Use of modulated MHC lactate formulation in the preparation of a medicament for promoting diseases associated with lactate levels in the body

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to the application of an MHC lactation modifier in the preparation of drugs for diseases related to promoting the body's lactation level. Specifically, it relates to the application of an MHC lactation modifier in the preparation of drugs for diseases related to promoting the body's lactation level, and the application of an MHC lactation modifier in the preparation of drugs for heart failure. The heart failure is defined as heart failure caused by decreased α-MHC-K1897 lactation due to Ang II-induced vasoconstriction. The invention also relates to the application of the aforementioned MHC lactation modifier in the preparation of drugs for heart failure caused by Ang II-induced vasoconstriction in humans, mice, or dogs. This invention also discovers new uses for p300 activators and SIRT1 inhibitors. Experimental studies have shown that treatment with p300 activators and SIRT1 inhibitors can affect the level of Myh6-K1897 lactation in the treatment of heart failure. The modification of Myh6-K1897 lactation as a therapeutic target plays an important role in the treatment of heart failure, providing a new approach to the treatment of heart failure.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of a MHC lactic acidification preparation in preparation of a drug for promoting a body lactic acidification level related disease. BACKGROUND

[0002] Heart failure is one of the important causes of human death, and although much progress has been made in the diagnosis and treatment of heart failure, the mortality rate of heart failure remains high. Epidemiological surveys show that 70% of heart failure is caused by hypertension. Long-term poor control of hypertension can cause changes in heart structure and function, mainly including left ventricular diastolic dysfunction, myocardial hypertrophy, and gradual development of myocardial systolic dysfunction. In addition, hypertension can also cause myocardial injury and arrhythmia, which are important causes of heart failure.

[0003] Studies have found that oxidative stress increases the risk of heart failure. Oxidative stress can cause reversible or irreversible oxidative post-translational modification (oxPTM) of cardiac proteins. Monitoring irreversible highly stable oxPTM can help the prognosis of heart failure [1]. Glycosylation modification can promote the progression of heart failure (cardiac hypertrophy) by increasing the transcriptional activity of the c-Myc gene [2]. In addition, the ubiquitin folding modifier 1 (Ufm1) specific ligase Ufl1 can regulate endoplasmic reticulum homeostasis and prevent heart failure. Ufl1 deficiency can induce age-dependent cardiomyopathy, aggravate pressure overload-induced myocardial hypertrophy, fibrosis and dysfunction. At the same time, Ufl1 deficiency also damages the endoplasmic reticulum homeostasis of myocardium and inhibits the PERK (PKR-like endoplasmic reticulum resident kinase) signal [3]. In summary, a large number of studies have shown that post-translational modification of proteins affects heart failure by regulating mitochondria, oxidative stress and endoplasmic reticulum homeostasis. Lysine modification, as a common form of post-translational modification, plays a very important role in heart failure. Studies have found that STAT3 deacetylation can reduce ischemia-reperfusion injury and hinder the progression of myocardial hypertrophy. Histone H3K9 can be deacetylated by SIRT6 to inhibit the IGF-AKT signaling pathway to regulate the progression of heart failure (cardiac hypertrophy). In addition, SIRT6 can also inhibit the expression of transcription factor STAT3 to prevent myocardial hypertrophy [4, 5]. Ubiquitination modification regulates heart failure by regulating the NF-KB pathway, the MAPK pathway and calcineurin [6]. CHIP is an E3 ubiquitin ligase that protects against angiotensin II-induced myocardial remodeling. CHIP has the ability to promote growth and resist apoptosis in CHIP gene-deficient mice, and can enhance the function of myocardial cells under pressure injury [7].

[0004] At present, the pathogenesis of heart failure is the focus of research, and elucidating the pathogenesis of heart failure will provide a strong theoretical basis for improving the clinical prevention and treatment strategies for the disease. SUMMARY

[0005] The application of the MHC lactylation regulating preparation in the preparation of a drug for promoting the body's lactate level related diseases, and the application of the MHC lactylation regulating preparation in the preparation of a drug for heart failure.

[0006] The heart failure is caused by the decrease of alpha-MHC-K1897 lactylation due to the blood vessel contraction caused by Ang II.

[0007] The application of the MHC lactylation regulating preparation in the preparation of a drug for heart failure caused by the blood vessel contraction caused by Ang II in human, mouse or dog.

[0008] The MHC lactylation regulating preparation is a preparation for regulating the lactate level of alpha-MHC-K1897 caused by the blood vessel contraction caused by Ang II.

[0009] The MHC lactylation regulating preparation is p300 lactyltransferase or SIRT1 de-lactyltransferase.

[0010] The application has the advantages that the application finds a new use of p300 activator and SIRT1 inhibitor, and through experimental research, it is found that the treatment of p300 activator and SIRT1 inhibitor can affect the level of Myh6-K1897 lactylation in heart failure treatment, and the lactylation modification of Myh6-K1897 as a treatment target plays an important role in the treatment of heart failure, and provides a new idea for the treatment of heart failure. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, X, Y, Z, AA, AB, AC, AD, AE, AF, AG, AH, AI, AJ, AK, AL, AM, AN, AO, AP, AQ, AR, AS, AT, AU, AV, AW, AX, AY, AZ, BA, BB, BC, BD, BE, BF, BG, BH, BI, BJ, BK, BL, BM, BN, BO, BP, BQ, BR, BS, BT, BU, BV, BW, BX, BY, BZ, CA, CB, CC, CD, CE, CF, CG, CH, CI, CJ, CK, CL, CM, CN, CO, CP, CQ, CR, CS, CT, CU, CV, CW, CX, CY, CZ, DA, DB, DC, DD, DE, DF, DG, DH, DI, DJ, DK, DL, DM, DN, DO, DP, DQ, DR, DS, DT, DU, DV, DW, DX, DY, DZ, EA, EB, EC, ED, EE, EF, EG, EH, EI, EJ, EK, EL, EM, EN, EO, EP, EQ, ER, ES, ET, EU, EV, EW, EX, EY, EZ, FA, FB, FC, FD, FE, FF, FG, FH, FI, FJ, FK, FL, FM, FN, FO, FP, FQ, FR, FS, FT, FU, FV, FW, FX, FY, FZ, GA, GB, GC, GD, GE, GF, GG, GH, GI, GJ, GK, GL, GM, GN, GO, GP, GQ, GR, GS, GT, GU, GV, GW, GX, GY, GZ, HA, HB, HC, HD, HE, HF, HG, HH, HI, HJ, HK, HL, HM, HN, HO, HP, HQ, HR, HS, HT, HU, HV, HW, HX, HY, HZ, IA, IB, IC, ID, IE, IF, IG, IH, II, IJ, IK, IL, IM, IN, IO, IP, IQ, IR, IS, IT, IU, IV, IW, IX, IY, IZ, JA, JB, JC, JD, JE, JF, JG, JH, JI, JJ, JK, JL, JM, JN, JO, JP, JQ, JR, JS, JT, JU, JV, JW, JX, JY, JZ, KA, KB, KC, KD, KE, KF, KG, KH, KI, KJ, KK, KL, KM, KN, KO, KP, KQ, KR, KS, KT, KU, KV, KW, KX, KY, KZ, LA, LB, LC, LD, LE, LF, LG, LH, LI, LJ, LK, LL, LM, LN, LO, LP, LQ, LR, LS, LT, LU, LV, LW, LX, LY, LZ, MA, MB, MC, MD, ME, MF, MG, MH, MI, MJ, MK, ML, MM, MN, MO, MP, MQ, MR, MS, MT, MU, MV, MW, MX, MY, MZ, NA, NB, NC, ND, NE, NF, NG, NH, NI, NJ, NK, NL, NM, NN, NO, NP, NQ, NR, NS, NT, NU, NV, NW, NX, NY, NZ, OA, OB, OC, OD, OE, OF, OG, OH, OI, OJ, OK, OL, OM, ON, OO, OP, OQ, OR, OS, OT, OU, OV, OW, OX, OY, and OZ.

[0012] Figure 2 p300 is a lactyltransferase of a-MHC-K1897, wherein,

[0013] A, B are endogenous proteins immunoprecipitated (IP) from H9c2 cell lysates (A) or myocardial tissue lysates (B) using control immunoglobulin G (IgG) or anti-p300 antibody. Western blotting was performed with anti-a-MHC and anti-p300 antibodies. C, D are IP of endogenous a-MHC of H9c2 cells treated with or without p300 activator (C) or p300 inhibitor (D) using control IgG or anti-a-MHC antibody followed by detection of a-MHC and a-MHC-K1897. E, F are IP of endogenous proteins of myocardial tissue treated with or without p300 activator (E) or with or without p300 inhibitor (F) using control IgG or anti-a-MHC antibody followed by detection of a-MHC and a-MHC-K1897. G, H are IP of endogenous p300 of H9c2 cells (G) or myocardial tissue (H) treated or not with control IgG or anti-p300 antibody followed by Western blotting analysis of a-MHC and p300. I is a schematic representation of the inhibitory effect of p300 on the interaction between Titin and a-MHC at cellular and mouse levels. J, K are IP of endogenous Titin from H9c2 cells (J) or myocardial tissue (K) treated with Ang II and p300 inhibitor. Control IgG or anti-Titin antibody was used for IP followed by Western blotting analysis of a-MHC. L is a quantitative determination of a-MHC interaction with Titin and relative expression level of a-MHC-K1897-lysine in k.Tubulin (n=3 per group). M is a schematic representation of the analysis of p300 activation on the interaction between Titin and a-MHC at cellular and mouse levels. N, O are IP of endogenous Titin from H9c2 cells (N) or myocardial tissue (O) treated with Ang II and p300 activator. Control IgG or anti-Titin antibody was used for IP followed by Western blotting analysis of a-MHC. P is a quantitative determination of a-MHC interaction with Titin and relative expression level of a-MHC-K1897-lysine in O.Tubulin was used as internal reference (n=3 per group). 1 pg of control IgG, anti-a-MHC antibody or anti-Titin antibody was used per immunoprecipitation sample. Data are expressed as mean ± SD. Statistical significance was assessed by two-way ANOVA and Bonferroni’s multiple comparison test (p values adjusted by 6 comparisons, *p<0.05; **p<0.01; ***p<0.001).

[0014] Figure 3 p300 is a lactosyltransferase of a-MHC-K1897

[0015] A Acetylation of a-MHC. HEK293T cells were transfected with plasmids expressing Myc-a-MHC and Flag-p300, Flag CBP, Myc-GCN5, or Flag PCAF. Equal amounts of lysates were prepared for IP with control IgG or anti-Myc magnetic beads, and then probed for Pan-Kla. B IP analysis of HEK293T cells transfected with plasmids expressing Myc-a-MHC. Control IgG or anti-p300 antibody was used for IP, and then Myc was detected. The domains of ca-MHC required for binding p300 were determined by IP analysis. HEK293T cells were transfected with plasmids expressing Myc-a-MHC-WT (full length), Myc-a-MHC-ASFI, Myc-a-MHC-ASclassll, Myc-a-MHC-ASpec, Myc-a-MHC-ASmmCoA, Myc-β-MHCAMIT CorA, and Myc-aMHC-TMPIT (truncations). Equal amounts of lysates were prepared for IP with anti-Myc magnetic beads, and then p300 was detected. D IP analysis of a-MHC acetylation. HEK293T cells were transfected with the indicated plasmids (Flag-p300 and Myc-a-MHC). Equal amounts of IP lysates were prepared with control IgG or anti-Pan-Kla antibody, and then Myc was detected. e IP analysis of a-MHC acetylation. HEK293T cells were transfected with the indicated plasmids (Myc-a-MHC-WT, Myc-a-MHC-K1897R, and Flag-p300). Equal amounts of lysates were prepared for IP with anti-Pan-Kla antibody, and then Myc was detected. F IP analysis of protein interactions in HEK293T cells transfected with the indicated plasmids (Myc-a-MHC and Flag-p300) using anti-Myc magnetic beads, and then a-MHC-K1897-lysine was detected. G Representative immunohistochemical (IHC) staining for BNP (top, using anti-BNP antibody diluted 1 :200) and p300 (bottom, using anti-p300 antibody diluted 1 :2000) protein in heart tissue from normal controls and heart failure patients. Scale bar, 50 μm. Negative controls were performed with normal rabbit IgG diluted 1 :200. H, I Quantification of the relative BNP and p300 expression scores. (n = 5 per group). J Western blot analysis to assess the expression level of p300 in H9c2 cells with or without Ang II treatment. K Quantification of the relative p300 expression in H9c2 cells. L Western blot analysis to assess the expression level of p300 in mouse myocardial tissue after NaCl or Ang II treatment for 2 weeks. M Quantification of the relative p300 expression in myocardial tissue. (a, c, f) 20 μl of anti-Myc magnetic beads were added to each immunoprecipitation sample. (b) Anti-p300 antibody or control IgG was used at 1 μg per immunoprecipitation sample.(d, e) μg for each immunoprecipitated sample. (h, i, k, m) Data are expressed as mean ± standard deviation. Statistical significance was assessed by Student's t-test (***P < 0.001).

[0016] Figure 4 SIRT1 is a delactylase of α-MHC-K1897.

[0017] a, b: Intracytoplasmic protein assays (IPs) of endogenous proteins from H9c2 cell lysates (a) or myocardial tissue lysates (b) using control IgG or anti-SIRT1 antibody. Anti-α-MHC and anti-SIRT1 antibodies were used for Western blot detection. c, d: IPs of endogenous α-MHC from H9c2 cells treated with or without SIRT1 activator (c) or SIRT1 inhibitor (d), using control IgG or anti-α-MHC antibody, followed by detection of α-MHC and α-MHC-K1897. e, f: IPs of endogenous proteins from myocardial tissue treated with or without SIRT1 activator (e) or SIRT1 inhibitor (f), using control IgG or anti-α-MHC antibody, followed by detection of α-MHC and α-MHC-K1897. g, h: Intracellular plasma (IP) of endogenous SIRT1 from H9c2 cells (g) or myocardial tissue (h) treated with or untreated with control IgG or anti-SIRT1 antibody, followed by Western blot analysis of α-MHC and SIRT1. i: Schematic diagram of SIRT1 activation analysis of the interaction between Titin and α-MHC at the cellular and mouse levels. j, k: IP of endogenous Titin from H9c2 cells (j) or myocardial tissue (k) treated with Ang II and SIRT1 activator. Control IgG or anti-Titin antibody was used for IP, followed by Western blot analysis of α-MHC. l: Quantitative determination of the interaction between α-MHC and Titin and the relative expression level of α-MHC-K1897-lysine in k. Tubulin (n=3 per group). m: Schematic diagram of SIRT1 inhibition of the interaction between Titin and α-MHC at the cellular and mouse levels. IP of endogenous Titin from H9c2 cells (n) or myocardial tissue (o) treated with Ang II and SIRT1 inhibitors, n and o, as shown. Control IgG or anti-Titin antibody was used for IP, followed by Western blot analysis of α-MHC. Quantification of pα-MHC-Titin interaction and relative expression levels of α-MHC-K1897-lysine in o. Tubulin was used as an internal reference (n=3 per group). (a, b, g, h) 1 μg in each immunoprecipitation sample. (cf, j, k, n, o) 4 μg of control IgG, anti-α-MHC antibody, or anti-Titin antibody in each immunoprecipitation sample.

[0018] Figure 5 SIRT1 is a delactylase of α-MHC-K1897.

[0019] a, b: α-MHC lactation was determined by IP analysis. HEK293T cells were transfected with plasmids expressing Myc-α-MHC and Myc-Flag-SIRT1, Flag-SIRT2, Flag-SIRT3, Flag-SIR T4, Flag-Sir T5, Flag-SIRCT6, or Flag-SIRT7. Equal volumes of lysates were prepared using anti-Flag affinity gel (a) and anti-Pan-Kla antibody (b) for IP, followed by detection of Myc. c: IP analysis of HEK293T cells transfected with plasmids expressing Myc-α-MHC and Flag-SIRT1. Anti-Myc magnetic beads were used for IP, followed by detection of Flag. The dα-MHC domain required for SIRT1 binding was determined by IP analysis. HEK293T cells were transfected (truncated) with plasmids expressing Myc-α-MHC-WT (full-length), Myc-α-MHC-ΔSH3, Myc-α-MHC-ΔclassII, Myc-a-MHC-ΔSpec, Myc-a-MHC-ΔmmCoA, Myc-β-MHCΔMIT CorA, and Myc-α-MHC-TMPIT. Equal volumes of lysate were prepared for IP using anti-Myc magnetic beads, and SIRT1 was then detected. e, f α-MHC lactylated IP analysis. HEK293T cells were transfected with indicated plasmids (Flag-SIRT1, Flag-p300, and Myc-α-MHC). Equal volumes of lysate were prepared for IP using anti-Pan-Kla antibody (e) or anti-Myc magnetic beads (f), and Myc or Pan-Kla was then detected. g, hIP analysis using SIRT1 activator or inhibitor-induced α-MHC lactylation. HEK293T cells were transfected with the specified plasmids (Flag-p300 and Myc-α-MHC), with or without SIRT1 activator (g, 5 μM; 24 h) and SIRT1 inhibitor (h, 10 μM; 24 h). Equal volumes of lysates were prepared using anti-Pan-Kla antibody for IP assays, followed by detection of Myc. jα-MHC-K1897 lactacylation. HEK293T cells were transfected with the indicated plasmids (Myc-α-MHC-WT, Myc-α-MHC-K1897R, Flag-p300, and Flag-SIRT1). Equal volumes of lysates were prepared using anti-Pan-Kla antibody for IP assays, followed by detection of Myc. jα-MHC-K1897 lactacylation. HEK293T cells were transfected with indicated plasmids (Myc-α-MHC, Flag-p300, and Flag-SIRT1) using anti-Myc magnetic beads, and then the effects of α-MHC-K1897-lysine on α-MHC, K1897-lysine (IP) assays were performed. SIRT1 activators and inhibitors were used to analyze the effects of their effects on α-MHC-K1897 lactylation.HEK293T cells were transfected with specified plasmids (Myc-α-MHC, Flag-p300, and Flag-SIRT1) with or without SIRT1 activator (k, 5 μM; 24 h) and SIRT1 inhibitor (l, 10 μM; 24 h). Equal volumes of lysates were prepared using anti-Myc magnetic beads for IP, followed by detection of α-MHC-K1897-lactate lysine. Representative immunohistochemical (IHC) staining of BNP (top, using 1:200 dilution of anti-BNP antibody) and SIRT1 (bottom, using 1:2000 dilution of anti-SIRT1 antibody) proteins from cardiac tissues of normal controls and heart failure patients was performed. Scale bar, 50 μm. Negative controls were performed using 1:200 dilution of normal rabbit IgG. Quantification of relative BNP and SIRT1 expression scores (n=5 per group). Western blot analysis was used to assess SIRT1 expression levels in H9c2 cells with and without Ang II treatment. q. Quantification of relative SIRT1 expression in H9c2 cells. r. Western blot analysis to assess SIRT1 expression levels in mouse myocardial tissue after 2 weeks of treatment with NaCl or Ang II. s. Quantification of relative SIRT1 expression in myocardial tissue. (a) 20 μl of anti-Flag affinity gel was added to each immunoprecipitation sample. (b, e, gi) 4 μg of anti-Myc magnetic beads were added to each immunoprecipitation sample. (c, d, f, jl) 20 μl of anti-Myc magnetic beads were added to each immunoprecipitation sample. (n, o, q, s) Data are expressed as mean ± standard deviation. Statistical significance was assessed by Student's t-test (**P < 0.01; ***P < 0.001). Detailed Implementation

[0020] 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.

[0021] Example 1: Clarifying the role of lactation modification of Myh6-K1897 in heart failure.

[0022] Construction of point mutant animals

[0023] The Myh6 α-MHC-K1897R mutant mice were designed and manufactured by [Company Name] (Shanghai, China). In short, the targeting construct for the α-MHC-K1897R mutant mice was designed with point mutations flanking exons 38 and 39 and a pGK Neomycine polyA box. The targeting vector was electroporated into C57BL / 6J embryonic stem cells (ES), and G418-resistant colonies were screened according to standard protocols. Homologous recombinant ES cell clones were identified by PCR and confirmed by Southern blotting analysis. Genotyping of tail DNA was performed using a Myh6-(K1897R) mouse mutation region-specific primer set. Highly chimeric mice were bred from Flp mice to obtain the F1 generation, and their genotypes were identified by PCR and sequencing using primer pairs.

[0024] F1:5'-tgtgtgcagaaacccccaagctg-3';

[0025] R1:5'-gaggacactcaccttggcacc-3'.

[0026] α-MHC-K1897R mice and wild-type mice were anesthetized with inhaled isoflurane / oxygen (2%, ~1500 mL / min). Adequate depth of anesthesia was confirmed by the absence of paw withdrawal reflex after anesthesia in the mid-scapula. An osmotic micropump (Alzet) was incised and subcutaneously implanted according to the manufacturer's instructions. Animals were treated for 14 days with Alzet (Alzet, Model 2002; 0.5 μL / h) via infusion of angiotensin II (3 mg / kg / day), a model of heart failure induced by cardiomyocyte damage and apoptosis. Blood pressure was measured daily using the tail cuff method (BP-2010 series sphygmomanometer, Softron, Japan). Cardiac EF% and FS% were measured using ultrasound. Cervical dislocation following isoflurane inhalation was used to euthanize the mice. The effects of different treatment factors on the heart were assessed by endpoint blood pressure, left ventricular EF%, and left ventricular FS%. Animals were handled according to the Animal Welfare Regulations of China Medical University (CMU2023020). All experiments involving animals were approved by the Animal Science Committee of China Medical University. This work was conducted in accordance with the "Guideline for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH Publication No. 85-23, revised in 1985).

[0027] Histopathology staining

[0028] Heart tissue samples were fixed in formalin (4%) for 48 hours, embedded in paraffin, and sectioned to 5 μm. They were then dewaxed with xylene, rehydrated with fractionated ethanol, and stained with hematoxylin and eosin (G1120, Solarbio, China), Masson's trichrome reagent (G1340; Solarbio (China)), and wheat germ lectin (L-1021, fluorescein F).

[0029] Electron microscopy

[0030] The excised heart was fixed in 2.5% glutaraldehyde, then in 2% osmium tetroxide, embedded in resin, and sectioned. The ultrastructure of the heart tissue was studied using transmission electron microscopy.

[0031] Cell culture

[0032] 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.

[0033] Plasmid construction and transfection

[0034] 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.

[0035] Western blotting and immunoprecipitation

[0036] 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 α-MHC and α-MHC-K1897-lysine in mouse myocardial tissue was performed using 10 μg of protein extract, and analysis of α-SMA, Col-1, Cleaved-caspase 3, and Cleaved-PARP1 in mouse heart 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.

[0037] Statistical analysis

[0038] 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.

[0039] Through the above process, our study confirms that the α-MHC-K1897R mutation exacerbates Ang II-induced heart failure, suggesting that the α-MHC-K1897 site may be a key factor in regulating the severity of heart failure.

[0040] We used Ang II to induce 2 weeks of intervention in α-MHC-WT and α-MHC-K1897R mice to simulate mouse heart failure. Figure 1 a) It was observed that, compared with α-MHC-WT mice, α-MHC-K1897R mutant mice showed significantly reduced α-MHC-Titin interaction under both physiological and pathological conditions. Figure 1 (b, 1c). Next, we evaluated the physiological effects of the α-MHC-K1897R mutation. Echocardiography showed that, under physiological conditions, α-MHC-K1897R mutant mice had slightly lower ejection fraction (EF%) and fractional shortening (FS%) compared to α-MHC-WT mice. Under Ang II-induced heart failure conditions, α-MHC-K1897R mutant mice showed significantly impaired EF% and FS% compared to α-MHC-WT mice. Figure 1 d, 1e, 1f). Masson staining showed that, compared with α-MHC-WT mice, α-MHC-K1897R mutant mice under physiological conditions had mild myocardial fibrosis, while Ang II-induced α-MHC-K1897R mutant mice showed significantly aggravated myocardial fibrosis. Figure 1 (g, 1h). Electron microscopy revealed that, compared to α-MHC-WT mice, α-MHC-K1897R mutant mice exhibited slightly disordered myofibril arrangement and myofibril swelling. Ang II stimulation significantly exacerbated these changes. Figure 1i). We examined changes in cardiac hypertrophy using H&E staining and WGA staining and observed that Ang II caused significant cardiac hypertrophy in α-MHC-WT mice, while α-MHC-K1897R mutant mice showed only mild hypertrophic changes. Figure 1 At the molecular level, we evaluated fibrosis markers (α-SMA, Col-1) and myocardial apoptosis-related damage (Cleaved-PARP1, Cleaved-caspase3). In α-MHC-K1897R mutant mice, all these markers were slightly upregulated at baseline, but significantly upregulated in Ang II-induced heart failure. Figure 1 (lo). In summary, we hypothesize that the α-MHC-K1897R mutation may directly lead to heart failure, but without significant myocardial hypertrophy.

[0041] Example 2, verification that p300 can alleviate heart failure caused by site 1897.

[0042] Cell culture

[0043] 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.

[0044] Plasmid construction and transfection

[0045] All plasmids used in this study were confirmed by sequencing. Following the manufacturer's instructions, Flag-p300, Flag-CBP, Myc-GCN5, Flag-PCAF, and Myc-α-MHC plasmids were transfected into H9c2 and HEK293T cells using Lipofectamine 3000 (Invitrogen, US), HiGene (Applygen, China), or jetPRIME (Polyplus, France). Cells were collected 48–72 hours post-transfection.

[0046] Western blotting and immunoprecipitation

[0047] 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 α-MHC and α-MHC-K1897-lysine in mouse myocardial tissue was performed using 10 μg of protein extract, and analysis of α-SMA, Col-1, Cleaved-caspase3, 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.

[0048] Through the above experimental methods, we found that among the four major lactyltransferases p300, CBP, GCN5 and PCAF, only p300 can act as a lactyltransferase for α-MHC and significantly upregulate α-MHC lactylation.

[0049] Animal handling

[0050] In an angiotensin II infusion model, wild-type mice were intraperitoneally injected with either 30 mmol / kg of p300 activator (CTB) or 30 mmol / kg of p300 inhibitor C646 for 14 consecutive days, followed by anesthesia with inhaled isoflurane / oxygen (2%, ~1500 mL / min). Adequate depth of anesthesia was confirmed by the absence of paw withdrawal reflex after anesthesia of the mid-scapula. An osmotic micropump (Alzet) was incised and subcutaneously implanted according to the manufacturer's instructions. Animals were treated with Alzet (Alzet, model 2002; 0.5 μL / hour) via infusion of angiotensin II (3 mg / kg / day) for 14 days, a model of heart failure induced by cardiomyocyte damage and apoptosis. Blood pressure was measured daily using the tail cuff method (BP-2010 series sphygmomanometer, Softron, Japan). Effusion percent (EF%) and FS% (FS%) of the mouse heart were measured using ultrasound. Cervical dislocation following isoflurane inhalation was used to euthanize the mice. The effects of different treatment factors on the heart were assessed using endpoint blood pressure, left ventricular EF%, and left ventricular FS%. Animals were handled in accordance with the Animal Welfare Regulations of China Medical University (CMU2023020). All experiments involving animals were approved by the Animal Science Committee of China Medical University. This work was conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised in 1985).

[0051] Ethical approval

[0052] 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).

[0053] Immunohistochemistry of human heart

[0054] 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 SP IHC kit (MXB, China) was used for immunostaining. After blocking, slides were incubated with primary antibody against BNP (Abcam, USA) or P300 antibody at 37°C. After washing, sections were soaked in biotin-conjugated immunoglobulin G secondary antibody from an immunohistochemistry kit for 20 minutes, then soaked in DAB (MXB, China) for 2 minutes, and then stained with hematoxylin for 2 minutes. Through the above experimental methods, we found that p300 can alleviate heart failure caused by the 1897 site. In the rescue verification, p300 activator significantly enhanced α-MHC-K1897 lactation and α-MHC-Titin interaction. However, compared with Ang II stimulation alone, the combination of p300 activator and Ang II treatment partially rescued the lactylation modification of α-MHC-K1897 and the α-MHC–Titin interaction.

[0055] Statistical analysis

[0056] 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.

[0057] Given the crucial role of α-MHC-K1897 lactylation in heart failure, we set out to determine the enzymatic mechanism of α-MHC lactylation. We tested enzymes including p300, CBP, GCN5, and PCAF. Only p300 overexpression significantly upregulated α-MHC lactylation. No effect was observed with CBP, GCN5, or PCAF. Figure 3 a).

[0058] Then, we verified the interaction between p300 and α-MHC through immunoprecipitation at the cellular and tissue levels. Figure 2 a, Figure 2 b; Figure 3 b). We also found that the α-MHC class II, Spec, and mmCoA regions are necessary for interaction with p300. Figure 3 c). Furthermore, p300 overexpression leads to enhanced α-MHC lactation ( Figure 3 d). The degree of lactylation modification at α-MHC-WT and α-MHC-K1897 sites increased with p300 overexpression, while lactylation of α-MHC-C1897R remained low regardless of p300 expression levels. Figure 3 e, Figure S4f). We validated the results in H9c2 cells and in vivo using p300 activators and p300 inhibitors. In H9c2 cells, p300 activators enhanced the degree of α-MHC-K1897 lactation, while p300 inhibitors attenuated the degree of α-MHC-K1897 lactation (e, Figure S4f). Figure 2 c, Figure 2 d). Similar results were observed when p300 activators or inhibitors were administered intraperitoneally to mice for 2 weeks. Figure 2 e, Figure 2 f).

[0059] Next, we sought to explore the role of p300 in regulating α-MHC lactylation modification in heart failure and myocardial injury. We found that p300 expression was upregulated in cardiac tissues of heart failure patients and mice, and also upregulated in Ang II-induced H9c2 cells in vitro. Figure 3 gm), but regardless of Ang II stimulation, the interaction between p300 and α-MHC did not show any change in H9c2 cells and mouse myocardial tissue. Figure 2 g, Figure 2 h). To analyze the effects of p300 on α-MHC-K1897 lactation and α-MHC-Titin interaction in Ang II-induced heart failure, we used p300 inhibitors and p300 activators in vitro and in vivo. H9c2 cells and mouse myocardial tissue treated with p300 inhibitors (h). Figure 2i) showed reduced baseline α-MHC-K1897 lactation and reduced α-MHC-Titin interaction (i) Figure 2 j). Ang II reduced the degree of α-MHC-K1897 lactation and the interaction between α-MHC and Titin. Compared with Ang II stimulation alone, combined stimulation with p300 inhibitors significantly reduced the degree of α-MHC-K1897 lactation and the interaction between α-MHC and Titin. Figure 2 jl). In the p300 activator experimental group ( Figure 2 Both cellular and animal results showed that p300 activator significantly enhanced α-MHC-K1897 lactation and α-MHC-Titin interaction. Ang II stimulation downregulated α-MHC-K1897 lactation and Titin binding to α-MHC. However, compared with Ang II stimulation alone, the combination of p300 activator and Ang II stimulation partially rescued α-MHC-K1897 lactation and α-MHC–Titin interaction. Figure 3 These results indicate that p300 is a lactyltransferase of α-MHC-K1897. However, p300 is not central to the reduction of α-MHC-K1897 lactation in heart failure.

[0060] Example 3 is a verification of how lactase can alleviate heart failure caused by site 1897.

[0061] Cell culture

[0062] 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.

[0063] Plasmid construction and transfection

[0064] All plasmids used in this study were confirmed by sequencing. Following the manufacturer's instructions, Flag-SIRT1, Flag-SIRT2, Flag-SIRT3, Flag-SIRT4, Flag-SIRT5, Flag-SIRT6, Flag-SIRT7, and Myc-α-MHC plasmids were transfected into H9c2 and HEK293T cells using Lipofectamine 3000 (Invitrogen, US), HiGene (Applygen, China), or jetPRIME (Polyplus, France). Cells were collected 48–72 hours post-transfection.

[0065] Western blotting and immunoprecipitation

[0066] 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 α-MHC and α-MHC-K1897-lysine in mouse myocardial tissue was performed using 10 μg of protein extract, and analysis of α-SMA, Col-1, Cleaved-caspase3, 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 using tubulin assays. Antibodies were used at a 1:1000 dilution to detect protein expression in Western blotting and immunoprecipitation. Using these methods, we found that among the seven major delactyltransferases SIRT1-7, only SIRT1 can act as a delactyltransferase of α-MHC, significantly downregulating α-MHC lactylation.

[0067] Animal handling

[0068] In an angiotensin II infusion model, wild-type mice were intraperitoneally injected with 20 mg / kg of the Sirtuin 1 activator SRT1270 or 1 mg / kg of the Sirtuin 1 inhibitor EX527 for 14 consecutive days, followed by anesthesia with inhaled isoflurane / oxygen (2%, ~1500 mL / min). Adequate depth of anesthesia was confirmed by the absence of paw withdrawal reflex after anesthesia of the mid-scapula. An osmotic micropump (Alzet) was incised and subcutaneously implanted according to the manufacturer's instructions. Animals were treated with Alzet (Alzet, model 2002; 0.5 μL / hour) via angiotensin II infusion (3 mg / kg / day) for 14 days, a model of heart failure induced by cardiomyocyte damage and apoptosis. Blood pressure was measured daily using the tail cuff method (BP-2010 series sphygmomanometer, Softron, Japan). Cardiac EF% and FS% were measured using ultrasound. Cervical dislocation following isoflurane inhalation was used to euthanize the mice. The effects of different treatment factors on the heart were assessed using endpoint blood pressure, left ventricular EF%, and left ventricular FS%. Animals were handled in accordance with the Animal Welfare Regulations of China Medical University (CMU2023020). All experiments involving animals were approved by the Animal Science Committee of China Medical University. This work was conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised in 1985).

[0069] Immunohistochemistry of human heart

[0070] 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 TMThe SP IHC kit (MXB, China) was used for immunostaining. After blocking, slides were incubated with primary antibody against BNP (Abcam, USA) or P300 antibody at 37°C. After washing, sections were soaked in biotin-conjugated immunoglobulin G secondary antibody from an immunohistochemistry kit for 20 minutes, then soaked in DAB (MXB, China) for 2 minutes, and then stained with hematoxylin for 2 minutes. Using the above experimental methods, we found that SIRT1 can alleviate heart failure caused by the 1897 site. In the rescue verification, SIRT1 inhibitors significantly enhanced α-MHC-K1897 lactation and α-MHC-Titin interaction. However, compared with Ang II stimulation alone, the combination of SIRT1 inhibitor and Ang II treatment partially rescued the lactylation modification of α-MHC-K1897 and the α-MHC–Titin interaction.

[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] Our results indicate that intervention with p300 lactyltransferase activators failed to rescue the degree of α-MHC-K1897 lactation and the interaction between α-MHC and Titin in an Ang II-induced heart failure mouse model. To further elucidate the regulatory mechanism, we attempted to identify the relevant α-MHC-K1897 delay enzymes. Non-histone deacetylation is primarily dependent on the sirtuins family;21 Therefore, we selected the Sirtuin enzyme family (SIRT1–7) as candidate proteins for α-MHC delay enzyme activity. We found that only SIRT1 expression significantly reduced the degree of α-MHC lactation ( Figure 5 a, Figure S5b).

[0074] Subsequently, we verified the interaction between SIRT1 and α-MHC in vivo and in vitro using immunoprecipitation.Figure 4 a, Figure 4 b; Figure 5 c). We identified the α-MHC domains interacting with SIRT1 as mmCoA, MITCorA, and TMPIT ( Figure 5 d). Consistent with the above results, overexpression of SIRT1 reduced the degree of α-MHC lactylation ( Figure 5 e, Figure S5f). Similarly, treatment of cells with SIRT1 activators reduced the degree of α-MHC lactation, while treatment with SIRT1 inhibitors (which interacted with α-MHC lactation) Figure 5 g, Figure S5h). Next, we used anti-Pan-Kla antibody and anti-α-MHC-K1897 lactyl lysine antibody to investigate SIRT1 delay at α-MHC-C1897. Overexpression of SIRT1 or addition of SIRT1 activator reduced lactylation at the α-MHC-K1897 site (g, Figure S5h). Figure 5 SIRT1 inhibitors increased lactylation at the K1897 site (auxiliary figure S5l). We observed similar results in cardiomyocytes treated with either SIRT1 activators or inhibitors. Figure 4 c, Figure 4 d). Myocardial examination showed similar results 2 weeks after intraperitoneal administration of SIRT1 activators or inhibitors. Figure 4 e, Figure 4 f).

[0075] Further supporting the role of SIRT1-regulated α-MHC lactylation modification, we found that SIRT1 expression was decreased in cardiac tissues of heart failure patients and mice, as well as in Ang II-induced H9c2 cells in vitro. Figure 5 ms). Ang II stimulation attenuated the interaction between SIRT1 and α-MHC in cardiomyocytes and myocardial tissue. Figure 4 g, Figure 4 To further investigate the effects of SIRT1 on α-MHC-K1897 lactation and α-MHC-Titin interaction in Ang II-induced heart failure, cardiomyocytes were treated in vitro and in vivo with SIRT1 activators and SIRT1 inhibitors. As expected, in H9c2 cells and mouse heart tissue, treatment with either SIRT1 activator or Ang II downregulated lactation at the α-MHC-K1897 site and reduced α-MHC–Titin interaction. Compared with the Ang II stimulation group, administration of Ang II in combination with a SIRT1 activator attenuated the degree of lactation at the α-MHC-K1897 site and reduced α-MHC–Titin interaction. Figure 4Conversely, the addition of a SIRT1 inhibitor led to upregulation of α-MHC-K1897 lactation and increased α-MHC-Titin interaction. However, when Ang II stimulation was combined with a SIRT1 inhibitor and compared with Ang II stimulation alone, the SIRT1 inhibitor partially rescued the reduction in α-MHC-K1897 lactation and α-MHC-Titin interaction. Figure 4 (mp).

[0076] These results indicate that SIRT1 is a delayed octylase of α-MHC-K1897. However, SIRT1 is not the underlying cause of decreased α-MHC-K1897 lactation in patients with heart failure.

Claims

1. The application of an MHC-regulating lactation agent in the preparation of drugs for diseases related to promoting the level of lactation in the body, characterized in that: Application of MHC lactation agents in the preparation of drugs for heart failure; The heart failure lesion described is heart failure caused by decreased α-MHC-K1897 lactation due to vasoconstriction induced by Ang II; The agent regulating MHC lactation is p300 lactyltransferase or SIRT1 delacyltransferase.

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