Use of an agent that promotes expression of or enhances the function of atad3a in the preparation of a medicament for preventing or treating diabetic cardiomyopathy

Abstract

This application discloses the use of reagents that promote ATAD3A expression or enhance ATAD3A function in the preparation of drugs for the prevention or treatment of diabetic cardiomyopathy. Experiments in this application revealed that ATAD3A was significantly downregulated in myocardial samples and models of diabetic cardiomyopathy. Further in vivo animal experiments confirmed that increasing ATAD3A expression or enhancing ATAD3A function improved cardiac function, reduced myocardial hypertrophy and fibrosis, and significantly reduced the number and volume of myocardial lipid droplets in diabetic cardiomyopathy models. ATAD3A also inhibits excessive enhancement of stress-induced myocardial hypertrophy (MAM) and alleviates mitochondrial Ca²⁺ oxidase. + It overloads and restores mitochondrial energy homeostasis. Therefore, ATAD3A can intervene at key pathological stages of diabetic cardiomyopathy, with a clear target basis and therapeutic value.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to the use of reagents that promote ATAD3A expression or enhance ATAD3A function in the preparation of drugs for the prevention or treatment of diabetic cardiomyopathy. Background Technology

[0002] Diabetic cardiomyopathy is a common and insidiously progressive cardiovascular complication in diabetic patients. Its core pathological features include myocardial metabolic abnormalities, lipid deposition, mitochondrial dysfunction, oxidative stress, myocardial hypertrophy, fibrosis, and impaired systolic and diastolic function. Current clinical treatment mainly focuses on controlling blood glucose and blood pressure and improving heart failure symptoms, lacking specific interventions directly targeting myocardial metabolic rigidity and lipotoxic damage in diabetic patients. In diabetic states, cardiomyocytes often exhibit increased fatty acid uptake but decreased oxidative utilization efficiency, leading to lipid droplet accumulation, abnormal accumulation of acylcarnitine, lipid peroxidation, and decreased mitochondrial membrane potential. Abnormal enhancement of the mitochondrial-associated endoplasmic reticulum membrane (MAM) and mitochondrial Ca²⁺ are also observed. + Overload is also considered an important mechanism driving mitochondrial stress and functional decompensation in diabetic cardiomyocytes. ATAD3A is an AAA+ ATPase protein located at mitochondrial membrane and organelle contact sites, with the potential to regulate mitochondrial structure, organelle interactions, and metabolic homeostasis. Although current technologies suggest that ATAD3A is associated with myocardial ischemia-reperfusion injury, its expression changes and cardioprotective effects in diabetic cardiomyopathy have not been systematically disclosed. Summary of the Invention

[0003] The purpose of this application is to provide an intervention strategy for diabetic cardiomyopathy with ATAD3A as the core target, specifically providing a new use of reagents that promote ATAD3A expression or enhance ATAD3A function in the preparation of drugs for the prevention and / or treatment of diabetic cardiomyopathy.

[0004] To achieve the above objectives, this application adopts the following technical solution:

[0005] This application provides the use of reagents that promote ATAD3A expression or enhance ATAD3A function in the preparation of drugs for the prevention and / or treatment of diabetic cardiomyopathy.

[0006] Furthermore, the reagents for promoting ATAD3A expression or enhancing ATAD3A function include at least one of the following: small molecule compounds, recombinant ATAD3A protein, ATAD3A gene, ATAD3A gene delivery vector, ATAD3A gene overexpression vector, ATAD3A gene overexpression cells, ATAD3A mRNA liposomes, and CRISPR-Cas9 gene knock-in system.

[0007] Furthermore, the reagent that promotes ATAD3A expression or enhances ATAD3A function is a CRISPR-Cas9 gene knock-in system, and the sgRNA sequence of the CRISPR-Cas9 gene knock-in system is shown in SEQ ID NO: 1.

[0008] Furthermore, the reagents for the CRISPR-Cas9 gene knock-in system include expression vectors for the Cas9 protein and the ATAD3A gene.

[0009] Furthermore, the drug includes a pharmacologically active ingredient and pharmaceutically acceptable excipients, wherein the pharmacologically active ingredient is a reagent that promotes ATAD3A expression or enhances ATAD3A function.

[0010] Further, the pharmaceutically acceptable excipients are one or more of the following: diluents, binders, wetting agents, lubricants, disintegrants, solvents, emulsifiers, cosolvents, preservatives, pH adjusters, osmotic pressure adjusters, surfactants, coating materials, antioxidants, and buffers.

[0011] Furthermore, the dosage form of the drug is an oral formulation or an injectable formulation.

[0012] Compared with the prior art, this application has the following beneficial effects: This application is the first to discover and confirm ATAD3A as a therapeutic target for diabetic cardiomyopathy. In a mouse model of diabetic cardiomyopathy, administration significantly improved cardiac function, reduced myocardial hypertrophy and fibrosis, and significantly decreased the number and volume of myocardial lipid droplets. ATAD3A enhances ATAD3A function, inhibits abnormally enhanced myocardial hypertrophy (MAM) under diabetic stress, and alleviates mitochondrial calcium deficiency. + Overload reduces myocardial lipid deposition and lipotoxic damage, thereby improving myocardial remodeling and cardiac dysfunction associated with diabetic cardiomyopathy. Attached Figure Description

[0013] Figure 1This study aims to detect changes in ATAD3A expression in diabetic cardiomyopathy. A shows the immunofluorescence and immunohistochemical staining of ATAD3A expression in myocardial samples from non-diabetic cardiomyopathy (Non-DbCM) and diabetic cardiomyopathy (DbCM). B shows the immunofluorescence staining of ATAD3A expression in myocardial tissue from normal diet (NCD) mice and HFD-STZ-induced diabetic mice. C shows the statistical results of the relative intensity of ATAD3A immunofluorescence in A. D shows the statistical results of the integrated optical density of ATAD3A immunohistochemistry in A. E shows the statistical results of the relative intensity of ATAD3A immunofluorescence in B. F shows the Western blot detection and quantification results of ATAD3A protein in myocardial tissue from NCD and HFD-STZ mice. G shows the Western blot detection and quantification results of ATAD3A protein in HL-1 cardiomyocytes treated with normal control (NC) and high glucose-high lipid (HGPA).

[0014] Figure 2 ATAD3A enhancement significantly improves HFD-STZ-induced diabetic cardiomyopathy; A shows representative echocardiograms of wild-type (WT) and ATAD3A-enhanced mice under normal diet (NCD) or HFD-STZ treatment conditions; B shows Masson staining (top) and WGA staining (bottom) of myocardial tissue from the WT group, WT-HFD-STZ group, ATAD3A-enhanced group, and ATAD3A-enhanced-HFD-STZ group. Detailed Implementation

[0015] To make the technical solution of this application clearer and easier to understand, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0016] Unless otherwise specified, the experimental or testing methods described in the following examples are conventional methods; the reagents and materials described are obtained from conventional commercial sources unless otherwise specified.

[0017] Example 1: Detection of ATAD3A expression changes in diabetic cardiomyopathy This embodiment examines the expression changes of ATAD3A in diabetic cardiomyopathy from three levels: human myocardial samples, animal models, and cell models.

[0018] Human myocardial samples included non-diabetic cardiomyopathy control samples and diabetic cardiomyopathy samples. According to a joint statement issued by the European Heart Failure Association (HFA) and the European Society of Cardiology (ESC), diabetic cardiomyopathy is defined as myocardial systolic and / or diastolic dysfunction occurring in the context of diabetes, and this definition is not limited by the presence or absence of other risk factors or cardiovascular disease. Myocardial tissue sections were subjected to ATAD3A immunofluorescence staining and ATAD3A immunohistochemical staining, and the fluorescence intensity and immunohistochemical integrated optical density of ATAD3A were quantified using ImageJ. The results are as follows: Figure 1 A, Figure 1 C and Figure 1 As shown in D.

[0019] The animal model used was a type 2 diabetic mouse model induced by a high-fat diet combined with streptozotocin (HFD-STZ). Control group mice were fed a normal diet and injected with an equal volume of the solvent. Experimental group mice were fed a high-fat diet for 4 weeks, followed by intraperitoneal injection of a low dose of streptozotocin (STZ). Mice were fasted for 8-10 hours before each injection, and the STZ dose was 50 mg / kg, administered for 5 consecutive days. Random blood glucose was measured three times within 2 weeks after injection. If blood glucose was >16.7 mmol / L in all three cases, the diabetic model was considered successfully established. Individuals that did not meet the standard received an additional STZ injection, with the same criteria; those still not meeting the standard were excluded. After successful modeling, mice continued to be fed a high-fat diet for 8 weeks for subsequent phenotypic evaluation and mechanistic studies. Blood glucose, weight, diet, and general condition were monitored regularly during the modeling period. After modeling, myocardial tissue was collected from mice, and ATAD3A protein expression levels were detected using ATAD3A immunofluorescence staining and Western blot. Fluorescence intensity and protein band gray-scale quantitative analysis were performed, and the results are shown below. Figure 1 B Figure 1 E and Figure 1 As shown in F.

[0020] Cell models were established using HL-1 cardiomyocytes in vitro. HL-1 cells were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. An in vitro glucose / lipotoxicity model was established using high-glucose-high-lipid (HGPA) treatment, with a glucose concentration of 40 mM and a final palmitic acid (PA) concentration of 250 µM for 48 h. The control group was treated with isotonic / iso-volume BSA vector. Normal control (NC) cells were cultured under standard conditions. Cellular proteins were collected after treatment, and ATAD3A protein expression levels were detected by Western blot. Quantitative analysis was performed using GAPDH as an internal control. Results are shown below. Figure 1 As shown in G.

[0021] The results showed that, compared with the corresponding controls, ATAD3A was significantly decreased in myocardial samples from diabetic cardiomyopathy patients, HFD-STZ-induced mouse myocardial tissue, and HGPA-treated HL-1 cardiomyocytes, suggesting that the decrease in ATAD3A is associated with myocardial injury in diabetic patients.

[0022] Example 2: ATAD3A Enhancement Improves HFD-STZ-Induced Diabetic Cardiomyopathy This embodiment uses a high-fat diet combined with streptozotocin (HFD-STZ) to induce a diabetic cardiomyopathy model and evaluates the effect of ATAD3A enhancement on improving cardiac function and myocardial remodeling in diabetic cardiomyopathy.

[0023] In this embodiment, based on the mouse Atad3a gene structure and transcript information, the mouse Atad3a-201 transcript (ENSMUST00000030903.11) was selected as the target transcript for gene knock-in model construction. This transcript encodes the 591-amino acid (aa) Atad3a protein. When constructing the C-terminal 6×Flag fusion expression cassette, the stop codon of the Atad3a coding sequence was removed, and the Atad3a coding sequence and the 6×Flag tag coding sequence were kept in the same reading frame, thus obtaining the Atad3a-6×Flag fusion expression sequence. The 6×Flag tag was used for Atad3a protein expression detection and subsequent experimental studies. This embodiment uses the CAG promoter to drive exogenous Atad3a gene expression. The CAG promoter is approximately 1.7 kb in length and can drive the expression of the target gene in various mouse tissues and cells. Its reference nucleic acid sequence is recorded in the GenBank database, accession number GU299216.1 (positions 3-1664). An expression cassette containing the CAG promoter, the Atad3a coding sequence, a 6×Flag tag, and a PolyA termination signal was used as the donor fragment to construct an Atad3a overexpression knock-in mouse model. In this embodiment, the mouse H11 locus was selected as the site-specific knock-in site for the exogenous gene. The H11 locus is located on mouse chromosome 11 and is a safe site for the integration of the exogenous gene. By integrating the target expression cassette into the H11 locus, stable expression of the exogenous Atad3a gene is achieved, minimizing the impact on the normal function of the endogenous gene. In this embodiment, CRISPR / Cas9 gene editing technology was used to insert the CAG-Atad3a-6×Flag-PolyA expression cassette into the mouse H11 locus. Specifically, sgRNA targeting the H11 site was obtained through in vitro transcription. A donor vector containing the CAG-Atad3a-6×Flag-PolyA expression cassette was constructed. Cas9, sgRNA, and the donor vector were microinjected into C57BL / 6JGpt mouse zygotes, allowing the CAG-Atad3a-6×Flag-PolyA expression cassette to integrate specifically into the H11 site. F0 generation mice were obtained via embryo transfer. Genotyping of the F0 generation mice was performed, and the correct insertion and genome integration of the exogenous expression cassette were verified using PCR, sequencing, and Southern blot methods. Positive F0 generation mice with the correct genotype were selected. These positive F0 generation mice were then mated with C57BL / 6JGpt mice, and the offspring genotypes were identified to obtain stably heritable F1 generation positive mice, thus constructing a CAG-Atad3a-6×Flag-PolyA H11 site knock-in mouse model.The sgRNA targeting sequence used for CRISPR / Cas9 gene editing is 5′-CTGAGCCAACAGTGGTAGTA-3′ (SEQ ID NO: 1), and the PAM sequence adjacent to this sgRNA targeting sequence on the genomic target DNA is 5′. AGG 3′, This sgRNA, together with Cas9 and a donor vector containing the CAG-Atad3a-6×Flag-PolyA expression cassette, was used for the above-mentioned site knock-in.

[0024] Specific experimental protocol and results: Eight-week-old male WT mice or male ATAD3A-KI mice were randomly divided into four groups: WT-NCD, WT-HFD-STZ, ATAD3A-KI-NCD, and ATAD3A-KI-HFD-STZ. The HFD-STZ model was established as follows: mice were induced to have metabolic abnormalities through a high-fat diet, followed by streptozotocin treatment to induce a diabetic state; mice in the normal diet group were given a standard diet and injected with an equal volume of solvent. For implementations using drugs or gene delivery vectors to enhance ATAD3A, reagents that promote ATAD3A expression or enhance ATAD3A function can be administered before, during, or after modeling. Administration methods include intravenous injection, intraperitoneal injection, local myocardial delivery, or other methods suitable for myocardial targeted delivery. After modeling, ejection fraction, fractional shortening, and diastolic function indices were detected by echocardiography; cardiomyocyte hypertrophy was detected by WGA staining; and myocardial fibrosis was detected by Masson staining. The results showed that HFD-STZ treatment resulted in impaired cardiac function and myocardial remodeling in WT mice; compared with the WT-HFD-STZ group, representative echocardiograms of the ATAD3A-KI-HFD-STZ group showed improved cardiac motion status, such as... Figure 2 As shown in Figure A; Masson staining and WGA staining results showed a reduction in the degree of myocardial fibrosis and cardiomyocyte hypertrophy, as... Figure 2 As shown in Figure B. These results indicate that enhanced ATAD3A can improve HFD-STZ-induced diabetic cardiomyopathy-related cardiac dysfunction and myocardial remodeling.

[0025] The above description is merely a preferred embodiment of this application and is not intended to limit this application in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from this application, and these improvements and additions should also be considered within the scope of protection of this application.

Claims

1. The use of reagents that promote ATAD3A expression or enhance ATAD3A function in the preparation of drugs for the prevention and / or treatment of diabetic cardiomyopathy.

2. The application according to claim 1, characterized in that, The reagents that promote ATAD3A expression or enhance ATAD3A function include at least one of the following: small molecule compounds, recombinant ATAD3A protein, ATAD3A gene, ATAD3A gene delivery vector, ATAD3A gene overexpression vector, ATAD3A gene overexpression cells, ATAD3A mRNA liposomes, and CRISPR-Cas9 gene knock-in system.

3. The application according to claim 1, characterized in that, The reagent that promotes ATAD3A expression or enhances ATAD3A function is a CRISPR-Cas9 gene knock-in system, and the sgRNA sequence of the CRISPR-Cas9 gene knock-in system is shown in SEQ ID NO:

1.

4. The application according to claim 3, characterized in that, The reagents used in the CRISPR-Cas9 gene knock-in system include the Cas9 protein and the donor vector for the ATAD3A gene expression cassette.

5. The application according to claim 1, characterized in that, The drug comprises a pharmacologically active ingredient and pharmaceutically acceptable excipients, wherein the pharmacologically active ingredient is a reagent that promotes ATAD3A expression or enhances ATAD3A function.

6. The application according to claim 5, characterized in that, The pharmaceutically acceptable excipients are one or more of the following: diluents, binders, wetting agents, lubricants, disintegrants, solvents, emulsifiers, cosolvents, preservatives, pH adjusters, osmotic pressure adjusters, surfactants, coating materials, antioxidants, and buffers.

7. The application according to any one of claims 1 to 6, characterized in that, The drug is available in oral or injectable form.

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