CMs, Cpeb3 was knocked down (shCpeb3) and subjected to myocardial infarction (hypoxia and starvation for 1 hour), stimulated with ES_Cu (50 nM) for 24 hours, and treated in the presence or absence of Com2 (5 μM). (D) Cell viability was detected by CCK-8. (E) Oxygen consumption rate (OCR) was analyzed by Seahorse mitochondrial stress test. (F) Extracellular acidification rate (ECAR) was analyzed. Data are presented as mean ± standard deviation. P values were calculated by one-way ANOVA, ****p < 0.001, ns indicates no statistical difference;

[0023] Figure 4 To study the experimental results of the interaction between Cpeb3 and HK2 through LLPS and m6A modification. (A) The binding of Cpeb3 and HK2 in rat Gpc6 was detected by immunoprecipitation-western blotting (IP-western blotting) + CMs under the stimulation conditions of hypoxia and starvation for 1 hour, with or without ES_Cu (50 nM) treatment. (B) Rat Gpc6 CMs with Cpeb3 knockdown (shCpeb3) or HK2 overexpression (HK2-OE), + subjected to myocardial infarction (hypoxia and starvation for 1 hour), stimulated with ES_Cu (50 nM) for 24 hours, and in the presence or absence of Com2 (5 μM), the fluorescence redistribution of the photobleached area of HK2-mCherry condensates was quantitatively measured (n = 10). (C) The affinity of recombinant rat Cpeb3 and HK2 was detected by SPR experiment. Rat Gpc6 + CMs were subjected to myocardial infarction (hypoxia and starvation for 1 hour), stimulated with ES_Cu (50 nM) for 24 hours, and in the presence or absence of Com2 (5 μM). (D) The binding of HK2 mRNA and Cpeb3 was measured by RIP experiment. (E) The m 6 6A modification of HK2 mRNA was detected by MeRIP qPCR. (F) Whether Cpeb3 was knocked down, the binding effect of HK2 mRNA and ALKBH5 / YTHDC1 was tested and detected by RNA pull-down experiment. (G) Further with or without Com2 (5 μM), actinomycin D (5 μg / mL) was added to block de novo RNA synthesis, and the stability of HK2 mRNA was detected by the fold change after 0 hours by qRT-PCR. (H) The proposed model summarizes the Cpeb3-HK2 signaling pathway in Gpc6 +Role of cuproptosis related to metabolic reprogramming in CMs. Data are presented as mean ± standard deviation. P values were calculated by one-way analysis of variance (ANOVA), *p < 0.05, ****p < 0.001, ns indicates no statistical difference;

[0024] Figure 5 To detect copper ions and Gpc6 in cardiomyocytes and myocardial tissues in a myocardial ischemia-reperfusion injury (MIRI) model established in Bama minipigs and rats + Figure showing the detection results of cardiomyocyte proportion; A myocardial ischemia-reperfusion injury (MIRI) model was established in (C) Bama minipigs and (F) rats by ligating the left anterior descending artery, and the successful establishment of the model was confirmed by ST-segment elevation in the electrocardiogram; (A) Detect the copper ion content in myocardial tissues of Bama minipigs at different time points after MIRI. (B) Analyze single-cell sequencing data from the hearts of mice after myocardial infarction 3 and quantify the composition of clustered cardiomyocytes. (D, E) Isolate primary cardiomyocytes from adult Bama minipigs and (G, H) rats at different time points after MIRI, and determine Gpc6 + cardiomyocytes (Gpc6 + CMs) proportion, and perform (D, G) statistical analysis. Data are presented as mean ± standard deviation (mean ± SD). P values were calculated by one-way analysis of variance (one-way ANOVA), *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, ns indicates no statistical difference;

[0025] Figure 6 For 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 respectively, or stimulated for 24 hours under the condition of adding TTM (10 μM), Z-VAD-FMK (20 μM), Nec-1 (20 μM) or Fer-1 (10 μM) for cell viability test results graph; (A) Detect cell viability by CCK-8 method; (B, C) Detect cell death by PI (propidium iodide) staining combined with flow cytometry, (C) and perform statistical analysis. Gpc6 isolated from ARPCs +CMs were subjected to infarction treatment (hypoxia and starvation for 1 hour), and (D) stimulated with different concentrations of Elesclomol-Cu (ratio = 1:1, ES_Cu) or Disulfiram-Cu (ratio = 1:1, DS_Cu) for 24 hours, and cell viability was detected using the CCK-8 method; (E) stimulated with ES_Cu (50 nM) for 24 hours, and cell viability was detected using the CCK-8 method under the conditions of whether to add 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 subjected to infarction treatment (hypoxia and starvation for 1 hour), and then Seahorse mitochondrial stress tests were performed with or without stimulation with ES_Cu (50 nM) 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 subjected to infarction treatment (hypoxia and starvation for 1 hour), and then Seahorse mitochondrial stress tests were performed with or without stimulation with ES_Cu (50 nM) for 24 hours: (H) OCR was detected and (I) ECAR was detected, and related indexes were calculated respectively. 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;

[0026] Figure 7 To establish a MIRI model using Bama minipigs, samples were collected from myocardial tissues in different regions for protein extraction or paraffin sectioning, and then (A) Western blot and (B) immunofluorescence experiments were performed 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 either subjected to infarction treatment (IF, hypoxia and starvation for 1 hour) or untreated, and then stimulated with ES_Cu (50 nM) for 24 hours or not, and the expression levels of Cpeb3 and HK2 were detected by Western blot. Gpc6 isolated from ARPCs +CMs were stimulated with or without ES_Cu (50 nM) for 24 hours under the condition of (D) knocking down Cpeb3 (shCpeb3) or (E) knocking down HK2 (shHK2), and western blotting was performed to evaluate the expression levels of (D) HK2 and (E) Cpeb3. According to the published single-cell sequencing data after myocardial infarction in mice, the whole-genome expression data of high expression of Cpeb3 and low expression of HK2 in Gpc6 + CMs were extracted for KEGG pathway analysis to show the related 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 cuproptosis and positively correlated with glycolysis; (I) on the contrary, Cpeb3 was positively correlated with cuproptosis and negatively correlated with glycolysis;

[0027] Figure 8 Gpc6 isolated from adult rat primary cardiomyocytes (ARPCs) + CMs were knocked down for Cpeb3 (shCpeb3) or overexpressed for HK2 (HK2-OE), and then underwent infarction treatment (IF, hypoxia and starvation for 1 hour), and were stimulated with or without ES_Cu (50 nM) for 24 hours in the presence or absence of TTM (10 μM). (A, B) Cell death rate was detected by PI (propidium iodide) staining combined with flow cytometry, and (B) statistical analysis was performed; (C) Mitochondrial ROS level was detected by flow cytometry. Seahorse mitochondrial stress test was performed to detect (D) mitochondrial oxygen consumption rate (OCR), and spare respiratory capacity, basal respiration, proton leak and ATP production were calculated; (E) Extracellular acidification rate (ECAR) was detected, and glycolytic capacity, glycolysis level and glycolytic reserve were further calculated; the effects of shCpeb3, HK2-OE or TTM on glucose-derived metabolites and the percentage of total metabolites in Gpc6 + CMs after infarction and stimulated with ES_Cu were analyzed. Cells were incubated with [U-13C] glucose (1.5 mM) for 24 hours, and (F) glucose-6-phosphate (Glc-6P), (G) glycolysis, (H) pentose phosphate pathway (PPP) and (I) tricarboxylic acid cycle (TCA) related metabolites were detected. Data were 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 indicated no statistical difference;

[0028] Figure 9To analyze single-cell RNA sequencing data (snRNA-seq) from the human heart 12-14, images drawn using the Uniform Manifold Approximation and Projection (UMAP) method show: (A) all cells and (B) clustered cardiomyocytes, with clusters annotated according to gene expression patterns. The data are from representative samples at different age stages (young group, age ≤ 45; middle-aged group, 45 < age < 60; elderly group, age ≥ 60). (C) In cardiomyocytes of different clusters, violin plots are used to show the expression of the specific cardiomyocyte subset marker Gpc6 in the snRNA-seq data;

[0029] Figure 10 Conditional knockout mice with cardiomyocyte-specific knockout of Cpeb3 (C57BL / 6J-Cpeb3em1flox purchased from Cyagen Biosciences, Guangzhou) were used, with n = 10 in each group. The mice underwent myocardial ischemia-reperfusion injury (MIRI), and after 24 hours, the following were detected: (A) plasma creatine kinase-MB (CK-MB) levels, (B) reperfusion injury was evaluated by Evans Blue and TTC (2,3,5-triphenyltetrazolium chloride) staining, and (C) AAR / LV was calculated, where AAR is the area at risk and LV is the left ventricular area. Four weeks later, echocardiography was used to detect: (F, left) Doppler flow map, (F, middle) M-mode, (F, right) two-dimensional speckle tracking map; (D) left ventricular ejection fraction (LVEF) and (E) heart rate (HR) were calculated; (G) Masson staining was performed to detect the fibrotic area. Optical mapping technology was used to detect: (H, upper) action potential and (H, lower) calcium transient duration, and statistical analysis was performed on (I) APD90 (action potential duration at 90% repolarization) and (J) CTD90 (calcium transient duration at 90% recovery). The data are expressed as mean ± standard deviation (mean ± SD). P values were calculated by one-way ANOVA, *p < 0.05, ***p < 0.005, ****p < 0.001, ns indicates no statistical difference;

[0030] Figure 11 (A) Chemical structural formula of Com1; (B) Binding of Com1 to recombinant rat Cpeb3 protein was determined by SPR. Gpc6 isolated from ARPCs +CMs were subjected to ischemia-reperfusion injury (hypoxia and starvation for 1 hour) and stimulated with ES_Cu (50 nM) for 24 hours in the absence or presence of Com1 (5 μM) or Com2 (5 μM): (C) Cell viability was detected by the CCK-8 assay, (D) Cell death was detected by flow cytometry with PI (propidium iodide) staining, (E) And statistical analysis was performed, (F) Mitochondrial ROS levels were evaluated by flow cytometry. The Seahorse mitochondrial stress test was used to detect: (G) Extracellular acidification rate (ECAR), and glycolytic capacity, glycolysis, and glycolytic reserve were further calculated, (H) Mitochondrial oxygen consumption rate (OCR), and spare respiratory capacity, basal respiration, proton leak, and ATP production 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;

[0031] Figure 12 (A) Schematic diagram of the docking interaction between complex Com2 and rat-derived Cpeb3 protein. (B) The binding effect of Com2 to wild-type or specifically amino acid-mutated rat-derived Cpeb3 recombinant protein was determined by SPR. The binding of Com2 to (C) human, (D) porcine, and (E) mouse-derived Cpeb3 recombinant proteins was determined by SPR. Gpc6 isolated from ARPCs + CMs were subjected to ischemia-reperfusion injury (hypoxia and starvation for 1 hour) and stimulated with ES_Cu (50 nM) for 24 hours in the absence or presence of Com2 (5 μM) or Cpeb3 gene knockdown (shCpeb3): (F) Cell death was detected by flow cytometry with PI (propidium iodide) staining, (G) And statistical analysis was performed, (H) Mitochondrial ROS levels were evaluated by flow cytometry. The Seahorse mitochondrial stress test was used to detect: (J) Mitochondrial oxygen consumption rate (OCR) and (I) extracellular acidification rate (ECAR), and related indexes 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;

[0032] Figure 13 (A) Gpc6 isolated from ARPCs +After CMs were knocked down for Cpeb3 gene (shCpeb3), they were subjected to ischemia (hypoxia and starvation for 1 hour), and then in the absence or presence of Com2 (5 μM), or stimulated with ES_Cu (50 nM) for 24 hours. To study the effects of shCpeb3 or Com2 on Gpc6 + the percentages of glucose-derived metabolites and total metabolites in CMs. After ischemia and ES_Cu stimulation, the cells were cultured with [U-13C] glucose (1.5 mM) for 24 hours, and the following metabolites were detected: (A) Glc-6P, (B) glycolysis, (C) tricarboxylic acid cycle (TCA), (D) pentose phosphate pathway (PPP)-related metabolites. 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;

[0033] Figure 14 (A) Binding simulation diagrams of Cpeb3 and HK2 across species. (B) Gpc6 isolated from ARPCs + CMs, after being knocked down for Cpeb3 gene (shCpeb3), were subjected to ischemia (hypoxia and starvation for 1 hour), and then in the absence or presence of Com2 (5 μM), or stimulated with ES_Cu (50 nM) for 24 hours. The expressions of Cpeb3 and HK2, and their co-localization with mitochondria and nuclei were detected by immunofluorescence. The nuclei were stained with Hoechst 33342, scale bar = 20 μm. Gpc6 isolated from ARPCs + CMs, subjected to ischemia (hypoxia and starvation for 1 hour), and then stimulated with ES_Cu (50 nM) for 24 hours, (C) time-lapse images of Cpeb3-GFP (left) and HK2-mcherry (right), (D) FRAP analysis of condensates of Cpeb3-GFP (top) and HK2-mcherry (bottom), (E) quantitative analysis of FRAP of the bleached area in condensates of Cpeb3-GFP and HK2-mcherry;

[0034] Figure 15Representative fluorescence images of HK2-mcherry droplets in the presence or absence of Cpeb3 or Com2, scale bar = 10 μm. The affinity assay of recombinant rat mutant Cpeb3 for HK2 was performed by (B) SPR experiments and (C) fluorescence polarization (FP) experiments. (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 was performed on HK2-mcherry condensates. Data are presented as mean ± standard deviation (SD). P values were calculated by one-way ANOVA, ***p < 0.005, ns indicates no statistical difference;

[0035] Figure 16 Gpc6 isolated from ARPCs + CMs, subjected to ischemia (hypoxia and starvation for 1 h) or not, and then the expression levels of cytoplasmic Cpeb3 (CytCpeb3) and nuclear Cpeb3 (NucCpeb3) were detected by Western blot experiments after stimulation with ES_Cu (50 nM) for 24 h in the absence or presence. (B) (a) HK2 mRNA sequence information from different species, including the start positions of 5'-UTR, CDS, and 3'-UTR; (b) mouse, (c) rat, (d) pig, and (e) human HK2 mRNA 3'-UTR CPE sequence information; and the predicted m6A sites and their corresponding secondary structures by SRAMP (http: / / www.cuilab.cn / sramp). (C) Rat Gpc6 + CMs, after being subjected to ischemia (hypoxia and starvation for 1 h), were stimulated with ES_Cu (50 nM) for 24 h, and the binding of HK2mRNA and Cpeb3 was measured using RIP experiments. In rat Gpc6 +In CMs, (D) the binding effects of wild-type and mutant HK2 mRNA 3'-UTR with Cpeb3 protein were evaluated using luciferase reporter gene assays; the binding regions and (F) sequences of HK2 mRNA with Cpeb3 protein were detected by RNA pull-down assays. Data are presented as mean ± standard deviation (SD). P values were calculated by one-way ANOVA, ***p < 0.005, ****p < 0.001, ns indicates no statistical difference;

[0036] Figure 17 is for rat Gpc6 + After CMs were subjected to ischemia (hypoxia and starvation for 1 hour), (B, C, D) with or without Cpeb3 gene knockout (shCpeb3) or (A), they were then stimulated with ES_Cu (50 nM) for 24 hours, and MeRIP assays were used to detect the m6A RNA modification of HK2 mRNA; proteins bound to Cpeb3 were collected by immunoprecipitation with Cpeb3-coated magnetic beads, mass spectrometry (MS) was used to evaluate the most significantly changed proteins, and statistical analyses were performed on two proteins related to m6A modification, (C) ALKBH5 and (D) YTHDC1, which showed the most significant changes in abundance. (E) Rat Gpc6 + CMs were subjected to ischemia (hypoxia and starvation for 1 hour) and either Cpeb3 gene knockout (shCpeb3) or YTHDC1 gene overexpression (YTHDC1-OE), and then stimulated with ES_Cu (50 nM) for 24 hours, and qRT-PCR was used to detect the nuclear and cytoplasmic fractions of HK2 mRNA. (F) Rat Gpc6 + After CMs were subjected to ischemia (hypoxia and starvation for 1 hour), they were stimulated with ES_Cu (50 nM) for 24 hours, and the co-localization of Cpeb3, YTHDC1, and YTHDC1 in the nucleus was detected by confocal microscopy in the absence or presence of Com2 (5 μM), scale bar = 10 μm. Data are presented as mean ± standard deviation (SD). P values were calculated by one-way ANOVA, **p < 0.01, ****p < 0.001, ns indicates no statistical difference. Detailed implementation methods

[0037] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided by way of explanation and not limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the present invention. For example, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0038] In a first aspect, the present invention provides the use of an inhibitor of the Cpeb3 gene and / or an inhibitor of the Cpeb3 protein in the preparation of a medicament for preventing and / or treating myocardial ischemia-reperfusion injury. The Cpeb3 gene inhibitor is a substance that can inhibit the level of the Cpeb3 gene in cells, tissues or organs, and the inhibitor of the Cpeb3 protein is a substance that can inhibit the level or activity of the Cpeb3 protein in cells, tissues or organs. The cells are selected from cardiomyocytes, and the organ or tissue is the heart.

[0039] The inventors have found through research that by inhibiting the expression of the Cpeb3 gene, knocking down or knocking out the level of the Cpeb3 protein in cells, tissues or organs, it is possible to significantly alleviate myocardial injury induced by ischemia / reperfusion injury (IRI). It is manifested that the diastolic and systolic functions of the hearts of animal models with Cpeb3 knockout are both improved, and the deformability of longitudinal myocardial fibers is enhanced. It shows that knocking out Cpeb3 can protect the overall and local systolic functions of the heart. In addition, Cpeb3 knockout effectively reduces myocardial fibrosis. Cpeb3 knockout significantly inhibits the activation of action potentials and calcium transients in cardiomyocytes, inhibits the prolongation of cardiomyocyte repolarization, and reduces the susceptibility of animal models to ventricular arrhythmia (VA); it increases the threshold of the animal model for evaluating ventricular fibrillation (VF) by burst stimulation. Since the increased incidence of VA is associated with higher mortality, specifically reducing or knocking out the expression of Cpeb3 can significantly promote myocardial repair, improve action potential stability, and reduce mortality. Therefore, Cpeb3 is Gpc6 + A specific intervention target for cuproptosis in cardiomyocytes (CMs). By inhibiting the level of the Cpeb3 gene and / or the Cpeb3 protein, myocardial ischemia-reperfusion injury can be prevented and / or treated.

[0040] In a preferred embodiment of the application of the present invention, the cardiomyocytes are Gpc6-positive cardiomyocytes. The inventors have found that cuproptosis occurs in specific Gpc6 + cardiomyocytes (CMs).

[0041] In a preferred embodiment of the application of the present invention, the inhibitor of the IGFBP3 gene is selected from at least one of the following substances: at least one of modified or unmodified RNA molecules or a recombinant vector comprising a coding RNA molecule, and the RNA molecule is selected from siRNA, sgRNA, snoRNA, shRNA or microRNA.

[0042] The recombinant vector includes, but is not limited to: recombinant lentiviral vector, recombinant adenoviral vector, recombinant adeno-associated viral vector, etc.

[0043] In addition to the coding sequence of the above-mentioned RNA molecule, the recombinant vector further includes a regulatory sequence and a coding sequence of a Cas protein, and the coding sequence of the RNA molecule, and the coding sequence of a Cas nuclease or a Cas nickase are operably linked to the regulatory sequence.

[0044] In some embodiments, the Cas nuclease is a type II Cas nuclease. In some embodiments, the Cas nuclease is Cas9, Cpfl, C2cl, C2c2 and C2c3 or a modified protein thereof. In some embodiments, the Cas nuclease is Streptococcus pyogenes or Staphylococcus aureus Cas9 nuclease or a modified protein thereof. In some embodiments, the Cas nuclease is from a type II CRISPR / Cas system.

[0045] In a preferred embodiment of the application of the present invention, the regulatory sequence includes: a promoter and a terminator corresponding to the promoter. For example, a type II promoter is paired with a terminator capable of terminating the type II promoter, and a type III promoter is paired with a terminator capable of terminating the type III promoter.

[0046] In a preferred embodiment of the application of the present invention, the promoter is selected from a constitutive promoter, an inducible promoter, a universal promoter, a tissue-specific promoter, a cell type-specific promoter, or a development stage-specific promoter.

[0047] In a preferred embodiment of the application of the present invention, the promoter is a type II promoter or a type III promoter; in a preferred embodiment of the application of the present invention, the type II promoter is selected from the CMV promoter, the CAG promoter, the PGK promoter, and the EF1α promoter.

[0048] In a preferred embodiment of the application of the present invention, the terminator is selected from SV40 polyA, bGH polyA, BETAPolyA or 6XT.

[0049] In a preferred embodiment of the application of the present invention, the recombinant vector further includes a selectable marker gene or a reporter gene.

[0050] The term "selectable marker" refers to a gene that will facilitate the selection of cells that actively express a nucleic acid sequence. Examples of suitable selectable markers include enzymes that encode resistance to antibiotics (i.e., antibiotic resistance genes), such as kanamycin, neomycin, puromycin, hygromycin, blastocidin, or bleomycin.

[0051] In addition, selectable marker genes also include amplifiable selectable marker genes. The reference to "amplifiable selectable marker gene" as used herein refers to a gene that allows the amplification of the gene under appropriate growth conditions. The amplifiable selectable marker gene is capable of increasing the expression product (i.e., the expression of the protein encoded by the amplifiable selectable marker gene) by amplification in response to the lack of an inhibitor or an essential metabolite. In one embodiment, the amplifiable selectable marker gene can be characterized as being capable of complementing a auxotrophic host.

[0052] In a preferred embodiment of the application of the present invention, the drug has at least one of the following uses:

[0053] (1) Alleviating myocardial injury induced by IRI (ischemia / reperfusion injury);

[0054] (2) Improving the diastolic and systolic functions of the whole or local heart of a subject;

[0055] (3) Enhancing the deformability of longitudinal myocardial fibers;

[0056] (4) Reducing myocardial fibrosis;

[0057] (5) Inhibiting the activation of action potentials and calcium transients in cardiomyocytes and inhibiting the prolongation of cardiomyocyte repolarization;

[0058] (6) Reducing the susceptibility of a subject to ventricular arrhythmia (VA);

[0059] (7) Increasing the threshold of a subject for assessing ventricular fibrillation (VF) in response to a sudden stimulus.

[0060] In a preferred embodiment of the application of the present invention, the drug further comprises a pharmaceutically acceptable carrier. The drug further comprises a pharmaceutically acceptable carrier, including but not limited to fillers, lubricants, disintegrants, binders, glidants, etc.

[0061] In a preferred technical solution of the present invention, the pharmaceutically acceptable carrier includes but not limited to one or a combination of polyvinylpyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methylcellulose and its derivatives, ethylcellulose and its derivatives, hydroxypropylcellulose 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, silica, titanium dioxide, talc, indigo.

[0062] In a second aspect, the present invention also provides the use of a copper death inhibitor in the preparation of a medicament for preventing and / or treating myocardial ischemia-reperfusion injury, and the copper death inhibitor is selected from molybdenum tetrasulfide (TTM), UK5099, Antimycin A, the compound shown in Formula I or its salt, and the compound shown in Formula II or its salt.

[0063] Formula I is: , CAS: 152684-55-4, named Com2 in this application; Formula II is: , CAS: 1228690-19-4, named Com1 in this application. UK5099 CAS NO. 56396-35-1.

[0064] The present invention utilizes the unique advantages of small molecule compounds and identifies two potential small molecule inhibitors of Cpeb3, Com1 and Com2, through high-throughput drug screening. Com2 has a stronger binding affinity for Cpeb3 (KD = 3.29 μM) and exhibits a robust binding ability to the 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, and this pocket is composed of TYR-7, ARG-1, LYS-82, THR-83, SER-85, and ASP-86, where the hydrogen bonds and π-π interactions are weak non-covalent interactions. In Gpc6 + CMs, Com2 significantly inhibits ES_Cu (Elesclomol-Cu (ratio = 1:1))-induced copper death and mitochondrial ROS production, and its effect is better than that of Com1 and is comparable to the effect of Cpeb3 knockdown. Compared with Com1, Com2 more effectively reverses the changes in aerobic respiration and glycolysis induced by ES_Cu, and its effect is similar to that of Cpeb3 knockdown. In addition, after Com2 treatment, the changes in glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle metabolites stimulated by ES_Cu are significantly reversed, and there is no significant difference from the effect of Cpeb3 knockdown. These results indicate that Com2 is an excellent small molecule inhibitor of Cpeb3, which can effectively counteract the changes in aerobic respiration and glycolysis in Gpc6-positive cardiomyocytes (Gpc6 + CMs) caused by high expression of Cpeb3, thereby regulating metabolic reprogramming and inhibiting the occurrence of copper death.

[0065] In a preferred embodiment of the application of the present invention, the medicament is administered by intravenous injection or gavage.

[0066] In a preferred embodiment of the application of the present invention, the medicament has at least one of the following uses:

[0067] (1) Inhibit Cu-induced cuproptosis and mitochondrial ROS production;

[0068] (2) Reverse the changes in aerobic respiration and / or glycolysis induced by Cu;

[0069] (3) Reverse the changes in metabolites of glycolysis, pentose phosphate pathway and tricarboxylic acid cycle induced by Cu stimulation;

[0070] (4) Inhibit the binding of Cpeb3 to HK2 and the formation of LLPS between Cpeb3 and HK2;

[0071] (5) Increase the m6A modification level on HK2 mRNA;

[0072] (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.

[0073] In a preferred embodiment of the application of the present invention, use (1) refers to inhibiting cuproptosis in Gpc6-positive cardiomyocytes induced by Cu.

[0074] In a preferred embodiment of the application of the present invention, the drug further comprises a pharmaceutically acceptable carrier.

[0075] The drug further comprises a pharmaceutically acceptable carrier, including but not limited to fillers, lubricants, disintegrants, binders, glidants, etc.

[0076] In a preferred technical solution of the present invention, the pharmaceutically acceptable carrier includes but not limited to one or a combination of polyvinylpyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methylcellulose and its derivatives, ethylcellulose and its derivatives, hydroxypropylcellulose 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 powder, indigo.

[0077] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0078] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0079] Experimental materials and methods:

[0080] (I) Patient information and blood samples.

[0081] In this study, 40 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 were selected. All patients underwent corresponding laboratory tests. Inclusion criteria: (1) Clinically diagnosed with acute STEMI; (2) PCI was performed within 12 hours after the onset of symptoms; (3) The patients and their families signed the relevant informed consent form. Exclusion criteria: (1) Patients with advanced malignant tumors; (2) Patients with multiple organ dysfunction; (3) Patients with severe coagulation disorders; (4) Poor treatment compliance of the patients. Peripheral blood was collected 24 hours after PCI to detect the copper ion content. Healthy people who underwent physical examinations 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 the informed consent form, and this study was approved by the Ethics Committee of Sichuan Provincial People's Hospital.

[0082] (II) Plasma and tissue copper detection.

[0083] Plasma samples were collected with heparin as an anticoagulant, centrifuged at 1000 g for 5 minutes at 4°C, and the supernatant was placed on ice for measurement. According to the manufacturer's instructions, a serum copper content assay kit (#BC5645, Solarbio, Beijing, China) was used to detect the copper ion level in peripheral blood. Approximately 0.1 g of heart tissue was measured and 1 mL of distilled water was added. Then the mixture was homogenized on ice. Then the homogenate was centrifuged at 10000 g 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).

[0084] (III) MIRI model.

[0085] The MIRI model was established according to previous reports. Spf-grade mice (8 weeks old), SD rats (180 - 230 g), and Bama miniature pigs (15 - 20 kg) were obtained from Chengdu Dashuo Laboratory Animal Co., Ltd. (Chengdu, China). All animal procedures were approved by the Animal Care and Use Committee of Sichuan Provincial People's Hospital. SD rats or mice were induced to anesthesia by intraperitoneal injection of sodium pentobarbital (50 mg / kg), tracheally intubated and connected to a small animal ventilator to ensure respiration. Electrocardiogram monitoring was used to evaluate the degree of myocardial ischemia. The chest was opened at the fourth left intercostal space to expose the heart, and the left anterior descending coronary artery (LCA) was ligated with 8-0 (for mice) or 6-0 (for rats) silk suture, and a slipknot was made 2 - 3 mm distal to the origin of the LCA. The inversion of the T wave and elevation of the ST segment were observed on the electrocardiogram, indicating ischemia. The slipknot was released after 45 minutes to reperfuse the affected area. For Bama miniature pigs, they were fasted for 12 hours before surgery to minimize the risk of aspiration. Anesthesia was induced with isoflurane and maintained with 1 - 2% isoflurane via tracheal intubation. Vital signs, including heart rate, respiratory rate, and oxygen saturation, were continuously monitored using electrocardiogram. The left chest was shaved and disinfected with povidone-iodine solution. Median sternotomy was performed to expose the heart, and the LCA coronary artery (the second diagonal branch) was pre-occluded for 45 minutes before ligation. The chest cavity was closed in layers with absorbable suture. Postoperatively, antibiotics (cefazolin, 25 mg / kg) and analgesics (lidocaine, 2 mg / kg) were administered and closely monitored during the recovery period. Myocardial infarction was confirmed by electrocardiogram immediately after surgery. At the conclusion of the study, these animals were humanely euthanized with an overdose of pentobarbital and tissues were harvested from the major organs.

[0086] (IV) Bioinformatics analysis.

[0087] All bioinformatics analyses were performed using the CeleLens Cloud platform provided by Singleron (https: / / www.celelenscloud.cn / # / my_analyzes). The Sc / snRNA-seq data of the mouse heart after myocardial infarction were obtained from the Gene Expression Omnibus (GSE214611). The Sc / snRNA-seq data of the 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), respectively. The Seurat package was used to classify and annotate the data, identify different cell types, and extract the marker genes of each subgroup. Subsequently, DESeq2 was used for differential expression analysis between the two groups of cells, and volcano plots were generated to visualize the significantly different 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 ferroptosis pathway and glycolysis pathway to evaluate the enrichment of related gene sets. All analyses were carried out in the R environment to ensure the reliability of data processing and the reproducibility of results, thus revealing the potential functions and mechanisms of different cell subgroups in ferroptosis and metabolic regulation.

[0088] (5) Cell viability and death.

[0089] To mimic the injury of MIRI to cardiomyocytes in vitro, adult rat primary cardiomyocytes (ARPCs) were used to isolate Gpc6+CMs / Gpc6-CMs by flow cytometry. All cells were ischemic (hypoxic starvation for 1 h) to mimic MIRI and then treated with different drugs. CCK8 assay 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 by flow cytometry.

[0090] (6) Flow cytometry.

[0091] Single-cell suspensions were obtained from myocardial tissues using enzymatic digestion (#abs9482, Absin, Shanghai, China), and washed with PBS buffer to remove residual enzymes. After adjusting the cell suspension to an appropriate concentration, anti-cTnI antibodies (rat: #MG270057, Abmart, Shanghai, China; pig: #ab231064, Abcam, Boston, Massachusetts, USA) and Gpc6 (mouse: #PC22025, Abmart, Shanghai, China; pig: Abconal custom antibody added) were gently mixed. The mixture was incubated at 4 °C for 1 hour to ensure sufficient binding of the antibodies to the target cells. Then the cells were washed three times with PBS to remove unbound primary antibodies. Fluorescently labeled secondary antibodies (FITC: #F-2761, Invitrogen, USA; PE: #12-4739-81, Invitrogen, USA added) were incubated at 4 °C for 30 minutes to ensure specific binding of the secondary antibodies to the primary antibodies. The cells were washed three times with PBS to remove unbound secondary antibodies. Subsequently, the cells were analyzed by flow cytometry. In the flow cytometer, fluorescent dyes were excited by lasers, and forward scatter and side scatter data were collected to determine the size and complexity of the cells. Specific fluorescence channels were used to detect the expression of cTnI and Gpc6. Positive and negative cells were distinguished according to the fluorescence intensity, and finally the number of gpc6-positive cardiomyocytes and their proportion in the total number of cells were recorded.

[0092] (VII) Seahorse analysis.

[0093] According to the manufacturer's instructions, the mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of isolated cardiomyocytes were evaluated using a Seahorse XF analyzer (Agilent, Santa Clara, CA). Cell-Tak (22 μg / ml, pH 6.5–8, #354241, Corning) was coated in the wells of a Seahorse XFe96 spherical microplate (#102959-100, Agilent) and incubated at 37 °C for 20 minutes. Then, the microplate was neutralized with deionized water, rinsed twice, air-dried and stored at 4 °C. The isolated cardiomyocytes were then transferred to the wells, placed in the medium and incubated overnight at 37 °C for attachment. The next day, it was replaced with 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.

[0094] For OCR assessment, measurements were performed after the addition of oligomycin (4 μmol / L), FCCP (4 μmol / L), and a combination of rotenone (2 μmol / L) and antimycin A (2 μmol / L) using the Seahorse XF Mitochondrial Stress Test Kit (#103015-100, Agilent, Santa Clara, CA). ECAR was measured using the Seahorse XF Glycolysis Stress Test Kit (#103020-100, Agilent, Santa Clara, CA) under baseline conditions and after sequential addition of glucose (20 mmol / L), oligomycin (4 μmol / L), and 2-deoxyglucose (2-DG, 100 mmol / L). Each injection port of the sensor cartridge was filled with 25 μl of the test compound or solvent diluted in DMEM / F12 buffer. After calibration, the cell cartridge was analyzed in the XF96 instrument with the measurement protocol set to 3 minutes of mixing followed immediately by 3 minutes of measurement. Results read by the Seahorse XF96 analyzer were normalized by the total protein level per well.

[0095] (VIII) Western blot and co-immunoprecipitation (Co-IP).

[0096] Tissues or cells were lysed with RIPA lysis buffer (#R0010, Solarbio, Beijing, China), and protein concentration was determined using the BCA Protein Assay Kit (#PC0020, Solarbio, Beijing, China). Proteins were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated with the primary antibody overnight at 4°C and then with the secondary antibody for 1 hour at 37°C. Band visualization was performed using the Amersham ImageQuant™ 800 system (Cytiva, Sweden).

[0097] In the Co-IP experiment, using the Pierce™ Classic Magnetic IP / Co-IP Kit (Thermo Scientific™, USA), after cell lysis, specific antibodies were used to capture the target protein, allowing other interacting proteins to co-precipitate. Subsequently, Protein A / G agarose beads were used to immobilize the antibody-protein complex and remove unbound components. Finally, the bound proteins were eluted and analyzed by Western blot to confirm the interaction between the target protein and other proteins. 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).

[0098] (IX) Immunofluorescence.

[0099] 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 and incubated at room temperature for 5 to 10 minutes, followed by three washes with PBS. Non-specific binding was blocked with 3% BSA for 30 to 60 minutes. The primary antibody diluted 1:500 was added and incubated overnight at 4°C. Then, it was washed three times with PBS, the fluorescently labeled secondary antibody diluted 1:1000 was added, incubated for 60 minutes in the dark, and washed three times again with PBS. Finally, the nuclei were stained with DAPI for 5 minutes, and after washing, it was sealed with an anti-fluorescence quenching mounting medium and observed under a microscope.

[0100] For tissue samples, fresh tissues were fixed with 4% paraformaldehyde for 24 hours, dehydrated with gradient alcohol, cleared with xylene, and then embedded in paraffin. The section thickness was 5 μm and loaded onto slides. After baking at 60°C for 30 minutes, the slides were dewaxed with xylene and alcohol, and then washed with distilled water. The sections were placed in citrate buffer and incubated at 95°C for 20 minutes, cooled, and washed three times with PBS. Non-specific binding was blocked with 3% BSA for 30 minutes. The primary antibody was added and incubated overnight at 4°C. Subsequently, it was washed three times with PBS, the fluorescently labeled secondary antibody was added, incubated for 1 hour in the dark, and washed three times again. The nuclei were stained with DAPI for 5 minutes, and after washing, it was sealed with a mounting medium and observed under a microscope.

[0101] The antibodies used in the experiment 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).

[0102] (X) Metabolomics.

[0103] The detection of metabolites was analyzed based on the methods in previous studies. To quantitatively detect metabolites in glycolysis, pentose phosphate pathway (PPP), and tricarboxylic acid cycle (TCA), two complementary experiments were conducted. The first experiment used ion chromatography-mass spectrometry (IC-MS) to analyze the levels of pyruvate and glucose-6P in the culture medium; the second experiment used ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) to separate hexoses, including glucose.

[0104] The isolated cardiomyocytes were co-incubated with 10 mM [U-13C]-labeled glucose for 6 hours. Metabolites were extracted with 80% methanol and concentrated by Labconco CentriVap. The dried metabolites were resuspended in 50% acetonitrile-aqueous solution and loaded onto a Luna 3µm NH2 100A column (150 × 2.0 mm, Phenomenex). Chromatographic separation was performed on a Vanquish Flex system (Thermo Scientific) at a flow rate of 200 µL / min, using a linear gradient from 15% to 95% of mobile phase A (5 mM NH4AcO, pH 9.9) for 18 minutes, followed by isocratic elution at 95% A for 9 minutes.

[0105] Metabolites were detected by a Q Exactive mass spectrometer (Thermo Scientific) in full scan mode, switching polarities (+3.5 kV / −3.5 kV), and the detected mass-to-charge ratio range was 65–975 m / z. Quantitative analysis was performed using TraceFinder 4.1 software, based on peak area, expected retention time, and accurate mass (error <5 ppm). The detected values were normalized to cell count, and the relative amounts of metabolites were calculated by summing all isotopologues of each metabolite and correcting for natural C13 abundance.

[0106] (XI) Mitochondrial reactive oxygen species (ROS) detection.

[0107] Mitochondrial reactive oxygen species were measured using the MitoSOX™ mitochondrial superoxide indicator (#M36005, Invitrogen™, USA). First, cell samples were cultured under appropriate conditions and then collected. The cells were washed once with PBS to remove the culture medium, and then 1 μl of 5 mM MitoSOX Green was diluted in 0.5 ml of PBS to prepare the working solution. 1 ml 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. Then, the cells were analyzed using a flow cytometer, and appropriate parameters were set to detect the fluorescence signal of MitoSOX. Through this analysis, the mitochondrial ROS level in each cell could be quantitatively determined, and the differences between different treatment groups could be compared. The obtained data could be used to evaluate the mitochondrial function and oxidative stress status within the cells.

[0108] (XII) Detection of cTnI and CK-MB.

[0109] The levels of cardiac troponin I (cTnI) were determined using specific ELISA kits, which were the porcine TNNI3 ELISA kit (#ELK5656, ELK Biotechnology, Wuhan, China), the rat cTnI ELISA kit (#D731143, Sangon Biotech, Shanghai, China), and the mouse cTnI ELISA kit (#D721149, Sangon Biotech, Shanghai, China) for different species, and the operations were carried out according to the manufacturer's instructions. Similarly, the levels of creatine kinase-MB (CK-MB) were determined using the relevant kits for each species: the kit for pigs (#CB10105-Pg, Coibo, Shanghai, China), the kit for rats (#SEKR-0059, Solarbio, Beijing, China), and the kit for mice (#SEKM-0152, Solarbio, Beijing, China). After successfully establishing the model for each group, 0.2 mL of venous blood was collected to measure the cTnI concentration.

[0110] (XIII) Evans Blue & TTC staining and Masson staining

[0111] At 24 hours after myocardial ischemia-reperfusion injury, the degree of myocardial injury was evaluated by double staining with Evans Blue (#E8010, Solarbio, Beijing, China) and TTC (2,3,5-triphenyltetrazolium chloride, #T8170, Solarbio, Beijing, China). First, a 0.5% Evans Blue solution was perfused for 10 minutes to label necrotic myocardium. Subsequently, the heart was removed, cut into slices approximately 1 cm thick, and stained with a 1% TTC solution in a 37°C water bath for 30 minutes. Healthy myocardial tissue stained red with TTC, while the ischemic injury area remained pale. After the experiment, the staining results were quantitatively evaluated using image analysis software, and the proportion of the infarct area to the total myocardial area was calculated to objectively evaluate the degree of ischemia-reperfusion injury.

[0112] At 4 weeks after myocardial ischemia-reperfusion injury, Masson staining (#G1346, Solarbio, Beijing, China) was used to evaluate the degree of myocardial fibrosis. The rat hearts were quickly rinsed with normal saline and then fixed in 10% neutral buffered formaldehyde for 24 hours. After fixation, the hearts were cut into 5-μm-thick slices and dehydrated and cleared. Subsequently, Masson staining solution was used for staining. The specific steps included: staining with Weigert iron solution (containing 1% potassium ferricyanide and 1% ferric chloride) at room temperature for 10 minutes, then rinsing with distilled water to stain the blue of the fiber background. Then, acid fuchsin solution was added and stained for 2 minutes, and the 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 the myocardial tissue were analyzed.

[0113] (XIV) Echocardiogram

[0114] The hearts of rats, mice, and pigs were examined by echocardiography using FUJIFILM Vevo 3100LT and Finno Ultrasound VINNO ULTIMUS 9E. Before the experiment, all animals received appropriate anesthesia and fixation. For rats and mice, isoflurane was used for anesthesia, and the anesthesia 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 a small anesthesia machine was used to monitor vital signs. For pigs, tiletamine-zolazepam was first used for induction at a dose of 10 mg / kg body weight. After anesthesia induction, pigs were maintained in an appropriate anesthetic state with 2-3% isoflurane. During anesthesia, the heart rate, respiratory rate, and body temperature of pigs were continuously monitored to ensure safety and anesthetic effect.

[0115] Using the Vevo 3100LT, the operator obtains cardiac images of rats and mice through a high-frequency probe and calculates important cardiac function parameters. The E / A ratio is obtained from the mitral valve Doppler flow velocity spectrum, and the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) are calculated by measuring the dynamic changes in the cardiac chamber contour using M-mode echocardiography of the left ventricular short axis. Subsequently, the VINNO ULTIMUS9E is used to perform ultrasonic examinations on rats, mice, and pigs. This device focuses on obtaining strain data of the long-axis section of the left ventricle (PLAXL), and myocardial strain is calculated by dynamically tracking the long-axis view. In addition, for pigs, the E / A ratio, LVEF, LVFS, and heart rate (HR) are also recorded to comprehensively evaluate cardiac function. All ultrasonic images and parameters are carefully analyzed to ensure the accuracy of the data, providing a reliable basis for cardiac function assessment in subsequent studies.

[0116] (XV) Electrophysiological Integration Detection

[0117] Electrophysiological integration indexes are detected using an Optical Mapping System. First, heparin sodium injection (3000 U / kg) is administered intraperitoneally. After waiting for 15 minutes, isoflurane anesthesia is induced. The skin is disinfected with alcohol, an incision is made on the chest skin to expose the xiphoid process, the xiphoid process is lifted with forceps, the ribs are incised along the left and right midlines, the chest cavity is opened, and the heart is exposed. The lung tissue is clamped with forceps, and the heart is quickly excised and rinsed in a calcium-free preoxygenated (95% O2, 5% CO2) modified Krebs buffer (118 mmol / L NaCl, 4.2 mmol / L KCl, 1.2 mmol / L KH2PO4, 1.2 mmol / L MgSO4, 23 mmol / L NaHCO3, 20 mmol / L D-glucose, 2 mmol / L sodium pyruvate, 1.8 mmol / L CaCl2, pH 7.35–7.45) at 4°C. The aorta is quickly inserted into the perfusion needle using a horizontal Langendorff perfusion device and fixed with surgical sutures, and it is observed whether there are air bubbles entering the heart. The heart is fixed in a silicone rubber chamber, retrograde perfusion is performed, and carbogen gas is introduced for ventilation. After the Langendorff-isolated heart is stabilized for 10 minutes, the heart is allowed to resume its normal rhythm, and fluorescent dyes are loaded in sequence.

[0118] After 10 minutes of stabilization, turn off all room lights and perform continuous perfusion with Krebs buffer. Inject the contractility inhibitor Blebbistatin from the Y-shaped inlet to abolish the mechanical contraction of the heart, and stop after about 1 - 2 minutes. Remove part of the perfusion fluid, reduce the oxygen supply from the circulation reservoir, and add Pluronic F127 to the remaining perfusion fluid. After 10 minutes of continuous perfusion, slowly inject the calcium indicator Rhod-2AM from the Y-shaped inlet and continue circulation for 15 minutes. Then, slowly inject the voltage-sensitive dye RH237, and then return the previously removed Krebs buffer to the perfusion reservoir and continue circulation.

[0119] 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 ground in the bath. Connect to a biological signal acquisition system to record the electrocardiogram (ECG), and adjust the sampling rate for continuous monitoring automatically. Use an 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 an appropriate imaging area. Record three action potentials and calcium ion signals under sinus rhythm.

[0120] Insert a pacing electrode approximately 1 mm to the right ventricle and deliver pacing stimuli. The initial current intensity is 0 mA, the frequency is 6 Hz, and the pulse width is 2 ms. Closely observe the ECG. When the ECG shows a clear 1:1 pacing corresponding to the stimulus, this current intensity is the diastolic threshold current. Set the pacing current to twice the diastolic threshold current and record the action potentials and calcium ion signals at this 6 Hz pacing rate. After eight consecutive S1 stimuli, apply one premature extra 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 fails to trigger a QRS waveform. At this time, record the S1S2 interval as the ventricular effective refractory period.

[0121] Induce ventricular arrhythmia using 50 Hz high-frequency pulses, gradually increase the stimulus current intensity from 5 mA to 10 mA, then to 15 mA, and finally to 20 mA, with a 3-minute interval between each stimulus, and record the occurrence, duration, and fibrillation threshold of ventricular arrhythmia after each stimulus. Record the ECG of all hearts under sinus rhythm, normal pacing, and induced ventricular arrhythmia, and observe the optical measurements of the membrane potential and calcium ion signals of the dye-loaded rat hearts before and after ischemia-reperfusion, and analyze the action potential depolarization, repolarization time, action potential duration, and calcium transient.

[0122] (XVI) Protein Purification

[0123] Plasmids containing different species of Cpeb3 and its specific amino acid mutants, Cpeb3-GFP, and HK2-mcherry were synthesized by Tsingke Biotechnology (Beijing, China) using the pGEX-4T-1 vector and subsequently transformed into competent Escherichia coli cells. Transformed bacteria were inoculated onto LB agar plates supplemented with antibiotics and cultured overnight to select plasmids containing the ampicillin resistance gene. A positive clone was selected and inoculated into LB liquid medium containing antibiotics and cultured with shaking at 37°C overnight.

[0124] Subsequently, a small amount of the overnight culture was transferred to fresh LB medium containing antibiotics and cultured with shaking at 37°C until the optical density at 600 nm (OD600) reached 0.6 - 0.8. At this stage, isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to induce protein expression, and the culture was continued to be cultured with shaking at 37°C for 6 hours. After induction, bacterial cells were collected by centrifugation at 6000 rpm for 10 minutes. The obtained cell pellet was resuspended in lysis buffer containing protease inhibitors to prevent protein degradation. The cells were lysed by sonication to effectively release the target protein into the solution.

[0125] After cell lysis, cell debris was removed by centrifugation, and the supernatant containing the soluble protein was collected for purification. The target protein with a His tag was separated using a Ni-NTA affinity chromatography column and eluted with elution buffer containing different concentrations of imidazole. The purity of the protein in each fraction collected during the elution process was analyzed by SDS-PAGE. If the purity was insufficient, the protein was further concentrated by dialysis or ultrafiltration techniques. Finally, the purified protein was stored in a suitable buffer for subsequent experimental use.

[0126] (XVII) Virtual Docking

[0127] Virtual docking was performed using AutoDock software. This study aimed to explore the binding interactions 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 box was defined, and docking simulations were carried out. By analyzing the binding affinity and visualizing the interactions, key hydrogen bonds and hydrophobic interactions were identified, and the distances between interacting groups were measured. Finally, the binding mechanism between Cpeb3 and the small molecule compound was revealed, providing a theoretical basis for subsequent drug design.

[0128] (XVIII) High-Throughput Drug Screening

[0129] Using the molecular modeling software AutoDock, the three-dimensional structure of Cpeb3 (UniProt ID: D4AD99) was constructed and energy minimized to ensure the accuracy of the model. Subsequently, compounds with specific chemical properties and bioactivity information were screened from 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. By calculating key parameters such as binding energy, number of hydrogen bonds, and hydrophobic interactions, potential high-affinity compounds were screened. During the virtual screening process, the PLANET model based on an image neural network was used to score the compounds to capture the complex relationship between molecular structure and bioactivity. Combining the results of molecular docking, 300 candidate compounds were finally selected.

[0130] Next, primary Gpc6-positive cardiomyocytes (Gpc6 + CMs) and cardiac fibroblasts (CMFs) were isolated from rats. 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 were cultured in DMEM / F12 medium, and cardiac fibroblasts were cultured in DMEM medium. Both media were supplemented with 10% fetal bovine serum and antibiotics, and the cells were cultured in an environment of 37°C and 5% CO 2 To simulate the myocardial infarction environment, the cells were treated under hypoxic (1% O 2 ) and starvation (serum-free medium) conditions for 1 hour to induce a stress response. Subsequently, 50 nM of ES_Cu and 5 μM of different candidate compounds were added to the medium and incubated for 24 hours.

[0131] The CCK8 kit was used to evaluate cell viability according to the manufacturer's instructions, and appropriate control groups (such as untreated group and ES_Cu-only treatment group) were set to ensure the reliability of the experiment. In the Gpc6 + CMs group, the cell viability increased significantly by 1.5-fold, 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 apoptosis signals. In the CMFs group, the increase in cell viability was no more than 1.1-fold, indicating that these compounds have no significant promoting effect on cardiac fibroblasts, highlighting their specificity and selectivity. Based on this result, compounds with a cell viability increase of more than 1.5-fold in the Gpc6 + CMs group and no significant increase in cell viability in the CMFs group were defined as "hits", demonstrating their superiority in targeting cardiomyocytes.

[0132] For the selected hit compounds, surface plasmon resonance (SPR) experiments were conducted. In the SPR experiment, the Cpeb3 protein was prepared by a recombinant expression system and immobilized on a sensor chip. Different concentrations of the selected compounds were injected, and their binding and dissociation processes were monitored in real time. The affinity constant (KD) was calculated through data analysis, and compounds with KD ≤ 50 μM were defined as effective hit compounds.

[0133] (XIX) Surface Plasmon Resonance (SPR) Experiment

[0134] The SPR experiment was performed using Octet® 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 nickel NTA sensor chip to ensure uniform binding of the protein, thereby improving the reproducibility and reliability of the experiment.

[0135] During the experiment, different concentrations of candidate compounds or proteins were sequentially injected onto the sensor surface by a flow injection method. The instrument continuously monitored the change in the reflected light intensity during the binding process, generating sensorgrams. These response curves showed the rate of the binding reaction and the changes during the dissociation process, clearly reflecting the interaction between the mobile phase and the immobilized protein.

[0136] In the data analysis stage, a dedicated software was used for baseline correction to eliminate background noise and extract the true signal. By fitting the binding and dissociation curves at different concentrations, the equilibrium dissociation constant (KD) was calculated.

[0137] (XX) RNA Immunoprecipitation (RIP) and Methylated RNA Immunoprecipitation (MeRIP)

[0138] The RIP and MeRIP experiments were performed according to the methods described in previous literature. The RIP experiment was carried out using the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (#17-701, Sigma-Aldrich, Billerica, MA, USA) and following the manufacturer's instructions. Briefly, cell lysis was achieved using the provided RIP lysis buffer to extract RNA-binding proteins. Immunoprecipitation was performed by binding anti-Cpeb3 antibody or control IgG to magnetic beads to capture the target RNA-binding protein. To detect the enrichment of HK2 mRNA in the immunoprecipitated RNA samples, qRT-PCR analysis was conducted. Additionally, to analyze the m6A modification level in HK2 mRNA, the MeRIP experiment was carried out based on the established method. In this step, 5 μg of anti-m6A antibody (#A19841, ABclonal, Wuhan, China) or control IgG was bound to the 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 overnight at 4°C with the anti-m6A antibody in the RIP immunoprecipitation buffer (provided by the Magna RIP kit) to specifically bind m6A-modified RNA. After incubation, the samples were treated with proteinase K to remove residual proteins, ensuring purified RNA samples. Subsequently, the RNA was purified using the RNeasy Mini Kit (#74104, Qiagen, Germany). The enrichment of HK2 mRNA in the m6A immunoprecipitated RNA fraction was quantitatively detected by qRT-PCR analysis.

[0139] (XXI) RNA Pull-Down Experiment

[0140] Biotin-labeled HK2 mRNA was synthesized by in vitro transcription using the pcDNA3.1 vector as a template. During transcription, Biotin RNA Labeling Mix (#11685597910, Merck, Germany) containing biotin-UTP and T7 RNA polymerase (#RPOLT7-RO, Merck, Germany) were added to drive the reaction. To remove residual DNA that might contaminate the RNA, the synthesized product was treated with RNase-free DNase I (#69182, Merck, Germany) at a concentration of 1 U / μg RNA at 37 °C for 30 minutes and then inactivated by heating at 75 °C for 10 minutes. The biotin-labeled RNA was purified using the RNeasy Mini Kit (#74104, Qiagen, Germany) and eluted with 30 μL of RNase-free water to obtain a pure and concentrated RNA product. Subsequently, the purified biotin-labeled RNA was added to a pre-prepared cell lysate (prepared from cultured cells using RIPA buffer containing protease inhibitors) and incubated at 4 °C for 2 hours. This incubation step promoted the interaction between RNA and proteins, forming RNA-protein complexes. After incubation, the biotin-labeled RNA-protein complexes were captured using Dynabeads™ M-270 Streptavidin (#65305, Invitrogen, USA). 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 the proteins that interacted with the biotin-labeled HK2 mRNA, thus revealing the functional dynamics of HK2 in the cellular environment.

[0141] (XXII) qRT-PCR Experiment

[0142] Total RNA was extracted using the RNAsimple Total RNA Kit (#DP419, Tiangen, Beijing, China), strictly following the manufacturer's instructions to ensure high-quality RNA for subsequent experiments. For cDNA synthesis, 1,000 ng of the extracted total RNA was used as a template, and reverse transcription was performed using the PrimeScript™ RT Reagent Kit with gDNA Eraser (#RR047, TaKaRa, Dalian, China). This kit includes a gDNA removal step to effectively eliminate genomic DNA contamination, thereby improving the accuracy and reliability of downstream experimental results. After cDNA synthesis, qPCR amplification was performed using a one-step reverse transcription 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 the internal reference gene to normalize the expression data and compensate for variations in cDNA input and overall PCR efficiency. The relative gene expression level was calculated by the 2 −(ΔΔCt) -ΔΔCT method to enable a robust comparison of gene expression between samples.

[0143] 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 RNAsimple Total RNA Kit, and the quality and concentration of the resulting RNA samples were evaluated using a Nanodrop spectrophotometer. By comparing the expression levels at different time points, the degradation kinetics of the target mRNA under transcriptional inhibition conditions were analyzed. Primer sequences

[0144] HK2 Forward: 5'- CTGCCCTTCTGCTCTTCTG -3' (SEQ ID NO:1);

[0145] HK2 Reverse: 5'- GCTGGTGGTGAGGTGATG -3' (SEQ ID NO:2).

[0146] (XXIII) Luciferase reporter gene assay

[0147] To identify the binding sites of Cpeb3 in the HK2 mRNA 3′-UTR region, the wild-type and mutant sequences of the HK2 3′-UTR were cloned into the pmirGLO luciferase vector. Rat Gpc6 +CMs cells were seeded into 96-well plates at a density of 5 × 10³ cells per well, with three replicates for each condition. The cells were co-transfected with the pmirGLO-HK2 wild-type 3′-UTR or pmirGLO-HK2 mutant 3′-UTR vector and shRNA against Cpeb3 to inhibit the expression of Cpeb3. After 48 h of transfection, the luciferase activity was measured using a dual-luciferase assay system (#RG029, Beyotime, Beijing, China) according to the manufacturer's instructions. The activities of firefly and Renilla luciferases were quantified, and the relative luciferase activity was determined by calculating the ratio of firefly luciferase to Renilla luciferase activity, thereby standardizing the transfection efficiency. This method enabled in-depth understanding of the interaction between Cpeb3 and the 3′-UTR region of HK2 mRNA.

[0148] (XXIV) Fluorescence polarization

[0149] To detect the binding of Cpeb3 protein to HK2 protein, fluorescence polarization technology was employed using a SpectraMax iD5 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 fluorescence polarization detection, the excitation wavelength was set at 488 nm and the emission wavelength was 520 nm to accommodate the fluorescence characteristics of GFP. The fluorescence polarization values under different conditions were recorded, and the polarization changes when wild-type and mutant GFP-Cpeb3 proteins bound to HK2 protein were compared to evaluate the binding affinity between the two.

[0150] (XXV) Fluorescence recovery after photobleaching (FRAP) experiment

[0151] Before imaging, different treatment strategies were applied to the cultured cells. Using a ZEISS LSM 980 confocal microscope, the region of interest (ROI) containing GFP or mCherry signals was completely photobleached using a 488 nm laser beam focused treatment. 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. The fluorescence intensity was measured before bleaching and at the start of recovery to comprehensively analyze the dynamic process of movement and redistribution of fluorescent proteins after the application of treatment.

[0152] (XXVI) RNA Pulldown / mass spectrometry (MS)

[0153] In rat Gpc6 + In CMs, Cpeb3 protein was either knocked down or not. Nuclear protein was extracted using a nuclear and cytoplasmic protein extraction kit (#P0027, Beyotime, Beijing, China), and an RNA pull-down assay for HK2 mRNA was 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 digested, and the resulting peptides were purified using a C18 solid-phase extraction column and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). In the mass spectrometry data analysis, the obtained mass spectrometry data were processed using MaxQuant software and compared with a protein database. Based on the mass spectra of the peptides, m6A-modified proteins interacting with Cpeb3 were identified, with particular attention paid to changes in relative abundance to distinguish differences between the control group and the Cpeb3 knockdown group.

[0154] (XXVII) Statistical analysis

[0155] All data are presented as mean ± standard deviation (mean ± s.d.). For normally distributed data, Student's unpaired t-test was used for statistical analysis, and for non-normally distributed data, the non-parametric Mann–Whitney rank sum test was used to compare differences between samples. Some experiments used one-way analysis of variance (One-way ANOVA) combined with Bonferroni post hoc test for specific analysis.

[0156] 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, ranging from -1 to 1, to evaluate the linear relationship between two continuous variables, with 0 indicating 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 two groups of data, with the results expressed as the Z statistic. For the comparison of two groups of percentages, 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.

[0157] Example 1

[0158] Based on the aforementioned experimental methods, the research results showed that copper overload induced Cpeb3-related metabolic reprogramming in Gpc6 + CMs, leading to cuproptosis in MIRI.

[0159] Previous studies have shown that iron ion overload during the MIRI process can induce ferroptosis. However, it is still unclear whether copper can trigger a similar phenomenon. Population data and animal experiment results (Table 1) show that the copper ion level increases after MIRI ( Figure 1 in A, Figure 5 in A), and is positively correlated with myocardial injury ( Figure 1 in B). Previous studies analyzed the heterogeneity of cardiomyocytes after myocardial infarction in mice. After re-analysis in the present invention, it was found that the change in the cell number of a type of Gpc6-positive cardiomyocytes (Gpc6 + CMs) was negatively correlated with the copper ion concentration ( Figure 1 in C, D, Figure 5 in B). Similar trends were observed in Bama minipigs ( Figure 5 in C, Figure 5 in D, Figure 5 in E) and rats ( Figure 5 in F, Figure 5 in G, Figure 5 in H), suggesting that the state of Gpc6 + CMs may be related to the copper concentration.

[0160] Table 1. General information of myocardial infarction and healthy populations ± s , M ( P 25 , P 75 )]

[0161]

[0162] Experiments showed that ES_Cu and DS_Cu promoted cuproptosis of Gpc6 + CMs in a dose-dependent manner ( Figure 1 in E, Figure 6 in B, Figure 6 in C), rather than other forms of cell death ( Figure 6 in A), and had no significant effect on Gpc6-negative cardiomyocytes (Gpc6-CMs) ( Figure 6 in B, Figure 6 in C, Figure 6 in D, Figure 6 in E). Spatial transcriptomics studies of human myocardial infarction showed that the specificity of disease injury highly depends on the heterogeneity of cardiomyocytes, and the metabolic characteristics of different cell types are different under the same stimulus. Our data further revealed that ES_Cu significantly inhibited the mitochondrial respiration ( + of Gpc6 Figure 1 CMs in F, Figure 6 in F) and glycolysis (Figure 1 in G, Figure 6 in G), but had no significant effect on Gpc6-CMs ( Figure 6 in H, Figure 6 in I).

[0163] At 24 hours after MIRI, Gpc6 + CMs showed high expression of Cpeb3 and low expression of HK2 in the infarct border zone ( Figure 1 in H, Figure 7 in A, Figure 7 in B) phenomenon, and this expression pattern was induced by ES_Cu ( Figure 7 in C), and the expression of HK2 was regulated by Cpeb3 ( Figure 7 in D, Figure 7 in E). KEGG and GSEA analyses showed that both Cpeb3 and HK2 were involved in cuproptosis and glycolysis ( Figure 7 in F, Figure 7 in G), and the correlation between the two was significant ( Figure 7 in H, Figure 7 in I). The enriched RNA-binding proteins could induce ferroptosis and also regulate the cell metabolic level, including glycolysis.

[0164] The results confirmed that the cuproptosis and metabolic changes activated by ES_Cu in Gpc6 + CMs were mainly regulated by Cpeb3, while the role of HK2 was relatively weak ( Figure 1 in I, J, K, Figure 8 in A, Figure 8 in B, Figure 8 in C, Figure 8 in D, Figure 8 in E). This process affected the generation of metabolic by-products in glycolysis, the tricarboxylic acid cycle (TCA), and the pentose phosphate pathway ( Figure 8 in F, Figure 8 in G, Figure 8 in H, Figure 8 in I), resulting in metabolic reprogramming and triggering cuproptosis ( Figure 1 in L).

[0165] As is well known, myocardial infarction usually causes more serious damage to the elderly population. Single-cell sequencing data of the human heart in different age groups showed that Gpc6 + the proportion of CMs increased with age ( Figure 1 in M, Figure 9 in A, Figure 9 in B, Figure 9 in C), which may explain the phenomenon that the elderly population is more prone to myocardial injury.

[0166] Example 2

[0167] In this example, Cpeb3 is specifically reduced or knocked out, and myocardial injury test and electrical conduction test are performed.

[0168] In the MIRI model, specific knockout of Cpeb3 in the myocardium significantly alleviated IRI-induced myocardial injury ( Figure 2 A, B, Figure 10 Middle A, Figure 10 Middle B, Figure 10 Middle C). Echocardiographic assessment showed that Cpeb3 knockout mice had reduced diastolic Figure 2 middle C) and contractile function ( Figure 2 Middle D, Figure 10 D) have been improved, as shown in the Doppler flow mode ( Figure 10 Middle F, left), M mode ( Figure 10 F, middle) and 2D speckle tracking ( Figure 10 F, right), while heart rate showed no significant change ( Figure 10 More importantly, the deformability of longitudinal myocardial fibers was enhanced ( Figure 2 Middle E), indicating that knocking out Cpeb3 can protect the overall and local contractile function of the heart. In addition, Cpeb3 knockout effectively reduced myocardial fibrosis ( Figure 2 Middle F, Figure 10 Middle G).

[0169] In MIRI, the duration of action potential and Ca²⁺ transient of left ventricular cardiomyocytes is prolonged, leading to an increase in the refractory period, which affects myocardial contraction and overall cardiac function. Fibrotic myocardium changes cardiac structure and electrical signal conduction, indirectly affecting the activation of action potential and calcium transients. The experimental results showed that Cpeb3 knockout significantly inhibited the activation of action potential and calcium transients and prolonged repolarization ( Figure 2 Middle G, 2H, Figure 10 Middle H, Figure 10 Middle I, Figure 10 Middle J).

[0170] The prolongation of action potential duration and heterogeneity of repolarization process increase the possibility of spontaneous myocardial excitation, thereby increasing the risk of ventricular arrhythmias; while the prolongation of calcium transient time leads to uncoordinated myocardial contraction and relaxation, which further increases the risk of arrhythmias and lowers the threshold of ventricular fibrillation. The susceptibility to ventricular arrhythmias (VA) was assessed by controlled stimulation, and the threshold of ventricular fibrillation (VF) was assessed by burst stimulation. MIRI significantly increased the susceptibility to VA, manifested as an increase in VA induction rate and a decrease in VF threshold, and these changes were significantly alleviated after Cpeb3 knockout ( Figure 2in I, J). An increased VA induction rate is associated with higher mortality ( Figure 2 in K).

[0171] These results indicate that Cpeb3 exacerbates myocardial injury in IRI, leading to prolonged action potential duration and abnormal calcium transients, thereby triggering arrhythmogenic death. Specifically reducing or knocking out Cpeb3 expression can significantly promote myocardial repair, improve action potential stability, and reduce mortality.

[0172] Example 3

[0173] In this example, small molecule inhibitors of Cpeb3 were screened and identified.

[0174] Taking advantage of the unique properties of small molecule compounds, we identified two potential small molecule inhibitors of Cpeb3, Com1 ( Figure 3 in A) and Com2 ( Figure 11 in A) through high-throughput drug screening ( Figure 3 in B). Com2 has a stronger binding affinity for Cpeb3 (KD = 3.29 μM) ( Figure 3 in C, Figure 11 in B), and exhibits robust binding ability to Cpeb3 in multiple species ( Figure 12 in C, Figure 12 in D, Figure 12 in E). Com2 binds to the molecular pocket of Cpeb3 through hydrogen bonds and π-π conjugation. This pocket is composed of TYR-7, ARG-1, LYS-82, THR-83, SER-85, and ASP-86 ( Figure 12 in A, Figure 12 in B), where the hydrogen bonds and π-π interactions are weak non-covalent interactions. In Gpc6 + CMs, Com2 significantly inhibited ES_Cu-induced cuproptosis and mitochondrial ROS production, with an effect superior to Com1 and comparable to shCpeb3 (Cpeb3 knockdown) ( Figure 3 in D, Figure 11 in C, Figure 11 in D, Figure 11 in E, Figure 11 in F, Figure 12 in F, Figure 12 in G, Figure 12 in H). Compared with Com1, Com2 more effectively reversed the changes in aerobic respiration and glycolysis induced by ES_Cu, and its effect was similar to that of shCpeb3 ( Figure 3 in E, 3F, Figure 11 in G, Figure 11 in H, Figure 12In I, Figure 12 In J). In addition, after Com2 treatment, the changes in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle metabolites caused by ES_Cu stimulation were significantly reversed, and there was no significant difference from the effect of shCpeb3 ( Figure 13 In A, Figure 13 In B, Figure 13 In C, Figure 13 In D). These results indicate that Com2 is an excellent small molecule inhibitor of Cpeb3, which can effectively counteract the aerobic respiration and glycolysis changes in Gpc6 + CMs caused by high expression of Cpeb3, thereby regulating metabolic reprogramming and inhibiting the occurrence of cuproptosis.

[0175] Example 4

[0176] Cpeb3 and HK2 regulate the interaction between metabolic reprogramming and cuproptosis through liquid-liquid phase separation (LLPS) and m6A modification.

[0177] In Gpc6 + CMs, Cpeb3 regulates the expression of HK2, and HK2 catalyzes glucose phosphorylation by correctly localizing to the outer mitochondrial membrane, thereby controlling energy production during glycolysis. The prediction results of Haddock2.4 show that Cpeb3 and HK2 have strong interaction ability between different species (Table 2, Figure 14 In A). ES_Cu significantly enhances the binding between Cpeb3 and HK2 ( Figure 4 In A), resulting in the translocation of HK2 from the outer mitochondrial membrane, and at the same time some Cpeb3 also translocates to the nucleus ( Figure 14 In B). Com2 can inhibit the binding between Cpeb3 and HK2 without affecting the translocation of Cpeb3 to the nucleus ( Figure 14 In B). PKM2 and PFK1, as regulators of glycolysis, can also undergo liquid-liquid phase separation (LLPS). At the same time, the RNA-binding protein FXR1 activates mRNA translation through LLPS. Our results show that both Cpeb3 and HK2 exhibit fusogenic and lytic liquid properties ( Figure 14 In C), and rapidly recover after photobleaching ( Figure 4 In B, Figure 14 In D, Figure 14 In E), which indicates that they have strong affinity through LLPS ( Figure 4 In C, KD = 47.4 nM), and the amino acid binding site is consistent with the prediction of Haddock2.4 ( Figure 15 In B, Figure 15 In C), while Com2 effectively inhibits the formation of LLPS between Cpeb3 and HK2 ( Figure 4B among, Figure 15 A among, Figure 15 D among). LLPS can lead to the translocation of proteins, and Cpeb3 also controls this process by regulating the expression of HK2 ( Figure 15 D among), and upon stimulation by ES_Cu, Cpeb3 translocates to the nucleus ( Figure 14 B among, Figure 16 A among).

[0178] Table 2. Prediction of the binding ability between Cpeb3 and HK2 in different species using HADDOCK 2.4

[0179]

[0180] Cpeb3 mainly binds to the U-rich cytoplasmic polyadenylation element (CPE), including sequences such as UUUUUAU, which are present in the 3' untranslated region (3'-UTR) of mRNA and regulate the polyadenylation and translation processes. The 3'-UTR of HK2 mRNA from different species is rich in CPE ( Figure 16 B among), and Cpeb3 can bind to the CPE region on the 3'-UTR of HK2 mRNA, and ES_Cu promotes this binding ( Figure 16 C among, Figure 16 D among, Figure 16 E among, Figure 16 F among), while Com2 effectively inhibits their interaction ( Figure 4 D among).

[0181] 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 "writer" complex, or recognize m6A as "reader" proteins to mediate downstream functions, or affect demethylation through interactions with "eraser" proteins. The SRAMP prediction server (http: / / www.cuilab.cn / sramp) indicates that the 3'-UTR of HK2 from different species is rich in m6A modification sites ( Figure 16 B among), and ES_Cu inhibits the m6A modification level of HK2 mRNA ( Figure 17 A among), and this effect is attributed to the activation of Cpeb3 ( Figure 17 B among), while Com2 can prevent this inhibition of m6A modification ( Figure 4 E among).

[0182] Mass spectrometry experiments confirmed that the abundances of the m6A eraser protein ALKBH5 and the reader protein YTHDC1 on HK2 mRNA are significantly regulated by Cpeb3 ( Figure 17 C among,Figure 17 In D). Cpeb3 can promote the binding of ALKBH5 to the m6A modification site on HK2 mRNA in the nucleus, thereby preventing the recognition by the reader protein YTHDC1 ( Figure 4 In F, Figure 17 In F). The reader protein YTHDC1 regulates the nuclear export of mRNA, thereby affecting the stability of RNA, but does not affect Gpc6 + The nuclear export of HK2 mRNA in cardiomyocytes ( Figure 17 In E). Treatment with actinomycin D showed that Cpeb3 could reduce the stability of HK2 mRNA through m6A-mediated RNA degradation, while Com2 could prevent the degradation of HK2 mRNA ( Figure 4 In G). These findings indicate that in Gpc6 + In cardiomyocytes, under copper ion stimulation, Cpeb3 forms a liquid with HK2 in the form of LLPS, resulting in the translocation of HK2 protein from the outer mitochondrial membrane to the cytoplasm. The HK2 protein dissociates from the outer mitochondrial membrane 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 trigger cuproptosis due to metabolic reprogramming ( Figure 4 In H).

[0183] In summary, during myocardial ischemia-reperfusion injury (MIRI), the overload of metal ions can trigger specific cell death. In addition to the known ferroptosis, the present invention also confirms that cuproptosis occurs in specific Gpc6+ cardiomyocytes (CMs). The induction of cuproptosis is related to the activation and nuclear translocation of Cpeb3. Cpeb3 further regulates the intracellular localization and expression of HK2 through liquid-liquid phase separation (LLPS) and m6A modification, thereby triggering metabolic reprogramming and then triggering copper-specific cell death. Small molecule inhibitors of Cpeb3 identified by high-throughput drug screening can significantly reverse the cuproptosis triggered by Cpeb3 activation. Therefore, the present invention provides a new solution for the prevention and treatment of myocardial ischemia-reperfusion injury and has good application prospects.

[0184] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a small molecule inhibitor of Cpeb3 in the preparation of a drug for preventing and / or treating myocardial ischemia-reperfusion injury, characterized in that: 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; Formula I is: ; Formula II is: .

2. The use according to claim 1, characterized in that: The drug is administered by intravenous injection or oral gavage.

3. The use according to claim 1 or 2, characterized in that: The drug has at least one of the following uses: (1) Inhibit Cu-induced copper death and mitochondrial ROS production; (2) Reversal of Cu-induced changes in aerobic respiration and / or glycolysis; (3) Reversing the changes in glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle metabolites caused 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.

4. The use according to claim 3, characterized in that: The use (1) is to inhibit Cu-induced copper death of Gpc6-positive cardiomyocytes.

5. The use according to claim 1 or 2, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

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