Use of siah2 inhibitors for treating heart failure or improving cardiomyocyte function

Abstract

The application discloses an application of an SIAH2 inhibitor to treating heart failure or improving myocardial cell function, relates to the technical field of medicine, and is used for researching the treatment effect and mechanism of SIAH2 on heart failure. The application provides an application of an SIAH2 gene or protein inhibitor to preparing a heart failure medicine, uses the SIAH2 inhibitor to intervene in a TAC-induced mouse heart failure model, finds that the SIAH2 inhibitor can improve myocardial energy metabolism and protect heart function, and provides a new target and strategy for clinically diagnosing and treating heart failure or improving myocardial cell function.

Description

Technical Field

[0001] This invention relates to the field of medical technology, and in particular to the application of a SIAH2 inhibitor for the treatment of heart failure or to improve myocardial cell function. Background Technology

[0002] The heart is a high-energy-demand organ and must continuously produce ATP to maintain its contractile function. The ATP stored in the heart can only sustain the heartbeat for 2-10 seconds.

[0003] Mitochondrial oxidative metabolism is the primary energy source for the heart. In a healthy heart, energy is supplied by various energy substrates, with approximately 40%-60% from fatty acid oxidation and 20%-40% from glucose metabolism. However, in a failing heart, energy metabolism is altered. Compared to a healthy heart, ATP levels in the end-stage of heart failure decrease by about 30%, leading to insufficient energy supply and ultimately impaired myocardial contractility. This alteration in energy metabolism may be due to impaired mitochondrial oxidative metabolism, changes in cardiac energy substrate preferences, and reduced cardiac efficiency. Metabolic remodeling plays a crucial role in regulating cardiac energy substrate utilization, ion and redox homeostasis, and maintaining ATP levels, and is essential for maintaining cardiac contractile function. Furthermore, the accumulation of certain metabolites during cardiac metabolic remodeling further exacerbates metabolic disorders. However, the signaling pathways involved in cardiac metabolic remodeling during the pathogenesis of heart failure are highly complex, and the regulatory mechanisms are not yet fully understood; current research is insufficient.

[0004] The ubiquitin-proteasome system (UPS) is a key intracellular protein degradation pathway responsible for regulating various cellular processes, including the cell cycle, signal transduction, gene expression, stress response, and metabolic homeostasis. In recent years, research on E3 ubiquitin ligases in cardiovascular diseases has increased. By specifically recognizing substrate proteins and promoting their ubiquitination, degradation, or stabilization, they regulate cardiovascular signal transduction, apoptosis, and inflammatory responses. Given the crucial role of E3 ubiquitin ligases in cardiovascular diseases, further exploration of their specific mechanisms in the occurrence and development of cardiovascular diseases, as well as their potential clinical application value as therapeutic targets, is essential.

[0005] In our study, we conducted a joint analysis of the transcriptomics of cardiomyocytes with β-receptor overactivation (ISO stimulation), mouse heart failure transcriptomics, and human heart failure transcriptomics. We found that among the genes with common changes, signaling pathways related to ubiquitination were significantly enriched. Further analysis of differentially expressed genes in the ubiquitination pathway revealed that SIAH2 is one of the most significantly changed E3 ubiquitin ligases.

[0006] SIAH2 (Seven in Absentia Homolog2) is an E3 ubiquitin ligase belonging to the SIAH family (Siah proteins), and its role in various cellular processes is gradually being revealed. It primarily participates in cell cycle, transcriptional regulation, metabolic regulation, and cellular stress responses by regulating protein ubiquitination. The mechanism of action and function of SIAH2 in different diseases are receiving increasing attention, especially in cardiovascular diseases, cancer, neurodegenerative diseases, and metabolic diseases, where its role as a key regulatory factor is becoming increasingly clear. For example, in adipogenesis, SIAH2 mediates the ubiquitination and degradation of ZFP521, promoting adipogenesis in preadipocytes; SIAH2 can also regulate DNA damage repair by promoting CtIP ubiquitination; and SIAH2 enhances the stability of HIF-1α by degrading PHD2 and PHD3, thereby promoting hypoxic adaptation of cancer cells and enhancing their invasive and metastatic abilities. Therefore, based on the above-mentioned studies, SIAH2 has been confirmed to be closely related to cellular stress, inflammatory response and apoptosis in a variety of disease states. However, the role of SIAH2 in heart failure remains unclear. Summary of the Invention

[0007] Based on the above analysis, the present invention aims to provide an application of SIAH2 inhibitors for the treatment of heart failure or to improve cardiomyocyte function, in order to study the therapeutic effect and mechanism of SIAH2 on heart failure.

[0008] The objective of this invention is mainly achieved through the following technical solutions:

[0009] This invention provides the application of an inhibitor of the SIAH2 gene or protein in the preparation of a drug for heart failure.

[0010] Furthermore, in the aforementioned applications, the inhibitor includes a specific small molecule inhibitor, the chemical structural formula of which is shown below, with the molecular formula being C0. 28 H 44 N4O8, with a molecular weight of 564.67 and CAS number 666843-10-3;

[0011]

[0012] Furthermore, in the aforementioned applications, the inhibitor also includes a SIAH2 gene knockout reagent.

[0013] Furthermore, in the aforementioned application, the SIAH2 gene knockout reagent is siRNA, the sense strand nucleotide sequence of which is shown in SEQ ID NO:1, and the antisense strand nucleotide sequence of which is shown in SEQ ID NO:2.

[0014] A second aspect of the present invention also provides the application of an inhibitor of the SIAH2 gene or protein in suppressing the expression level of heart failure marker mRNA in heart tissue.

[0015] Furthermore, in the aforementioned applications, the heart failure biomarker mRNA includes ANP, BNP, β-MHC, col1a1, and / or col3a1.

[0016] A third aspect of the present invention also provides the application of an inhibitor of the SIAH2 gene or protein in inhibiting the decrease in the expression of mitochondrial functional markers in heart tissue of heart failure.

[0017] Furthermore, in the aforementioned applications, the mitochondrial functional markers are Nudfa11, SDHB, Uqcrb, Cox6a2, and / or ATP5e.

[0018] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects:

[0019] The application of the SIAH2 inhibitor provided by this invention for the treatment of heart failure or the improvement of cardiomyocyte function, using the SIAH2 inhibitor to intervene in a TAC-induced mouse heart failure model, it was found that the SIAH2 inhibitor can improve myocardial energy metabolism and protect cardiac function. This invention provides new targets and strategies for the clinical diagnosis and treatment of heart failure or the improvement of cardiomyocyte function.

[0020] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention may be realized and obtained by means of the structures particularly pointed out in the written description and the accompanying drawings. Attached Figure Description

[0021] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts.

[0022] Figure 1 This figure shows the experimental results of detecting the expression level of SIAH2 protein in a TAC-induced mouse heart failure model and sham surgery in this embodiment using Western blotting.

[0023] Figure 2 This figure shows the experimental results of Western blotting detection of SIAH2 protein expression levels in the cardiac tissue of patients with heart failure and in the control group of patients with non-cardiac diseases in this embodiment.

[0024] Figure 3This study presents a quantitative analysis of the expression of ANP, BNP, β-MHC, col1a1, and col3a1, markers of central failure, in TAC-WT and TAC-KO model mice, using qRT-PCR.

[0025] Figure 4 This is a graph showing the quantitative analysis of qRT-PCR expression of mitochondrial functional markers Nudfa11, SDHB, Uqcrb, Cox6a2 and ATP5e in TAC-WT and TAC-KO model mice in this embodiment.

[0026] Figure 5 This is a transmission electron microscopy comparison of mitochondria in cardiomyocytes of TAC-WT and TAC-KO model mice in this embodiment;

[0027] Figure 6 In this embodiment, Seahorse was used to monitor the effects of shSIAH2 and ISO treatments on cellular mitochondrial respiratory function.

[0028] Figure 7 This figure shows the results of Seahorse monitoring of the effect of SIAH2 expression regulation on mitochondrial function in primary myocardial cells of ISO-stimulated lactating mice in this embodiment.

[0029] Figure 8 RT-PCR quantitative analysis of heart failure-related gene expression was performed after treating a wild-type mouse model with a specific small molecule inhibitor, TAC. Detailed Implementation

[0030] The present invention will be further illustrated below with reference to the accompanying drawings and embodiments, but this is not intended to limit the invention. Specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and not intended to limit the invention. Materials of the same or similar type, model, quality, properties, or functions as the reagents and instruments described below can be used to implement the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0031] This invention provides the application of an inhibitor of the SIAH2 gene or protein in the preparation of a drug for heart failure.

[0032] Furthermore, the inhibitor includes a specific small molecule inhibitor, the chemical structural formula of which is shown below, with the molecular formula being C0. 28 H 44 N4O8, with a molecular weight of 564.67 and CAS number 666843-10-3;

[0033]

[0034] Furthermore, the inhibitor also includes a SIAH2 gene knockout reagent.

[0035] Furthermore, the SIAH2 gene knockout reagent is siRNA, the sense strand nucleotide sequence of which is 5′-CUGAUAAAGAGUUAUGCCATT-3′ (SEQ ID NO:1), and the antisense strand nucleotide sequence of which is 5′-UGGCAUAACUCUUUAUCAGTT-3′ (SEQ ID NO:2).

[0036] The second invention also provides the application of an inhibitor of the SIAH2 gene or protein in suppressing the expression level of heart failure marker mRNA in cardiac tissue.

[0037] Furthermore, the heart failure biomarker mRNA includes ANP, BNP, β-MHC, col1a1, and / or col3a1.

[0038] A third aspect of the present invention also provides the application of an inhibitor of the SIAH2 gene or protein in inhibiting the decrease in the expression of mitochondrial functional markers in heart tissue of heart failure.

[0039] Furthermore, the mitochondrial functional markers are Nudfa11, SDHB, Uqcrb, Cox6a2 and / or ATP5e.

[0040] Example

[0041] 1. Establishment of a mouse model of heart failure induced by transverse aortic constriction (TAC):

[0042] TAC-induced heart failure models in mice include: TAC-induced wild-type heart failure model (TAC-WT model) and TAC-induced SIAH2 knockout mouse heart failure model (TAC-KO model). The TAC-WT model uses 8-week-old adult male C57BL / 6 mice weighing about 20–25g, while the TAC-KO model uses SIAH2 knockout mice constructed using CRISPR technology (SIAH2 knockout mice KO, sourced from Cyagen (Suzhou) Biotechnology Co., Ltd.).

[0043] The specific process for constructing a mouse model of heart failure induced by aortic arch coarctation is as follows:

[0044] Mice were anesthetized and fixed in a supine position. Hair was shaved from the anterior chest, and the skin was disinfected routinely. A longitudinal incision of approximately 1 cm was made along the midline of the sternum towards the tail, extending to the second intercostal space. The fascia in the suprasternal notch was bluntly dissected to expose the trachea. The thymus tissue was then separated laterally with small curved forceps to expand the surgical field. A self-made syringe needle (with the tip cut off and bent at a right angle) was inserted through the brachiocephalic trunk using a No. 5 surgical suture, pre-tying a knot for later ligation. A 4mm 27G needle with a modified bend was then inserted below the suture insertion site, and the ligation was completed. After ligation, the 27G needle was slowly removed, and the muscle layer and skin were sutured. The mice were then placed in a warm environment to recover, thus establishing the surgical model.

[0045] Mice in the sham-operated group were treated with the same procedure, but only the sutures were threaded without ligating the blood vessels.

[0046] 2. Collect cardiac samples from patients with clinical heart failure:

[0047] Following ethical review and with informed consent, left ventricular myocardial tissue (HFrEF) was collected from four patients clinically diagnosed with heart failure at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (located in Wuhan). Simultaneously, left ventricular myocardial tissue was collected from three control groups of patients without heart disease and without heart failure.

[0048] 3. Protein imprinting analysis of cardiac samples from clinical heart failure, TAC-induced mouse heart failure model, and sham-operated group:

[0049] Extraction of total protein from myocardial tissue: Myocardial tissue preserved in liquid nitrogen was ground in a mortar with liquid nitrogen. Two-thirds of the ground tissue (the other one-third was used to extract RNA) was added to tissue lysis buffer (20 mmol / L Tris-HCl pH 7.4, 150 mmol / L NaCl, 2.5 mmol / L EDTA, 50 mmol / L NaF, 0.1 mmol / L Na4P2O7, 1 mmol / L Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 1% deoxycholic acid, 1 mmol / L PMSF, 1 μg / mL aprotinin). After mixing, the mixture was placed on ice for 15 minutes. Approximately 800 μL of lysis buffer was added for every 50 mg of myocardial tissue. Collect the homogenate, sonicate it (45%, turn on for 5 seconds, turn off for 5 seconds, repeat 4 times), centrifuge at 12000 rpm for 15 minutes at 4℃, transfer a portion of the supernatant to a new EP tube, quantify the protein, freeze at -80℃, add a quarter volume of 5X loading buffer to a portion of the supernatant, mix well, boil at 100℃ for 5 minutes, freeze, and reserve for subsequent protein blot detection of related proteins.

[0050] Western blot assay: After electrophoresis on a 10% SDS-PAGE gel, the membrane was transferred to a nitrocellulose membrane, blocked with 5% skim milk at room temperature for 1 hour, and incubated overnight at 4°C with primary antibodies. The primary antibodies were: fibronectin (ab2413, abcam, Cambridge, MA, USA), αSMA (ab32575, abcam, Cambridge, MA, USA), ColI (203002, MDBiosciences), SIAH2 (ab75105, abcam, Cambridge, MA, USA), and GAPDH (2118S, CST). The membrane was washed three times with TBST, then replaced with the corresponding species-specific secondary antibody, incubated at room temperature for 1 hour, washed again with TBST, and developed. The membrane was placed in developing solution (Millipore Corporation), drained, and then exposed in a chemiluminescence immunoassay analyzer. Band intensity was quantified using NIH ImageJ software. GAPDH was used as a control.

[0051] The results of the protein imprinting experiment show that, for example Figure 1 As shown, compared with the sham-operated group, the expression level of SIAH2 was increased in the TAC-induced mouse heart failure model during the pathogenesis of heart failure. Figure 2 As shown, compared with the control group of patients without heart disease, the expression level of SIAH2 in the cardiac tissue of patients with heart failure was increased.

[0052] 4. Detect the expression levels of heart failure markers after surgery and SIAH2 knockout in TAC-induced mouse heart failure model mice.

[0053] The severity of heart failure in TAC-WT and TAC-KO model mice was verified using polymerase chain reaction (PCR), and the results were as follows: Figure 3 As shown, the mRNA expression levels of heart failure markers ANP, BNP, β-MHC, Col1a1, and Col3a1 in TAC-KO model mice were significantly decreased (primer sequences are shown in Table 1), indicating that cardiac function was restored and TAC-induced heart failure in mice was alleviated.

[0054] Mitochondrial function in the hearts of TAC-WT and TAC-KO model mice was verified using polymerase chain reaction (PCR), and the results are as follows: Figure 4 As shown, the expression levels of heart failure markers Nudfa11, SDHB, Uqcrb, Cox6a2 and ATP5e (primer sequences are shown in Table 1) in TAC-KO model mice were significantly increased, indicating that cardiac function was restored and TAC-induced heart failure in mice was alleviated.

[0055] Transmission electron microscopy was used to verify the mitochondrial morphology of cardiomyocytes in TAC-WT and TAC-KO mouse models, such as... Figure 5As shown, the mitochondria of the cardiomyocytes in the TAC-KO model mice have clear morphological cristae, indicating a protective function.

[0056] Table 1 Primer sequence list

[0057]

[0058] 5. Seahorse Experimental Study on the Effect of SIAH2 Expression Regulation on Mitochondrial Function in Primary Cardiac Cardiac Cells of Lactating Mice Stimulated by ISO.

[0059] 5.1 Primary cardiac cells from newborn mice: SPF-grade newborn mice aged 1-3 days were selected, and primary cardiac cells were isolated by trypsin digestion. They were cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator.

[0060] 5.2 SIAH2 gene knockout reagent (siRNA reagent): The siRNA sequences used to knock down SIAH2 are 5′-GCAGUUCUGUUUCCCUGUATT-3′ (SEQ ID NO:1) of the sense strand and 5′-UACAGGGAAACAGAACUGCTT-3′ (SEQ ID NO:2) of the antisense strand, with a purity ≥98%.

[0061] 5.3 Other reagents and instruments: Isoproterenol (ISO, purity ≥98%); Agilent Seahorse XFe24 cell energy metabolism analyzer and matching Seahorse cell culture microplates; DMEM medium containing 10% fetal bovine serum.

[0062] 5.4 Specific operations

[0063] 5.4.1 SIAH2 knockdown treatment: When the primary myocardial cells of neonatal mice grew to 70%-80% confluence, the above siRNA was transfected into the myocardial cells according to the instructions of Lipofectamine 3000 transfection reagent. A blank control group without transfection and a negative control group transfected with irrelevant sequence siRNA were set up. After transfection, the cells were cultured for another 24 hours.

[0064] 5.4.2 Cell Seeding and Stimulation: Transfected cardiomyocytes and control group cells were seeded at 5 × 10⁻⁶ cells / year. 4 The cells were evenly seeded at a density of cells / well in Seahorse cell culture microplates. After culturing for 24 hours, isoproterenol (ISO) was added to each well to a final concentration of 10 μmol / L, and the cells were cultured for another 48 hours.

[0065] 5.4.3 Seahorse Assay: The cell culture microplates were removed and replaced with Seahorse XF basal medium (containing 25 mmol / L glucose, 1 mmol / L pyruvate, and 2 mmol / L glutamine), and equilibrated at 37°C in a CO2-free environment for 1 hour. The microplates were then placed in an Agilent Seahorse XFe24 instrument, and cellular oxygen consumption was measured according to the instrument's instruction manual. The measured parameters included basal respiratory oxygen consumption, idle respiratory oxygen consumption, oxygen consumption for ATP production, and maximum respiratory oxygen consumption.

[0066] 5.5 Results

[0067] like Figure 6 As shown, knocking down SIAH2 enhances mitochondrial function in primary myocardial cells of lactating mice stimulated by ISO (maximum respiration, ATP production, and basal oxygen consumption all increase), indicating that the SIAH2 gene knockout reagent (SIAH2 knockdown) may affect the energy metabolism response of myocardial cells to ISO by regulating mitochondrial respiration.

[0068] Figure 7 The results showed that overexpression of SIAH2 impaired mitochondrial function in cardiomyocytes, indicating a significant disruption in mitochondrial function. Seahorse experiments confirmed that overexpression of SIAH2 led to a decrease in mitochondrial maximal oxygen consumption, idle oxygen consumption, ATP production oxygen consumption, and basal respiratory oxygen consumption in cardiomyocytes.

[0069] 6. Polymerase chain reaction was used to verify the effect of a specific small molecule inhibitor (Mizagliflozin) on TAC-induced mental exhaustion.

[0070] Aortic coarctation (TAC) was performed as described above to establish a TAC-induced heart failure model in wild-type mice caused by pressure overload. On the third day after surgery, the experimental mice were randomly divided into a treatment group and a control group.

[0071] The treatment group received a specific small molecule inhibitor (Mizagliflozin) via gavage at 20 mg / kg / day; the control group received DMSO solvent at 20 mg / kg / day. Administration continued for 2 weeks until the 4th week post-surgery. Subsequently, the expression levels of heart failure-related marker genes and genes related to collagen synthesis and extracellular matrix deposition were detected by RT-qPCR, such as... Figure 8 As shown, the specific small molecule inhibitor (Mizagliflozin) can reduce the mRNA levels of heart failure biomarkers ANP, BNP, β-MHC, Col1a1, and Col3a1, and inhibit the progression of stress overload-induced heart failure.

[0072] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of inhibitors of the SIAH2 gene or protein in the preparation of drugs for heart failure, characterized in that, The inhibitor acts directly on the SIAH2 gene or protein, and the inhibitor is a SIAH2 gene knockout reagent. The SIAH2 gene knockout reagent is siRNA, the nucleotide sequence of the sense strand of siRNA is shown in SEQ ID NO:1, and the nucleotide sequence of the antisense strand of siRNA is shown in SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The inhibitor is used to suppress the mRNA expression level of heart failure biomarkers in the cardiac tissue of heart failure patients.

3. The application according to claim 2, characterized in that, The heart failure biomarker mRNAs include ANP, BNP, β-MHC, col1a1, and / or col3a1.

4. The application according to claim 1, characterized in that, The inhibitor is used to suppress the decline in the expression of mitochondrial functional markers in heart tissue of patients with heart failure.

5. The application according to claim 4, characterized in that, The mitochondrial functional markers are Nudfa11, SDHB, Uqcrb, Cox6a2 and / or ATP5e.

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