CN114344296A

CN114344296A - Application of NAT10 inhibitor in diagnosis and treatment of heart failure caused by pressure overload

Info

Publication number: CN114344296A
Application number: CN202210064755.2A
Authority: CN
Inventors: 葛均波; 赵永超; 孙爱军; 魏子伦; 宋帅; 熊卫东
Original assignee: Zhongshan Hospital Fudan University
Current assignee: Zhongshan Hospital Fudan University
Priority date: 2022-01-20
Filing date: 2022-01-20
Publication date: 2022-04-15
Anticipated expiration: 2042-01-20
Also published as: CN114344296B

Abstract

The invention discloses application of a NAT10 inhibitor in diagnosis and treatment of heart failure caused by pressure overload. According to the invention, by establishing a heart failure animal model after aortic arch constriction of a mouse and gastric perfusion administration of a NAT10 inhibitor, the heart function, the ventricular remodeling condition and the expression of related marker molecules are evaluated by utilizing the heart super-section staining and molecular biology technologies, the fact that the heart function can be improved after the NAT10 inhibitor is dried is found, the ventricular remodeling is improved, the related marker of the heart failure is reduced, the deposition of collagen and extracellular matrix is reduced, ac4C is obviously inhibited after the Remodelin intervention, and the heart remodeling and the heart function of the mouse induced by TAC can be obviously improved by the NAT10 inhibitor, so that a new target and a new strategy are provided for clinically diagnosing and treating the heart failure caused by pressure overload.

Description

Application of NAT10 inhibitor in diagnosis and treatment of heart failure caused by pressure overload

Technical Field

The invention relates to application of a NAT10 inhibitor in diagnosis and treatment of heart failure caused by pressure overload, belonging to the field of biological medicines.

Background

Heart failure, as the terminal stage of progression of various cardiovascular diseases, has a high incidence and a poor prognosis. Although the survival rate of the heart failure patients is improved by the drug treatment of inhibiting the excessive activation of neuroendocrine such as 'golden triangle' and 'new tetrad', the life quality and the long-term prognosis are still poor, and the 1-year death rate of the heart failure patients is still as high as 17%.

Heart failure is classified into different types due to different mechanisms of occurrence. From the pathophysiological point of view, heart failure can be roughly divided into two main categories, i.e., the primary myocardial damage and the long-term volume (or) pressure overload of the heart, which leads to the final development of the pump function from compensation to decompensation. The heart failure is mainly the ventricular remodeling, that is, the pathological remodeling of the ventricles, including the change of the size, the shape, the tissue structure and the functional state of the ventricles, caused by the maladaptive change of the myocardium in the process of the heart failure, is the basic pathological process of the development of the heart failure.

Cardiac remodeling is an important pathophysiological basis for the development and progression of heart failure. At the gross morphological level, myocardial remodeling is mostly manifested by thickening, mass increase of local or most or even the entire ventricular wall or/and atrial wall. At the cellular level, single cardiomyocytes that are unable to divide become hypertrophic, interstitial components such as fibroblasts proliferate, collagen synthesis and secretion increase, and myocardial tissue components change with apoptosis. At the molecular level, the gene expression promoting cardiac hypertrophy is increased, such as connective tissue growth factor CTGF, transforming growth factor TGF beta, type I and type III collagen, etc., which increases the stiffness of the ventricular wall and reduces cardiac muscle compliance. Although cardiac remodeling is initially a compensatory mechanism, long-lasting cardiac remodeling can significantly increase the incidence and mortality of heart failure. At present, no effective prevention and treatment method for heart failure caused by cardiac remodeling exists clinically. Therefore, the discovery of specific molecules and molecular pathways for blocking heart remodeling to cause heart failure and the search of drug targets for preventing and treating heart failure have very important theoretical significance and clinical significance.

Epigenetics is increasingly receiving attention from researchers in the field of heart failure as an important means for regulating gene expression. RNA epigenetic post-transcriptional modification is an important link in epigenetics, both the protective role in the progression of various diseases by targeting 6-methyladenosine suggests the potential of RNA epigenetic post-transcriptional modification mechanisms in the treatment of heart failure. 4-acetyl cytidine (N4-acetylcytidine, ac4C) is a conserved posttranscriptional chemically modified nucleoside type present on a variety of RNAs. The formation of ac4C is mainly catalyzed by N-acetyltransferase 10 (NAT 10), and is involved in the onset and progression of various diseases, including immune inflammatory response, metabolic diseases, autoimmune diseases, and cancer. However, no report is known about the effect of NAT10 inhibitor in preparing medicine for treating heart failure caused by pressure overload.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to improve the curative effect and prognosis of heart failure caused by pressure overload such as cardiac remodeling.

In order to solve the technical problems, the invention provides application of an inhibitor of NAT10 gene or protein in preparing a medicament for treating heart failure caused by pressure overload.

Preferably, the inhibitor of the NAT10 gene or protein comprises Remodelin, which has the formula C₁₅H₁₄N₄S, molecular weight of 282.36, chemical structural formula

The invention also provides a medicament or a pharmaceutical composition for treating heart failure caused by pressure overload, which comprises a pharmaceutically acceptable carrier and an effective amount of active ingredients, wherein the active ingredients comprise NAT10 gene or protein inhibitor.

Preferably, the inhibitor of the NAT10 gene or protein comprises Remodelin.

The invention also provides application of the reagent for detecting the expression or activity of the NAT10 gene or protein in preparing a reagent or a kit for diagnosing the heart failure caused by the pressure overload.

Preferably, the reagent for detecting the expression or activity of the NAT10 gene or protein comprises NAT10 antibody.

The invention also provides the use of an agent for detecting the level of modification of ac4C in the preparation of a reagent or kit for the diagnosis of heart failure due to pressure overload.

Compared with the prior art, the invention has the beneficial effects that:

according to the invention, the specific inhibitor Remodelin of NAT10 is creatively used for interfering TAC-induced pathological myocardial remodeling and heart failure of mice, and the fact that the Remodelin is significantly inhibited after ac4C is found, the TAC-induced cardiac remodeling and heart function of the mice can be significantly improved, so that a new target and a new strategy are provided for clinically diagnosing and treating the heart failure caused by pressure overload.

Drawings

FIG. 1 is ac4C modification and NAT10 expression levels in human heart failure heart tissue and TAC-induced mouse heart failure heart tissue; wherein, A is the ac4C modification level in heart failure heart tissue and normal tissue of human, B is the NAT10 expression level in heart failure heart tissue and normal tissue of human, C is the ac4C modification level in heart failure heart tissue of mouse induced by TAC and heart tissue of sham operated mouse, D is the NAT10 expression level in heart failure heart tissue of mouse induced by TAC and heart tissue of sham operated mouse;

fig. 2 is a 2-week intragastric gavage of NAT10 inhibitor based on constructed TAC-induced heart failure mice, and Remodelin intervention was found to significantly reduce the level of modification of ac4C (a), reduce the ejection fraction ef (b), and reduce the fractional shortening fs (c);

FIG. 3 is a graph of HE staining, Masson staining and WGA staining of mouse heart tissue 4 weeks after TAC operation (A), myocardial fibrosis detection (B), myocardial cell hypertrophy level detection (C), RT-qPCR detection of expression level of heart failure-related marker molecules (D), and RT-qPCR detection of expression level of collagen and extracellular matrix deposition-related genes (E);

in the above figures, the symbol indicates that the significant difference between the two groups is shown by statistical analysis, P is less than 0.05; the expression shows that the two groups have significant difference by statistical analysis, and P is less than 0.01; trepresents that the two groups have significant difference through statistical analysis, and P is less than 0.001.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.

In a first aspect of the invention, there is provided the use of an inhibitor of the NAT10 gene or protein in the manufacture of a medicament for the treatment of heart failure.

The heart failure is caused by pressure overload.

The inhibitor of the NAT10 gene or protein is an inhibitor for instructing the NAT10 protein to lose activity or reduce activity.

The inhibitor of the NAT10 gene or protein is selected from small molecular compounds or biological macromolecules.

The small molecular compound is Remodelin with a molecular formula C₁₅H₁₄N₄S, molecular weight of 282.36, chemical structural formula

In a second aspect of the present invention, a pharmaceutical composition for treating heart failure is provided, wherein the pharmaceutical composition comprises an inhibitor of NAT10 gene or protein as an active ingredient, and further comprises a pharmaceutically acceptable carrier.

The heart failure is caused by pressure overload.

The inhibitor of the NAT10 gene or protein is an inhibitor for promoting the loss or reduction of the activity of the NAT10 protein.

The dosage form of the pharmaceutical composition is injection, capsule, granule, tablet, pill or oral liquid, etc.

In a third aspect of the present invention, there is provided a use of NAT10 as a diagnostic marker in the preparation of a reagent or kit for the diagnosis of heart failure.

In a fourth aspect of the invention, there is provided the use of an agent for detecting the level of modification of ac4C in the manufacture of a reagent or kit for the diagnosis of heart failure.

It should be noted that heart failure is classified into different types due to different mechanisms. From the pathophysiological point of view, heart failure can be roughly divided into two main categories, i.e., the primary myocardial damage and the long-term volume (or) pressure overload of the heart, which leads to the final development of the pump function from compensation to decompensation. The heart failure is mainly the ventricular remodeling, that is, the pathological remodeling of the ventricles, including the change of the size, the shape, the tissue structure and the functional state of the ventricles, caused by the maladaptive change of the myocardium in the process of the heart failure, is the basic pathological process of the development of the heart failure. As used herein, the term "NAT 10 inhibitor" includes antagonists, down-regulators, blockers, etc., as long as they are capable of reducing or losing the activity of NAT10 protein, or down-regulating the expression level of NAT10 or inhibiting its activity. They may be chemical compounds, chemical small molecules, biological molecules. The biological molecules can be nucleic acid level (including DNA and RNA) or virus products for inhibiting the expression of NAT 10. By way of example, the inhibitors are: compound Remodelin, formula C₁₅H₁₄N₄S, molecular weight 282.36. Nucleic acid inhibitors, protein inhibitors, nucleases, nucleic acid binding molecules, as long as they are capable of reducing or abolishing the activity of NAT10 protein, or down-regulating the expression of NAT 10.

The mice referred to in the following examples were purchased from the collective pharmaceutical biotechnology company, the NAT10 specific inhibitor Remodelin was purchased from the mce (medchemexpress) company, and the animal experiments were approved by the ethical committee of the subsidiary zhongshan hospital of the university of counterden.

Examples

This example provides the use of NAT10 inhibitors in the diagnosis of heart failure due to stress overload:

1. constructing a mouse aortic arch constriction (TAC) model:

adult male C57bl/6 mice, 8 weeks old and 20-25g in weight, were selected, anesthetized, fixed, depilated, prepared, and then the skin was cut from the superior border of the suprasternal fossa and the median line of the sternum to the second intercostal space (approximately 1cm incision). After the fascia is separated at the suprasternal fossa in a blunt way and the air outlet pipe is exposed, the small-curve forceps enter the suprasternal fossa to push the thymus tissue to two sides. The field of vision is exposed by adopting a retractor, and a No. 5 thread is penetrated into a blood vessel at the head and arm trunk through the front section of a self-made syringe needle (the front section is cut off and made into a right angle) and is knotted in advance. Then pad with a 4 mm 27G needle (made square) and tie off. And (4) slowly pulling out the 4 mm 27G needle after ligation, suturing muscle tissues, placing in a warm environment after skin sewing for reviving, and completing model construction. The prescription and method of the pseudo-operation are consistent, and only the threading is needed without ligation.

2. And (3) performing NAT10 expression and ac4C acetylation modification detection on clinical heart failure heart specimens and heart failure mouse heart specimens caused by TAC:

through ethical examination and informed consent, 3 cases of left ventricle myocardial tissues of patients who need heart transplantation operation and have final end-stage heart failure caused by primary dilated cardiomyopathy in clinical diagnosis of Zhongshan hospital affiliated to the university of Compound Dan are collected. 3 cases of left ventricular myocardial tissue of a control group of non-heart failure patients were also collected. In addition, 6 cases of the heart tissues of the mice in the sham-operated group and the TAC-induced heart failure group were collected. Respectively extracting total RNA of the samples, detecting the content of ac4C acetylation modification by liquid chromatography-tandem mass spectrometry (LC-MS), and detecting the expression of NAT10 by an RT-qPCR method.

3. NAT10 inhibitor remodelain intervention:

TAC surgery induced stress overload induced heart failure in mice was performed as described above. The treatment group is randomly grouped at 3 days after TAC operation, NAT10 inhibitor Remodelin (100 mg/kg/d) is given to the treatment group in a gastric perfusion mode, DMSO with the same volume is given to the control group for intragastric administration for 2 weeks continuously until 4 weeks after TAC, heart ultrasonic detection of cardiac function, HE staining detection of myocardial morphological change, Masson staining detection of fibrosis, WGA staining detection of myocardial cell hypertrophy level, and RT-qPCR detection of heart failure related marker molecules, collagen and extracellular matrix deposition related gene expression.

4. Cardiac ultrasonic detection of cardiac structure and function:

after dehairing, mice were anesthetized with isoflurane and mounted on a heating pad (1% -2%). The isoflurane flow was carefully controlled to maintain the heart rate around 450 beats/min. The geometry and function of the heart were assessed using a two-dimensional (2-D) guided M-mode echocardiogram (VisualSonics Vevo 2100, Canada) equipped with a 30mHz linear transducer. A parasternal long axis view was obtained while recording the heart rate. Obtaining the geometric shape and functional indexes of the heart: ejection Fraction (EF), Fractional Shortening (FS). Each measurement indicator is averaged over at least 5 consecutive cardiac cycles.

5. Staining and checking a heart specimen section:

heart material was obtained after TAC 4 weeks, fixed with paraformaldehyde, and then embedded in normal paraffin and sectioned. After the section is made, the section is dewaxed and hydrated conventionally, and the morphology of the heart tissue, the fibrosis degree and the surface area of the myocardial cells are observed by HE, Masson and WGA staining.

6. The RT-qPCR method is used for analyzing the expression of heart failure related markers such as Nppa, Nppb, Myh7, Col1a1, Col3a1, Ctgf and Lox, collagen and extracellular matrix deposition related genes, and the improvement of the TAC-induced heart failure related marker molecule expression by the NAT10 inhibitor is proved.

7. The experimental results are as follows:

(1) in the experiment, ac4C modification and NAT10 expression of collected heart tissues clinically diagnosed as heart failure caused by dilated cardiomyopathy and mouse heart failure heart tissues induced by TAC are detected by LC-MS and RT-qPCR methods, and the ac4C modification and NAT10 expression are found to be remarkably up-regulated in human heart specimens or mouse heart specimens, as shown in FIG. 1.

(2) By performing intragastric administration of the NAT10 inhibitor for 2 weeks on the basis of the constructed TAC-induced heart failure mice, the fact that Remodelin intervention can significantly reduce the modification level of ac4C and improve the cardiac function index is found, as shown in figure 2, the NAT10 inhibitor can also improve TAC-induced cardiac insufficiency and ventricular remodeling, reduce the expression of myocardial hypertrophy and heart failure-related marker molecules and improve the deposition of collagen and extracellular matrix, as shown in figure 3.

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way and substantially, it should be noted that those skilled in the art may make several modifications and additions without departing from the scope of the present invention, which should also be construed as a protection scope of the present invention.

Claims

Use of an inhibitor of the NAT10 gene or protein for the preparation of a medicament for the treatment of heart failure due to stress overload.
2. The use of claim 1, wherein the inhibitor of the NAT10 gene or protein comprises Remodelin, having the formula C₁₅H₁₄N₄S, molecular weight of 282.36, chemical structural formula
3. A medicament or pharmaceutical composition for the treatment of heart failure due to stress overload, comprising a pharmaceutically acceptable carrier and an effective amount of an active ingredient comprising an inhibitor of the NAT10 gene or protein.
4. The drug or pharmaceutical composition for use in the treatment of pressure overload heart failure as claimed in claim 3, wherein the inhibitor of the NAT10 gene or protein comprises Remodelin.
5. Application of a reagent for detecting the expression or activity of NAT10 gene or protein in preparing a reagent or a kit for diagnosing heart failure caused by pressure overload.
6. The use of claim 5, wherein the reagent for detecting the expression or activity of NAT10 gene or protein comprises NAT10 antibody.
7. Use of an agent for detecting the level of modification of ac4C in the manufacture of a reagent or kit for the diagnosis of heart failure due to pressure overload.