Application of OMA1 inhibitor in preparation of medicine for treating heart failure

Abstract

The invention provides application of an OMA1 inhibitor in preparation of a medicine for treating heart failure, relates to the technical field of biomedicine, and is technically characterized in that the OMA1 inhibitor is provided, preferably ZINC14880369, and the effect of the Oma1 small-molecule inhibitor ZINC14880369 in the heart failure is verified by adopting a heart failure model in an experiment. The heart failure model is constructed through aortic arch constriction. In-vitro experiments in the application verify that Oma1mRNA and protein expression in the heart of a mouse after heart failure are increased, the small-molecule inhibitor ZINC14880369 is designed and proved to be capable of effectively inhibiting Oma1 gene expression, and further research is carried out, so that a potential treatment strategy is provided for drug research and development. The Oma1 small-molecule inhibitor in the heart can relieve the heart failure course of a mouse and lower the expression of Oma1 mRNA at the same time. Therefore, down-regulation or inhibition of Oma1 gene and protein expression by using a small-molecule inhibitor is an effective way for treating heart failure.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of an OMA1 inhibitor in preparing a drug for treating heart failure. Background Art

[0002] Heart failure (HF) is a chronic, progressive syndrome caused by abnormalities in cardiac structure or function and is one of the most common cardiovascular diseases worldwide. Globally, the prevalence of HF is approximately 1-2%, particularly in aging societies, with the prevalence exceeding 10% in people aged 65 and older. With the accelerated aging of the population and changes in lifestyle, the incidence of HF continues to rise, with the number of new patients increasing year by year. This has placed a significant economic burden and resource pressure on the global healthcare system. Despite recent advances in drug therapy (e.g., beta-blockers, RAAS inhibitors, SGLT2i inhibitors) and mechanical support (e.g., cardiac resynchronization therapy, left ventricular assist devices), the mortality and hospitalization rates for HF remain high, making it difficult to significantly improve patients' long-term survival and quality of life. Summary of the Invention

[0003] The purpose of the present invention is to solve the problem of high mortality and hospitalization rate of heart failure in the prior art.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] An OMA1 inhibitor, wherein the OMA1 inhibitor is at least one of ZINC14880369, ZINC14880389, ZINC14881286, ZINC14880272, ZINC14881215, ZINC14611917, ZINC16052056, ZINC02046955, ZINC14880926, and ZINC12504443.

[0006] The present application also provides use of the above-mentioned OMA1 inhibitor in the preparation of a drug for treating heart failure.

[0007] Preferably, the OMA1 inhibitor is ZINC14880369.

[0008] Preferably, the drug inhibits the expression of OAM1 gene and protein through the small molecule inhibitor ZINC14880369 to achieve the purpose of treating heart failure.

[0009] The present application also provides a drug for treating heart failure, comprising the above-mentioned OMA1 inhibitor.

[0010] Preferably, the drug further comprises a medically acceptable adjuvant

[0011] Compared with the prior art, this application has the following beneficial effects:

[0012] In this application, a heart failure model is used to verify the role of the Oma1 small molecule inhibitor ZINC14880369 in heart failure. The heart failure model is constructed by aortic arch constriction. This application verifies through in vitro experiments that the expression of Oma1 mRNA and protein in the heart of mice with heart failure increases. The results of the present invention design and prove that the small molecule inhibitor ZINC14880369 can effectively inhibit the expression of the Oma1 gene, and further research is carried out to provide potential treatment strategies for drug development. The Oma1 small molecule inhibitor in the heart can alleviate the course of heart failure in mice and at the same time downregulate the expression of Oma1 mRNA. Therefore, downregulating or inhibiting the expression of the Oma1 gene and protein by using small molecule inhibitors is an effective way to treat heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The binding mode of the optimal small molecule ZINC14880369 in the receptor binding pocket in one embodiment of the present invention;

[0014] Figure 2 These are the cardiac ultrasound, heart weight / body weight, and heart weight / tibia length changes of mice with heart failure induced by the TAC model (aortic arch constriction model) in one embodiment of the present invention;

[0015] Figure 3 These are the cardiac ultrasound, heart weight / body weight, and heart weight / tibia length changes of mice with heart failure induced by the TAC model (aortic arch constriction model) in one embodiment of the present invention;

[0016] Figure 4 Figure 2 shows changes in Oma1 mRNA expression in the hearts of mice with heart failure induced by the TAC model (aortic arch constriction model) in one embodiment of the present invention. Nine mice were included in each group. Real-time PCR results were analyzed using the Student's T-test. ***P<0.001 compared with the vehicle group at the corresponding time point.

[0017] Figure 5 The figure shows the changes in Oma1 protein expression in the hearts of mice with heart failure induced by the TAC model (aortic arch constriction model) in one embodiment of the present invention;

[0018] Figure 6 The figure shows the changes in Oma1 protein expression in the hearts of mice with heart failure induced by the TAC model (aortic arch constriction model) in one embodiment of the present invention;

[0019] Figure 7 Figure 2 shows changes in Oma1 mRNA expression in an ISO-stimulated H9C2 cell model of cardiac hypertrophy, according to one embodiment of the present invention. Nine replicate wells were used in each group. Real-time PCR results were compared with the solvent group at the corresponding time point using the Student's t-test. ***P < 0.001.

[0020] Figure 8 The changes in ATP production in the ISO-stimulated H9C2 cell cardiac hypertrophy model according to one embodiment of the present invention;

[0021] Figure 9 The changes of mitochondrial Mitotracker in the ISO-stimulated H9C2 cell cardiac hypertrophy model in one embodiment of the present invention;

[0022] Figure 10 The changes of mitochondrial mt-Keima in the H9C2 cell cardiac hypertrophy model stimulated by ISO in one embodiment of the present invention;

[0023] Figure 11 The changes of mitochondrial Mitosox in the H9C2 cell cardiac hypertrophy model stimulated by ISO in one embodiment of the present invention;

[0024] Figure 12 The changes of mitochondrial Fura2 in the H9C2 cell cardiac hypertrophy model stimulated by ISO in one embodiment of the present invention;

[0025] Figure 13 The changes of mitochondrial JC-1 in the ISO-stimulated H9C2 cell cardiac hypertrophy model in one embodiment of the present invention;

[0026] Figure 14 This is an in vitro experiment verifying the inhibitory effect of the small molecule ZINC14880369 on Oma1 in H9C2 cells according to one embodiment of the present invention. H9C2 cells were stimulated to hypertrophy by ISO, and then the small molecule ZINC14880369 was used to inhibit Oma1 for 48 hours. Oma1 protein expression was then detected by Western blotting.

[0027] Figure 15 This is an M-mode echocardiogram of a mouse in one embodiment of the present invention.

[0028] Figure 16 This is a graph showing the results of treating heart failure in mice after interference with the Oma1 small molecule inhibitor ZINC14880369 in one embodiment of the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below with reference to specific embodiments.

[0030] Use of an OMA1 inhibitor in the preparation of a drug for treating heart failure, wherein the OMA1 inhibitor includes at least one of ZINC14880369, ZINC14880389, ZINC14881286, ZINC14880272, ZINC14881215, ZINC14611917, ZINC16052056, ZINC02046955, ZINC14880926, and ZINC12504443. Preferably, the OMA1 inhibitor is ZINC14880369.

[0031] The small molecule inhibitor ZINC14880369 is used to inhibit the expression of OAM1 gene and protein to achieve the purpose of treating heart failure.

[0032] The above contents are described below with reference to specific embodiments:

[0033] 1. Experimental materials and sources:

[0034] 1. Main reagents and manufacturers in this application

[0035]

[0036]

[0037] 2. Main instruments and manufacturers in this application

[0038] Serial number instrument source 1 PCR instrument Applied Biosystems, USA 2 Real-time fluorescence quantitative PCR instrument BIO-RAD, USA 3 DNA electrophoresis apparatus BIO-RAD, USA 4 Gel imaging instrument BIO-RAD, USA 5 Horizontal shaker Hualida Experimental Equipment Company 6 Skipping work rocking bed Hualida Experimental Equipment Company 7 cell culture incubator Thermo Fisher Scientific, USA 8 NanodropOne Micro-Volume UV Spectrophotometer Thermo Fisher Scientific, USA 9 4℃, -20℃ refrigerator Samsung, South Korea 10 Ultra-low temperature refrigerator SANYO Corporation, Japan 11 Ice Maker SANYO Corporation, Japan 12 Different specifications of pipettes and tips Eppendorf, Germany 13 Low-temperature high-speed centrifuge Eppendorf, Germany 14 Electrophoresis and transfer apparatus BIO-RAD, USA 15 microplate reader BIO-TEK, USA 16 Constant temperature incubator Shanghai Hengyi Scientific Instrument Co., Ltd. 17 Constant temperature water bath Shanghai Hengyi Scientific Instrument Co., Ltd. 18 Blast drying oven Shanghai Hengyi Scientific Instrument Co., Ltd. 19 High temperature and high pressure sterilizer TOMY, USA 20 Desktop handheld centrifuge TOMY, USA 21 Chemiluminescent gel imaging instrument Shanghai Tianneng 22 Inverted fluorescence microscope Leica, Germany 23 Water Purifier Millipore Corporation, USA 24 cell counter Invitrogen 25 Upright fluorescence microscope Olympus, Japan 26 Confocal microscopy Olympus, Japan

[0039] 3. Consumables and sources:

[0040] Serial number Consumables Name Production Company 1 Various types of gun heads KIRGEN 2 Various types of pipettes Corning 3 Various models of multi-well cell culture plates Corning 4 Various types of cell culture dishes Corning 5 Cryogenic tubes Corning 6 Various types of centrifuge tubes NEST 7 Transparent 8-piece PCR tube Shanghai Bioengineering 8 96-plex PCR plate Shanghai Bioengineering 9 Optical sealing film Shanghai Bioengineering

[0041] 4. Main reagent configuration:

[0042] (1) Complete culture medium: 1% double antibody was added to DMEM high glucose medium containing 10% FBS and stored in a refrigerator at 4°C.

[0043] (2) Small molecule compound ZINC14880369 stock solution: Weigh the small molecule compound ZINC14880369 (1 mg) and dissolve it in (1.9882 ml DMSO) and store at -20°C in the dark.

[0044] (3) Preparation of 10% SDS-PAGE separation gel:

[0045]

[0046] Continued Table 3-1

[0047]

[0048] 4) Preparation of PAGE stacking gel:

[0049]

[0050] (5) Preparation of 10× electrophoresis buffer:

[0051]

[0052] (6) Preparation of 10× transfer buffer:

[0053] Tris 30.2g

[0054] Glycine 188g

[0055] Double distilled water to 1L

[0056] (7) Preparation of 1×TBST buffer:

[0057] Take a bag of TBS powder, add it to 1L of double-distilled water, and add 2ml of Tween-20.

[0058] (8) Preparation of blocking solution: Add 2.5 g of skim milk powder to 50 ml of 1× TBST buffer.

[0059] (9) Antibody preparation: Use dedicated primary and secondary antibody diluents and prepare the required concentrations according to the antibody instructions.

[0060] 2. Experimental steps

[0061] Example 1: Design of small molecule compounds that inhibit Oma1 expression

[0062] In one embodiment, Oma1 was processed using UCSF chimera (version 1.14), which included adding hydrogen atoms and standard charges to the entire receptor. The first step of the docking process was performed using a grid-based scoring method. The top 100 molecules with the lowest gridscores were then subjected to Hawkins scoring in the second step. The top 10 Hawkins-scoring small molecules were finally selected for further analysis.

[0063] ZINC12 database hawkin_score(kcal / mol) PubChem database ZINC14880369 -57.067825 10579415 ZINC14880389 -49.513336 657149 ZINC14881286 -48.285492 18431979 ZINC14880272 -47.239639 5180209 ZINC14881215 -47.06031 3396483 ZINC14611917 -44.884117 54723986 ZINC16052056 -40.842224 24916826 ZINC02046955 -39.36113 2440 ZINC14880926 -39.013535 46936791 ZINC12504443 -38.721802 5287728

[0064] Example 2:

[0065] In this example, see Figure 1 , the UCSF chimera was used to show the three-dimensional diagram of the interaction between Oma1 and the ligand, and the Ligplus software was used to show the binding mode of OMA1 and the optimal binding small molecule ZINC14880369 in the receptor binding pocket.

[0066] Example 3: Changes in Oma1 mRNA and protein expression in heart failure

[0067] 3.1 Preparation of aortic arch constriction heart failure model mice:

[0068] The experimental animals were C57BL / 6J mice (female, weighing 20-25 g), which were housed under a 12-hour light / dark cycle for one week. The mice were induced with general anesthesia using isoflurane anesthesia, and the anesthesia concentration was maintained at 1.5%-2%. Under sterile conditions, a midline chest incision was performed to expose the aortic arch. A 7-0 silk suture was used to bypass the aortic arch and narrow the aortic arch to approximately 50% of its diameter by ligation to form a continuous blood flow load. The recovery period of the mice after surgery was 1-2 hours, and vital signs were observed.

[0069] In this application, the experimental animals were divided into four groups: Sham group, TAC group, TAC+Vehicle group, and TAC+ZINC14880369 treatment group.

[0070] 3.2 Animal tissue collection, RNA extraction, reverse transcription of cDNA and real-time PCR:

[0071] 2.2.1 Tissue sampling

[0072] (1) Use a Reward anesthesia mask to maintain the mouse in a stable breathing anesthesia state, cut the chest cavity, insert the perfusion needle into the left apex of the heart, and perform cardiac perfusion with normal saline;

[0073] (2) The liver was observed to be whitish and the mesentery was transparent, indicating that the perfusion was complete. A midline incision was made in the chest to expose the heart. Part of the heart tissue was taken out and placed in an EP tube containing 200 μL Trizol RNAase Free and placed in an ice box.

[0074] 2.2.2 RNA extraction

[0075] (1) Homogenize the sample using a homogenizer once every 30 seconds for 4 times until no tissue fragments are visible. Add 800 μL of Trizol and let it stand for 5 minutes.

[0076] (2) Add 200 μL of chloroform to each EP tube, shake rapidly and vigorously for 1 min, and let it stand for 5 min;

[0077] (3) Centrifugation, conditions: 4°C, 12000 rpm, 15 min;

[0078] (4) Gently pipette the upper clear liquid into an RNAase-Free EP tube, add an equal volume of isopropanol, gently invert the tube upside down until no visible filaments remain, and let it stand for 10 minutes;

[0079] (5) Centrifugation, conditions: 4°C, 12000 rpm, 15 min;

[0080] (6) Discard the supernatant, add 1 ml of anhydrous ethanol, mix gently, and centrifuge at 4°C, 12,000 rpm, for 15 min.

[0081] (7) Discard the supernatant, invert the EP tube onto filter paper, and allow to dry at room temperature until a translucent precipitate appears at the bottom of the tube;

[0082] (8) Add 20 μL RNase-free H2O to each tube, heat to 60°C, and dissolve in a water bath for 10 min. After dissolution, place the tube in an ice box.

[0083] (9) Measure RNA concentration using an OD meter.

[0084] 2.2.3 Reverse transcription of total RNA into cDNA

[0085] (1) Removal of DNA impurities in samples (10 μL system)

[0086]

[0087] (2) RNA reverse transcription to cDNA (20 μL system)

[0088]

[0089] After reverse transcription into cDNA, add triple-distilled water to dilute 8 times and store at -20℃ for future use.

[0090] (3) Primer sequence

[0091] Primer design: This patent uses the mouse Ccl2 mRNA sequence in the NCBI database. Primers were designed on the NCBI website and verified for specificity using BLAST. The size of the amplified product was predicted and the primer specificity was specifically verified based on the melting curve and agarose gel running results. The primer sequences were synthesized by OBiO (Shanghai Heyuan).

[0092] The primer sequences required in the experiment are as follows

[0093]

[0094] 2.2.4 Real-time PCR experiment

[0095] (1) Prepare the real-time PCR reaction system (10 μL) according to the following table:

[0096]

[0097] (2) PCR reaction (Life Technology) under the following conditions:

[0098]

[0099] 3.3 Animal tissue collection, protein extraction and Western blotting

[0100] ① After anesthesia, C57BL / 6 mice were washed with cold PBS by cardiac perfusion to remove blood. Subsequently, the heart tissue was quickly removed and cut in ice-cold PBS. After anesthesia, the heart tissue was washed with cold PBS by cardiac perfusion, and the heart tissue was quickly removed and cut in ice-cold PBS. The tissue was quickly placed in lysis buffer containing protease inhibitors (PMSF) and ground using a tissue grinder. After cold centrifugation (12,000×g, 4°C, 10 minutes), the supernatant was collected to obtain the protein sample.

[0101] ② The extraction steps of neonatal rat cardiomyocytes were the same as above. After the intervention, the cells were washed with PBS and the total protein was extracted using a lysis buffer containing a protease inhibitor (PMSF). The cell samples were centrifuged at 12000×g, 4°C, for 10 minutes, and the supernatant was collected to obtain the protein sample.

[0102] ③ Quantify protein concentration using BCA (Bicinchoninic Acid Assay);

[0103] ④ The protein samples were subjected to SDS-PAGE (12% polyacrylamide gel). After running the gel at 80V, the proteins were transferred from the gel to the PVDF membrane using the wet transfer method. The transfer conditions were 200mA for 2 hours. After transfer, the membrane was blocked with 5% skim milk + TBS-T (TBS containing 0.1% Tween-20) buffer for 1 hour at room temperature, and then incubated with the primary antibody overnight. The membrane was washed with TBST for 10 minutes three times, and the secondary antibody was used at room temperature for one hour and TBST for 10 minutes three times. The enhanced chemiluminescence (ECL) reagent was used for color development, and the images were captured using the chemiluminescence imaging system Bio-Rad ChemiDoc.

[0104] 3.4 Mouse cardiac function test:

[0105] ① All mice were anesthetized with isoflurane through a mask to ensure an appropriate depth of anesthesia and avoid movement interference;

[0106] ② After anesthesia, place the mouse on a warming bed and fix it in a supine position. Apply ultrasound coupling to the mouse's chest to reduce air interference and ensure good contact between the probe and the skin.

[0107] ③ A high-resolution Vevo 2100 small animal cardiac ultrasound device was used for two-dimensional imaging, and various functional indicators of the mouse heart were recorded by M-mode scanning, including left ventricular ejection fraction (EF), left ventricular fractional shortening (FS), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), left ventricular end-diastolic wall thickness (LVWTd), and left ventricular end-systolic wall thickness (LVWTs).

[0108] 3.5 Masson staining of mouse heart

[0109] ① Anesthesia and heart removal: After the mouse was anesthetized with isoflurane, a midline chest incision was performed to expose the heart;

[0110] ② Heart isolation and washing: quickly remove the heart and place it in cold PBS (phosphate buffered saline, pH 7.4) for washing to remove blood residue;

[0111] ③ The heart tissue was dewaxed, hydrated, stained, differentiated, collagen stained, nuclear stained, dehydrated and sealed;

[0112] ④ Sections Masson-stained heart tissue sections were observed using an optical microscope (Olympus BX51) and images were taken.

[0113] 3.6 H&E staining of mouse heart

[0114] ① Anesthesia and heart removal: After the mouse was anesthetized with isoflurane, a midline chest incision was performed to expose the heart;

[0115] ② Heart isolation and washing: quickly remove the heart and place it in cold PBS (phosphate buffered saline, pH 7.4) for washing to remove blood residue;

[0116] ③ The heart tissue was dewaxed and hydrated, stained with hematoxylin, differentiated and blued, stained with eosin, dehydrated and transparentized, and then mounted;

[0117] ④ Sections The H&E-stained heart tissue sections were observed using an optical microscope (Olympus BX51) and images were taken.

[0118] 3.7 TEM staining of mouse heart

[0119] ① Myocardial tissue fixation: Use 2.5% glutaraldehyde (paraformaldehyde) and 2.5% glutaraldehyde / 1% chromium oxide (OsO4) fixative at 4°C for 2 hours, wash the tissue with PBS (pH 7.4) for 10 minutes each time, and cut the fixed heart tissue into 1mm 3 small pieces;

[0120] ② Dehydration: The fixed heart tissue blocks were dehydrated using the following gradient ethanol solutions: 30%, 50%, 70%, 80%, 90% and 100% ethanol, each treatment lasting 20 minutes. After dehydration, the tissue blocks were immersed in pure acetone for 20 minutes.

[0121] ③ Sample embedding and sectioning: The dehydrated tissue blocks were immersed in epoxy resin for embedding. After curing in a 60°C oven for 48 hours, they were cut into ultrathin sections with a thickness of 70-90 nm using an ultramicrotome.

[0122] ④ Transmission electron microscopy observation: Ultrathin sections were placed on copper grids and contrast stained with 1% lead acetate solution for 10 minutes. The sections were observed and photographed using a Hitachi H-7650 transmission electron microscope.

[0123] 3. Construction of 8H9C2 cardiomyocyte hypertrophy model

[0124] ① Anesthesia and heart removal: After the mouse was anesthetized with isoflurane, a midline chest incision was performed to expose the heart;

[0125] ② Heart isolation and washing: quickly remove the heart and place it in cold PBS (phosphate buffered saline, pH 7.4) for washing to remove blood residue;

[0126] ③ Cardiomyocyte digestion: Cut the heart tissue into small pieces and place them in a digestion solution containing 0.1% collagenase I (Sigma-Aldrich, C0130) and 0.1% trypsin (Sigma-Aldrich, T4665). Digest at 37°C with shaking for 30-40 minutes. Gently shake every 10 minutes to ensure complete tissue dissociation. Terminate the digestion reaction with DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS).

[0127] ④Cell filtration and collection: Filter the digested cell suspension through a 70 μm cell sieve to remove undigested tissue fragments. After the cells are collected, they are precipitated by centrifugation (1000 rpm, 5 minutes);

[0128] ⑤ Cell resuspension and inoculation: Resuspend the cells in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin), and inoculate them into 6-well or 24-well plates at a concentration of 100,000-200,000 cells per well. Culture the cells in a constant temperature incubator at 37°C and 5% CO2. Later, ISO drug 100umol / L was used for cell intervention to construct a cardiomyocyte hypertrophy model.

[0129] 3.9 Cardiomyocyte Mitotracker Probe Detection

[0130] ① After 48 hours of myocardial cell extraction and plating, wash once with PBS to remove residual culture medium;

[0131] ② Add 100 μL Mitotracker Red solution (100 nM) to each well and gently shake the culture plate to ensure that the cells are evenly exposed to the dye. Incubate in a 4°C, 5% CO2 incubator for 30 minutes. After staining, wash the cells three times with PBS and add Hoechst in a 37°C, 5% CO2 incubator for 10 minutes. After incubation, wash the cells three times with PBS.

[0132] ③Use Olympus IX83 confocal microscope with excitation wavelength of 577nm and emission wavelength of 590nm to observe and take pictures of mitochondrial morphology of cardiomyocytes.

[0133] 3.10 Cardiomyocyte Mitosox Probe Detection

[0134] ① After 48 hours of myocardial cell extraction and plating, wash once with PBS to remove residual culture medium;

[0135] ②Add an appropriate amount of MitoSOX working solution (5 μM) to each well and incubate in a 37°C, 5% CO2 incubator for 30 minutes. After incubation, wash three times with PBS and add Hoechst and incubate in a 37°C, 5% CO2 incubator for 10 minutes. After incubation, wash three times with PBS.

[0136] ③ Use an Olympus IX83 confocal microscope with an excitation wavelength of 510 nm and an emission wavelength of 580 nm to observe and photograph the distribution and intensity of red fluorescence in the cells.

[0137] 3.11 JC-1 probe detection in cardiomyocytes

[0138] ① After 48 hours of myocardial cell extraction and plating, wash once with PBS to remove residual culture medium;

[0139] ② Add an appropriate amount of 2 μM JC-1 dye solution to each well and incubate in a 37°C incubator for 30 minutes. After incubation, wash three times with PBS and add Hoechst and incubate in a 37°C, 5% CO2 incubator for 10 minutes. After incubation, wash three times with PBS.

[0140] ③ Use an Olympus IX83 confocal microscope for observation. The red fluorescence (high membrane potential) excitation wavelength is 490 nm / emission wavelength is 590 nm. The green fluorescence (low membrane potential) excitation wavelength is 490 nm / emission wavelength is 530 nm. Observe and photograph the green and red fluorescence in the cells and calculate the ratio.

[0141] 3.12 Cardiomyocyte Fura2 probe detection

[0142] ① After 48 hours of myocardial cell extraction and plating, wash once with PBS to remove residual culture medium;

[0143] ② Add an appropriate amount of Fura-2 working solution (10 μM) to each well and incubate in a 37°C, 5% CO2 incubator for 15 minutes. After incubation, wash three times with PBS and add Hoechst and incubate in a 37°C, 5% CO2 incubator for 10 minutes. After incubation, wash three times with PBS.

[0144] ③Use confocal microscope Olympus IX83 for observation, with excitation wavelength of 340nm / emission wavelength of 510nm for bound calcium and excitation wavelength of 380nm / emission wavelength of 510nm for free calcium to observe and photograph the fluorescence intensity of Fura-2 in cells, and use the fluorescence intensity ratio (F340 / F380) to calculate the changes in intracellular calcium ion concentration.

[0145] 3.13 Myocardial cell ATP detection

[0146] ① ATP extraction: After extracting mouse heart tissue, the heart tissue was homogenized on ice using pre-chilled physiological saline (0.9% NaCl). 1 mL of the extract was added to every 100 mg of heart tissue and homogenized on ice using a glass homogenizer. The homogenized sample was centrifuged (12,000 rpm, 4°C, 10 minutes) to remove cell debris and undissolved tissue, and the supernatant was collected as the ATP extract.

[0147] ② ATP quantitative analysis by HPL method: ATP was separated using a C18 reverse phase column. The injection volume of each sample was 50 μL. The ATP detection wavelength was 254 nm. The ATP content was calculated based on the ATP standard curve.

[0148] 3.14 Detection of mtKeima in cardiomyocytes

[0149] ① mtKeima plasmid transfection: Mix plasmid DNA and transfection reagent Lipofectamine 3000 at a 1:1 ratio and add to cardiomyocyte culture medium. The transfection time is 4-6 hours. After the transfection is completed, replace the culture medium with DMEM containing 10% FBS and continue to culture at 37°C for 48 hours. Then add Hoechst and incubate in a 37°C, 5% CO2 incubator for 10 minutes. After the incubation, wash three times with PBS.

[0150] ② mtKeima fluorescence microscopy: Detection was performed using an Olympus IX83 fluorescence microscope. mtKeima exhibits distinct fluorescence properties under different pH conditions. Its emission wavelength is 570 nm at neutral pH and 450 nm in acidic environments. Using a 488 nm excitation light source, emission light at 570 nm (neutral pH) and 450 nm (acidic pH) was collected to observe and image the fluorescence intensity and location of mtKeima in cells.

[0151] 3. Experimental Conclusions

[0152] See also Figure 2 , M-mode echocardiography of mice: Figures A-C show that compared with the mice in the sham group, the EF and FS of the mice in the TAC group were decreased, and Figure D shows that compared with the mice in the sham group, the HW / BW and HW / TL were increased, **p<0.01, ***p<0.001, ****p<0.0001 compared with the control group, the data are expressed as the mean ± SEM, and the statistical significance was evaluated by one-way analysis of variance followed by LSD, n=6).

[0153] like Figure 3 As shown, Figure 3 A and Figure 3 B, Compared with the mice in the Sham group, the myocardial fibrosis in the TAC group was more obvious as shown by Masson staining. Figure 3 C and Figure 3 D Compared with the mice in the Sham group, H&E staining showed that the cell volume was enlarged, the cell morphology was irregular, the arrangement was disordered, and the cell nuclei were pyknotic and necrotic (scale bar: 50 μm, magnification, ×20). Figure 3 E and Figure 3 Transmission electron microscopy (TEM) showed the ultrastructure of myocardial mitochondria: compared with the sham group, the myocardial mitochondrial sections of the TAC group mice showed a series of damages, the normal mitochondrial structure was destroyed, the mitochondria were swollen, the cristae disappeared significantly, and the mitochondrial autophagosomes were reduced (n=3, scale bar: 2μm).

[0154] like Figure 4As shown in the figure, real-time PCR results show that Oma1 mRNA expression in heart failure mice is significantly elevated after heart failure modeling. Each group consisted of 9 mice. Real-time PCR results were compared with the vehicle group at the corresponding time point using the Student's t-test. ***P < 0.001.

[0155] See also Figure 5 ,Westerblotting results showed that the protein expression of Oma1 in heart failure was significantly increased in the TAC group compared with the Sham group.

[0156] like Figure 6 As shown, Western blotting results showed that the protein expressions of Opa1 and PINK1 in heart failure were significantly decreased in the TAC group compared with the sham group.

[0157] See also Figure 7 Compared with the Con group, the expression of Oma1 mRNA in the ISO group was significantly increased. There were 6 replicates in each group. The real-time PCR results were analyzed using the Student's T-test. Compared with the solvent group at the corresponding time point, ***P<0.001.

[0158] like Figure 8 As shown in the figure, compared with the Con group, the ATP expression in the ISO group was significantly reduced. The results of 6 replicate wells in each group were tested using the Student's T-test. Compared with the solvent group at the corresponding time point, ***P<0.001.

[0159] like Figure 9 As shown, compared with the Con group, the mitochondria in ISO-stimulated NRCMs were less elongated and more short, thick or round rod-shaped, indicating reduced fusion and increased fission (representative confocal microscopy images are shown, scale bar: 50 μm, the data presented are representative data of three independent experiments).

[0160] like Figure 10 As shown, Figure 10 AB, Compared with the Con group, the red fluorescence of mitochondria in the ISO group decreased, indicating a significant decrease in mitophagosomes, ****P < 0.0001. (Representative confocal microscopy images are shown, scale bar: 50 μm, data presented are representative of three independent experiments).

[0161] like Figure 11 As shown, Figure 11AB, The red fluorescence in the ISO group was significantly increased compared with that in the Con group (representative confocal microscopy images are shown, scale bar: 50 μm, the data presented are representative of three independent experiments, **p < 0.01 relative to the control, data are expressed as mean ± SEM, and statistical significance was assessed using one-way ANOVA followed by LSD).

[0162] like Figure 12 As shown, Figure 12 AB, Compared with the Con group, the mitochondrial calcium level in the ISO group was decreased, and the red fluorescence was reduced (representative confocal microscopy images are shown, scale bar: 50 μm, the data presented are representative data of three independent experiments, ***p < 0.001 versus control, data are expressed as mean ± SEM, and statistical significance was assessed using one-way ANOVA followed by LSD).

[0163] like Figure 13 As shown, compared with the Con group, the mitochondrial membrane potential level of the ISO group decreased, the green monomers increased, and the red aggregates decreased (representative confocal microscopy images are shown, scale bar: 50 μm, the data presented are representative data of three independent experiments, ****p < 0.0001 relative to the control, data are expressed as mean ± SEM, and statistical significance was assessed using one-way ANOVA followed by LSD).

[0164] See also Figure 14 Compared with the TAC+Vehicle group, the Oma1 protein expression level in the TAC+ZINC14880369 group was decreased. Western blotting results showed that the small molecule ZINC14880369 significantly inhibited the expression of Oma1 protein.

[0165] See also Figure 15 M-mode echocardiography of mice: Figures A to D showed increased EF and F in the TAC+Vehicle group compared with the mice in the TAC+ZINC14880369 group, and decreased HW / BW and HW / TL in Figures E and F, **p<0.01 ****p<0.0001 compared with the control group. Data are expressed as mean ± SEM, and statistical significance was assessed using one-way ANOVA followed by LSD, n=6).

[0166] In addition, Figure 6As shown, in this application, a TAC model was established to induce heart failure. The small molecule inhibitor ZINC14880369 was gavage-injected on the second day after TAC, and then whether the inhibitor alleviated the course of heart failure was detected. Masson, H&E, and TEM results showed that the Oma1 small molecule inhibitor ZINC14880369 could alleviate the progression of TAC-induced heart failure. Masson and H&E staining: Figures A and B, compared with the TAC+Vehicle group of mice, the myocardial fibrosis of the TAC+ZINC14880369 group mice was significantly improved by Masson staining. Figures C and D, compared with the TAC+Vehicle group of mice, H&E staining showed that the cell volume was reduced, the cell morphology was regular, and the nuclear pyknosis and necrosis were reduced (scale bar: 50μm, magnification, ×20). Figures E and F show the ultrastructure of myocardial mitochondria by transmission electron microscopy (TEM): compared with the mice in the TAC+Vehicle group, the myocardial mitochondrial sections of the mice in the TAC group showed less damage, normal mitochondrial knots, reduced mitochondrial swelling, obvious cristae, and increased mitochondrial autophagosomes (n=3, scale bar: 2 μm).

[0167] In summary, in the present application, after targeted inhibition of Oma1 by the small molecule inhibitor ZINC14880369 in the hearts of mice with heart failure, examinations showed that cardiac enlargement was significantly reduced, cardiac function was significantly improved, and perspective electron microscopy showed that mitochondrial dynamics changed, fission was reduced, and autophagy was increased. In the present application, in a cardiomyocyte hypertrophy model, after inhibition of Oma1 by the small molecule inhibitor ZINC14880369, mitochondrial dynamics changed significantly, mitochondrial fission was reduced, fusion was increased, and the mitochondrial fusion and fission proteins also underwent the same changes. It is worth noting that the fusion protein Opa1 underwent hydrolysis changes after Oma1 inhibition, L-Opa1 expression increased, and S-Opa1 expression decreased. The state of mitochondrial autophagy was also detected in the present application. After Oma1 inhibition, mitochondrial autophagy was improved, and Pink1 expression increased. Mitochondrial function was further detected in the present application. After inhibition of Oma1, mitochondrial ATP production increased, calcium overload improved, membrane potential levels increased, and reactive oxygen species production decreased. In summary, the Oma1 small molecule inhibitor ZINC14880369 can improve the simultaneous hydrolysis of Opa1 and Pink1 to affect mitochondrial dynamics, thereby increasing mitochondrial energy production and reducing ROS generation, thereby improving the course of heart failure.

Claims

1. An OMA1 inhibitor, characterized in that: The OMA1 inhibitor is at least one of ZINC14880369, ZINC14880389, ZINC14881286, ZINC14880272, ZINC14881215, ZINC14611917, ZINC16052056, ZINC02046955, ZINC14880926, and ZINC12504443.

2. Use of an OMA1 inhibitor according to claim 1 in the preparation of a medicament for treating heart failure.

3. The use according to claim 2, characterized in that: The OMA1 inhibitor ZINC14880369.

4. The use according to claim 3, characterized in that: The drug inhibits the expression of OAM1 gene and protein through the small molecule inhibitor ZINC14880369 to achieve the purpose of treating heart failure.

5. A drug for treating heart failure, characterized in that: The invention comprises the OMA1 inhibitor according to claim 1.

6. A drug for treating heart failure according to claim 5, characterized in that: The medicine further comprises a medically acceptable adjuvant.

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