Application of NDUFA13 mutant and NDUFA13 inhibitor in preparing medicament for treating heart failure

Abstract

The present invention relates to the field of biomedicine, and discloses the use of NDUFA13 mutants and NDUFA13 inhibitors in the preparation of drugs for treating heart failure. The present invention for the first time discovers that NDUFA13 mutants and NDUFA13 inhibitors can play a role in maintaining the mitochondrial cristae structure and promoting mitochondrial fatty acid-related metabolism in heart failure through the following pathways: (1) alleviating the disruption of the PHB1 / 2 superpolymer to maintain the mitochondrial cristae structure; (2) further promoting fatty acid-related metabolism by maintaining the activity of carnitine palmitoyltransferase II dependent on the mitochondrial cristae structure.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the application of NDUFA13 mutants and NDUFA13 inhibitors in the preparation of drugs for treating heart failure. Background Art

[0002] Heart failure is a serious heart disease, and its prevalence shows a continuous upward trend globally. The latest research findings indicate that the prevalence of heart failure has increased by 44% in the past 15 years, with an increase of more than 9 million people globally. This data makes people realize that heart failure has become a major public health problem in the world today. Heart failure has a serious impact on the survival time and quality of life of patients. In patients with heart failure, the ventricular structure and systolic and diastolic functions are abnormal, resulting in the heart's inability to effectively pump blood, thereby causing stasis in the systemic and pulmonary circulations. This stasis causes symptoms such as dyspnea, shortness of breath, and fatigue in patients, seriously affecting their daily life and working ability. At the same time, heart failure also increases the risk of death caused by cardiac events in patients. According to statistics, the mortality rate of heart failure patients within 5 years after discharge is as high as 42%. To address this public health challenge, we need to deeply understand the pathological mechanism of heart failure and develop targeted effective prevention and treatment measures to reduce the incidence of heart failure, improve the quality of life of patients, and increase their survival rate.

[0003] NDUFA13 plays a key role in the mitochondrial respiratory chain and is a component of respiratory chain complex I. Complex I is a key protein complex in mitochondria and is involved in the process of intracellular energy production. The NDUFA13 gene is located in the long arm region of the human genome and is closely related to the normal function of the mitochondrial respiratory chain. Research shows that mutations in NDUFA13 may lead to abnormal function of the mitochondrial respiratory chain, thereby affecting the energy production and metabolic processes of cells. Mutations in NDUFA13 are related to the occurrence and development of various diseases. For example, in some studies, it has been found that mutations in NDUFA13 are related to mitochondrial diseases associated with defects in mitochondrial respiratory chain complex I, such as mitochondrial myopathy. In addition, the abnormal expression of NDUFA13 is also related to the occurrence of some nervous system-related diseases such as Parkinson's disease and Alzheimer's disease. The pathological process of heart failure involves disorders of multiple molecular and cellular processes. Although initial progress has been made in the research on heart failure, the role of NDUFA13 in the pathological process of heart failure is still unclear, especially its pathophysiological role in cardiomyocytes. If the association between NDUFA13 and heart failure can be revealed, it can provide new targets and strategies for the diagnosis and treatment of heart failure.

[0004] Prohibitin 2 (PHB2) is a highly conserved membrane protein that is widely expressed in the cytoplasm and mitochondria. As an important cytokine, early studies have found that PHB2 plays a key role in multiple cellular processes. First, PHB2 is involved in regulating cell proliferation and apoptosis in the cytoplasm. Studies have shown that PHB2 regulates the progression of the cell cycle and the occurrence of apoptosis by interacting with other proteins. PHB2 polymerizes with PHB1 (Prohibitin 1) to form PHB1 / 2 (Prohibitin 1 / 2) to exert physiological functions. Previous literature reports that PHB1 / 2 is related to the stability of mitochondrial cristae structure, but its specific regulation in the pathological process of heart failure is still unclear. In addition, PHB2 is also involved in regulating biological processes such as lipid metabolism and cell migration. Although the functions of PHB2 have been confirmed in multiple cellular processes, its specific role in the pathological process of heart failure is still unclear. Heart failure is a complex heart disease, and its pathological mechanism involves disorders of multiple molecular and cellular processes. Recent studies have shown that PHB2 may play an important role in the occurrence and development of heart failure. Further research is needed to explore the specific mechanism of action of PHB2 in the pathological process of heart failure to provide new targets and strategies for the prevention and treatment of heart failure. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides the use of NDUFA13 mutants and NDUFA13 inhibitors in the preparation of drugs for the treatment of heart failure. The present invention discovers for the first time that NDUFA13 mutants and NDUFA13 inhibitors can play a role in maintaining the mitochondrial cristae structure and promoting mitochondrial fatty acid-related metabolism in heart failure through the following pathways: (1) alleviating the destruction of PHB1 / 2 superpolymers to maintain the mitochondrial cristae structure; (2) further promoting fatty acid-related metabolism by maintaining the activity of carnitine palmitoyltransferase II dependent on the mitochondrial cristae structure.

[0006] The specific technical solutions of the present invention are as follows:

[0007] In the first aspect, the present invention provides the use of NDUFA13 mutants or NDUFA13 inhibitors as protectants for PHB1 / 2 superpolymers (superpolymers composed of PHB2 and PHB1). In the second aspect, the present invention provides the use of NDUFA13 mutants or NDUFA13 inhibitors as protectants for the mitochondrial cristae structure of cardiomyocytes.

[0008] Preferably, the NDUFA13 mutant is NDUFA13-K7R, that is, the K7 site of NDUFA13 is mutated to arginine (inhibiting the acetylation modification at the K7 site). The amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.3; the nucleotide sequence is as shown in SEQ ID NO.2 or SEQ ID NO.4. The NDUFA13 inhibitor is a reagent that can inhibit the acetylation modification at the K7 site of NDUFA13.

[0009] Both NDUFA13 and PHB2 are present in mitochondria. The present invention first discovers the regulatory relationship between the two. And the present invention finds that the mutation of NDUFA13 may lead to the destruction of mitochondrial cristae structure. Therefore, the present invention intervenes in the NDUFA13-PHB2 regulatory axis. The above-mentioned NDUFA13-K7R mutant and NDUFA13 inhibitor of the present invention can effectively alleviate the destruction of PHB1 / 2 (a superpolymer composed of PHB2 and PHB1), thereby playing a role in maintaining the mitochondrial cristae structure.

[0010] Previous literature reports that NDUFA13 is a subunit protein in mitochondrial respiratory chain complex I, but the exact role of NDUFA13 in maintaining mitochondrial OXPHOS function has not been clarified. In the pathological process of heart failure, our research finds that the acetylation modification at the NDUFA13-K7 site promotes the destruction of PHB1 / 2 and simultaneously inhibits the activity of carnitine palmitoyltransferase II (CPT II), ultimately leading to damage to mitochondrial cristae structure and mitochondrial fatty acid-related metabolism. Therefore, the NDUFA13-K7R mutant, that is, the 7th lysine site is mutated to arginine (this mutation inhibits acetylation modification), can improve the above pathological changes and ultimately alleviate the pathological process of heart failure.

[0011] Furthermore, the NDUFA13 inhibitor is selected from honokiol, SZC-6, and the lentivirus overexpressing the NDUFA13 mutant.

[0012] In the third aspect, the present invention provides the application of the NDUFA13 mutant or the NDUFA13 inhibitor as a promoter for the activity of carnitine palmitoyltransferase II (CPT II) in cardiomyocyte mitochondria. In the fourth aspect, the present invention provides the application of the NDUFA13 mutant or the NDUFA13 inhibitor as a promoter for mitochondrial fatty acid-related metabolism in cardiomyocytes.

[0013] The present invention first discovers that the NDUFA13-K7R mutant and the NDUFA13 inhibitor can play a role in promoting mitochondrial fatty acid-related metabolism in cardiomyocytes in heart failure through the following pathways: (1) alleviating the disruption of the PHB1 / 2 superpolymer to maintain the mitochondrial cristae structure; (2) further promoting fatty acid-related metabolism by maintaining the activity of carnitine palmitoyltransferase II (CPT II) dependent on the mitochondrial cristae structure.

[0014] In a fifth aspect, the present invention provides the use of an NDUFA13 mutant or an NDUFA13 inhibitor in the preparation of a therapeutic drug for heart failure.

[0015] The present invention first discovers that there is a regulatory relationship between NDUFA13 and PHB2 in the pathological process of heart failure. Therefore, the intervention of the present invention on the NDUFA13-PHB2 regulatory axis can be used as an effective strategy to alleviate the pathological process of heart failure. A series of results provide new targets and strategies for the treatment of heart failure. On this basis, it is beneficial to further develop treatment methods targeting the NDUFA13-PHB2 regulatory axis to improve the cardiac function and quality of life of heart failure patients.

[0016] Preferably, the drug comprises an NDUFA13 mutant and / or an NDUFA13 inhibitor, as well as a pharmaceutically acceptable carrier, excipient or solvent.

[0017] Preferably, the drug is an oral preparation or an injection preparation.

[0018] Furthermore, the dosage form of the oral preparation is a capsule, tablet, solution or powder; the injection preparation is a vesicle, injection syringe or powder.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention first discovers that there is a regulatory relationship between NDUFA13 and PHB2 in the pathological process of heart failure. The present invention intervenes on the NDUFA13-PHB2 regulatory axis and discovers that the NDUFA13 mutant and the NDUFA13 inhibitor can play a role in maintaining the mitochondrial cristae structure and promoting mitochondrial fatty acid-related metabolism in heart failure. Therefore, the NDUFA13 mutant and the NDUFA13 inhibitor can be used as an effective strategy to alleviate the pathological process of heart failure, providing new targets and strategies for the treatment of heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Destruction of the PHB polymer and abnormal mitochondrial structure in heart failure, and it is related to the promotion of the interaction between NDUFA13 / PHB2 by NDUFA13-K7 acetylation:

[0021] (a) After glutaraldehyde fixation of non-heart failure and heart failure tissues, transmission electron microscopy (TEM) was used to observe the mitochondrial structure in cardiomyocytes; (b) The mitochondrial area and cristae density in (a) were calculated (n = 200); (c) BN-PAGE analysis was performed on heart tissues to detect PHB1 / 2 and PHB1 / 2-SC; (d) Mitochondria in myocardial tissues were isolated and lysed, and then Western blot detection was performed using an anti-pan-acetyl-lysine antibody to evaluate the acetylation level of mitochondrial proteins; (e) Protein profiling and post-translational modification mass spectrometry detection methods were used to identify the acetylated lysine sites on NDUFA13 and PHB1 / 2. The spatial distribution and structural characteristics of the protein domains corresponding to the acetylation sites were shown in the schematic diagram; (f) After lysis of non-heart failure and heart failure myocardial tissues, Western blot and co-IP detections were performed to analyze the correlation between NDUFA13-K7 acetylation and the interaction between NDUFA13 / PHB2;

[0022] Figure 2 : The NDUFA13-K7R mutation (lysine at position 7 mutated to arginine) maintains the stability of PHB1 / 2 and PHB1 / 2-SC, and maintains mitochondrial function:

[0023] (a) NDUFA13fl / fl-MEFs expressed NDUFA13-WT, NDUFA13-K7Q, and NDUFA13-K7R, and PHB1 / 2 and PHB1 / 2-SC were detected by BN-BAGE; (b) After treatment with Oligomycin, NDUFA13fl / fl-MEFs expressed NDUFA13-WT, NDUFA13-WT, and NDUFA13-K7R, and the TMRM signal was detected by flow cytometry, and mitochondrial function was evaluated by the mitochondrial membrane potential level; (c) After treatment with Oligomycin, NDUFA13fl / fl-MEFs expressed NDUFA13-WT, NDUFA13-K7Q, and NDUFA13-K7R, and the Mito-SOX signal was detected by flow cytometry, and mitochondrial oxidative stress was evaluated by the ROS level;

[0024] Figure 3 : The NDUFA13-K7R mutation alleviates the maintenance of mitochondrial structure and alleviates the pathological process of heart failure:

[0025] (a) Mitochondrial size was expressed as the average surface area, and mitochondria in the NDUFA13-WT (n = 2000) and NDUFA13-K7R (n = 2000) groups treated with TAC were imaged by transmission electron microscopy; (b) The frequency distribution of mitochondrial surface area was calculated, and the Kruskal-Wallis test was performed, **P < 0.01, and the data were median values; (c) After Sham and TAC treatments, echocardiography was performed on NDUFA13-WT and NDUFA13-K7R mice, and typical axial sectional images were shown; (d) Left ventricular fractional shortening (LVFS), left ventricular end-diastolic diameter (LVIDd), and left ventricular mass (LVMass) were measured to evaluate cardiac morphology and function; (e) After the human AC16 cardiomyocyte line was treated with honokiol for 24 hours, mitochondrial structure was detected by electron microscopy, and mitochondrial area and mitochondrial cristae density were calculated.

[0026] Figure 4 : NDUFA13-K7R maintains the mitochondrial tricarboxylic acid cycle (TCA cycle) and improves mitochondrial lipid metabolism:

[0027] (a) shows the levels of TCA cycle intermediate metabolites in the heart tissues of NDUFA13-WT and NDUFA13-K7Q mice detected by metabolic mass spectrometry after TAC treatment, and the differences and fold changes are presented in the form of numerical values and heat maps; (b) The relevant results in (a) were mapped to the glycolysis and TCA cycle metabolic pathways, with red indicating increased relative content and blue indicating decreased relative content; (c) Schematic diagram of the cytoplasmic-to-mitochondrial fatty acid transport mechanism. CPT-II depends on the mitochondrial cristae structure to promote the transport of free fatty acids (FFAs) and acyl carnitines from the cytoplasm to the mitochondria, where they are further oxidized to generate energy; (d) The contents of Carnitine-C16, Carnitine-C18:1, and Carnitine-C2, as well as the ratio of Carnitine-(C16 + C18:1) / C2, were calculated to evaluate CPT-II activity. Detailed implementation methods

[0028] The present invention will be further described below in conjunction with the embodiments.

[0029] General embodiments

[0030] Use of NDUFA13 mutants or NDUFA13 inhibitors as protectants for PHB1 / 2 superassemblies, protectants for mitochondrial cristae structures in cardiomyocytes, promoters for the activity of mitochondrial carnitine palmitoyltransferase II (CPT II) in cardiomyocytes, and promoters for mitochondrial fatty acid-related metabolism in cardiomyocytes.

[0031] Preferably, the NDUFA13 mutant is NDUFA13-K7R, and its amino acid sequence is shown in SEQ ID NO.1 (mouse) or SEQ ID NO.3 (human), and the nucleotide sequence is shown in SEQ ID NO.2 (mouse) or SEQ ID NO.4 (human).

[0032] SEQ ID NO.1 (mouse): MAASKVRQDMPPPGGYGPIDYKRNLPRRGLSGYSMFAVGIGALIFGYWRMMRWNQERRRLLIEDLEARIALMPLFQAEKDRRTLQILRENLEEEAIIMKDVPNWKVGESVFHTTRWVPPLIGEMYGLRTKEEMSNANFGFTWYT;

[0033] SEQ ID NO.3 (human): MAASKVRQDMPPPGGYGPIDYKRNLPRRGLSGYSMLAIGIGTLIYGHWSIMKWNRERRRLQIEDFEARIALLPLLQAETDRRTLQMLRENLEEEAIIMKDVPDWKVGESVFHTTRWVPPLIGELYGLRTTEEALHASHGFMWYT;

[0034] SEQ ID NO.2 (mouse):

[0035] ATGGCGGCGTCGAAGGTGCGCCAGGACATGCCCCCGCCAGGGGGCTACGGCCCCATCGACTACAAGCGGAACCTGCCCCGCCGGGGACTGTCGGGGTACAGCATGTTTGCTGTGGGCATCGGGGCCTTGATCTTTGGCTACTGGAGAATGATGAGGTGGAACCAGGAGCGCAGGCGCCTGCTGATTGAGGACTTGGAGGCCAGGATCGCCCTCATGCCGCTCTTCCAGGCAGAGAAGGACCGGAGGACCCTGCAGATTCTCCGGGAAAACCTGGAGGAGGAAGCCATCATCATGAAGGATGTGCCCAACTGGAAGGTGGGCGAGTCTGTGTTCCATACCACACGATGGGTGCCACCCCTCATTGGCGAGATGTATGGGTTGCGCACCAAGGAGGAGATGAGCAATGCCAACTTCGGCTTCACCTGGTACACTTAG;

[0036] SEQ ID NO.4 (Human):

[0037] ATGGCGGCGTCAAAGGTGCGCCAGGACATGCCTCCGCCGGGGGGCTATGGGCCCATCGACTACAAACGGAACTTGCCGCGTCGAGGACTGTCGGGCTACAGCATGCTGGCCATAGGGATTGGAACCCTGATCTACGGGCACTGGAGCATAATGAAGTGGAACCGTGAGCGCAGGCGCCTACAAATCGAGGACTTCGAGGCTCGCATCGCGCTGTTGCCACTGTTACAGGCAGAAACCGACCGGAGGACCTTGCAGATGCTTCGGGAGAACCTGGAGGAGGAGGCCATCATCATGAAGGACGTGCCCGACTGGAAGGTGGGGGAGTCTGTGTTCCACACAACCCGCTGGGTGCCCCCCTTGATCGGGGAGCTGTACGGGCTGCGCACCACAGAGGAGGCTCTCCATGCCAGCCACGGCTTCATGTGGTACACGTAG。

[0038] The NDUFA13 inhibitor is a reagent that can inhibit the acetylation modification of the K7 site of NDUFA13, and is further preferably selected from honokiol, SZC-6, and lentivirus overexpressing NDUFA13 mutants.

[0039] A therapeutic drug for heart failure, comprising the above-mentioned NDUFA13 mutant and / or NDUFA13 inhibitor, and a pharmaceutically acceptable carrier, excipient or solvent; preferably, the drug is an oral preparation or an injection preparation. Further, the dosage form of the oral preparation is a capsule, tablet, solution or powder; the injection preparation is a vesicle, injection needle or powder.

[0040] Example 1

[0041] Preparation of lentivirus overexpressing NDUFA13-K7R mutant:

[0042] The lentivirus overexpressing NDUFA13-K7R mutant (Ubi-NDUFA13-K7R mutant-3FLAG-CBh-gcGFP-IRES-puromycin) used in the examples of the present invention, negative control lentivirus

[0043] (Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin), all purchased from GeneChem Co., Ltd. in Shanghai;

[0044] Amino acid sequence of mouse NDUFA13-K7R mutant (SEQ ID NO.1).

[0045] Example 2

[0046] I. Cells and cell culture conditions

[0047] MEF cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU / mL penicillin, and 100 μg / mL streptomycin. The cells were maintained in a humidified incubator at 37°C and 5% CO2.

[0048] II. Animal model and transverse aortic constriction (TAC) surgery

[0049] Myh6Cre+NDUFA13-WT-flox / flox and Myh6Cre+ NDUFA13-K7R-flox / - mice were bred in a laminar flow ultra-clean animal breeding standard laboratory, allowed to eat and drink freely, and maintained in a room with controlled temperature (22±1°C) and humidity (65 - 70%) according to a 12:12 hour light / dark cycle. All procedures were approved by the Animal Ethics Review Committee of Zhejiang University and conducted in accordance with the guidelines of NIH Publication No. 85 - 23 (revised in 1996). Male mice at 6 - 8 weeks of age underwent TAC surgery, resulting in the occurrence of cardiac afterload overload. The mice were anesthetized with sodium pentobarbital (50 mg / kg intraperitoneal injection), and then a left thoracotomy was performed at the second intercostal space. 7-0 silk thread was wrapped around the aorta and tied to a 26-gauge blunt needle, and then the needle was removed, thus forming a narrowed area. Mice in the sham surgery group underwent a similar surgical procedure but did not receive TAC surgery.

[0050] III. Immunoprecipitation and Western blotting

[0051] The cultured cells were lysed in immunoprecipitation lysis buffer, and then the cell lysate was incubated overnight with the corresponding antibody in immunoprecipitation wash buffer with gentle shaking at 4°C. Subsequently, the protein-antibody immune complex was precipitated after incubation with protein A / G plus agarose enzyme (Santa Cruz Biotechnology, Shanghai, China) for 1 hour. The protein A / G plus agarose enzyme adsorbed with the protein-antibody immune complex was washed 5 times with lysis buffer before SDS-PAGE and immunoblotting, and immunoblotting was performed using the designated antibody.

[0052] IV. Mitochondrial Isolation

[0053] Before measuring the respiration of mitochondrial respiratory chain complexes, mitochondria were isolated using a mitochondrial isolation kit (Shanghai Boyuan Biotechnology Co., Ltd.) according to the manufacturer's instructions. Briefly, mice were sacrificed by cervical dislocation, and the hearts were removed and weighed. Each heart was washed in ice-cold BIOPS and minced in 1 ml of BIOPS, and then the tissue and 2 ml of isolation buffer were transferred to a pre-cooled glass Potter homogenizer. The tissue was homogenized 6 times at medium speed and then centrifuged at 800 g for 10 minutes at 4°C. The supernatant was then transferred to a new centrifuge tube and centrifuged at 10,000 g for 10 minutes at 4°C. After centrifugation, the supernatant was carefully poured off. The mitochondrial pellet was washed with 2 ml of isolation buffer and then resuspended in 100 µl of isolation buffer. Some isolated heart mitochondria were stored on ice until use, while other freshly isolated mitochondria were immediately used to measure oxygen consumption.

[0054] V. Native Gel Detection

[0055] The isolated mitochondrial fraction was lysed on ice for 15 minutes in BN-PAGE buffer (20 mM Bis-Tris, pH 7.0, 2 mM NaCl, 500 mM aminocaproic acid, 1 mM EDTA, 10% (v / v) glycerol, and protease inhibitor cocktail) containing 0.5% (w / v) digitonin. The lysate was clarified by centrifugation, and 0.5% (w / v) Coomassie Brilliant Blue G-250 was added. Proteins were separated on a 4-12% BN-PAGE gel and immunoblotted using the designated antibody.

[0056] VI. Echocardiography Detection

[0057] Transthoracic echocardiography was performed on the 56th day after TAC surgery. Mice were anesthetized by inhalation of isoflurane. A comprehensive echocardiographic study was performed using a Vevo 2100 system (VisualSonics, Toronto, Canada), and two-dimensional and M-mode images were acquired and analyzed to evaluate cardiac morphology and function.

[0058] VII. Quantitative mass spectrometry analysis of small molecule metabolites

[0059] All standard compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA), Steraloids Inc. (Newport, RI, USA), and TRC Chemicals (Toronto, ON, Canada). These standard substances were accurately weighed and dissolved to prepare stock solutions with a concentration of 5.0 mg / mL. Appropriate amounts of each stock solution were mixed to prepare calibration solutions. To minimize degradation, samples were thawed on ice. Approximately 10 mg of each sample was placed into a 1.5 mL tube, and 20 μL of deionized water was added. Ten zirconia beads were added, and the samples were homogenized for 3 minutes. Then, 120 μL of pre-cooled methanol solution containing internal standard was added, and homogenization was performed again for 3 minutes. After that, the samples were centrifuged at 18,000 g for 20 minutes, and 20 μL of the supernatant was transferred to a 96-well plate. The following steps were performed using an Eppendorf epMotion workstation (Eppendorf Inc., Hamburg, Germany). Freshly prepared derivatization reagent (20 μL) was added to each well, and the well plate was sealed for derivatization at 30°C for 60 minutes. Then, 330 μL of pre-cooled 50% methanol solution was added to each well, and the well plate was centrifuged (4°C, 4000 g, 30 minutes). Then, 135 μL of the supernatant was aspirated and transferred to a new 96-well plate. Finally, 10 μL of internal standard was added to each well, and a gradient dilution of the derivatization standard stock solution was added to the left wells. After the well plate was sealed, it was used for LC-MS analysis.

[0060] To quantify functional small molecule metabolites, analysis was performed using ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS / MS) (ACQUITY UPLC-Xevo TQ-S, Waters Corp., Milford, MA, USA). The raw data files generated by UPLC-MS / MS were processed using QuanMET software (v2.0, Metabo-Profile, Shanghai, China) for peak area integration, calibration, and quantitative analysis of metabolites. Statistical analysis was performed using the R studio software package.

[0061] VIII. Flow Cytometry Analysis of Mitochondrial Membrane Potential and Mitochondrial Oxidative Stress

[0062] After cardiomyocytes were treated with TMRM (200 nmol / L) or Mito-SOX (50 nmol / L) for 30 minutes, mitochondrial membrane potential (ψmt) and mitochondrial oxidative stress were measured. The average fluorescence intensity (arbitrary unit) obtained was used as the evaluation value.

[0063] IX. Transmission Electron Microscopy (TEM)

[0064] Mouse heart specimens of the designated genotype were fixed in 2% formaldehyde and 2.5% (V / V) glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.4) at room temperature for 2 hours, and then incubated overnight at 4°C. Then, the sections were washed three times in 0.1 M PBS for 15 minutes each, and then post-fixed in 1% potassium permanganate for 1 hour. The sections were stained with uranyl acetate, dehydrated through a gradient ethanol solution, washed with propylene aldehyde, and then embedded in epoxy resin. Then, they were trimmed and cut into ultra-thin sections (120 nm), and subsequently observed under a transmission electron microscope ((H7500 TEM, Hitachi, Tokyo, Japan, http: / / www.hitachi.com). The mitochondrial area and mitochondrial cristae density were measured using the multi-measure ROI tool developed by the ImageJ software manufacturer.

[0065] X. Data Analysis

[0066] 1. In heart failure, the disruption of PHB polymers and abnormal mitochondrial structure are related to the promotion of the interaction between NDUFA13 and PHB2 by NDUFA13-K7 acetylation

[0067] Left ventricular free wall myocardial tissues were selected and examined by transmission electron microscopy. It was found that the mitochondrial cristae density was significantly decreased ( Figure 1 a&b). In mitochondria, Prohibitin1 (PHB1) and Prohibitin2 (PHB2) form a polymer, namely PHB1 / 2; PHB1 / 2 continues to form a super-polymer with related proteins such as AFG3L2, namely PHB1 / 2-SC; and PHB1 / 2 is the key to maintaining mitochondrial cristae. We lysed heart failure myocardial tissues and detected PHB1 / 2 and PHB1 / 2-SC using non-denaturing gel (BN-PAGE) after lysis. The results showed that PHB1 / 2 and PHB1 / 2-SC were significantly disrupted in heart failure ( Figure 1 c).

[0068] We further isolated mitochondria from heart failure myocardial tissues. After lysing the mitochondria, we used the anti-pan-acetyl-lysine antibody to detect the acetylation level of mitochondrial proteins. The results showed that in heart failure, mitochondrial proteins were significantly acetylated ( Figure 1 d). Further, using proteomics to detect protein-protein interactions and post-translational modifications, we found that in heart failure, NDUFA13 interacted with PHB2, the NDUFA13-K7 site was acetylated, and the PHB2-K202 / K224 sites were acetylated ( Figure 1 e). By using co-immunoprecipitation, we found that in heart failure, NDUFA13 / PHB2 interaction occurred when NDUFA13-K7 was acetylated. In contrast, in normal hearts (non-heart failure), the NDUFA13-K7 was in a non-acetylated state and no NDUFA13 / PHB2 interaction occurred ( Figure 1 f).

[0069] The NDUFA13-K7R mutation (lysine 7 mutated to arginine) maintains the stability of PHB1 / 2 and PHB1 / 2-SC and mitochondrial function

[0070] PHB1 / 2 and PHB1 / 2-SC are key protein complexes for the formation and stability of mitochondrial cristae structures. Therefore, in vitro experiments were further conducted to detect the effects of NDUFA13-K7R and NDUFA13-K7Q mutations on PHB1 / 2 and PHB1 / 2-SC. Lentiviruses carrying the NDUFA13-K7R mutation were constructed, in which lysine 7 was mutated to arginine to inhibit the acetylation modification at the NDUFA-K7 site. At the same time, lentiviruses carrying the NDUFA13-K7Q mutation were constructed, in which lysine 7 was mutated to glutamine to mimic the acetylation modification at the NDUFA13-K7 site. Lentiviruses carrying NDUFA13-WT, that is, the wild-type NDUFA13 (non-mutated sequence), were used as a control (both mutations (NDUFA13-K7Q mimicking acetylation and NDUFA13-K7R inhibiting acetylation) were carried out in in vitro experiments, and one mutation (NDUFA13-K7R inhibiting acetylation) was carried out in in vivo experiments). The results showed that under the stress state mediated by Oligomycin, compared with MEF cells expressing NDUFA13-WT, the NDUFA13-K7R mutation alleviated the disruption of PHB1 / 2 and PHB1 / 2-SC aggregates. In contrast, the NDUFA13-K7Q mutation promoted the disruption of PHB1 / 2 and PHB1 / 2-SC aggregates ( Figure 2 a).

[0071] TMRM staining was further used to detect mitochondrial membrane potential to evaluate the mitochondrial function status. It was found that under the stress state mediated by Oligomycin, compared with MEF cells expressing NDUFA13-WT, NDUFA13-K7R could significantly alleviate the impaired mitochondrial function ( Figure 2 b). In addition, Mito-SOX staining was used to detect mitochondrial oxidative stress to evaluate mitochondrial oxidative stress. It was found that NDUFA13-K7R had no significant effect on the mitochondrial oxidative stress state ( Figure 2 c).

[0072] 3. NDUFA13-K7R Mutation Alleviates the Maintenance of Mitochondrial Structure and Alleviates the Pathological Process of Heart Failure

[0073] The transverse aortic constriction (TAC) surgery was used to construct a heart failure model, and sham was used as a sham operation control. Finally, transmission electron microscopy examination showed that the NDUFA13-K7R mutation in heart failure alleviated the maintenance of mitochondrial volume and alleviated the abnormal cristae structure ( Figure 3 a&b), as follows: 2000 mitochondria from NDUFA13-WT and NDUFA13-K7R mice after TAC surgery were analyzed to evaluate the distribution of mitochondrial area. The kurtosis of the peak of the mitochondrial area distribution in the NDUFA13-WT group was mainly concentrated in the range of 0.2-0.6 μm 2 , suggesting that the mitochondrial volume was small and the density of its mitochondrial cristae structure was also low; on the contrary, in the NDUFA13-K7R group, the number of mitochondria with an area of 0.2-0.6 μm 2 was significantly reduced, while the number of mitochondria with an area below 0.8 μm 2 increased, suggesting that the cardiomyocyte mitochondria of NDUFA13-K7R mice were larger and the density of their mitochondrial cristae structure was also higher.

[0074] Echocardiography showed that there was no difference in the left ventricular end-diastolic diameter (LVIDd) and left ventricular fractional shortening (LVFS) measurements at 8 weeks after surgery between NDUFA13-WT and NDUFA13-K7R mice receiving sham surgery; in NDUFA13-WT and NDUFA13-K7R mice receiving TAC surgery, the LVIDd of NDUFA13-WT mice increased significantly and the LVFS decreased significantly, while NDUFA13-K7R mice could significantly alleviate the above pathological changes ( Figure 3 c&d). Based on the above echocardiography data, the NDUFA13-K7R mutation could significantly alleviate the deterioration of cardiac function in heart failure.

[0075] While inhibiting the acetylation of the NDUFA13-K7 site through the above NDUFA13 mutants, the small molecule compound honokiol was used to inhibit the acetylation of NDUFA13-K7. After honokiol inhibited the acetylation of the NDUFA13-K7 site, it could significantly increase the mitochondrial area and significantly increase the mitochondrial cristae density ( Figure 3 e).

[0076] 4. NDUFA13-K7R maintains the mitochondrial tricarboxylic acid cycle (TCA cycle) and improves mitochondrial lipid metabolism

[0077] After TAC surgery, compared with NDUFA13-WT mice, the levels of malate and fumarate in the TCA cycle of NDUFA13-K7R mice were significantly increased, suggesting that the NDUFA13-K7R mutation maintains the TCA cycle metabolic reaction ( Figure 4 a&b). The activity of carnitine palmitoyltransferase II (CPT II) depends on the integrity of the mitochondrial cristae structure. According to the mechanism diagram description, after TAC surgery, the damaged mitochondrial ridge structure may lead to abnormal CPT II function, thereby inhibiting the transfer of free fatty acids (FFAs) and carnitines from the cytoplasm to the mitochondria, resulting in blocked oxidative metabolism and increased concentrations of them ( Figure 4 c). Previous studies have shown that Carnitine-(C16+C18:1) / C2 is a key indicator reflecting the activity of the CPT II metabolic enzyme. To verify the above speculation, the metabolite content in myocardial tissue was detected using metabolic mass spectrometry. The results showed that after TAC surgery, compared with NDUFA13-WT mice, the levels of Carnitine-C16 and Carnitine-C18:1 in NDUFA13-K7R mice were significantly decreased, and the ratio of Carnitine-(C16+C18:1) / C2 was also significantly decreased ( Figure 4 d). The above data all indicate that NDUFA13-K7R significantly improves the function of CPT II, thereby alleviating the impaired fatty acid metabolism in cardiomyocytes in heart failure.

[0078] The raw materials and equipment used in the present invention are all common raw materials and equipment in the art unless otherwise specified; the methods used in the present invention are all conventional methods in the art unless otherwise specified.

[0079] The above are only the preferred embodiments of the present invention, and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent transformations made to the above embodiments according to the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. Use of NDUFA13 mutants in the preparation of drugs for treating heart failure, characterized in that: The amino acid sequence of the NDUFA13 mutant is shown in SEQ ID NO.1 or SEQ ID NO.3; the nucleotide sequence is shown in SEQ ID NO.2 or SEQ ID NO.

4.

2. The application according to claim 1, wherein: The drug comprises the NDUFA13 mutant and a pharmaceutically acceptable carrier.

3. The application according to claim 1, characterized in that: The drug comprises the NDUFA13 mutant and a pharmaceutically acceptable excipient.

4. The application according to claim 1, wherein: The drug comprises the NDUFA13 mutant and a pharmaceutically acceptable solvent.

5. The application according to claim 1, characterized in that: The drug is an oral preparation or an injection preparation.

6. The application according to claim 5, wherein: The dosage form of the oral preparation is capsule, tablet, solution or powder.

7. The application according to claim 5, characterized in that: The injection preparation is a powder.

8. Use of lentivirus overexpressing NDUFA13 mutant in the preparation of a therapeutic drug for heart failure, characterized in that: The amino acid sequence of the NDUFA13 mutant is shown in SEQ ID NO.1 or SEQ ID NO.3; the nucleotide sequence is shown in SEQ ID NO.2 or SEQ ID NO.

4.

9. The application according to claim 8, wherein: The drug comprises a lentivirus overexpressing the NDUFA13 mutant and a pharmaceutically acceptable excipient.

10. The application according to claim 8, wherein: The drug comprises a lentivirus overexpressing the NDUFA13 mutant and a pharmaceutically acceptable solvent.

11. The application according to claim 8, wherein: The drug is an oral preparation or an injection preparation.

12. The application according to claim 11, wherein: The dosage form of the oral preparation is capsule, tablet, solution or powder.

13. The application according to claim 11, wherein: The injection preparation is a powder.

Images