Application of NSun2 gene or its encoded protein in the treatment of pressure-overload heart failure

Abstract

The present invention relates to the field of biomedicine technology, and to the application of NSun2 gene or the protein encoded by it in the treatment of pressure-load heart failure. The present invention provides a drug for treating pressure-load heart failure, wherein the drug contains NSun2 gene, NSun2 protein or NSun2 gene expression promoter; the promoter is a substance that increases the expression amount of NSun2 gene, and further, the promoter contains an expression vector carrying a nucleotide sequence encoding NSun2 protein or a recombinant virus containing a nucleotide sequence encoding NSun2 protein. The present invention first discovered that NSun2 is down-regulated in the myocardial cells of pressure-load heart failure, and by overexpressing the NSun2 gene in the myocardial cells of pressure-load heart failure, myocardial cell apoptosis can be slowed down and cardiac function can be effectively restored. Therefore, NSun2 can be used as a target gene in drug or gene therapy, and applied to the prevention, relief or / and treatment of pressure-load heart failure.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to application of NSun2 gene or protein encoded by it in treating pressure-overload heart failure. Background Art

[0002] Heart failure (HF) is the final result of various cardiovascular events and the cumulative effect of various cardiac abnormalities, which eventually leads to a decline in the heart's pumping function. Primary myocardial damage and abnormal cardiac load are the main causes of HF. The abnormal cardiac load that can lead to HF mainly includes pressure load and volume load. Among them, excessive pressure load (afterload) is common in diseases such as hypertension and aortic / pulmonary valve stenosis that cause increased ventricular ejection resistance; excessive volume load (preload): seen in valvular heart disease or diseases that cause systemic hyperdynamic state (anemia, renal failure, sepsis and pregnancy, etc.). In addition, constrictive pericarditis, pericardial effusion and even excessive iatrogenic fluid infusion can also cause heart failure.

[0003] In the adult heart, cardiomyocytes remain undifferentiated. In response to increased cardiac load, individual cardiomyocytes no longer proliferate but instead enlarge, and the heart hypertrophies to reduce ventricular wall tension and maintain cardiac output to cope with increased cardiac load. Cardiac hypertrophy is initially induced as an adaptive response to various pathological stimuli, such as chronic hypertension, myocardial infarction, aortic stenosis, mitral regurgitation, storage diseases, and mutations in genes encoding myofibrillar proteins (hypertrophic cardiomyopathy, HCM). Unless these stresses are promptly eliminated at an early stage, the hypertrophic heart can easily enter a state of decompensation, ultimately leading to heart failure.

[0004] It is generally believed that pathological conditions promote cardiac hypertrophy and remodeling mainly through neuroendocrine hormones and mechanical forces, accompanied by the activation of a large number of downstream effector pathways, and PKA signaling plays an important role in regulating cardiac performance and morphology. Under hypertrophic stress, PKA phosphorylates target proteins in discrete microdomains to increase cardiac volume and improve myocardial contraction and relaxation.

[0005] NSun2 was originally considered to be a tRNA cytosine methyltransferase, and its ability to methylate mRNA (5-methylcytosine, m5C) has been widely studied, affecting almost all aspects of RNA metabolic processes, including mRNA maturation, translation, and degradation. However, it is still unknown whether NSun2 is involved in the adaptive remodeling of the myocardium and the regulation of myocardial function under cardiac pressure overload. Summary of the invention

[0006] In view of the problems and shortcomings in the prior art, the present invention aims to provide an application of the NSun2 gene or the protein encoded by it in the treatment of pressure-overload heart failure.

[0007] To achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides the use of NSun2 gene or the protein encoded by it in the preparation of a drug for preventing, alleviating or / and treating pressure-overload heart failure.

[0009] The second aspect of the present invention provides the use of a promoter of NSun2 gene expression in the preparation of a drug for preventing, alleviating or / and treating pressure-overload heart failure.

[0010] According to the above application, preferably, the promoter is a substance that increases the expression level of the NSun2 gene.

[0011] According to the above application, preferably, the promoter comprises an expression vector carrying a nucleotide sequence encoding NSun2 protein.

[0012] According to the above application, preferably, the promoter comprises a recombinant virus containing a nucleotide sequence encoding NSun2 protein. More preferably, the recombinant virus is an adeno-associated virus overexpressing the NSun2 gene. Most preferably, the recombinant virus is an adeno-associated virus type 9 overexpressing the NSun2 gene.

[0013] The third aspect of the present invention provides a drug for treating pressure-overload heart failure, wherein the drug contains NSun2 gene, NSun2 protein or NSun2 gene expression promoter.

[0014] According to the above-mentioned medicine, preferably, the promoter is a substance that increases the expression level of NSun2 gene.

[0015] According to the above-mentioned drug, preferably, the promoter comprises an expression vector carrying a nucleotide sequence encoding NSun2 protein or a recombinant virus containing a nucleotide sequence encoding NSun2 protein.

[0016] According to the above-mentioned medicine, preferably, the recombinant virus is an adeno-associated virus overexpressing the NSun2 gene. More preferably, the recombinant virus is an adeno-associated virus type 9 overexpressing the NSun2 gene.

[0017] According to the above-mentioned medicine, preferably, the medicine also contains a pharmaceutically acceptable carrier / excipient.

[0018] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0019] The present invention first discovered that NSun2 is downregulated in myocardial cells of pressure-load heart failure. By overexpressing the NSun2 gene in myocardial cells of pressure-load heart failure, the ejection fraction in the heart failure stage can be increased, myocardial cell apoptosis can be slowed down, and cardiac function can be effectively restored. Therefore, NSun2 can be used as a target gene in drug or gene therapy, and applied to the prevention, relief and / or treatment of pressure-load heart failure, which can provide a new strategy for the prevention and treatment of pressure-load heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of HE and Masson staining of the hearts of mice after TAC induction treatment; A is the result of HE staining; B is the result of Masson staining; Sham represents the sham operation group, and TAC represents TAC induction treatment;

[0021] Figure 2 The Western Blot test results of NSun2 expression in myocardial cells of mice with TAC-induced heart failure are shown in Figure 1, where A is the Western Blot test result 4 weeks after TAC surgery; B is the Western Blot test result 16 weeks after TAC surgery; Figure C is the statistical result of A and B;

[0022] Figure 3 To construct the schematic diagram of NSun2 cKO mice;

[0023] Figure 4 NSun2 cKO mice flox / flox Western Blot detection results of NSun2 expression level in mouse cardiomyocytes; NSun2 cKO represents NSun2 cKO mice, NSun2f / f represents NSun2 flox / flox Mice;

[0024] Figure 5 The results of ultrasound cardiography detection of NSun2 cKO mice; NSun2 cKO means NSun2 cKO mice, and NSun2f / f means NSun2 flox / flox Mice;

[0025] Figure 6 HE staining of the heart of NSun2 cKO mice, where NSun2 cKO means NSun2 cKO mice, and NSun2f / f means NSun2 flox / flox Mice;

[0026] Figure 7 The results of ultrasound cardiology testing of NSun2 cKO mice 4 weeks after TAC induction treatment;

[0027] Figure 8 The results of Western Blot detection of NSun2 expression in cardiomyocytes of mice in the FF group, FT group, and CT group; A is the expression level of NSun2, MYH7, Caspase3, and GAPDH detected by Western Blot, and B is the statistical result of A;

[0028] Fig. 9 HE staining results of the hearts of mice in the FF group, FT group, and CT group;

[0029] Fig.10 The results of Masson staining of the hearts of mice in the FF, FT, and CT groups;

[0030] Fig.11 The results of the analysis of the methylation modification of PRKACA gene mRNA by NSun2 and its effect on its expression and PKA activity are shown in Figure 1. A is the result of screening differentially expressed protein-coding genes and performing GO analysis and functional annotation. B is the result of visualizing the m5C peaks enriched in PRKACA in different groups using GV software. C is the result of detecting the methylation level of PRKACA using m5C-RIP. D is the result of detecting the protein expression level of PRKACA using Western Blot. E is the result of detecting the activity of PKA. **, P≤0.01.

[0031] Fig.12 These are the results of ultrasound cardiology testing of mice in the CT4, CT16, and NT16 groups;

[0032] Fig.13 Figure 2 is the detection of apoptosis in mouse heart tissue by TUNEL staining and its statistical results, wherein A is the image of cell apoptosis by TUNEL staining, and B is the statistical result of cell apoptosis by TUNEL staining. DETAILED DESCRIPTION

[0033] The following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0034] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. In addition, it should also be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates that there are features, steps, operations, parts and / or combinations thereof.

[0035] The experimental methods in the following examples without specifying specific conditions all adopt conventional techniques in the art or follow the conditions recommended by the manufacturers; the reagents or instruments used without specifying the manufacturers are all conventional products that can be obtained commercially.

[0036] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0037] Example 1: Study on the expression of NSun2 in myocardial cells of TAC-induced heart failure mouse model

[0038] Western Blot was used to detect the expression level of NSun2 in cardiomyocytes of mice with TAC-induced heart failure.

[0039] 1. Selection and grouping of experimental mice

[0040] Wild-type c57 mice aged 8-10 weeks (weight: 20-25 g) were purchased from Gempharmatech Technology Co., Ltd. (Nanjing, Jiangsu, China) and received adaptive feeding for 1 week before the start of the study. All mice were housed in individual ventilated cages under specific (temperature: 20-25°C; humidity: 50±5%) barrier conditions.

[0041] The experimental mice were randomly divided into TAC-induced heart failure group and sham operation group, with 6 mice in each group. The mice in TAC-induced heart failure group underwent TAC surgery, and the mice in sham operation group (Sham) underwent the same surgery but without ligation.

[0042] 2. Construction of TAC-induced heart failure mouse model

[0043] The mice in the TAC-induced heart failure group underwent TAC surgery to construct a mouse model of TAC-induced heart failure. The specific operation steps were as follows: the mice were anesthetized by intraperitoneal injection of ketamine / xylazine (55 mg / kg / 3.3 mg / kg). When the mice were deeply anesthetized, the skin in front of the trachea was incised and the muscle tissue was separated. The second rib was gently lifted and incised, pulled to both sides, and the aortic arch was fully exposed. The 26-gauge needle was tightened and ligated with a 6-0 nylon suture to complete the transverse aortic constriction between the right innominate and left common carotid arteries, and then the sternum and skin were sutured. Doppler echocardiography showed that the transaortic transection speed was greater than 4 m / s, confirming the success of the TAC surgery. The mice were transferred to a heating pad and closely monitored. When the mice regained consciousness, buprenorphine (1 mg / kg subcutaneous injection) was given twice a day for 2 consecutive days. All operators and analysts were blindly randomized to avoid mouse genotype preference.

[0044] The mice in the sham group underwent the same surgery but without ligation.

[0045] 3. HE and Masson staining of mouse hearts after TAC induction

[0046] The hearts of mice were harvested 4 and 16 weeks after successful TAC surgery and processed for HE and Masson staining.

[0047] The specific operation of HE and Masson staining is as follows: fix the mouse heart with 4% paraformaldehyde at room temperature, perform paraffin sectioning within 24 hours or cryosectioning within 2 hours. After dehydration, embed it with paraffin (paraffin section) or OCT (cryosection), and slice it continuously along the short axis by 5 μm until the maximum short axis cross-section of the heart is reached. HE and Masson staining were used to evaluate the heart morphology and degree of fibrosis. After scanning using the Olympus digital scanning imaging system FV1000 (Olympus, Tokyo, Japan), the captured images were quantitatively analyzed using Image J software (NIH, Bethesda, MD, USA). The specific results are as follows Figure 1 shown.

[0048] Depend on Figure 1 It can be seen that four weeks after TAC surgery, the mouse hearts showed symptoms of ventricular wall thickening and the hearts entered the stage of myocardial hypertrophy; 16 weeks after TAC surgery, the mouse hearts showed symptoms of ventricular wall thinning and ventricular cavity enlargement and the hearts entered the stage of heart failure.

[0049] 4. Western Blot detection of NSun2 expression in myocardial cells of mouse heart tissue after TAC induction

[0050] (1) Isolation of mouse cardiomyocytes:

[0051] The hearts of mice were removed 4 and 16 weeks after successful TAC surgery, and the cardiomyocytes of mice were isolated using a Langendorff perfusion apparatus. The specific steps are as follows:

[0052] Mice were intraperitoneally injected with 1000U / kg of heparin 30min before the start of surgery. Subsequently, they were anesthetized with 5% isoflurane and killed by cervical dislocation. The heart, including about 2-3mm of the aorta, was removed and placed in a Langendorff perfusion system. The heart was perfused with calcium-free medium (135mM NaCl, 4mM KCl, 1mM MgCl2, 10mM HEPES, 0.33mM NaH2PO4, 10mM D-(+)-Glucose, 10mM BDM, 5mM taurine, adjusted to pH 7.4), and enzymatic cardiomyocyte isolation buffer (calcium-free medium, 525U / ml type II collagenase and 0.67g / L BSA) at 37° for 10-15min. Once the heart reached a soft, swollen, pink state, the heart was removed from the cannulated syringe needle, excess tissue and blood vessels were removed, and the left ventricle was cut into small pieces in enzymatic cardiomyocyte isolation buffer. Gently pump the pipette tip up and down until all cells are separated. Filter the cell suspension through a 100-mesh cell strainer. Let it rest for 10 minutes, repeat the filtration 3 times, and collect the naturally settled cardiomyocytes.

[0053] (2) Western Blot

[0054] The specific steps for detecting NSun2 expression in cardiomyocytes by Western Blot are as follows:

[0055] 1) Protein extraction and quantification

[0056] Discard the culture medium in the cells, wash the cell surface twice with PBS, digest with trypsin or collect the cells into a centrifuge tube with a cell scraper, and centrifuge at 4°C, 3000 rpm for 5 minutes; discard the supernatant, add 200 μl of RIPA lysis buffer (containing 100× cocktail) (taking 6-well plate as an example), lyse on ice for 30 minutes, and shake and mix every 10 minutes; centrifuge at 4°C, 12000 rpm for 10 minutes, and discard the precipitate.

[0057] After the protein sample was diluted at a certain multiple, 25 μl was taken and placed in a 96-well plate; another 25 μl H2O was taken as a blank control, and 25 μl was taken from 5 different concentrations of bovine serum albumin standard solutions (125 μg / ml, 250 μg / ml, 500 μg / ml, 1000 μg / ml, 2000 μg / ml) and added to the 96-well plate, and then 200 μl of BCA reaction mixture (A solution: B solution = 50:1) was added to each well, incubated at 37°C for 30 minutes, and the absorption peak at 570 nm was detected on an ELISA instrument. The standard curve was fitted according to the readings of protein standard solutions of different concentrations, and then the protein concentration of the sample was calculated according to the absorbance value of the sample.

[0058] 2) SDS-PAGE electrophoresis

[0059] According to the protein concentration, take an appropriate amount of cell lysate, add 6×SDS protein loading buffer (100mM Tris-HCl, 200mM DTT pH=6.8, 4% SDS, 0.01% bromophenol blue, 20% glycerol), mix well, incubate at 95℃ for 5 minutes, load the sample, and prepare for electrophoresis. The electrophoresis system is as follows:

[0060] Table 1 Composition of concentrated gel

[0061] Stacking gel: 4ml 30% Acr-Bis (Acr:Bis=29:1) 0.67ml 1M Tris-HCl, pH 6.8 0.5ml 10% SDS 0.04ml 10% AP 0.04ml TEMED 0.004ml <![CDATA[dddH 2 The]]> 2.7ml

[0062] Table 2 Separation gel composition

[0063] Separation gel: 10ml 30% Acr-Bis (Acr:Bis=29:1) 4ml 1.5M Tris-HCl, pH 8.8 2.5ml 10% SDS 0.1ml 10% AP 0.1ml TEMED 0.005ml <![CDATA[dddH 2 The]]> 3.3ml

[0064] Table 3 5×Tris-glycine electrophoresis buffer composition

[0065] Tris 15.1g Glycine 94g 10% SDS 50ml <![CDATA[Add ddH 2 O to]]> 1000ml

[0066] The samples were electrophoresed at 10 V / cm in the stacking gel and 15 V / cm in the separation gel until the bromophenol blue was 1 cm from the bottom of the gel.

[0067] 3) Wet transfer

[0068] Take out the gel, remove the concentrated gel part, cut 4 filter papers of the same size as the gel to be transferred and 1 nitrocellulose membrane (NC membrane), soak the membrane in transfer buffer (48mM Tris, 39mM glycine, 20% methanol) for more than 5 minutes. Stack 4 filter papers soaked with transfer buffer neatly on the cathode plate, and align the gel on it. Cover the membrane on the gel, mark it, place another 4 soaked filter papers on the membrane, cover the anode plate, and pay attention to expel bubbles during the whole process. According to the molecular weight of the protein, use 250mA constant current transfer for 90-120 minutes. Remove the nitrocellulose membrane and perform the following hybridization reaction.

[0069] 4) Hybridization and result processing

[0070] The transferred NC membrane was rinsed with TBST (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% Tween 20) for 5 minutes. The membrane was blocked with TBST containing 5% skim milk powder for 1 hour on a shaker at room temperature; rinsed with TBST twice, and primary antibodies (concentration 0.2 μg / ml) NSun2 (#36103S, Cell Signaling Technology, Beverly, MA, USA)), MYH7 (#sc-53090, Santa Cruz, CA, USA), BNP (A2179, Abclonal, Wuhan, Hubei, China), GAPDH (#AC001, Abclonal, Wuhan, Hubei, China) were added according to the antibody instructions, and incubated overnight at 4°C. Rinse with TBST for 5 minutes × 4 times at room temperature, add the corresponding HRP-labeled secondary antibody (1:2000) or fluorescent labeled secondary antibody (1:2000), and incubate at room temperature for 1 hour in the dark. Rinse with TBST for 5 minutes × 4 times, expose in the dark room with ECL supersensitive luminescent liquid or scan with Odyssey two-color infrared laser imaging system. Use Image-J software to perform grayscale scanning and analysis of Western Blot exposure results.

[0071] Western Blot results of NSun2 in mouse cardiomyocytes are shown in Figure 2. Figure 2 shown.

[0072] Depend on Figure 2 It can be seen that 4 weeks after TAC surgery, the mouse heart was in the stage of myocardial hypertrophy, at which time, the expression of NSun2 and myocardial hypertrophy marker MYH7 in mouse cardiomyocytes increased; 16 weeks after TAC surgery, the mouse heart was in the stage of heart failure, at which time, the expression of NSun2 and myocardial hypertrophy marker MYH7 in mouse cardiomyocytes decreased. This shows that NSun2 is downregulated in cardiomyocytes of pressure-overload heart failure.

[0073] Example 2: Study on the knockout of Nsun2 gene in mouse cardiomyocytes and the effect of knockout on the myocardium

[0074] 1. Mouse selection:

[0075] NSun2 flox / flox Mice were grown on a C57 / BL6 background using CRISPR / Cas9 targeting and homology-directed repair from Cyagen Biosciences (Suzhou, Jiangsu, China). ERT2They were purchased from Gempharmatech Technology Co., Ltd. (Nanjing, Jiangsu, China) and received adaptive feeding for 1 week before the start of the study. All mice were housed in individual ventilated cages under specific (temperature: 20-25°C; humidity: 50±5%) barrier conditions.

[0076] 2. Construction of cardiomyocyte Nsun2 gene knockout mouse (NSun2 cKO mouse) model:

[0077] NSun2 flox / flox / Myh6-Cre ERT2 (NSun2 cKO) mice were expressed through NSun2 flox / flox Mice and Myh6-Cre ERT2 Mating and breeding between mice (eg Figure 3 The genotyping was verified by PCR amplification. At 8 weeks of age, NSun2 cKO mice with conditional knockout of NSun2 gene were intraperitoneally injected with 75 mg / kg tamoxifen (T-5648, Sigma) every day for 5 consecutive days to activate the cre recombinase in male mice.

[0078] 3. Western Blot detection of Nsun2 expression in cardiomyocytes of NSun2 cKO mice:

[0079] Four weeks after the last injection of tamoxifen, cardiomyocytes of NSun2 cKO mice were isolated using a Langendorff perfusion apparatus. The specific operation was the same as in Example 1 and will not be described again.

[0080] Western Blot was used to detect the expression of NSun2 cKO mice and NSun2 flox / flox Expression level of NSun2 in mouse cardiomyocytes The specific operation steps of Western Blot detection are the same as those in Example 1 and will not be repeated here.

[0081] Western Blot test results are as follows Figure 4 shown.

[0082] Depend on Figure 4 It can be seen that NSun2 is almost completely absent in the myocardium of NSun2 cKO mice, indicating that the NSun2 cKO mouse model was successfully established.

[0083] 4. Echocardiography of NSun2 cKO mice:

[0084] The specific operation of ultrasonic cardiology detection of NSun2 cKO mice is the same as that in Example 3, and will not be repeated here. Figure 5 shown.

[0085] Depend on Figure 5 It can be seen that, with NSun2 flox / flox Compared with mice, knockout of NSun2 did not affect cardiac function or ventricular wall thickness, diameter, and HW / TL index in mice.

[0086] 5. HE staining of NSun2 cKO mouse heart:

[0087] The specific experimental method of HE staining of NSun2 cKO mouse heart is the same as that in Example 1, which will not be repeated here. Figure 6 shown.

[0088] Depend on Figure 6 It can be seen that after the NSun2 gene was knocked out in the mouse heart, the heart size and morphology were almost unchanged. The above experiments show that the heart-specific knockout of NSun2 does not affect the size of mouse cardiomyocytes or heart function.

[0089] Example 3: Study on the effect of TAC induction on the myocardium of NSun2 cKO mice

[0090] NSun2 cKO mice were treated with TAC to study the compensatory response of myocardial hypertrophy after knockout of NSun2 gene in mouse cardiomyocytes.

[0091] 1. Mouse selection and experimental grouping:

[0092] The NSun2 cKO mice and NSun2 flox / flox The experiment set up three groups, namely FF group, FT group, and CT group, with 9 mice in each group; the FF group used NSun2 cKO mice as experimental subjects and subjected NSun2 cKO mice to TAC induction treatment; the FT group used NSun2 flox / flox Mice were used as experimental subjects, and NSun2 flox / flox Mice were treated with TAC; the CT group was treated with NSun2 flox / flox Mice were used as experimental subjects, but NSun2 flox / flox Mice were treated with TAC induction and sham surgery only (sham surgery means that mice underwent the same surgery but without ligation).

[0093] 2. TAC induction treatment:

[0094] One week after the last tamoxifen injection in NSun2 cKO mice, TAC surgery was performed on NSun2 cKO mice. The specific operation was as follows:

[0095] Mice were anesthetized by intraperitoneal injection of ketamine / xylazine (55 mg / kg / 3.3 mg / kg). When the mice were deeply anesthetized, the skin in front of the trachea was incised and the muscle tissue was separated. The second rib was gently lifted and incised and pulled to both sides to fully expose the aortic arch. A 26-gauge needle was tightened and ligated with a 6-0 nylon suture to complete the transverse aortic constriction between the right innominate and left common carotid arteries, and then the sternum and skin were sutured. Doppler echocardiography showed a transaortic transection velocity greater than 4 m / s, confirming the success of the TAC procedure. The mice were transferred to a heating pad and closely monitored. When the mice regained consciousness, buprenorphine (1 mg / kg subcutaneous injection) was given twice a day for 2 consecutive days. All operators and analysts were blinded to randomize to avoid mouse genotype preference.

[0096] 3. Echocardiographic examination of the hearts of NSun2 cKO mice after TAC induction:

[0097] Four weeks after TAC, echocardiography was performed on mice induced by TAC using a Vevo 3100 system with an MS400C probe. The systolic and diastolic ventricular diameters and wall thicknesses were determined by measuring the two-dimensional muscle pattern at the level of the papillary muscles. The ejection fraction (EF), short-axis contraction ratio (FS), ventricular wall thickness (LVAW, s left ventricular anterior wall thickness at end-systole; LVPW, s left ventricular posterior wall thickness at end-systole) and heart weight / tibia length ratio (HW / TL), as well as ventricular cavity size (LVID, s left ventricular diameter at end-systole; LVID, d left ventricular diameter at end-diastole) were calculated. Specific results are shown in Table 1. Figure 7 shown.

[0098] Depend on Figure 7 It can be seen that after NSun2 knockout, the heart enters the decompensation stage prematurely, and the ejection fraction and short-axis contraction rate decrease significantly, indicating that the heart enters the heart failure stage.

[0099] 4. Western Blot Detection

[0100] Four weeks after TAC surgery, 6 mice were randomly selected from each of the FF, FT, and CT groups, and the cardiomyocytes of the 18 mice selected from the FF, FT, and CT groups were separated using a Langendorff perfusion instrument, and the expression levels of NSun2, MYH7, Caspase3, and GAPDH in the cardiomyocytes of the mice were detected by Western Blot. The specific operation of separating the cardiomyocytes of the FF, FT, and CT group mice using a Langendorff perfusion instrument is the same as in Example 1, and will not be repeated here; the specific operation steps of Western Blot detection are the same as in Example 1, and will not be repeated here.

[0101] Western Blot test results are as follows Figure 8 shown.

[0102] Depend on Figure 8 It can be seen that after knocking out NSun2, the myocardial hypertrophy-related marker MYH7 was significantly reduced, and cardiac function entered the decompensation stage.

[0103] 5. HE staining and Masson staining of mouse heart:

[0104] Four weeks after TAC surgery, the hearts of mice in the FF, FT, and CT groups were stained with HE and Masson staining. The specific experimental methods of HE staining and Masson staining were the same as those in Example 1 and will not be repeated here. Fig. 9 The results of Masson staining of mouse heart are shown in Fig.10 shown.

[0105] Depend on Fig. 9 It can be seen that in the case of NSun2 gene knockout, TAC-induced treatment does not induce cardiac hypertrophy response.

[0106] Depend on Fig.10 It can be seen that myocardial fibrosis is aggravated after knocking out NSun2.

[0107] The above experimental results indicate that myocardial knockout of NSun2 will aggravate the development of heart failure under conditions of sustained pressure overload.

[0108] Example 4: Study on the mechanism of NSun2 regulating cardiomyocyte hypertrophy and heart failure

[0109] m5C-RIP sequencing was used to detect the mRNA m5C methylation level of PRKACA in the cardiomyocytes of mice in the FF group, FT group, and CT group in Example 3.

[0110] 1. m5C-RIP-seq detection:

[0111] Four weeks after TAC surgery, 6 mice were randomly selected from each of the FF, FT, and CT groups. The cardiomyocytes of 18 mice selected from the FF, FT, and CT groups were isolated using a Langendorff perfusion apparatus, and total RNA was extracted from the Langendorff-isolated cardiomyocytes using Trizol reagent (Invitrogen, #15596026). RNA was extracted with DNase I and then DNA was digested. A260 / A280 was detected using a NanodropTMOneC spectrophotometer (Thermo Fisher Scientific) to detect RNA quality. RNA integrity (RQN) was confirmed using Qsep 100 (BIOptic Inc). 200 μg of total RNA was enriched for polyadenylated RNA (VAHTS, cat. NO. N401-01 / 02) using VAHTS mRNA capture beads. 20 mM ZnCl 2 Add to mRNA and incubate at 95°C for 5-10 minutes until the RNA fragments are mainly distributed within 100-200nt. Then save 10% of the RNA fragments as "input" and the rest for m5C immunoprecipitation. Use specific anti-m5C antibody (Abcam, ab10805) for m5C immunoprecipitation. Use Trizol reagent to prepare RNA samples for input and IP. Screen differentially expressed protein-encoding genes through MeRIP experiments and high-throughput sequencing, and perform GO analysis and functional annotation of differentially expressed proteins. The results are as follows Fig.11 Shown in A. MeRIP experiments and high-throughput sequencing and data analysis were performed by Seqhealth Technology Co., Ltd. (Wuhan, China).

[0112] Depend on Fig.11 As shown in Figure A, NSun2 methylates PRKACA gene mRNA, and knockout of NSun2 inhibits PRKACA protein expression and PKA activity.

[0113] The PRKACA-enriched m5C peaks of different groups were visualized using IGV software, as shown in Fig.12 As shown in B.

[0114] Depend on Fig.11 As shown in Figure B, compared with the control group FF, TAC induction increased the m5C methylation level of the PRKACA gene, while NSun2 gene knockout significantly reduced the enrichment of the TAC-induced PRKACA gene m5C peak.

[0115] 2. Verify methylation on PRKACA mRNA using m5C-RIP-qPCR:

[0116] Four weeks after TAC, total RNA from cardiomyocytes isolated from mice in the FT and CT groups (three mice in each group) was treated with DNaseI (ThermoFisher Scientific), cut into 100-200 nucleotide fragments using RNA fragmentation reagent (ThermoFisher Scientific, AM8740), and purified by ethanol precipitation. Five micrograms of IgG and m5C antibodies (Abcam, ab10805) were pre-bound to 50uL protein A / G magnetic beads at 4°C for 2 hours. Fragmented RNA was mixed with MeRIP buffer (10mM Tris-HCl, 150mM NaCl, 0.1% NP-40, pH7.4, 0.4U / μL RNase inhibitor) and incubated with protein A / G magnetic beads at 4°C for 4 hours. Samples were washed four times in IP wash buffer. Bound RNA was extracted by proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation. Input and m5C-enriched RNA were reverse transcribed using random hexamers with Superscript III and analyzed by quantitative PCR. Calculate the methylation modification level, such as Fig.11 As shown in C.

[0117] Depend on Fig.11 As shown in C, knocking out NSun2 significantly reduced the m5C methylation level of PRKACA mRNA in mouse cardiomyocytes. This indicates that RNA m5C modification plays an important role in regulating cardiac function and homeostasis, and NSun2-mediated m5C methylation level of PRKACA mRNA increases during cardiac hypertrophic remodeling.

[0118] 3. Detection of PRKACA protein expression and PKA activity in the myocardium of mice in the FT and CT groups

[0119] Four weeks after TAC surgery, the cardiomyocytes of mice in the FT and CT groups were isolated using a Langendorff perfusion apparatus, and the expression levels of PRKACA in the cardiomyocytes of mice in the FT and CT groups were detected by Western Blot. PRKACA antibody was purchased from Abclonal, Wuhan, Hubei, China (A18603). The results of Western Blot detection are shown in Figure 2. Fig.11 As shown in D.

[0120] PKA activity was detected using a PKA colorimetric activity kit (EIAPKA; ThermoFisher Scientific) by measuring the phosphorylation level of specific PKA substrates. 30 μg of protein sample from Langendorff-isolated cardiomyocytes was used for each assay. The results of PKA activity assay are shown in Fig.11 It can be known from E.

[0121] Depend on Fig.11 As shown in Figure D, the expression level of PRKACA protein was significantly decreased in NSun2 knockout mice; Fig.11 As shown in Figure E, the TAC model can increase PKA activity, while knocking out NSun2 significantly decreases PKA activity.

[0122] This suggests that NSun2 can methylate PRKACA mRNA, regulate PRKACA protein expression, affect PKA signaling activity, and thus affect the progression of cardiomyocyte hypertrophy and heart failure.

[0123] Example 5: Effects of NSun2 gene supplementation on myocardium in mice at the heart failure stage

[0124] 1. Mouse selection

[0125] C57BL / 6 male mice aged 6-7 weeks were selected (purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Beijing, China).

[0126] 2. Experimental Grouping and Treatment

[0127] The experimental mice were randomly divided into three groups: CT4, CT16, and NT16, with 6 mice in each group.

[0128] The mice in the CT4 group underwent TAC surgery (the specific operation of TAC surgery was the same as that in Example 1), and ultrasound cardiography (the specific operation of ultrasound cardiography was the same as that in Example 1) and cardiac TUNEL staining were performed 4 weeks after TAC surgery.

[0129] The mice in the CT16 group underwent TAC surgery (the specific operation of TAC surgery was the same as that in Example 1), and were injected with adeno-associated virus AVV9 empty vector through the tail vein 8 weeks after TAC surgery; then, ultrasound cardiography (the specific operation of ultrasound cardiography was the same as that in Example 1) and cardiac TUNEL staining were performed 16 weeks after TAC surgery.

[0130] The mice in the NT16 group underwent TAC surgery (the specific operation of TAC surgery was the same as that in Example 1), and 1×10 adeno-associated virus AVV9-Nsun2 (cardiomyocyte-specific overexpression of Nsun2, purchased from Hanheng Biotechnology Co., Ltd.) was injected into the tail vein 8 weeks after TAC surgery. 11 PFU, and then ultrasound cardiography detection (the specific operation of ultrasound cardiography is the same as that in Example 1) and cardiac TUNEL staining were performed 16 weeks after TAC surgery.

[0131] The specific operation of TUNEL staining is as follows: TUNEL staining was performed according to the instructions of TMR (red) Tunel apoptosis detection kit (Cat#G1502-100T, Servicebio Technologies, China). After dewaxing and hydration, the heart tissue sections were subjected to antigen extraction. After permeabilization with proteinase K, the images were labeled with TdT incubation buffer, and the TUNEL-positive spots were counted in multiple randomly selected fields of view at 20× magnification using the Olympus FV1000 digital scanning imaging system (Olympus, Tokyo, Japan) to measure the apoptotic index.

[0132] 3. Experimental results:

[0133] The results of ultrasound cardiology of mice in the CT4, CT16, and NT16 groups are shown in Fig.12 shown.

[0134] Depend on Fig.12 It can be seen that aortic constriction in mice causes their hearts to first hypertrophy (4 weeks) and then enter the heart failure stage (16 weeks). During the process of pressure-induced myocardial hypertrophy, supplementing NSun2 can effectively enhance cardiac function and delay the progression of heart failure.

[0135] The experimental results of TUNEL staining are as follows Fig.13 shown.

[0136] Depend on Fig.13 It can be seen that aortic constriction in mice leads to a gradual increase in cardiac cell apoptosis (4 to 16 weeks), and supplementation of NSun2 can effectively reduce apoptotic cardiomyocytes and delay cardiomyocyte death.

[0137] The above experiments show that pressure overload causes compensatory hypertrophy phenotype in the mouse heart (4 weeks), and as time goes on, it enters the heart failure stage (16 weeks). Supplementing NSun2 can effectively restore heart function, delay the progression of heart failure, and alleviate cardiomyocyte apoptosis.

[0138] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of the NSun2 gene or the protein encoded by it in the preparation of a drug for alleviating and / or treating pressure-overload heart failure.

2. Use of a promoter of NSun2 gene expression in the preparation of a drug for alleviating and / or treating pressure-overload heart failure, the promoter comprising an expression vector carrying a nucleotide sequence encoding the NSun2 protein or a recombinant virus containing a nucleotide sequence encoding the NSun2 protein.

3. The use according to claim 2, characterized in that: The recombinant virus is an adeno-associated virus that overexpresses the NSun2 gene.

4. The use according to claim 3, characterized in that: The adeno-associated virus type 9 is adeno-associated virus.

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