Application of ACLY, PDHB and BCAT2 targets in heart failure drug screening

Abstract

The invention belongs to the technical field of medicines, and discloses application of three drug targets in anti-heart failure treatment drug screening. The target spot is closely related to energy metabolism change occurring in acute heart failure heart tissue with heart injury caused by mechanical stimulation or external force, and the energy metabolism change comprises ACLY, PDHB and BCAT2. The medicine has the beneficial effects that the treatment target of the medicine is obviously changed after the heart failure occurs, and is changed in different areas of heart tissues. The target spot relates to tricarboxylic acid cycle, branched chain amino acid metabolism and fatty acid metabolism, and has important significance in overall regulation and control of cardiac metabolism. The three targets have good application and development prospects in guiding clinical metabolic therapy-based anti-heart failure new drug screening and combined medication strategies. The composition can be used for improving metabolism remodeling of acute heart failure.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to drug action targets for heart failure and related applications in heart failure drug screening. Background Art

[0002] With the accelerating pace of population aging, heart failure (HF) has gradually become the main cause of increasing morbidity and mortality worldwide. In 2022, there were 8.9 million HF patients in China, and this number is expected to double by 2030. According to the left ventricular ejection fraction (LVEF), it is divided into heart failure with reduced ejection fraction (HFrEF), heart failure with preserved ejection fraction (HFpEF), and heart failure with intermediate ejection fraction (HFmrEF). According to the time and speed of onset of heart failure, it is divided into chronic HF and acute HF. Most patients with acute HF have partial relief of symptoms after hospitalization and develop chronic HF; patients with chronic HF often require hospitalization due to acute exacerbations caused by various factors, resulting in considerable medical costs and social burden.

[0003] Neurohormonal blockade has been the mainstay of clinical treatment for HF for decades. Although it alleviates symptoms, it cannot fundamentally alter the pathological conditions of HF, including organ damage, microcirculatory disturbances, and ischemia-reperfusion injury. Furthermore, it reduces myocardial metabolic efficiency, leading to high rates of rehospitalization and mortality. Drugs that promote mitochondrial utilization of glucose and non-fat substrates may improve the metabolic efficiency and function of failing hearts. Therefore, strategies such as improving myocardial energy metabolism, reducing chronic low-grade inflammation, and alleviating immunosuppression may help reduce the risk of HF. Identifying targets related to energy metabolism after HF development is crucial for guiding the screening of anti-HF drugs.

[0004] ATP citrate lyase (ACLY), pyruvate dehydrogenase (PDHB), and branched-chain amino acid transaminase 2 (BCAT2) are involved in the remodeling of energy metabolism after HF. In a rat model of HF established by ligation of the left anterior descending coronary artery, ACLY was significantly elevated in both the infarcted and non-infarcted areas of the heart compared with the sham-operated rats. PDHB was decreased in both the infarcted and non-infarcted areas, and BCAT2 was decreased in the infarcted area. ACLY generates acetyl-CoA from citrate, which contributes to fatty acid and cholesterol synthesis, and is a key gene linking carbohydrate metabolism and lipid metabolism. PDHB catalyzes the overall conversion of pyruvate to acetyl-CoA and carbon dioxide, and is a key gene linking glycolysis and the tricarboxylic acid cycle. BCAT2 is a key gene catalyzing the first step in the catabolism of branched-chain amino acids (BCAAs). Therefore, these three genes play an important role in the metabolic remodeling of cardiac tissue after HF.

[0005] Currently, there are few reports on anti-HF drugs that primarily target metabolic therapies. Therefore, to investigate changes in key metabolic targets in cardiac tissue following cardiac damage, ischemic myocardial infarction, and subsequent HF, we established a HF rat model using left anterior descending artery ligation. Spatial metabolomics and bulk RNA-seq analyses were performed on cardiac tissue from sham-operated, model-treated, Qishen Yiqi Dropping Pill (QDP), and valsartan (V)-treated rats. By analyzing metabolites and key regulatory genes in related metabolic pathways between the infarcted and non-infarcted regions of cardiac tissue across the different groups of rats, we ultimately identified three targets for anti-HF drugs. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide three new drug treatment targets for HF drug screening, which are used for acute HF induced by mechanical stimulation and external injury, wherein the ejection fraction is reduced and left ventricular ischemic infarction triggers HF.

[0007] In order to solve the technical problems of the present invention, the present invention provides the following technical solutions:

[0008] The present invention provides the following drug targets: After the occurrence of HF, insufficient energy supply to the cardiac tissue leads to a series of energy metabolic remodeling in the cardiac tissue, which in turn triggers compensatory changes in related metabolites. Targeting this metabolic change, relevant drug targets are sought, and the targets are: ACLY, PDHB, and BCAT2.

[0009] The present invention uses left anterior descending coronary artery ligation surgery to establish a HF model caused by acute ischemia and reduced left ventricular ejection fraction due to external mechanical stimulation or injury. The present invention uses echocardiography to assess HF modeling, measuring the rats' ejection fraction (EF) and fractional shortening (FS). An EF less than 60% ± 2% is defined as successful modeling. Modeled rats are divided into sham-operated, model, QDP-administered, and V-administered groups based on echocardiographic results.

[0010] In the present invention, the sampling of rat hearts is mainly divided into two parts. First, the rat heart is taken out after perfusion with normal saline, and after distinguishing the atria and ventricles, the atrial tissue is removed along the ligature line using a scalpel according to the coronal plane, retaining the left and right ventricles. After distinguishing the left and right ventricles, the cavity is slowly filled with a pipette containing Laica embedding glue until the heart basically recovers to its pre-perfusion shape. It is then quickly frozen in liquid nitrogen for two seconds to quickly fix the heart shape. The quick-frozen heart is placed in a six-well plate for frozen tissue sectioning. This part is sliced, and adjacent complete slices are cut for mass spectrometry imaging analysis and corresponding histopathological staining analysis. Secondly, the part below the ligature line of the heart tissue is taken according to the above method, and then 100 mg each of the infarct area of ​​the heart tissue (observe the ischemic white part below the ligature line of the heart) and the distal end of the coronal plane of the infarct area are taken and placed in an EP tube and stored at -80°C for Bulk RNA-seq sequencing.

[0011] The present invention uses spatially resolved metabolomics based on aerodynamically assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) to perform differential metabolite analysis on the heart tissue of HF rats. Comparing with the corresponding H&E and Masson pathological tissue staining, the characteristics of cardiac tissue damage and pathological changes are distinguished. The mass spectrometry imaging map is regionalized into infarcted and non-infarcted areas. After that, 3×3 mass spectrometry imaging data of the corresponding parts are obtained. This operation is repeated three times, with three replicates in each group, for a total of four groups.

[0012] The present invention uses Bulk RNA-seq to sequence total RNA in myocardial tissue. According to the above method, the cardiac tissue is divided into infarcted and non-infarcted areas and sequenced separately to obtain the mRNA transcription status of cardiac tissue in the infarcted and non-infarcted areas of each group. Since myocardial cells account for 75% of cardiac tissue, this method can, to a certain extent, reflect the transcription status of myocardial cells with vigorous energy metabolism.

[0013] In this study, SIMCA was used to screen significantly differentially expressed metabolites based on a VIP > 1. T-tests and Anova tests were performed on the differentially expressed metabolites using SPSS to analyze intergroup differences. Metaboanalyst 6.0 was used to enrich the differentially expressed metabolites based on the KEGG database to identify differentially expressed metabolic pathways. The differentially expressed metabolic pathways between the model and sham groups were primarily related to the tricarboxylic acid cycle and amino acid metabolism, including pathways such as alanine, aspartate, and glutamate metabolism, branched-chain amino acid metabolism, and taurine metabolism.

[0014] This study used R programming language to perform pathway enrichment analysis on differentially expressed genes between the model and sham-operated groups in bulk RNA-seq data, identifying relevant pathways including fatty acid oxidation, the tricarboxylic acid cycle, and valine catabolism. By integrating metabolite changes with transcript-level mRNA changes, three relevant targets were identified: ACLY, PDHB, and BCAT2, which are involved in the tricarboxylic acid cycle, fatty acid metabolism, and branched-chain amino acid catabolism. The effects of drugs on these targets were observed, and changes in ACLY, PDHB, and BCAT2 in cardiac tissue following mechanical stimulation and injury were assessed, as well as the extent to which the drugs improved these three targets.

[0015] A characteristic of the present invention is that after the onset of HF, ACLY levels increase in both infarcted and non-infarcted areas of cardiac tissue, while PDHB decreases. BCAT2 levels also decrease in the infarcted area. This indicates that the aforementioned drug targets undergo significant changes after the onset of HF. Drug intervention significantly improves HF, with a significant reduction in the aforementioned targets.

[0016] The present invention also relates to combination medications with the drug targets involved in the present invention as the main targets. The drug Qishen Yiqi Dropping Pills involved in the present invention can reverse ACLY, PDHB and BCAT2 in the infarcted and non-infarcted areas of the heart in HF, and valsartan can improve PDHB and BCAT2 in the infarcted area and ACLY, PDHB and BCAT2 in the non-infarcted area. Combination medications can be used to achieve comprehensive treatment of the infarcted and non-infarcted areas by targeting the characteristics of different areas of cardiac tissue. Combination medications can also take into account multiple targets and other target designs. For example, if the drug valsartan is an angiotensin II receptor inhibitor, the target of action takes into account both angiotensin receptors and the targets related to the present invention.

[0017] Beneficial technical effects

[0018] 1. The drug targets of this invention involve the tricarboxylic acid cycle, fatty acid synthesis, and branched-chain amino acid metabolism, which play a key role in metabolic remodeling after the onset of acute heart failure. There are no reports on the development of anti-HF drugs or combination drug strategies targeting these three targets.

[0019] 2. With the continued development of acute HF, chronic HF will inevitably become a trend. After the onset of acute HF, targeting the insufficient energy supply, in-depth metabolic therapy is needed to improve targets related to energy metabolism after heart failure, control the continuous deterioration of acute HF, and reduce the high rehospitalization rate caused by chronic heart failure.

[0020] 3. Although neurohormonal blockade can alleviate patients' symptoms, it cannot fundamentally change the pathological conditions such as organ damage, microcirculatory disorders and ischemia-reperfusion injury caused by HF. Developing clinical metabolic therapies for heart failure and finding metabolic-related targets are of beneficial value.

[0021] 4. The drug valsartan involved in the present invention takes into account both angiotensin receptors and the relevant targets of the present invention, and has beneficial value in solving the problem that clinical HF is often accompanied by multiple chronic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Definition: QDP (Qishen Yiqi Dropping Pills) refers to Qishen Yiqi Dropping Pills; V (Valsartan) refers to valsartan.

[0023] Figure 1 A HF rat model was established by left anterior descending artery ligation, and ultrasound was used to assess model establishment and improvement after drug administration. The normal group underwent the same surgical procedure as the model group, with the suture passed through the heart but not ligated. The model group underwent left anterior descending artery ligation. The drug administration groups were divided into the QDP group (QDP 270 mg / kg / day administered orally by gavage) and the V group (V 30 mg / kg / day administered orally by gavage). This study investigated changes in left ventricular echocardiographic parameters, including ejection fraction and fractional shortening, after left anterior descending artery ligation in rats, and their improvement after drug intervention. Before drug administration, the ejection fraction of rats in each model group was statistically significantly lower than that in the sham-operated group. After drug administration, the left ventricular ejection fraction of rats in each drug group recovered significantly compared to the model group, indicating successful HF model establishment and improved HF with drug administration. n = 12, mean ± SD. Compared with the sham-operated group: ****p < 0.0001, compared with the model group: # p<0.05.

[0024] Figure 2 H&E staining showed the pathological damage of cardiac tissue in each group. In this experiment, frozen sections of cardiac tissue from rats in each group were stained with H&E. The cardiac tissue was divided into infarcted and non-infarcted areas based on cell morphology and nuclear arrangement. Among them, the sham-operated group had more cells in the infarcted and non-infarcted areas, and the cell nuclei were neatly arranged. In the model group, the infarcted area had fewer cells, disordered nuclear arrangement, and unclear cell morphology. In the non-infarcted area, the nuclei were disordered and the cell boundaries were unclear, but the number of cells was relatively large. After drug administration, the number of cells increased, the cell boundaries were clear, and the cell nuclei were neatly arranged. According to the above description, the infarcted and non-infarcted areas were marked as areas ① and ②, respectively. n = 3.

[0025] Figure 3Spatially resolved metabolomics characterizes metabolite changes after HF. In this study, metabolomics analysis of cardiac tissue metabolites after HF was performed using SIMCA and Metaboanalyst 6.0, and differential pathway enrichment was performed. Significant abnormalities were observed in metabolites related to the tricarboxylic acid cycle and branched-chain amino acid metabolism, with statistically significant differences compared to the sham group. QDP and V administration resulted in a partial recovery, with statistically significant differences compared to the model group. n = 3, mean ± SD. Compared to the model group: *p < 0.05, **p < 0.01.

[0026] Figure 4 Bulk RNA-seq was performed on HF cardiac tissue to obtain mRNA transcription information after HF development. Differentially expressed genes and differentially expressed metabolic pathways were screened using R language to identify potential targets for HF. After the onset of heart failure, ACLY increased in the infarcted region, while PDHB and BCAT2 decreased, with statistically significant differences compared to the sham group. ACLY increased and PDHB decreased in the infarcted margin, with statistically significant differences compared to the sham group. After QDP intervention, ACLY and PDHB in the non-infarcted region returned to levels close to those of the sham group, with statistically significant differences compared to the model group. After valsartan administration, ACLY in the non-infarcted region returned to levels close to those of the sham group, with statistically significant differences compared to the model group. n = 3, mean ± SD. Compared to the model group: *p < 0.05, **p < 0.01.

[0027] Figure 5 Western blot was used to validate the screened targets at the protein level. After HF modeling, ACLY increased in both the infarcted and non-infarcted regions of the model group, while PDHB and BCAT2 decreased, which was generally consistent with the bulk RNA-seq results. Compared with the sham group, there was a statistically significant difference. After QDP administration, the abundance of these proteins in both the infarcted and non-infarcted regions returned to levels close to those of the sham group. After V administration, PDHB and BCAT2 were restored in the infarcted region, while the abundance of ACLY, PDHB, and BCAT2 proteins in the non-infarcted region was improved. Compared with the model group, there was a statistically significant difference. n = 3, mean ± SD. Compared with the model: *p < 0.05, **p < 0.01. DETAILED DESCRIPTION

[0028] The following further illustrates the role of ACLY, PDHB and BCAT2 in the target sites of anti-heart failure drugs in conjunction with the present invention. The following examples illustrate the present invention in more detail and are not intended to limit the present invention in any way.

[0029] Experimental Example 1: Establishment of HF model in rats

[0030] Experimental Principle

[0031] Ligation of the left anterior descending coronary artery can cause acute heart failure due to localized left ventricular ischemia.

[0032] Experimental methods

[0033] Model establishment: Rats were placed in a gas anesthesia apparatus for initial anesthesia with isoflurane gas, followed by anesthesia with 1.5% sodium pentobarbital injected sublingually. The rats were then immediately intubated and connected to a ventilator to maintain normal breathing. The left chest was depilated with depilatory cream, and the limbs were immobilized in a supine position. A thoracotomy was performed at the 3rd or 4th intercostal space below the left axilla. The pericardium was opened, and the left anterior descending coronary artery was ligated with a suture needle. Following ligation, the skin was sutured, and the surgical site and surrounding skin tissue were wiped with iodine to reduce the risk of wound infection. The rats were observed on a ventilator for half an hour. Once vital signs stabilized, the intubation tube was removed and the rats were placed in a cage for observation.

[0034] Imaging examinations: Two weeks after surgery and four weeks after drug administration, rats were fasted for 12 hours and anesthetized with 3% sodium pentobarbital intraperitoneally. The left chest was depilated with depilatory cream and the rats were fixed in the supine position. An ultrasound probe was used in cardiac testing mode to obtain M-mode ultrasound images of the maximum long-axis section of the left ventricle. Parameters such as ejection fraction (EF) and fractional shortening (FS) were calculated from the M-mode images of the left ventricular long-axis.

[0035] Experimental results

[0036] In this example, after the ligation of the anterior descending coronary artery, echocardiography showed that the left ventricular EF and FS of the model group rats were significantly reduced, which was statistically significant compared with the sham operation group. This shows that the HF rat acute myocardial infarction model was successfully established. The results are shown in Tables 1-4 and Figure 1 .

[0037] Table 1 Left ventricular echocardiographic EF values ​​(%) in the sham-operated group and the model group two weeks after modeling.

[0038]

[0039] Table 2 FS values ​​(%) of left ventricle echocardiogram in sham-operated group and model group two weeks after modeling.

[0040]

[0041] Table 3. Left ventricular echocardiographic EF values ​​(%) in the sham group, model group, and each treatment group two weeks after administration. (The sham group and model group died during anesthesia before ultrasound, so n = 11.)

[0042]

[0043] Table 4 FS values ​​(%) of left ventricle echocardiogram in the sham operation group, model group and each drug-treated group two weeks after drug administration.

[0044]

[0045] Experimental Example 2: H&E and Masson staining to delineate different damaged areas of cardiac tissue

[0046] Experimental Principle

[0047] After HF occurs, pathological changes occur in the damaged area of ​​cardiac tissue. H&E and Masson histopathological staining can observe cell morphological damage and tissue fibrosis, respectively. In H&E staining, hematoxylin stain is alkaline, and the chromatin in the cell nucleus and the cytoplasmic nucleic acid are purplish-blue. Eosin stain is an acidic dye, which makes the components in the cytoplasm and extracellular matrix red. Masson stain is a connective tissue stain, which is mostly used to observe the proliferation and distribution of fibrous connective tissue in diseased tissue. Collagen fiber tissue has a buffering effect and can protect the surrounding blood vessels and nerves. Collagen fiber proliferation is usually due to local skin inflammation or tissue hyperplasia. The test results show that collagen fibers are blue, muscle fibers are stained red, and cell nuclei are stained blue-black.

[0048] Experimental methods

[0049] Hematoxylin and Erythrocyte Embryo Staining: Sections were allowed to warm to room temperature for 5 minutes, then fixed with 10% neutral formalin for 5 minutes. The sections were then washed three times with tap water and then with double-distilled water. The following staining procedure was used: hematoxylin staining for 10 minutes, followed by a 1-minute tap water rinse; differentiation in 0.5% hydrochloric acid for 2 seconds, followed by a 10-minute tap water rinse; bluing with 1% HCl–EtOH, followed by a 10-minute tap water rinse. Eosin staining was performed for 5 minutes, followed by differentiation in 80% ethanol for 60 seconds; and dehydration in 90% ethanol for 3 minutes. Sections were then placed in 100% ethanol I for 5 minutes, 100% ethanol II for 5 minutes, 100% ethanol III for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes to clear the sections. The sections were then mounted with neutral gum and observed under a microscope. Staining results were recorded using a NIKON DS-U3 imaging system (Nikon, Japan).

[0050] Masson staining: Sections were allowed to warm to room temperature for 5 minutes, then fixed with 10% neutral formalin for 5 minutes. The sections were then rinsed three times with tap water and then with double-distilled water. The following staining procedure was used: Ponceau staining for 5 minutes, followed by a 1-minute tap water rinse; immersion in phosphomolybdic acid for 5 minutes, staining with aniline blue for 30 seconds, and rinsing with acetic acid for 30 seconds. The sections were then differentiated in 80% ethanol for 60 seconds and dehydrated in 90% ethanol for 3 minutes. The sections were then placed in 100% ethanol I for 5 minutes, 100% ethanol II for 5 minutes, and 100% ethanol III for 5 minutes, followed by xylene I for 5 minutes, and xylene II for 5 minutes to clear the sections. The sections were then mounted with neutral gum and observed under a microscope. The staining results were recorded using a NIKON DS-U3 imaging system (Nikon, Japan).

[0051] Experimental results

[0052] According to the staining results, the left ventricle of the heart tissue was divided into two areas, including the left ventricular infarction area and the non-infarction area. The results showed that compared with the sham operation group, the number of cells in the infarction area of ​​the model group was reduced, the cell nuclei were disordered, and severe morphological damage and fibrosis occurred. The cell nuclei in the non-infarction area were neatly arranged, the cell boundaries were clear, the pathological damage was restored, and the fibrosis was reduced, but there were certain pathological changes compared with the sham operation group. The results of pathological tissue staining showed significant tissue heterogeneity in HF hearts. Figure 2 .

[0053] Experimental Example 3: Detection of Differential Metabolites and Metabolic Pathways in HF Heart Tissue Based on Spatial Resolution Metabolomics

[0054] Experimental Principle

[0055] After the onset of heart failure (HF), the heart experiences insufficient energy supply, leading to compensatory changes in some metabolites. Spatially resolved metabolomics can provide information on in situ cardiac metabolite changes and reveal differential metabolic pathways in HF hearts.

[0056] Experimental methods

[0057] Cardiac tissue collection: 4 weeks after administration, the chest cavity was opened to expose the heart. Forceps were used to expose the heart. A 2 mL syringe with a blunted needle was inserted into the left ventricle from the apex of the heart. The inferior vena cava was cut. The perfusion was completed when the fluid flowing out of the right atrial appendage was clear and transparent. The heart was then removed and rinsed again in normal saline 2-3 times. It was blotted dry with absorbent paper. The area above the ligature was removed to expose the left and right ventricles. The heart was filled with Leica Cryo-Gel embedding gel using a pipette to maintain the heart morphology. The heart was then quickly frozen in liquid nitrogen for 3-5 seconds, placed in a six-well plate, and stored at -80°C for mass spectrometry imaging analysis.

[0058] Slice preparation: Heart tissue was removed and embedded in Leica Cryo-Gel embedding gel. Cryofixation was performed on a Leica CM1860 microtome at -20°C for 5 minutes. Twelve SD rat hearts (sham-operated group (S), model group (M), Qishen Yiqi Dripping Pill group (Q), and valsartan group (V)) were cut into 15-μm frozen sections along the coronal plane. Morphologically intact sections from the same location between the apex and the middle of the ligature were selected from each group and mounted on positively charged, anti-shedding slides. The sections were dried in a -20°C vacuum desiccator for 0.5 h and then at room temperature for 0.5 h before AFADESI-MSI analysis.

[0059] Mass spectrometry imaging parameters: Mass spectrometry imaging experiments were performed using the AFADESI platform and a quadrupole orbitrap mass spectrometer (QExactive, Thermo Science, Bremen, Germany) as previously reported. To obtain high spatial resolution images, cardiac tissue sections were analyzed using a 20-μm inner diameter spray needle. Acetonitrile:water (8:2, v / v) was used as the spray solvent with a flow rate of 5 μL / min, supplemented by 0.7 MPa spray gas. The spray voltage was set to ±7.0 kV. The extraction gas flow rate was 45 L / min. The three-dimensional motorized translation stage (Beijing Optical Instrument Factory, Beijing, China) was set at a constant rate of 0.15 mm / s in the x-direction and separated by vertical steps of 0.15 mm / s in the y-direction. AFADESI-MSI analysis used full MS scans in negative ion mode. Mass spectrometer parameters: capillary temperature 350°C, maximum injection time 200 ms, automatic gain control target 3e 6 , resolution 70000, m / z range 70-1000.

[0060] Data analysis: The collected MS data files (.raw format) were converted into (.cdf) format data, and image reconstruction and background subtraction were performed using MassImager Pro. The average mass spectrum data (.txt) of the corresponding tissue area was obtained based on the pathological staining results. The mass spectrometry data files (.txt) from different samples were imported into Markerview. TMPeak alignment was achieved using software 1.2.1 (AB SCIEX, Framingham, MA, USA). The data set matrix was then further imported into the SIMCA-P 14.0 software package (Umetrics AB, Sweden) for multivariate statistical data analysis. Distinguishable metabolites were screened from MassImager ProTM and Simca14.1.0 software. The P value of each metabolite between the two groups was analyzed using an unpaired two-tailed t-test and anova test, with a confidence interval of 0.95. The obtained data matrix was screened using EXCEL, and [M+H]+, [M+Na]+, [M+K]+, [M+NH4]+, [MH]- and [M-Cl]- ions were selected using the Human Metabolome Database (HMDB). https: / / hmdb.ca / ) and Liped ( https: / / www.lipidmaps.org / ) for preliminary metabolite identification. Metaboanalyte 6.0 was used for differential pathway enrichment.

[0061] Experimental results

[0062] In this experiment, the levels of glucose, citric acid, malic acid, succinic acid and fumaric acid in the infarcted area of ​​the heart tissue of HF model rats were significantly decreased, and the relative intensities of isoleucine and leucine were significantly increased, which were statistically significantly different from those in the sham operation group. The levels of citric acid, malic acid, glucose, isoleucine and leucine in the non-infarcted area were abnormally increased, and succinic acid and fumaric acid were significantly decreased, which were statistically different from those in the sham operation group. After QDP administration, the above metabolites in the non-infarcted area returned to levels close to those of the sham operation group, which were statistically different from those in the model group. After V administration, the abnormal changes of citric acid, malic acid, leucine and isoleucine in the infarcted area were improved, which were statistically significantly different from those in the model group. The above results indicate that after the occurrence of acute HF, energy metabolism-related metabolites and metabolic pathways will undergo metabolic remodeling, and changes in energy metabolism suggest the feasibility of metabolic therapy for HF. Results are shown in Figure 3 .

[0063] Experimental Example 4: Obtaining HF Heart Tissue Transcriptional Data Based on Bulk RNA-seq

[0064] Experimental Principle

[0065] Total tissue RNA information was extracted to obtain the RNA composition of the infarcted and non-infarcted areas of cardiac tissue and after drug administration.

[0066] Experimental methods

[0067] Tissue collection: Heart tissue was harvested from the ligature, and 100 mg of each of the infarcted and non-infarcted areas was separated. After washing with saline, the tissue was placed in a 1.5 mL EP tube and frozen at -80°C for transcriptomic analysis.

[0068] Data Analysis: Bulk RNA-seq data were acquired using an Illumina Novaseq 6000 system in PE150 mode and analyzed by Beijing Novogene Technology Co., Ltd. Differential expression analysis was performed using the R package edgeR. Fold changes in differentially expressed genes were defined as >1.3 or <0.77. Gene Ontology (GO) and KEGG enrichment analyses were performed using the R package clusterProfiler. Pathway analysis was performed using Ingenuity Pathway Analysis (IPA) software, provided by Shanghai Bohao Biotechnology Co., Ltd. A predicted gene ontology z-score >2 and p-value <0.05 indicated an activated molecular interaction, while a z-score <-2 and p-value <0.05 indicated an inhibited interaction.

[0069] Experimental results

[0070] In this experiment, the model group showed up-regulated mRNA expression of ACLY in the infarcted and non-infarcted areas, while down-regulated mRNA expression of PDHB and BCAT2 in the infarcted and non-infarcted areas, with statistically significant differences compared to the sham-operated group. After QDP administration, the mRNA expression of ACLY, PDHB, and BCAT2 in the non-infarcted areas returned to levels close to those of the sham-operated group, with statistically significant differences compared to the model group. After V administration, the mRNA expression of ACLY in the non-infarcted areas and BCAT2 in the infarcted areas returned to levels close to those of the sham-operated group, with statistically significant differences compared to the model group ( Figure 4 ).

[0071] Experimental Example 5: Verification of protein abundance of ACLY, PDHB, and BCAT2 using Western blot

[0072] Experimental Principle

[0073] This method utilizes SDS-PAGE technology to separate proteins in a biological sample based on their molecular weight on a gel. These proteins are then transferred to a solid-phase membrane via electrophoresis. The proteins on the membrane act as antigens, reacting with the corresponding antibodies, which then react with an enzyme-labeled secondary antibody. Finally, the proteins expressed by the specific target gene separated by electrophoresis are detected using methods such as substrate development or fluorescence imaging.

[0074] Experimental methods

[0075] The rapidly frozen heart tissue was dissolved in RIPA buffer containing 1 mM protein inhibitors and homogenized on ice for 30 seconds using a manual tissue homogenizer. After centrifugation at 12,000 g for 10 minutes, the supernatant was collected as total protein. The sample was diluted to a concentration of 30 μg / μL. It was heated and boiled at 95°C for 5 minutes in a 5× loading tank. The proteins were then separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and then transferred to a 0.45 μm polyvinylidene difluoride membrane. After washing with room temperature water for 2 minutes, the derivatized No Stain was added. TM Protein labeling reagent (Thermo Fisher Scientific, Waltham, MA, USA) was added and incubated for 10 min. A shaking flask containing 0.5 g / L BSA powder was sealed in TBST solution for 1 h. Subsequently, the membrane was incubated at 4°C with anti-ACLY (A22273, Abclonal, Wuhan, China) (1:1000), PDHB (A4645, Abclonal, Wuhan, China) (1:2000), BCAT2 (A23793, Abclonal, Wuhan, China) (1:5000). After washing with TBST, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (A0208, Beyotime, Shanghai, China) (1:5000) at room temperature for 1 h. Subsequently, total protein was visualized using a multifunctional gel imager and detected with ECL chemiluminescent solution (Thermo Fisher Scientific, Waltham, MA, USA) in a scanner and then normalized.

[0076] Experimental results

[0077] Western Blot verification was performed on PDHB, a key enzyme that regulates the entry of pyruvate into the TCA cycle, ACLY, a key enzyme that connects glycolipid metabolism, and BCAT2, a rate-limiting enzyme in branched-chain amino acid catabolism. The results showed that compared with the sham operation group, the expression of PDHB and BCAT2 in the infarct area and non-infarct area of ​​the model group was downregulated, and ACLY expression was upregulated. After QDP administration, the abundance of ACLY, PDHB, and BCAT2 proteins in the infarct area and non-infarct area returned to levels close to those of the sham operation group, with statistical differences compared with the model group. After V administration, PDHB and BCAT2 in the infarct area returned to the levels of the sham operation group, with statistical differences compared with the model group ( Figure 5 ).

Claims

1. Application of the following drug targets in screening for drugs for the treatment of heart failure, wherein the drug targets for heart failure are: ATP-citrate lyase (ACLY), pyruvate dehydrogenase (PDHB) and branched-chain amino acid transaminase 2 (BCAT2).

2. The use according to claim 1, characterized in that The heart failure mentioned refers to acute heart failure.

3. The use according to claim 2, characterized in that The acute heart failure is caused by acute ischemia due to external mechanical stimulation, injury, etc., which reduces the left ventricular ejection fraction, resulting in myocardial infarction and secondary heart failure.

4. The use according to claim 1, characterized in that The drug targets play a key role in the alterations in energy metabolism pathways following the onset of heart failure as follows: the tricarboxylic acid cycle, fatty acid synthesis, and branched-chain amino acid catabolism.

5. The use according to claim 4, characterized in that The energy metabolism pathway change includes a detection unit and a data analysis unit, wherein: The detection unit is the infarcted area and the non-infarcted area of ​​the heart tissue of the rat with heart failure; the detection unit performs detection and obtains the detection results according to the following operations: Spatially resolved metabolomics based on aerodynamically assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) detects metabolite information, and transcriptomics based on bulk RNA-seq obtains transcriptome information.

6. The use according to claim 5, wherein the detection criteria for rat heart failure are as follows: The left ventricle of the rat heart was detected by echocardiography after modeling. The modeling was determined to be successful when the EF value was less than 62%±2%, that is, the rat had heart failure.

7. The application according to claim 5, wherein the data analysis unit comprises: SPSS statistical analysis software was used to obtain differential metabolites, and SIMCA and Metaboanalyst 6.0 software were used to perform enrichment analysis on metabolomics data based on the KEGG database to obtain differential metabolic pathways; SPSS statistical analysis software and R language software package were used to perform statistical analysis on the differentially expressed genes in the obtained heart tissues and enrich differential metabolic pathways to obtain transcriptional information of heart failure heart tissues.

Images