Application of Cpeb3 gene inhibitor and copper death inhibitor in preparation of medicine for treating myocardial ischemia-reperfusion injury

Abstract

The invention discloses application of a Cpeb3 gene inhibitor and a copper death inhibitor in preparation of a medicine for treating myocardial ischemia-reperfusion injury, and relates to the technical field of myocardial ischemia-reperfusion injury. By specifically reducing or knocking out Cpeb3 expression, myocardial repair can be remarkably promoted, action potential stability is improved, and the death rate is reduced. Therefore, Cpeb3 is a specific intervention target spot of copper death in Gpc6 + CMs. The small molecule inhibitor of Cpeb3 can remarkably reverse changes of copper death, aerobic respiration and / or glycolysis caused by Cpeb3 activation. In addition, the copper death inhibitor can improve the survival rate of myocardial cells, reverse the change of aerobic respiration and / or glycolysis induced by Cu, and reverse the change of glycolysis, pentose phosphate pathway and tricarboxylic acid cycle metabolites caused by Cu stimulation. Therefore, the invention provides a new solution for prevention and treatment of myocardial ischemia reperfusion injury, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of myocardial ischemia-reperfusion injury, and in particular to application of a Cpeb3 gene inhibitor and a copper death inhibitor in the preparation of a drug for treating myocardial ischemia-reperfusion injury. Background Art

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

[0003] During MIRI, the metabolic pattern of cardiomyocytes changes from fatty acid metabolism to glucose metabolism dominated by glycolysis. ATP generated by glycolysis not only maintains cell activity, but also supports the function of Na⁺ / K⁺-ATPase and Ca²⁺-ATPase pumps, which is essential for maintaining myocardial potential gradient and action potential propagation. Hexokinase 2 (HK2) is a key enzyme in the glycolysis pathway, responsible for catalyzing the phosphorylation of glucose to produce glucose-6-phosphate. It is mainly located in the outer membrane of mitochondria and regulates mitochondrial energy supply. Studies have shown that amino acid metabolism and related proteins play an important role in regulating glycolysis, including proteins such as c-Myc, Sirt1 and Akt, which connect glycolysis with amino acid metabolism by affecting HK2. 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, ATF4-mediated stress response and GCN2 / eIF2α pathway.

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

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

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

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

[0008] The present invention is achieved in that: In the first aspect, the present invention provides a use of a Cpeb3 gene inhibitor and / or a Cpeb3 protein inhibitor 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 Cpeb3 gene level in cells, tissues or organs, and the Cpeb3 protein inhibitor is a substance that can inhibit the Cpeb3 protein level or activity in cells, tissues or organs.

[0009] In a second aspect, the present invention further provides the use of a small molecule inhibitor of Cpeb3 or a copper death inhibitor in the preparation of a drug for preventing and / or treating myocardial ischemia-reperfusion injury, wherein the copper death inhibitor is selected from molybdenum tetrasulfide, UK5099 and Antimycin A; the small molecule inhibitor of Cpeb3 is selected from a compound or a salt thereof represented by formula I and a compound or a salt thereof represented by formula II: Formula I is: , CAS: 152684-55-4, named Com2 in this application; Formula II is: , CAS: 1228690-19-4, this application is named Com1.

[0010] The present invention has the following beneficial effects: The present invention confirms that copper overload exists during myocardial ischemia-reperfusion injury (MIRI) and induces Gpc6-positive cardiomyocytes (Gpc6 +Copper overload promotes high expression of Cpeb3, which interacts with HK2 through liquid-liquid phase separation (LLPS) and m6A modification, regulates metabolic reprogramming and induces copper death.

[0011] In the MIRI model, specific knockout of Cpeb3 in cardiomyocytes can significantly alleviate IRI-induced myocardial injury. The animal model with Cpeb3 knockout showed improved cardiac diastolic and systolic functions, and enhanced deformation capacity of longitudinal myocardial fibers. This indicates that knockout of Cpeb3 can protect the overall and local contractile function of the heart. In addition, Cpeb3 knockout effectively reduced myocardial fibrosis.

[0012] Cpeb3 knockout significantly inhibited the activation of action potential and calcium transients in cardiomyocytes, inhibited the prolongation of cardiomyocyte repolarization, reduced the susceptibility of animal models to ventricular arrhythmias (VA), and increased the threshold of ventricular fibrillation (VF) assessed by burst stimulation in animal models. Since increased VA induction rates are 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 Gpc6 + Specific intervention targets for copper death in CMs.

[0013] The present invention identifies a small molecule inhibitor of Cpeb3 through high-throughput drug screening, and the small molecule inhibitor of Cpeb3 can significantly reverse the changes in copper death, aerobic respiration and / or glycolysis caused by Cpeb3 activation. In addition, copper death inhibitors such as TTM and other substances have the effects of improving the survival rate of cardiomyocytes, reversing the changes in aerobic respiration and / or glycolysis induced by Cu, and reversing the changes in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle metabolites caused by Cu stimulation.

[0014] Therefore, the present invention provides a new solution for the prevention and treatment of myocardial ischemia-reperfusion injury and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 To investigate how copper overload occurs in MIRI and acts on Gpc6 via Cpeb3-mediated metabolic reprogramming +CMs, leading to subsequent copper death experimental results; (A) Peripheral blood was collected from healthy individuals (n=20) and myocardial infarction patients (24 hours; n=40), and copper levels and cTnI were measured. (B) Correlation analysis between copper and cTnI was performed. Single-cell sequencing data from myocardial infarction mouse hearts were analyzed, and clustered cardiomyocytes were displayed using unified manifold approximation and projection (UMAP) maps (snRNA-seq), which were annotated based on spatial localization and gene expression patterns. (C) Quantification of the composition of clustered cardiomyocytes; (D) Violin plots using the specific cardiomyocyte marker Gpc6 were used to display snRNA-seq data in different cardiomyocyte clusters. Gpc6 was isolated by flow cytometry using adult rat primary cardiomyocytes (ARPCs) + CMs, subjected to myocardial infarction (hypoxia and starvation for 1 h): (E) CCK-8 assay was used to detect cell survival after 24 h of treatment with different concentrations of elesclomol-Cu (ratio 1:1, ES_Cu) or disulfiram-Cu (ratio 1:1, DS_Cu); (F) Mitochondrial oxygen consumption rate (OCR) was analyzed by hippocampal model mitochondrial stress test after 24 h of ES_Cu (50 nM) treatment; (G) Extracellular acidification rate (ECAR) was analyzed by hippocampal model mitochondrial stress test; Oligo (oligomycin), FCCP (carbon cyano trifluoromethoxyphenylhydrazine), Rot (rot), Ant (antimycin A), 2-DG (2-deoxyglucose), Rot+Ant represents a mixture of equal concentrations of Rot and Ant. (H) Volcano plot shows Gpc6 24 h after myocardial infarction + Differentially expressed genes (DEGs) between CMs and Gpc6-CMs, threshold for differentially expressed genes: padj < 0.05, Gpc6 isolated from ARPCs + CMs were knocked down (shCpeb3) or overexpressed (HK2-OE) Cpeb3 and subjected to myocardial infarction (hypoxia and starvation for 1 h), stimulated with ES_Cu (50 nM) for 24 h in the absence or presence of TTM (10 μM; molybdenum tetrasulfide, a copper death inhibitor), (I) cell survival was measured by CCK-8 assay; (J) OCR was analyzed by hippocampal model mitochondrial stress test; (K) ECAR was analyzed. (L) The proposed model summarizes the role of copper-Cpeb3 signaling in Gpc6 +Role of copper death associated with metabolic reprogramming of CMs. (M) Single-cell sequencing data of human hearts at different ages were analyzed to quantitatively cluster the composition of cardiomyocytes. Clusters 1 and 3 were marked by high expression of Gpc6. Data are presented as mean ± standard deviation. A was determined by two-tailed t-test, B was calculated by Pearson correlation, and I was calculated by one-way ANOVA. **p<0.01, ****p<0.001, ns indicates no statistical difference; Figure 2 Cpeb3 functions as a key regulator in the MIRI mouse model. Conditional knockout mice (cardiac-specific Cpeb3 gene-deficient mice, C57BL / 6J-Cpeb3em1flox) were used, with n=10 per group. 24 hours after mice were subjected to MIRI treatment, (A) plasma cTnI levels were measured, (B) reperfusion injury, AAR (area at risk), IF (infarct size) were detected by Evans blue and TTC (2,3,5-triphenyltetrazolium chloride) staining. After 4 weeks, echocardiography was performed, including Doppler blood flow, M-mode ultrasound, and two-dimensional speckle tracking, and (C) E / A (early peak / atrial peak) ratio, (D) LVFS (left ventricular ejection fraction), (E) PLAXL (left ventricular parathoracic long axis view) strain; (F) fibrosis area was tested by Masson staining; (G) Action potentials were detected by optical mapping, and (H) the duration of Ca2+ transient changes was detected. Electrocardiograms were performed to assess ventricular electrophysiological stability and arrhythmia (VA) susceptibility, and (I) VA inducibility and (J) VF threshold were measured. (K) Kaplan-Meier survival curves show the survival rate of mice throughout the whole process. Data are expressed as mean ± standard deviation. P values ​​were calculated by one-way analysis of variance (ANOVA), *p<0.05, **p<0.01, ***p<0.005, ****p<0.001, ns indicates no statistical difference. Survival rate was analyzed by log-rank (Mantel-Cox) test; Figure 3 Com2 is an inhibitor of Cpeb3. (A) Flow chart of screening small molecule Cpeb3 inhibitors. IF (1 hour of hypoxia and starvation treatment), Gpc6 isolated from adult rats + CMs (cardiomyocytes) and CMFs (cardiac fibroblasts). Cell viability was detected by CCK-8 and SPR (surface plasmon resonance) analysis. (B) Chemical structure of Com2. (C) Com2 binding to rat Cpeb3 recombinant protein detected by SPR assay. Gpc6 isolated from ARPCs +CMs, Cpeb3 was knocked down (shCpeb3) and subjected to myocardial infarction (hypoxia and starvation for 1 h), stimulated with ES_Cu (50 nM) for 24 h, treated in the presence or absence of Com2 (5 μM), (D) cell survival was detected by CCK-8, (E) OCR (oxygen consumption rate) was analyzed by Seahorse mitochondrial stress test, (F) ECAR (extracellular acidification rate) was analyzed. Data are expressed as mean ± SD. P values ​​were calculated by one-way analysis of variance (ANOVA), ****p<0.001, ns indicates no statistical difference; Figure 4 To study the interaction between Cpeb3 and HK2 through LLPS and m6A modification. (A) Immunoprecipitation-Western blotting (IP-western blotting) was used to detect the interaction between Cpeb3 and HK2 in rat Gpc6 + Binding in CMs stimulated by hypoxia and starvation for 1 h in the presence or absence of ES_Cu (50 nM) treatment. (B) Gpc6 in rats with Cpeb3 knockdown (shCpeb3) or HK2 overexpression (HK2-OE) + CMs, subjected to myocardial infarction (hypoxia and starvation for 1 h), were stimulated with ES_Cu (50 nM) for 24 h, and the fluorescence redistribution of the photobleached regions of HK2-mCherry aggregates was quantitatively measured in the presence or absence of Com2 (5 μM) (n = 10). (C) The affinity of recombinant rat Cpeb3 and HK2 was detected by SPR assay. Rat Gpc6 + CMs were subjected to myocardial infarction (hypoxia and starvation for 1 h) and stimulated with ES_Cu (50 nM) for 24 h in the presence or absence of Com2 (5 μM). (D) The binding of HK2 mRNA to Cpeb3 was measured by RIP assay, and (E) the mRNA expression of HK2 mRNA was detected by MeRIP qPCR assay. 6 A modification, (F) Whether Cpeb3 was knocked down, the binding effect of HK2 mRNA on ALKBH5 / YTHDC1 was tested by RNA pull-down assay. (G) Com2 (5 μM) or not was further added, and new RNA synthesis was blocked with actinomycin D (5 μg / mL), and the stability of HK2 mRNA was tested, which was measured by qRT-PCR fold change after 0 hours. (H) The proposed model summarizes the Cpeb3-HK2 signaling pathway in Gpc6 +Role of cuproptosis in CMs associated with metabolic reprogramming. Data are presented as mean ± SD. P values ​​were calculated by one-way analysis of variance (ANOVA), *p < 0.05, ****p < 0.001, ns indicates no statistical difference; Figure 5 To establish myocardial ischemia-reperfusion injury (MIRI) models in Bama miniature pigs and rats, we investigated the expression of copper ions and Gpc6 in myocardial cells and myocardial tissues. + Results of myocardial cell ratio detection; Myocardial ischemia-reperfusion injury (MIRI) model was established in (C) Bama miniature pigs and (F) rats by ligating the left anterior descending artery, and ST segment elevation in the electrocardiogram confirmed the successful establishment of the model; (A) Detection of copper ion content in myocardial tissue of Bama miniature pigs at different time points after MIRI. (B) Analysis of single-cell sequencing data from mouse hearts after myocardial infarction3 to quantify the composition of clustered cardiomyocytes. (D, E) Primary cardiomyocytes of adult Bama miniature pigs and (G, H) rats were isolated at different time points after MIRI, and Gpc6 was determined by (E, H) flow cytometry. + Cardiomyocytes (Gpc6 + The proportion of CMs was analyzed (D, G). Data are expressed as mean ± SD. P values ​​were calculated by one-way ANOVA, *p<0.05, **p<0.01, ***p<0.005, ****p<0.001, ns indicates no statistical difference; Figure 6 Gpc6 isolated from adult rat primary cardiomyocytes (ARPCs) + Cardiomyocytes (Gpc6 + CMs) were treated with infarction (IF, hypoxia and starvation for 1 hour), and then stimulated with ES_Cu (50 nM) for 24 hours, or stimulated with TTM (10 μM), Z-VAD-FMK (20 μM), Nec-1 (20 μM) or Fer-1 (10 μM) for 24 hours for cell survival test results; (A) CCK-8 method was used to detect cell survival; (B, C) PI (propidium iodide) staining combined with flow cytometry was used to detect cell death, and (C) statistical analysis was performed. Gpc6 isolated from ARPCs +CMs were treated with infarction (hypoxia and starvation for 1 h), (D) stimulated with different concentrations of Elesclomol-Cu (ratio = 1:1, ES_Cu) or Disulfiram-Cu (ratio = 1:1, DS_Cu) for 24 h, and the cell survival rate was measured by CCK-8 method; (E) stimulated with ES_Cu (50 nM) for 24 h, and the cell survival rate was measured by CCK-8 method in the presence or absence of TTM (10 μM), Z-VAD-FMK (20 μM), Nec-1 (20 μM), or Fer-1 (10 μM). Gpc6 isolated from ARPCs by flow cytometry + CMs were treated with infarction (hypoxia and starvation for 1 hour) and then subjected to hippocampal mitochondrial stress test with or without ES_Cu (50 nM) stimulation for 24 hours: (F) mitochondrial oxygen consumption rate (OCR) was detected, and spare respiratory capacity, basal respiration, proton leak and ATP production capacity were calculated; (G) extracellular acidification rate (ECAR) was detected, and glycolytic capacity, glycolytic level and glycolytic reserve capacity were further calculated. Gpc6-CMs isolated from ARPCs were treated with infarction (hypoxia and starvation for 1 hour) and then subjected to hippocampal mitochondrial stress test with or without ES_Cu (50 nM) stimulation for 24 hours: (H) OCR and (I) ECAR were detected, and the relevant indicators were calculated. Data are expressed as mean ± 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; Figure 7 To establish the MIRI model using Bama miniature pigs, samples were collected from myocardial tissues in different regions for protein extraction or paraffin sectioning, followed by (A) Western blot and (B) immunofluorescence assays to detect the expression levels of Cpeb3 and HK2, scale bar = 50 μm. (C) Gpc6 isolated from adult rat primary cardiomyocytes (ARPCs) + CMs and Gpc6-CMs were stimulated with ES_Cu (50 nM) for 24 h or not after infarction treatment (IF, hypoxia and starvation for 1 h) or not, and the expression levels of Cpeb3 and HK2 were detected by Western blotting. +CMs were stimulated with or without ES_Cu (50 nM) for 24 h under conditions of (D) Cpeb3 knockdown (shCpeb3) or (E) HK2 knockdown (shHK2), and Western blot was performed to evaluate the expression levels of (D) HK2 and (E) Cpeb3. Gpc6 was extracted based on published single-cell sequencing data after myocardial infarction in mice. + The whole genome expression data of high-expressed Cpeb3 and low-expressed HK2 in CMs were subjected to KEGG pathway analysis to show the relevant pathways of (F) high expression of Cpeb3 and (G) low expression of HK2. Gene set enrichment analysis (GSEA) showed that: (H) HK2 was negatively correlated with copper death and positively correlated with glycolysis; (I) In contrast, Cpeb3 was positively correlated with copper death and negatively correlated with glycolysis; Figure 8 Gpc6 isolated from adult rat primary cardiomyocytes (ARPCs) + CMs knocked down Cpeb3 (shCpeb3) or overexpressed HK2 (HK2-OE), then underwent infarction (IF, 1 h of hypoxia and starvation), and were stimulated with or without ES_Cu (50 nM) for 24 h in the presence or absence of TTM (10 μM). (A, B) Cell death rate was detected by PI (propidium iodide) staining combined with flow cytometry, (B) and statistical analysis was performed; (C) Mitochondrial ROS levels were detected by flow cytometry. Seahorse mitochondrial stress test was performed to detect (D) mitochondrial oxygen consumption rate (OCR), calculate spare respiratory capacity, basal respiration, proton leak, and ATP production; (E) extracellular acidification rate (ECAR) was detected, and glycolytic capacity, glycolytic level, and glycolytic reserve were further calculated; the effects of shCpeb3, HK2-OE, or TTM on Gpc6 after infarction and ES_Cu stimulation were analyzed. + Effect of glucose-derived metabolites and percentage of total metabolites in CMs. Cells were incubated with [U-13C]glucose (1.5 mM) for 24 h, and metabolites related to (F) glucose-6-phosphate (Glc-6P), (G) glycolysis, (H) pentose phosphate pathway (PPP), and (I) tricarboxylic acid cycle (TCA) were detected. Data are presented as mean ± 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; Fig. 9To analyze single-cell sequencing data (snRNA-seq) from human hearts, 12-14 images were drawn using the unified manifold approximation and projection (UMAP) method to show: (A) all cells and (B) clustered cardiomyocytes, with clusters annotated according to gene expression patterns. Data are from representative samples of different age groups (young group, age ≤ 45; middle-aged group, 45 < age < 60; elderly group, age ≥ 60). (C) In different clusters of cardiomyocytes, violin plots are used to show the expression of Gpc6, a marker for specific cardiomyocyte subsets, in snRNA-seq data; Fig.10 Conditional knockout mice with cardiomyocyte-specific knockout of Cpeb3 (C57BL / 6J-Cpeb3em1flox purchased from Guangzhou Saiye) were used, with n=10 per group. Mice underwent myocardial ischemia-reperfusion injury (MIRI), and 24 hours later: (A) plasma creatine kinase-MB (CK-MB) levels were measured, (B) reperfusion injury was assessed 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 performed to detect: (F, left) Doppler blood 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 fibrosis area. Optical mapping was used to detect: (H, upper) action potential and (H, lower) calcium transient duration, and statistical analysis was performed for (I) APD90 (action potential duration at 90% repolarization) and (J) CTD90 (calcium transient duration at 90% recovery). Data are presented as mean ± SD. P values ​​were calculated by one-way ANOVA, *p<0.05, ***p<0.005, ****p<0.001, ns indicates no statistical difference; Fig.11 (A) Chemical structure of Com1; (B) SPR determination of the binding of Com1 to rat Cpeb3 recombinant protein. Gpc6 isolated from ARPCs +CMs were subjected to ischemia-reperfusion injury (hypoxia and starvation for 1 h) and stimulated with ES_Cu (50 nM) for 24 h in the absence or presence of Com1 (5 μM) or Com2 (5 μM): (C) Cell survival was detected by CCK-8 assay, (D) Cell death was detected by PI (propidium iodide) staining flow cytometry, (E) and statistical analysis was performed, (F) Mitochondrial ROS levels were assessed by flow cytometry. The Seahorse Mitochondrial Stress Test was used to detect: (G) Extracellular acidification rate (ECAR), which was further calculated for glycolytic capacity, glycolysis, and glycolytic reserve, and (H) Mitochondrial oxygen consumption rate (OCR), which was used to calculate spare respiratory capacity, basal respiration, proton leak, and ATP production. Data are expressed as mean ± 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; Fig.12 Schematic diagram of the docking interaction between (A) the complex Com2 and rat Cpeb3 protein. (B) SPR was used to measure the binding effect of Com2 with wild-type or specific amino acid mutant rat Cpeb3 recombinant protein. The binding of Com2 with (C) human, (D) pig and (E) mouse Cpeb3 recombinant protein was measured by SPR. Gpc6 isolated from ARPCs + CMs were subjected to ischemia-reperfusion injury (hypoxia and starvation for 1 h) and stimulated with ES_Cu (50 nM) for 24 h in the absence or presence of Com2 (5 μM) or Cpeb3 knockdown (shCpeb3): (F) Cell death was detected by PI (propidium iodide) staining flow cytometry, (G) and statistical analysis was performed, (H) Mitochondrial ROS levels were assessed 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 the relevant index was calculated. Data are expressed as mean ± SD. P values ​​were calculated by one-way ANOVA, *p<0.05, **p<0.01, ***p<0.005, ****p<0.001, ns indicates no statistical difference; Fig.13 (A) Gpc6 isolated from ARPCs +CMs were subjected to ischemia (hypoxia and starvation for 1 h) after Cpeb3 knockdown (shCpeb3) and then stimulated with ES_Cu (50 nM) for 24 h in the absence or presence of Com2 (5 μM). + Effects of glucose-derived metabolites and percentage of total metabolites in CMs. After ischemia and ES_Cu stimulation, cells were cultured with [U-13C]glucose (1.5 mM) for 24 h, and the following metabolites were detected: (A) Glc-6P, (B) glycolysis, (C) tricarboxylic acid cycle (TCA), (D) pentose phosphate pathway (PPP)-related metabolites. Data are expressed as mean ± SD. P values ​​were calculated by one-way ANOVA, *p<0.05, **p<0.01, ***p<0.005, ****p<0.001, ns indicates no statistical difference; Fig.14 (A) Simulation of the binding of Cpeb3 and HK2 across species. (B) Gpc6 isolated from ARPCs + CMs, after Cpeb3 knockdown (shCpeb3), were subjected to ischemia (hypoxia and starvation for 1 h) and then stimulated with ES_Cu (50 nM) for 24 h in the absence or presence of Com2 (5 μM). The expression of Cpeb3 and HK2, as well as their colocalization with mitochondria and nuclei, were detected by immunofluorescence. Nuclei were stained with Hoechst 33342, scale bar = 20 μm. Gpc6 isolated from ARPCs + CMs, subjected to ischemia (hypoxia and starvation for 1 h) and then stimulated with ES_Cu (50 nM) for 24 h, (C) Time-lapse images of Cpeb3-GFP (left) and HK2-mcherry (right), (D) FRAP analysis of Cpeb3-GFP (top) and HK2-mcherry (bottom) aggregates, (E) FRAP quantitative analysis of bleached areas in Cpeb3-GFP and HK2-mcherry aggregates; Fig.15 (A) Representative fluorescence images of HK2-mcherry droplets in the presence or absence of Cpeb3 or Com2, scale bar = 10 μm. The affinity of recombinant rat mutant Cpeb3 to HK2 was determined by (B) SPR assay and (C) fluorescence polarization (FP) assay. (D) Gpc6 isolated from ARPCs+ CMs, after Cpeb3 knockdown (shCpeb3) or overexpression (Cpeb3-OE), were subjected to ischemia (hypoxia and starvation for 1 h) and then stimulated with ES_Cu (50 nM) for 24 h in the absence or presence of Com2 (5 μM). FRAP analysis of HK2-mcherry aggregates was performed. Data are presented as mean ± standard deviation (SD). P values ​​were calculated by one-way ANOVA, ***p<0.005, ns indicates no statistical difference; Fig.16 (A) Gpc6 isolated from ARPCs + CMs were subjected to ischemia (hypoxia and starvation for 1 h) or not, and then stimulated in the absence or presence of ES_Cu (50 nM) for 24 h, and the expression levels of cytoplasmic Cpeb3 (CytCpeb3) and nuclear Cpeb3 (NucCpeb3) were detected by Western blotting. (B) (a) HK2 mRNA sequence information from different species, including the start position of 5'-UTR, CDS, and 3'-UTR; (b) CPE sequence information in the 3'-UTR of HK2 mRNA from mouse, (c) rat, (d) pig, and (e) human; and the m6A site predicted by SRAMP (http: / / www.cuilab.cn / sramp) and its corresponding secondary structure. (C) Rat Gpc6 + CMs were subjected to ischemia (hypoxia and starvation for 1 h) and then stimulated with ES_Cu (50 nM) for 24 h, and the binding of HK2 mRNA and Cpeb3 was measured using RIP assay. + In CMs, (D) the binding effect of wild-type and mutant HK2 mRNA 3'-UTR with Cpeb3 protein was evaluated by luciferase reporter gene assay; (E) HK2 mRNA binding region and (F) sequence binding with Cpeb3 protein were detected by RNA pull-down assay. Data are expressed as mean ± standard deviation (SD). P value was calculated by one-way ANOVA, ***p<0.005, ****p<0.001, ns indicates no statistical difference; Fig.17 Rat Gpc6 +After CMs were subjected to ischemia (hypoxia and starvation for 1 h), (B, C, D) Cpeb3 gene knockout (shCpeb3) or (A) non-knockout were performed, followed by stimulation with ES_Cu (50 nM) for 24 h, and the m6A RNA modification of HK2 mRNA was detected by MeRIP assay; proteins bound to Cpeb3 were collected by immunoprecipitation with Cpeb3-coated magnetic beads, and the most significantly changed proteins were evaluated by mass spectrometry (MS), and statistical analysis was performed on (C) ALKBH5 and (D) YTHDC1, two proteins associated with m6A modification, which showed the most significant changes in abundance changes. (E) Rat Gpc6 + CMs were subjected to ischemia (hypoxia and starvation for 1 h) and subjected to Cpeb3 knockout (shCpeb3) or YTHDC1 overexpression (YTHDC1-OE) and stimulated with ES_Cu (50 nM) for 24 h, and the nuclear and cytoplasmic fractions of HK2 mRNA were detected by qRT-PCR. (F) Rat Gpc6 + CMs were subjected to ischemia (hypoxia and starvation for 1 h) and stimulated with ES_Cu (50 nM) for 24 h. Cpeb3, YTHDC1, and colocalization of YTHDC1 in the nucleus were detected by confocal microscopy in the absence or presence of Com2 (5 μM). Barcode = 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 DESCRIPTION

[0017] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0018] In a first aspect, the present invention provides a use of a Cpeb3 gene inhibitor and / or a Cpeb3 protein inhibitor in the preparation of a drug for preventing and / or treating myocardial ischemia-reperfusion injury, wherein the Cpeb3 gene inhibitor is a substance that can inhibit the Cpeb3 gene level in cells, tissues or organs, and the Cpeb3 protein inhibitor is a substance that can inhibit the Cpeb3 protein level or activity in cells, tissues or organs. The cells are selected from myocardial cells, and the organ or tissue is the heart.

[0019] The inventors have found that by inhibiting the expression of the Cpeb3 gene, knocking down or knocking out the level of Cpeb3 protein in cells, tissues or organs, myocardial damage induced by ischemia / reperfusion injury (IRI) can be significantly alleviated. The manifestation is that the diastolic and systolic functions of the heart in the animal model with Cpeb3 knockout are improved, and the deformability of longitudinal myocardial fibers is enhanced. It shows that knocking out Cpeb3 can protect the overall and local contractile function of the heart. In addition, Cpeb3 knockout effectively reduces myocardial fibrosis. Cpeb3 knockout significantly inhibits the activation of action potential and calcium transients of cardiomyocytes, inhibits the prolongation of cardiomyocyte repolarization, reduces the susceptibility of animal models to ventricular arrhythmias (VA); and increases the threshold of ventricular fibrillation (VF) assessed by burst stimulation in animal models. Since an increased VA induction rate is associated with a higher mortality rate, specifically reducing or knocking out Cpeb3 expression can significantly promote myocardial repair, improve action potential stability, and reduce mortality. Therefore, Cpeb3 is Gpc6 + Specific intervention targets for copper death in cardiomyocytes (CMs). Myocardial ischemia-reperfusion injury can be prevented and / or treated by inhibiting the levels of Cpeb3 gene and / or Cpeb3 protein.

[0020] In a preferred embodiment of the present invention, the cardiomyocytes are Gpc6 positive cardiomyocytes. The inventors have found that in specific Gpc6 + Copper death occurs in cardiomyocytes (CMs).

[0021] In a preferred embodiment 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 encoding an RNA molecule, and the RNA molecule is selected from siRNA, sgRNA, snoRNA, shRNA or microRNA.

[0022] Recombinant vectors include but are not limited to: recombinant lentiviral vectors, recombinant adenoviral vectors, recombinant adeno-associated viral vectors, and the like.

[0023] In addition to the coding sequence of the above-mentioned RNA molecule, the recombinant vector also includes a regulatory sequence and a coding sequence of the Cas protein. The coding sequence of the RNA molecule and the coding sequence of the Cas nuclease or Cas nickase are operably connected to the regulatory sequence.

[0024] In some embodiments, the Cas nuclease is a Class 2 Cas nuclease. In some embodiments, the Cas nuclease is Cas9, Cpf1, C2cl, C2c2, and C2c3, or a modified protein thereof. In some embodiments, the Cas nuclease is a 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.

[0025] In a preferred embodiment of the present invention, the regulatory sequence includes: a promoter and a terminator corresponding to the promoter, such as a type II promoter coordinated with a terminator capable of terminating the type II promoter, and a type III promoter coordinated with a terminator capable of terminating the type III promoter.

[0026] In a preferred embodiment of the present invention, the promoter is selected from a constitutive promoter, an inducible promoter, a ubiquitous promoter, a tissue-specific promoter, a cell type-specific promoter, or a developmental stage-specific promoter.

[0027] In a preferred embodiment of the present invention, the promoter is a type II promoter or a type III promoter; in a preferred embodiment of the present invention, the type II promoter is selected from CMV promoter, CAG promoter, PGK promoter, and EF1α promoter.

[0028] In a preferred embodiment of the present invention, the terminator is selected from SV40 polyA, bGH polyA, BETAPolyA or 6XT.

[0029] In a preferred embodiment of the present invention, the recombinant vector further comprises a selection marker gene or a reporter gene.

[0030] The term "selectable marker" refers to a gene that will facilitate selection of cells that actively express a nucleic acid sequence. Examples of suitable selectable markers include enzymes encoding resistance to antibiotics (i.e., antibiotic resistance genes), such as kanamycin, neomycin, puromycin, hygromycin, blasticidin, or bleomycin.

[0031] In addition, the selectable marker gene also comprises an amplifiable selectable marker gene. Mentioning " amplifiable selectable marker gene " as used herein refers to the gene that allows to increase this gene under suitable growth conditions. The amplifiable selectable marker gene can respond to the shortage of inhibitor or essential metabolite by amplification to increase the expression product (that is, by the expression of the protein encoded by the amplifiable selectable marker gene). In one embodiment, the amplifiable selectable marker gene can be characterized as being able to complement with an auxotrophic host.

[0032] In a preferred embodiment of the present invention, the drug has at least one of the following uses: (1) Alleviate myocardial damage induced by IRI (ischemia / perfusion reinjury); (2) Improve the overall or local diastolic and systolic function of the subject's heart; (3) Enhance the deformation capacity of longitudinal myocardial fibers; (4) Reduced myocardial fibrosis; (5) Inhibit the activation of action potential and calcium transient of cardiomyocytes, and inhibit the prolongation of cardiomyocyte repolarization; (6) Reduce the susceptibility of subjects to ventricular arrhythmias (VA); (7) Improve the subject's threshold for burst stimulation assessment of ventricular fibrillation (VF).

[0033] In a preferred embodiment of the present invention, the drug further comprises a pharmaceutically acceptable carrier, including but not limited to a filler, a lubricant, a disintegrant, a binder, a glidant, and the like.

[0034] In the preferred technical scheme of the present invention, the pharmaceutically acceptable carrier includes but is not limited to polyvinyl pyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methyl cellulose and its derivatives, ethyl cellulose and its derivatives, hydroxypropyl cellulose and its derivatives, starch and its derivatives, polyethylene glycol and its derivatives, lactose, sucrose, mannitol, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, calcium hydrogen phosphate, calcium stearate, sodium stearyl fumarate, silicon dioxide, titanium dioxide, talc, indigo or a combination thereof.

[0035] In a second aspect, the present invention also provides the use of a copper death inhibitor in the preparation of a drug for preventing and / or treating myocardial ischemia-reperfusion injury, wherein the copper death inhibitor is selected from molybdenum tetrasulfide (TTM), UK5099, Antimycin A, a compound shown in formula I or a salt thereof, and a compound shown in formula II or a salt thereof.

[0036] Formula I is: , CAS: 152684-55-4, named Com2 in this application; Formula II is: , CAS: 1228690-19-4, this application is named Com1. UK5099 CAS NO. 56396-35-1.

[0037] The present invention utilizes the unique advantages of small molecule compounds and identifies two potential Cpeb3 small molecule inhibitors, Com1 and Com2, through high-throughput drug screening. Com2 has a stronger binding affinity to Cpeb3 (KD=3.29μM) and exhibits robust binding ability to Cpeb3 protein in multiple species (such as humans, pigs and mice). Com2 binds to the molecular pocket of Cpeb3 through hydrogen bonds and π-π conjugation, which is composed of TYR-7, ARG-1, LYS-82, THR-83, SER-85 and ASP-86, among which hydrogen bonds and π-π interactions are medium-weak non-covalent interactions. In Gpc6 + In CMs, Com2 significantly inhibited ES_Cu (Elesclomol-Cu (ratio = 1:1))-induced copper death and mitochondrial ROS production, and its effect was better than Com1 and comparable to that of Cpeb3 knockdown. Compared with Com1, Com2 more effectively reversed the changes in aerobic respiration and glycolysis induced by ES_Cu, and its effects were similar to those of Cpeb3 knockdown. In addition, Com2 treatment significantly reversed the changes in glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle metabolites induced by ES_Cu stimulation, with no significant difference from the effect of Cpeb3 knockdown. These results indicate that Com2 is an excellent small molecule inhibitor of Cpeb3, which can effectively counteract the Gpc6-positive cardiomyocytes (Gpc6 + Aerobic respiration and glycolysis in CMs were altered, thereby regulating metabolic reprogramming and inhibiting the occurrence of copper death.

[0038] In a preferred embodiment of the present invention, the drug is administered by intravenous injection or oral administration.

[0039] In a preferred embodiment of the present invention, 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.

[0040] In a preferred embodiment of the present invention, use (1) refers to: inhibiting Cu-induced copper death of Gpc6-positive cardiomyocytes.

[0041] In a preferred embodiment of the present invention, the drug further comprises a pharmaceutically acceptable carrier.

[0042] The drug also includes pharmaceutically acceptable carriers, including but not limited to fillers, lubricants, disintegrants, binders, glidants, etc.

[0043] In the preferred technical scheme of the present invention, the pharmaceutically acceptable carrier includes but is not limited to polyvinyl pyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methyl cellulose and its derivatives, ethyl cellulose and its derivatives, hydroxypropyl cellulose and its derivatives, starch and its derivatives, polyethylene glycol and its derivatives, lactose, sucrose, mannitol, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, calcium hydrogen phosphate, calcium stearate, sodium stearyl fumarate, silicon dioxide, titanium dioxide, talc, indigo or a combination thereof.

[0044] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0045] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0046] Experimental Materials and Methods (A) Patient information and blood samples.

[0047] This study enrolled patients with acute ST-segment elevation myocardial infarction (STEMI) who underwent percutaneous coronary intervention (PCI) in Sichuan Provincial People's Hospital from January 2022 to December 2023 (n=40). All patients underwent corresponding laboratory tests. Inclusion criteria: (1) clinical diagnosis of acute STEMI; (2) PCI within 12 hours of symptom onset; (3) patients and their families signed the relevant consent forms for inclusion. Exclusion criteria: (1) patients with advanced malignant tumors; (2) patients with multiple organ dysfunction; (3) patients with severe coagulation dysfunction; (4) patients with poor treatment compliance. Peripheral blood was collected 24 hours after PCI to detect copper ion content. Healthy subjects who underwent physical examination in Sichuan Provincial People's Hospital during the same period were selected as the control group (n=20), and peripheral blood was collected for copper ion detection. All enrolled subjects signed informed consent forms, and this study was approved by the Ethics Committee of Sichuan Provincial People's Hospital.

[0048] (B) Plasma and tissue copper detection.

[0049] Plasma samples were collected with heparin as an anticoagulant, centrifuged at 1000 g for 5 min at 4°C, and the supernatant was placed on ice for measurement. Copper ion levels in peripheral blood were measured using a serum copper content assay kit (#BC5645, Solarbio, Beijing, China) according to the manufacturer's instructions. Approximately 0.1 g of heart tissue was measured and 1 mL of distilled water was added. The mixture was then homogenized on ice. The homogenate was then centrifuged at 10,000 g for 10 min at 4°C, and the resulting supernatant was collected. Copper content was measured according to the instructions of the tissue copper content assay kit (#BC5565, Solarbio, Beijing, China).

[0050] (iii) MIRI model.

[0051] The MIRI model was established according to a previous report. Spf mice (8 weeks), SD rats (180–230 g), and Bama miniature pigs (15–20 kg) were obtained from Chengdu Dashuo Experimental Animal Co., Ltd. (Chengdu, China), and all animal procedures were approved by the Animal Care and Use Committee of Sichuan Provincial People’s Hospital. SD rats or mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg), intubated, and connected to a small animal ventilator to ensure breathing. The degree of myocardial ischemia was evaluated by electrocardiographic monitoring. The heart was exposed by thoracotomy at the fourth intercostal space on the left side, and the left anterior descending coronary artery (LCA) was occluded with 8-0 (mice) or 6-0 (rat) silk sutures, and a slipknot was tied 2–3 mm distal to the origin of the LCA. T wave reversal and ST-segment elevation were observed on the electrocardiogram, indicating ischemia, and the slipknot was released 45 minutes later to allow reperfusion of the affected area. For Bama miniature pigs, fasting for 12 h before surgery was performed to minimize the risk of aspiration. Anesthesia was initiated with isoflurane and maintained with 1–2% isoflurane via endotracheal intubation. Vital signs, including heart rate, respiratory rate, and oxygen saturation, were continuously monitored using an electrocardiogram. The left chest was shaved and disinfected with povidone-iodine solution. A midline thoracotomy was performed to expose the heart, and the LCA coronary artery (second diagonal branch) was preoccluded for 45 minutes before ligation. The thorax was closed in layers using absorbable sutures. Antibiotics (cefazolin, 25 mg / kg) and analgesics (lidocaine, 2 mg / kg) were administered postoperatively and closely monitored during recovery. Myocardial infarction was confirmed by electrocardiogram immediately postoperatively. At the conclusion of the study, the animals were humanely euthanized with an overdose of pentobarbital, and tissues were removed from major organs.

[0052] (IV) Bioinformatics analysis.

[0053] All bioinformatics analyses were performed using the CeleLens Cloud platform provided by Singleron (https: / / www.celelenscloud.cn / # / my_analyzes). Sc / snRNA-seq data of mouse heart after myocardial infarction were obtained from the Gene Expression Omnibus (GSE214611), and Sc / snRNA-seq data of human heart were obtained from the Human Cell Atlas Data Coordination Platform (ERP123138), the European Genome-phenome Archive (EGAS00001006374), and cellxgene (https: / / cellxgene.cziscience.com / collections / 8191c283-0816-424b-9b61-c3e1d6258a77). The Seurat package was used to classify and annotate the data, identify different cell types, and extract marker genes for each subpopulation. Subsequently, DESeq2 was used to perform differential expression analysis between the two groups of cells, and a volcano plot was generated to visualize significantly differentially expressed genes. For the expression levels of CPEB3 and HK2, KEGG analysis was performed to identify their related biological pathways. In addition, GSEA was used to analyze the copper death pathway and glycolysis pathway to evaluate the enrichment of related gene sets. All analyses were performed in the R environment to ensure the reliability of data processing and the reproducibility of results, thereby revealing the potential functions and mechanisms of different cell subpopulations in copper death and metabolic regulation.

[0054] (V) Cell activity and death.

[0055] To simulate the injury of MIRI to cardiomyocytes in vitro, adult rat primary cardiomyocytes (ARPCs) were used to separate Gpc6+CMs / Gpc6-CMs by flow cytometry, and all cells were subjected to ischemia (hypoxia and starvation for 1 h) to simulate MIRI and then treated with different drugs. CCK8 detection was performed according to the protocol provided by cell Counting Kit-8 (#C0037, Beyotime, Beijing, China) to evaluate cell death. In addition, propidium iodide (#C1062S, Beyotime, Beijing, China) staining was used to identify cells with impaired membrane integrity and analyzed using flow cytometry.

[0056] (vi) Flow cytometry.

[0057] Single cell suspension was obtained from myocardial tissue by enzyme digestion (#abs9482, Absin, Shanghai, China) and washed with PBS buffer to remove residual enzymes. After the cell suspension was adjusted to the appropriate concentration, anti-cTnI antibody (rat: #MG270057, Abmart, Shanghai, China; pig: #ab231064, Abcam, Boston, MA, USA) and Gpc6 (mouse: #PC22025, Abmart, Shanghai, China; pig: Abconal custom antibody) were added and mixed gently. The mixture was incubated at 4°C for 1 hour to ensure that the antibody was fully bound to the target cells. The cells were then washed three times with PBS to remove unbound primary antibody. Fluorescently labeled secondary antibodies (FITC: #F-2761, Invitrogen, USA; PE: #12-4739-81, Invitrogen, USA), incubated at 4°C for 30 minutes to ensure that the secondary antibody specifically binds to the primary antibody. Wash the cells three times with PBS to remove unbound secondary antibody. Subsequently, the cells were analyzed by flow cytometry. In the flow cytometer, the fluorescent dye was excited with a laser, and forward scatter and side scatter data were collected to determine the size and complexity of the cells. The expression of cTnI and Gpc6 was detected using specific fluorescence channels. Positive cells and negative cells were distinguished according to the fluorescence intensity, and the number of gpc6-positive cardiomyocytes and the proportion of total cells were finally recorded.

[0058] (vii) Seahorse analysis.

[0059] Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of isolated cardiomyocytes were assessed by Seahorse XF Analyzer (Agilent, Santa Clara, CA) according to the manufacturer’s instructions. Cell-Tak (22 μg / ml, pH 6.5–8, #354241, Corning) was applied to the wells of Seahorse XFe96 spheroid microplates (#102959-100, Agilent) and incubated at 37 °C for 20 min. Afterwards, the wells were neutralized with deionized water, rinsed twice, air-dried, and stored at 4 °C. Isolated cardiomyocytes were then transferred to the wells, placed in culture medium, and incubated overnight at 37 °C to allow attachment. The next day, the buffer was changed to low-phosphate DMEM / F12 pre-warmed to 37 °C in a CO2-free incubator. The sensor chip was calibrated with the provided Seahorse buffer the day before the assay.

[0060] For OCR evaluation, measurements were performed after the addition of oligomycin (4 µmol / L), FCCP (4 µmol / L), and a combination of rotundone (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 by the Seahorse XF Glycolysis Stress Test Kit (#103020-100, Agilent, Santa Clara, CA) under baseline conditions and after the 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 plate was filled with 25 μl of the test compound or solvent diluted in DMEM / F12 buffer. After calibration, the cell plate was analyzed in the XF96 instrument with the measurement protocol set to 3 min mixing followed by 3 min measurement. Results read on the Seahorse XF96 Analyzer were normalized by the total protein level in each well.

[0061] (VIII) Western blot and co-immunoprecipitation (Co-IP).

[0062] Tissues or cells were lysed with RIPA lysis buffer (#R0010, Solarbio, Beijing, China), and protein concentration was determined by BCA protein quantification kit (#PC0020, Solarbio, Beijing, China). Proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with primary antibodies overnight at 4°C and then incubated with secondary antibodies for 1 hour at 37°C. The bands were visualized using the Amersham ImageQuant™ 800 system (Cytiva, Sweden).

[0063] In the Co-IP experiment, using the Pierce™ Classic Magnetic IP / Co-IP Kit (Thermo Scientific™, USA), the target protein is captured using a specific antibody after cell lysis, allowing other interacting proteins to co-precipitate. Subsequently, the antibody-protein complex is immobilized using Protein A / G agarose beads and unbound components are removed. Finally, the bound proteins are eluted and analyzed by Western blot to confirm the interaction between the target protein and other proteins. The antibodies used in the experiments include: 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).

[0064] (ix) Immunofluorescence.

[0065] The cells were fixed with 4% paraformaldehyde for 10 to 20 minutes and washed three times with PBS. Then, 0.2%-0.5% Triton X-100 (prepared in PBS) was added, incubated at room temperature for 5 to 10 minutes, and then washed three times with PBS. Nonspecific binding was blocked with 3% BSA for 30 to 60 minutes. Primary antibodies were added at a dilution of 1:500 and incubated overnight at 4°C. Then, the cells were washed three times with PBS, and fluorescently labeled secondary antibodies were added at a dilution of 1:1000, incubated for 60 minutes in the dark, and washed again with PBS three times. Finally, the nuclei were stained with DAPI for 5 minutes, washed, and sealed with anti-fluorescence attenuation mounting medium for microscopic observation.

[0066] For tissue samples, fresh tissues were first fixed with 4% paraformaldehyde for 24 hours, dehydrated with gradient alcohol, and then paraffin-embedded after being transparentized with xylene. The slices were sliced ​​to a thickness of 5 μm and mounted on glass slides. After baking at 60°C for 30 minutes, the slides were dewaxed with xylene and alcohol, and then washed with distilled water. The slices were placed in citrate buffer, incubated at 95°C for 20 minutes, and washed three times with PBS after cooling. Nonspecific binding was blocked with 3% BSA for 30 minutes. Primary antibodies were added and incubated overnight at 4°C. Subsequently, the sections were washed three times with PBS, fluorescently labeled secondary antibodies were added, incubated for 1 hour under light-proof conditions, and washed again three times. The nuclei were stained with DAPI for 5 minutes, and after washing, the sections were sealed with sealing agents and observed under a microscope.

[0067] The antibodies used in the experiments included: Cpeb3 (#PC11340, Abmart, Shanghai, China), HK2 (#TD6176, #MG680856, Abmart, Shanghai, China), Gpc6, YTHDC1 and ALKBH5 (customized by Abconal), His-Tag (#M30111, Abmart, Shanghai, China) and Flag-Tag (#TT0053, Abmart, Shanghai, China).

[0068] 10. Metabolomics.

[0069] Metabolite detection was analyzed based on methods from previous studies. Two complementary experiments were performed to quantify metabolites in glycolysis, the pentose phosphate pathway (PPP), and the tricarboxylic acid cycle (TCA). The first experiment used ion chromatography-mass spectrometry (IC-MS) to analyze pyruvate and glucose-6P levels in the culture medium; the second experiment used ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) to separate hexose sugars, including glucose.

[0070] Isolated cardiomyocytes were incubated with 10 mM [U-13C]-labeled glucose for 6 h. Metabolites were extracted using 80% methanol and concentrated by Labconco CentriVap. Dried metabolites were resuspended in 50% acetonitrile-water 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% mobile phase A (5 mM NH4AcO, pH 9.9) over 18 min, followed by isocratic elution at 95% A for 9 min.

[0071] Metabolites were detected by a Q Exactive mass spectrometer (Thermo Scientific) in full scan mode with switched polarity (+3.5 kV / −3.5 kV) over a mass-to-charge range of 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). Detection values ​​were normalized to cell counts, and relative amounts of metabolites were calculated by summing all isotopologues of each metabolite and correcting for natural C13 abundance.

[0072] (XI) Mitochondrial reactive oxygen species (ROS) detection.

[0073] Mitochondrial reactive oxygen species were measured using MitoSOX™ Mitochondrial Superoxide Indicator (#M36005, Invitrogen™, USA). First, the cell samples were cultured under appropriate conditions and then the cells were collected. The cells were washed once with PBS to remove the culture medium, and then 1 µl of 5 mM MitoSOX Green was diluted in 0.5 ml PBS to prepare a 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. The cells were then analyzed using a flow cytometer with appropriate parameters set to detect the fluorescence signal of MitoSOX. This analysis allows the quantification of mitochondrial ROS levels in each cell and comparisons between different treatment groups. The data obtained can be used to evaluate mitochondrial function and oxidative stress status in cells.

[0074] (XII) Detection of cTnI and CK-MB.

[0075] Cardiac troponin I (cTnI) levels were determined using specific ELISA kits for different species: porcine TNNI3 ELISA kit (#ELK5656, ELK Biotech, Wuhan, China), rat cTnI ELISA kit (#D731143, Sanger Biotech, Shanghai, China), and mouse cTnI ELISA kit (#D721149, Sanger Biotech, Shanghai, China) according to the manufacturer's instructions. Similarly, creatine kinase-MB (CK-MB) levels were determined using the relevant kits for each species: porcine kit (#CB10105-Pg, Coibo, Shanghai, China), rat kit (#SEKR-0059, Solarbio, Beijing, China), and mouse kit (#SEKM-0152, Solarbio, Beijing, China). After the model was successfully established for each group, 0.2 mL of venous blood was collected to determine the cTnI concentration.

[0076] 13. Evans Blue & TTC staining and Masson staining At 24 hours after myocardial ischemia-reperfusion injury, the degree of myocardial injury was assessed by double staining with Evans Blue (#E8010, Solarbio, Beijing, China) and TTC (diphenyltetrazolium chloride, #T8170, Solarbio, Beijing, China). First, 0.5% Evans Blue solution was perfused for 10 minutes to mark the necrotic myocardium. The heart was then removed, cut into slices of approximately 1 cm thickness, and stained with 1% TTC solution in a 37°C water bath for 30 minutes. Healthy myocardial tissue appeared red after TTC staining, while the ischemic injury area remained pale. After the experiment was completed, the staining results were quantitatively evaluated using image analysis software, and the proportion of infarct area to total myocardial area was calculated to objectively evaluate the degree of ischemia-reperfusion injury.

[0077] Masson staining (#G1346, Solarbio, Beijing, China) was used to assess the degree of myocardial fibrosis 4 weeks after myocardial ischemia-reperfusion injury. The rat hearts were quickly rinsed with saline and fixed in 10% neutral buffered formaldehyde for 24 h. After fixation, the hearts were cut into 5-µm-thick sections, dehydrated and transparentized. Masson staining was then performed. The specific steps included staining with Weigert iron solution (containing 1% potassium ferrocyanide and 1% ferric chloride) at room temperature for 10 minutes, followed by rinsing with distilled water to stain the fiber background blue. Next, acid fuchsin solution was added for staining for 2 minutes, and the excess dye was washed off. Finally, the sections were rinsed with 1% acetic acid and sealed with resin. After staining, the degree of myocardial fibrosis was observed under a microscope, and the distribution and proportion of blue collagen fibers in myocardial tissue were analyzed.

[0078] 14. Echocardiography Echocardiography of the hearts of rats, mice, and pigs was performed using FUJIFILM Vevo 3100LT and Finno Ultrasound VINNO ULTIMUS 9E. All animals were properly anesthetized and immobilized before the experiment. For rats and mice, isoflurane was used for anesthesia, and the anesthetic concentration was maintained at 2-3% to ensure the depth and stability of anesthesia. After anesthesia, the animals were placed in a warm environment to prevent hypothermia, and vital signs were monitored using a small anesthesia machine. For pigs, Tiletamine-Zolazepam was first used for induction at a dose of 10 mg / kg body weight. After anesthesia induction, the pigs were maintained in an appropriate state of anesthesia with 2-3% isoflurane. During anesthesia, the heart rate, respiratory rate, and body temperature of the pigs were continuously monitored to ensure safety and anesthetic efficacy.

[0079] Using Vevo 3100LT, operators acquired cardiac images of rats and mice through high-frequency probes and calculated important cardiac function parameters. The E / A ratio was obtained through the mitral Doppler flow velocity spectrum, and the left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVFS) were calculated by measuring the dynamic cardiac chamber contour changes through the left ventricular short-axis M-mode. Subsequently, VINNO ULTIMUS9E was used to perform ultrasound examinations on rats, mice, and pigs. The device focuses on obtaining strain data of the left ventricular long axis section (PLAXL) and calculates myocardial strain by dynamically tracking the long axis view. In addition, for pigs, the E / A ratio, LVEF, LVFS, and heart rate (HR) were also recorded to comprehensively evaluate cardiac function. All ultrasound images and parameters were carefully analyzed to ensure the accuracy of the data, providing a reliable basis for cardiac function evaluation for subsequent studies.

[0080] 15. Electrophysiological integration testing Electrophysiological integration indices were detected by an optical mapping system. First, isoflurane anesthesia was induced by intraperitoneal injection of heparin sodium injection (3000 U / kg) and 15 minutes later. The skin was disinfected with alcohol, and an incision was made on the chest skin to expose the xiphoid process. The xiphoid process was lifted with forceps, and the ribs were cut along the left and right midline to open the chest cavity and expose the heart. The lung tissue was clamped with forceps, and the heart was quickly removed and placed in 4°C calcium-free pre-filled oxygenated (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 sodiumpyruvate, 1.8 mmol / L CaCl2, pH 7.35–7.45) for flushing. The aorta was quickly inserted into the perfusion needle using a horizontal Langendorff perfusion apparatus and fixed with surgical sutures to observe whether there were any bubbles entering the heart. The heart was fixed in a silicone rubber chamber for retrograde perfusion and ventilation with carbogen gas. After the Langendorff-isolated heart was stabilized for 10 minutes, the heart was allowed to resume normal rhythm and loaded with fluorescent dyes in sequence.

[0081] After 10 minutes of stabilization, all room lights were turned off and continuous perfusion with Krebs buffer was performed. The cardioplegic agent Blebbistatin was injected from the Y-shaped inlet to eliminate the mechanical contraction of the heart, and it stopped after about 1-2 minutes. Part of the perfusate was removed, the oxygen supply was reduced from the circulating reservoir, and Pluronic F127 was added to the remaining perfusate. After 10 minutes of continuous perfusion, the calcium indicator Rhod-2AM was slowly injected from the Y-shaped inlet, and the circulation was continued for 15 minutes. Then, the voltage-sensitive dye RH237 was slowly injected, and the previously removed Krebs buffer was returned to the perfusion reservoir and the circulation continued.

[0082] Transfer the dye-loaded heart to the bath and place ECG electrodes on both sides of the heart, attaching the red electrode to the tip of the left ventricle, the blue electrode to the right atrium, and the black electrode to the ground in the bath. Connect to the biosignal acquisition system to record the electrocardiogram (ECG), and adjust the sampling rate to automatic for continuous monitoring. Using the optical imaging system microscope, focus the excitation light on the heart, adjust the distance between the lens and the heart, select the maximum resolution and frame the appropriate imaging area. Record three action potentials and calcium ion signals in sinus rhythm.

[0083] Insert the pacing electrode approximately 1 mm into the right ventricle and deliver the pacing stimulus. The initial current intensity is 0 mA, the frequency is 6 Hz, the pulse width is 2 ms, and the ECG is closely observed. When the ECG shows a clear 1:1 pacing corresponding to the stimulus, the current intensity is the diastolic threshold current. Set the pacing current to twice the diastolic threshold current, and record the action potential and calcium ion signal at this 6 Hz pacing rate. After eight consecutive S1 stimuli, apply an early additional stimulus S2, the S1S1 interval is reduced by 20 ms compared to the inherent RR interval, and the S1S2 interval is gradually reduced by 5 ms until S2 cannot induce a QRS waveform. At this time, the S1S2 interval is recorded as the effective ventricular reentry period.

[0084] Ventricular arrhythmias were induced using 50 Hz high-frequency pulses, and the intensity of the stimulation current was gradually increased from 5 mA to 10 mA, then to 15 mA, and finally to 20 mA. The interval between each stimulation was 3 minutes, and the occurrence, duration, and fibrillation threshold of ventricular arrhythmias were recorded after each stimulation. ECGs of all hearts were recorded under sinus rhythm, normal pacing, and induced ventricular arrhythmias, and optical measurements of membrane potential and calcium ion signals of dye-loaded rat hearts were observed before and after ischemia-reperfusion, and action potential depolarization, repolarization time, action potential duration, and calcium transients were analyzed.

[0085] 16. Protein purification Plasmids containing Cpeb3 from different species and its specific amino acid mutants, Cpeb3-GFP, and HK2-mcherry were synthesized by Qingke Biotechnology (Beijing, China) using the pGEX-4T-1 vector and subsequently transformed into competent Escherichia coli cells. The transformed bacteria were inoculated on LB agar plates supplemented with antibiotics and cultured overnight to select plasmids containing the ampicillin resistance gene. One positive clone was inoculated into LB liquid medium containing antibiotics and cultured at 37°C overnight with shaking.

[0086] Subsequently, a small amount of the overnight culture was transferred to fresh LB medium containing antibiotics and cultured at 37°C with shaking 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 at 37°C with shaking for 6 hours. After induction, the 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 broken by ultrasound to effectively release the target protein into the solution.

[0087] After cell lysis, centrifugation is performed to remove cell debris, and the supernatant containing dissolved proteins is collected for purification. The target protein with a His tag is separated using a Ni-NTA affinity column and eluted with an elution buffer containing different concentrations of imidazole. The individual fractions collected during the elution process are analyzed for protein purity by SDS-PAGE. If the purity is insufficient, the protein is further concentrated by dialysis or ultrafiltration. Finally, the purified protein is stored in a suitable buffer for subsequent experiments.

[0088] 17. Virtual Docking Virtual docking was completed by AutoDock software. This study aims to explore the binding interaction between rat Cpeb3 and small molecule compounds using molecular docking technology. First, the three-dimensional structure of Cpeb3 was obtained, and the ligand structure of the small molecule compound was prepared. Then the docking grid frame was defined, and the docking simulation was performed. By analyzing the binding affinity and visualizing the interaction, the key hydrogen bonds and hydrophobic interactions were identified, and the distances between the interacting groups were measured. Finally, the binding mechanism of Cpeb3 with small molecule compounds was revealed, providing a theoretical basis for subsequent drug design.

[0089] 18. High-throughput drug screening Using the molecular modeling software AutoDock, the three-dimensional structure of Cpeb3 (UniProt number: D4AD99) was constructed and its energy was minimized to ensure the accuracy of the model. Subsequently, compounds with specific chemical properties and biological activity information were screened from the 19 million compounds provided by Topscience to establish a compound database. Using molecular docking technology, each compound was docked with the binding site of Cpeb3 to evaluate its binding affinity. Potential high-affinity compounds were screened by calculating key parameters such as binding energy, number of hydrogen bonds, and hydrophobic interactions. During the virtual screening process, the PLANET model based on image neural network was used to score the compounds to capture the complex relationship between molecular structure and biological activity. Combined with the results of molecular docking, 300 candidate compounds were finally selected.

[0090] Next, primary Gpc6-positive cardiomyocytes (Gpc6 + CMs) and cardiac fibroblasts (CMFs). First, the heart tissue was digested with collagenase to extract cells, and then the expression of Gpc6 was detected by flow cytometry. During cell culture, cardiomyocytes used DMEM / F12 medium, and cardiac fibroblasts used DMEM medium. The medium was supplemented with 10% fetal bovine serum and antibiotics, and the cells were cultured at 37°C and 5% CO2. In order to simulate the myocardial infarction environment, the cells were placed in hypoxia (1% O2) and starvation (serum-free medium) for 1 hour to induce stress response. Subsequently, 50 nM ES_Cu and 5 μM of different candidate compounds were added to the culture medium and incubated for 24 hours.

[0091] Cell viability was assessed using the CCK8 kit according to the manufacturer's instructions, and appropriate control groups (such as untreated group and ES_Cu-only treated group) were set up to ensure the reliability of the experiment. + In the CMs group, cell viability increased significantly by 1.5 times, indicating that these compounds have good activity in promoting cardiomyocyte survival and functional recovery, which may be related to their regulation of cell metabolism, antioxidant pathways or inhibition of apoptotic signals. In the CMFs group, the increase in cell viability did not exceed 1.1 times, indicating that these compounds had no significant promoting effect on cardiac fibroblasts, highlighting their specificity and selectivity. Based on this result, the Gpc6 + Compounds with cell viability increased by more than 1.5 times in the CMs group and no significant viability increase in the CMFs group were defined as “hits”, demonstrating their superiority in targeting cardiomyocytes.

[0092] For the hit compounds screened, surface plasmon resonance (SPR) experiments were performed. In the SPR experiment, Cpeb3 protein was prepared by a recombinant expression system and immobilized on a sensor chip. Different concentrations of the screening compounds were injected, and their binding and dissociation processes were monitored in real time. The affinity constant (KD) was calculated by data analysis, and compounds with KD ≤ 50 μM were defined as effective hit compounds.

[0093] 19. Surface Plasmon Resonance (SPR) Experiment Octet® R4 (Sartorius, Germany) was used for SPR experiments. The target protein was produced by a recombinant expression system, carrying a histidine tag, and then purified. The protein was immobilized on a pre-treated Ni-NTA sensor chip to ensure uniform binding of the protein, thereby improving the reproducibility and reliability of the experiment.

[0094] During the experiment, different concentrations of candidate compounds or proteins are injected into the sensor surface in sequence by flow injection. The instrument continuously monitors the changes in reflected light intensity during the binding process and generates sensor response curves (senorgrams). These response curves show the rate of the binding reaction and the changes during the dissociation process, clearly reflecting the interaction between the mobile phase and the fixed protein.

[0095] In the data analysis stage, dedicated software is used to perform baseline correction to eliminate background noise and extract the real signal. The equilibrium dissociation constant (KD) is calculated by fitting the binding and dissociation curves at different concentrations.

[0096] 20. RNA immunoprecipitation (RIP) and methylated RNA immunoprecipitation (MeRIP) RIP and MeRIP experiments were performed according to the methods described previously. RIP experiments were performed using the EZ-Magna RIP™ RNA Binding Protein Immunoprecipitation Kit (#17-701, Sigma-Aldrich, Billerica, MA, USA) according to the manufacturer's instructions. Briefly, RNA-binding proteins were extracted by cell lysis using the provided RIP lysis buffer. Target RNA-binding proteins were captured by immunoprecipitation using magnetic beads conjugated to anti-Cpeb3 antibodies or control IgG. To detect the enrichment of HK2 mRNA in the immunoprecipitated RNA samples, qRT-PCR analysis was performed. In addition, to analyze the m6A modification level in HK2 mRNA, MeRIP experiments were performed based on established methods. In this step, 5 μg of anti-m6A antibody (#A19841, ABclonal, Wuhan, China) or control IgG was conjugated to magnetic beads in the Magna RIP kit (#MAGNARIP01, Merck, Germany). Then, 50 μg of total RNA (extracted from cells by standard RNA isolation techniques) was incubated with anti-m6A antibody in RIP immunoprecipitation buffer (provided by Magna RIP kit) at 4°C overnight to specifically bind to m6A-modified RNA. After incubation, samples were treated with proteinase K to remove residual proteins to ensure purified RNA samples. Subsequently, RNA was purified using RNeasy Mini Kit (#74104, Qiagen, Germany). The enrichment of HK2 mRNA in the m6A immunoprecipitated RNA fraction was quantitatively detected by qRT-PCR analysis.

[0097] 21. RNA Pull-Down Experiment Biotin-labeled HK2 mRNA was synthesized by in vitro transcription using the pcDNA3.1 vector as a template. Biotin RNA Labeling Mix (#11685597910, Merck, Germany) and T7 RNA polymerase (#RPOLT7-RO, Merck, Germany) containing biotin-UTP were added to drive the reaction during transcription. To remove residual DNA that may contaminate RNA, the synthesized product was treated with RNase-free DNase I (#69182, Merck, Germany) at a concentration of 1 U / μg RNA at 37 °C for 30 minutes and then inactivated by heating at 75 °C for 10 minutes. Biotin-labeled RNA was purified using RNeasy Mini Kit (#74104, Qiagen, Germany) and eluted with 30 μL of RNase-free water to obtain pure and concentrated RNA products. Subsequently, the purified biotin-labeled RNA was added to the previously prepared cell lysate (prepared from cultured cells using RIPA buffer containing protease inhibitors) and incubated at 4 °C for 2 hours. This incubation step promotes RNA-protein interaction to form RNA-protein complexes. After incubation, biotinylated 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, bound proteins were eluted from the magnetic beads using 2× Laemmli sample buffer and denatured at 95 °C for 5 minutes. The eluted samples were analyzed by Western blot to identify proteins that interact with biotinylated HK2 mRNA, thereby revealing the functional dynamics of HK2 in the cellular environment.

[0098] 22. qRT-PCR experiment Total RNA was extracted using RNAsimple Total RNA Kit (#DP419, Tiangen, Beijing, China) and the manufacturer's instructions were strictly followed to ensure high-quality RNA to meet the requirements of subsequent experiments. For cDNA synthesis, 1,000 ng of extracted total RNA was used as a template and reverse transcribed using PrimeScript™ RT Reagent Kit with gDNA Eraser (#RR047, TaKaRa, Dalian, China) to synthesize cDNA. This kit includes a gDNA removal step to effectively remove genomic DNA contamination, thereby improving the accuracy and reliability of downstream experimental results. After cDNA synthesis, qPCR amplification was performed using a One-step Reverse Transcription PCR Kit (#FP313-01, Tiangen, Beijing, China) to quantitatively evaluate the expression level of the target gene. GAPDH was selected as an internal reference gene to normalize the expression data to compensate for changes in cDNA input and overall PCR efficiency. Relative gene expression levels were calculated by 2 −(ΔΔCt) Methods were calculated to enable robust comparison of gene expression between samples.

[0099] 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 assessed using a Nanodrop spectrophotometer. By comparing expression levels at different time points, the degradation kinetics of the target mRNA under transcription inhibition conditions were analyzed. Primer sequences HK2 Forward: 5'- CTGCCCTTCTGCTTCTCTG -3' (SEQ ID NO: 1); HK2 Reverse: 5'- GCTGGTGGTGAGGTGATG -3' (SEQ ID NO: 2).

[0100] (XXIII) Luciferase reporter gene experiment To identify the binding site of Cpeb3 in the 3′-UTR region of HK2 mRNA, the wild-type and mutant sequences of HK2 3′-UTR were cloned into the pmirGLO-luciferase vector. +CMs cells were seeded in 96-well plates at a density of 5 × 10³ cells per well, and each condition was performed in triplicate. Cells were co-transfected with pmirGLO-HK2 wild-type 3′-UTR or pmirGLO-HK2 mutant 3′-UTR vectors and shRNA against Cpeb3 to knock down the expression of Cpeb3. Luciferase activity was measured 48 hours after transfection using the Dual Luciferase Assay System (#RG029, Beyotime, Beijing, China) according to the manufacturer’s instructions. Firefly and Renilla luciferase activities were quantified, and the relative luciferase activity was determined by calculating the ratio of firefly luciferase to Renilla luciferase activity to normalize the transfection efficiency. This approach provided insight into the interaction between Cpeb3 and the 3′-UTR region of HK2 mRNA.

[0101] 24. Fluorescence polarization method To detect the binding of Cpeb3 protein to HK2 protein, fluorescence polarization technology was used, using a SpectraMaxiD5 fluorescence polarization detector (Molecular Devices). First, recombinant HK2 protein and Cpeb3 protein (including wild type and mutant) labeled with green fluorescent protein (GFP) were prepared. In each experimental group, different concentrations of HK2 protein (including control group and HK2 overexpression group) were set. An equal amount of Cpeb3 protein was added to each reaction tube and gently mixed, followed by measurement using a fluorescence polarization detector. In the fluorescence polarization detection, the excitation wavelength was set to 488 nm and the emission wavelength was set to 520 nm to adapt to the fluorescence characteristics of GFP. The fluorescence polarization values ​​under different conditions were recorded, and the polarization changes of wild-type and mutant GFP-Cpeb3 proteins when binding to HK2 protein were compared to evaluate the binding affinity between the two.

[0102] 25. Fluorescence recovery after photobleaching (FRAP) experiment Different treatment strategies were applied to cultured cells before imaging. Using a ZEISS LSM 980 confocal microscope, regions of interest (ROIs) containing GFP or mCherry signals were completely photobleached and treated with a focused 488 nm laser beam. After photobleaching, the recovery of fluorescence intensity within the ROI was monitored in real time, and data was captured at predetermined intervals to quantify the recovery kinetics. Fluorescence intensity was measured before bleaching and at the beginning of recovery, allowing a comprehensive analysis of the dynamics of the movement and redistribution of fluorescent proteins after treatment.

[0103] (XXVI) RNA Pulldown / Mass Spectrometry (MS) In rat Gpc6 +Cpeb3 protein was knocked down or not in CMs. Nuclear proteins were extracted using a nuclear and cytoplasmic protein extraction kit (#P0027, Beyotime, Beijing, China), and RNA pull-down experiments of HK2 mRNA were performed. After overnight incubation at 4°C, Protein A / G magnetic beads were added to capture the antibody-antigen complex, and non-specifically bound proteins were removed by washing. Subsequently, the bound proteins were enzymatically 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). For mass spectrometry data analysis, the obtained mass spectrometry data were processed using MaxQuant software and compared with the protein database. Based on the mass spectra of the peptides, the m6A-modified proteins that interacted with Cpeb3 were identified, with special attention paid to the changes in relative abundance to distinguish the differences between the control group and the Cpeb3 knockdown group.

[0104] 27. Statistical Analysis All data are presented as mean ± SD. Normally distributed data were statistically analyzed using Student's unpaired t test, while non-normally distributed data were compared using the non-parametric Mann–Whitney rank sum test. Some experiments were analyzed using one-way ANOVA combined with Bonferroni post hoc test.

[0105] Animal survival analysis was performed using the Kaplan–Meier method, and differences were detected by the log-rank (Mantel–Cox) test. Linear correlation analysis used the Pearson correlation coefficient, which ranged from -1 to 1 to evaluate the linear relationship between two continuous variables, and 0 indicated no correlation. For non-normally distributed data, the median (25% and 75% quantiles) was used for descriptive statistics, and the Mann-Whitney U test (nonparametric test) was used to compare the two groups of data, and the results were expressed as Z statistics. For the comparison of the percentages between the two groups, a 2×2 contingency table analysis was used, and the Fisher exact test was used to calculate the significant differences. The threshold for statistical significance was set at P < 0.05. All statistical analyses were performed using GraphPad Prism software.

[0106] Example 1 Based on the aforementioned experimental methods, the results showed that copper overload induced Gpc6 + Cpeb3-dependent metabolic reprogramming in CMs leads to copper death in MIRI.

[0107] Previous studies have shown that iron overload during MIRI can induce ferroptosis, but it is unclear whether copper can induce a similar phenomenon. Human data and animal experimental results (Table 1) show that copper levels increase after MIRI ( Figure 1 Middle A, Figure 5 A), and was positively correlated with myocardial injury ( Figure 1 In the previous study, the heterogeneity of cardiomyocytes after myocardial infarction in mice was analyzed. The present invention re-analyzed the heterogeneity of cardiomyocytes and found that a type of Gpc6-positive cardiomyocytes (Gpc6 + The change in cell number of CMs was negatively correlated with the copper ion concentration ( Figure 1 C, D, Figure 5 Middle B). In Bama miniature pigs ( Figure 5 Middle C, Figure 5 Middle D, Figure 5 Middle E) and rats ( Figure 5 Middle F, Figure 5 Middle G, Figure 5 A similar trend was observed in (H), suggesting that Gpc6 + The status of CMs may be related to the copper concentration.

[0108] Table 1. General information of myocardial infarction and healthy subjects ± s , M ( P 25 , P 75 )]

[0109] The experiments showed that ES_Cu and DS_Cu promoted Gpc6 in a dose-dependent manner + Copper death in CMs ( Figure 1 Middle E, Figure 6 Middle B, Figure 6 C), rather than other forms of cell death ( Figure 6 A), and had no significant effect on Gpc6-negative cardiomyocytes (Gpc6-CMs) ( Figure 6 Middle B, Figure 6 Middle C, Figure 6 Middle D, Figure 6 E). Spatial transcriptomic studies of human myocardial infarction showed that the specificity of disease damage is highly dependent on the heterogeneity of cardiomyocytes, and different cell types have different metabolic characteristics under the same stimulation. Our data further revealed that ES_Cu significantly inhibited Gpc6 + Mitochondrial respiration of CMs ( Figure 1 Middle F, Figure 6 F) and glycolysis ( Figure 1 Middle G, Figure 6 G), but had no significant effect on Gpc6-CMs ( Figure 6 Middle H, Figure 6 Middle I).

[0110] 24 hours after MIRI, Gpc6 + CMs showed high expression of Cpeb3 and low expression of HK2 in the infarct edge area ( Figure 1 Middle H, Figure 7 Middle A, Figure 7 B) phenomenon, this expression pattern is induced by ES_Cu ( Figure 7 C), and the expression of HK2 is regulated by Cpeb3 ( Figure 7 Middle D, Figure 7 Middle E). KEGG and GSEA analysis showed that both Cpeb3 and HK2 were involved in copper death and glycolysis ( Figure 7 Middle F, Figure 7 The correlation between the two is significant ( Figure 7 Middle H, Figure 7 (center I). The enriched RNA-binding proteins can induce ferroptosis and regulate cellular metabolism, including glycolysis.

[0111] The results confirmed that ES_Cu in Gpc6 + Activated copper death and metabolic changes in CMs are mainly regulated by Cpeb3, while HK2 plays a weaker role ( Figure 1 I, J, K, Figure 8 Middle A, Figure 8 Middle B, Figure 8 Middle C, Figure 8 Middle D, Figure 8 This process affects the production of metabolic byproducts in glycolysis, the tricarboxylic acid cycle (TCA), and the pentose phosphate pathway ( Figure 8 Middle F, Figure 8 Middle G, Figure 8 Middle H, Figure 8 I), leading to metabolic reprogramming and copper death ( Figure 1 Middle L).

[0112] It is well known that myocardial infarction is usually more serious in the elderly. Single-cell sequencing data of human heart in different age groups showed that Gpc6 + The proportion of CMs increases with age ( Figure 1 Medium M, Fig. 9 Middle A, Fig. 9 Middle B, Fig. 9 This may explain the increased susceptibility of the elderly to myocardial injury.

[0113] Example 2 In this example, Cpeb3 is specifically reduced or knocked out, and myocardial injury test and electrical conduction test are performed.

[0114] In the MIRI model, specific knockout of Cpeb3 in the myocardium significantly alleviated IRI-induced myocardial injury ( Figure 2 A, B, Fig.10 Middle A, Fig.10 Middle B, Fig.10 Middle C). Echocardiographic assessment showed that Cpeb3 knockout mice had reduced diastolic Figure 2 middle C) and contractile function ( Figure 2 Middle D, Fig.10 D) have been improved, as shown in the Doppler flow mode ( Fig.10 Middle F, left), M mode ( Fig.10 F, middle) and 2D speckle tracking ( Fig.10 F, right), while heart rate showed no significant change ( Fig.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, Fig.10 Middle G).

[0115] 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, Fig.10 Middle H, Fig.10 Middle I, Fig.10 Middle J).

[0116] 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 2 I, J). Increased VA induction rates are associated with higher mortality ( Figure 2 Middle K).

[0117] These results suggest that Cpeb3 exacerbates myocardial injury in IRI, leading to prolonged action potential duration and abnormal calcium transients, which in turn trigger arrhythmic death. Specific reduction or knockout of Cpeb3 expression significantly promotes myocardial repair, improves action potential stability, and reduces mortality.

[0118] Example 3 This example screens and identifies small molecule inhibitors of Cpeb3.

[0119] Taking advantage of the unique advantages of small molecule compounds, we use high-throughput drug screening ( Figure 3 Middle A) Two potential small molecule inhibitors of Cpeb3 were identified, Com1 ( Fig.11 ComA) and Com2 ( Figure 3 Middle B). Com2 has a stronger binding affinity to Cpeb3 (KD=3.29μM) ( Figure 3 Middle C, Fig.11 B), and showed robust binding to Cpeb3 in multiple species ( Fig.12 Middle C, Fig.12 Middle D, Fig.12 Com2 binds to the molecular pocket of Cpeb3 through hydrogen bonds and π-π conjugation, which is composed of TYR-7, ARG-1, LYS-82, THR-83, SER-85 and ASP-86 ( Fig.12 Middle A, Fig.12 In B), hydrogen bonds and π-π interactions are medium-weak non-covalent interactions. + In CMs, Com2 significantly inhibited ES_Cu-induced copper death and mitochondrial ROS production, which was superior to Com1 and comparable to shCpeb3 (Cpeb3 knockdown) ( Figure 3 Middle D, Fig.11 Middle C, Fig.11 Middle D, Fig.11 Middle E, Fig.11 Middle F, Fig.12 Middle F, Fig.12 Middle G, Fig.12 Compared with Com1, Com2 more effectively reversed the changes in aerobic respiration and glycolysis induced by ES_Cu, and its effects were similar to those of shCpeb3 ( Figure 3 Middle E, 3F, Fig.11 Middle G, Fig.11 Middle H, Fig.12 Middle I, Fig.12In addition, Com2 treatment significantly reversed the changes in glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle metabolites induced by ES_Cu stimulation, with no significant difference from the effect of shCpeb3 ( Fig.13 Middle A, Fig.13 Middle B, Fig.13 Middle C, Fig.13 These results indicate that Com2 is an excellent small molecule inhibitor of Cpeb3 and can effectively counteract the Gpc6 induced by overexpression of Cpeb3. + Aerobic respiration and glycolysis were altered in CMs, thereby regulating metabolic reprogramming and inhibiting the occurrence of copper death.

[0120] Example 4 Cpeb3 and HK2 regulate the crosstalk between metabolic reprogramming and copper death via liquid-liquid phase separation (LLPS) and m6A modification.

[0121] In Gpc6 + In CMs, Cpeb3 regulates the expression of HK2, which controls energy production during glycolysis by catalyzing glucose phosphorylation by properly localizing in the outer membrane of mitochondria. The prediction results of Haddock2.4 showed that Cpeb3 and HK2 have strong interaction ability between different species (Table 2, Fig.14 Middle A). ES_Cu significantly enhances the binding between Cpeb3 and HK2 ( Figure 4 A), resulting in HK2 translocation from the mitochondrial outer membrane and some Cpeb3 translocation to the nucleus ( Fig.14 Middle B). Com2 can inhibit the binding of Cpeb3 to HK2 without affecting the translocation of Cpeb3 to the nucleus ( Fig.14 (B). PKM2 and PFK1, regulators of glycolysis, are also capable of liquid-liquid phase separation (LLPS). Meanwhile, the RNA-binding protein FXR1 activates mRNA translation via LLPS. Our results show that both Cpeb3 and HK2 exhibit liquid-state properties of fusion and cleavage ( Fig.14 C) and recovers quickly after photobleaching ( Figure 4 Middle B, Fig.14 Middle D, Fig.14 E), indicating that they have a strong affinity for LLPS ( Figure 4 C, KD = 47.4 nM), and the amino acid binding site is consistent with the prediction of Haddock2.4 ( Fig.15 Middle B, Fig.15 C), while Com2 effectively inhibits the LLPS formation between Cpeb3 and HK2 ( Figure 4 Middle B, Fig.15 Middle A, Fig.15 Middle D). LLPS can lead to protein translocation, and Cpeb3 also controls this process by regulating the expression of HK2 ( Fig.15 D), and under the stimulation of ES_Cu, Cpeb3 translocated to the nucleus ( Fig.14 Middle B, Fig.16 Middle A).

[0122] Table 2. Prediction of the binding ability of Cpeb3 and HK2 in different species using HADDOCK 2.4

[0123] Cpeb3 mainly binds to U-rich cytoplasmic polyadenylation elements (CPEs), including sequences such as UUUUUUAU, which are present in the 3' untranslated region (3'-UTR) of mRNA and regulate polyadenylation and translation processes. The 3'-UTR of HK2 mRNA from different species is rich in CPEs ( Fig.16 Middle B), Cpeb3 is able to bind to the CPE region on the 3'-UTR of HK2 mRNA, and ES_Cu promotes this binding ( Fig.16 Middle C, Fig.16 Middle D, Fig.16 Middle E, Fig.16 F), while Com2 effectively inhibited their interaction ( Figure 4 Middle D).

[0124] m6A modification is widely present in RNA, and RNA-binding proteins can directly or indirectly participate in the addition, recognition, and removal of m6A. They can help add m6A as part of the "writing" complex, or recognize m6A as a "reading" protein to mediate downstream functions, or affect demethylation through interaction with "erasing" proteins. The SRAMP prediction server (http: / / www.cuilab.cn / sramp) shows that the 3'-UTR of HK2 in different species is rich in m6A modification sites ( Fig.16 B), and ES_Cu inhibited the m6A modification level of HK2 mRNA ( Fig.17 This effect is attributed to the activation of Cpeb3 ( Fig.17 Com2 can prevent the inhibition of m6A modification ( Figure 4 Middle E).

[0125] Mass spectrometry experiments confirmed that the abundance of m6A eraser protein ALKBH5 and reader protein YTHDC1 on HK2 mRNA was significantly regulated by Cpeb3 ( Fig.17 Middle C, Fig.17Cpeb3 can promote ALKBH5 to bind to the m6A modification site on HK2 mRNA in the nucleus, thereby preventing the recognition of the reader protein YTHDC1 ( Figure 4 Middle F, Fig.17 Middle F). The reader protein YTHDC1 regulates mRNA nuclear export, thereby affecting RNA stability but not Gpc6. + Nuclear export of HK2 mRNA in cardiomyocytes ( Fig.17 Treatment with actinomycin D showed that Cpeb3 could reduce the stability of HK2 mRNA through m6A-mediated RNA degradation, while Com2 could prevent HK2 mRNA degradation ( Figure 4 These findings suggest that Gpc6 + In cardiomyocytes, under the stimulation of copper ions, Cpeb3 forms a liquid with HK2 in the form of LLPS, causing the HK2 protein to translocate from the outer membrane of the mitochondria to the cytoplasm. The HK2 protein detaches from the outer membrane of the mitochondria where it should be located, resulting in the inhibition of the mitochondrial glycolysis pathway it promotes, thereby inhibiting mitochondrial function. In addition, Cpeb3 can enter the nucleus, promote the binding of ALKBH5 to the m6A modification site on HK2 mRNA, reduce the m6A modification level, inhibit the recognition of m6A by the reader protein YTHDC1, promote the degradation of HK2 mRNA, inhibit the expression of HK2, further inhibit mitochondrial function, and ultimately induce copper death due to metabolic reprogramming ( Figure 4 Middle H).

[0126] In summary, during myocardial ischemia-reperfusion injury (MIRI), overload of metal ions can trigger specific cell death. In addition to the known ferroptosis, the present invention also confirmed that copper death occurred in specific Gpc6+ cardiomyocytes (CMs). The induction of copper death is associated with the activation and nuclear translocation of Cpeb3, which further regulates the intracellular localization and expression of HK2 through liquid-liquid phase separation (LLPS) and m6A modification, thereby inducing metabolic reprogramming and triggering copper-specific cell death. Small molecule inhibitors of Cpeb3 identified by high-throughput drug screening can significantly reverse copper death caused 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.

[0127] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a Cpeb3 gene inhibitor and / or a Cpeb3 protein inhibitor in the preparation of a drug for preventing and / or treating myocardial ischemia-reperfusion injury, characterized in that: The Cpeb3 gene inhibitor is a substance that can inhibit the Cpeb3 gene level in cells, tissues or organs, and the Cpeb3 protein inhibitor is a substance that can inhibit the Cpeb3 protein level or activity in cells, tissues or organs. The cells are selected from myocardial cells, and the organ or tissue is the heart.

2. The use according to claim 1, characterized in that: The cardiomyocytes are Gpc6 positive cardiomyocytes.

3. The use according to claim 1, characterized in that: The inhibitor of the Cpeb3 gene is selected from at least one of the following substances: at least one of modified or unmodified RNA molecules or a recombinant vector encoding the RNA molecule, wherein the RNA molecule is selected from siRNA, sgRNA, snoRNA, shRNA or microRNA.

4. The use according to any one of claims 1 to 3, characterized in that: The drug has at least one of the following uses: (1) Alleviate myocardial damage induced by IRI; (2) Improve the overall or local diastolic and systolic function of the subject's heart; (3) Enhance the deformation capacity of longitudinal myocardial fibers; (4) Reduced myocardial fibrosis; (5) Inhibit the activation of action potential and calcium transient of cardiomyocytes, and inhibit the prolongation of cardiomyocyte repolarization; (6) Reduce the susceptibility of subjects to ventricular arrhythmias (VA); (7) Improve the subject's threshold for burst stimulation assessment of ventricular fibrillation (VF).

5. The use according to any one of claims 1 to 3, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

6. 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: .

7. The use according to claim 6, characterized in that: The drug is administered by intravenous injection or oral gavage.

8. The use according to claim 6 or 7, 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.

9. The use according to claim 8, characterized in that: The use (1) refers to: inhibiting Cu-induced copper death of Gpc6-positive cardiomyocytes.

10. The use according to claim 6 or 7, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

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