Myh6 protein modified by milk acylation and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a milk acylation modified Myh6 protein and application thereof. The antigen polypeptide for identifying the heart failure marker has an amino acid sequence as shown in SEQ ID NO: 1, lysine located at the 9th site is subjected to milk acylation modification, and the heart failure marker is milk acylation modified Myh6 protein; the milk acylation modified Myh6 protein is a lysine milk acylation modified protein (Myh6-K1897) which is located at the 1897 site of a wild type Myh6 protein. The Myh6-K1897 can be used as a marker for identifying heart failure as a target spot, is a set of novel and perfect protein milk acylation molecular regulation and control system, can judge the interaction between Myh6 and Tin, and is used for treating the core and key of heart failure. The research provides a new and powerful treatment strategy for the treatment of human heart failure diseases.

Description

[0001] This application is a divisional application of a Chinese invention patent application. The original application was filed on June 6, 2023, with application number 202310662290.5, invention title: a lactylated Myh6 protein and its application, publication number: CN116751276A. Due to the unity of invention issue pointed out by the examiner in the original application, the applicant filed this divisional application. Technical Field

[0002] This invention belongs to the field of pharmaceutical technology, specifically relating to a lactylated Myh6 protein and its applications. Background Technology

[0003] Cardiovascular disease has become the leading cause of death in humans [1]. Heart failure is the final pathway for damage to the structure and function of the heart caused by cardiovascular and related diseases [2]. In heart failure, the heart's contractile function is reduced, and the sarcomere is the basic unit of the heart's contractile function, the core of which is actin and myosin filaments [3,4]. With the development of electron microscopy, the main microstructures of the sarcomere have been successively confirmed. Cryo-electron microscopy and transmission electron microscopy can observe the basic structure of the sarcomere, such as thick filaments, thin filaments and cross-bridges, which can infer the rationality of the myofibril sliding theory [4,5]. However, the key questions of how myosin is metabolized and dynamically maintained in balance to ensure its contractile ability in heart failure, and how these metabolic balance changes affect cardiac function, are still unclear.

[0004] Myosin (myh6 and myh7) is the backbone of the coarse filaments and acts as the molecular engine of the heart, converting the chemical energy of ATP into kinetic energy to induce myocardial fiber contraction [6]. Therefore, the amount of myosin is directly related to myocardial contractility, and it is the most critical protein affecting myocardial contractility. Titin is the largest known protein to date and mainly acts as a scaffold for coarse filament assembly, binding tightly to myosin [7]. Electron microscopy has shown that coarse filaments exhibit lower myocardial fiber density and significant disordered arrangement, suggesting abnormal binding of myosin and titin. However, the underlying causes of these metabolic changes and the mechanisms by which they lead to heart failure are completely unknown. At rest, the heart is the main organ in mammals that produces and metabolizes lactic acid [8]. Lactic acid has long been considered a metabolic waste product, but recently there has been a growing view that lactic acid may play a role in cardiac metabolism and disease [9]. Inhibition of cardiomyocyte monocarboxylic acid transporter 4 (MCT4) promotes lactate dehydrogenase to accumulate pyruvate in the opposite direction, prompting it to enter the mitochondria via the mitochondrial pyruvate carrier (MPC), maintaining the TCA cycle, which helps reduce cardiomyocyte hypertrophy and induce myocardial recovery in heart failure patients

[10] . In addition, the lack of lactate dehydrogenase A (LDHA) in cardiomyocytes can lead to myocardial hypertrophy defects and accelerate the development of heart failure. Lactate, as a product of LDHA, plays an important role in cardiac hypertrophy and the early development of heart failure

[11] .

[0005] Lactic acidification is a histone lysine modification associated with metabolic stress that can directly stimulate gene transcription in chromatin

[12] . The transcription factor Glis1 can activate glycolysis, leading to increased levels of acetylation and lactation, and promoting the reprogramming of senescent cells

[13] . Lactic acid-induced UPS subunit PTM, in the form of lysine lactation (Kla), reduces UPS activity by affecting its assembly

[14] . Kla is a pervasive modification that transcends histone and transcriptional regulation, preferentially affecting enzymes involved in metabolic pathways to regulate cellular metabolism

[15] . As the heart is a vital organ for lactate metabolism and production, it is unclear whether Kla plays a role in heart failure, and the regulatory mechanism of Kla is currently completely unknown. Summary of the Invention

[0006] The purpose of this invention is to provide a lactylated Myh6 protein and its application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A lactylated Myh6 protein, with lactylation of the lysine residue at position 1897 in the wild-type Myh6 protein.

[0009] The lysine lactylation at position 1897 is achieved by adding a lactylation group to the lysine residue.

[0010] An application of the lactacylated Myh6 protein, specifically its use as a biomarker for identifying heart failure.

[0011] The application of the lactylated Myh6 protein as a biomarker for identifying heart failure in humans or mice.

[0012] The heart failure described is caused by Ang II-induced vasoconstriction, which increases cardiac afterload, accompanied by Ang II-directly induced myocardial cell damage, ultimately leading to heart failure.

[0013] An antigenic polypeptide for recognizing a heart failure biomarker, the antigenic polypeptide having the amino acid sequence shown in SEQ ID NO:1, wherein the lysine at position 9 is lactylated, and the heart failure biomarker is the lactylated Myh6 protein as described in claim 1.

[0014] The amino acid sequence shown in SEQ ID NO:1 is CQANTNLSKFRK.

[0015] An antibody that identifies heart failure biomarkers, wherein the antibody is an antibody obtained by screening after immunization with the antigenic polypeptide.

[0016] A kit for identifying heart failure biomarkers, the kit containing antibodies obtained by screening after immunization with the said antigen peptide.

[0017] One application of the described antibody or kit is the use of it in identifying heart failure biomarkers.

[0018] A method for diagnosing heart failure involves using the lactylated Myh6 protein (Myh6-K1897) as a target and measuring its lactylation level using the antibody or kit described above. Low lactylation levels in the system indicate severe heart failure, while high lactylation levels indicate alleviated heart failure.

[0019] An formulation that causes lactylation modification of Myh6 protein, wherein the lactylated Myh6 protein is a wild-type Myh6 protein with lysine lactylation modification at position 1897.

[0020] Advantages of this invention:

[0021] This invention identifies Myh6-K1897 as a target biomarker for identifying heart failure. This novel and comprehensive protein lactation molecular regulatory system can determine the interaction between Myh6 and Titin, and is a core and key component in the treatment of heart failure. This research provides a new and powerful therapeutic strategy for the treatment of human heart failure. Attached Figure Description

[0022] Figure 1 The following are the proteomic and lactylation modification analysis results of wild-type control mice and hypertensive heart failure model mice provided in this embodiment of the invention. A is a schematic diagram of the proteomic and lactylation modification analysis of each group of mice. B is a heatmap of protein histone expression in heart samples from the control group and the hypertensive heart failure group. C is a bar chart of omics data for proteins identified by mass spectrometry, lactylated proteins, and lactylation modification sites. D is a quantitative distribution map of lactylation modification sites in proteins identified by mass spectrometry in the control group and the hypertensive heart failure group. E is a Venn diagram of common and specific lactylation modified proteins in the control group and the hypertensive heart failure group. F is a heatmap of differential expression of common lactylation modified proteins in heart samples from the control group and the hypertensive heart failure group. G is a heatmap of common and specific lactation sites in heart proteins from the control group and the hypertensive heart failure group. H is a Venn diagram showing significant differential expression of lactylation modification sites between the control group and the hypertensive heart failure group.

[0023] Figure 2 The mass spectra of α-MHC modification sites provided in this embodiment of the invention are shown below. A represents the potential lactylation sites of α-MHC revealed by mass spectrometry. B, C, and D represent the lactylation sites of α-MHC identified by mass spectrometry: K1249 (B), K1533 (C), and K1897 (D). E and F represent the lactation of sites K1897, K1533, and K1249 on α-MHC under physiological and hypertensive conditions, as determined by immunoprecipitation analysis. G shows the alignment of the amino acid sequences surrounding the K1897 site in α-MHC homologs from different species.

[0024] Figure 3 Preliminary ELISA detection of 6 α-MHC-K1897 lactylation site-specific antibodies provided in this embodiment of the invention.

[0025] Figure 4 ELISA detection of four purified α-MHC-K1897 lactylation site-specific antibodies provided in this embodiment of the invention.

[0026] Figure 5 Dotblot assay of four purified α-MHC-K1897 lactylation site-specific antibodies provided in this embodiment of the invention.

[0027] Figure 6 Westren blot analysis of four purified α-MHC-K1897 lactylation site-specific antibodies provided in this embodiment of the invention.

[0028] Figure 7This invention provides a method for detecting the lactylation level of α-MHC and its K1897 site under hypertensive heart failure conditions. Specifically, A represents immunoprecipitation and Western blotting of α-MHC lactylation modification under physiological conditions; B represents immunoprecipitation and Western blotting of α-MHC lactylation modification under both physiological and hypertensive heart failure conditions; C represents immunoprecipitation and Western blotting of α-MHC-K1897 lactylation site-specific antibodies under physiological conditions; D represents immunoprecipitation and Western blotting of α-MHC-K1897 lactylation site-specific antibodies under both physiological and hypertensive heart failure conditions; E represents immunohistochemical staining of human BNP and α-MHC-K1897; F represents the quantification of BNP expression levels in immunohistochemical staining; and G represents the quantification of α-MHC-K1897 expression levels in immunohistochemical staining. Detailed Implementation

[0029] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0030] Example 1

[0031] Obtaining the fully lactylated fragment:

[0032] A heart failure model was established in mice by subcutaneously implanting a microosmolar pump and infusing angiotensin II (3 mg / kg / day) via the pump over 14 days. This method has been previously demonstrated to induce cardiomyocyte damage and apoptosis, thereby inducing heart failure. Hearts were then collected from control animals or animals with heart failure (i.e., the amino acid sequence of wild-type heart α-myosin heavy chain protein (α-MHC) in control animals is NCBI Reference Sequence: NM_001164171.1; https: / / www.ncbi.nlm.nih.gov / nuccore / NM_001164171.1), and lactylated proteomics analysis was performed. Figure 1 A, B); specifically:

[0033] Proteomics and Lactic Proteomics

[0034] Protein extraction

[0035] The sample was pulverized in liquid nitrogen and placed in a 5-mL tube. Four volumes of lysis buffer (8M urea, 1% protease inhibitor mixture, 3μM aspergillus A (TSA), 50mM nicotinamide (NAM)) were added, and the sample was sonicated three times on ice using a Scientz ultrasonic homogenizer. The sample was then centrifuged at 12,000 rpm for 30 minutes at 4°C. The protein concentration of the supernatant was measured using a dioctanoic acid assay kit.

[0036] trypsin digestion Equal amounts of total protein were enzymatically digested in each sample, and the volume was adjusted to be consistent throughout the sample group. TCA was added dropwise to a final concentration of 20%, and the precipitate was vortexed at 4°C for 2 hours. The granules were centrifuged at 4500g for 5 minutes and washed twice with pre-cooled acetone. After drying, the granules were resuspended in 200 mM MTEAB by sonication, and trypsin was added to each sample at a ratio of 1:50 (protease:protein, w / w) for overnight digestion. Dithiothreitol was added at 5 mM, and the mixture was incubated at 56°C for 30 minutes. Then, 11 mM iodoacetamide was added, and the mixture was incubated in the dark at room temperature for 15 minutes.

[0037] Enrichment of post-translational modified peptides

[0038] The peptides were resuspended in immunoprecipitation buffer (100 mM sodium chloride [NaCl], 1 mM EDTA [EDTA], 50 mM Tris hydrochloride [HCl], and 0.5% NP-40; pH 8.0) mixed with pre-washed anti-lysine lactase residual antibody resin (PTM-1401; Hangzhou Jingjie PTM Biotechnology) and incubated overnight with gentle shaking at 4°C. The antibody resin was washed with immunoprecipitation buffer and deionized water. Finally, the enriched peptides were eluted three times with 0.1% trifluoroacetic acid and washed with C18 ZipTips.

[0039] Liquid chromatography-tandem mass spectrometry analysis

[0040] Trypsin peptides were dissolved in liquid chromatography mobile phase A and separated on a NanoElute ultra-high performance liquid chromatography system. Mobile phases A and B were 0.1% formic acid and 2% acetonitrile in water and 0.1% formic acid in acetonitrile, respectively. Peptides were eluted using a gradient set to 0–72 min, 7%–24% B; 72–84 min, 24%–32% B; 84–87 min, 32%–80% B; 87–90 min, 80% B, at a constant flow rate of 450 nL / min. Peptides (internal ID, particle size) were separated on a capillary column before injection into a capillary ion source for ionization and a timsTOF Pro mass spectrometer (ion source voltage, 1.6 kV; scan range, 100–1700 Da). Data acquisition was performed using the Parallel Accumulated Serial Segmentation (PASEF) mode. Precursors with charge states of 0–5 were selected for lysis, and 10 PASEF tandem mass spectrometry scans were acquired per cycle. The dynamic exclusion time for tandem mass spectrometry scanning is 30 seconds to prevent multiple scans of the same parent ion.

[0041] Database search

[0042] Raw mass spectrometry data were searched using Maxquant (v1.6.6.0) with reverse bait entries and common contaminant proteins, against the Swissprot protein sequence database (Mus_musculus_10090_SP.fasta). Trypsin / P digestion allowed a maximum of two missing cleavages, and each peptide required at least seven amino acids. The mass error tolerance for precursor ions was 10 ppm, and the mass deviation tolerance for product ions was 20 ppm. Cysteine ​​alkylation (carbamoylmethyl [C]) was considered a fixed modification. Variable modifications were methionine oxidation and N-terminal acetylation. Lysine lactylation and diglycine on lysine were also set as variable modifications for enrichment analysis of the corresponding modifications. The false discovery rate for both protein and PSM identification was 1%.

[0043] Of the 2902 proteins identified in the control group, 159 lactylated proteins and 576 lactylation sites were detected. Of the 3350 proteins resolved in the angiotensin II-induced heart failure group, 160 lactylated proteins and 551 lactylation sites were identified. The control and angiotensin II-induced heart failure groups together contained 142 lactylated proteins and 483 lactylation sites. Figure 1 CG). Heatmap analysis showed statistically significant differences in the levels of these shared lactylated proteins and sites between the control group and the angiotensin II-induced heart failure group (fold change > 1.5, p < 0.05), with the substrate sites showing significant lactylation changes being K1897, K1533, and K1249 on α-MHC. Figure 1H), among which the most significant changes in cardiac α-MHC lactylation in heart failure animals compared to wild-type mice were observed at site K1897.

[0044] Furthermore, in the angiotensin II-induced heart failure group, the lactylation levels at K1533 and K1249 sites on the α-MHC were also reduced. Figure 1 H, Figure 2 In addition to AD, we further investigated the changes in lactylation levels of K1897, K1533, and K1249 under physiological conditions and angiotensin II treatment; the specific methods were as follows:

[0045] Cell culture

[0046] H9c2 and HEK293T cells were cultured in high-glucose Dulbecco modified Eagle medium. Both H9c2 and HEK293T cells were obtained from the American Type Culture Collection (a non-profit organization that collects, stores, and distributes standard reference cell lines). All cells were cultured with 10% fetal bovine serum, penicillin (100 μg / mL), and streptomycin (100 μg / mL) in a humidified atmosphere at 37°C and 5% carbon dioxide.

[0047] Plasmid construction and transfection

[0048] All plasmids used in this study were confirmed by sequencing. Following the manufacturer's instructions, plasmids were transfected into H9c2 and HEK293T cells using Lipofectamine 3000 (Invitrogen, US), HiGene (Applygen, China), and jetPRIME (Polyplus, France). Cells were collected 48–72 hours post-transfection.

[0049] Western blot and immunoprecipitation

[0050] Mouse heart tissues and cells were lysed using lysis buffer (137 mM NaCl, 10 mM NaF, 50 mM Tris-HCl [pH 7.6], 1 mM EDTA, 0.1 mM sodium orthovanadate, 10% glycerol, 1% Nonidet P-40 [NP-40], and 1 mM protease inhibitor). For Western blot analysis, protein samples were quantified, and the total mass and volume of each protein sample were adjusted according to the expression of the target protein. Western blot analysis of LDHA, α-MHC, and α-MHC-K1897-lysine in mouse myocardial tissue was performed using 10 μg of protein extract, while analysis of α-SMA, Col-1, Cleaved-caspase 3, and Cleaved-PARP1 in mouse heart tissue was performed using 40 μg of protein extract. Western blot analysis of p300 and SIRT1 in H9c2 cells and mouse myocardial tissue was performed using 40 μg of protein extract. For co-immunoprecipitation and lactylated immunoprecipitation, 40 μg of protein lysate was administered in H9c2 cells and 10 μg in mouse myocardial tissue. 30 μL of pre-washed magnetic beads were added to 1 mg of protein lysate, and the mixture was incubated by rotation at room temperature for 20 minutes. Subsequently, the beads were separated from the lysate using a magnetic separator, and the pre-cleaned lysate was transferred to a clean tube. Then, 1 mg of protein lysate was incubated with primary antibodies (anti-Pan Kla, 4 μg, PTMBIO; anti-Titin, 4 μg Santa Cruz; anti-α-MHC, 4 μg Santa Cruz; anti-MHC-K1897 lactosyllysine, 4 μg / PTMBIO) bound to 30 μL of protein A / g magnetic beads (Cat#B23202, Biotool, US) at 4°C for 12 hours. The complex was then washed with cold lysis buffer and eluted with sodium dodecyl sulfate (SDS) loading buffer. The complex was separated by SDS-polyacrylamide gel electrophoresis and then electrotransferred to a polyvinylidene fluoride (PVDF) membrane. PVDF membranes were incubated with 5% bovine serum albumin at room temperature for 1 hour, followed by incubation with primary (4°C, overnight) and secondary (room temperature, 1 hour) antibodies sequentially. Protein expression was quantified using Image J v1.46 (National Institutes of Health). Protein expression was normalized by tubulin assay. Antibodies were used at a 1:1000 dilution to detect protein expression in both Western blotting and immunoprecipitation.

[0051] Statistical analysis

[0052] Data are expressed as mean ± standard deviation. Homogeneity of variance was assessed using the F-test (two groups) or the Brown-Forsyth test (three or more groups). The Shapiro-Wilk test was used to assess the normality of the data. Student's t-test and Welch's t-test were used for equal and unequal variances (two groups), respectively. Two-way ANOVA with Bonferroni multiple comparisons was used when considering both cases between two groups. P-values ​​were adjusted for multiple comparisons where appropriate. Detailed methods used to assess significance and the number of biological replicates in each experimental group are indicated in each figure legend. All statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad, US) and SPSS 22.0 software (SPSS, US), and a p-value < 0.05 was considered statistically significant.

[0053] Under physiological conditions, compared with α-MHC-WT, the lactylation level in α-MHC-K1897R [the substitution of lysine (K) at position 1897 for arginine (R)] was significantly reduced, while the lactylation levels in α-MHC-K1533R and α-MHC-K1249R were only slightly reduced. Figure 2 E) indicates that lactylation at the K1897 site may be a pivotal part of α-MHC lactylation. Under Ang II treatment conditions, the lactylation level of α-MHC-WT decreased the most, and the lactylation degrees of α-MHC-K1533R and α-MHC-K1249R also decreased significantly. Figure 2 F). Furthermore, the lactylation level of α-MHC-K1897R remained low and did not significantly decrease upon treatment with angiotensin II. Figure 2 These results indicate that the decrease in α-MHC lactylation levels after angiotensin II intervention is mainly due to a reduction in lactylation modification at the K1897 site. Notably, α-MHC-K1897 residues and their surrounding environment are conserved across different species, including humans, mice, rats, blind moles, hamsters, cattle, and African clawed frogs. Figure 2 G).

[0054] Example 2

[0055] As can be seen from the above examples, the lactylated Myh6 protein (Myh6-K1897) as a marker for recognizing heart failure involves specific antibodies that recognize it, specifically:

[0056] Specific antibodies were designed and purified by Jinjie PTM BioLabs using conventional methods. The α-myosin heavy chain protein sequence and lactylation site were analyzed, along with two antigenic peptides (A: CQANTNLS-(lactyl)K-FRK; B: NTNLS-(lactyl)K-FRKVQHEC, both designed antigenic peptides having lactate modification at the K site) and one unmodified control peptide (NTNLSKFRKVQHUC).

[0057] The designed peptides were used to immunize six SPF New Zealand rabbits multiple times (three immunizations in this example). Peptide A was administered to rabbits 1-3, labeled R1, R2, and R3, respectively; peptide B was administered to rabbits 4-6, labeled R4, R5, and R6. A small amount of serum was then collected, and the serum titer and specificity were preliminarily evaluated using ELISA (see [link to ELISA]). Figure 3-6 ).

[0058] In the initial evaluation, R1, R3, R5, and R6 identified modified peptides with titers exceeding 1:10K (OD>1) and demonstrated good efficacy. We selected sufficient amounts of rabbit serum with high efficacy against protein A and performed immunogenic peptide column affinity purification. The antibodies purified from R1, R3, R5, and R6 were labeled Ab1, Ab3, Ab5, and Ab6, respectively.

[0059] The purified antibodies were tested using ELISA, dot blot, and Western blotting. Based on all the results, antibody Ab3 showed the highest quality, and we selected Ab3, which had the most stable results, as the final antibody for detecting α-myosin heavy chain-K1897 lactation. Therefore, the specificity and efficacy of the Myh6-K1897 lactation antibody in our study can be confirmed.

[0060] Example 3: Validation of the relationship between antibody levels and heart failure:

[0061] Further in vivo verification confirmed the presence of lactylation modification on α-MHC. Specifically: Ethical approval

[0062] This study included five male patients and five age- and sex-matched controls. Failed heart samples were obtained from patients with end-stage heart failure at the time of heart transplantation (mean ejection fraction 20 ± 5%) (General Hospital of the Northern Theater Command). Disease-free hearts were obtained from donors with normal cardiac contractility who died in accidents, through echocardiographic or autopsy analysis (Department of Forensic Medicine, China Medical University; Organ Transplant Center, First Affiliated Hospital of China Medical University). The procurement of heart tissue complied with the principles outlined in the Declaration of Helsinki and was approved by the Institutional Ethics Committee of the First Affiliated Hospital of China Medical University (Protocol No.: AF-SOP-07-1.1-01;

[2022] 504).

[0063] Immunohistochemistry of the human heart

[0064] Immunohistochemical examination was performed on myocardial specimens from five male patients and five age- and sex-matched controls. Heart samples were obtained from patients with end-stage heart failure at the time of heart transplantation (mean EF 20% ± 5%) (Shenyang General Hospital of the PLA). Failure-free hearts were obtained by echocardiography from donors with normal cardiac systolic function who died in accidents (Department of Forensic Medicine, China Medical University; Organ Transplant Center, First Affiliated Hospital of China Medical University). Formalin-fixed heart tissue was embedded in paraffin and cut into 4 μm sections. Sections were dewaxed in xylene, rehydrated in a series of decreasing percentages of ethanol, and boiled in Tris-EDTA solution for antigen recovery. Ultrasensitive TM The SPIHC kit (MXB, China) was used for immunostaining. After blocking, the slides were incubated at 37°C with either a primary antibody against BNP (Abcam, USA) or an α-MHC-K1897-lac antibody (the finally obtained effective purified antibody in Example 2). After washing, the sections were immersed in biotin-conjugated immunoglobulin G secondary antibody from the immunohistochemistry kit for 20 minutes, then infused with DAB (MXB, China) for 2 minutes, and finally stained with hematoxylin for 2 minutes.

[0065] Cell culture

[0066] H9c2 and HEK293T cells were cultured in high-glucose Dulbecco modified Eagle medium. Both H9c2 and HEK293T cells were obtained from the American Type Culture Collection (a non-profit organization that collects, stores, and distributes standard reference cell lines). All cells were cultured with 10% fetal bovine serum, penicillin (100 μg / mL), and streptomycin (100 μg / mL) in a humidified atmosphere at 37°C and 5% carbon dioxide.

[0067] Plasmid construction and transfection

[0068] All plasmids used in this study were confirmed by sequencing. Following the manufacturer's instructions, plasmids were transfected into H9c2 and HEK293T cells using Lipofectamine 3000 (Invitrogen, US), HiGene (Applygen, China), and jetPRIME (Polyplus, France). Cells were collected 48–72 hours post-transfection.

[0069] Western blot and immunoprecipitation

[0070] Mouse heart tissues and cells were lysed using lysis buffer (137 mM NaCl, 10 mM NaF, 50 mM Tris-HCl [pH 7.6], 1 mM EDTA, 0.1 mM sodium orthovanadate, 10% glycerol, 1% Nonidet P-40 [NP-40], and 1 mM protease inhibitor). For Western blot analysis, protein samples were quantified, and the total mass and volume of each protein sample were adjusted according to the expression of the target protein. Western blot analysis of LDHA, α-MHC, and α-MHC-K1897-lysine in mouse myocardial tissue was performed using 10 μg of protein extract, while analysis of α-SMA, Col-1, Cleaved-caspase 3, and Cleaved-PARP1 in mouse heart tissue was performed using 40 μg of protein extract. Western blot analysis of p300 and SIRT1 in H9c2 cells and mouse myocardial tissue was performed using 40 μg of protein extract. For co-immunoprecipitation and lactylated immunoprecipitation, 40 μg of protein lysate was administered in H9c2 cells and 10 μg in mouse myocardial tissue. 30 μL of pre-washed magnetic beads were added to 1 mg of protein lysate, and the mixture was incubated by rotation at room temperature for 20 minutes. Subsequently, the beads were separated from the lysate using a magnetic separator, and the pre-cleaned lysate was transferred to a clean tube. Then, 1 mg of protein lysate was incubated with primary antibodies (anti-Pan Kla, 4 μg, PTMBIO; anti-Titin, 4 μg Santa Cruz; anti-α-MHC, 4 μg Santa Cruz; anti-MHC-K1897 lactosyllysine, 4 μg / PTMBIO) bound to 30 μL of protein A / g magnetic beads (Cat#B23202, Biotool, US) at 4°C for 12 hours. The complex was then washed with cold lysis buffer and eluted with sodium dodecyl sulfate (SDS) loading buffer. The complex was separated by SDS-polyacrylamide gel electrophoresis and then electrotransferred to a polyvinylidene fluoride (PVDF) membrane. PVDF membranes were incubated with 5% bovine serum albumin at room temperature for 1 hour, followed by incubation with primary (4°C, overnight) and secondary (room temperature, 1 hour) antibodies sequentially. Protein expression was quantified using Image J v1.46 (National Institutes of Health). Protein expression was normalized by tubulin assay. Antibodies were used at a 1:1000 dilution to detect protein expression in both Western blotting and immunoprecipitation.

[0071] Statistical analysis

[0072] Data are expressed as mean ± standard deviation. Homogeneity of variance was assessed using the F-test (two groups) or the Brown-Forsyth test (three or more groups). The Shapiro-Wilk test was used to assess the normality of the data. Student's t-test and Welch's t-test were used for equal and unequal variances (two groups), respectively. Two-way ANOVA with Bonferroni multiple comparisons was used when considering both cases between two groups. P-values ​​were adjusted for multiple comparisons where appropriate. Detailed methods used to assess significance and the number of biological replicates in each experimental group are indicated in each figure legend. All statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad, US) and SPSS 22.0 software (SPSS, US), and a p-value < 0.05 was considered statistically significant.

[0073] Consistent with our proteomics data, we validated α-MHC lactylation modification by immunoprecipitation of heart tissue proteins from wild-type mice using anti-Pan-Kla antibody, followed by detection of α-MHC via Western blotting. Figure 7 A). Similarly, by immunoprecipitating cardiac tissue proteins from wild-type mice and hypertensive heart failure model mice with α-MHC antibodies, and then detecting α-MHC lactylation levels by immunoblotting and anti-Pan-Kla antibody, it was found that α-MHC lactylation levels were decreased in myocardial tissue. Figure 7 B). Next, to observe changes in α-MHC lactylation levels at site 1897, we generated an antibody that specifically recognizes this modification site. Consistent with our proteomics analysis, we used purified anti-α-MHC-K1897-Lactyl Lysine antibody to immunoprecipitate proteins from wild-type mouse heart tissue, followed by Western blotting to detect α-MHC and validate lactylation modification at site α-MHC-K1897. Figure 7 C). Similarly, by immunoprecipitation of cardiac tissue proteins from wild-type mice and hypertensive heart failure model mice using anti-α-MHC antibodies, and then detecting the lactylation level of the α-MHC-K1897 site by Western blotting and anti-α-MHC-K1897-Lactyl Lysine antibody, it was found that the lactylation level of the α-MHC-K1897 site in myocardial tissue was decreased. Figure 7D). We also found that in human cardiac pathological samples, immunohistochemical (IHC) staining using BNP (left, using 1:200 diluted anti-BNP antibody) and α-MHC-K1897-lysine (right, using 1:2000 diluted anti-α-MHC-C1897-lysine antibody) revealed a negative correlation between the level of the heart failure marker BNP and the level of lactylation of α-MHC K1897. Figure 1 EG).

[0074] The above-mentioned wild-type cardiac α-myosin heavy chain protein (α-MHC) amino acid sequence is NCBI Reference Sequence: NM_002471.4 https: / / www.ncbi.nlm.nih.gov / nuccore / NM_002471.4.

Claims

1. An antigenic polypeptide for recognizing heart failure biomarkers, characterized in that, The antigenic polypeptide has the amino acid sequence shown in SEQ ID NO:1, wherein the lysine at position 9 is lactylated, and the heart failure marker is the lactylated Myh6 protein. The lactylated Myh6 protein is a lysine lactylated modification at position 1897 of the wild-type Myh6 protein (Myh6-K1897).

2. An antibody that identifies heart failure biomarkers, characterized in that: The antibody is the antibody obtained by screening after immunization with the antigen polypeptide as described in claim 1.

3. A reagent kit for identifying heart failure biomarkers, characterized in that: The kit contains antibodies obtained by screening after immunization with the antigen peptide described in claim 1.

4. An application as described in claim 2 or 3, characterized in that: The application of the antibody or kit in identifying heart failure biomarkers.

5. A method for diagnosing heart failure, characterized in that: Using the lactylated Myh6 protein (Myh6-K1897) as described in claim 1 as the target, the lactylation level is measured by the antibody or kit described in claim 2 or 3. Low lactylation level in the system indicates severe heart failure, while high lactylation level in the system indicates relief of heart failure.

6. A formulation, characterized in that: The formulation induces lactylation modification of the Myh6 protein, wherein the lactylated Myh6 protein is a wild-type Myh6 protein with lysine lactylation modification at position 1897.

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