CN117122684A - Use of formulations for knockdown or inhibition of METTL4 in the preparation of a medicament for the treatment of heart failure - Google Patents

Abstract

The present invention belongs to the field of biomedical technology, and specifically relates to the application of preparations that knock down or inhibit METTL4 in the preparation of drugs for the treatment of heart failure. The present invention proves that the expression of METTL4 in heart mitochondria of heart failure is increased; at the same time, studies have concluded that knocking out METTL4 expression can alleviate heart failure and mitochondrial lesions. Therefore, METTL can be used as a candidate target for the development of heart failure drugs. Inhibiting the activity or expression of METTL4 using drugs or genetic means can be used as a potential strategy to prevent and treat heart failure.

Description

The use of agents that knock down or inhibit METTL4 in the preparation of drugs for the treatment of heart failure application

Technical field

The present invention belongs to the field of biomedical technology, and specifically relates to the application of preparations that knock down or inhibit METTL4 in the preparation of drugs for the treatment of heart failure.

Background technique

The heart is the organ with the greatest energy demand in the body. Mitochondria are important organelles that ensure normal contraction-diastolic function of cardiomyocytes and heart health. Mitochondrial dysfunction can lead to insufficient energy supply to cardiomyocytes, resulting in systolic-diastolic dysfunction and heart failure. Correcting mitochondrial dysfunction is an important direction in the prevention and treatment of heart failure, but there is currently a lack of effective intervention targets and means. METTL4 is a DNA methyltransferase, but its function in cardiomyocytes and its pathophysiological significance in heart diseases have not been reported.

In view of this, the present invention is proposed.

Contents of the invention

The technical problem to be solved by the present invention is to explore the function of METTL4 in cardiomyocytes and its pathophysiological significance in heart diseases, in order to provide candidate targets for the development of heart failure therapeutic drugs.

The present invention adopts the following technical solutions to solve the above technical problems:

In one aspect, the present invention provides the use of a preparation that knocks down or inhibits METTL4 in the preparation of a drug for treating heart failure.

Further, the heart failure is manifested as cardiac systolic-diastolic dysfunction.

Further, the heart failure is manifested as pathological remodeling of the heart.

Further, the heart failure manifests as cardiac mitochondrial dysfunction.

Further, the agent for knocking down or inhibiting METTL4 is selected from small molecule compounds or biological macromolecules.

On the other hand, the present invention provides a pharmaceutical composition for treating heart failure, which pharmaceutical composition uses a preparation that knocks down or inhibits METTL4 as an active ingredient, and further includes a pharmaceutically acceptable carrier.

Further, the heart failure is manifested by cardiac systolic-diastolic dysfunction, cardiac pathological remodeling, and/or cardiac mitochondrial dysfunction.

In another aspect, the present invention provides the use of METTL4 detection reagents in the preparation of products for diagnosis and/or prognosis of heart failure.

Further, the heart failure is manifested by cardiac systolic-diastolic dysfunction, cardiac pathological remodeling, and/or cardiac mitochondrial dysfunction.

The invention has the following beneficial effects:

The present invention provides the use of a preparation that knocks down or inhibits METTL4 in the preparation of drugs for treating heart failure. Animal experiments show that knocking down METTL4 can alleviate AngII/PE-induced cardiac systolic-diastolic dysfunction, cardiac pathological remodeling, and cardiac mitochondrial dysfunction. The present invention clarifies for the first time that METTL4 can be used as a candidate target for the development of heart failure drugs, using drugs Or genetic means to inhibit METTL4 activity or expression can be used as a potential strategy to prevent and treat heart failure.

Description of the drawings

Figure 1 shows the localization of METTL4 in neonatal and adult C57BL/6J cardiomyocytes.

Figure 2 shows the comparison of METTL4 expression in cardiomyocytes of heart failure mice and wild-type mice.

Figure 3 shows the levels of METTL4 gene mRNA (A) and METTL4 protein (B) in wild-type mouse cardiomyocytes after stimulation with vehicle (vehicle) or AngII/PE for 24 hours.

Figure 4 is a schematic diagram of the construction strategy of Mettl4 cKO mice.

Figure 5 is the plasmid map of the homologous recombination vector.

Figure 6 shows the comparison of METTL4 protein expression in cardiomyocytes (A) and non-cardiomyocytes (B) between Mettl4 cKO mice and wild-type mice.

Figure 7 shows the left ventricular end-diastolic pressure (A), left ventricular end-systolic pressure (B), and maximum ventricular pressure rising rate (C) of Mettl4 cKO mice and wild-type mice induced by vehicle (vehicle) or AngII/PE. Compare with the rate of maximum ventricular pressure reduction (D).

Figure 8 shows the heart weight/tibia length ratio (A), cardiomyocyte cross-sectional area (B), and cardiomyocyte hypertrophy and fibrosis levels of Mettl4 cKO mice and wild-type mice induced by vehicle (vehicle) or AngII/PE ( C) Typical comparison chart.

Figure 9 is a typical comparison chart of mitochondrial morphological lesions in Mettl4 cKO mice and wild-type mice induced by vehicle (vehicle) or AngII/PE.

Figure 10 is a comparison of the mitochondrial respiratory function of cardiomyocytes in Mettl4 cKO mice and wild-type mice induced by vehicle (vehicle) or AngII/PE.

Detailed ways

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but should not be understood as limitations of the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following examples can all be obtained from commercial sources, unless otherwise specified.

The experimental animals used in the following examples were 8-10 week old healthy male C57BL/6J adult mice and newborn mice, which were provided by the Animal Experiment Center of the Air Force Medical University of the Chinese People's Liberation Army.

Example 1: Localization of METTL4

The myocardial tissues of adult C57BL/6J mice and newborn mice were taken, paraffin sections were made and stained, and cardiomyocyte mitochondria were observed under a transmission electron microscope.

As shown in Figure 1, METTL4 is localized in cardiomyocyte mitochondria. Therefore, METTL4 is a DNA methylase localized in cardiomyocyte mitochondria.

Example 2: Expression of METTL4 in the hearts of mice with heart failure

Angiotensin II/phenylephrine (AngII/PE, 1.5 μg/g/d and 50 μg/g/d) was pumped into adult C57BL/6J mice for 4 weeks to construct heart failure mice (Failing group) using an osmotic pump. Myocardial tissue was taken, paraffin sections were made and stained to evaluate the expression of METTL4 in the hearts of mice with heart failure.

As shown in Figure 2, METTL4 expression is increased in the hearts of mice with heart failure.

Example 3: Effect of AngII/PE-incubated cardiomyocytes on METTL4 protein that promotes heart failure

After cardiomyocytes of adult C57BL/6J mice were stimulated with vehicle (vehicle) or AngII/PE for 24 hours, the METTL4 gene mRNA level and METTL4 protein expression level were detected.

As shown in Figure 3, AngII/PE stimulation significantly up-regulated the expression level of METTL4.

Example 4: Construction of cardiomyocyte-specific METTL4 knockout mouse model

The cardiomyocyte-specific METTL4 knockout mouse model was constructed by Shanghai Southern Model Biotechnology Co., Ltd. (South Model Biotech). The Mettl4 gene (ENSMUSG00000055660) was modified by flox using the principle of homologous recombination and fertilized egg homologous recombination. (Figure 4). The brief process is as follows:

Cas9 mRNA and gRNA were obtained through in vitro transcription; a homologous recombination vector (donor vector, Figure 5) was constructed through In-Fusion cloning. The vector contains a 3.0kb 5' homology arm, a 1.0kb flox region and a 3.0 kb 3' homology arm. Cas9 mRNA, gRNA and donor vector were microinjected into fertilized eggs of C57BL/6J mice to obtain F0 generation mice. The F0 generation mice identified as positive by PCR amplification and sequencing were crossed with tamoxifen-induced cardiomyocyte-specific Cre overexpression (MerCreMer) mice (available for purchase at Nanmo Biotech) to obtain positive F1 generation mice. Positive F1 Generation of mice was bred to obtain F2 homozygous mice. The above mice were fed a tamoxifen-containing diet (400 mg/kg) for 7 days to construct cardiomyocyte-specific METTL4 knockout (Mettl4 cKO) mice. Western blot detected METTL4 protein expression in cardiomyocytes and non-cardiomyocytes.

The sequence of the gRNA is as follows:

gRNA1 (SEQ ID NO.1): CACTCTACACTTAATAAAGGTGG;

gRNA2 (SEQ ID NO. 2): AACAACTGAATAGTTTACAT GGG.

As shown in Figure 6, METTL4 protein expression in cardiomyocytes of Mettl4 cKO mice was significantly reduced.

Example 5: METTL4 knockout alleviates AngII/PE-induced cardiac systolic-diastolic dysfunction

Wild-type (WT) C57BL/6J mice and METTL4 cKO mice were pumped with small doses of angiotensin II/phenylephrine (AngII/PE, 1.5 μg/g/d and 50 μg/g/d) via vehicle (vehicle). After 4 weeks of rats, floating catheters were used to detect left ventricular end-diastolic pressure (LVEDP), left ventricular end-systolic pressure (LVESP), maximum ventricular pressure rise rate (dp/dtmax) and maximum ventricular pressure fall rate (-dp/dtmax).

As shown in Figure 7, METTL4 cKO mice had significantly reduced left ventricular systolic-diastolic dysfunction induced by AngII/PE compared with WT mice.

Example 6: METTL4 knockout alleviates AngII/PE-induced pathological cardiac remodeling

Wild-type (WT) C57BL/6J mice and METTL4 cKO mice were pumped with small doses of angiotensin II/phenylephrine (AngII/PE, 1.5 μg/g/d and 50 μg/g/d) via vehicle (vehicle). After 4 weeks, the mice were tested for pathological remodeling phenomena such as cardiac hypertrophy. The detection indicators were mainly heart weight/tibia length ratio (HW/TL) and cardiomyocyte cross-sectional area (CSA), which were used to evaluate cardiac hypertrophy and cardiomyocyte hypertrophy. Degree, Masson staining and WGA staining were used to evaluate cardiomyocyte hypertrophy and fibrosis.

As shown in Figure 8, the symptoms of AngII/PE-induced cardiac hypertrophy and other pathological remodeling were significantly alleviated in METTL4 cKO mice.

Example 7: METTL4 knockout alleviates AngII/PE-induced cardiac mitochondrial dysfunction

Wild-type (WT) mice and METTL4 cKO mice were pumped with vehicle or angiotensin II/phenylephrine (AngII/PE, 1.5 μg/g/d and 50 μg/g/d) for 4 weeks. Afterwards, the mouse myocardial tissue was taken to make paraffin sections and stained. Myocardial mitochondria were observed under a transmission electron microscope to evaluate mitochondrial morphological lesions such as cardiac hypertrophy. Mouse cardiomyocytes were isolated, and the mitochondrial base (State-2), maximum (State-3) and ADP of complex I substrate glutamate and complex II and III substrates succinate were measured using an O2k mitochondrial respiratory function detector. Exhausted (State-4) respiratory function.

As shown in Figure 9, AngII/PE-induced cardiac hypertrophy, mitochondrial vacuolation, mitochondrial cristae disappearance and other mitochondrial morphological lesions were significantly alleviated in METTL4 cKO; Figure 10 shows that AngII/PE caused significant impairment of mitochondrial respiratory function in cardiomyocytes. This change was significantly alleviated in the METTL4 cKO group.

Although the preferred embodiments of the present invention have been described, those skilled in the art will be able to make additional changes and modifications to these embodiments once the basic inventive concepts are apparent. Therefore, it is intended that the appended claims be construed to include the preferred embodiments and all changes and modifications that fall within the scope of the invention.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the invention. In this way, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims (9)

1. Use of a formulation that knocks down or inhibits METTL4 in the manufacture of a medicament for treating heart failure.

2. The use according to claim 1, wherein the heart failure is manifested as a systolic-diastolic dysfunction.

3. The use according to claim 1, wherein the heart failure is manifested as a pathological remodeling of the heart.

4. The use according to claim 1, wherein the heart failure is manifested as cardiac mitochondrial dysfunction.

5. The use according to claim 1, wherein the agent that knocks down or inhibits METTL4 is selected from a small molecule compound or a biological macromolecule.

6. A pharmaceutical composition for treating heart failure, wherein the pharmaceutical composition comprises a METTL4 knockdown or inhibition formulation as an active ingredient and further comprises a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, wherein the heart failure is manifested by systolic-diastolic dysfunction, pathological remodeling of the heart and/or mitochondrial dysfunction of the heart.

Use of a detection reagent for mettl4 for the preparation of a product for the diagnosis and/or prognosis of heart failure.

9. The use according to claim 8, wherein the heart failure is manifested by a systolic-diastolic dysfunction, a pathological remodeling of the heart and/or a mitochondrial dysfunction of the heart.