ECHS1 K43 and its application in the preparation of products for the treatment or detection of myocardial infarction.

Abstract

The application belongs to the field of biological medicine, and particularly relates to ECHS1 K43 and application thereof in preparation of myocardial infarction treatment or detection products. The nucleotide sequence of the ECHS1 K43 is shown as SEQ ID NO. 1, and the nucleotide sequence of the ECHS1 mutant ECHS1 K43R is shown as SEQ ID NO. 2. The application finds through experiments that the level of lysine lactylation modification (ECHS1 K43la) of the 43th lysine of the ECHS1 protein is significantly increased in myocardial infarction, and the existence of the modification is confirmed for the first time through a specific lactylation modification antibody. It is further found that the K43 lactylation modification can mediate the myocardial infarction process, and the in-vivo function experiment proves the potential of the ECHS1 K43 delactylation modification in treating cardiac remodeling after myocardial infarction.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to ECHS1 K43 and its application in the preparation of myocardial infarction treatment or detection. Applications in products. Background Technology Cardiovascular disease is one of the leading causes of death worldwide. According to the "China Cardiovascular Health and Disease Report 2024," in 2021, cardiovascular disease accounted for 48.98% and 47.35% of deaths in rural and urban areas, respectively, meaning that two out of every five deaths were caused by cardiovascular disease, highlighting the significant burden of cardiovascular disease in China. Cardiac remodeling after myocardial infarction is the core pathological basis of heart failure, contributing to high mortality and morbidity rates globally.

[0002] Post-translational modifications (PDMs) refer to the chemical modifications that proteins undergo after translation, and they are widely involved in the regulation of myocardial infarction mechanisms. In the context of myocardial infarction, cardiomyocytes undergo a series of complex physiological and pathological changes, including PDMs such as phosphorylation, acetylation, and ubiquitination. These modifications regulate protein function, stability, activity, localization, and interactions, thereby controlling cardiomyocyte signaling and stress responses, and ultimately influencing cardiomyocyte responses to injury and stress, thus regulating cardiomyocyte function and fate. PDMs play a crucial role in myocardial remodeling, particularly in the interaction between the endoplasmic reticulum and mitochondria, which is essential for cardiomyocyte energy metabolism and calcium signaling. Studies have shown that PDMs in cardiovascular disease are not limited to traditional phosphorylation and acetylation, but also include novel modifications such as succinylation, S-nitrosylation, and lactylation. Palmitoylation, a type of post-translational modification, refers to the process by which palmitic acid is linked to cysteine ​​residues in proteins via a thioester bond. It participates in the development and progression of pyroptosis by regulating the membrane localization of the N-terminal pore-forming domain of the GSDMD protein, providing a novel intervention strategy for treating inflammation-related diseases such as sepsis. These novel modifications have potential roles in the diagnosis and treatment of cardiovascular diseases, and may become new biomarkers and therapeutic targets. By deeply studying the mechanisms of these post-translational modifications, we can better understand the pathophysiological processes of cardiac remodeling and pyroptosis after myocardial infarction and develop more effective treatment strategies.

[0003] Lactation modification is an emerging post-translational modification that plays a crucial role in various pathophysiological processes. In recent years, increasing research has revealed the key roles of lactation modification in diseases such as tumor immune escape, cardiovascular disease, and osteoarthritis. Lactation modification plays a vital regulatory role in the tumor microenvironment; lactation-driven METTL3-mediated RNA m6A modification can promote the immunosuppressive function of tumor-infiltrating myeloid cells, thereby affecting tumor growth and immune escape. Furthermore, lactate accumulation in the tumor microenvironment further enhances this immunosuppressive effect by inducing METTL3 upregulation through H3K18 lactation. Lactation modification also plays a significant role in cardiovascular diseases. In ischemic myocardial diseases, lactation modification participates in the regulation of gene expression and cell proliferation, influencing pathological processes such as myocardial infarction, myocardial fibrosis, and heart failure.

[0004] Targeting glycolysis and lactation modification may offer novel treatment strategies for cardiovascular diseases. The progression of osteoarthritis is closely related to glycolytic dysregulation, and lactate, a major metabolite of glycolysis, plays a detrimental role in osteoarthritis. Studies have found that lactation modification of UDP-glucose dehydrogenase inhibits its enzymatic activity, leading to reduced glycosaminoglycan synthesis and exacerbating chondrocyte apoptosis and extracellular matrix degradation by activating the MAPK signaling pathway. Lactic acidification also plays an important regulatory role in ocular diseases. Lactic acidification signaling functions in regulating ocular morphogenesis and retinal homeostasis and is associated with various ophthalmic diseases such as myopia, intraocular malignancies, and retinal angiogenesis. During retinal neovascularization, lactation modification promotes angiogenesis by upregulating FGF2 expression. As an important post-translational modification, lactation modification plays a crucial role in various pathophysiological processes; in-depth research into its mechanisms will provide new ideas and strategies for the diagnosis and treatment of related diseases. Previous studies have shown that ECHS1, also known as crotonylase, can also lead to hypertrophic cardiomyopathy due to gene mutations. Loss of ECHS1 function increases histone crotonylation and promotes the expression of Nppb genes associated with myocardial hypertrophy. Investigating the biological role and related mechanisms of ECHS1 lactation in myocardial infarction may provide new targets for the diagnosis and treatment of myocardial infarction. Summary of the Invention

[0005] Based on the aforementioned background, this invention, through integrated analysis of lactation modification omics, discovered that the lactation modification level at lysine 43 of short-chain enoyl-CoA hydratase 1 (ECHS1) is significantly decreased in myocardial infarction tissue. Researching the biological role and related mechanisms of ECHS1 lactation modification in myocardial infarction holds promise for providing new targets for the diagnosis and treatment of myocardial infarction.

[0006] In order to overcome the shortcomings of the prior art, the present invention provides a target for the treatment of myocardial infarction and its application.

[0007] This invention is achieved through the following technical solution: In a first aspect, the present invention provides the application of ECHS1 K43 in the preparation of products for the treatment or detection of myocardial infarction, characterized in that: the nucleotide sequence of the ECHS1 K43 is as shown in SEQ ID NO.1.

[0008] Secondly, the present invention provides a target for the treatment of myocardial infarction, wherein the target is ECHS1 K43la, which is lactated by lysine at position 43 of ECHS1, and its nucleotide sequence is shown in SEQ ID NO.1.

[0009] Thirdly, this invention provides the application of ECHS1 K43la, which is lactated by lysine at position 43 of ECHS1, as a target in the preparation of drugs for treating myocardial infarction.

[0010] Fourthly, this invention provides the application of a reagent for detecting ECHS1 K43la expression modified by lactation of lysine at position 43 of ECHS1 in the preparation of diagnostic products for myocardial infarction.

[0011] This invention provides the application of ECHS1 K43la as a clinical biomarker in the preparation of products for detecting myocardial infarction.

[0012] This invention provides the application of ECHS1 K43la as a target in the preparation of drugs for treating myocardial infarction.

[0013] This invention provides the application of ECHS1 K43la expression reagents in the preparation of myocardial infarction diagnostic products.

[0014] This invention provides the application of ECHS1 K43la in the preparation of products for the treatment or detection of myocardial hypertrophy.

[0015] Fifthly, the present invention provides a specific antibody against lactation at site 43 of the ECHS1 protein, the antibody sequence of which is shown in SEQ ID NO.3, EKKGK(L-Lactic acid)NSSC.

[0016] In a sixth aspect, the present invention provides a mutant ECHS1 K43R targeting ECHS1, having a nucleotide sequence as shown in SEQ ID NO.2.

[0017] This invention also provides the use of the mutant ECHS1 K43R in the preparation of drugs for the prevention or treatment of myocardial infarction.

[0018] The ECHS1 nucleotide sequence is derived from mice, humans, or other mammals.

[0019] Compared with the prior art, the advantage of this invention is that it has been experimentally discovered that the level of lactation modification of lysine 43 of the ECHS1 protein (ECHS1 K43la) is significantly increased in myocardial infarction, and the existence of this modification has been confirmed for the first time by a specific lactation antibody. Mutations in ECHS1 K43 reduce the lactation level at this site, thereby inhibiting the development of myocardial hypertrophy, myocardial fibrosis, and even cardiac remodeling after myocardial infarction.

[0020] Compared with the prior art, the beneficial effects of the present invention include: 1. This invention is the first to discover that ECHS1 K43 lactation modification is increased during myocardial infarction, and that ECHS1 K43 mutations significantly reduce the infarct size and cardiac function impairment; 2. Based on the regulatory mechanism of ECHS1 K43 lactation in myocardial infarction, this invention provides the application of ECHS1 K43 lactation as a clinical biomarker and provides a product for detecting myocardial infarction; 3. This invention relies on ECHS1 K43 lactation antibody and its ECHS1 K43R mutant, which can be used as a method for diagnosing myocardial infarction and provides a drug for the prevention and / or treatment of heart disease. Attached Figure Description

[0021] Figure 1 The expression levels of ECHS1 and K43 lactation (K43la) in the hearts of mice in the Sham and MI groups were adjusted. Figure 2 Evolutionary conservation analysis of ECHS1 K43; Figure 3 The expression levels of ECHS1 and K43 lactation in cardiac tissues of patients with non-heart failure (CTRL) and heart failure (HF) patients; Figure 4 Construction of ECHS1 K43R mutant mice; Figure 5 Quantitative echocardiographic analysis of left ventricular ejection fraction (EF) in wild-type (WT) and K43R mice after sham surgery or myocardial infarction (MI); Figure 6 The expression levels of genes related to myocardial hypertrophy after ECHS1 K43R mutation following MI surgery-induced myocardial infarction; Figure 7 This represents the expression levels of genes related to myocardial fibrosis following ECHS1 K43R mutation after MI surgery-induced myocardial infarction. Detailed Implementation

[0022] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the invention are merely illustrative examples of specific implementations of the invention and are intended to explain the invention, and do not constitute a limitation thereof. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0023] The endpoints of the ranges and any values ​​disclosed herein are not limited to the exact ranges or values, which should be understood to include those close to them. Example 1: Expression of ECHS1 K43la in mouse myocardial infarction tissue

[0024] 1) Construction of a mouse model of myocardial infarction All animal experiments were conducted in accordance with protocols approved by the Animal Ethics Committee of Qingdao University. We used 5-8 week old C57BL / 6 mice as experimental subjects. A dedicated inhalation anesthesia machine equipped with a pure oxygen-mixed anesthetic was used, with isoflurane volatilized through an oxygen flow; typically, a 2-3% concentration was used for anesthesia, maintained at 1.5-2%, with an anesthesia duration of 2-3 minutes. The mice's chest fur was surgically removed (ensuring complete exposure of the surgical area), and the surgical site was disinfected with 75% ethanol. The ventilator was turned on and parameters were set (respiratory rate 110 breaths / min). An incision was made in the third to fourth intercostal space using ophthalmic scissors to fully expose the heart and the left anterior descending coronary artery region. A 6-0 suture needle, along with needle forceps, was inserted 2 mm below the lower edge of the left atrial appendage, passing through the left anterior descending coronary artery to completely occlude its blood flow. After ligation, the chest opening was completely closed with 4-0 sutures (ensuring no gaps and accurate alignment), and the muscles and skin were sutured layer by layer from the inside out.

[0025] 2) Expression of ECHS1 K43la in the sham-operated group and the myocardial infarction group Total protein was extracted from mouse tissues using RIPA and PMSF. The total protein concentration was measured using a BCA kit, and the OD value was read using a microplate reader. Protein loading buffer was added to the protein samples according to the specified concentration, and the samples were incubated at 95°C for 10 min. The protein samples were then subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. Protein expression was detected using specific antibodies. The antibodies used were: ECHS1 K43la. (Shanghai Qiangyao, 1:1000); GAPDH (ABclonal, Cat: A19056, 1:5000).

[0026] 3) Preparation of ECHS1 K43la specific antibody Immunization process: (1) The polypeptide “EKKGK(L-Lactic acid)NSSC” (abbreviated as lactic acid) was conjugated with KLH as an antigen, and the antigen was aliquoted and frozen at -20 ℃. (2) On day 1, 2,000 μg of antigen was added to 2 mL of Freund's complete adjuvant, and PBS was added to bring the volume to 4 mL. The mixture was then emulsified (to test the degree of emulsification: drop a drop of the emulsified antigen solution into physiological saline; if it does not disperse, the requirement has been met). The mixture was then injected subcutaneously at multiple points (at least 8 points) on the back of the neck. Each antigen was used to immunize 4 New Zealand white rabbits. (3) On day 15, 1,200 μg of antigen was added to 2 mL of Freund's incomplete adjuvant, and PBS was added to bring the volume to 4 mL. The mixture was then emulsified (to test the degree of emulsification: drop a drop of the emulsified antigen solution into physiological saline; if it does not disperse, the requirement has been met). The mixture was then injected subcutaneously at multiple points (at least 8 points) on the back of the neck. Each antigen was used to immunize 4 New Zealand white rabbits. (4) On day 29, 1,200 μg of antigen was added to 2 mL of Freund's incomplete adjuvant, and PBS was added to bring the volume to 4 mL. The mixture was then emulsified (to test the degree of emulsification: drop a drop of the emulsified antigen solution into physiological saline; if it does not disperse, the requirement has been met). The mixture was then injected subcutaneously at multiple points (at least 8 points) on the back of the neck. Each antigen was used to immunize 4 New Zealand white rabbits. (5) On day 43, 1,200 μg of antigen was added to 2 mL of Freund's incomplete adjuvant, and PBS was added to bring the volume to 4 mL. The mixture was then emulsified (to test the degree of emulsification: drop a drop of the emulsified antigen solution into physiological saline; if it does not disperse, the requirement has been met). The mixture was then injected subcutaneously at multiple points (at least 8 points) on the back of the neck. Each antigen was used to immunize 4 New Zealand white rabbits. (6) On day 53, blood was collected from the carotid artery. The whole blood of the euthanized rabbit was placed at 4 ℃ overnight, centrifuged (4 ℃, 10,000 rpm) for 30 min, and the serum was collected.

[0027] ELISA process: (1) Coating: The original lactated-BSA and non-lactated-BSA were diluted with CBS to 2 μg / mL and added to the microplate, 100 μl per well, and incubated overnight at 4 °C. (2) Sealing: Discard the coating solution, add 200 μL of blocking solution (5% skim milk powder) to each well, and incubate at 37 °C for 2 h. (3) Sample addition: Discard the blocking solution, add sample (serum or rabbit polyclonal antibody), 100 μl per well to the ELISA plate, and incubate at 37°C for 1 h. (4) Washing: Wash 3 times with detergent, pat dry, (5) Add secondary antibody: Dilute enzyme-labeled goat anti-rabbit antibody to working concentration with PBS, add 100 μl to each well of the ELISA plate, and incubate at 37°C for 40 min. (6) Washing: Wash 3 times with detergent, pat dry. (7) Add TMB chromogenic substrate: Add 90 μl to each well of the microplate and incubate at 37 °C for 15 min. (8) Add stop solution: Add 50 μl of 0.5 M H2SO4 to each well. (9) Reading: OD 450 nm - OD 630 nm readings from the microplate reader. Purification process (antigen affinity purification): Rabbit serum was diluted 3-fold with PBS, filtered through a 0.45 μm filter membrane, and set aside. Two mL of NHS packing material was placed in two gravity columns, and the proto-lactated-BSA and non-lactated-BSA were coupled onto the packing material, named the lactated column and the non-lactated column. Equilibration: The column bed was washed three times with 3 column volumes of PBS. Sample loading: Pass the processed serum through a lactation column at a flow rate of 1.5 mL / min. Washing: After sample loading, rinse the column with PBS until there is almost no color reaction when detected at G250. Elution: Elute the column bed with eluent at a flow rate of 1.5 mL / min, collect the eluent, and neutralize the eluent with neutralizing solution to approximately pH 7. Dialysis: Place the eluent in a dialysis bag and dialyze with 50 times the volume of PBS. Concentration: Centrifuge the dialyzed solution at 3,500 rpm to concentrate it. Sample loading: Pass the concentrated rabbit polyclonal antibody solution through a non-lactated column at a flow rate of 0.5 mL / min. Concentration: Collect the flow-through and centrifuge at 3,500 rpm for concentration, then perform SDS-PAGE analysis.

[0028] result: To investigate ECHS1 expression in myocardial infarction tissue, hearts from mice in the Sham and MI groups were collected. Western blot analysis was used to determine whether ECHS1 K43 is a key lactation site for cardiac remodeling after myocardial infarction. The results showed a significant increase in K43 lactation in myocardial infarction samples (Figure 1). Conservation analysis of ECHS1 indicated that K43 is highly conserved across different species (Figure 2). Western blot analysis using a specific antibody targeting ECHS1 K43 lactation modification further confirmed that K43 lactation modification was significantly upregulated in myocardial tissue from heart failure patients compared to normal tissue (Figure 3). These data collectively suggest that ECHS1 K43 lactation may play a role in cardiac remodeling after myocardial infarction. Example 2: ECHS1 K43R mutation regulates cardiac remodeling after myocardial infarction

[0029] 1) Construction of ECHS1 K43R mutant mice All animal experiments were approved by the Institutional Review Board (IRB) of Qingdao University, and the experimental procedures strictly followed the relevant guidelines of Qingdao University. The gene knock-in mouse was constructed at Cyagen Biosciences in China using CRISPR / Cas-mediated genome engineering technology. Researchers co-injected guide RNA targeting the ECHS1 gene, homologous recombination template DNA, and CRISPR protein into fertilized mouse oocytes, successfully cultivating offspring with the target genotype. Positive mice were finally selected using PCR technology.

[0030] 2) Real-time quantitative PCR (RT-qPCR) Total RNA was extracted from mouse cardiomyocytes or tissues using TRIZOL reagent, and its concentration and purity were determined using nanodrop One. The AG Evo M-MLV RT Kit was used to convert the total RNA into cDNA. Quantitative real-time PCR was performed using the SYBR Green Kit. RT-qPCR was performed using a Thermo Fisher Scientific real-time quantitative PCR system. Reaction conditions: 95℃ for 10 min; 95℃ for 30 s; 95℃ for 10 s, 60℃ for 30 s, for a total of 40 cycles. The results were analyzed qualitatively for relative expression levels using the 2-ΔΔCt method.

[0031] result: To evaluate the effect of ECHS1 lactation at the K43 site on cardiac remodeling after myocardial infarction, we mutated the lysine AAG codon to the arginine CGT codon and constructed a mutant mouse model expressing delactated ECHS1 (K43R) (Figure 4). ECHS1 K43R mutant mice and their wild-type control group underwent myocardial infarction surgery. Compared with wild-type mice, ECHS1 K43R mutant mice exhibited better cardiac function after myocardial infarction (Figure 5). Simultaneously, detection of myocardial hypertrophy-related genes showed that the degree of myocardial hypertrophy in these mutant mice was improved (Figure 6). qPCR detection of myocardial fibrosis-related genes showed that the ECHS1 K43 mutation could alleviate cardiac fibrosis induced by myocardial fibrosis after myocardial infarction (Figure 7).

Claims

1. The application of ECHS1 K43 in the preparation of products for the treatment or detection of myocardial infarction, characterized by: The nucleotide sequence of ECHS1 K43 is shown in SEQ ID NO.

1.

2. A target ECHS1 K43la for the treatment of myocardial infarction, characterized in that: The target ECHS1 K43la is a lactation modification of lysine 43 of the ECHS1 protein, and its nucleotide sequence is shown in SEQ ID NO.

1.

3. The application of ECHS1 K43la as a clinical biomarker as described in claim 2 in the preparation of products for detecting myocardial infarction.

4. The use of ECHS1 K43la as a target as described in claim 2 in the preparation of a drug for treating myocardial infarction.

5. The application of the ECHS1 K43la expression reagent as described in claim 2 in the preparation of myocardial infarction diagnostic products.

6. The use of ECHS1 K43la as described in claim 2 in the preparation of products for the treatment or detection of myocardial hypertrophy.

7. An antibody specifically targeting the lactation of ECHS1 K43la at site 43 of the ECHS1 protein as described in claim 2, characterized in that: The antibody sequence targeting ECHS1 K43la is shown in SEQ ID NO.3, EKKGK(L-Lactic acid)NSSC.

8. A mutant ECHS1 K43R targeting the ECHS1 of claim 2, characterized in that: The ECHS1 K43R has a nucleotide sequence as shown in SEQ ID NO.

2.

9. The use of the mutant ECHS1 K43R according to claim 8 in the preparation of drugs for the prevention or treatment of myocardial infarction.

Images